Sex differences in effects of mild chronic stress on seizure risk and GABAA receptors in rats

Social stress is a common occurrence in our society that can negatively impact health. Therefore, we wanted to study the effects of a mild stressor designed to model social stress on seizure susceptibility and GABAA receptors in male and female rats. The mild chronic stress of individual housing consistently decreased bicuculline (but not pentylenetetrazol, PTZ) seizure thresholds by 10-15% in both sexes. Housing conditions did not alter the anticonvulsant activity of diazepam or ethanol, although the anticonvulsant effect of ethanol was significantly greater against PTZ-induced seizures. Experiments testing the addition of an acute restraint stress unmasked sex differences in seizure induction. The acute stress also selectively decreased the potency of GABA to modulate GABAA receptor-mediated chloride uptake in group-housed females. There were additional sex differences by housing condition for GABAA receptor-gated chloride uptake but no differences in [3H]flunitrazepam binding. We also found significant effects of sex and housing on ethanol-induced increases in corticosterone (CORT) levels. In summary, there were complex and sex-selective effects of mild chronic stress on seizure induction and GABAA receptors. Gaining a better understanding of mechanisms underlying interactions between sex and stress has important implications for addressing health concerns about stress in men and women.     

GABAA receptors in the rat stomach may mediate mucoprotective effects

The occurrence and characteristics of binding sites specific for gamma-aminobutyric acid (GABA) and muscimol in the rat stomach were examined by biochemical and autoradiographic techniques, and the effects of GABAergic model compounds on gastric ulceration induced by chemical irritation was studied in intact and unilaterally vagotomized rats. Specific binding sites for [3H]GABA and [3H]muscimol, which showed the characteristics of GABAA receptors, were demonstrated on gastric membranes. Specific muscimol binding sites were found in all regions of the stomach and were present in both the mucosal layer and the remaining tissue of the stomach. Oral pretreatment of the rats with GABA, selective GABAA receptor agonists, or inhibitors of GABA degradation protected the gastric mucosa against the ulcers induced by acidified ethanol (chemical irritant), in both intact and vagotomized rats. These findings are consistent with the view that a subpopulation of GABAA receptors in the rat stomach may mediate the anti-ulcer effect.     

Behavioral and neurochemical effects of caffeine in normal and aggressive mice

Caffeine is known to exert psychostimulant effects both in man and in animals and it has been shown to modify the levels of brain neurotransmitters. In normal and isolated-aggressive mice, caffeine induces modifications of both the level and the turnover of brain serotonin. Such modifications are however more evident in normal than in aggressive mice. Moreover, caffeine shows an antiaggressive effect and increases the exploratory activity of aggressive mice, without altering the performance of normal animals. It seems possible to conclude that the biochemical and behavioral effects of this drug differ in extent and intensity depending upon the emotional baseline on which it acts.     

The role of GABAA receptors in the acute and chronic effects of ethanol

GABA_A receptors are sensitive to ethanol in distinct brain regions and are clearly involved in the acute actions of ethanol, ethanol tolerance, ethanol dependence and ethanol self-administration. Data from a variety of perspectives such as molecular, cellular and behavioral analysis have elucidated the role of GABA_A receptors in these processes. GABA_A receptor activation mediates many of the behavioral effects of ethanol including motor incoordination, anxiolysis and sedation. The actions of ethanol at GABA_A receptors are influenced by endogenous modulators such as the neuroactive steroids. Sensitization to these compounds influences ethanol dependence and withdrawal and may explain gender differences in the molecular effects of ethanol. Furthermore, GABA_A receptors may also play a role in ethanol self-administration via the mesolimbic reward system. Ethanol tolerance and dependence may be explained, in part, by changes in the function of GABA_A receptors. We have proposed that alterations in native GABA_A receptor subunit assembly could alter the functional properties of these receptors. However, post-translational modifications or other post-synaptic mechanisms may also explain changes in GABA_A receptor function. Genetic animal models of ethanol dependence have also identified GABA_A receptor genes as likely mediators of the behavioral adaptations associated with ethanol dependence and withdrawal. A better understanding of the effects of ethanol at GABA_A receptors has highlighted important potential mechanisms involved in the development of alcoholism. Received: 17 December 1997/Final version: 6 February 1998     

GABAA and GABAB receptors in locus coeruleus: effects of blockers

Racemic baclofen, (-)-baclofen and muscimol depressed all spontaneously firing locus coeruleus neurons tested in a slice preparation. Racemic phaclofen (100 microM; 1 mM) moderately antagonized the effects of racemic baclofen without antagonizing those of muscimol. Bicuculline (10, 30, 100 microM) potently antagonized the action of muscimol without affecting the inhibition of baclofen. Phaclofen and bicuculline had no pronounced effect on the spontaneous discharge rate of cells. The results suggests that there are GABAA and GABAB receptors in the locus coeruleus.     

