In vivo‐synthesized radioactively labelled α‐methyl serotonin as a selective tracer for visualization of brain serotonin neurons

Abstract

To investigate the use of alpha-[3H]methyl tryptophan (alpha-[3H]MTrp) as a tracer for the in vivo study of brain serotonergic neurons, we examined whether alpha-[3H]MTrp and its metabolite alpha-[3H]methyl serotonin (alpha-[3H]M5-HT) selectively label serotonergic neurons and whether once accumulated in these neurons, the radioactive metabolite behaves like endogenous serotonin. Rats received a systemic injection of 1-5 mCi of alpha-[3H]MTrp and 24 h later their brains were immediately removed or fixed by perfusion before removal. Tissue sections in which serotonergic neurons had been immunostained for 5-HT or its synthesizing enzyme, tryptophan hydroxylase, were processed for radioautography at the light and electron microscopic level. In another group of rats, the release of radioactivity from different brain areas was studied both under basal and depolarizing conditions. In the dorsal raphe nucleus, the light microscopic examination revealed almost complete colocalization between serotonergic neurons and those that accumulated radioactivity, with a heterogeneity in the content of alpha-[3H]M5-HT among the various cells. At the ultrastructural level, immunoidentified serotonergic perikarya and dendritic processes in the dorsal raphe nucleus, as well as nerve terminals in the cerebral cortex were also found to contain alpha-[3H]M5-HT. Under basal conditions, radioactivity was released from the brainstem raphe region and from projection areas such as the striatum and hippocampus. The basal output of alpha-[3H]M5-HT increased approximately twofold after a depolarizing 50 mM KCl solution was added to the perfusion fluid. These findings suggest that newly synthesized alpha-[3H]M5-HT can be released both at somatodendritic and terminal sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Origin of the serotonergic innervation to the rat dorsolateral hypothalamus: Retrograde transport of cholera toxin and upregulation of tryptophan hydroxylase mRNA expression following selective nerve terminals lesion

“…Sequential incubation of free-floating sections using a combination of methods previously described for CTb (Luppi et al, 1990;Morin and Beaudet, 1998) or TPH (Cohen et al, 1995) immunocytochemistry were used. Sections were first incubated overnight (4°C) under gentle stirring with a polyclonal goat choleragenoid antiserum (List) diluted 1:20,000 in 0.1M phosphate buffered saline (PBS) containing 0.1% Triton X-100 (PBST).…”

Densities of Serotonergic Projections as Revealed by In Situ Synthesised Labelled α-Methyl-Serotonin: an Autoradiographic Evaluation

“…In vivo synthesized a-M5-HT is stored in vesicles and it can be released through depolarization with potassium [6]. A released fraction of labelled a-M5-HT is approximately the same as a released fraction of neuronal 5-HT [6]. a-M5-HT has been identified as a non-selective agonist for several 5-HT receptors [13].…”

“…This was confirmed by the HPLC in one of the rats; there was no measurable amount of labelled a-MTrp present in the brain after 14 days (Diksic et al unpublished observation). In vivo synthesized a-M5-HT is stored in vesicles and it can be released through depolarization with potassium [6]. A released fraction of labelled a-M5-HT is approximately the same as a released fraction of neuronal 5-HT [6].…”

“…From the experimental data collected so far there is only one metabolic pathway in the normal brain responsible for trapping the labelled compound. The tracer levels of a-M5-HT produced in situ are stored in the 5-HT neuronal pool and can be released from the brain tissue slices upon depolarization with potassium [6]. This accumulation in the tissue serotonergic pool and at tracer levels (in situ produced tracer for 5-HT) permits the localization and determination of the distribution of the natural congener (5-HT) without any pharmacological intervention.…”

“…This accumulation in the tissue serotonergic pool and at tracer levels (in situ produced tracer for 5-HT) permits the localization and determination of the distribution of the natural congener (5-HT) without any pharmacological intervention. In this mode, the procedure is able to evaluate 5-HT distribution related to the in vivo activity of TPH [6], because it has been shown that there is an excellent co-localization between the labelled tracer (both a-MTrp and a-M5-HT) [8,22], TPH and endogenous 5-HT [6]. On the assumption that the vesicles are packed with the same number of molecules of the neurotransmitter and because of the constant ratio between the tracer and endogenous neurotransmitter, one can estimate the number of neuronal projections in a particular brain region.…”