Previously, we reported (Cagetti, Liang, Spigelman, and Olsen, 2003) that chronic intermittent ethanol (CIE) treatment leads to signs of alcohol dependence, including anxiety and hyperactivity, accompanied by reduced synaptic gamma-aminobutyric acid (A) receptor (GABAAR) function and altered sensitivity to its allosteric modulators consistent with a measured switch in subunit composition. In this study, we separated the synaptic and extrasynaptic components of GABAAR activation in recordings from pyramidal CA1 cells of hippocampal slices and demonstrated marked differences in the responsiveness of synaptic and extrasynaptic GABAARs to agonists and allosteric modulators in control rats, and in the way they are altered following CIE treatment. Notably, tonic inhibition mediated by extrasynaptic GABAARs was differentially sensitive to the partial agonist gaboxadol (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol; THIP) and the allosteric modulator zolpidem, compared with the miniature inhibitory synaptic currents (mIPSCs) in the same cells from saline-treated rats. After CIE treatment, potentiation of tonic currents by diazepam and zolpidem was lost, whereas potentiation by the alpha4 subunit-preferring benzodiazepine Ro15-4513 (ethyl 8-azido-6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a]-[1,4]benzodiazepine-3-carboxylate) and THIP was only partially reduced. Potentiation of synaptic GABAAR currents by zolpidem was eliminated after CIE, whereas THIP slightly inhibited mIPSCs from control rats and greatly enhanced them after CIE treatment. These results are consistent with alpha1 subunit decreases at synaptic and extrasynaptic GABAARs, whereas alpha4 subunits are increased at synaptic and decreased at extrasynaptic GABAARs. Behaviorally, THIP was active as a hypnotic and anxiolytic but not as an anti-convulsant against pentylenetetrazol seizures in control rats. Only slight tolerance was observed to the sleep time, but not to the anxiolytic, effect of THIP after CIE. Thus, differential alterations in synaptic and extrasynaptic GABAARs appear to play an important role in the brain plasticity of alcohol dependence, and withdrawal signs may be profitably treated with GABAergic drugs such as THIP, which does not show cross-tolerance with ethanol.

The National Institute for Health and Clinical Excellence (NICE) has issued guidance on cost-effectiveness analyses, suggesting that preference-based health-related quality of life (HRQL) weights or utilities be based on UK community preferences, preferably using the EQ-5D; ideally all analyses would use the same system for deriving HRQL weights, to encourage consistency and comparability across analyses. Development of a catalogue of EQ-5D scores for a range of health conditions based on UK preferences would help achieve many of these goals.

. To provide a UK-based catalogue of EQ-5D index scores.

.

s were consistent with the previously published catalogue of EQ-5D scores for the US. Community-based UK preferences were applied to EQ-5D descriptive questionnaire responses in the US-based Medical Expenditure Panel Survey (MEPS). Ordinary least squares (OLS), Tobit, and censored least absolute deviations (CLAD) regression methods were used to estimate the 'marginal disutility' of each condition controlling for covariates.

. Pooled MEPS files (2000-2003) resulted in 79,522 individuals with complete EQ-5D scores. Marginal disutilities for 135 chronic ICD-9 and 100 CCC codes are provided. Unadjusted descriptive statistics including mean, median, 25th and 75th percentiles are also reported.

. This research provides community-based EQ-5D index scores for a wide variety of chronic conditions that can be used to estimate QALYs in cost-effectiveness analyses in the UK. Although using EQ-5D questionnaire responses from the US-based MEPS is less than ideal, the estimates approximate HRQL guidelines by NICE and provide an easily accessible"off-the-shelf" resource for cost-effectiveness and public-health applications.

BACKGROUND

Reliably mapping from generic or disease-specific health status measures into health state utilities would assist health economists. Existing studies mainly use ordinary least squares (OLS) regression equations to predict utility values for particular health states. The authors examine an alternative approach to map between 2 generic health status instruments, the SF-12 and the EQ-5D.

METHODS

Multinomial logit regression is used to estimate the probability that a respondent will select a particular level of response to questions in the EQ-5D, using individual question responses and summary scores from the SF-12 as predictors. Monte Carlo simulation methods are used to generate predicted EQ-5D responses, and utility scores (tariffs) are then attached. Results are compared with an alternative approach based on direct mapping to utility scores using OLS.

