Applying small quantities of multiple compounds to defined locations of in vitro brain slices

When studying in vitro brain slices, rapidly applying multiple agonists, antagonists, drugs, or modulatory compounds is a significant technical problem. There are three major ways that multiple compounds are applied to slices: by bath, via a U-tube device, or by pressure application using a "puffer" pipette. Each of these methods has advantages and disadvantages, making each more appropriate for particular purposes. Because puffer pipettes have a small, sharp tip, they are best suited to apply a small quantity of a compound to a well-defined location within the slice. When used in this way, puffer pipettes have two shortcomings. Solution leaking from the tip of the pipette can contaminate the signal, and it is difficult to apply more than one test solution to exactly the same local area of the slice. We describe methods and newly designed devices aimed at overcoming those limitations. Relatively inexpensive approaches are described to apply eight different solutions to the same exact location deep within a brain slice. The validity of the approach is verified by measuring ligand-gated channel currents activated by glutamate (Glu), acetylcholine (ACh), and gamma-amino butyric acid (GABA).