2.2.1 Fixation and visualisation of slices
Slices were transferred, individually, to the glass-bottomed recording chamber of an upright microscope (BX50WI, Olympus, Japan). To provide mechanical stability, the microscope was placed on a vibration isolation air table (Newport Corporation, CA, U.S.A.), and the slice was held in position by a grid o f 5 - 8 parallel nylon threads glued to a flattened, U-shaped piece of platinum wire (Edwards et aL, 1989).
The recording chamber (volume ~ 1 ml) was continuously superfused with Krebs solution, at a flow rate of 1 - 3 ml / minute. The solution was fed to the recording chamber via gravity flow from a syringe barrel, in which the supply o f solution was continuously bubbled with 95 % O2 / 5 % CO2. Solution was removed from the
suction pump (HYFLO, MedCalf Bros. Ltd., Potters Bar, UK). All drugs, unless otherwise stated, were bath-applied. The effects of drugs were measured when the effects has stabilised; this was typically 3 - 8 minutes after the drug solution first arrived at the recording chamber.
Slices were viewed on a television screen, with a video camera (Hamamatsu C2400, Japan, or Hitachi KP-MIE/K, Japan), using either a 40 x or a 60 x water immersion objective and a 0.9 numerical aperture condenser, under infrared differential interference contrast microscopy.
Purkinje cells were easily identified by their shape, size and position in the slice. Putative Lugaro cells were identified, by their position in the slice and the shape of their proximal dendrites, whilst viewing the slice with a 60 x objective. Firstly, their somata lay in or immediately below the Purkinje cell layer; secondly, their somata were elliptical in shape, and large diameter processes emerged from their poles, in the sagittal plane. As explained in Chapter 6, identifying Lugaro cells in this way does not always distinguish them from other cell types occupying similar positions in the slice, and definitive identification of Lugaro cells was made using confocal imaging (see section 2.4). The molecular layer intemeurones were identified by the size of their somata and their position in the slice. The only other neuronal somata that are found in the molecular layer are those of migrating granule cells, which, in neonatal rats, have not yet reached their positions in the granule cell layer. However, the somata of basket cells and stellate cells ( 7 - 1 0 pm diameter) are larger than those of granule cells (Vincent and Marty, 1996).
2.2.2 Patching procedure
All recordings were made at room temperature (21 - 24 °C). Thick-walled borosilicate glass electrodes (World Precision Instruments, FA, U.S.A.) were pulled on a two-stage electrode puller (PP-83 microelectrode puller, Narishige, Japan), and filled with intracellular solution using a MicroFil (World Precision Instruments, FL, U.S.A.). The resistances of the electrodes were 2 - 4 MQ for Purkinje cell recordings, and 4 - 6 MQ for recordings from Lugaro cells and molecular layer intemeurones. The exact electrode resistance depended upon the intracellular solution used. The composition of the intracellular solutions is given in Table 2.2.
The microscope was placed inside a Faraday cage, in order to prevent externally derived electrical noise from interfering with recordings. A filled recording electrode was inserted into a Teflon electrode holder containing a silver wire, which had been coated with AgCl by previously immersing the wire in bleach for 2 - 6 hours. The headstage of the patch-clamp amplifier was mounted on a micromanipulator (Marzhauser, Wetzlar, Germany; or LNSMl, Luigs and Neumann, Ratingen, Germany). The ground electrode was a AgCl pellet, which was submerged in the Krebs solution in the recording chamber. All recordings were made using either an Axopatch 1-D (Axon Instruments, CA, U.S.A.), or an EPC9/2 (HEKA Elektronik, Lambrecht, Germany) patch-clamp amplifier.
Offset potentials were set to zero when the tip of the recording electrode was submerged in the Krebs solution in the recording chamber. Whilst the electrode was advanced towards the surface of the slice, positive pressure was applied to the
interior of the electrode via a tube connected to the electrode holder. This prevented the tip of the electrode from becoming blocked, and blew away any tissue obscuring the cell of interest. All recordings were made by patching the somata of cells that lay at, or near to, the surface of the slice. Upon touching the membrane o f the cell of interest, the positive pressure was removed and slight suction was applied. The potential of the recording electrode was then set to - 70 mV. The resistance of the electrode was monitored by applying - 10 mV test pulses of 50 ms duration, at a frequency of 2 Hz. The suction gradually (over tens of seconds) resulted in the formation of a high resistance seal (> 1 GQ) between the cell membrane and the tip of the recording electrode. At this point, the suction was released and the recording was in the ‘cell-attached’ configuration. Pipette capacitance compensation was performed, using the pipette capacitance compensation circuitry of the patch-clamp amplifier; the pipette capacitance was typically 5 - 8 pF. In this configuration, action potentials arising from the patched cell could be recorded.
