5-HT 3 receptor antagonist ICS 205-930 alters the discriminative effects of ethanol.

The ability of a selective 5-hydroxytryptamine (5-HT(3)) receptor antagonist, ICS 205-930 (3-tropanyl-indole-1-carboxylate, tropisetron), to block the discriminative stimulus effects of ethanol was investigated in rats that were trained to discriminate ethanol (1.25 g/kg ip) from saline with food as the reinforcement. Prior administration of ICS 205-930, at the dose of 0.01 mg/kg, significantly decreased ethanol's discriminative stimulus effect at ED(75) dose of ethanol, while higher doses of ICS 205-930 (10 and 17 mg/kg) showed enhancement of ethanol's discriminative effects at ED(0), ED(25), and ED(50) doses of ethanol. Under conditions where ICS 205-930 (10, 17 mg/kg) was tested alone, rats responded exclusively on the saline-appropriate lever. These effects occurred without significantly altering response rates or blood ethanol concentrations. The results suggest that the 5-HT(3) antagonist ICS 205-930 at lower concentration decreases, and at higher concentration enhances the discriminative stimulus effects associated with a lower to moderate dose of ethanol.

Opiate delta-2-receptor antagonist naltriben does not alter discriminative stimulus effects of ethanol.

The ability of a selective 2-opiate receptor antagonist, naltriben, to modulate ethanol discrimination was investigated in a rat model using a drug discrimination procedure. Rats were trained to discriminate ethanol (1.25 g/kg, IP) from saline on a fixed-ratio schedule, FR10. Once rats had acquired the ethanol-saline discrimination, ethanol dose-response tests were conducted with 15-min pretest injections. Following the characterization of the ethanol dose-response curve, the effect of naltriben on ethanol's discriminative stimulus was assessed by administering naltriben (0. 032-5.6 mg/kg, IP) 15 min before the ethanol administration. In the present study, naltriben did not have any modulatory effect on ethanol discrimination, suggesting that either Delta(2)-opiate receptors are not involved in the formation of ethanol's discriminative stimulus or the antagonism of Delta(2)-opiate receptors is not sufficient to alter ethanol's compound discriminative stimulus.

Age-related differences in the quality of life of breast carcinoma patients after treatment.

BACKGROUND: The objective of this study was to compare the quality of life (QOL) of younger (< or =50 years) versus older (>50 years) women on recent completion of treatment of breast carcinoma. METHODS: Data reported herein were obtained from a baseline assessment of 304 breast carcinoma patients. These patients were enrolled in a multiinstitutional, randomized trial testing a psychosocial telephone counseling intervention for breast carcinoma patients immediately after treatment. The assessment was made using a self-administered (mail) questionnaire, with an overall response rate of 86%. Included in this questionnaire were standardized measures of QOL using the Functional Assessment of Cancer Therapy-Breast instrument, the Center for Epidemiologic Studies Depression Scale, and the Impact of Event Scale. RESULTS: Comparisons of baseline data analyzed according to age approximating menopausal status (< or =50 years and >50 years) indicated that younger women reported significantly greater QOL disturbance. QOL was significantly worse for younger women globally (P = 0.021), and with regard to domains of emotional well-being (P = 0.0002) and breast carcinoma specific concerns (P = 0.022). Furthermore, symptoms of depression (P = 0.041) and disease specific intrusive thoughts (P = 0.013) were significantly worse for younger women. No significant sexual dysfunction or body image differences were noted. CONCLUSIONS: Results from this analysis suggest that younger women with breast carcinoma should be considered to be at high risk for QOL disruption and significant clinical distress. Targeted interventions for this cohort are recommended.

Genotypic differences between C57BL/6 and A inbred mice in anxiolytic and sedative actions of diazepam.

