Aspen shaving versus chip bedding: effects on breeding and behavior.

The choice of laboratory cage bedding material is often based on both practical and husbandry issues, whereas behavioral outcomes rarely appear to be considered. It has been noted that a breeding success difference appears to be associated with the differential use of aspen chip and aspen shaving bedding in our facility; therefore, we sought to analyze breeding records maintained over a 20-month period. In fact, in all four mouse strains analyzed, shaving bedding was associated with a significant increase in average weanlings per litter relative to chip bedding. To determine whether these bedding types also resulted in differences in behaviors associated with wellbeing, we examined nest building, anxiety-like, depressive-like (or helpless-like), and social behavior in mice housed on chip versus shaving bedding. We found differences in the nests built, but no overall effect of bedding type on the other behaviors examined. Therefore, we argue that breeding success, perhaps especially in more challenging strains, is improved on shaving bedding and this is likely due to improved nest-building potential. For standard laboratory practices, however, these bedding types appear equivalent.

Canagliflozin, a novel inhibitor of sodium glucose co-transporter 2, dose dependently reduces calculated renal threshold for glucose excretion and increases urinary glucose excretion in healthy subjects.

Canagliflozin, a potent, selective sodium glucose co-transporter 2 inhibitor in development for treatment of type 2 diabetes, lowers plasma glucose (PG) by lowering the renal threshold for glucose (RT(G) ) and increasing urinary glucose excretion (UGE). An ascending single oral-dose phase 1 study investigated safety, tolerability and pharmacodynamics of canagliflozin in healthy men (N = 63) randomized to receive canagliflozin (n = 48) or placebo (n = 15). Canagliflozin (10, 30, 100, 200, 400, 600 or 800 mg q.d. or 400 mg b.i.d.) was administered to eight cohorts (six subjects/cohort: canagliflozin; two subjects/cohort: placebo). Dose dependently, canagliflozin decreased calculated 24-h mean RT(G) with maximal reduction to approximately 60 mg/dl, and increased mean 24-h UGE. At doses >200 mg administered before breakfast, canagliflozin reduced postprandial PG and serum insulin excursions at that meal. Canagliflozin was generally well tolerated; most adverse events were mild and no hypoglycaemia was reported. These results support further study of canagliflozin.

Pharmacological profile of a novel, non-TZD PPARgamma agonist.

AIM: The purpose of this study was to characterize a novel, non-thiazolidinedione (TZD) peroxisome proliferator-activated receptor (PPAR)gamma agonist, RWJ-348260, via both in vitro and in vivo approaches. METHODS: The in vitro PPARgamma activities of RWJ-348260 were assessed in PPARgamma-GAL4 co-transfection assay, PPARgamma receptor binding assay, aP2 gene induction assay and preadipocyte differentiation assay. The in vivo efficacy of the compound was determined in rodent genetic diabetes models [ob/ob mouse, db/db mouse and Zucker diabetic fatty (ZDF) rat] following multiple days of oral administration. RESULTS: RWJ-348260 selectively activated PPARgammain vitro. In vivo, RWJ-348260 produced significant decreases in plasma glucose, HbA1c, insulin and triglyceride levels. RWJ-348260 also dose-dependently improved oral glucose tolerance. In db/db mice, the compound up-regulated PPARgamma target genes in white adipose tissues. RWJ-348260 produced a lower extent of hepatocyte lipid deposition and a smaller increase in liver weight compared to rosiglitazone in db/db mice. While RWJ-348260 effectively normalized hyperglycaemia and dyslipidaemia, it did not change haematocrit, transaminase, alkaline phosphatase, total bilirubin levels or liver weights in ZDF rats. CONCLUSIONS: RWJ-348260 is a potent PPARgamma agonist with efficacious antidiabetic activity in diabetic animal models. The compound has an improved side-effect profile compared to rosiglitazone.

Topiramate ameliorates hyperglycaemia and improves glucose-stimulated insulin release in ZDF rats and db/db mice.

