Immunochemical Localization of GABAA Receptor Subunits in the Freshwater Polyp Hydra vulgaris (Cnidaria, Hydrozoa).

γ-aminobutyric acid (GABA) receptors, responding to GABA positive allosteric modulators, are present in the freshwater polyp Hydra vulgaris (Cnidaria, Hydrozoa), one of the most primitive metazoans to develop a nervous system. We examined the occurrence and distribution of GABAA receptor subunits in Hydra tissues by western blot and immunohistochemistry. Antibodies against different GABAA receptor subunits were used in Hydra membrane preparations. Unique protein bands, inhibited by the specific peptide, appeared at 35, 60, ∼50 and ∼52 kDa in membranes incubated with α3, ß1, γ3 or δ antibodies, respectively. Immunohistochemical screening of whole mount Hydra preparations revealed diffuse immunoreactivity to α3, ß1 or γ3 antibodies in tentacles, hypostome, and upper part of the gastric region; immunoreactive fibers were also present in the lower peduncle. By contrast, δ antibodies revealed a strong labeling in the lower gastric region and peduncle, as well as in tentacles. Double labeling showed colocalization of α3/ß1, α3/γ3 and α3/δ immunoreactivity in granules or cells in tentacles and gastric region. In the peduncle, colocalization of both α3/ß1 and α3/γ3 immunoreactivity was found in fibers running horizontally above the foot. These data indicate that specific GABAA receptor subunits are present and differentially distributed in Hydra body regions. Subunit colocalization suggests that Hydra GABA receptors are heterologous multimers, possibly sub-serving different physiological activities.

Neonatal exposure to estradiol decreases hypothalamic allopregnanolone concentrations and alters agonistic and sexual but not affective behavior in adult female rats.

Exposure of developing female rats to estradiol during the perinatal period induced long-lasting dysregulation of gonadal axis and decreased cerebrocortical and plasma concentrations of allopregnanolone. We have now examined the effects of neonatal estradiol administration in female rats on hypothalamic allopregnanolone concentrations and on exploratory, affective, agonistic and sexual behaviors as well as social learning. A single administration of ß-estradiol 3-benzoate (EB, 10µg) on the day of birth resulted in a delay of vaginal opening, acyclicity and ovarian failure. These alterations were associated with a significant decrease in the concentrations of allopregnanolone in the hypothalamus at 21 and 60days, but not at 7days, after birth. Neonatal administration of EB also increased agonistic behaviors in adult rats, such as dominant behaviors and following of an ovariectomized intruder, while living attacks unaffected. EB-treated rats showed also an increase in anogenital investigation, associated with a drastic reduction in spontaneous and induced female sexual behaviors (receptivity and proceptivity). In contrast, neonatal administration of EB did not affect locomotor activity, anxiety- and mood-related behaviors, the social transmission of flavor preferences, and seizures sensitivity. These effects of estradiol suggest that it plays a major role in regulation of both the abundance of allopregnanolone and the expression of agonistic and sexual behaviors, while failing to influence affective behaviors and social learning. Thus, the pronounced and persistent decrease in hypothalamic allopregnanolone concentration may be related to the manifestation of agonistic and sexual behaviors.

Estimation of differential pathlength factor from NIRS measurement in skeletal muscle.