The role of genotype in susceptibility to the behavioral actions of benzodiazepines is not well characterized. To develop a model for such studies, we have characterized the anxiolytic and sedative activities of diazepam in C57BL/6J and A/J inbred mice. C57BL/6J mice were more responsive than A/J mice to diazepam-induced anxiolytic-like activity in the mirrored chamber aversion assay and the elevated plus-maze assay. Basal activity of the two strains did not differ in either assay. In contrast, the two strains were equally responsive to the anxiolytic effects of the 5-HT1A receptor partial agonist, buspirone. C57BL/6J mice were also more susceptible to the sedative effects of diazepam than were A/J mice. Flumazenil blocked the effects of diazepam in these behavioral assays. Measurement of diazepam and nordiazepam in blood and brain suggested that the response differences are of a pharmacodynamic rather than a pharmacokinetic nature. Taken together, these findings indicate that C57BL/6J and A/J mice provide a valuable tool for behavioral genetic studies of the mechanisms underlying the pharmacological actions of benzodiazepines.

Enhancement of gamma-aminobutyric acidA receptor activity by alpha-chloralose.

alpha-Chloralose is widely used as an anesthetic in the laboratory due to its minimal effects on autonomic and cardiovascular systems, yet little is known about its mechanism of action. We examined the effects of alpha-chloralose on gamma-aminobutyric acid type A (GABAA) receptor activity because recent studies have shown that several classes of general anesthetics modulate the function of this receptor. GABAA receptor activity was assayed by measuring the GABA-induced current in Xenopus oocytes expressed with human GABAA receptor alpha-1, beta-1 and gamma-2L subunits. alpha-Chloralose produced a concentration-dependent potentiation of the GABA-induced current with an EC50 value of 49 microM and a maximal effect of 239% of control. Membrane current was not affected by alpha-chloralose in the absence of GABA. alpha-Chloralose (100 microM) increased the affinity for GABA 5-fold and produced a small (17%) increase in the efficacy of GABA. Measurement of the reversal potentials for the alpha-chloralose response suggested that the effect is mediated through increased Cl- conductance. Studies of alpha-chloralose interactions with other allosteric modulators determined that alpha-chloralose binds to a site on the GABAA receptor complex distinct from the benzodiazepine, neurosteroid and barbiturate sites. Chloral hydrate, trichloroethanol and urethane also augmented GABA-induced currents. alpha-Chloralose had no effect on the hydroxytryptamine-induced currents in oocytes expressed with the 5-hydroxytryptamine3 receptor. These data extend the number of classes of anesthetics that allosterically modulate GABAA receptor activity and indicate that GABAA receptors may be a common site of action for diverse classes of general anesthetics.

Telephone counseling of breast cancer patients after treatment: a description of a randomized clinical trial.

The Telephone Counseling Trial for Breast Cancer Survivors is a randomized, controlled study designed to test the impact of a telephone-based counseling intervention on quality of life of early-stage breast cancer patients who have completed adjuvant treatment. A psychoeducational counseling model is utilized to promote adaptive coping to re-entry stressors and survivorship issues. Adaptation is fostered through the exploration of thematic materials, application of active coping strategies, encouragement of a personal expression of the breast cancer experience and the provision of psychological support. Patients are being recruited in collaboration with two NCI-designated clinical cooperative oncology groups: the Eastern Cooperative Oncology Group (ECOG) and the Southwest Cooperative Oncology Group (SWOG). The recruitment goal is 400 breast cancer survivors with Stage 1, Stage 2 and Stage 3 disease (with no greater than 10 positive lymph nodes involved). Patients are being enrolled by data managers on-site during their last treatment visit. The intervention is being delivered by the Cancer Information and Counseling Line (CICL) of the AMC Cancer Research Center. It includes 16 telephone outcalls which are delivered over a 12-month period. Primary outcome measures are quality of life, mood, social support, self-efficacy, and sexual functioning, assessed at baseline, 3, 6, 12 and 18 months follow-up. This article provides a description of the intervention protocol and study design. It is argued that this study could provide a model for developing and testing other psychosocial interventions within clinical cooperative groups nationwide.

Diazepam-sensitive GABA(A) receptors in the NTS participate in cardiovascular control.