METHODS

The authors estimate equations using 12,967 adult survey responses-from the 2000 US Medical Expenditure Panel Survey. They report mean squared error (MSE) and mean absolute error (MAE) of their predicted utilities within this sample, and out-of-sample using 13,304 adults from the 1996 Health Survey for England.

RESULTS

The authors obtain an in-sample and out-of-sample MSE of 0.03, compared with 0.02 for the OLS approach. Their MAE of 0.11 is similar to OLS results. The authors' method predicts group mean utility scores and differentiates between groups with or without known existing illness.

CONCLUSIONS

The authors' approach has higher MSE than the direct OLS approach but gives more descriptive data on domains of health effects. Further out of sample prediction work will help test the validity of these methods.

Oxidative mechanisms of injury are important in many neurological disorders, including hypoxic-ischemic brain damage. Cerebral palsy after preterm birth is hypothesized to be caused by hypoxic-ischemic injury of developing oligodendrocytes (OLs). Here we examined the developmental sensitivity of OLs to exogenous hydrogen peroxide (H2O2) with stage-specific rat oligodendrocyte cultures. We found that H2O2 itself or that generated by glucose oxidase was more toxic to developing than to mature OLs. Mature OLs were able to degrade H2O2 faster than developing OLs, suggesting that higher antioxidant enzyme activity might be the basis for their resistance. Catalase expression and activity were relatively constant during oligodendrocyte maturation, whereas glutathione peroxidase (GPx) was upregulated with a twofold to threefold increase in its expression and activity. Thus, it appeared that the developmental change in resistance to H2O2 was caused by modulation of GPx but not by catalase expression. To test the relative roles of catalase and GPx in the setting of oxidative stress, we measured enzyme activity in cells exposed to H2O2 and found that H2O2 induced a decrease in catalase activity in developing but not in mature OLs. Inhibition of GPx by mercaptosuccinate led to an increase in the vulnerability of mature OLs to H2O2 as well as a reduction in catalase activity. Finally, H2O2-dependent inactivation of catalase in developing OLs was prevented by the GPx mimic ebselen. These data provide evidence for a key role for GPx-catalase cooperativity in the resistance of mature OLs to H2O2-induced cell death.

Volatiles released from corn seedlings on which beet armyworm larvae were feeding were attractive to females of the parasitoid,Cotesia marginiventris (Cresson), in flight tunnel bioassays. Analyses of the collected volatiles revealed the consistent presence of 11 compounds in significant amounts. They were: (Z)-3-hexenal, (E)-2-hexenal, (Z)-3-hexen-1-ol, (Z)- 3-hexen-1-yl acetate, linalool, (3E)-4,8-dimethyl-1,3,7-nonatriene, indole, α-trans-bergamotene, (E)-β-farnesene, (E)-nerolidol, and (3E,7E)-4,8,12-trimethyl-1, 3,7,ll-tridecatetraene. A synthetic blend of all 11 compounds was slightly less attractive to parasitoid females than an equivalent natural blend. However, preflight experience with the synthetic blend instead of experience with a regular plant-host complex significantly improved the response to the synthetic blend. Our results suggest thatC. marginiventris females, in their search for hosts, use a blend of airborne semiochemicals emitted by plants on which their hosts feed. The response to a particular odor blend dramatically increases after a parasitoid experiences it in association with contacting host by-products.