By applying brief, strong pulses of suction to the recording electrode, the patch of cell membrane beneath the electrode tip was ruptured, such that the interior of the electrode was in continuity with the interior of the cell. The recording was then in the ‘whole-cell’ configuration.
2.2.3 Whole-cell voltage-clamp recordings
The holding potential in whole-cell voltage-clamp recordings was set at - 70 mV, unless otherwise stated. The current transients associated with the charging and discharging of the cell membrane capacitance during each - 10 mV test pulse were
cancelled using the whole-cell capacitance compensation circuitry of the patch-clamp amplifier, and the series resistance was then usually compensated by 50 - 80 % (Llano et aL, 1991b). Series resistances in whole-cell voltage-clamp recordings from
Purkinje cells, as estimated during compensation of the cell capacitance, were typically between 10 and 20 MQ. The quality o f the recording was regularly checked throughout experiments, by examining the current response to the - 10 mV test pulse in the absence of cell capacitance compensation and series resistance compensation. If the series resistance changed by more than approximately 20 % during a recording, the recording was terminated.
2.2.4 Quality of whole-cell voltage-clamp control
Whole-cell voltage-clamp experiments on cells that are not spherical, but emit processes such as the extensive dendritic trees of Purkinje cells, suffer from problems of imperfect ‘space-clamp’. This means that, due to the access resistances between the interior of the recording electrode and the cell’s processes, the voltage set in the electrode is not controlled uniformly over the whole cell membrane. The access resistances consist of the resistance between the interior o f the electrode and the cell soma (‘series resistance’), and the axial resistances between the morphological compartments of the cell. Since these resistances are in series with the resistance of the cell membrane, they result in a voltage error, increasing with distance from the recording electrode, in the potential at which the cell membrane is held. Imperfect space-clamp can alter both the apparent amplitude and time course of synaptic currents. Three methods were adopted in the experiments described in this thesis in order to minimise space-clamp problems. Firstly, the cerebella were taken from
young animals. At 14 days after birth, the dendritic trees of rat Purkinje cells are not fully developed, and synaptic currents therefore arise at locations on the cell membrane that are electrotonically closer to the recording electrode than would be the case in Purkinje cells of older animals. Secondly, when possible, a cæsium-based intracellular solution was used; this reduces space-clamp problems by inhibiting potassium ion conductances in the membrane of the recorded cell, increasing the membrane resistance and, thereby, increasing the proportion o f the electrode voltage that is held across the membrane. Finally, as noted above, series resistance compensation was employed. This technique reduces the effective series resistance, allowing the membrane of the recorded cell to be held at a potential closer to that set in the preamplifier.
2.2.5 Extracellular stimulation of svnaptic currents
Stimulating electrodes were pulled, and filled, as described above for recording electrodes (section 2.2.2). In some early experiments, the stimulating electrode contained Krebs solution, but filling the stimulating electrode with 1 M NaCl allowed the use of lower stimulating voltages and gave smaller stimulus artefacts, and therefore NaCl was used in most experiments. The ground electrode of the stimulating circuit was a silver wire, which was submerged in the Krebs solution in the recording chamber. The stimulating electrode was inserted into an electrode holder containing a silver wire, which was coated in AgCl as described in section 2.2.2. The electrode holder was mounted in a micromanipulator (MP-285, Sutter Instrument Co., CA, U.S.A. or MHW-103, Narishige, Japan). Before patching a cell, the tip of the stimulating electrode was placed on the surface o f the slice, such that it
was near to the cell of interest and could be manoeuvred with minimal disturbance of the subsequent recording. No stimuli were yet applied. Having patched the cell, synaptic currents were evoked by applying bipolar, rectangular voltage pulses (100 - 200 ps duration) to the stimulating electrode, at a frequency of 0.5 Hz (Grass SD9 stimulator, Astro-Med, Inc, RI, U.S.A. or A-M Systems stimulator model 200, A-M Systems Inc., WA, U.S.A.).