The present study employed neuropharmacological and receptor binding protocols to determine if diazepam-sensitive (DS) gamma-aminobutyric acid-A (GABA(A)) receptors in the nucleus tractus solitarius (NTS) participate in autonomic regulation of cardiovascular function. The first set of protocols was designed to determine if GABA(A) receptors in the NTS were functionally modulated by the benzodiazepine agonist, diazepam. Mean arterial pressure and heart rate responses to microinjection of GABAergic substances into the NTS were examined in urethane-anesthetized rats. Microinjection of the GABA(A) agonist isoguvacine into the NTS increased mean arterial pressure and heart rate, and these effects were blocked by the GABA(A) receptor antagonist, bicuculline. Preadministration of diazepam into the NTS potentiated the pressor actions of isoguvacine and had variable effects on heart rate changes. Flumazenil, a benzodiazepine antagonist, blocked the diazepam-induced potentiation of the pressor response to isoguvacine. The second protocol employed receptor autoradiography to examine the presence of DS and diazepam-insensitive (DI) GABA(A) receptors in the NTS. Autoradiography confirmed that DS GABA(A) receptors were present in the NTS; however, no measurable levels of DI GABA(A) receptors were detected. We conclude that GABA(A)-mediated integration of central autonomic control in the NTS is mediated solely by DS GABA(A) receptors.

Neurosteroid modulation of [3H]flunitrazepam binding in the medulla: an autoradiographic study.

Neurosteroids bind to unique sites on the GABA(A) receptor complex and modulate receptor function. The effects of neurosteroids on GABA(A) receptors have been well characterized in forebrain regions. However, little is known about their effects on GABA(A) receptors in the medulla, especially those areas involved in autonomic reflex pathways. Stimulation of [3H]flunitrazepam binding to the GABA(A) receptor by two progesterone metabolites, 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha-OH-DHP) and 3beta-hydroxy-5alpha-pregnan-20-one (3beta-OH-DHP), was studied using autoradiographic methods in the medulla and cerebellum of female rats at estrus. [3H]Flunitrazepam binding was enhanced by 3alpha-OH-DHP in every nucleus examined in the medulla and cerebellum. This effect was stereoselective since 3beta-OH-DHP had no effect on binding in any region. No differences were observed in the degree of stimulation of [3H]flunitrazepam binding by 3alpha-OH-DHP among medullary brain regions. However, in the cerebellum, the stimulation of binding was significantly greater in the granular layer than in the molecular layer. Stimulation of [3H]flunitrazepam binding by 3alpha-OH-DHP in nuclei involved in the baroreflex pathways supports previous studies which report that neurosteroids modulate autonomic regulation of blood pressure. These actions may also underlie alterations in autonomic function during pregnancy.

The GABAA receptor alpha 6 subunit gene (Gabra6) is tightly linked to the alpha 1-gamma 2 subunit cluster on mouse chromosome 11.

We have established that the GABAA receptor alpha 6 (Gabra6) and alpha 1 (Gabra 1) subunit genes are tightly linked on mouse chromosome 11 by analysing the strain distribution patterns of RFLPs for the two genes and microsatellite markers flanking these genes in 26 BXD recombinant inbred strains. These results further demonstrate clustering of the GABAA receptor subunit genes on mouse chromosomes and the synteny for these clusters between the mouse and human genomes.

Anxiolytics by ethanol, diazepam and buspirone in a novel murine behavioral assay.

This study extends the pharmacological characterization of a novel quantitative murine behavioral method, the mirrored chamber aversion assay, which appears to be selectively sensitive to anxiolytic agents. Behavioral effects of acute diazepam administration were compared with those of the 5-HT1A anxiolytic buspirone and those of ethanol in C57BL/6J mice. These known anxiolytics produced a dose-dependent reduction in avoidance behavior of large magnitude, as evidenced by statistically significant decreases in latency to enter and increases in time spent in the mirrored chamber. Anxiolytic-like effects of these compounds in the mirrored chamber assay differed from those observed by the elevated plus-maze method. The behavioral effects of the test compounds were not due to alteration of locomotor activity. These findings indicate that the murine mirrored chamber assay responds to several agents known to be anxiolytic in man but differs from the plus-maze in the pharmacological spectrum of the anxiolytics to which it is sensitive.