We have cloned and sequenced the structural genes encoding the delta 5,6 sterol desaturase (ERG3 gene) and the 14 alpha-methyl sterol demethylase (ERG11 gene) from Candida glabrata L5 (leu2). Single and double mutants of these genes were created by gene deletion. The phenotypes of these mutants, including sterol profiles, aerobic viabilities, antifungal susceptibilities, and generation times, were studied. Strain L5D (erg3 delta::LEU2) accumulated mainly ergosta-7,22-dien-3 beta-ol, was aerobically viable, and remained susceptible to antifungal agents but had a slower generation time than its parent strain. L5LUD (LEU2 erg11 delta::URA3) strains required medium supplemented with ergosterol and an anaerobic environment for growth. A spontaneous aerobically viable mutant, L5LUD40R (LEU erg11 delta::URA3), obtained from L5LUD (LEU2 erg11 delta::URA3), was found to accumulate lanosterol and obtusifoliol, was resistant to azole antifungal agents, demonstrated some increase in resistance to amphotericin B, and exhibited a 1.86-fold increase in generation time in comparison with L5 (leu2). The double-deletion mutant L5DUD61 (erg3 delta::LEU2 erg11 delta::URA3) was aerobically viable, produced mainly 14 alpha-methyl fecosterol, and had the same antifungal susceptibility pattern as L5LUD40R (LEU2 erg11 delta::URA3), and its generation time was threefold greater than that of L5 (leu2). Northern (RNA) analysis revealed that the single-deletion mutants had a marked increase in message for the undeleted ERG3 and ERG11 genes. These results indicate that differences in antifungal susceptibilities and the restoration of aerobic viability exist between the C. glabrata ergosterol mutants created in this study and those sterol mutants with similar genetic lesions previously reported for Saccharomyces cerevisiae.

OBJECTIVE

In this study we examined the uptake of circulating lipoproteins into the retina, using a naturally fluorescent cholesterol analog for imaging and deuterated cholesterol for quantification by mass spectroscopy. The purpose of this study was to better understand cholesterol uptake, transport and homeostasis in the retina.

METHODS

Human low density lipoprotein (LDL) and high density lipoprotein (HDL) were labeled with the fluorescent cholesterol analog cholesta-5,7,9(11)-trien-3beta-ol (CTL) and deuterated cholesterol (25,26,26,26,27,27,27-[2H]cholesterol, D7Ch). Rats were injected intravenously with CTL-LDL, CTL-HDL and D7Ch-LDL. Fluorescent confocal microscopy was used to image the uptake of CTL and mass spectroscopy was used to quantify D7Ch. Immunohistochemistry and fluorescent confocal microscopy were used to localize apoB (an LDL marker protein) and LDL receptor (LDLR) protein in rat and monkey retinas.

RESULTS

CTL-specific fluorescence was imaged by confocal microscopy in the retinal pigment epithelium (RPE), choriocapillaris and parts of the neural retina within 2 h post-injection and was visualized in the photoreceptor outer segments by 4 h. Replacing LDL with HDL as the CTL carrier gave a less robust and more delayed labeling of retinal layers. Human apolipoprotein B (apoB) was also localized in the rat choriocapillaris and RPE by 4 h post-injection. Human apoB was detected by immunoblot analysis in the rat retina primarily as a about 70 kDa protein, suggesting proteolytic degradation. LDL-mediated uptake of cholesterol was quantified by mass spectroscopy using deuterated cholesterol in place of CTL. In addition, apoB and LDLR were localized in monkey retina by immunohistochemistry.

CONCLUSIONS

The retina is capable of rapid uptake of circulating LDL via an LDLR-mediated process primarily occurring in the RPE and also possibly Müller cells. Despite the dominance of HDL over LDL in rat serum, LDL appears to be the preferred carrier for cholesterol transport to and uptake by the retina. The results also suggest that blood-borne LDL represents a significant contributor to the steady-state levels of cholesterol and possibly other lipids in the retina.

5 alpha-Pregnan-3 alpha-ol-20-one (3 alpha-OH-DHP) is a naturally occurring metabolite of progesterone that can modulate brain excitability through a specific steroid recognition site on the GABA/benzodiazepine receptor-chloride ionophore complex. The anticonvulsant properties of 3 alpha-OH-DHP were determined using standardized anticonvulsant screening tests in mice. This steroid was found to be effective against metrazol-, (+)-bicuculline- and picrotoxin-induced seizures. The steroid has maximum potency against (+)-bicuculline-induced convulsions and no activity against maximal electroshock and strychnine-induced seizures. These findings support the hypothesis that therapeutically useful anticonvulsant steroids active at the putative steroid recognition site associated with the GABA/benzodiazepine receptor-chloride ionophore complex can be identified.