Statistical evaluation for stability studies under stress storage conditions.

During the pharmaceutical development of a new drug, it is necessary to select as soon as possible the formulation with the best stability characteristics. The current International Commission for Harmonisation (ICH) regulations regarding stability testing requirements for a Registration Application provide the stress testing conditions with the aim of assessing the effect of severe conditions on the drug product. In practice, the well-known Arrhenius theory is still used to make a rapid stability prediction, to estimate a drug product shelf life during early stages of its pharmaceutical development. In this work, both the planning of a stress stability study to obtain a correct stability prediction from a temperature extrapolation and the suitable data treatment to discern the reliability of the stability results are discussed. The study was focused on the early formulation step of a very stable drug, Mitonafide (antineoplastic agent), formulated in a parenteral solution and in tablets. It was observed, for the solid system, that the extrapolated results using Arrhenius theory might be statistically good, but far from the real situation if the stability study is not designed in a correct way. The statistical data treatment and the stress-stability test proposed in this work are suitable to make a reliable stability prediction of different formulations with the same drug, within its pharmaceutical development.     

Low level lead effects on activity under varying stress conditions in the developing rat

The study was designed to determine whether lead ingestion by nursing rats would affect the way offspring reacted to the stress inducing properties of the test environment both as juveniles and mature rats. Dams were exposed to diets with 0.0, 0.2, 0.4 or 1.0 percent by weight metallic lead. Mean blood-lead levels of pups at weaning were 4, 25, 36 and 55 micrograms/100 ml of blood respectively. The stress factor was varied by (1) changing the test apparatus, i.e., forcing rats to occupy an open field or allowing the animal to be a free agent in the start box of a T-maze; (2) testing rats under a longitudinal and a cross-sectional experimental design to vary familiarity with the apparatus; and (3) comparing behavior in the presence or absence of noise. Reactivity was assessed by examining the inter-and intra-session pattern of ambulations and defecations. Analysis of data revealed that lead treated rats demonstrated the greater response to stress. This response was generally dose related although recovery was dependent upon the test applied and measures taken. The findings provide a conceptual framework to account for varied results across previous studies.     

Characterization of the discriminative stimulus effects of GABAA receptor ligands in Macaca fascicularis monkeys under different ethanol training conditions

Rationale: Some evidence suggests an involvement of nucleus accumbens in spatial learning. However, it is controversial whether the mesoaccumbens dopaminergic pathways play a specific role in the acquisition of spatial information. Objective: The goal of these experiments was to investigate the effect of dopaminergic manipulations in the nucleus accumbens on a non-associative task designed to estimate the ability to encode/transmit spatial and non-spatial information. Methods: The effects of focal administrations of the D1 and D2 dopamine receptor antagonists, SCH 23390 (6.25, 12.5, 50 ng/side) and sulpiride (12.5, 50, 100 ng/side), respectively, and dopamine (DA; 1.25 and 2.5 μg/side) into the nucleus accumbens were studied on reactivity to spatial and non-spatial changes in an open field with objects. Results: Both SCH 23390 and sulpiride impaired reactivity to spatial change. However, several differences were found in the effects induced by the two DA antagonists. SCH 23390 did not affect locomotor activity and only slightly impaired exploration of the novel object. On the contrary, the D2 antagonist, induced a general, dose-dependent, impairment on all variables measured. Local administration of DA increased locomotor activity, but did not affect reactivity to spatial and non-spatial changes. Conclusions: These results demonstrate a facilitatory role of mesoaccumbens dopamine in the acquisition of spatial information. Moreover, they suggest that nucleus accumbens D1 DA receptors, play a more selective role in the modulation of spatial learning than accumbens D2 DA receptors. Electronic Publication     

The effects of pesticides, pH, and predatory stress on amphibians under mesocosm conditions

