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.

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.

Experimental analysis and modelling of in vitro HUVECs proliferation in the presence of various types of drugs.

OBJECTIVES: This study focuses on experimental analysis and corresponding mathematical simulation of in vitro HUVECs (human umbilical vein endothelial cells) proliferation in the presence of various types of drugs. MATERIALS AND METHODS: HUVECs, once seeded in Petri dishes, were expanded to confluence. Temporal profiles of total count obtained by classic haemocytometry and cell size distribution measured using an electronic Coulter counter, are quantitatively simulated by a suitable model based on the population balance approach. Influence of drugs on cell proliferation is also properly simulated by accounting for suitable kinetic equations. RESULTS AND DISCUSSION: The models' parameters have been determined by comparison with experimental data related to cell population expansion and cell size distribution in the absence of drugs. Inhibition constant for each type of drug has been estimated by comparing the experimental data with model results concerning temporal profiles of total cell count. The reliability of the model and its predictive capability have been tested by simulating cell size distribution for experiments performed in the presence of drugs. The proposed model will be useful in interpreting effects of selected drugs on expansion of readily available human cells.

In vitro ovine articular chondrocyte proliferation: experiments and modelling.

This study focuses on analysis of in vitro cultures of chondrocytes from ovine articular cartilage. Isolated cells were seeded in Petri dishes, then expanded to confluence and phenotypically characterized by flow cytometry. The sigmoidal temporal profile of total counts was obtained by classic haemocytometry and corresponding cell size distributions were measured electronically using a Coulter Counter. A mathematical model recently proposed (1) was adopted for quantitative interpretation of these experimental data. The model is based on a 1-D (that is, mass-structured), single-staged population balance approach capable of taking into account contact inhibition at confluence. The model's parameters were determined by fitting measured total cell counts and size distributions. Model reliability was verified by predicting cell proliferation counts and corresponding size distributions at culture times longer than those used when tuning the model's parameters. It was found that adoption of cell mass as the intrinsic characteristic of a growing chondrocyte population enables sigmoidal temporal profiles of total counts in the Petri dish, as well as cell size distributions at 'balanced growth', to be adequately predicted.

Experimental analysis and modelling of in vitro proliferation of mesenchymal stem cells.

OBJECTIVES: Stem cell therapies based on differentiation of adult or embryonic stem cells into specialized ones appear to be effective for treating several human diseases. This work addresses the mathematical simulation of proliferation kinetics of stem cells. MATERIALS AND METHODS: Sheep bone marrow mesenchymal stem cells (phenotype characterized by flow cytometry analysis) seeded at different initial concentrations in Petri dishes were expanded to confluence. Sigmoid temporal profiles of total counts obtained through classic haemocytometry were quantitatively interpreted by both a phenomenological logistic equation and a novel model based on a one-dimensional, single-staged population balance approach capable of taking into account contact inhibition at confluence. The models' parameters were determined by comparison with experimental data on population expansion starting from single seeding concentration. Reliability of the models was tested by predicting cell proliferation carried out starting from different seeding concentrations. RESULTS AND DISCUSSION: It was found that the proposed population balance modelling approach was successful in predicting the experimental data over the whole range of initial cell numbers investigated, while prediction capability of phenomenological logistic equation was more limited.

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.

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.

Role of allopregnanolone in regulation of GABA(A) receptor plasticity during long-term exposure to and withdrawal from progesterone.

Here we summarize recent data from our laboratory pertaining to the effects of fluctuations in the brain concentrations of the progesterone (PROG) metabolite allopregnanolone (3alpha,5alpha-TH PROG) on the expression and function of gamma-aminobutyric acid type A (GABA(A)) receptors. The effects of long-term exposure to progesterone and of its sudden withdrawal on the activity of GABA(A) receptors and on the abundance of receptor subunit mRNAs were examined in cultured rat cerebellar granule cells and cortical neurons. The effects of a persistent reduction in the brain concentration of 3alpha,5alpha-TH PROG on GABA(A) receptor function and gene expression were examined in vivo in rats subjected to long-term administration of oral contraceptives. Our results demonstrate that long-lasting changes in the exposure of GABA(A) receptors to this PROG metabolite induce marked effects on receptor structure and function. These effects of 3alpha,5alpha-TH PROG appear to be mediated through modulation of GABA(A) receptor signaling mechanisms that control the expression of specific receptor subunit genes. Furthermore, the specific outcomes of such signaling appear to differ among neurons derived from different regions of the brain. Neuroactive steroids such as 3alpha,5alpha-TH PROG might thus exert differential actions on GABA(A) receptor plasticity in distinct neuronal cell populations, likely accounting for some of the physiological effects induced by these compounds.