In this study, 48 h administration of 3alpha-OH-5beta-pregnan-20-one (3alpha,5beta-THP) or 17beta-estradiol (E2)+progesterone (P) to female rats increased expression of the delta subunit of the GABA(A) receptor (GABAR) in CA1 hippocampus. Coexpression of alpha4 and delta subunits was suggested by an increased response of isolated pyramidal cells to the GABA agonist 4,5,6,7- tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), following 48 h steroid treatment, and nearly complete blockade by 300 microM lanthanum (La3+). Because alpha4betadelta GABAR are extrasynaptic, we also recorded pharmacologically isolated GABAergic holding current from CA1 hippocampal pyramidal cells in the slice. The La3+-sensitive THIP current, representative of current gated by alpha4betadelta GABAR, was measurable only following 48 h steroid treatment. In contrast, the bicuculline-sensitive current was not altered by steroid treatment, assessed with or without 200 nM gabazine to block synaptic current. However, 48 h steroid treatment resulted in a tonic current insensitive to the benzodiazepine agonists lorazepam (10 microM) and zolpidem (100 nM). These results suggest that 48 h steroid treatment increases expression of alpha4betadelta GABAR which replace the ambient receptor population. Increased anxiolytic effects of THIP were also observed following 48 h steroid treatment. The findings from the present study may be relevant for alterations in mood and benzodiazepine sensitivity reported across the menstrual cycle.

BRL 34915 [6-cyano-3,4-dihydro-2,2-dimethyl-trans-4-(2-oxo-1-pyrrolidyl) 2H-benzo(b) pyran-3-ol], minoxidil sulfate and diazoxide may relax vascular smooth muscle via hyperpolarization due to an opening of membrane potassium channels. We therefore examined the effects of several potassium channel antagonists on the relaxation response to these vasodilators in isolated rat portal venous strips which were mounted in vitro for detecting changes in isometric force. BRL 34915 (IC50 = 4.7 X 10(-8) M), minoxidil sulfate (IC50 = 1.4 X 10(-7) M) and diazoxide (IC50 = 5 X 10(-6) M) elicited concentration-dependent relaxations of the spontaneous, myogenic contractions in venous strips. The relatively nonselective potassium channel antagonists tetraethylammonium ion (0.3-10 X 10(-3) M) and 4-aminopyridine (1-10 X 10(-3) M) caused concentration-dependent shifts (5- to 50-fold) in the relaxation responses to each vasodilator. Charybdotoxin (up to 10(-7) M) and apamin (up to 10(-7) M), known to be antagonists of high and low conductance calcium-activated potassium channels, respectively, had no inhibitory effect on the relaxation-response curves to BRL 34915, minoxidil sulfate or diazoxide. Glyburide (10(-7) to 3 X 10(-5) M), a sulfonylurea which has been shown to block the ATP-modulated potassium channel in insulin-secreting cells, caused concentration-dependent shifts to the right (up to 100-fold) of the IC50 value for BRL 34915 and diazoxide, and at 10(-6) M, abolished the relaxation response to minoxidil sulfate.(ABSTRACT TRUNCATED AT 250 WORDS)

Spatial activation patterns within the olfactory bulb are believed to contribute to the neural representation of odorants. In this study, we attempted to predict the perceptions of odorants from their evoked patterns of neural activity in the olfactory bulb. We first describe the glomerular activation patterns evoked by pairs of odorant enantiomers based on the uptake of [(14)C]2-deoxyglucose in the olfactory bulb glomerular layer. Using a standardized data matrix enabling the systematic comparison of these spatial odorant representations, we hypothesized that the degree of similarity among these representations would predict their perceptual similarity. The two enantiomers of carvone evoked overlapping but significantly distinct regions of glomerular activity; however, the activity patterns evoked by the enantiomers of limonene and of terpinen-4-ol were not statistically different from one another. Commensurate with these data, rats spontaneously discriminated between the enantiomers of carvone, but not between the enantiomers of limonene or terpinen-4-ol, in an olfactory habituation task designed to probe differences in olfactory perception.

Epicatechin is a flavan-3-ol that is commonly present in green teas, red wine, cocoa products, and many fruits, such as apples. There is considerable interest in the bioavailability of epicatechin after oral ingestion. In vivo studies have shown that low levels of epicatechin are absorbed and found in the circulation as glucuronides, methylated and sulfated forms. Recent research has demonstrated protective effects of epicatechin and one of its in vivo metabolites, 3'-O-methyl epicatechin, against neuronal cell death induced by oxidative stress. Thus, we are interested in the ability of ingested epicatechin to cross the blood brain barrier and target the brain. Rats were administered 100 mg/kg body weight/d epicatechin orally for 1, 5, and 10 d. Plasma and brain extracts were analyzed by HPLC with photodiode array detection and LC-MS/MS. This study reports the presence of the epicatechin glucuronide and 3'-O-methyl epicatechin glucuronide formed after oral ingestion in the rat brain tissue.