Topiramate (TPM) is a novel neurotherapeutic agent. Clinical studies reported that TPM treatment reduced body weight and decreased fasting blood glucose levels in obese patients with or without type 2 diabetes. It is unclear whether the blood glucose-normalizing phenomenon observed during TPM treatment is a primary effect or the consequence of reduced food intake and weight loss. In the present studies, we chronically treated female Zucker diabetic fatty (ZDF) rats (fed with a diabetogenic diet) and db/db mice with TPM (30-300 mg/kg/day) to examine the effect of TPM on hyperglycaemia and its relationship with food intake and body weight gain. Our data showed that TPM treatment markedly reduced blood glucose levels in both ZDF rats and db/db mice without a significant reduction in body weight gain. Pair-fed db/db mice treated with the vehicle alone did not exhibit a significant decrease in blood glucose levels compared with mice fed ad libitum. TPM treatment increased glucose-stimulated insulin release by 2-3-fold during an oral glucose tolerance test in both ZDF rats and db/db mice. We also observed a 1.4-fold increase of pancreatic insulin content and heightened insulin immunostaining in pancreatic beta cells in db/db mice treated with TPM. Our data suggest that the antidiabetic effect of TPM is independent of the changes in body weight gain and food intake. Improved glucose-induced insulin release may, in part, underlie the mechanisms by which TPM ameliorates the hyperglycaemia.

Multiple cross mapping (MCM) markedly improves the localization of a QTL for ethanol-induced activation.

This study examines the use of multiple cross mapping (MCM) to reduce the interval for an ethanol response QTL on mouse chromosome 1. The phenotype is the acute locomotor response to a 1.5-g/kg i.p. dose of ethanol. The MCM panel consisted of the six unique intercrosses that can be obtained from the C57BL/6J (B6), DBA/2J (D2), BALB/cJ (C) and LP/J (LP) inbred mouse strains (N > or = 600/cross). Ethanol response QTL were detected only with the B6xD2 and B6xC intercrosses. For both crosses, the D2 and C alleles were dominant and decreased ethanol response. The QTL information was used to develop an algorithm for sorting and editing the chromosome 1 Mit microsatellite marker set (http://www.jax.org). This process yielded a cluster of markers between 82 and 85cM (MGI). Evidence that the QTL was localized in or near this interval was obtained by the analysis of a sample (n = 550) of advanced cross heterogenous stock animals. In addition, it was observed that one of the BXD recombinant inbred strains (BXD-32) had a recombination in the interval of interest which produced the expected change in behavior. Overall, the data obtained suggest that the information available within existing genetic maps coupled with MCM data can be used to reduce the QTL interval. In addition, the MCM data set can be used to interrogate gene expression data to estimate which polymorphisms within the interval of interest are relevant to the QTL.

Quantitative analysis of agonist-dependent parathyroid hormone receptor trafficking in whole cells using a functional green fluorescent protein conjugate.

Many G-protein coupled receptors (GPCRs) undergo ligand-dependent internalization upon activation. The parathyroid hormone (PTH) receptor undergoes endocytosis following prolonged exposure to ligand although the ultimate fate of the receptor following internalization is largely unknown. To investigate compartmentalization of the PTH receptor, we have established a stable cell line expressing a PTH receptor-green fluorescent protein (PTHR-GFP) conjugate and an algorithm to quantify PTH receptor internalization. HEK 293 cells expressing the PTHR-GFP were compared with cells expressing the wild-type PTH receptor in whole-cell binding and functional assays. 125I-PTH binding studies revealed similar Bmax and kD values in cells expressing either the PTHR-GFP or the wild-type PTH receptor. PTH-induced cAMP accumulation was similar in both cell lines suggesting that addition of the GFP to the cytoplasmic tail of the PTH receptor does not alter the ligand binding or G-protein coupling properties of the receptor. Using confocal fluorescence microscopy, we demonstrated that PTH treatment of cells expressing the PTHR-GFP conjugate produced a time-dependent redistribution of the receptor to the endosomal compartment which was blocked by pretreatment with PTH antagonist peptides. Treatment with hypertonic sucrose prevented PTH-induced receptor internalization, suggesting that the PTH receptor internalizes via a clathrin-dependent mechanism. Moreover, co-localization with internalized transferrin showed that PTHR-GFP trafficking utilized the endocytic recycling compartment. Experiments using cycloheximide to inhibit protein synthesis demonstrated that recycling of the PTHR-GFP back to the plasma membrane was complete within 1-2 h of ligand removal and was partially blocked by pretreatment with cytochalasin D, but not nocodazole. We also demonstrated that the PTH receptor, upon recycling to the plasma membrane, is capable of undergoing a second round of internalization, a finding consistent with a role for receptor recycling in functional resensitization.

