Patch-clamp recording filling

Since the neurons targeted by whole-cell patch clamp recording were also filled with biocytin (0.2 %) we were also able to identify the shape and position of these P -GFP neurons within the cortical network. We used streptavidin conjugated to a red fluorescent probe (Alexa 568). Streptavidin reacts directly with biotin (biocytin) within the filled neuron, labeling only the filled neuron in the red fluorescence channel of the wide-field microscope. Typically we obtained recovery percentages of 80-100 % of neurons using the protocol below. 

Figure 4. (a) Planar bilayer membrane system for single-channel currents measurement. Soybean lecithin in n-decane was applied to a hole separating two aqueous chambers. Chambers were filled with metal chloride salt at pH 7.2. The voltage was applied to the outer cell with respect to the inner. The currents across the bilayer were recorded on a PCM recorder through a patch-clamp amplifier and a lowpass filter, (b) Typical records of current observed at -t-50.0 mV (symmetrical 0.5 M solution). Currents increase upward from the zero level shown by the dotted line in each panel. 

Measuring the electrode resistance The filled patch pipette is mounted on the holder. At this point, a positive pressure is applied to the pipette, to avoid the tip collecting dust when it crosses the air-water interface when it is introduced into the bath, or collecting debris from the preparation when it is already in the bath. Meanwhile, a small voltage signal, such as 2 mV and 2 ms pulses, is input to the patch-clamp amplifier command circuit. The recorded current signal will mainly correspond to the holder and pipette associated capacity, and no resistive current is observed (Fig. 5A). 

Direct CNS Stimulation. An alternative to the standard suction electrode stimulation of the peripheral nerve is direct stimulation of the CNS (Nishikawa and Kidokoro 1995). A microelectrode filled with 3-4 M KCl or potassium acetate is inserted into the middle of the ventral ganglion and positive pulses of approximately 2 pA in intensity and approximately 2 msec in duration are delivered (Dietcher et al. 1998). Synaptic transmission is recorded in the patch-clamped muscle in the standard configuration. 

Focal recording electrodes are pulled from glass capillary tubes (e.g., 75 il, 1.5-mm outer diameter) and then fire polished and shaped on a microforge to allow perpendicular approach to the muscle for obtaining better seal resistance. The loose-patch electrodes are made with an inner diameter of 5-10 pm. When filled with the appropriate bath solution, they have resistances of 0.5-2 M 2. Seals around the boutons are made by applying mild suction and usually increase the resistance two to six times (seal factor Rivosecchi et al. 1994). Recordings are made with a loose patch-clamp amplifier and contain a calibration pulse to measure electrode series and seal resistances. These measurements are used to correct for attenuated current amplitudes at the electrode tip. 

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