Flavonoids are polyphenolic compounds that are abundant in fruits and vegetables, and increasing evidence demonstrates a positive relationship between consumption of flavonoid-rich foods and disease prevention. Epidemiological, in vitro and animal studies support the beneficial effects of dietary flavonoids on glucose and lipid homeostasis. It is encouraging that the beneficial effects of some flavonoids are at physiological concentrations and comparable to clinically-used anti-diabetic drugs; however, clinical research in this field and studies on the anti-diabetic effects of flavonoid metabolites are limited. Flavonoids act on various molecular targets and regulate different signaling pathways in pancreatic β-cells, hepatocytes, adipocytes and skeletal myofibers. Flavonoids may exert beneficial effects in diabetes by (i) enhancing insulin secretion and reducing apoptosis and promoting proliferation of pancreatic β-cells; (ii) improving hyperglycemia through regulation of glucose metabolism in hepatocytes; (iii) reducing insulin resistance, inflammation and oxidative stress in muscle and fat and (iv) increasing glucose uptake in skeletal muscle and white adipose tissue. This review highlights recent findings on the anti-diabetic effects of dietary flavonoids, including flavan-3-ols, flavanones, flavonols, anthocyanidins, flavones and isoflavones, with particular emphasis on the studies that investigated the cellular and molecular mechanisms involved in the beneficial effects of the compounds.

This article proposes and evaluates a method to test for mediation in multilevel data sets formed when an intervention administered to intact groups is designed to produce change in individual mediator and outcome variables. Simulated data of this form were used to compare ordinary least squares (OLS) and two multilevel estimators of the mediated effect. OLS and multilevel standard error approximations were also evaluated and recommendations given for optimal estimator choice. These methods were applied to data from an existing substance use intervention to show the impact multilevel mediation modeling can have on the conclusions drawn from real-world evaluation studies.

Anthocyanins, flavan-3-ols, and flavonols are the three major classes of flavonoid compounds found in grape berry tissues. Several viticultural practices increase flavonoid content in the fruit, but the underlying genetic mechanisms responsible for these changes have not been completely deciphered. The impact of post-veraison sunlight exposure on anthocyanin and flavonol accumulation in grape berry skin and its relation to the expression of different transcriptional regulators known to be involved in flavonoid synthesis was studied. Treatments consisting of removing or moving aside the basal leaves which shade berry clusters were applied. Shading did not affect sugar accumulation or gene expression of HEXOSE TRANSPORTER 1, although in the leaf removal treatment, these events were retarded during the first weeks of ripening. Flavonols were the most drastically reduced flavonoids following shading and leaf removal treatments, related to the reduced expression of FLAVONOL SYNTHASE 4 and its putative transcriptional regulator MYB12. Anthocyanin accumulation and the expression of CHS2, LDOX, OMT, UFGT, MYBA1, and MYB5a genes were also affected. Other regulatory genes were less affected or not affected at all by these treatments. Non-transcriptional control mechanisms for flavonoid synthesis are also suggested, especially during the initial stages of ripening. Although berries from the leaf removal treatment received more light than shaded fruits, malvidin-3-glucoside and total flavonol content was reduced compared with the treatment without leaf removal. This work reveals that flavonol-related gene expression responds rapidly to field changes in light levels, as shown by the treatment in which shaded fruits were exposed to light in the late stages of ripening. Taken together, this study establishes MYB-specific responsiveness for the effect of sun exposure and sugar transport on flavonoid synthesis.