Further characterization and high-resolution mapping of quantitative trait loci for ethanol-induced locomotor activity.

Differential sensitivity to the stimulant effects of ethanol on locomotor activity is determined in part by genetic differences. Among inbred strains of mice, moderate doses of ethanol (1-2 g/kg) stimulate locomotor activity in some strains, e.g., the DBA/2J (D2), but only mildly affect activity in other strains, e.g., C57BL/6J (B6) (Crabbe et al., 1982, 1983; Crabbe, 1986; Dudek and Phillips, 1990; Dudek et al., 1991; Dudek and Tritto, 1994). Quantitative trait loci (QTL) for the acute ethanol (1.5 g/kg) locomotor response has been identified in the BXD recombinant inbred (RI) series (N = 25 strains), a C57BL/6J x DBA/2J (B6D2) F2 intercross (N = 1800), and heterogeneous stock (HS) mice (N = 550). QTLs detected (p < .01) in the RI series were found on chromosomes 1, 2, and 6 and these QTLs were expressed in a time-dependent fashion. The QTLs on chromosomes 1 and 2 were confirmed in the F2 intercross at p < 10(-7) or better. HS mice from G32 to G35 were used to fine-map the chromosome 2 QTL. Compared to the consensus map, the genetic map in the HS animals was expanded 10- to 15-fold. Over the region flanked by D2Mit94 to D2Mit304, three separate QTLs were detected in the HS animals. The data obtained confirm the usefulness of HS mice for the fine-mapping of QTLs to a resolution of 2 cM or less.

Supersensitivity to anandamide and enhanced endogenous cannabinoid signaling in mice lacking fatty acid amide hydrolase.

The medicinal properties of marijuana have been recognized for centuries, but clinical and societal acceptance of this drug of abuse as a potential therapeutic agent remains fiercely debated. An attractive alternative to marijuana-based therapeutics would be to target the molecular pathways that mediate the effects of this drug. To date, these neural signaling pathways have been shown to comprise a cannabinoid receptor (CB(1)) that binds the active constituent of marijuana, tetrahydrocannabinol (THC), and a postulated endogenous CB(1) ligand anandamide. Although anandamide binds and activates the CB(1) receptor in vitro, this compound induces only weak and transient cannabinoid behavioral effects in vivo, possibly a result of its rapid catabolism. Here we show that mice lacking the enzyme fatty acid amide hydrolase (FAAH(-/-)) are severely impaired in their ability to degrade anandamide and when treated with this compound, exhibit an array of intense CB(1)-dependent behavioral responses, including hypomotility, analgesia, catalepsy, and hypothermia. FAAH(-/-)-mice possess 15-fold augmented endogenous brain levels of anandamide and display reduced pain sensation that is reversed by the CB(1) antagonist SR141716A. Collectively, these results indicate that FAAH is a key regulator of anandamide signaling in vivo, setting an endogenous cannabinoid tone that modulates pain perception. FAAH may therefore represent an attractive pharmaceutical target for the treatment of pain and neuropsychiatric disorders.

Discovery of the first non-peptide antagonist of the motilin receptor.

A first-in-class non-peptide antagonist of the motilin receptor was identified through electronic screening of our corporate database against a 3D pharmacophore. The pharmacophore was developed from the motilin 22 residue endogenous peptide using NMR structural data, principles of peptide folding, and peptide structure activity relationships. The NMR data supported helical content within the peptide, and both the hydrophobic staple and N-capping box motifs were identified in the motilin sequence. The conformational features of these motifs were imposed on the peptide structure, providing a constrained conformer as a starting point for database searching. A trisubstituted cyclopentene lead was identified directly from the electronic search. Compounds in this series inhibit the binding of 125I-motilin to human antral smooth muscle membrane and antagonize motilin-induced intracellular calcium mobilization in cells expressing the human motilin receptor. A potent compound developed through optimization, RWJ 68023, is active in binding and cell-based functional assays and is also effective in inhibiting motilin-induced contractility in segments of rabbit duodenum. This orally active compound is currently undergoing clinical evaluation for the treatment of gastrointestinal disorders associated with altered motility.

