Protocol

Dye Loading with Patch Pipettes

Adapted from Imaging in Neuroscience and Development (eds. Yuste and Konnerth). CSHL Press, Cold Spring Harbor, NY, USA, 2005.

INTRODUCTION

This protocol describes the loading of individual cells with fluorescent probes via patch pipettes. The patch-clamp methodology has been successfully used for single-cell dye labeling in cultured neurons, brain slices, and in vivo preparations. A broad range of dyes can be used with this loading technique. Markers for morphological reconstruction (e.g., Lucifer yellow); ion-sensitive indicator dyes for monitoring second-messenger cascades (e.g., fura-2); and dye-labeled proteins for fluorescence resonance energy transfer (FRET), fluorescence correlation spectroscopy (FCS), and fluorescence recovery after photobleaching (FRAP) studies are all suitable for patch-clamp loading. The most widespread application of this technique has been for Ca²⁺ imaging. Whole-cell patch-clamp recordings represent a versatile loading technique that allows combined electrophysiological and optical measurements at a quantitative level.

RELATED INFORMATION

This protocol assumes familiarity with the standard procedures of whole-cell patch-clamp recordings (see, e.g., Marty and Neher 1995). For details on the standard electrophysiological equipment required, see Penner (1995). A protocol for Preparation of Rodent Hippocampal Slice Cultures (Fuller and Dailey 2007) is available.

MATERIALS

Reagents

Biological sample

Acutely dissociated cells, cultured cells, acute brain slices (see Preparation of Rodent Hippocampal Slice Cultures [Fuller and Dailey 2007]), or in vivo preparations are suitable. In some preparations, white matter, glia cells, and/or the perineural net may hinder successful patch-clamp recordings.

Pipette solution for current-clamp recordings

Pipette solution for voltage-clamp recordings can be used as an alternative (see Step 1).

Standard external saline

Equipment

Electrophysiological equipment (e.g., amplifier, manipulator, pipette holder, etc.)

Microloaders (Eppendorf)

Microscope, epifluorescence

Pipettes

It is important to choose the right kind of pipette for this technique. Dye should be loaded with borosilicate pipettes (e.g., from Hilgenberg) rather than soft, soda glass pipettes. The latter release trace amounts of cations that can interfere with voltage-gated channels and, potentially, ion-sensitive dyes.

METHOD

1. Using microloaders, fill the chosen pipettes with 5-10 µL of the appropriate dye-containing solution.
Do not use syringe needles to load the pipettes because these may release heavy metals.

2. Use standard techniques to lower the pipette into the recording chamber containing the chosen slice. Apply positive pressure to prevent dust floating on the surface from entering the pipette.

3. Once the pipette tip is in the standard external saline, set the positive pressure to a level that prevents the ejection of too much dye (see Fig. 1A ).

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Figure 1 . (A) Minimal pressure should be applied to the patch pipette while approaching the tissue. A schematic drawing showing the effect of different pressure settings, as viewed through fluorescence optics. (Aa) If no pressure is applied, the standard external saline enters the pipette because of capillary forces. Thus, the composition of the pipette solution is modified, and successful seal formation may become impossible. (Ab) If the pressure applied to the pipette is too high, a strong jet of dye-containing solution is expelled from the pipette tip, and surrounding tissue may become heavily stained. (The standard external saline flows from the top to the bottom.) (Ac) At the correct pressure setting, only a minimal amount of dye is ejected. (B) Dye-loading of distal dendrites. A Purkinje neuron in a cerebellar slice preparation was loaded with the calcium indicator dye fura-2 via a somatic patch pipette. During the whole-cell recording, repeated measurements of the dendritic fluorescence at the calcium-insensitive wavelength were performed. From these data, the times of half-maximal loading were estimated to be ~2.5 min, 16 min, and 30 min for regions 1, 2, and 3, respectively. (Modified, with permission, from Rexhausen 1992.)

4. Apply a brief pulse of stronger pressure immediately before seal formation to clean the surface of the cell.

5. Perform seal formation and establish the whole-cell configuration as per standard electrophysiological recordings
Immediately after the patch is ruptured, the dye will start to diffuse into the cell. The time required to reach a certain cytosolic dye concentration will depend on the mobility of the dye molecule, the dye concentration in the pipette, the series resistance (R_s) of the pipette, and the cell geometry.
See Troubleshooting.

TROUBLESHOOTING

Problem: A difficulty frequently encountered, especially in brain slices, is that R_s tends to increase gradually during whole-cell recordings to levels that seriously hinder dye loading and proper voltage control.

