Perfusion System

A low-noise peristaltic perfusion pump (PPS2, Multi Channel Systems MCS GmbH, flow rate: 0.3 ml/min) was fed into a micro ML-6 manifold (Warner Instruments, Harvard apparatus) that received also 5 PE-50 tubing from a 6-valve computer-controlled gravity system (VC-6M Valve Control System, Warner Instruments, Harvard Apparatus). Each input was connected to a 20 ml syringe filled with treatment solutions (Figure 1). Each of the channels of the gravity system was height adjusted to match the flow rate of the peristaltic pump (0.3 ml/min). The outlet of the micro manifold was then connected to the animal’s head through the PE-50 tubing as described above.

In our initial experiments with the suction-system provided by the PPS2 pump system to maintain a constant level of fluid in the head capsule, we found both vibration and electrical noise to be problematic as the suction system cyclically made and broke contact with the meniscus in the perfused area. Subsequently we solved this problem to maintain a constant fluid level in the head capsule through a capillary-based system, whereby we placed the tip of a thin cotton thread on the right side of the head with the other extremity in a glass container to collect the liquid (Figure 1). The constant capillary action in this thread gently draws fluid away from the perfused area at a rate high enough to allow the entire volume of the interior head capsule to be changed approximately every 10 s (0.3 ml/min). After experimenting with several different threads for this purpose, we found that a pre-washed and wetted tea-bag string (Twinings, United Kingdom) provided an ideal compromise between size and capillary action.