Identification and time dependence of quantitative trait loci for basal locomotor activity in the BXD recombinant inbred series and a B6D2 F2 intercross.

A complimentary two-phase strategy was used to detect and map quantitative trait loci (QTLs) associated with the basal locomotor response to a saline challenge (10 ml/kg). In phase 1, putative QTLs, significant at p < 0.01 or better, were identified by analysis of the strain means for 25 strains of the B x D recombinant inbred series. QTLs were identified on chromosomes 1, 3, 5, 9, 10, 16, and 18. Some of these QTLs were detected across the entire experimental period (0-20 min), while others were associated with specific 5-min blocks. Eighteen hundred C57BL/6J (B6) x DBA/2J (D2) F2 intercross animals were phenotyped for the basal locomotor response, and of this group, 500 to 700 individuals, pseudo-randomly selected, were used for a genomewide scan to confirm the RI-generated QTLs and to detect new QTLs. No new QTLs were detected but the QTLs on chromosome 1 were confirmed at p < 10(-5) to p < 10(-9), depending on the time interval. In addition, the QTLs on chromosomes 5 and 9 were confirmed at p < 0.001, providing a combined probability (RI + F2) which exceeds the threshold for a significant association. Two additional phenotypes which showed significant RI strain differences were examined--adaptation and thigmotaxis. Adaptation mapped to the same region of chromosome 9 and thigmotaxis to the same region of chromosome 1 as the distance-traveled QTL. Overall, the data presented here and elsewhere (Flint et al., 1995; Gershenfeld et al., 1997) illustrate that QTLs for basal activity are both robust and reliable.

Cell-based, high-content screen for receptor internalization, recycling and intracellular trafficking.

A variety of physiologically important receptors are internalized and then recycled back to the plasma membrane by the endocytic recycling compartment. These include the transferrin receptor and many G-protein coupled receptors (GPCRs). The internalization of GPCRs is a result of agonist stimulation. A cell-based fluorescent imaging assay is described that detects and quantifies the presence of fluorescently labeled receptors and macromolecules in the recycling compartment. This High Content Screening application is conducted on the ArrayScan II System that includes fluorescent reagents, imaging instrumentation and the informatics tools necessary to screen for compounds that affect receptor internalization, recycling and GPCR activation. We demonstrate the Receptor Internalization and Trafficking application by quantifying (i) the internalization and recycling of the transferrin receptor using a fluorescently labeled ligand and (ii) the internalization of a physiologically functional model GPCR, a GFP-parathyroid hormone receptor chimera. These assays give high signal-to-noise ratios, broad dynamic ranges between stimulated and unstimulated conditions and low variability across different screening runs. Thus, the Receptor Internalization and Trafficking application, in conjunction with the ArrayScan II System, forms the basis of a robust, information-rich and automated screen for GPCR activation.

A branched DNA signal amplification assay to quantitate messenger RNA of human uncoupling proteins 1, 2, and 3.

Uncoupling proteins (UCP) are inner mitochondrial membrane transporters which dissipate the proton gradient, releasing stored energy as heat. Three subtypes of UCP have been identified so far. The regulation of UCP expression is mainly controlled at the transcriptional level, thus making the measurement of UCP mRNA beneficial for both diagnosis and research of weight disorders and diabetes. We have developed an assay using the branched DNA signal amplification assay (bDNA assay) to quantitatively measure the mRNA levels for human UCP1, 2, and 3. UCP-subtype-specific primers were designed for the assay. RNA transcripts of each UCP generated by in vitro transcription were used to validate the specificity and sensitivity of the assay. The quantitative measurement of UCP mRNA was further demonstrated with cultured cells and human tissue. A comprehensive survey of UCP expression from 17 human tissues measured by the newly developed assay is provided. The method described here offers a rapid, sensitive, specific, and quantitative assay for measurement of human UCP mRNA.