Synaptic currents were evoked using ‘minimal stimulation’. The stimulating voltage was gradually increased from zero until synaptic currents were observed that displayed an all-or-none relation to stimulus strength. The stimulating electrode was then moved, and this process repeated, until the position was found at which the synaptic currents could be evoked by the weakest stimulus. At stimulating voltages below threshold, no synaptic currents were detectable; the currents appeared at full amplitude when the stimulating voltage reached a threshold level. This relation between the amplitude of evoked synaptic currents and stimulus strength can be used as an indicator that the currents arise through the activation o f a single presynaptic input, or a small number of inputs running very closely together (Schneggenburger and Konnerth, 1992). An example of an input / output curve for minimal stimulation is shown in Figure 5.1C. Synaptic currents were evoked throughout each experiment using a stimulating voltage just above threshold, so as to stimulate only the one input (for example, for the cell from which the data shown in Figure 5.1C were taken, 14 V stimulation was used). Finally, the position of the tip of the stimulating electrode was marked on the television screen. Both this position and the stimulation threshold (as assessed by the input / output curve for the synaptic currents) were regularly
checked throughout each experiment, in an attempt to ensure that the evoked synaptic currents arose from the original input.
2.2.6 U-tube application of drues
Fast application of receptor agonists to slices was achieved using a solenoid valve- controlled U-tube system (Fenwick et aL, 1982). The U-tube was made from
borosilicate electrode glass, bent when heated with a Bunsen burner into a U - shape (angle at bend ~ 45 °). 4 - 5 short pieces of electrode glass were glued from side to side across the U-tube, and a thick metal wire (4 - 6 cm long) was glued across these glass supports. The metal wire formed the handle for the U-tube. In order to form the aperture in the U-tube through which solutions were applied to the slice, a piece of rubber tubing was attached to each end of the U-tube, and one piece of tubing was tied off, A syringe was inserted into the other piece of tubing. The U-tube was then clamped in a retort stand, and placed close to the flame of a Bunsen burner. Application of strong positive pressure to the U-tube, using the syringe, caused a hole to blow out in the bend in the glass, which had been softened by the flame. The U-tube aperture formed in this manner was approximately 100 pm across.
During experiments, the U-tube was mounted, by its metal handle, in an electrode holder on a micromanipulator. Rubber tubing was connected to each end of the U- tube. One piece of tubing led to a beaker, and the other was connected to 4 syringe barrels, each fitted with a tap, such that opening the tap allowed solution to flow, by gravity, through the U-tube to the beaker. The composition of the U-tube solution is given in Table 2.3. Upon switching a solenoid valve, the outlet to the beaker was
closed off for 500 ms, forcing the solution to flow out of the U-tube aperture. Before patching a cell, the U-tube aperture was placed above the soma, and, with a flow of control solution, the U-tube was advanced slowly, until operating the switch caused tissue on the surface of the slice to move slightly. This indicated that the solution from the U-tube was being applied adequately to the slice. In this position, the aperture was usually 50 - 100 pm above the soma of the cell of interest.
Having patched the cell, a 500 ms pulse of U-tube solution was applied and, in order to eliminate mechanical artefacts, the recording was discarded if the cell showed any response. Correct operation of the U-tube was verified by applying GAB A, which always resulted in an inward current (an example is shown in Figure 4.4D). Drugs were then applied in three 500 ms pulses, separated by at least 20 s. During these experiments, both the Krebs solution in the recording chamber and the solution in the U-tube itself contained 0.5 pM tetrodotoxin, in order to prevent presynaptic effects of the applied drugs. When the effects of antagonists were tested on U-tube-evoked responses, the antagonists were present in both the recording chamber solution and the U-tube solution.
2.2.7 Dual whole-cell current-clamp and voltage-clamp recordings
All dual recordings were made using an EPC9/2 patch-clamp amplifier. For dual recording experiments, both headstages were joined to one ground electrode, rather than using one ground electrode per headstage; this was found to reduce the noise in the recordings. The slice was first scanned for two Purkinje cell somata lying within approximately 200 pm of each other. Areas were sometimes found that contained
several Purkinje cells suitable for patching, with superficial somata; in this case, recordings were made from Purkinje cells lying in close proximity to each other. In other cases, suitable Purkinje cells could only be found at greater distances from each other. The distances between recorded cells were estimated by eye, and are reported as the distance between the centres of the two somata. The KCl-based intracellular solution (solution C, see Table 2.2) was used in both recording electrodes. In some cases, as noted in Chapter 8, the concentration of calcium in the Krebs solution was raised to 5 mM.