The objective of this work is to review data from epidemiological and preclinical studies addressing the potential benefits of diets based on flavonoids for cancer prevention. Flavonoids are subdivided into subclasses including flavonols, flavones, flavanones, flavan-3-ols, anthocyanidins, and isoflavones. Epidemiological studies suggest dietary intake of flavonoids may reduce the risk of tumors of the breast, colon, lung, prostate, and pancreas. However, some studies have reported inconclusive or even harmful associations. A major challenge in the interpretation of epidemiological studies is that most of the data originate from case-control studies and retrospective acquisition of flavonoid intake. Differences in agricultural, sociodemographics, and lifestyle factors contribute to the heterogeneity in the intake of flavonoids among populations residing in the United States, Europe, and Asia. Dose and timing of exposure may influence the anticancer response to flavonoid-rich diets. A limited number of intervention trials of flavonoids have documented cancer preventative effects. Proposed anticancer mechanisms for flavonoids are inhibition of proliferation, inflammation, invasion, metastasis, and activation of apoptosis. Prospective studies with larger sample sizes are needed to develop biomarkers of flavonoid intake and effect. Mechanistic studies are needed to ascertain how flavonoid-rich diets influence gene regulation for cancer prevention.

Many investigators use the reduced major axis (RMA) instead of ordinary least squares (OLS) to define a line of best fit for a bivariate relationship when the variable represented on the X-axis is measured with error. OLS frequently is described as requiring the assumption that X is measured without error while RMA incorporates an assumption that there is error in X. Although an RMA fit actually involves a very specific pattern of error variance, investigators have prioritized the presence versus the absence of error rather than the pattern of error in selecting between the two methods. Another difference between RMA and OLS is that RMA is symmetric, meaning that a single line defines the bivariate relationship, regardless of which variable is X and which is Y, while OLS is asymmetric, so that the slope and resulting interpretation of the data are changed when the variables assigned to X and Y are reversed. The concept of error is reviewed and expanded from previous discussions, and it is argued that the symmetry-asymmetry issue should be the criterion by which investigators choose between RMA and OLS. This is a biological question about the relationship between variables. It is determined by the investigator, not dictated by the pattern of error in the data. If X is measured with error but OLS should be used because the biological question is asymmetric, there are several methods available for adjusting the OLS slope to reflect the bias due to error. RMA is being used in many analyses for which OLS would be more appropriate.

Promoting remyelination, a major goal of an effective treatment for demyelinating diseases, has the potential to protect vulnerable axons, increase conduction velocity, and improve neurologic deficits. Strategies to promote remyelination have focused on transplanting oligodendrocytes (OLs) or recruiting endogenous myelinating cells with trophic factors. Ig-based therapies, routinely used to treat a variety of neurological and autoimmune diseases, underlie our approach to enhance remyelination. We isolated two human mAbs directed against OL surface antigens that promoted significant remyelination in a virus-mediated model of multiple sclerosis. Four additional OL-binding human mAbs did not promote remyelination. Both human mAbs were as effective as human i.v. Ig, a treatment shown to have efficacy in multiple sclerosis, and bound to the surface of human OLs suggesting a direct effect of the mAbs on the cells responsible for myelination. Alternatively, targeting human mAbs to areas of central nervous system (CNS) pathology may facilitate the opsonization of myelin debris, allowing repair to proceed. Human mAbs were isolated from the sera of individuals with a form of monoclonal gammopathy. These individuals carry a high level of monoclonal protein in their blood without detriment, lending support to the belief that administration of these mAbs as a therapy would be safe. Our results are (i) consistent with the hypothesis that CNS-reactive mAbs, part of the normal Ig repertoire in humans, may help repair and protect the CNS from pathogenic immune injury, and (ii) further challenge the premise that Abs that bind OLs are necessarily pathogenic.

The naturally occurring progesterone metabolites 5 beta-pregnan-3 alpha-ol-20-one and 5 beta-pregnane-3,20-dione reversibly enhance membrane currents elicited by locally applied GABA in bovine adrenomedullary chromaffin cells. Such potentiation was not influenced by the benzodiazepine antagonist Ro 15-1788. At concentrations in excess of those necessary to evoke potentiation of GABA currents, 5 beta-pregnan-3 alpha-ol-20-one and 5 beta-pregane-3,20-dione directly activated a membrane conductance. The resulting currents were potentiated by phenobarbitone and diazepam, and abolished by the GABAA-receptor antagonist, bicuculline. On outside-out membrane patches, 5 beta-pregnan-3 alpha-ol-20-one and 5 beta-pregnane-3,20-dione activated single channel currents of similar amplitude to those evoked by GABA. The results suggest that certain naturally occurring steroids potentiate the actions of GABA and, additionally, directly activate the GABAA receptor.