Effect of genetic cross on the detection of quantitative trait loci and a novel approach to mapping QTLs.

A genome-wide scan was conducted in two F(2) intercrosses, C57BL/6J (B6)xDBA/2J (D2) and BALB/cJ (C)xLP/J (LP), for three different phenotypes: basal locomotor activity, ethanol-induced locomotor activity, and haloperidol-induced catalepsy. For basal activity, significant quantitative trait loci (QTLs, LOD> or =4.3) were detected on chromosomes 9 and 19 for the CxLP intercross and chromosome 1 for the B6xD2 intercross. Significant QTLs for ethanol-induced activation were detected on chromosome 6 for the CxLP intercross, and on chromosomes 1 and 2 for the B6xD2 intercross. For haloperidol-induced catalepsy, significant QTLs were detected on chromosome 14 (two different QTLs) in the CxLP intercross, and chromosomes 1 and 9 in the B6xD2 intercross. These data illustrate the importance of the genetic cross for QTL detection. Finally, the data reported here, and elsewhere, are also used to demonstrate a novel approach to QTL detection and localization.

Characterization of the progestin receptors in the human TE85 and murine MC3T3-E1 osteoblast-like cell lines.

Progestins are believed to exert positive effects on bone density through receptors located in osteoblasts. In the present studies, the binding characteristics and regulation of the progestin receptors in two osteoblast-like cell lines were compared with those in human breast lines. Human TE85 and murine MC3T3-E1 osteoblast-like cells contain a single, high-affinity progestin binding site whose affinity and concentration are lower than in human breast cells. The osteoblastic progestin binding sites showed the expected steroid specificity and associated with the cell nuclei when occupied by ligand. The progestin receptors in osteoblastic cells also had sedimentation coefficients similar to those receptors in breast cells. The regulation of the progestin receptor in the osteoblast-like cells was explored by treating them with estradiol. In contrast to the large, rapid change seen in the breast cells, the progestin receptor levels in the MC3T3-E1 cells showed only a small, delayed up-regulation with estradiol treatment. The progestin receptor number in the TE85 cells was unaffected by estradiol. Down-regulation of the progestin receptors was explored by treating the cells with the progestin, norethindrone (NET). NET administration produced a rapid drop in progestin binding sites in the breast cells and a smaller, more gradual decline in MC3T3-E1 progestin binding. While the maximal decrease in receptor number occurred within 24 h in the breast cells, the receptor number was still continuing to fall after 72 h in the MC3T3-E1 cells. The data presented here demonstrate that both human and murine osteoblast-like cells contain a functional progestin receptor whose binding characteristics and regulation are similar, but not identical, to those receptors in other progestin target tissues such as the breast.

PPARgamma activation induces the expression of the adipocyte fatty acid binding protein gene in human monocytes.

The peroxisome-proliferator activated receptor gamma (PPARgamma), a member of the nuclear receptor superfamily of ligand activated transcription factors, plays a key role in the anti-diabetic actions of the thiazolidinediones (TZDs). PPARgamma induces the expression of many genes involved in lipid anabolism, including the adipocyte fatty acid binding protein (aP2), and is a key regulator of adipocyte differentiation. PPARgamma is also expressed in hematopoietic cells and is up-regulated in activated monocytes/macrophages. Activation of PPARgamma may play a role in the induction of differentiation of macrophages to foam cells that are associated with atherosclerotic lesions. We report that both natural and synthetic PPARgamma agonists induce time- and dose-dependent increases in aP2 mRNA in both primary human monocytes and the monocytic cell line, THP-1. These data suggest that PPARgamma activation may play a role in monocyte differentiation and function analogous to its well-characterized role in adipocytes.

A novel method for analysis of nuclear receptor function at natural promoters: peroxisome proliferator-activated receptor gamma agonist actions on aP2 gene expression detected using branched DNA messenger RNA quantitation.