The utilization of continuous wave (CW) near-infrared spectroscopy (NIRS) device to measure non-invasively muscle oxygenation in healthy and disease states is limited by the uncertainties related to the differential path length factor (DPF). DPF value is required to quantify oxygenated and deoxygenated heme groups' concentration changes from measurement of optical densities by NIRS. An integrated approach that combines animal and computational models of oxygen transport and utilization was used to estimate the DPF value in situ. The canine model of muscle oxidative metabolism allowed measurement of both venous oxygen content and tissue oxygenation by CW NIRS under different oxygen delivery conditions. The experimental data obtained from the animal model were integrated in a computational model of O2 transport and utilization and combined with Beer-Lambert law to estimate DPF value in contracting skeletal muscle. A 2.1 value was found for DPF by fitting the mathematical model to the experimental data obtained in contracting muscle (T3) (Med.Sci.Sports.Exerc.48(10):2013-2020,2016). With the estimated value of DPF, model simulations well predicted the optical density measured by NIRS on the same animal model but with different blood flow, arterial oxygen contents and contraction rate (J.Appl.Physiol.108:1169-1176, 2010 and 112:9-19,2013) and demonstrated the robustness of the approach proposed in estimating DPF value. The approach used can overcome the semi-quantitative nature of the NIRS and estimate non-invasively DPF to obtain an accurate concentration change of oxygenated and deoxygenated hemo groups by CW NIRS measurements in contracting skeletal muscle under different oxygen delivery and contraction rate.

Blood volume versus deoxygenated NIRS signal: computational analysis of the effects muscle O2 delivery and blood volume on the NIRS signals.

Near-infrared spectroscopy (NIRS) signals quantify the oxygenated (ΔHbMbO2) and deoxygenated (ΔHHbMb) heme group concentrations. ΔHHbMb has been preferred to ΔHbMbO2 in evaluating skeletal muscle oxygen extraction because it is assumed to be less sensitive to blood volume (BV) changes, but uncertainties exist on this assumption. To analyze this assumption, a computational model of oxygen transport and metabolism is used to quantify the effect of O2 delivery and BV changes on the NIRS signals from a canine model of muscle oxidative metabolism (Sun Y, Ferguson BS, Rogatzki MJ, McDonald JR, Gladden LB. Med Sci Sports Exerc 48: 2013-2020, 2016). The computational analysis accounts for microvascular (ΔHbO2, ΔHHb) and extravascular (ΔMbO2, ΔHMb) oxygenated and deoxygenated forms. Simulations predicted muscle oxygen uptake and NIRS signal changes well for blood flows ranging from resting to contracting muscle. Additional NIRS signal simulations were obtained in the absence or presence of BV changes corresponding to a heme groups concentration changes (ΔHbMb = 0-48 µM). Under normal delivery (Q = 1.0 L·kg-1·min-1) in contracting muscle, capillary oxygen saturation (So2) was 62% with capillary ΔHbO2 and ΔHHb of ± 41 µΜ for ΔHbMb = 0. An increase of BV (ΔHbMb = 24 µΜ) caused a ΔHbO2 decrease (16µΜ) almost twice as much as the increase observed for ΔHHb (9 µΜ). When So2 increased to more than 80%, only ΔHbO2 was significantly affected by BV changes. The analysis indicates that microvascular So2 is a key factor in determining the sensitivity of ΔHbMbO2 and deoxygenated ΔHHbMb to BV changes. Contrary to a common assumption, the ΔHHbMb is affected by BV changes in normal contracting muscle and even more in the presence of impaired O2 delivery.NEW & NOTEWORTHY Deoxygenated is preferred to the oxygenated near-infrared spectroscopy signal in evaluating skeletal muscle oxygen extraction because it is assumed to be insensitive to blood volume changes. The quantitative analysis proposed in this study indicates that even in absence of skin blood flow effects, both NIRS signals in presence of either normal or reduced oxygen delivery are affected by blood volume changes. These changes should be considered to properly quantify muscle oxygen extraction by NIRS methods.

Mobility of heavy metals from tailings to stream waters in a mining activity contaminated site.

In this paper the results of a recent characterization of Rio Piscinas (SW of Sardinia, Italy) hydrological basin are reported. In such area (about 50 km2), previous mining activities caused a serious heavy metal contamination of surface waters, groundwater, soils and biota. Acid mine drainage phenomena were observed in the area. The main sources of contamination are the tailings stored in mine tunnels and abandoned along fluvial banks. A methodological approach was adopted in order to identify relations between tailings and water contamination. Representative samples of tailings and stream sediments samples were collected. XRD analyses were performed for mineralogical characterization, while acid digestion was carried out for determining metal contents. Batch sequential leaching tests were performed in order to assess metal mobility. Also groundwater and stream water were sampled in specific locations and suitably characterized. All information collected allowed the understanding of the effect of tailings on water contamination, thus contributing to the qualitative prediction of pollution evolution on the basis of metal mobility. Finally, a potential remediation strategy of stream water is proposed.

