In the monkey dorsal raphe, we reported that 1-month (mo) of estrogen replacement, with or without progesterone supplementation for 14 days, significantly increased tryptophan hydroxylase-1 (TPH-1) mRNA; decreased serotonin reuptake transporter (SERT) mRNA and decreased monoamine oxidase (MAO)-A mRNA, but had no effect on MAO-B mRNA. Here, we questioned what effect would 1 or 5 mo of ovarian hormones or the selective estrogen receptor modulator (SERM), raloxifene, have on TPH protein and phosphorylation, and on protein expression of SERT, MAO-A or MAO-B? Raloxifene antagonizes estrogen in breast or uterus, but estrogen-like activities in the brain have been reported. Cytoplasmic and membrane extracts of the dorsal raphe region were processed for Western blotting. TPH, phosphoserine, SERT, MAO-A, and MAO-B were detected with specific antibodies. The optical densities of the signals were measured with NIH image and analyzed by ANOVA. Both 1 and 5 mo of estrogen, with or without progesterone, and 5 mo of raloxifene significantly increased TPH protein. Administration for 5 mo of estrogen plus progesterone and raloxifene also increased TPH phosphorylation. Estrogen, with or without progesterone, for 1 mo had no effect on SERT protein. However, 5 mo of estrogen and 5 mo of raloxifene increased SERT protein. Estrogen alone or combined with progesterone for 1 mo caused a significant reduction in MAO-A. Yet, after 5 mo of the same treatments, MAO-A was not different from spayed controls. Estrogen alone had no effect on MAO-B. However, the addition of progesterone significantly increased MAO-B. Raloxifene for 5 mo had no effect on MAO-A or MAO-B. Thus, to various extents, estrogen, progesterone, and raloxifene may increase serotonin production and transport. The expression of the degradative enzymes suggests a complex combination of gene transcription, post-transcriptional processing, and substrate feedback mechanisms.

The results clearly indicate that laboratory-determined enzyme concentrations in feeding tube aspirates are helpful in predicting tube location. Thus, it is desirable that inexpensive, simple bedside tests be developed so that they can be used in conjunction with pH measurements to help predict tube position.

Preliminary laboratory-based data indicate that appropriate use of the proposed algorithm could significantly reduce the number of x-rays needed to exclude respiratory placement and to distinguish between gastric and intestinal placement.

No abstract

The newly developed VBIL scale is effective in determining the bilirubin content in feeding tube aspirates. Furthermore, compared with predicting tube location from pH-paper alone, dichotomized readings from the VBIL scale/bilirubin teststrip used in combination with a pH-teststrip improved the ability to differentiate between respiratory and gastrointestinal tube placement, as well as between gastric and intestinal tube placement. Refinement of the VBIL scale is indicated to increase its accuracy.

Objective: Nuclear factor kappa B (NFκB) is a transcription factor that activates gene expression in response to proinflammatory cytokines, and elevated cytokines are associated with depression, which has a serotonergic component. We questioned (1) whether serotonin neurons contain NFκB, (2) whether NFκB detection with immunocytochemistry is changed in the dorsal raphe nucleus (DRN) by ovarian hormone treatment and (3) whether ovarian hormones regulate midbrain NFκB gene or protein expression. Methods: Monkeys were spayed and treated with placebo, estrogen (E), progesterone (P) or E+P for 1 month (n = 4 animals/treatment group), and the midbrain was harvested for immunocytochemistry and stereology. An antibody that detects nuclear location-specific (NLS)-NFκB p65 was applied, and the numbers of NLS-NFκB-immunopositive cells were counted in 9 sections of the DRN. Additional monkeys were used for Western blot analysis and quantitative reverse transcription-polymerase chain reaction (RT-PCR) for NFκB p65. Results: In placebotreated macaques, neurons were double-immunostained for serotonin and nuclear NFκB p65 throughout the DRN. The mean total number of NFκB-positive cells equalled 2178 (and standard error of the mean [SEM] 129) in the placebo group, 1631 (SEM 221) in the Etreated group, 2314 (SEM 186) in the P-treated group and 1162 (SEM 100) in the E+P-treated group (analysis of variance p = 0.003). The E-treated and E+P-treated groups had a significantly lower density of cells stained positive for NFκB than the placebo or P-treated groups (post hoc). Unmasking of NLS-NFκB immunostaining in the DRN revealed dense immunostaining in the cytoplasm of large dorsal raphe neurons. There was no difference between treatment groups in the amount of NFκB p65 detected by Western blot or in the relative expression of NFκB p65 mRNA with quantitative RT-PCR. Conclusions: These observations are consistent with the notion that gene and protein expression of NFκB are constitutive but that ovarian hormones can decrease the nuclear location of NFκB in dorsal raphe neurons and, thereby, decrease the ability of NFκB to drive gene expression in response to cytokines.Objectif : Le facteur nucléaire kappa B (NFκB) est un facteur de transcription qui active l'expression génique en réponse à des cytokines pro-inflammatoires, et l'élévation des cytokines est associée à la dépression, qui comporte un élément constituant sérotoninergique. Nous avons cherché à savoir (1) si les neurones à sérotoninergiques contiennent du NFκB, (2) si un traitement aux hormones ovariennes modifie la détection du NFκB par immunocytochimie dans le noyau du raphé dorsal (NRD) et (3) si les hormones ovariennes assurent la régulation de l'expression génique ou protéique du NFκB dans le mésencéphale. Méthodes : On a pratiqué une ovariectomie sur des guenons auxquelles on a administré un placebo, de l'oestrogène (E), de la progestérone (P) ou une combinaison E+P pendant un mois (n = 4 animaux/groupe de traitement). On a prélevé ensuite le mésencéphale pour effectuer ...

No abstract

Estrogen and progesterone act on gene and protein expression in serotonin neurons in a manner that suggests serotonin neurotransmission should increase. However, measurement of extracellular serotonin in macaques was lacking. Elevated prolactin secretion can be an indicator of increased serotonergic function and prolactin is increased by combined estrogen and progesterone treatment. We examined extracellular serotonin by microdialysis in a well-characterized macaque model of steroid-induced prolactin secretion. Monkeys were fitted with 2 guide tubes directed to the arcuate nucleus of the hypothalamus. Samples (75μl/15-min interval) were obtained via a tether-swivel device through sample lines into an adjoining room. Serotonin was measured with a modified commercial enzyme linked immunoassay (ELISA) kit. Fenfluramine infused through the probe (300 μM for 2 h; n=2 trials) or administered intravenously (2.5 mg/kg; n=2 trials) caused a marked increase in extracellular serotonin and verified the efficacy of the procedure. Three monkeys were maintained with an estrogen implant for 2 weeks. Each monkey was injected with 20 mg of progesterone s.c. in oil at 1500h; microdialysis was initiated the next morning and samples were obtained for 24 h. There was a significant increase in serotonin between 40 and 43 h after the progesterone injection (P < 0.001, ANOVA). Serotonin averaged 59±1 pg/sample from 18-30 h post-progesterone injection, and averaged 76±2 pg/sample from 30-48 h post-progesterone injection (P < 0.0001; t-test). Since the increase in serotonin is delayed by ∼40 h after progesterone-injection, we speculate that the action of progesterone may involve either nuclear progestin receptors or membrane progestin receptors.