Two Purkinje cells were first patched as described above for whole-cell voltage- clamp recording, and compensation of both series resistances and cell membrane capacitances was performed (see sections 2.2.2 and 2.2.3). The Purkinje cell in which action potentials were evoked will be called ‘cell 1’, whilst the Purkinje cell which was recorded for synaptic activity will be called ‘cell 2 ’. The recording mode of cell 1 was switched to current-clamp, and the holding current of cell 1 was set so as to hold the membrane potential at - 60 to - 80 mV. The usual protocol for evoking action potentials in cell 1 was to inject a current pulse of + 1 nA amplitude, for 2 - 5 ms, once every second. The duration of the current pulse was varied so as to limit the response to only one action potential. In some experiments, paired-pulse stimulation was used: two identical current pulses were given with an inter-pulse interval of 30 ms, every 1 s. In cases when the recordings of both cells remained sufficiently stable, the recording modes of the two cells were reversed, such that cell 1 was switched back to whole-cell voltage-clamp and cell 2 was switched into whole-cell current-clamp. The stimulating protocols were then repeated. Thus, in
some cases, synaptic connections could be investigated in both directions between the two Purkinje cells.
2.3 Analysis of electrophvsiological recordings
2.3.1 Single cell recordings: data acquisition
Currents were recorded from the amplifier at a bandwidth of 10 kHz (4-pole Bessel filter for the Axopatch 1-D, 3-pole Bessel filter for the EPC9/2). Recordings were either sampled directly onto computer, or were first digitised at 44 kHz onto tape (Fuji Magnetics, Kleve, Germany) with a modified video cassette recorder (Vetter model 200, A.R. Vetter Co. Inc., PA, U.S.A.). For recordings made using the EPC9/2, all recordings were digitised at 32 kHz onto DAT tape (Model VDAT2, Vetron Technology Inc., PA, U.S.A.). Recordings were resampled from tape onto computer through a 2 kHz filter (8-pole Bessel, Frequency Devices, MA, U.S.A.), at a sampling frequency of 10 kHz, with a CED 1401 interface (CED, Cambridge, UK) or an Axon Digidata 1200 interface. In experiments involving the U-tube or extracellular stimulation of synaptic currents, the program WinWCP (kindly supplied by Dr. J. Dempster, University of Strathclyde Electrophysiology Software: www.strath.ac.uk/Departments/PhysPharm/ses.htm) was used for the acquisition and analysis of recordings. WinWCP was triggered to acquire data by the stimulus artefact, or, in U-tube experiments, by the trigger that switched the solenoid valve. 40 ms of pre-artefact baseline were acquired, and each record was 200 ms (evoked synaptic currents) or 5 -1 0 s (U-tube-evoked responses) in total length.
For analysis of spontaneous synaptic currents, recordings were resampled from tape, at a sampling frequency of 10 kHz, using the programme WinCDR (also supplied by Dr. J. Dempster, University of Strathclyde Electrophysiology Software). WinCDR acquired the data as a continuous record, and then performed automatic detection of spontaneous synaptic currents using the following detection criteria: to be classified as a spontaneous current, an event had to cross an amplitude threshold of - 3 pA (+ 3 pA when detecting outward currents) for a duration of 5 ms, with a dead time between consecutive events of 15 ms. The same detection criteria were used for each phase of a recording (for example, in the absence and presence of a drug). Detected events were then inspected by eye, to avoid inclusion of artefacts. Events were accepted if they did not overlap with other synaptic events, their rise times were faster than approximately 10 ms, and their decay phases were slower and appeared approximately exponential. Detected events were saved in the format of a WinWCP file.
2.3.2 Single cell recordings: data analvsis
WinWCP was used for all analyses of synaptic currents from single cell recordings, and for the analysis of U-tube-evoked responses. The zero current baseline was set by averaging 20 sample points approximately 30 ms before the onset of the synaptic current or U-tube-evoked response. Each set of 3 U-tube-evoked responses was averaged, and the peak amplitude of the average was measured with respect to the