Mechanically induced self-propagating reactions: analysis of reactive substrates and degradation of aromatic sulfonic pollutants.

Mechanochemical reactions have been identified as a valuable alternative to conventional methodologies for the degradation of toxic pollutants as well as for their abatement in contaminated matrices. This paper discusses the application of the mechanochemical technique to the degradation of sulfonic acids in a contaminated matrix. The degradation of the pollutant compound was carried out by taking advantage of combustive reactions on a suitable reactive system ignited under mechanical treatment conditions. Two systems have been investigated as possible reactive substrates. The first one was a Mg-SiO2 powder mixture while the second system was a Ca-SiO2 powder mixture. Milling trials performed under different mechanical processing conditions allowed one to characterise the reactivity of these chemical systems, which basically undergo a reduction/oxidation reaction involving the formation of MgO and Si phases when the Mg-SiO2 system is considered and CaO and Si phases when the Ca-SiO2 system is employed, respectively. The systematic change of the stoichiometric ratios Mg:SiO2 and Ca:SiO2 permitted to identify the minimum Mg or Ca content necessary for the ignition of the combustive reactions. The experimental runs performed with such systems have shown a greater effectiveness of the Mg-SiO2 because of less energy inputs required to ignite a combustion. For this reason the Mg-SiO2 has been considered as a reactive substrate in the following trials. Since the SiO2 amount in stoichiometric excess may be regarded as inert phase, it was substituted with a different phase consisting of the matrix contaminated by sulfonic acids. This aspect permitted to ignite a combustive reaction with the minimum possible content of Mg-SiO2 reacting mixture. The chemical analyses performed after the combustive reaction proved the complete removal of the sulfonic acid from the contaminated matrix.

GABAergic and dopaminergic transmission in the rat cerebral cortex: effect of stress, anxiolytic and anxiogenic drugs.

Benzodiazepines produce their pharmacological effects by regulating the interaction of GABA with its recognition site on the GABAA receptor complex. In fact, the anxiolytic effect of benzodiazepines may be considered the consequence of the activation of the GABAA receptors induced by these drugs. On the contrary, beta-carboline derivatives which bind with high affinity to benzodiazepine recognition sites modulate the GABAergic transmission in a manner opposite to that of benzodiazepines. Thus, these compounds reduce the function of the GABA-coupled chloride channel and produce pharmacological effects (anxiogenic, proconvulsant and convulsant) opposite to those of benzodiazepines. Taken together, these data strongly indicate that the GABAA receptor complex plays a major role in the pharmacology, neurochemistry and physiopathology of stress and anxiety. This conclusion is further supported by the finding that the function of the GABAA/benzodiazepine receptor complex may be modified by the emotional state of the animals before sacrifice. Accordingly, using an unstressed animal model, the 'handling-habituated' rats, it has been demonstrated that stress, like anxiogenic drugs, decreases the function of GABAA receptor complex, an effect mimicked by the in vivo administration of different inhibitors of GABAergic transmission and antagonized by anxiolytic benzodiazepines. Moreover, a long-lasting down regulation of GABAergic synapses can be obtained after repeated administration of anxiogenic, proconvulsant and convulsant negative modulators of GABAergic transmission. The latter finding further suggests that GABAergic synapses undergo rapid and persistent plastic changes when the GABAergic transmission is persistently inhibited. Finally, the evidence that the activity of mesocortical dopaminergic pathways is altered in opposite manner by drugs that either inhibit or enhance the GABAergic transmission indicates that GABA has a functional role in regulation of dopaminergic neurons in the rat cerebral cortex. Altogether these results suggest that cortical GABAergic and dopaminergic transmission play a major role in the pharmacology, neurochemistry and pathology of the emotional states and fear.

