Title: a Recording Chamber for Small Volume Slice Electrophysiology 3 4

41 42 43 Electrophysiological recordings from brain slices are typically performed in small 44 recording chambers that allow for the superfusion of the tissue with artificial 45 extracellular solution (ECS), while the chamber holding the tissue is mounted in 46 the optical path of a microscope to image neurons in the tissue. ECS itself is 47 inexpensive and thus, superfusion rates and volumes of ECS consumed during 48 an experiment using standard ECS are not critical. However, some experiments 49 require the addition of expensive pharmacological agents or other chemical 50 compounds to the ECS, creating a need to build superfusion systems that 51 operate on small volumes while still delivering appropriate amounts of oxygen 52 and other nutrients to the tissue. We developed a closed circulation tissue 53 chamber for slice recordings that operates with small volumes of bath solution in 54 the range of 1.0 ml to 2.6 ml, and a constant oxygen/carbon dioxide delivery to 55 the solution in the bath. In our chamber, the ECS is oxygenated and re-circulated 56 directly in the recording chamber, eliminating the need for tubes and external 57 bottles/containers to recirculate and bubble ECS, and greatly reducing the total 58 ECS volume required for superfusion. At the same time, the efficiency of tissue 59 oxygenation and health of the section are comparable to standard superfusion 60 methods. We also determined that the small volume of ECS contains a sufficient 61 amount of nutrients to support the health of a standard brain slice for several 62 hours without concern for either depletion of nutrients or accumulation of waste 63 products. 64 65