Susan P. Rohrer

17β-Estradiol-induced antidepressant-like effect in the Forced Swim Test is absent in estrogen receptor-β knockout (BERKO) mice

RationaleThe decrease in levels of estrogens (ER) that occurs in menopause has been correlated with depressive disorders, probably due to ER direct and/or indirect effects in the brain, where these hormones act through both genomic (i.e. interaction as transcription factors with nuclear receptors ER-α and ER-β) and non-genomic (i.e. binding with cell-membrane receptors) mechanisms. With respect to mood related disorders the interaction between ER-β and the serotonin (5-HT) system is highly relevant. 17β-Estradiol (E2) induces expression of the enzyme implicated in 5-HT synthesis - tryptophan hydroxylase (TPH), and this effect is mediated through ER-β located in 5-HT cell bodies of the dorsal raphe nucleus (DRN).ObjectiveThe present studies tested the hypothesis that E2 induces antidepressant-like effects in female ovariectomized (OVX) mice, and that expression of ER-β is mandatory for such effects.MethodsThe Forced Swim Test (FST) was used in three experiments to assess (a) dose response effect of E2 in outbred and inbred mouse strains, (b) length of treatment necessary for effect, (c) and role of ER-β receptors.ResultsE2 (100 or 200 μg/kg), as well as the antidepressant desipramine (DMI), significantly reduced total duration of immobility in the FST in mice from different strains. Four consecutive daily doses (200 μg/kg) were required for such effect, which was absent in mice lacking the gene coding for ER-β (BERKO mice).ConclusionThese data suggest that E2-induced antidepressant-like effects in mice are mediated through activation of ER-β. They offer preliminary support to the hypothesis that specific compounds acting at ER-β may influence mood in postmenopausal women.

Involvement of sst2 somatostatin receptor in locomotor, exploratory activity and emotional reactivity in mice.

Somatostatin (SRIF) controls many physiological and pathological processes in the central nervous system but the respective roles of the five receptor isotypes (sst1–5) that mediate its effects are yet to be defined. In the present study, we attempted to identify functions of the sst2 receptor using mice with no functional copy of this gene (sst2 KO mice). In contrast with control 129Sv/C57Bl6 mice, sst2 mRNA was no longer detectable in the brain of sst2 KO mice; 125I‐labeled Tyr0DTrp8‐SRIF14 binding was also greatly reduced in almost all brain structures except for the hippocampal CA1 area, demonstrating that sst2 accounts for most SRIF binding in mouse brain. Invalidation of this subtype generated an increased anxiety‐related behaviour in a number of behavioural paradigms, while locomotor and exploratory activity was decreased in stress‐inducing situations. No major motor defects could be detected. sst2 KO mice also displayed increased release of pituitary ACTH, a main regulator of the stress response. Thus, somatostatin, via sst2 receptor isotype pathways, appears involved in the modulation of locomotor, exploratory and emotional reactivity in mice.

Estrogen receptor-β regulates tryptophan hydroxylase-1 expression in the murine midbrain raphe

BACKGROUND Distinct expression patterns of estrogen receptor (ER)-alpha and ER-beta are displayed in the murine central nervous system. ER-beta is the predominant form of the receptor expressed in the murine midbrain dorsal raphe nucleus (DRN). Tryptophan hydroxylase (TPH) is abundantly expressed in the serotonergic neurons of the DRN and is regulated by estrogen in both the monkey and the guinea pig. METHODS In this study we used immunocytochemistry to show that ER-beta and TPH are colocalized in the serotonergic cells of the murine DRN. We utilized the ER-alpha and ER-beta gene deletion mouse models and in situ hybridization to demonstrate that ER-beta is responsible for regulating TPH1 mRNA expression. RESULTS Estrogen increased TPH1 mRNA expression in the DRN of wild type and ER-alpha knockout mice (alpha-ERKO) but not ER-beta knockouts (beta-ERKO). CONCLUSIONS These data indicate that ER-beta is responsible for mediating estrogen regulated TPH1 expression in the murine DRN.

Ivermectin binding sites in sensitive and resistant Haemonchus contortus.

Membranes from both ivermectin-sensitive and -resistant Haemonchus contortus L3 larvae were examined for the presence of high affinity [3H]ivermectin binding sites. Both tissue preparations displayed high affinity drug binding sites (Kd = 0.13 nM). Receptor density (Bmax = 0.4 pmol/mg) was the same in both the sensitive and resistant nematodes suggesting that target site modification was not involved in the development of drug resistance in this particular strain of H. contortus. The H. contortus ivermectin binding site appeared to be similar to the well characterized Caenorhabditis elegans ivermectin binding site with respect to affinity for ivermectin and receptor density.

