For electrophysiological recordings, an EPC-9/2 patch-clamp amplifier (C-board version, HEKA) was used. Electrical currents were sampled on a Macintosh Quadra 950 computer (256 MB RAM) using the software Pulse (V8.50 with lock-in extension, HEKA) running on a Mac OS D2-9.1 operating system. For further analysis, the data was transferred to a Dell Optiplex GX260 PC (2.4 GHz, 512 MB RAM; Dell), and loaded into the analysis software Igor Pro (Wavemetrics Inc., Lake Oswego OR, USA).
The two headstages containing the preamplifiers were mounted on piezoelectric micromanipulators (Piezosystems Jena, Jena, Germany), and the coupled pipette holders were airtight connected to air pressure sensors, and a mouthpiece for oral pressure adjustment. Ag/AgCl electrodes connected the preamplifiers with the intracellular solution in the patch pipette, and via a bath electrode with the extracellular solution.
Patch-pipettes were pulled from borosilicate capillaries (2 mm diameter, 0.3 mm wall thickness) with a vertical two-step pipette puller (HEKA Electronik, Lambrecht/Pfalz, Germany). The heat-polished pipette tips had a diameter of about 1 µm, giving a pipette resistance of 4.0 – 5.5 MΩ, and 2.5 – 3.5 MΩ for pre- and postsynaptic recordings, respectively. To minimize the pipette capacitance, and to slow down capacitive transients for easier software capacitance compensation, the outer pipette wall was covered with an isolating synthetic resin (Sylgard Silicon RTV, Sinus Electronics GmbH, Untereisesheim, Germany; Edwards et al., 1989).
Calyces of Held were identified under infrared illumination, and also fluorescence images of Ca2+ indicator dye filled presynaptic terminals confirmed correct identifications. For paired pre- and postsynaptic recordings, pipettes were filled with the according intracellular solutions (see below), and since high concentrations of the Ca2+ chelator DM-nitrophen (DMN; chapter 2.3) make sealing difficult, presynaptic pipettes were dipped into a DMN-free intracellular solution, and then filled from the back with the DMN-containing solution. Slight pressure was applied to the pipette when approaching the cell, being smaller for presynaptic recordings, in order to prevent the dipping solution without DMN to be blown out too quickly.
Releasing the pressure, or for the presynapse an abrupt change to slightly negative values, led to tight and stable seals reaching a resistance of several GΩ. Having obtained a GΩ seal, short suction pulses ruptured an opening into the cell membrane underneath the tip of the patch pipette. The achieved access resistance was about two times larger than the mere pipette resistance (Marty and Neher, 1995), being 8 – 25 MΩ and 3 – 9 MΩ for pre- and postsynaptic recordings, respectively. Diffusion-driven exchange of soluble components led
to a replacement of the cytoplasm by the pipette solution (Pusch and Neher, 1988). In this way, fluorescent dyes, and photolabile Calcium chelators could be introduced into the cell.
During presynaptic sealing, the extracellular fluid was changed from standard to the wanted toxic composition, and after attaining paired whole-cell configurations, stimulation protocols were started with a delay of at least 3 minutes. This allowed intra- and extracellular toxins fully to take effect, and all components of the artificial intracellular solutions to properly wash-in.
For cellular recovery from activity, stimuli where separated by at least one minute for short depolarizations up to 16 ms, at least 1.5 minutes for depolarizations up to 50 ms, and at least 2 minutes for all other stimuli including Ca2+ uncaging.
Recordings were taken only as long as the leak current was smaller than 100 pA for presynaptic, or 500 pA for postsynaptic recordings, typically ranging between 50 pA and 80 pA, and between 100 pA and 300 pA, respectively. Also, rundown of cellular activity was always checked by comparison of peak EPSC amplitudes and presynaptic pool size estimates from deconvolved postsynaptic traces (chapter 2.4.1). As soon as the amount of vesicles released decreased compared to stimuli of similar intensity, data was discarded.
To compensate for charging transients of the pipette wall, and of the cell membrane, both were corrected for by using the internal, software controlled compensation circuits of the EPC-9/2 amplifier. In the cell-attached configuration, the pipette capacitance and series resistance could nicely be equilibrated, and in the whole-cell mode the cell membrane capacitance and membrane resistance were compensated, too. To correct for membrane voltage errors due to high access resistances to the cell, the automatic EPC-9/2 Rs -compensation has been used (time constant of 10 µs), and was set to 50 % for presynaptic, and to 50 % - 90 % for postsynaptic recordings. The recorded traces were corrected for this off-line. Also, charging transients, arising when the membrane potential was changed, were estimated using a standard p/4 protocol for each stimulus applied, and presynaptic passive components were subtracted off-line.
Under whole-cell voltage-clamp conditions, membrane currents were sampled at 50 kHz, the holding potential was -80 mV presynaptically, and -70 mV postsynaptically. For presynaptic sine+DC membrane capacitance measurements, a 2 kHz, 70 mV peak-to-peak sine wave was applied in the amplifier’s lock-in mode (Wölfel et al. 2003).
Depolarizing presynaptic stimuli always clamped the presynaptic voltage to 0 mV for efficient Ca2+ entry into the cell through voltage-gated Ca2+ channels. In many cases, steps to 0 mV were preceded by a 4 ms jump to +80 mV to open the Ca2+ channels at a voltage, where the driving force for Ca2+ to enter the cell is low. Thus, without triggering vesicle release beforehand, Ca2+ entry became more efficient for a subsequent step to 0 mV.
Intracellular solutions
The production of the intracellular solutions was partitioned into two steps. First, a two times concentrated stock solution without CaCl2, MgCl2, Fura-2FF or DMN was made for storage up to three month at -20°C. Using this stock solution, the final intracellular solution was mixed every one to two weeks, adding the other components remaining.
intracellular solutions presynaptic for
Ca2+ uncaging
presynaptic for depolarizations
postsynaptic
substance
Cs-gluconate 110 – 120 130 130
TEA-Cl 20 20 20
HEPES 20 10 10
EGTA - 0.2 5
Na2-phosphocreatine - 5 5
Mg-ATP - 4 4
Na2-ATP 5 - -
Na2-GTP 0.3 0.3 0.3
CaCl2 1.3 - -
MgCl2 0.5 - -
Fura-2FF (K+ salt) 0.1 - -
DMN (4 Na+ salt) 1.5 - -
pH 7.2 7.2 7.2
osmolarity (mOsm) 315 - 330 315 - 330 300 - 320
Table 2-2 Intracellular solutions (concentrations in mM)
Cesium-gluconate was not commercially available, and therefore self-made according to Meyer (1999). CaCl2 and MgCl2 were purchased from Merck (Darmstadt, Germany), Fura-2FF (K+ salt) from Tef-Labs (Austin, UK), and DMN (4 Na+ salt) from Calbiochem (Darmstadt, Germany). All other chemicals were bought from Sigma (Steinheim, Germany).