User manual

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10.3 Automated Single-Line Perfusion Patch-Clamp Experiments
10.3.1 Automated Fast Perfusion Changes During and Between Sweeps
In WinLTP 2.00 we have also added the capability of rapidly changing perfusion solutions DURING as
well as BETWEEN sweeps to enable automated perfusion control for patch-clamp experiments. The key
to this capability is that, In addition to rapidly changing solutions during sweeps, which most
electrophysiological data acquisition systems do, WinLTP also uses the Perfuse statement in the Protocol
Builder to also change solutions at specific times between sweeps. This capability, along with the
changes during the sweep, provides full rapid Automated Perfusion Changes for a patch-clamp
experiment, with the one exception of increment/decrement of stimulation values (which we are working
on).
In addition to WinLTP, Molecular Devices’ pClamp and Heka’s PatchMaster also have the basic capability
to automatically change perfusion solutions between sweeps. PClamp uses Sequence Keys to set
analog or digital outputs, and PatchMaster uses ‘Set DAC’ or ‘Set Digital Word’ events within its Protocol
Editor.
Slow perfusion changes BETWEEN sweeps, which is appropriate for extracellular slice experiments, has
already been discussed in Section 10.2. And the basic perfusion systems from ALA Scientific, Automate
Scientific, BioScience Tools and Warner have also been discussed in Section 10.2.
This section deals with rapid or fast perfusion changes DURING and BETWEEN sweeps which is more
appropriate for patch-clamp experiments where solutions changes not only need to be made between
sweeps, but also include rapid solution changes during the sweep. It is designed to work with the
standard perfusion system (not a pre-flush syem) where 8 or 16 tubes go into a manifold which goes a
short distance to a ca 100 uM pipette and onto a cell and into the chamber (Fig. 10.3.1.1). Pre-flushing is
not usually required in patch-clamp experiments because the solutions are not bicarbonate buffered, and
therefore loss of oxygen across the tubing wall and subsequent change in pH are not a problem. In
single-line patch-clamping experiments, this biggest problem is the dead volume is between the
manifold and the end of the pipette in the chamber (shown in red in Fig. 10.3.1.1), because this
affects how quickly perfusion solutions can be changed.
Fast Perfusion changes using a single perfusion line involve changes to a new solution lasting typically
tens of millseconds up to a second or more. This relatively long perfusion time is due to needing to wash
out the dead volume between the manifold and the tip of the pipette in the chamber. For faster solution
changes typically down to a millisecond in the new solution, a mechanical stepper device to move
between solution pipes is required (Section 10.4). Also, the single-line fast perfusion requires more
expensive fast valves such as Lee valves rather than the less expensive pinch valves that are suitable for
extracellular and stepper/pinch valve perfusion systems. There are some relatively inexpensive steppers
such as the Warner SF-77, which coupled with inexpensive pinch valve controller system, may not cost
much more than a fast valve single-line perfusion system, and have solution changes down to a
millisecond in the new solution for the fastest piezo steppers. Note: we have not tried the Warner SF-77
and do not recommend, we just suggest that you might want to consider it.