Positive allosteric modulators (PAMs) of receptors represent a class of pharmacologic agents having the desirable property of acting only in the presence of cognate ligands. Discovery and optimization of the structure activity relationships of PAMs is complicated by the requirement of a second ligand to manifest their action, and by the need to quantify both affinity and intrinsic efficacy. Multivariate regression analysis is a statistical method capable of simultaneously obtaining affinity and intrinsic efficacy parameters from curve fits of multiple agonist dose-response functions generated in the presence of varying concentrations of PAMs. Capitalizing on the advantages of multivariate regression analysis for PAM optimization requires a theoretical framework and a system that facilitates efficient flow of information from data generation through data analysis, storage, and retrieval. We describe here the experimental design, mathematical model and informatics workflow enabling a multivariate regression approach for rapidly obtaining affinity and intrinsic efficacy values for PAMs in a drug discovery setting.

Transient receptor potential melastatin-5 (TRPM5) is a calcium-gated monovalent cation channel expressed in highly specialized cells of the taste bud and gastrointestinal tract, as well as in pancreatic β-cells. Well established as a critical signaling protein for G protein-coupled receptor-mediated taste pathways, TRPM5 also has recently been implicated as a regulator of incretin and insulin secretion. To date, no inhibitors of practical use have been described that could facilitate investigation of TRPM5 functions in taste or secretion of metabolic hormones. Using recombinant TRPM5-expressing cells in a fluorescence imaging plate reader-based membrane potential assay, we identified triphenylphosphine oxide (TPPO) as a selective and potent inhibitor of TRPM5. TPPO inhibited both human (IC₅₀ = 12 μM) and murine TRPM5 (IC₅₀ = 30 μM) heterologously expressed in HEK293 cells, but had no effect (up to 100 μM) on the membrane potential responses of TRPA1, TRPV1, or TRPM4b. TPPO also inhibited a calcium-gated TRPM5-dependent conductance in taste cells isolated from the tongues of transgenic TRPM5(+/)⁻ mice. In contrast, TPP had no effect on TRPM5 responses, indicating a strict requirement of the oxygen atom for activity. Sixteen additional TPPO derivatives also inhibited TRPM5 but none more potently than TPPO. Structure-activity relationship of tested compounds was used for molecular modeling-based analysis to clarify the positive and negative structural contributions to the potency of TPPO and its derivatives. TPPO is the most potent TRPM5 inhibitor described to date and is the first demonstrated to exhibit selectivity over other channels.

Estrogens modulate critical homeostatic functions of the hypothalamus such as temperature regulation, sexual behavior and sleep with the most pronounced effects in rats occurring during the dark-phase. The neurochemical signals underlying estrogenic regulation of these hypothalamic functions have not been clearly identified, possibly due to the fact that previous studies have not explored the effects of estrogen treatments on neuronal signaling during the dark-phase. In the present study, ovariectomized rats received estradiol benzoate (5 μg/rat for 7 days, s.c.) and norepinephrine and dopamine levels were measured in the preoptic area of the hypothalamus across the light/dark cycle using in vivo microdialysis. Estradiol benzoate treatment increased extracellular norepinephrine and dopamine levels relative to vehicle treatment during the dark-phase. Increases in norepinephrine and dopamine were first detected by 30 min and 5.5 h after lights-off, respectively. Subsequent increases in norepinephrine and dopamine were also noted throughout the 9.5- h collection period. The effect of estradiol benzoate on catecholamine release did not correlate with increases in either tyrosine hydroxylase (TH) protein expression or activity levels in the anterior hypothalamus, although a marked decrease in TH activity correlated with a rise in extracellular norepinephrine at the beginning of the dark-phase. We conclude that subchronic estradiol benzoate treatment increases extracellular catecholamine levels in the preoptic area of the hypothalamus during the dark-phase without a concomitant increase in neurotransmitter biosynthesis. The estradiol benzoate-induced increases in norepinephrine and dopamine levels in the preoptic area during the dark-phase may play an important role in modulating critical hypothalamic functions.

Thyroid hormone receptors (TRs) are nuclear receptors that are activated by thyroid hormone ligands and co-regulator proteins. Two receptor subtypes, TRalpha and TRbeta, have been suggested to play a role in numerous physiological functions. However, specificity of receptor subtype function and co-regulator interaction is unclear due to the lack of TR subtype-specific ligands. Five TR ligands were evaluated for their selectivity and interaction with the TR subtypes. A multiplex assay was used to identify co-regulator peptide interaction, and biochemical assays were used to characterize ligand-receptor specificity. In the biochemical assay, rank order ligand potencies were similar in the presence of co-activator peptides, SRC1-2 and SRC3-2, and the co-repressor peptide, NCoR1-2, with T3 and Triac potencies greater in the presence of the co-repressor. The potency of Tetrac was similar regardless of the co-regulator used while T4 and rT3 demonstrated selectivity for TRalpha subtype. The rank order among TR ligands at either receptor subtype in the biochemical assay correlated with the multiplex assay. These assays can be used to identify new ligands that can provide further insight into TR biology.