Peroxisome proliferator-activated receptor-gamma (PPARgamma), a member of the nuclear hormone receptor superfamily, plays an essential role in the mediation of the actions of antidiabetic drugs known as thiazolidinediones (TZDs). PPARgamma activates many target genes involved in lipid anabolism including the adipocyte fatty acid binding protein (aP2). In this study, induction of aP2 gene expression by PPARgamma agonists was examined in both cultured cells and diabetic mice using branched DNA (bDNA)-mediated mRNA quantitation. bDNA technology allows for the direct measurement of a particular mRNA directly within cellular lysate using a 96-well plate format in a time frame comparable to a reporter gene assay. In cultured human subcutaneous preadipocytes, the TZDs, troglitazone and BRL-49653, both rapidly induced aP2 mRNA as detected with the bDNA method. In these cells, the effect of BRL-49653 on aP2 mRNA levels was detectable as early as 30 min after treatment (47% increase) and was maximal after 24 h of treatment (12-fold increase). The effects of troglitazone on aP2 mRNA induction were similar to those of BRL-49653 except that the maximal level of induction was consistently lower (e.g. 24 h treatment = 4-fold increase). Dose-response relationships for both of the TZDs were also determined using the 24-h treatment time point. EC50s for both BRL-49653 and troglitazone were estimated to be 80 nM and 690 nM, respectively. A natural PPARgamma ligand, 15-deoxy-delta12,14-PGJ2, was also active in this assay with a maximal induction of aP2 mRNA of approximately 5-fold when tested at 1 microM. Since the PPARgamma:retinoid X receptor (RXR) heterodimer has been characterized as a permissive heterodimer with respect to RXR ligands, the ability of 9-cis-retinoic acid (9-cis-RA) to induce aP2 mRNA was examined. Although 9-cis-RA had very low efficacy (2-fold induction), the maximal effect was reached at 100 nM. No synergism or additivity in aP2 mRNA induction was detected when 9-cis-RA was included with either of the TZDs used in this study. Significant induction of aP2 mRNA in bone marrow of db/db mice treated with either troglitazone or BRL-49653 was also detected, indicating that the bDNA assay may be a simple method to monitor nuclear receptor target gene induction in vivo.

Ethanol-induced expression of c-Fos differentiates the FAST and SLOW selected lines of mice.

The effect of ethanol on the number of Fos-like immunoreactive (Fos-li) neurons was previously studied in the C57BL/6J (B6) and DBA/2J (D2) inbred mouse strains (Hitzemann and Hitzemann, 1997). Data obtained suggested that the locomotor activation response to ethanol found in the D2 but not the B6 strain was associated with an increase in the number of Fos-li neurons (a putative measure of synaptic activity) in the central nucleus of the amygdala (CeA), but not in other brain regions, including the basal ganglia. Supporting results were obtained in B6D2 F2 intercross animals (Demarest et al., 1998) those animals exhibiting a marked locomotor activation response to ethanol also showed a significant increase in the number of Fos-li neurons in the CeA. The current study extends this line of investigation to the FAST and SLOW selected lines of mice (Shen et al., 1995). Twenty-eight SLOW and FAST mice (taken evenly from both replicate lines) were randomly assigned to receive either saline or ethanol (1.5 g/kg). One hour later, the animals were sacrificed, and the number of Fos-li neurons were determined using standard immunocytochemical techniques. Both the FAST and SLOW lines showed a marked increase (>300%) in the number of Fos-li neurons in the lateral aspect of the CeA; however, in the capsular division, only the FAST line showed an increase (>500%). In several brain regions, the basal (saline) response was markedly higher in the SLOW line; these regions included the subthalamic nucleus, the entopeduncular nucleus, the substantia nigra compacta, and the ventral tegmental area. Furthermore, it was found that ethanol decreased the number of Fos-li neurons in the ventral tegmental area of the SLOW but not FAST mice. These data suggest a substantial involvement of the basal ganglia in the segregation of the FAST and SLOW lines.

Identification of an acute ethanol response quantitative trait locus on mouse chromosome 2.