Intracellular recordings of membrane potassium current were made from rat locus coeruleus in vitro. The effects of agonists at mu-opioid receptors were studied on neurons from rats that had been chronically treated with morphine; these were compared with actions on neurons from control rats. Tolerance to the opioid-induced increase in potassium conductance was observed, and this was more pronounced for normorphine than for [Met5]enkephalin and [D-Ala2, Mephe4, Gly5-ol]enkephalin: experiments with the irreversible receptor blocker beta-chlornaltrexamine indicated that normorphine had lower intrinsic efficacy than [Met5]enkephalin and [D-Ala2 MePhe4, Gly5-ol]enkephalin. This adaptation was not due to any change of the properties of the potassium conductance activated by mu-receptors because both full and partial agonists at alpha 2-adrenoceptors, which couple to the same potassium conductance, were unchanged in their effectiveness; nor was it associated with any change in the affinity of mu-receptors for the antagonist naloxone. Naloxone had no effect on the neurons other than simple competitive reversal of the action of the mu-receptor agonists. These results demonstrate that 1) the mechanism responsible for tolerance in locus coeruleus neurons is specifically associated with mu-receptors and/or their coupling to potassium channels, 2) the intrinsic efficacy of an opioid determines the degree of tolerance observed, and 3) tolerance and physical dependence can be dissociated at the cellular level.

The purpose of these experiments was to test the hypothesis that attenuating the endogenous increase of the 5alpha-reduced progesterone metabolite 5alpha-pregnan-3alpha-ol-20-one (3alpha,5alpha-THP) in the hippocampus will alter anxiety and depression behavior of proestrous rats. In Experiment 1, anxiety (open field) and depression (forced swim test) behavior was compared of rats that should have high (proestrous) and low (diestrous and male rats) endogenous hippocampal 3alpha,5alpha-THP. Proestrous rats exhibited more anxiolytic-like (increased central entries in the open field) and anti-depressant-like (less immobility in the forced swim test) behavior than diestrous or male rats. In Experiments 2 and 3, respectively, systemic and intrahippocampal finasteride, a 5alpha-reductase inhibitor which attenuates progesterone's metabolism to 3alpha,5alpha-THP, versus vehicle administration to proestrous rats was compared for effects on open field and forced swim test behavior. Systemic or intrahippocampal finasteride decreased central entries in the open field and increased immobility in the forced swim tests compared to vehicle administration. In Experiment 4, the effects of systemic and intrahippocampal finasteride vs vehicle administration on hippocampal 3alpha,5alpha-THP of proestrous rats was examined. Finasteride, SC or intrahippocampally, reduced 3alpha,5alpha-THP in the hippocampus compared to vehicle administration. Together these data suggest that variations in 3alpha,5alpha-THP levels in the hippocampus may mitigate proestrous changes in anxiety and depressive behavior of cycling rats.

An HPLC analysis was performed on the concentrations of flavonoids in 42 species and cultivars of the Citrus genus and those of two Fortunella and one Poncirus species according to the classification system established by Tanaka. The composition of 8 flavanones and 9 flavone/ols for these species was determined in the albedo, flavedo, segment epidermis and juice vesicle tissues, and those in the fruit and peel tissues were calculated from the composition data of the tissues. A principal component analysis showed that such neohesperidosyl flavonoids as neoeriocitrin, naringin, neohesperidin, and rhoifolin had large factor loading values in the first principal component for each tissue. The flavonoid composition of citrus fruits was approximately the same within each section of Tanaka's system, except for the species in the Aurantium section and those with a peculiar flavonoid composition such as Bergamot (C. bergamia), Marsh grapefruit (C. paradisi), Sour orange (C. aurantium), and Shunkokan (C. shunkokan). The Aurantium section included both naringin-rich and hesperidin-rich species.