Serotonin and serotonin receptors in the central auditory system.

Immunohistochemical and ligand-binding techniques were used to visualize the neurotransmitter serotonin and one of its receptors, the 5-HT1A subtype, in auditory nuclei of the brainstem. Serotonergic fibers and terminal endings were found in all auditory nuclei extending from the cochlear nucleus to the inferior colliculus, including the superior olivary complex and the nuclei of the lateral lemniscus. The density of the innervation varied between and within each nucleus. All serotonergic cell bodies were located outside the auditory nuclei. The 5-HT1A receptor subtype was found in the cochlear nucleus as well as in the inferior colliculus. With no serotonergic cell bodies present in the auditory nuclei, the present neuroanatomic and neurochemical findings support behavioral and neurophysiologic findings that the serotonergic system may modulate central auditory processing.

Differential expression of gamma-aminobutyric acidA receptor subunits.

A 1.8-kilobase (kb) cDNA clone for a beta 1 subunit of the human gamma-aminobutyric acidA (GABAA) receptor has been isolated and sequenced. The longest open reading frame of the clone, pCLL610, contains nucleotide sequence encoding a portion of the putative signal sequence followed by 449 amino acids of the entire mature protein. The deduced amino acid sequence of pCLL610 differs from a recently described human beta 1 subunit by a single amino acid. The amino acid sequences of the human GABAA receptor beta 1 subunits share 98% identity with the beta 1 subunits of the bovine and rat GABAA receptor, with the majority of the differences occurring in the intracellular loop between the M3 and M4 transmembrane regions of the protein. A single 11-kb transcript is observed in Northern blots of poly(A)+ RNA isolated from rat brain probed with nick-translated pCLL610. In human brain, the pCLL610 probe recognized the 11-kb message, in addition to two other bands between 8 and 11-kb. Developmental studies of rat brain mRNA show that the message of the GABAA beta 1 subunit is highest at birth, rapidly decreases, and reaches adult levels of 5 to 7 days of age. This is in contrast to the development of the alpha 1 subunit, which is low from days 1 to 5 and increases to adult levels by days 14 to 25. Relative levels of the mRNA for the alpha 1 and beta 1 subunits vary among rat brain regions. The levels of mRNA for the alpha 1 subunit are similar in the cortex, hippocampus, and midbrain, whereas cerebellar levels are twice those in the cortex. The rank order of the relative amount of beta 1 subunit message is hippocampus greater than cortex = midbrain greater than cerebellum. These data, taken with our previous study of the alpha 1 subunits of the GABAA receptor, suggest that the differences in the distribution and regulation of the alpha 1 and beta 1 subunits may reflect a variety of subunit combinations forming the GABAA receptor. Heterogeneity in the GABAA receptor composition may provide a molecular basis for the diverse pharmacological properties associated with this receptor.

Effects of continuous diazepam administration on GABAA subunit mRNA in rat brain.

Rats treated chronically with diazepam develop tolerance to diazepam effects and show changes in sensitivity of GABAergic systems. In order to investigate possible molecular mechanisms associated with these changes, we have evaluated the effects of acute and chronic diazepam treatment on levels of mRNA for the alpha 1 and beta 1 subunits of the GABAA receptor. Northern blots were hybridized with 32P-labeled GABA alpha 1 and beta 1 cDNA probes, and resulting bands were quantified by autoradiography and densitometry. Levels of alpha 1 mRNA were significantly decreased in cerebral cortex but not in cerebellum or hippocampus of chronic diazepam-treated rats. Acute diazepam treatment did not change levels of alpha 1 mRNA in any of the brain regions. Levels of beta 1 mRNA were examined by Northern blot analysis and also by solution hybridization analysis using a 32P-labeled riboprobe. Both methods showed that beta 1 mRNA was not significantly changed by chronic diazepam treatment. These results demonstrate a specific change in alpha 1 subunit that is associated with a state of altered GABA sensitivity and provide further support for the regional heterogeneity of chronic diazepam effects.