Pesticides are applied throughout the world often with unintended consequences on ecological communities. In some regions, pesticides are associated with declining amphibians, but we have a poor understanding of the underlying mechanisms. Pesticides break down more slowly under low pH conditions and become more lethal to amphibians when combined with predatory stress, but these phenomena have not been tested outside of the laboratory. I examined how pH, predatory stress, and a single application of an insecticide (carbaryl) affected the survival and growth of larval bullfrogs (Rana catesbeiana) and green frogs (R. clamitans) in outdoor mesocosms. Decreased pH had no effect on survival, but caused greater tadpole growth. Low concentrations of carbaryl had no effect on either species, but high concentrations caused lower survival and greater growth in bullfrogs. Predatory stress and reduced pH did not make carbaryl more lethal likely due to the rapid breakdown rate of carbaryl in outdoor mesocosms. Thus, whereas the stress of pH and predators can make carbaryl (and other pesticides) more lethal under laboratory conditions using repeated applications of carbaryl, these stressors did not interact under mesocosm conditions using a single application of carbaryl.     

Opposing effects of activation of central GABAA and GABAB receptors on brown fat thermogenesis in the rat

Baclofen (a GABAB agonist) stimulates body temperature, metabolic rate and brown adipose tissue (BAT) in the rat through a central action, but no effects of gamma-aminobutyric acid (GABA) itself on these parameters were observed. In the present study, it was found that the central effects of (+/-)baclofen (0.5-2.0 micrograms injection i.c.v.) on the temperature (1.2 degrees C increase) and metabolic rate (44-76% increase) of brown adipose tissue were inhibited by previous treatment with the GABAA agonist, muscimol (0.05 micrograms). Injection of GABA alone (12 micrograms) did not significantly affect these parameters, but in the presence of the GABAA antagonist bicuculline (2.5 micrograms), GABA significantly increased the temperature (0.3 degrees C) and oxygen consumption (22%) of brown fat. (-)Baclofen was found to be approximately 50-times more effective in stimulating the temperature of brown adipose tissue than (+/-)baclofen. The results indicate that activation of central GABAB receptors stimulates the activity and hence metabolic rate of brown adipose tissue. However, activation of the GABAA receptors opposes the effects of GABAB stimulation on the thermogenesis of brown fat.     

Feeding conditions differentially affect the neurochemical and behavioral effects of dopaminergic drugs in male rats

The high co-morbidity of eating disorders and substance abuse suggests that nutritional status can impact vulnerability to drug abuse. These studies used rats to examine the effects of food restriction on dopamine clearance in striatum and on the behavioral effects of amphetamine (locomotion, conditioned place preference), the dopamine receptor agonist quinpirole (yawning), and the dopamine receptor antagonist raclopride (catalepsy). Amphetamine increased locomotion and produced conditioned place preference. Food restriction reduced dopamine clearance, which was restored by repeated treatment with amphetamine or by free feeding. Food restriction also decreased sensitivity to quinpirole-induced yawning and raclopride-induced catalepsy; normal sensitivity to both drugs was restored by free feeding. The same amphetamine treatment that normalized dopamine clearance, failed to restore normal sensitivity to quinpirole or raclopride, suggesting that in food-restricted rats the activity of dopamine transporters and dopamine receptors is differentially affected by pathways that are stimulated by amphetamine. These studies show that modest changes in nutritional status markedly alter dopamine neurotransmission and the behavioral effects of direct-acting dopamine receptor drugs (agonist and antagonist). These results underscore the potential importance of nutritional status (e.g., glucose and insulin) in modulating dopamine neurotransmission and in so doing they begin to establish a neurochemical link between the high co-morbidity of eating disorders and drug abuse.     