Age-related changes of benzodiazepine and GABA binding sites in the rat retina.

The changes in the number and sensitivity of benzodiazepine and GABA binding sites in the rat retina during postnatal development, adulthood and ageing and their functional relationship at different ages have been studied. Data indicate an increase in the total number of both GABA and benzodiazepine binding sites with age. In contrast, the activation of retinal benzodiazepine receptor binding by GABA is significantly reduced in aged rats with respect to young adult and newborn rats. Moreover, the activation of retinal benzodiazepine receptor binding induced by dark exposure of the animals is present in young adult rats but is lost in aged rats. These results suggest that in the retina of aged rats there is an increase of GABA and benzodiazepine receptors which have lost their functional connection.

Evidence for an involvement of GABA receptors in the mediation of the proconvulsant action of ethyl-beta-carboline-3-carboxylate.

The kinetic characteristics of binding of [3H]-GABA and the pattern of isoniazid-induced convulsions were studied in rats treated with repeated intraventricular injections of ethyl-beta-carboline-3-carboxylate (beta-CCE) (10 micrograms/rat, twice daily for 8 days). Thirty-six hours after the last injection, the total number of binding sites for [3H]-GABA was decreased (25%) in the cerebral cortex and hippocampus. On the other hand, there was no significant difference in the dissociation constant (KD) between beta-CCE and solvent-treated rats. The decrease in binding sites for [3H]-GABA was paralleled by a strong potentiation of the convulsant pattern elicited by isoniazid. The results suggest that the proconvulsant effect elicited by beta-CCE is mediated by the decrease in the total number of binding sites for GABA, secondary to the interaction between beta-CCE and the benzodiazepine receptor coupled to the GABA receptor.

Striato-nigral denervation increases type II benzodiazepine receptors in the substantia nigra of the rat.

The degeneration of the striato-nigral projection induced by the injection of kainic acid into the striatum produced a 30% increase in the density of type II benzodiazepine binding sites (measured as the proportion of [3H]flunitrazepam which remained after the addition of 2 X 10(-7) M CL 218872). The lesion did not change the number of type I benzodiazepine binding sites (measured using [3H]ethyl-beta-carboline-3-carboxylate). The increase of type II benzodiazepine binding sites persisted and was markedly enhanced in the substantia nigra, previously lesioned with kainic acid. In fact, the injection of kainic acid into the nigra caused, 3 weeks after the treatment, a 80% decrease in the total number of type I benzodiazepine binding sites, and no change in the number of type II benzodiazepine binding sites. The density of type II sites increased by 70% following a subsequent injection of kainic acid into the striatum, homolateral to the lesioned substantia nigra. The results suggest that type I benzodiazepine binding sites in the nigra are located on kainic acid-sensitive elements (probably intrinsic neurones), while type II benzodiazepine binding sites, the number of which increased after degeneration of the striato-nigral pathway, are localized on kainic acid-resistant structures (probably axons or terminals) that receive an input from striatal afferents and from interneurones in the nigra.

Changes in GABAA receptor gamma 2 subunit gene expression induced by long-term administration of oral contraceptives in rats.