Psychological stressors have a prominent effect on sleep in general, and rapid eye movement (REM) sleep in particular. Disruptions in sleep are a prominent feature, and potentially even the hallmark, of posttraumatic stress disorder (PTSD) (Ross, R.J., Ball, W.A., Sullivan, K., Caroff, S., 1989. Sleep disturbance as the hallmark of posttraumatic stress disorder. American Journal of Psychiatry 146, 697–707). Animal models are critical in understanding both the causes and potential treatments of psychiatric disorders. The current review describes a number of studies that have focused on the impact of stress on sleep in rodent models. The studies are also in Table 1, summarizing the effects of stress in 4-h blocks in both the light and dark phases. Although mild stress procedures have sometimes produced increases in REM sleep, more intense stressors appear to model the human condition by leading to disruptions in sleep, particularly REM sleep. We also discuss work conducted by our group and others looking at conditioning as a factor in the temporal extension of stress-related sleep disruptions. Finally, we attempt to describe the probable neural mechanisms of the sleep disruptions. A complete understanding of the neural correlates of stress-induced sleep alterations may lead to novel treatments for a variety of debilitating sleep disorders.

Temperature dysfunction, clinically described as hot flashes/flushes and night sweats, commonly occur in women transitioning through menopause. Research in this field has yet to fully elucidate the biological underpinnings explaining this dysfunction. The need to develop animal models that can be used to study hormone-dependent temperature regulation is essential to advancing this scientific area. Development of telemetric transmitters for monitoring tail-skin (TST) and core body (CBT) temperatures for animal research has increased the accuracy of data by reducing extraneous factors associated with previous methods. However, until recently, TST and CBT could not be simultaneously measured telemetrically within the same animal. In this report, new dual temperature monitoring transmitters were validated by simultaneously evaluating them with the single measurement transmitters using the ovariectomized (OVX) rat thermoregulatory dysfunction model. A major advantage of measuring TST and CBT in the same animal is the ability to relate temporal changes on these two temperature parameters. Comparative experimentation was performed by single administration of clonidine (alpha(2) adrenergic agonist), MDL-100907 (5-HT(2a) antagonist), or a 7-day treatment of 17alpha-ethinyl estradiol (EE). Clonidine caused decreases in TST and CBT, MDL-100907 caused increases in TST while decreasing CBT, and EE caused decreases in TST with minor CBT decreases only at the higher dose. Data from either probe type showed similar results on temperature parameters regardless of transmitter used. These findings support the use of the new dual temperature transmitters and should enhance the quality and interpretation of data being generated in thermoregulation studies.

Estrogen treatment decreases active phase rapid eye movement (REM) sleep in ovariectomized rats. Here we explored further the effect of 17α-ethinyl estradiol (17α-EE) on active phase REM sleep in ovariectomized rats by analyzing spectral properties and the number and length of REM sleep bouts. The greatest suppression of REM sleep occurred on day 4 of 17α-EE treatment, was due to decreases in bout length, and was accompanied by decreased EEG theta power. These results further elucidate 17α-EE's effects on REM sleep and provide greater understanding of the mechanisms by which estrogens alter sleep–wakefulness patterns.

In ovariectomized (OVX) Sprague-Dawley rats, estradiol benzoate (EB) has been reported to decrease rapid eye movement (REM) and non-REM (NREM) sleep during the dark phase for up to 3 days. It is unknown, however, if estrogenic effects on sleep extend beyond 3 days or if other estrogens could induce the same changes. Furthermore, it is unclear whether the increased wakefulness in the dark phase was due to changes in active or quiet wakefulness. Therefore, we examined the effects of daily injections of 17alpha-ethinyl estradiol (EE) for 6 days on sleep and wakefulness in the OVX rat. After 3 days of baseline recording using a telemetric system, rats were administered sesame oil (sc) for 3 days followed by injection with EE (20 mug/rat/day, sc) for 6 days. After treatment, sleep was recorded during hormone withdrawal for an additional 5 days. A few sporadic but statistically significant increases in light phase sleep occurred during the last 3 days of EE treatment. Starting on day 2 of the study, EE caused statistically significant decreases in dark phase REM sleep that were maintained throughout the treatment period and persisted until the 3rd day of hormone withdrawal. During the dark phase, statistically significant decreases in NREM sleep and increases in active wakefulness started on the second day of treatment and abated by the end of treatment. This study demonstrated that EE had similar effects on sleep-wakefulness to EB and demonstrates the utility of telemetric polysomnographic recording of the female OVX rat as a model for understanding the estrogen-induced changes on sleep-wakefulness.

