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10.4 Automated Dual- and Triple-Line/Stepper Perfusion Patch-
Clamp Experiments
In Section 10.2 we showed an automated multi-slice extracellular experiments using automated perfusion
control (with and without pre-flushing line, changing between sweeps) which typically is used to produce
solution changes in the minutes timeframe. In Section 10.3 we showed an automated single-line
perfusion control for patch-Clamp experiments which typically is used to produce solution changes in the
tens of milliseconds to seconds timeframe. In this section we show how to implement perfusion
changes as quick as a millisecond in duration by using a stepper in addition to two or three valve
controllers.
The Dual- and Triple-Line/Stepper Perfusion system need to use only the inexpensive pinch valves used
with Extracellular Slice Perfusion, and not the faster, more expensive valves such as Lee valves that are
required for Single-Line Perfusion. Although many piezo steppers can be quite expensive, some steppers
such as the Warner SF-77 are reasonably priced. If an inexpensive stepper and controllers using
inexpensive pinch valves are used, the price of such a system will be not much more than a Single-Line
Perfusion system. We do not necessarily recommend such a system, but it should be considered.
The addition of stepper control of tube placement allows dual- or triple-line perfusion patch-clamp
experiments for up to 48 solutions.
10.4.1 Automated Dual-Line/Stepper Perfusion
The way that WinLTP can control a Dual-Line/Stepper perfusion system is shown in Fig. 10.4.1.1. Slow0
Perfusion produces digital output on static Port1 to control one valve controller which in turn controls 4 to
16 valves. This Slow0 perfusion is for producing slow perfusion changes BETWEEN sweeps, and
controls the perfusion solution coming out of Tube 0, which usually contains several agonist or agonist +
antagonist test solutions.
Slow1 Perfusion similarly produces digital output on static Port2 to control one valve controller which in
turn controls 4 to 16 valves. This Slow1 perfusion is for producing slow perfusion changes BETWEEN
sweeps, and controls the perfusion solution coming out of Tube 1 which usually contains several
antagonist solutions possibly including normal ACSF (a no antagonist solution).
The Fast0 Stepper produces fast changing output from 4-bits of the high-speed digital Port0, or output
from AnalogOut 1 or AnalogOut 1 & 2 DURING the sweep. Fast0 output controls the Stepper which
quickly moves the two tubes from Tube 1 to Tube 0 and back by controlling a Stepper. If the stepper is a
fast piezo device, solution deliveries from Tube 0 can be as quick as one millisecond in duration.
As with Single-Line Perfusion (Section 10.3.1.1), when designing your experiment you have to determine
whether the antagonist is strongly or weakly bound to the receptor. If the antagonist is strongly bound
and is not displaced during agonist application, then one set of reservoirs can contain different
concentrations of antagonist and another minimal set of reservoirs can contain different concentrations of
agonist. If the antagonist is weakly bound and is displaced during agonist application, then one set of
reservoirs can contain different concentrations of antagonist and another much larger set of reservoirs