The effects of oral contraceptives (OCs) on neurosteroid concentrations were evaluated in female rats and women. In rats, ethynylestradiol and levonorgestrel (0.030 and 0.125 mg, respectively, subcutaneously) administered daily for 6 weeks reduced the concentrations of pregnenolone (-41%) progesterone (-74%) and allopregnanolone (-79%) in the cerebral cortex; the plasma concentrations of these steroids were also reduced but by smaller extents. OC administration for 3 months also reduced the serum concentrations of pregnenolone, progesterone and allopregnanolone in women. Chronic administration of OCs in rats increased the abundance of gamma-aminobutyric acid type A (GABA(A)) receptor gamma 2L and gamma 2S subunit mRNAs and the relative protein in the cerebral cortex, while the amounts of various alpha and beta subunit mRNAs were unaffected. Ovariectomy did not modify the effect of OCs administration on the concentrations of neurosteroids in the rat cerebral cortex (but not in the plasma) as well as on the GABA(A) receptor gene expression, suggesting a direct effect of OCs in brain. Finally, rats treated with OCs exhibited an anxiety-like behavior in the elevated plus-maze test. These results indicate that long-term treatment with OCs induced a persistent reduction in the concentrations of pregnenolone, progesterone and its GABA(A) receptor-active metabolite, allopregnanolone, both in rats and women. In rats this effect was associated with a plastic adaptation of GABA(A) receptor gene expression in the rat cerebral cortex.

Isoniazid-induced inhibition of GABAergic transmission enhances neurosteroid content in the rat brain.

Isoniazid (375 mg/kg, s.c.), a drug that decreases GABAA receptor-mediated transmission, elicited a time-dependent increase of neuroactive steroid (pregnenolone, progesterone and allotetrahydrodeoxycorticosterone) concentrations in rat brain and plasma. This treatment also time-dependently increased the plasma concentration of corticosterone. Brain and plasma neuroactive steroid levels peaked between 40 and 120 min after isoniazid administration, respectively, and returned to control values by 5 hr. Acute foot shock stress mimicked the effect of isoniazid by increasing in a time-dependent manner the same neuroactive steroids both in brain and plasma. Abecarnil (0.3 mg/kg, i.p.), a beta-carboline derivative with anxiolytic properties, antagonized the effect of both isoniazid and foot shock on brain and plasma neuroactive steroids and on plasma corticosterone level. These data indicate that an inhibition of central GABAergic transmission enhances the concentrations of THDOC and its precursors pregnenolone and progesterone in the rat brain and plasma as well as the plasma levels of corticosterone. This finding suggests that GABA exerts a tonic inhibitory action on the mechanisms involved in the regulation of the synthesis and release of these neuroactive steroids in the central nervous system and plasma.

Modulation of gamma-aminobutyric acid (GABA) receptors and the feeding response by neurosteroids in Hydra vulgaris.

Gamma-Aminobutyric acid (GABA) receptors are present in membrane preparations from Hydra vulgaris, one of the most primitive organisms with a nervous system. These receptors are sensitive to muscimol and benzodiazepines and appear to be important in the regulation of the feeding response. The effects of neurosteroids, general anaesthetics, and GABA antagonists on GABA(A) receptors in membranes prepared from Hydra and on the feeding response have now been investigated. The neurosteroids tetrahydroprogesterone and tetrahydrodeoxycorticosterone increased [3H]GABA binding to hydra membranes with nanomolar potency (EC50, 141+/-11 and 623+/-36 nM, respectively) and high efficacy (maximal increase 79+/-6.5 and 62+/-4%, respectively), whereas the 3beta-hydroxy epimer of tetrahydroprogesterone was ineffective. The benzodiazepine receptor ligands diazepam (100 microM), clonazepam (100 microM) and abecarnil (30 microM) enhanced [3H]GABA binding to Hydra membranes by 22, 20 and 24%, respectively; effects abolished by the specific benzodiazepine antagonist flumazenil (100 microM). On the contrary, the peripheral benzodiazepine receptor ligand 4'chlorodiazepam failed to affect [3H]GABA binding to Hydra membranes. The general anaesthetics propofol and alphaxalone similarly increased (+38% and +30% respectively) [3H]GABA binding. Moreover, [3H]GABA binding to Hydra membranes was completely inhibited by the GABA(A) receptor antagonist SR 95531, whereas bicuculline was without effect. The modulation of GABA(A) receptors in vitro by these various drugs correlated with their effects on the glutathione-induced feeding response in the living animals. Tetrahydroprogesterone and tetrahydrodeoxy-corticosterone (1 to 10 microM) prolonged, in a dose-dependent manner, the duration of mouth opening induced by 10 microM glutathione, with maximal effects of +33 and +29%, respectively, apparent at 10 microM neurosteroid. Alphaxalone (10 microM) similarly increased (+33%) the effect of glutathione. The effects of steroids on the feeding response were inhibited by SR 95531 in a dose-dependent manner; t-butylbyclophosphorothyonate (1 microM), a specific Cl- channel blocker, which per se, like picrotoxin but not bicuculline, shortened the duration of the response, also counteracted the steroids effects at 1 microM. These results suggest that the modulation of GABA(A) receptors by steroids is an ancient characteristic of the animal kingdom and that the pharmacological properties of these receptors have been highly conserved through evolution.

