A recording chamber for small volume slice electrophysiology

Abstract

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

Keywords: brain slice; in vitro; patch clamp; recording chamber; slice recordings.

Fig. 1.

Combining both slice superfusion and solution oxygenation directly in the recording chambers allows for a significant reduction of the total extracellular solution (ECS) volume required for an in vitro experiment. A: design of the small volume recording chamber showing an angled, near top view of the chamber. B: expanded view of the 3 parts that need to be assembled into a functional chamber. These are the main chamber (B1); the glass coverslip serving as the chamber's bottom (B2); and the gas inflow tube that needs to be inserted into one of the shorter side walls of the main chamber (B3). See materials and methods for the technical specifications of the parts. Scale bar = 24 mm.

Fig. 2.

Technical drawing of the chamber including all measurements in millimeters, and all design features with panels showing the chamber from all six sides. A: side view of the side opposite to the air inflow. B and D: side views of the longer side walls. C: side view of the side that contains the air inflow. E: top view. F: bottom view.

Fig. 3.

Fluid flow speeds varying over 3- to 4-fold range support the signature figure 8 flow pattern of the chamber while minimizing fluid velocity at and near the tissue section. Modeling the flow of solution in the chamber with different flow speeds at the solution outlet port (arrows in A and B). A: lowest fluid velocity (measured at the entrance to the chamber) that still produces the figure 8 liquid circulation, 0.3 ml/s. B: maximal fluid velocity that supports the figure “8” flow pattern. C: plot of various liquid velocities along a longitudinal cross section through the 30-mm long tissue placement compartment of the chamber, based on various initial fluid velocities at the fluid outlet port.

Fig. 4.

The oxygenation mechanism within the chamber is capable of oxygenating the solution in the chamber to supersaturation values and is nearly as effective as standard “bubbling” of solution in an external container with gas dispersion tubes. Dissolved oxygen (DO) concentrations in solution samples that were collected from the chamber after having been in the chamber and exposed to oxygenation for various amounts of time. The initial t = 0 value represents the oxygen concentration of deoxygenated (boiled) water. Dashed line indicates dissolved oxygen in water at equilibrium with atmospheric oxygen.

Fig. 5.

Incubating brain slices in the chamber for prolonged periods of time maintains tissue health, even when afferent fibers are chronically stimulated to trigger action potential firing. The figure shows current-clamp traces recorded from neurons in slices that were incubated in the chamber for various amounts of time (A and B) and had been electrically stimulated for prolonged periods of time (C–F). The traces show responses to current injections of −200 to +800 pA in 100-pA steps (A and B) or current injections of −300 to +800 pA in 100-pA steps (C–F) for a duration of 300 ms. The recordings were performed after tissue sections had been incubated in the chamber for 60 min (A) and 180 min (B) without electrical stimulation. The recordings shown in C and E were obtained directly after establishing a whole cell configuration. Subsequently, the recordings were held for 75 min (D) or 24 min (F) while afferent fibers were stimulated. In the case of E and F an intact direct synaptic connection was verified.

Fig. 6.

Electrical stimulation of brain slices did not deteriorate tissue health. Micrographs of 3 gerbil brain stem slices containing medial nucleus of the trapezoid body (MNTB) neurons. Images were taken at t = 0 (A and C), after 60 min of electrical stimulation (B, D, and E), and after 120 min of electrical stimulation (F). E and F also show a glass pipette that was used for additional patch-clamp recordings. The ages of the animals were postnatal day (P) 11 in A and B; P14 in C and D; and P15 in E and F. Scale bar = 20 μm for A–F.

Fig. 7.

Tissue could be successfully maintained at physiological temperature. Micrographs of a gerbil brain stem slice containing MNTB neurons that was incubated at 37°C. A: t = 0; B: t = 120 min. As a control, the aeration mechanism was turned off in C and D. The age of the animal was P11. Scale bar = 20 μm for A–D.

Fig. 8.

Chromatograms of compounds in ECS using mass spectrometry analysis. A: liquid chromatography-mass spectrometry (LC-MS) total ion chromatogram showing overlap of chamber buffer at T0 (n = 3), T10min (n = 3), and T3hr (n = 3). Levels of glucose and myo-inositol showed no statistically significant changes after 3 h (ANOVA, P = 0.455 and P = 0.367 respectively). B: LC-MS extracted ion chromatogram for ascorbic acid. Ascorbic acid was detected at T0 but it was below the instrument detection limit at T10min and T3hr. C: LC-MS extracted ion chromatogram for pyruvic acid. Pyruvic acid showed no statistically significant change over time, ANOVA, P = 0.183.