The methodology for labeling the distal cerebrospinal fluid-contacting neurons in rats

The distal cerebrospinal fluid-contacting neurons (dCSF-CNs) are one special type of neurons, whose bodies are in the parenchyma of the brain and processes extend into the ventricle, which suggested that dCSF-CNs might play the important roles in neuromodulation and neuroendocrinal regulation. Without special tracing method, the dCSF-CNs is hard to identify in parenchyma of the brain. Thus, up to present, these structure and function are seldom investigated. To explore dCSF-CNs, the cholera toxin subunit B conjugated with horseradish peroxidase (CB-HRP) as a tracer was injected into one of the rats' lateral ventricles, 48 h later following tracer injection rats were deeply anesthetized and fixed by intracardiac perfusion of 450 ml fixative at 25-30 ml/min. The brainstem were dissected out and stored in the fixative (postfix) for 4-6 h at 4 °C, then cryoprotected by immersion for 24-48 h in gradient sucrose (5%, 10%, 15%, 20%, and 30%) with 0.01 mol/l PBS at 4 °C. The brainstem segment was embedded, transverse or horizontal sections were cut with a cryostat and collected in phosphate-buffered saline (PBS). Three methodology were described to label the dCSF-CNs including tetramethylbenzidine-sodium tungstate (TMB-ST) method, CB-HRP-immunofluorescence procedures and CB-HRP-immunoelectron microscopic procedures.