Effect of halide ions on t-[35S]butylbicyclophosphorothionate binding.

The binding of t-[35S]butylbicyclophosphorothionate [( 35S]TBPS) to a site on the GABAA receptor complex is ion dependent. This study was conducted to determine the effects of ion species and concentration on the time course, affinity, and number of sites of [35S]TBPS binding. At a concentration of 200 mM ion, the time to equilibrium for [35S]TBPS binding was shortest for I-, followed by Br- less than Cl- less than F-. A similar rank order was observed for the concentration of ion required to produce half-maximal [35S]TBPS binding. Saturation binding experiments were conducted to evaluate the effect of increasing ion concentration on the KD and Bmax of [35S]TBPS binding. The Bmax was independent of both ion species and concentration. The receptor affinity, however, increased with increasing concentration for each ion. Calculated maximal affinity values were not different between ions; however, the EC50 to produce those values was different among ions and ranked in the same order as that for time course and maximal binding data. Association and dissociation rates for [35S]TBPS binding were greater in I- than in Cl-. These data emphasize the importance of ion selection and incubation times on [35S]TBPS binding.

Differential effects of iodide and chloride on allosteric interactions of the GABAA receptor.

t-[35S]Butylbicyclophosphorothionate [( 35S]TBPS) has been shown to bind to the GABAA receptor complex. The binding is modulated allosterically by drugs that interact at components of the receptor complex. The present studies were designed to evaluate the influence of ionic environment and state of equilibrium on the allosteric modification of [35S]TBPS binding. In both I- and Cl- under nonequilibrium conditions, diazepam, gamma-aminobutyric acid (GABA), and pentobarbital (PB) stimulate and methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) inhibits [35S]TBPS binding. In addition, there is an inhibitory component to the effect of GABA and PB at higher drug concentrations. These effects are blocked by the appropriate antagonists for each drug. In Cl-, the stimulation of [35S]TBPS binding by drugs disappears at equilibrium, whereas the inhibition by GABA and PB persists. The inhibitory effect of DMCM in Cl- also disappears at equilibrium. When assayed in I- at equilibrium, however, DMCM stimulates [35S]TBPS binding. In addition, bicuculline, which is without effect under nonequilibrium conditions in either Cl- or I-, stimulates [35S]TBPS binding in I- at equilibrium. The persistent effects of DMCM, bicuculline, and GABA in I- are accompanied by alterations in the affinity of [35S]TBPS for its receptor. In addition, the stimulation of [35S]TBPS binding by GABA is associated with a decreased number of [35S]TBPS binding sites. These data demonstrate that receptor complex interactions with anions influence the responsiveness to drug binding.

Isolation of a cDNA clone for the alpha subunit of the human GABA-A receptor.

A cDNA clone of an alpha subunit of the human GABA-A receptor has been isolated. The human clone (pCLL800) contains 1055 nucleotides in an open reading frame and 260 nucleotides in the 5' non-coding region. The 351 amino acid sequence of this human alpha subunit shows 97% homology with its bovine counterpart. Hybridization of pCLL800 to Northern blots shows a 3.9/4.3 Kb RNA doublet in human cortex, rat whole brain, cortex, hippocampus, midbrain, olfactory bulb and cerebellum. Developmental studies show that the levels of the rat alpha mRNA increase between one and three weeks of age in a manner similar to the development of the benzodiazepine binding sites.

Effects of prenatal phenobarbital on benzodiazepine receptor development.