The cellular and molecular processes that underlie the drives and functions of sleep have been the topic of many studies in the last few decades. Discovery-based techniques, such as cDNA microarrays, have increasingly been utilized in conjunction with sleep deprivation paradigms to examine the molecular mechanisms and functions of sleep. These studies have helped to validate and expand existing hypotheses, such as those on the roles of sleep in synaptic plasticity and in energy metabolism. The mechanisms underlying the highly prevalent changes in sleep architecture with age are not known, but likely reflect fundamental changes in the molecular basis of circadian timing and sleep homeostatic processes. We decided to explore the effects and interactions of sleep deprivation and aging utilizing the proteomic technique of difference in gel electrophoresis (DIGE). DIGE, which utilizes cyanine dye labeling of samples, allows for the comparison of multiple experimental groups within and across gels. In this study, we compared cerebral cortex tissue from young (2.5 months) and old (24 months) mice that had been sleep deprived for 6 h to tissue from undisturbed young and old control animals. Following DIGE, automatic image matching and spot identification, and statistical analysis, 43 unique proteins were identified. The proteins were grouped into seven functional classes based on published characteristics: cell signaling, cytoskeletal, energy metabolism, exocytosis, heat shock proteins, mRNA processing/trafficking, and serum proteins. The identity and characteristics of these proteins relevant to sleep and aging are discussed.

Psychological stressors have a prominent effect on rapid eye movement sleep (REMS) in humans and animals. We hypothesized that the stress-related neurochemical corticotropin-releasing factor (CRF), acting in the amygdala, could initiate neural events that lead to REMS alterations. Therefore, we made bilateral microinjections of three different doses of CRF into the central nucleus of the amygdala (CeA) in five rats. Only the lowest dose of CRF (1 ng) induced a change in sleep, specifically REMS, during the 4-h post-injection period. Thus, REMS alterations following psychological stress may depend, in part, on CRF release in the CeA.

Thermoregulation is a complex intercommunicative function requiring coordination between core body temperature (CBT), the central nervous system, and peripheral vasculature. In menopausal women, dysregulation of thermoregulatory mechanisms leads to hot flushes and night sweats. A previous study in ovariectomized (OVX) rats has suggested that mirtazapine can alleviate thermoregulatory dysfunction by blocking 5-HT(2A) receptor signaling. This is in opposition to other work in which 5-HT(2A) receptor blockade appeared to exacerbate thermoregulatory dysfunction in OVX rats. Thus, the goals of the present study were to reexamine the effects of mirtazapine on temperature regulation in OVX rat models and explore further the role of 5-HT(2A) receptor blockade. Mirtazapine exhibited potent functional antagonism (EC(50)=0.62 nM) at the cloned human 5-HT(2A) receptor. In the morphine-dependent model of thermoregulatory dysfunction, mirtazapine (10 mg/kg, i.p.) induced an increase in tail-skin temperature (TST) prior to naloxone administration. In the telemetry model, mirtazapine (0.3-3 mg/kg, i.p.) caused an increase in TST. However, at the highest dose tested (10 mg/kg, i.p.), mirtazapine induced a small but significant decrease in TST followed by an increase in TST. To examine this finding further, mirtazapine's effect on CBT was determined. Administration of mirtazapine (1-3 mg/kg, i.p.) resulted in a slight decrease in CBT but at the 10 mg/kg dose a dramatic decrease (-3.6 degrees C) in CBT was observed. These data support the concept that 5-HT(2A) receptors play a role in temperature regulation but that functional blockade of these receptors by mirtazapine is not a likely mechanism for restoring thermoregulatory processes in OVX rats.

Background: A prominent sleep disturbance, likely including a disruption of rapid eye movement sleep (REMS) continuity, characterizes posttraumatic stress disorder (PTSD). We set out to develop a fear conditioning paradigm in rats that displays alterations in sleep architecture analogous to those in PTSD.Methods: Baseline polysomnographic recordings of rats were performed in a neutral context to which the rats bad been habituated ,for several days. Rats were then shock- or mock-trained in a distinctly different context, and their sleep was studied the following day in that context. A separate group of rats was shock-trained and studied in the neutral context on the following 2 days.Results: Rats that slept in the neutral context exhibited a REMS-selective increase in sleep 24 hours after training and increases in REMS and non-REMS 48 hours after training. In contrast, rats that slept in the presence of situational reminders of the training context exhibited a REMS-selective decrease in sleep 24 hours later. Animals that were mock-trained showed no changes in sleep.Conclusions: Shock training induced days-long changes in sleep architecture that were disrupted when the animal was exposed to situational reminders of the training context.