The effects of inhibitors of GABAergic transmission and stress on brain and plasma allopregnanolone concentrations.

1. This study was undertaken to investigate the relationship between a reduction in brain GABAA receptor function and the cerebro-cortical content of 3 alpha-hydroxy-5 alpha-pregnan-20 one (allopregnanolone, AP), a potent endogenous positive modulator of 7-aminobutyric acid (GABA) action at GABAA receptors, with anticonflict and anticonvulsant effects in rodents. 2. An acute depletion of the cerebral content of GABA or an attenuation of GABAA receptor-mediated transmission by systemic injections of isoniazid (375 mg kg-1, s.c.) or FG 7142 (15 mg kg-1, i.p.) induced a transient increase in the cerebro-cortical and plasma concentrations of AP in handling-habituated (not stressed) rats. 3. Two stress paradigms, handling in naive rats and mild foot shock in handling-habituated rats, that reduce central GABAergic tone mimicked the effects of isoniazid and FG 7142 on cortical AP content; foot shock in handling-habituated rats, but not handling in naive animals, also increased plasma AP. Isoniazid, FG 7142, and foot shock also each increased the concentrations of the AP precursors, pregnenolone and progesterone, in both brain and plasma of handling-habituated rats, whereas handling in naive rats increased the concentrations of these steroids only in brain. 4. Pretreatment of handling-habituated rats with the anxiolytic beta-carboline derivative abecarnil, a positive allosteric modulator of GABAA receptors, which per se failed to affect the AP concentration in brain or plasma, prevented the increase in brain and plasma AP induced by foot shock or isoniazid. 5. In adrenalectomized and castrated rats foot shock or isoniazid failed to increase AP both in brain cortex and plasma. 6. These observations indicate that inhibition of GABAergic transmission, induced by foot shock or pharmacological manipulations, results in an increase in the concentrations of AP in brain and plasma, possibly via a modulation of hypothalamic-pituitary-adrenal (HPA) axis. 7. Given that AP enhances GABAA receptor function with high efficacy and potency, an increase in brain AP concentration may be important in the fine tuning of the GABA-mediated inhibitory transmission in the central nervous system.

Functional correlation between allopregnanolone and [35S]-TBPS binding in the brain of rats exposed to isoniazid, pentylenetetrazol or stress.

