A modification of the Falck Hillarp technique is described, which significantly improves the quality of 5 HT fluorescence in the rat. The method employs perfusion of the animal with a hypertonic formaldehyde solution in conjunction with drug pretreatment. There were various groups of rats, and the results of the various treatments were as follows: Brains which were not perfused or which were perfused with buffer alone showed weak 5 HT cell body fluorescence but almost no sign of fluorescence extending beyond the cell body into the axons. Hypertonic sucrose perfusion resulted in only marginal improvement in both cell body and axon fluorescence. Brains which were either perfused with 2.5% glutaraldehyde or glutaraldehyde in combination with 4% paraformaldehyde showed similar results. The perikaryial fluorescence appeared a very deep yellow, almost orange color. No clear signs of axon fluorescence were seen. In addition, the tissue presented an intense green background fluorescence with a perforated texture. Perfusion with 4% paraformaldehyde in hypertonic buffer resulted in a marked increase in perikarial fluorescence and yellow fiber bundles were now clearly seen leaving the raphe area in the rostral direction. Only the midbrain region was examined in these studies. Once 4% paraformaldehyde in hypertonic buffer was shown to be an advantageous perfusion technique, the procedure was repeated in a second group of animals treated with chloral hydrate in an attempt to further reduce diffusion. Animals were divided as follows: those receiving chloral hydrate (400 mg/kg i.p.) without perfusion; those perfused without chloral hydrate, or those treated with chloral hydrate followed by perfusion. Good fluorescence of both fibers and cell bodies was seen in all groups. However, the best perikarial fluorescence appeared to be in the group without chloral hydrate pretreatment, and the best fiber fluorescence appeared to be in the group which received both chloral hydrate and perfusion with 4% paraformaldehyde in hypertonic buffer.
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