A key distinction between neural pacemaker and conventional network models for the generation of breathing rhythm in mammals is whether phasic reciprocal inhibitory interactions between inspiratory and expiratory neurons are required. In medullary slices from neonatal rats generating respiratory-related rhythm, we measured the phasic inhibitory inputs to expiratory neurons with the use of whole cell patch clamp in the hypothesized rhythm generation site, the pre-Bötzinger complex (pre-BötC). Expiratory neurons, which generate tonic impulse activity during the expiratory period, exhibited inhibitory postsynaptic potentials (IPSPs) synchronized to the periodic inspiratory bursts of the hypoglossal nerve root (XIIn). Bath application of the glycine receptor antagonist strychnine (STR; 5-10 microM) reversibly blocked these inspiratory-phase IPSPs, whereas the gamma-aminobutyric acid-A (GABA(A)) receptor antagonist bicuculline (BIC; 10-100 microM) had no effect on these IPSPs. Replacing the control in vitro bathing solution with a Cl(-)-free solution also abolished these IPSPs. Respiratory-related rhythmic activity was not abolished when inspiratory-phase IPSPs were blocked. The frequency and strength of XIIn rhythmic activity increased and seizurelike activity was produced when either STR, BIC, or Cl(-)-free solution was applied. Inspiratory-phase IPSPs were stable after establishment of whole cell patch conditions (patch pipettes contained 7 mM Cl-). Under voltage clamp, the reversal potential of inspiratory-phase inhibitory postsynaptic currents (IPSCs) was -75 mV. The current-voltage (I-V) curve for IPSCs shifted to the right when extracellular Cl- concentration was reduced by 50% (70 mM) and the reversal potential was reduced to -60 mV, close to the new Cl- Nernst potential. In tetrodotoxin (0.5 microM) under voltage clamp (holding potential = -45 mV), local application of glycine (1 mM) over pre-BötC induced an outward current and an increase in membrane conductance in expiratory neurons. The effect was blocked by bath application of STR (0.8-1 microM). Local application of the GABA(A) receptor agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP, 1 mM) induced an outward current and an increase in membrane conductance that was blocked by BIC (10-100 mM). Under voltage clamp (holding potential = -45 mV), we analyzed spontaneous IPSCs during expiration in expiratory neurons. Bath application of BIC (10 microM) reduced the IPSC frequency (from 2.2 to 0.3 per s), whereas the inspiratory-phase IPSCs did not change. Bath application of STR (8-10 microM) abolished both IPSCs. These results indicate that 1) reciprocal inhibition of expiratory neurons is glycinergic and mediated by a glycine-activated Cl- channel that is not required for respiratory-related rhythm generation in neonatal rat medullary slices; 2) endogenous GABA and glycine modulate the excitability of respiratory neurons and affect respiratory pattern in the slice preparation; 3) both glycine and GABA(A) receptors are found on pre-BötC expiratory neurons, and these receptors are sensitive to STR and BIC, respectively; 4) glycine and GABA(A) inhibitory mechanisms play different functional roles in expiratory neurons: both glycine and GABA(A) receptors modulate neuronal excitability, whereas glycinergic transmission alone is responsible for reciprocal inhibition; and 5) intracellular Cl- concentration in these neonatal expiratory neurons is similar to that in adults.

Fibroblast growth factors (FGFs) have been implicated in numerous cellular processes, including proliferation, migration, differentiation, and survival. Whereas FGF-2, the prototypic ligand in a family of 22 members, activates all four tyrosine kinase FGF receptors (FGFR1-FGFR4), other members demonstrate a higher degree of selectivity. Oligodendrocytes (OLs), the myelin-producing cells of the CNS, are highly influenced by FGF-2 at all stages of their development. However, how other FGFs and their cognate receptors orchestrate the development of OLs is essentially undefined. Using a combination of specific FGF ligands and receptor blocking antibodies, we now show that FGF-8 and FGF-17 target OL progenitors, inhibiting their terminal differentiation via the activation of FGFR3, whereas FGF-9 specifically targets differentiated OLs, triggering increases in process growth via FGFR2 signaling; FGF-18 targets both OL progenitors and OLs via activation of both FGFR2 and FGFR3. These events are highly correlated with changes in FGF receptor expression from FGFR3 to FGFR2 as OL progenitors differentiate into mature OLs. In addition, we demonstrate that, although activation of FGFR1 by FGF-2 leads to proliferation of OL progenitors, it produces deleterious effects on differentiated OLs (i.e., aberrant reentry into cell cycle and down-regulation of myelin proteins with a loss of myelin membrane). These data suggest that ligand availability, coupled with changes in FGF receptor expression, yield a changing repertoire of ligand-receptor signaling complexes that contribute critically to the regulation of both normal OL development and potential OL/myelin pathogenesis.