Estimation of the Rate of Tryptophan Hydroxylation In Vivo : A Sensitive Microassay in Discrete Rat Brain Nuclei

Estimation of the Rate of Tryptophan Hydroxylation In Vivo : A Sensitive Microassay in Discrete... Abstract: The rate of tryptophan hydroxylation in vivo was estimated in discrete rat brain nuclei by measuring L‐5‐hydroxytryptophan (5‐HTP) accumulated after pharmacological blockade of L‐5‐hydroxytryptophan decarboxylase by NSD 1015, using a sensitive radioenzymatic microassay. Endogenous serotonin, a major contaminant in this assay, was quantitatively removed by cationexchange chromatography prior to analysis. In non‐treated animals, endogenous 5‐HTP could be detected in small but measurable amounts. Following NSD 1015, accumulation occurred linearly for at least 30 min. At this time the recorded figures were two to six times higher when compared to values obtained in the same discrete structure from non‐treated animals. This allows an accurate estimation of the rate of tryptophan hydroxylation in vivo in small fragments of grossly dissected brain regions (e.g. cortex) as well as in discrete nuclei containing either serotoninergic (5‐HT) cell bodies (brain stem raphe nuclei) or 5‐HT‐terminals (e.g. catecholaminergic group A l, A2, A6.,. etc). Parachlorophenylalanine drastically reduced the rate of tryptophan hydroxylation in vivo in both terminal regions and raphe nuclei, with similar figures, 3 h or 3 days after injection. Chloral hydrate anaesthesia was attended by a transient decrease which appeared delayed in the raphe nuclei. Finally, pargyline pretreatment led to an 80% decrease in the forebrain, while no significant change appeared in the raphe nuclei. Thus, as illustrated by these few pharmacological manipulations, this method allows the study of the regulation of tryptophan hydroxylation in vivo with an improved anatomical resolution. Investigations can be carried out in the various raphe nuclei and their corresponding terminals in discrete brain areas simultaneously. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Neurochemistry Wiley

Estimation of the Rate of Tryptophan Hydroxylation In Vivo : A Sensitive Microassay in Discrete Rat Brain Nuclei

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Publisher
Wiley
Copyright
Copyright © 1980 Wiley Subscription Services, Inc., A Wiley Company
ISSN
0022-3042
eISSN
1471-4159
DOI
10.1111/j.1471-4159.1980.tb09668.x
Publisher site
See Article on Publisher Site

Abstract

Abstract: The rate of tryptophan hydroxylation in vivo was estimated in discrete rat brain nuclei by measuring L‐5‐hydroxytryptophan (5‐HTP) accumulated after pharmacological blockade of L‐5‐hydroxytryptophan decarboxylase by NSD 1015, using a sensitive radioenzymatic microassay. Endogenous serotonin, a major contaminant in this assay, was quantitatively removed by cationexchange chromatography prior to analysis. In non‐treated animals, endogenous 5‐HTP could be detected in small but measurable amounts. Following NSD 1015, accumulation occurred linearly for at least 30 min. At this time the recorded figures were two to six times higher when compared to values obtained in the same discrete structure from non‐treated animals. This allows an accurate estimation of the rate of tryptophan hydroxylation in vivo in small fragments of grossly dissected brain regions (e.g. cortex) as well as in discrete nuclei containing either serotoninergic (5‐HT) cell bodies (brain stem raphe nuclei) or 5‐HT‐terminals (e.g. catecholaminergic group A l, A2, A6.,. etc). Parachlorophenylalanine drastically reduced the rate of tryptophan hydroxylation in vivo in both terminal regions and raphe nuclei, with similar figures, 3 h or 3 days after injection. Chloral hydrate anaesthesia was attended by a transient decrease which appeared delayed in the raphe nuclei. Finally, pargyline pretreatment led to an 80% decrease in the forebrain, while no significant change appeared in the raphe nuclei. Thus, as illustrated by these few pharmacological manipulations, this method allows the study of the regulation of tryptophan hydroxylation in vivo with an improved anatomical resolution. Investigations can be carried out in the various raphe nuclei and their corresponding terminals in discrete brain areas simultaneously.

Journal

Journal of NeurochemistryWiley

Published: Apr 1, 1980

References

  • Regional transport of tryptophan in rat brain
    Denizeau, Denizeau; Sourkes, Sourkes
  • A, sensitive microassay for tryptophan hydroxylase in brain
    Kizer, Kizer; Zivin, Zivin; Saavedra, Saavedra; Brownstein, Brownstein

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