Mechanism of action of benzodiazepines on GABAA receptors

Wild-type and mutant alpha1beta2gamma2 GABA(A) receptors were expressed in Xenopus laevis oocytes and examined using the two-electrode voltage clamp. Dose-response relationships for GABA were compared in the absence and presence of 1 microM diazepam (DZP) or methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM). The dose-current relationships yielded EC(50)'s (concentration for half-maximal activation) of 41.0+/-3.0, 21.7+/-2.7, and 118.3+/-6.8 microM for GABA, GABA plus DZP, and GABA plus DMCM, respectively.DZP- and DMCM-mediated modulation were examined in GABA(A) receptors in which the beta-subunit carries the L259S mutation. This mutation has been shown to produce spontaneous opening and impart a leftward shift in the dose-response relationship. In this case, neither DZP nor DMCM produced a significant alteration in the GABA dose-response relationship with GABA EC(50)'s of 0.078+/-0.005, 0.12+/-0.03, and 0.14+/-0.004 microM for GABA, GABA plus 1 microM DZP, and GABA plus 1 microM DMCM.DZP- and DMCM-mediated modulations were examined in GABA(A) receptors in which the alpha-subunit carries the L263S mutation. This mutation also produced spontaneous opening and a leftward shift of the GABA dose-response relation, but to a lesser extent than that of betaL259S. In this case, the leftward and rightward shifts for DZP and DMCM were still present with EC(50)'s=0.24+/-0.03, 0.14+/-0.02, and 1.2+/-0.04 microM for GABA, GABA plus 1 microM DZP, and GABA plus 1 microM DMCM, respectively.Oocytes expressing ultrahigh levels of wild-type GABA(A) receptors exhibited currents in response to 1 muM DZP alone, whereas DMCM decreased the baseline current. The DZP-mediated activation currents were determined in wild-type receptors as well as receptors in which the GABA binding site was mutated (beta2Y205S). The EC(50)'s for DZP-mediated activation were 72.0+/-2.0 and 115+/-6.2 nM, respectively, similar to the EC(50) for DZP-mediated enhancement of the wild-type GABA-activated current (64.8+/-3.7 nM).Our results support a mechanism in which DZP increases the apparent affinity of the receptor, not by altering the affinity of the closed state, but rather by shifting the equilibrium towards the high-affinity open state.     

Multiple actions of propofol on alphabetagamma and alphabetadelta GABAA receptors

GABAA receptors are predominantly composed of alphabetagamma and alphabetadelta isoforms in the brain. It has been proposed that alphabetagamma receptors mediate phasic inhibition, whereas alphabetadelta receptors mediate tonic inhibition. Propofol (2,6-di-isopropylphenol), a widely used anesthetic drug, exerts its effect primarily by modulating GABAA receptors; however, the effects of propofol on the kinetic properties of alphabetagamma and alphabetadelta receptors are uncertain. We transfected human embryonic kidney (HEK293T) cells with cDNAs encoding rat alpha1, alpha6, beta3, gamma2L, or delta subunits and performed whole-cell patch-clamp recordings to explore this issue. Propofol (3 microM) increased GABA concentration-response curve maximal currents similarly for both alpha1beta3gamma2L and alpha6beta3gamma2L receptors, but propofol increased those for alpha1beta3delta and alpha6beta3delta receptors differently, the increase being greater for alpha1beta3delta than for alpha6beta3delta receptors. Propofol (10 microM) produced similar alterations in alpha1beta3gamma2L and alpha6beta3gamma2L receptor currents when using a preapplication protocol; peak currents were not altered, desensitization was reduced, and deactivation was prolonged. Propofol enhanced peak currents for both alpha1beta3delta and alpha6beta3delta receptors, but the enhancement was greater for alpha1beta3delta receptors. Desensitization of these two isoforms was not modified by propofol. Propofol did not alter the deactivation rate of alpha1beta3delta receptor currents but did slow deactivation of alpha6beta3delta receptor currents. The findings that propofol reduced desensitization and prolonged deactivation of gamma2L subunit-containing receptors and enhanced peak currents or prolonged deactivation of delta subunit-containing receptors suggest that propofol enhancement of both phasic and tonic inhibition may contribute to its anesthetic effect in the brain.     

Electrophysiological effects of adenosine and adenosine triphosphate on sheep Purkinje fibres under normal and simulated ischaemic conditions.

1. The electrophysiological effects of adenosine and adenosine triphosphate (ATP) were examined in sheep Purkinje fibres, superfused in vitro with either a normal or a hypoxic, hyperkalaemic and acidotic physiological salt solution (PSS). The ability of adenosine to modify the effects of noradrenaline on action potential characteristics was also investigated. 2. The only statistically significant effects of adenosine (10(-6)-10(4) M) and of ATP (10(-6)-10(-4) M) on normal action potential characteristics were a slight dose-dependent shortening of the action potential by adenosine and a depolarization by ATP, 10(-4) M. 3. Superfusion with a hypoxic, hyperkalaemic and acidotic PSS caused marked reductions in resting membrane potential, upstroke and duration of the action potential. 4. Both adenosine and ATP attenuated the reduction in the rate of rise of the upstroke and the amplitude of the action potential caused by the modified PSS. 5. Adenosine did not alter the noradrenaline-induced effects on automaticity or on action potentials of normal or depressed Purkinje fibres. 6. Adenosine and ATP had electrophysiological effects on Purkinje fibres, exposed to conditions in vitro that mimic mild myocardial ischaemia, that were different from those observed on normally polarized fibres.     