Phenobarbital (PB) was administered to pregnant mice during days 9-21 of gestation. Forebrain and cerebellar [3H]flunitrazepam ([3H]FLU) binding was assayed in the offspring at birth and at 21 days of age. Prenatal treatment produced a decrease in the number (Bmax) of [3H]FLU receptors in both the forebrain and cerebellum at birth. A small decrease in the [3H]FLU dissociation constant (KD) values in the forebrain was also detected at birth, but no changes were seen in the [3H]FLU KD values in the cerebellum. No changes were observed in forebrain and cerebellar [3H]FLU Bmax or KD values at 21 days of age, indicating that the effects of prenatal exposure to PB on [3H]FLU binding are eliminated during the postnatal development of the forebrain and cerebellum. The receptor affinity for the triazolopyridazine CL 218,872, which distinguishes the type I and type II benzodiazepine (BDZ) receptors, was not altered by prenatal PB treatment. The coupling of the BDZ receptor to the gamma-aminobutyric acid and pentobarbital binding sites was unaffected by exposure to PB in utero.

Heterogeneity of benzodiazepine binding sites: a review of recent research.

This paper reviews selected aspects of benzodiazepine binding site heterogeneity. These include receptor heterogeneity revealed by biochemical determinations of receptor numbers, autoradiographic localization in histological sections of brain, lesion studies, solubilization of receptors, and photoaffinity labelling. The data summarized support the concept of benzodiazepine receptor multiplicity. In addition, we have reviewed recent work on peripheral-type benzodiazepine binding sites and suggest that further study of these sites may increase our understanding of both the central and peripheral actions of benzodiazepines and other ligands.

The development of type I and type II benzodiazepine receptors in the mouse cortex and cerebellum.

The postnatal development of benzodiazepine (BDZ) receptors was monitored in Heterogeneous Stock (HS) mice, and the BDZ receptors were characterized and categorized into Type I and Type II receptors. When the number of 3H-Flu binding sites (Bmax) was assessed at weekly intervals after the birth of the animal, the number of sites in both the cortex and cerebellum increased significantly if the data was expressed as fmol/mg tissue. On the other hand, no significant change in 3H-Flu binding sites was evidenced in the cortex, and the number of 3H-Flu binding sites in the cerebellum decreased during postnatal development if Bmax values were expressed as fmole/mg protein. When receptor binding data was analyzed for the presence of Type I and Type II BDZ receptors, the changes in KD values for 3H-Flu binding development could be accounted for by changes in relative proportions of Type I and Type II receptors present in the cortex and cerebellum during the maturation process. Type II receptors predominated in both cortex and cerebellum at birth, and Type I receptors proliferated primarily during the first two weeks of postnatal life. In the cortex of adult mice there were approximately equal numbers of Type I and Type II BDZ receptors. In the cerebellum of adult mice, computer assisted analysis of binding data could not distinguish the presence of two distinct BDZ binding sites. However, Hill coefficients and overall binding constants determined from data on CL-218,872 displacement of 3H-Flu binding to cerebellar membranes indicated that cerebellar tissue from adult mice did contain a heterogeneous array of BDZ receptors.

Substrate requirements and subcellular distribution of calcium transport activities in brain membranes.

Ca2+ transport activity in synaptosomal membranes has been identified as having two major components: Ca2+-stimulated ATP hydrolysis and ATP-dependent CA2+ uptake. Both processes exhibit similar affinities for Ca2+ and operate maximally under identical buffer conditions. Subcellular fractionation studies revealed the Ca2+/Mg2+ ATPase and ATP-dependent CA2+ uptake activities to be highest in synaptic plasma membrane fractions 1 and 2, with lesser activity in synaptic vesicles and mitochondria. Progressive treatment with Triton X-100 activated, then decreased Ca2+/Mg2+ ATPase, Mg2+ ATPase and Ca2+ ATPase. ATP-dependent Ca2+ uptake was progressively decreased by similar treatment with Triton X-100. These studies illustrate that Ca2+ ATPase and ATP-dependent Ca2+ uptake may provide two important mechanisms for buffering of cytosolic Ca2+ at the nerve terminal. These systems may function to rapidly sequester cytosolic Ca2+ following a rise during depolarization and then extrude Ca2+ from the terminal against a concentration gradient. This regulation of cytosolic Ca2+, represented by two processes of the type seen in other plasma membranes, may play critical roles in calcium homeostasis in nerve cells.