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2.3.1 M echanical Fixation of Preparations

Brain slices were fixed to the bottom of the recording chamber using a U- shaped ‘harp’ constructed from 0.5mm platinum wire flattened with a vice and strung with nylon threads (taken from nylon stockings). This device was laid on top of the slice, with the threads running in the Y direction, allowing the maximum possible area available for access by the recording electrode (which approached along the X axis). This configuration allowed the slices to remain stably positioned when superfused with solutions flowing at up to 10ml m in'\

Figure 2.3: Isolated Purkinje cell obtained as described in the text. The remains o f the cell’s primary dendrite and axon can be seen. The soma is approximately 20|im across.

Dendritic Stump

Cell Soma

Isolated neurons, glia or CHO cells were allowed to settle onto the bottom of the recording chamber for a period before perfusion was started, and became fixed to the glass surface. A large number of the cells isolated remained fixed in place after perfiision was begun, but were also loose enough to be pulled from the bottom after patching, in order to isolate them from disturbances due to vibration or electrode movement. This is especially important for recording from isolated cells, as the electrode tip is often less than 10|am from the bottom of the chamber at the time of patching.

2.3.2 Visualisation of Cells

For patch clamp recording experiments, the recording chamber was attached to the fixed stage of an upright microscope (Axioskop, Zeiss, Germany). The slices were viewed with a 40x water immersion objective with a 0.9 NA condenser using Differential Interference Contrast optics. After patching, cells were often monitored for the duration of the experiment using a TV (Hitachi, Japan) fed by a CCD camera (Radio Spares, Japan) attached to the microscope.

2.3.3 Patch Clamp Recording

The microscope and perfusion chamber were situated within a Faraday cage, and mechanically fixed to an air table to provide a vibration free environment in which to record. Patch pipettes were pulled from thin-walled borosilicate glass (GC150TF-10, Clark Electromedical, England) using a two stage cycle on a horizontal puller (BB-CH, Mechanex, Switzerland). The resistance o f the pipettes before sealing was 2-3 M fl when filled with solution F (Table 2.2) and bathed in solution C (Table 2.1). Patch pipettes were inserted into a double-seal polyacrylate holder with a chlorided silver wire forming the electrical connection (Dept, of Pharmacology, UCL,

Axon Instruments, USA). The amplifier head-stage was mounted on a mechanical micromanipulator (MX-1, Narishige, Japan), also fixed to the air table. An Ag/AgCl pellet was used for the earth electrode, except for zero chloride experiments, where the pellet was replaced with a 4M NaCl agar bridge to avoid bath potential changes. lOmV voltage step pulses were applied to the pipette by the patch clamp amplifier (Axopatch 200A, Axon Instruments, USA) to monitor its resistance. After the pipette was submerged in the perfusion medium, the current offset was cancelled to zero. A slight positive pressure was continuously applied, via a tube connected to a mouthpiece, until the pipette was close (around 1 -2pm) to the membrane of the cell to be patched. After a brief period during which the outflow of intracellular solution was used to clean the surface of the cell, the positive pressure was released and small negative pressure was applied until a high-resistance seal (> lGf2) was established. At this point, the electrode was hyperpolarized by 60-80mV and the application of brief pulses of suction to the patch electrode was used to rupture the cell membrane and enter the whole-cell configuration.

2.3.4 Series Resistance Errors

If the voltage drop across the resistance of the patch electrode is significant, series resistance errors will occur, leading to unreliable voltage and current measurements in whole-cell mode. This can occur if either the series resistance is high or the whole-cell currents are large. Table 2.6 (at the end of this chapter) lists typical series resistance (Rs) and holding current (I) values for the different cells I studied.

The voltage drop (I x R ) for the CHO and Müller cells was less than I.5mV, which is

a negligible voltage error, and was not corrected for in the analysis o f the results. However, the voltage error for neurons could be as much as lOmV, and series

resistance compensation was carried out using the controls provided on the patch amplifier, in order to reduce the voltage error to <2mV.

2.3.5 Junction Potential Measurements

The cancellation of the current offset prior to seal formation introduces an error into the measurement of voltages after the whole cell configuration is formed due to a phenomenon known as the liquid junction potential. This arises due to the charge separation which occurs when anions and cations of different mobilities diffuse across the boundary between two solutions. The result is that, after entering the whole cell mode, the actual value of zero volts differs from that indicated on the amplifier by the potential difference which existed between the two (intracellular and extracellular) solutions before seal formation. The measurement of the liquid junction potential was as described by Fenwick et al (1982). First, the recording chamber and pipette are both filled with the internal solution. The amplifier is set to current clamp mode, and the voltage adjusted to zero using the current offset control. The recording chamber is then emptied and filled with the external solution to be tested. The voltage change displayed on the amplifier indicates the potential difference between the two solutions, and can be used to calculate the true holding potential for all experiments in which that particular intracellular/extracellular solution combination was used. For all liquid junction potential measurements, a 4M NaCl agar bridge was used for the earth

electrode to prevent its junction voltage changing.