To examine the influence of conditioned fear stimuli on sleep-wake states, we recorded sleep in Sprague-Dawley rats after exposure to tones previously paired with footshock. After habituation to a recording chamber and the recording procedure, a baseline sleep recording was obtained the next day. One day later, experimental animals were exposed to shock training designed to induce conditioned fear (FC), consisting of five tone-footshock pairings. The 5-s tones (conditioned stimuli; CS) co-terminated with 1-s footshocks (unconditioned stimuli; US). The next day sleep was recorded for 4 h in the recording chamber after presentation of five CSs alone. Sleep efficiency (total sleep time/recording period) and REM sleep (REM) and non-REM (NREM) measures were determined. While sleep efficiency was not significantly changed after CS presentation, the percentage of total sleep time spent in REM (REM percentage) was reduced in the FC animals. The reduction in REM percentage in the FC animals was due to a decrease in the number of REM bouts. In a separate experiment, we repeated the procedures, except the tones and shocks were presented in an explicitly unpaired (UP) fashion. The next day, presentation of the tones increased REM percentage in the UP group. Results are discussed in terms of the decreases in REM as a response to conditioned fear, and the relevance of these findings to the sleep changes seen in post-traumatic stress disorder (PTSD).

This report details the development of a secure messaging add-on for use on public Instant Messaging (IM) networks. It is a continuation of the project discussed in [25]. Due to sociopolitical and monetary reasons, the growth of secure enterprise IM systems has not kept pace with the use of insecure public IM systems. A solution to this problem is to develop a free prototype that is able to secure communications over a public IM network, using a set of guidelines for information security and IM network compatibility. The prototype was able to successfully integrate with a third party IM client and conduct an encrypted session over the MSN Messenger Network. With more development and testing the prototype could become a viable option for use as a secure IM add-on.

Background: The interaction between lithium, a mood stabilizer, and the thyroid axis has been extensively studied; however the regulation of thyroid hormone receptors by lithium is yet to be investigatedMethods: To test whether lithium affects thyroid hormones at the receptor level, we examined the effects of lithium in combination with triiodothyronine (T3) on gene expression of thyroid hormone receptor isoforms in GH3 and B103 cells.Results: The pattern of expression expression as well as the magnitude of regulation of the different thyroid hormone receptor isoforms appeared to :be cell line specific. Whereas T3 regulated all Sour isoforms in GH3 cells at both time points, T3 did not alter thyroid hormone receptor TR alpha 1 and TR alpha 2 mRNA in B103 cells. Addition of lithium to thyroid hormone-deficient GH3 cells decreased TR alpha 1, alpha 2, and beta 2 expression without affecting TR beta 1 expression at 2 but not 5 days. Addition of lithium to T3-treated GH3 cells did not further modulate gene expression of TR alpha 1, alpha 2, beta 1, or beta 2 when compared to cells treated with T3 alone. The effects of lithium in B103 cells appeared to be isoform specific as well as time dependent, since TR alpha 1 expression was selectively decreased in B103 cells, when treated with T3 in the presence of lithium.Conclusions: The present study provides direct evidence that T3 and/or lithium regulate TR gene expression in vitro in a both time-dependent and cell line-specific manner. (C) 1999 Society of Biological Psychiatry.

Even though lithium has received wide attention in the treatment of manic depressive illness, the mechanisms underlying its mood stabilizing effects are not understood. Lithium is known to interact with the thyroid axis and causes hypothyroidism in a subgroup of patients, which compromises its mood stabilizing effects. Since lithium was recently reported to alter thyroid hormone metabolism in the rat brain, the present study investigated whether these effects were mediated through regulation of thyroid hormone receptor (THR) gene expression. Adult male euthyroid rats were either given a diet containing 0.25% lithium or one without lithium for 14 days. Rats were sacrificed in the evening and RNA was isolated from different brain regions to quantitate the isoform specific mRNAs of THRs. Following 14 days of lithium treatment, THR alpha1 mRNA levels were increased in the cortex and decreased in hypothalamus; THR alpha2 mRNA levels were increased in the cortex and THR beta mRNA levels were decreased in the hypothalamus. No significant difference in the expression of these THR isoforms was observed in the hippocampus or cerebellum. Thus, chronic lithium treatment appeared to regulate THR gene expression in a subtype and region specific manner in the rat brain. It remains to be determined whether the observed effects of lithium on THR gene expression are related to its therapeutic efficacy in the treatment of bipolar disorder.