1. The relation between changes in the cerebral cortical concentration of allopregnanolone and gamma-aminobutyric acid (GABA) type A receptor function after intracerebroventricular injection of this neurosteroid was investigated in male rats. 2. Intracerebroventricular administration of allopregnanolone (1.25 to 15 micrograms) produced a maximal increase (100 fold at the highest dose) in cortical allopregnanolone concentration within 5 min; the concentration remained significantly increased at 15 and 30 min, before returning to control values by 60 min. 3. The same treatment induced a rapid and dose-dependent decrease in the binding of t-[35S]-butylbicyclophosphorothionate ([35S]-TBPS) to cerebral cortical membranes measured ex vivo, an effect mimicked by the benzodiazepine midazolam but not by the 3 beta-hydroxyepimer of allopregnanolone. The time course of changes in [35S]-TBPS binding paralleled that of brain allopregnanolone concentration. 4. In a dose-dependent manner, allopregnanolone both delayed the onset of convulsions and inhibited the increase in [35S]-TBPS binding to cortical membranes induced by isoniazid. The potency of allopregnanolone in inhibiting [35S]-TBPS binding in isoniazid-treated rats was approximately four times that in control animals. 5. The ability of allopregnanolone to decrease [35S]-TBPS binding in isoniazid-treated rats also correlated with its anticonvulsant activity against pentylenetetrazol-induced seizures as well as its inhibitory effect on the increase in [35S]-TBPS binding induced by foot shock. 6. The results indicate that the in vivo administration of allopregnanolone enhances the function of GABAA receptors in rat cerebral cortex and antagonizes the inhibitory action of stress and drugs that reduce GABAergic transmission.

Stress and neurosteroids in adult and aged rats.

The progesterone derivative 3 alpha-hydroxy-5 alpha-pregnan-20 one (allopregnanolone/AP) and the deoxycorticosterone derivative 3 alpha-21-dihydroxy-5 alpha- pregnan-20 one (allotetra-hydrodeoxycorticosterone/THDOC) are endogenous neuroactive steroids endowed with neuromodulatory actions in the central nervous system. Their best-characterized membrane-receptor-dependent action consists in the amplification of GABA-gated chloride currents mediated by specific interactions with the GABAA receptor complex, which appears responsible for the pharmacological effects (anxiolytic, anticonvulsant, hypnotic/anaesthetic) of exogenously administered AP and THDOC. Several acute stress paradigms and different negative allosteric modulators (isoniazid and FG 7142) of GABAA receptors time dependently increase brain and plasma concentrations of AP and THDOC only in intact or sham-operated but not in adrenalectomized-orchiectomized rats. These results suggest that acute stress and inhibitors of GABAA receptors increase the brain and plasma neurosteroid concentrations via a reduction of the inhibitory action exerted by GABA on the hypothalamic-pituitary-adrenal axis. The comparison between the time course of the changes in GABAA receptor function and of their behavioral correlates (proconflict behavior) and that of the changes of endogenous neuroactive steroids are consistent with the view that AP and THDOC may play a role in restoring the GABAergic tone to prestress conditions, by limiting the duration and the extent of its stress-induced reduction. The acute stress-elicited increase of AP and THDOC is observed in adult as well as in aged rats, which show a reduced basal GABAergic transmission and a greater response to the effect of stress in terms of their brain cortical neuroactive steroid concentrations than adult rats.

Antagonism by pivagabine of stress-induced changes in GABAA receptor function and corticotropin-releasing factor concentrations in rat brain.

Pivagabine [4-(2.2-dimethyl-l-oxopropylamino) butanoic acid] (PVG) is a hydrophobic 4-aminobutyric acid derivative with neuromodulatory activity. The effects of subchronic treatment with PVG on stress-induced changes both on brain concentrations of corticotropin-releasing factor (CRF) and neurosteroids and on the function of the gamma-aminobutyric acid type A (GABAA) receptor complex were investigated in male rats. Subchronic treatment with PVG (100-200 mg/kg, i.p.) resulted in a dose-dependent inhibition of the foot shock-induced increase in the binding of t-[35S]butylbicyclophosphorothionate to unwashed membranes prepared from the cerebral cortex of rats killed immediately after stress; PVG treatment alone had no effect on this parameter. This antagonistic action of PVG was also shown in adrenalectomized-orchietomized rats. Foot-shock stress decreased by 74% and increased by 125% the CRF concentration in the hypothalamus and cerebral cortex, respectively. PVG prevented these effects of stress on CRF concentration in both brain regions; this drug per se reduced hypothalamic CRF concentration by 52% but had no effect in the cortex. Moreover, intracerebroventricular injection of CRF, like stress, induced a dose-dependent increase of [35S]TBPS binding to cerebral cortical membranes: an effect not prevented by subchronic treatment of PVG. Finally, PVG did not antagonize the stress-induced increases in the concentrations of neuroactive steroids in brain or plasma. These results suggest that the marked antistress action of PVG is mediated by antagonizing the effects of stress on GABA(A) receptor function and CRF concentrations in the brain, but not by altering the stress-induced increase in neurosteroid concentrations.