[Step 5]

Solution: Especially for long-lasting experiments, the first minutes of the whole-cell recordings should be devoted entirely to the establishment of a low and stable series resistance by applying suction pulses to the pipette whenever R_s tends to increase. Although sometimes this approach may destroy the whole-cell configuration, it is preferable to lose a cell during the first minutes than to load it for a prolonged time with an unfavorable R_s and compromise on the degree of dye loading.

DISCUSSION

The electrical resistance of the patch pipette (R_P) is of special importance for fast and efficient dye-loading. R_P (which should not be confused with the series resistance) is determined by the conductivity of the pipette solution and the geometry of the pipette (i.e., by the length of the pipette taper and the width of the pipette opening). Thus, R_P can be finely tuned by changing the settings of the pipette puller. Depending on the experimental requirements and the cell type under study, the optimal R_P value may vary considerably. A low R_P permits a fast dialysis of remote cellular regions (Fig. 1B). However, it is more difficult to maintain long-lasting recordings with low-resistance pipettes. A higher R_P should be chosen if multiple measurements at a steadily increasing dye concentration are needed, as for measurements of the calcium-binding ratio with the calcium flux approach.

­

The cell size usually imposes limits on the diameter of the pipette tip and, consequently, on R_P. As a general rule, large cells tolerate larger tips than small cells or fine cellular processes. For instance, patch pipettes with resistances of 2.0-3.5 M (measured with a KCl-based pipette solution) are well suited for long-lasting recordings from the large cerebellar Purkinje cell bodies, which have a diameter of 25-30 µm.

To load remote cellular compartments such as axons or terminal dendrites and spines of central neurons, the time that is required to reach a certain dye concentration depends strongly on their distance from the patch pipette and the geometrical characteristics of the connecting cellular structures. Figure 1B shows an example from a study of dye diffusion in cerebellar Purkinje neurons. In this experiment, the cell was loaded with the calcium-indicator dye fura-2 through a somatic patch pipette for 120 min (R_s = 5.5-6.7 M). The fluorescence of distinct dendritic compartments was monitored during the loading phase, and the time required to reach half of the dye concentration in the pipette solution was estimated (Fig. 1B). Thus, for remote compartments, ~30-40 min of whole-cell recordings may be needed before the dye concentration reaches levels that allow fluorometric recordings with a reasonable signal-to-noise ratio.

In comparison to other staining techniques such as dye loading with membrane-permeable dyes (e.g., acetoxymethyl-type) or dye injection via sharp electrodes, the patch-clamp dye loading technique offers important advantages. First, all cell types that can be studied with the whole-cell patch-clamp technique are suitable for staining with patch-clamp dye loading. This is of crucial importance, especially when studying cells that are too small to tolerate impalement with a sharp electrode, or neurons that are covered extensively by glia cells and therefore cannot be efficiently stained with acetoxymethyl dyes. A further advantage is that the intracellular dye concentration can be predicted precisely, either during the loading phase or after an appropriate loading time, when the cytosolic dye concentration is close to that of the pipette solution. Thus, quantitative studies relying on accurate knowledge of cytosolic dye concentrations are possible (Neher 1995).

There are drawbacks of dye loading via patch pipettes. These are generally the same as those associated with the difficulties of the whole-cell patch-clamp technique; staining tissue with membrane-permeable dyes is obviously an easier technique. Also, cytosolic constituents can wash out during prolonged whole-cell recordings. Finally, there are limits to the size of dye-labeled proteins that can be loaded via pipettes, although proteins of up to 150 kDa have been used successfully.

References

Fuller, L. and Dailey, M.E. 2007. Preparation of rodent hippocampal slice cultures. Cold Spring Harb. Protoc. doi: 10.1101/pdb.prot4848.[Abstract/Free Full Text]

Marty, A. and Neher, E. 1995. Tight-seal whole-cell recordings. In Single-channel recordings (eds. B. Sakmann and E. Neher), 2nd ed, pp. 31–52. Plenum, New York.

Neher, E. 1995. The use of fura-2 for estimating Ca buffers and Ca fluxes. Neuropharmacology 34: 1423–1442.[Medline]

Penner, R. 1995. A practical guide to patch-clamping. In Single-channel recordings (eds. B. Sakmann and E. Neher), 2nd ed, pp. 3–30. Plenum, New York.

Rexhausen, U. 1992. PhD thesis, University of Göttingen, Germany.