Localization of the amphibian respiratory rhythm generator using a multilayer PDMS microfluidic chamber

Abstract

This study used an in vitro brainstem preparation from North American bullfrog (Lithobates catesbeianus) tadpoles placed in a multilayer polydimethylsiloxane (PDMS) microfluidic chamber to localize the site(s) for respiratory rhythm generation. Brainstems from pre‐metamorphic tadpoles were placed in the microfluidic chamber and superfused with oxygenated artificial CSF (aCSF; 20–22 °C). Spontaneous respiratory motor output was measured by a suction electrode attached to cranial nerve (CN) V or XII. A synaptic blockade cocktail (SBC) solution (50 μM Bicuculline, 50 μM Strychnine, 25 μM CNQX, 25 μM APV) was introduced into the microfluidic chamber as a laminar flow stream to create a virtual ‘slice’ that reversibly blocked synaptic activity in specific brainstem regions. Introduction of the SBC solution blocked or reduced respiratory motor output when the solution reached the level of CN X (vagus n.). This occurred whether the solution was introduced from either the rostral or caudal direction. The disruption of respiratory motor output by the SBC solution was completely reversible upon superfusion with control aCSF. These results support the hypothesis that respiratory rhythm generation in the pre‐metamorphic tadpole brainstem requires intact synaptic activity in a small region of the brainstem that coincides with the level of the vagus nerve. Supported by NIH SO6 GM48135 & NIH NS051580.