Failure of gamma-hydroxybutyrate to alter the function of the GABAA receptor complex in the rat cerebral cortex.

The present study was designed to evaluate the possible interaction of gamma-hydroxybutyrate (GHB) with the GABAA receptor complex in the rat cerebral cortex. To this purpose we studied the effect of in vitro addition and in vivo administration of GHB on the biochemical parameters currently used to evaluate the function of the GABAergic system. In vitro addition of increasing concentrations of GHB failed to modify [3H]flunitrazepam ([3H]FNZ) binding and the modulatory action of GABA on this binding. Moreover, unlike diazepam, GHB did not modify in vitro both muscimol-stimulated 36Cl- uptake and t-[35S]butylbicyclophosphorothionate ([35S]TBPS) binding to rat cerebral cortex. In vivo administration of sedative and hypnotic doses of GHB (300-750 mg/kg IP) failed to induce in 60 min any significant change in the [35S]TBPS binding to unwashed cortical membranes. Moreover, GHB also failed to antagonize the increase in [35S]TBPS binding (+55%) induced by isoniazid (350 mg/kg SC). In contrast, at the highest doses used, this drug completely antagonized the seizure activity induced by isoniazid. In conclusion, our data show that GHB fails to alter the function of the GABAA/benzodiazepine/ionophore receptor complex in the rat cerebral cortex.

Increase of cyclic GMP in cerebellum by methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM).

The intraperitoneal administration of DMCM (0.5-3 mg/kg) produced a dose-related increase in the content of cyclic GMP in the rat cerebellar cortex. The effect of DMCM on cyclic GMP was abolished by pretreatment with benzodiazepine receptor ligands, diazepam and Ro15-1788 and by the GABA agonist muscimol. The results suggest that DMCM increases cerebellar cyclic GMP content through a direct action on benzodiazepine receptors located in the cerebellar cortex. The interaction between DMCM and the GABAergic system associated with benzodiazepine receptors is discussed. Cerebellar cyclic GMP content can be used as a biochemical index to differentiate agonists and antagonists for benzodiazepine receptors.

Involvement of benzodiazepine recognition sites in the foot shock-induced decrease of low affinity GABA receptors in the rat cerebral cortex.

The cerebral cortices of rats habituated to the handling manipulation that precedes sacrifice by guillotine (unstressed rats) have a higher number of low affinity GABA receptors than naive rats (stressed rats). Foot shock stress delivered to handling-habituated rats 5 min before sacrifice decreased the number of low affinity GABA receptors to the level found in naive animals, while leaving almost unchanged the [3H]GABA binding in the latter group. Since benzodiazepine (BZ) recognition sites are the target through which benzodiazepines modulate the emotional states of the animals, we investigated whether these receptors were involved in the action of foot shock stress on GABA binding. The in vitro addition of diazepam (5 X 10(-6) M) to cortical membranes from foot-shocked handling-habituated rats brought back the number of low affinity GABA receptors to the level found in cortical membranes from handling habituated rats. Moreover, the effect of foot shock on low affinity GABA receptors was completely antagonized in vivo by pretreatment with the specific benzodiazepine antagonist Ro15-1788 (30 mg/kg per os). Since the effect of foot shock on [3H]GABA binding is mimicked by the in vitro addition of beta-carbolines to cortical membranes from handling habituated rats, our working hypothesis is that an endogenous ligand for BZ recognition sites, possessing beta-carboline-like properties, is released during foot shock stress.