Differential hormonal regulation of tryptophan hydroxylase-2 mRNA in the murine dorsal raphe nucleus

BACKGROUND Recently a novel tryptophan hydroxylase isoform (TPH2) was identified and shown to be highly expressed in the central nervous system (CNS). Hormonal effects on TPH2 mRNA expression in the rodent dorsal raphe nucleus (DRN) are unknown. METHODS In situ hybridization histochemistry and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) were used to assess the effects of dexamethasone or estradiol on TPH2 mRNA levels in the DRN of C57/Bl6 mice. RESULTS Dexamethasone reduced TPH2 mRNA levels in the DRN of both ovx female and intact male mice. Reduction of TPH2 mRNA in the DRN was blocked by co-administration of mifepristone. Estradiol had no detectable effect on TPH2 mRNA levels in the DRN. CONCLUSIONS TPH2 mRNA is regulated by glucocorticoids but not estradiol in the mouse DRN. Glucocorticoid-mediated reduction of TPH2 message may have relevance to the etiology of major depression, psychotic major depression in particular, where elevated glucocorticoids are one hallmark of the disease.

Potent, orally bioavailable somatostatin agonists: Good absorption achieved by urea backbone cyclization

Backbone cyclization of urea-based somatostatin agonists resulted in novel, orally bioavailable agonists. Binding assays confirmed that the resulting conformationally constrained cyclic ureas retained the potency of their acyclic counterparts. SAR studies subsequently led to highly potent analogs, selective for receptor subtype 2, and having good oral bioavailability.

Identification and characterization of subtype selective somatostatin receptor agonists

High affinity, subtype selective non-peptide agonists of somatostatin receptor subtypes 1-5 were identified in combinatorial libraries constructed based on molecular modeling of known peptide agonists. Simultaneous traditional chemical synthesis yielded an additional series of somatostatin subtype-2 receptor (SSTR2) selective agonists. These compounds have been used to further define the physiological functions of the individual somatostatin receptor subtypes. In vitro experiments demonstrated the role of the SSTR2 in inhibition of glucagon release from mouse pancreatic alpha-cells and the somatostatin subtype-5 receptor (SSTR5) as a mediator of insulin secretion from pancreatic beta-cells. Both SSTR2 and SSTR5 regulated growth hormone release from the rat anterior pituitary gland. In vivo studies performed with SSTR2 receptor selective compounds demonstrated effective inhibition of pulsatile growth hormone release in rats. The SSTR2 selective compounds also lowered plasma glucose levels in normal and diabetic animal models. The availability of high affinity, subtype selective non-peptide agonists for each of the somatostatin receptors provides a direct approach to defining their physiological function both peripherally and in the central nervous system.

Ophiobolin M and analogues, noncompetitive inhibitors of ivermectin binding with nematocidal activity

A series of ophiobolins were isolated from a fungal extract based on their nematocidal activity. These compounds are non-competitive inhibitors of ivermectin binding to membranes prepared from the free-living nematode, Caenorhabditis elegans, with an inhibition constant of 15 microM. The ophiobolins which were most potent in the biological assays, ophiobolin C and ophiobolin M, were also the most potent compounds when evaluated in a C. elegans motility assay. These data suggest that the nematocidal activity of the ophiobolins is mediated via an interaction with the ivermectin binding site. The isolation, structure and biological activity of ophiobolins have been described.

A β-Lactamase-Dependent Gal4-Estrogen Receptor β Transactivation Assay for the Ultra-High Throughput Screening of Estrogen Receptor β Agonists in a 3,456-Well Format

Estrogen action is mediated via two estrogen receptor (ER) subtypes, ERα and ERβ. Selective ER modulators with balanced high affinity for ERα and ERβ have been developed as therapeutics for the tre...

Identification of neuron-specific ivermectin binding sites in Drosophila melanogaster and Schistocerca americana.

High affinity avermectin binding sites have been identified and partially characterized in membranes from two insect species, Drosophila melanogaster and the locus Schistocerca americana. There is a 10-fold increase in the density of ivermectin binding sites associated with membranes isolated from Drosophila heads (a neuronally enriched tissue source) compared to the bodies (Bmax values were 3.5 and 0.22 pmol/mg, respectively) with only a small difference in the apparent dissociation constant (Kd values of 0.20 and 0.34 nM for heads and bodies, respectively). Membranes prepared from metathoracic ganglia of the locust, Schistocerca americana, were highly enriched in high affinity avermectin binding sites (Kd = 0.2 nM and Bmax = 42 pmol/mg). Using an [125I]arylazido-avermectin analog as a photoaffinity probe, a 45 kDa protein was identified in both the Drosophila head and body tissue preparations. A 45 kDa protein was also specifically labeled with [125I]azido-avermectin in the locust neuronal membranes.