A two-stage strategy was used to identify and confirm quantitative trait loci (QTLs) associated with the changes in locomotor activity induced by a 1.5 gm/kg ethanol challenge. For stage 1, putative QTLs were identified by analysis of the strain means for 25 strains of the BXD recombinant inbred (RI) series (males only). QTLs were identified on chromosomes 1, 2, 4, and 6. The activity response to chlordiazepoxide generated similar QTLs on chromosomes 2 and 6. None of the QTLs were similar to those generated from analysis of the saline response data. For stage 2, 900 male C57BL/6J (B6) x DBA/2J (D2) F2 intercross animals were phenotyped for ethanol response, and the phenotypic extremes (those animals > and <1 SD from the mean) were identified. These extremes differed by >10,000 cm/15 min in their response to ethanol. The extreme progeny were used for a genome-wide scan both to confirm the putative RI-generated QTLs and to detect new QTLs. The F2 analysis generated no new QTLs with logarithm of the likelihood for linkage (LOD) scores >3. For RI-generated QTLs, only the QTL on chromosome 2 was confirmed (LOD = 5.3). The position of the peak LOD was estimated to be 47 cM with a 20 cM 1 LOD support interval; this QTL accounted for 6% of the phenotypic variance. The 1 LOD support interval overlaps with QTLs previously identified for alcohol preference and acute ethanol withdrawal (;; ).

Further evidence that the central nucleus of the amygdala is associated with the ethanol-induced locomotor response.

The effect of ethanol on the number of Fos-like immunoreactive (Fos-li) neurons was previously studied in the C57BL/6J (B6) and DBA/2J (D2) inbred mouse strains (Hitzemann and Hitzemann, Alcohol. Clin. Exp. Res. 21:1497-1507, 1997). The data obtained suggested that the locomotor activation response to ethanol found in the D2 but not the B6 strain was associated with an increase in the number of Fos-li neurons (a putative measure of synaptic activity) in the central nucleus of the amygdala (CeA), but not in other brain regions, including the basal ganglia. The current study was performed to obtain data supporting a role for the CeA in the locomotor response. B6D2 F2 intercross animals were phenotyped for their locomotor response to ethanol (1.5 g/kg). The animals from the extreme phenotypes (> 1 SD from the mean) were denoted as very high and very low activity (HH, LL) and differed in their ethanol response by >9,000 cm/15 min (baseline activity was similar in both phenotypes: 5,500 cm/15 min). These extremes especially differed from the parental strains in that the LL group showed a significant ethanol-induced inhibition of activity. After 2 weeks, HH and LL animals were rechallenged with 1.5 g/kg of ethanol or saline and the number of Fos-li neurons determined 1 hr later. In the HH group, ethanol increased the number of Fos-li neurons >600%, whereas in the LL group the increase was 170% (difference: p < 0.001). The increase in the HH group was principally located in the GABA neuron-rich lateral aspect of the CeA and not in the medial posterior-ventral division or the caps division. No significant difference was found between groups in the Fos response for the basolateral or lateral amygdala. Other brain regions were also examined, including the basal ganglia, the hippocampus (CA1, CA3, and dentate gyrus), the bed nucleus of the stria terminalis, and several cortical regions. In some regions (e.g., the bed nucleus), a significant ethanol effect was detected, but it did not differentiate the HH and LL groups. Overall, the data obtained further argue that the CeA has an important role in regulating the acute locomotor response to ethanol.

In vitro permeability of eight beta-blockers through Caco-2 monolayers utilizing liquid chromatography/electrospray ionization mass spectrometry.

It is demonstrated that the apparent permeability (P(app)) coefficients of beta-adrenoceptor antagonist drugs can easily be determined for Caco-2 cell culture intestinal models utilizing liquid chromatography/mass spectrometry (LC/MS). The LC/MS method with electrospray ionization in the single ion monitoring mode showed an increased sensitivity of 1000-fold compared with LC/UV detection and enhanced selectivity with respect to both LC/UV and radioactivity assays. The P(app) coefficients of beta-adrenoceptor antagonists determined by LC/MS have the same ranking order as those determined by LC/UV and radioactivity assays. However, the P(app) coefficients determined in this study showed significant discrepancies from those determined in other laboratories. There are several experimental factors that directly affect the absolute value of the P(app) coefficients, including pH gradients, additional diffusion barriers (i.e. unstirred water layer and type of filter support), analyte concentration, detection method and possibly cell culture variations. These parameters should be controlled when generating Caco-2 P(app) coefficients for different compounds.