Effects of pantoyl-GABA on GABAA and GABAB receptors in the rat brain

The interactions of the "antidementia drug" pantoyl-gamma-aminobutyric acid (pantoyl-GABA) with gamma-aminobutyric acid (GABA) receptors were investigated by studies on bindings of radiolabelled ligands in rat brain. Pantoyl-GABA inhibited the binding of [3H]GABA to GABAA receptors and those of [3H]baclofen and [3H]GABA to GABAB receptors in the rat cerebral cortex. These data suggest that pantoyl-GABA interacts with both types of GABA-receptors in the rat brain.     

Actions of long chain alcohols on GABAA and glutamate receptors: relation to in vivo effects.

1. The effects of n-alcohols on GABAA and glutamate receptor systems were examined, and in vitro effectiveness was compared with in vivo effects in mice and tadpoles. We expressed GABAA, NMDA, AMPA, or kainate receptors in Xenopus oocytes and examined the actions of n-alcohols on receptor function using two-electrode voltage clamp recording. 2. The function of GABAA receptors composed of alpha 1 beta 1 or alpha 1 beta 1 gamma 2L subunits was potentiated by all of the n-alcohols studied (butanol-dodecanol). 3. In contrast to GABAA receptors, glutamate receptors expressed from mouse cortical mRNA or from cRNAs encoding AMPA (GluR3)- or kainate (GluR6)-selective subunits were much less sensitive to longer chain alcohols. In general, octanol and decanol were either without effect or high concentrations were required to produce inhibition. 4. In contrast to the lack of behavioural effects by long chain alcohols reported previously, decanol produced loss of righting reflex in short- and long-sleep mice, indicating that the in vivo effects of decanol may be due in part to actions at GABAA receptors. Furthermore, butanol, hexanol, octanol, and decanol produce similar potentiation of GABAA receptor function at concentrations required to cause loss of righting reflex in tadpoles, an in vivo model where alcohol distribution is not a compromising factor. 5. Thus, the in vivo effects of long chain alcohols are not likely to be due to their actions on NMDA, AMPA, or kainate receptors, but may be due instead to potentiation of GABAA receptor function.     

Distribution of GABAA and glycine receptors in the mammalian retina

1. GABA and glycine mediate synaptic inhibition via specific neurotransmitter receptors. Molecular cloning studies have shown that there is a great diversity of receptors for these two neurotransmitters. In the present paper, the distribution of GABAA and glycine receptors in the mammalian retina is reviewed. 2. In situ hybridization, immunocytochemistry with subunit-specific antibodies and single cell injection were used to analyse the localization of receptor subunits. Specific subunits are expressed in characteristic strata of the inner plexi-form layer, suggesting that different functional circuits involve specific subtypes of neurotransmitter receptors. 3. Different cell types express different combinations of receptor subunits and an individual neuron can express several receptor isoforms at distinct post-synaptic sites.     

Effects of flavone 6-substitutions on GABAA receptors efficacy

Flavones have been studied for their activities via benzodiazepine site on the type-A γ-aminobutyric acid (GABA_A) receptors, for which knowledge on structure–efficacy relationships has been rather limited in comparison to that on structure–affinity relationships. The present study focused on flavone 6-substitution, implied in previous studies being relevant to efficacy. Structure analogs, each varying only at position 6, were compared, including 6-fluoroflavone, 6-chloroflavone, 6-bromoflavone, and 2′-hydroxyflavone analyzed in the present study, as well as 6,2′-dihydroxyflavone reported earlier. Radio-ligand binding assays, whole-cell patch-clamp, and mouse behavioral experiments were performed. In consistent with a previous report, the present whole-cell patch-clamp and animal behavior experiments demonstrated 6-bromoflavone to be a positive modulator at GABA_A receptors acting through flumazenil-sensitive high-affinity benzodiazepine site. In contrast, the other two 6-haloflavones were both neutralizing modulators. In vitro electrophysiological and in vivo animal experiments showed that 2′-hydroxyflavone was a neutralizing modulator, different in efficacy from its structural analog, 6,2′-dihydroxyflavone, a negative modulator of GABA_A receptors. The fact that flavone analogs differing only at position 6 showed drastically different pharmacological properties clearly points to 6-substitution being an important determinant of efficacy. The results suggest that a large width of the first atom on the 6-substituent favors a high binding affinity of the 6-substituted flavone, whereas a large overall volume of the 6-substituent favors positive modulator activity, which could be modified by, e.g., 2′-hydroxyl substitution. These findings have contributed to the understanding of quantitative structure–efficacy relationships for flavones acting at GABA_A receptors, and hence facilitation of flavone-based drug development.