In vitro hippocampal uptake of tritiated serotonin (3H-5HT): A morphological, biochemical, and pharmacological approach to specificity.

Abstract

The in vitro uptake of tritiated serotonin (3H-5HT) into hippocampal slices was measured in Ringer's solution (37#{176}C) containing pargyline, ascorbic acid, and dextrose. The specific uptake of 3H-5HT rose asymptotically as the 3H-5HT molarity was increased from 5 x 1O #{176}to 1.5 x lO M. Linear regression analysis gave a Km value for the specific uptake of 1.4 x 1O M. The nonspecific binding (NSB) was the amount of 3H-5HT retained by the slices following incubation in a medium with a very large excess of unlabeled 5-HT added to dilute the specific uptake of 3H-5HT. This NSB increased with increasing molarity of 3H-5HT, and was linearly related to 3H-5HT concentrations between 5 x 1O and 1.5 x 1O M. The ratio of specific uptake to NSB was highest at 5 x 1O M (2.75) and lowest at 1.5 x 1O M of3H-5HT (0.54). Competition studies with noradrenaline, desipramine (a noradrenergic uptake blocker), fluoxetine (a 5-HT uptake blocker), and tryptophan confirmed the specificity of the 3H-5HT uptake mechanism. Radioautographic studies of in vitro incubated hippocampal slices showed silver grain aggregates at 3H-5HT specific uptake sites. Addition of an excess of unlabeled 5-HT to the slices, or the use of 1 19.5 boutons/ 10,000 .tm2, in the dentate gyrus it was 67.4 boutons/ 10,000 m2, and in the subicular cortex it was 79.2 boutons/1O,000 am2. These results are consistent with previous quantitative results.

Interactions between Hippocampal Serotonin and the Pituitary-Adrenal Axis in the Septal Driving of Hippocampal Theta-Rhythm

“…The high-affinity uptake of 'H-5-HT was reduced to 15% of control in the whole hippocampus more than 40 days after lesion in both sham and bilaterally adrenalectomized rats. Previous re ports from our laboratory showed that dorsal hippocampal serotonergic fibers are almost completely eliminated by our triple-injection procedure [5]. The deafferentation of sero tonergic fibers leads to a shift in the septal driving of hippo campal theta rhythm to a minimal threshold at 6.9 Hz after 5,7-DHT injections.…”

“…Anatomical studies have shown that serotonergic fibers innervate both the granule and pyr amidal neurons in the hipocampus [5,6,17], These same cells have been shown to concentrate circulating 'H-corticosterone [12]. Thus, the same neurons may be responsive to changes in both endocrine and neuronal factors.…”

Ablation of serotonergic nerves in the rat pancreas. Lack of effects on hormone secretion and intrinsic blood flow

“…The specific serotonin neurotoxin, 5,7-dihydroxytryptamine (5,7-DHT) (Azmitia & Marovitz 1980) was dissolved to a final concentration of 5 x IO"4 mol/1 in Krebs Ringer bicarbonate buffer (KRBH), pH 7.4, supplemented with ascorbic acid (10~3 mol/1) as an antioxidant and the monoamine oxidase inhibitor, parglyline (10~4 mol/1). Rats were anaesthetized with ether and the pancreas was exposed through a midline laparatomy.…”

Effects of Microinjections of 5,7-Dihydroxytryptamine in the Suprachiasmatic Nuclei of the Rat on Serotonin Reuptake and the Circadian Variation of Corticosterone Levels

“…Similar sized pieces of parietal cortex were taken for use as unincubated controls. The assay procedure has been described previously [7,40].…”