It has been reported that distal cerebrospinal fluidcontacting neurons(dCSF-CNs)can be detected by immunohistochemical assay using cholera toxin subunit B-conjugated horseradish peroxidase(CBHRP).In the present study,another two methods with CB alone or CB-conjugated FITC(CB-FITC)were used,and the results from the three methods were compared.Adult Sprague-Dawley rats were randomly divided into three groups with CB-HRP,CB or CB-FITC.Tracers were diluted to 30%in artificial cerebrospinal fluid and injected separately(in a volume of 3μL)into the lateral ventricle.Animals from the CB-HRP and CB groups were perfused 48 h after surgery while animals from the CB-FITC group were perfused 1,3,6,12,24 or 48 h after surgery.The brain was sectioned(40μm)for immunofluorescence and five sections with positive neurons were selected from each rat for neuron counting.Three clusters of positive neurons in a'Y-like'distribution were detected ventral to the cerebral aqueduct of rats from the three groups.No significant difference was observed among the quantitative data.In the CB-FITC group,stable staining was detected even at 6 h after injection.Taken together,lateral ventricle injection of CB/CB-FITC is a useful method for labeling dCSFCNs in rats.The CB-FITC method makes dCSF-CNs labeling much simpler and more convenient.