GABAA-receptor plasticity during long-term exposure to and withdrawal from progesterone.

The subunit composition of native gamma-aminobutyric acid type A (GABAA) receptors is an important determinant of the role of these receptors in the physiological and pharmacological modulation of neuronal excitability and associated behavior. GABAA receptors containing the alpha 1 subunit mediate the sedative-hypnotic effects of benzodiazepines (Rudolph et al., 1999; McKernan et al., 2000), whereas the anxiolytic effects of these drugs are mediated by receptors that contain the alpha 2 subunit (Löw et al., 2000). In contrast, GABAA receptors containing the alpha 4 or alpha 6 subunits are insensitive to benzodiazepines (Barnard et al., 1998). Characterization of the functions of GABAA-receptors thus requires an understanding of the mechanisms by which the receptor subunit composition is regulated. The expression of specific GABAA-receptor subunit genes in neurons is affected by endogenous and pharmacological modulators of receptor function. The expression of GABAA-receptor subunit genes is thus regulated by neuroactive steroids both in vitro and in vivo. Such regulation occurs both during physiological conditions, such as pregnancy, and during pharmacologically induced conditions, such as pseudo-pregnancy and long-term treatment with steroid derivatives or anxiolytic-hypnotic drugs. Here, we summarize results obtained by our laboratory and by other groups pertaining to the effects of long-term exposure to, and subsequent withdrawal from, progesterone and its metabolite 3 alpha,5 alpha-tetrahydroprogesterone on both the expression of GABAA-receptor subunits and GABAA-receptor function.

Putative glycine receptors in Hydra: a biochemical and behavioural study.

Glycine acts as an inhibitory transmitter in the lower brain stem and spinal cord of vertebrate species, while very few data are yet available to support a similar role in invertebrate nervous systems. Here we report the identification and characterization of glycine receptors in the freshwater polyp Hydra vulgaris (Cnidaria, Hydrozoa) by biochemical and behavioural studies. Saturation experiments revealed the occurrence of one population of binding sites of nanomolar affinity (KD = 33 nm) and low capacity (Bmax = 79 fmol/mg protein) for [(3)H]strychnine. The addition of glycine or taurine (0.1 microm-1 mm) produced a dose-dependent inhibition of [(3)H]strychnine binding. Beta-alanine (0.1-1 mm) did not significantly affect [(3)H]strychnine binding. The pharmacological properties of these receptors compare with those of vertebrate glycine receptors. Stimulation of Hydra polyps by reduced glutathione resulted in a significant increase in the duration of mouth opening in the presence of glycine, taurine or beta-alanine. The enhancement of the response was related both to amino acid (10-100 microm) and to glutathione concentration (1-10 microm). The effects of glycine or its agonists were suppressed by strychnine (1-10 microm). D-serine, a glycine agonist at the vertebrate NMDA receptor, produced opposite effects to those of glycine. The effects of d-serine were suppressed by 5,7-dichlorokynurenic acid but not by strychnine. In vitro, [(3)H]strychnine binding was not displaced by d-serine. These results indicate a dual action of glycine in Hydra tissues. The hypothesis that NMDA receptors may also be present in this elementary nervous system is proposed.

Caffeine-induced increases in the brain and plasma concentrations of neuroactive steroids in the rat.

The effects of caffeine, a naturally occurring stimulant, on the brain and plasma concentrations of neuroactive steroids were examined in the rat. A single intraperitoneal injection of caffeine induced dose- and time-dependent increases in the concentrations of pregnenolone, progesterone, and 3alpha-hydroxy-5alpha-pregnan-20-one (allopregnanolone) in the cerebral cortex. The increases were significant at a caffeine dose of 25 mg/kg and greatest (+188, +388, and +71%, respectively) at a dose of 100 mg/kg in rats killed 30 min after caffeine administration. Caffeine also increased the plasma concentrations of pregnenolone and progesterone with a dose-response relation similar to that observed in the brain, whereas the caffeine-induced increase in the plasma concentration of allopregnanolone was maximal at a dose of 50 mg/kg. Caffeine increased the plasma concentration of corticosterone, but it had no effect on the brain or plasma concentrations of 3alpha, 21-dihydroxy-5alpha-pregnan-20-one and dehydroepiandrosterone. Moreover, the brain and plasma concentrations of pregnenolone, progesterone, and allopregnanolone were not affected by caffeine in adrenalectomized-orchiectomized rats. These results suggest that neuroactive steroids may modulate the stimulant and anxiogenic effects of caffeine.

Physiological modulation of GABA(A) receptor plasticity by progesterone metabolites.

The possible functional relation between changes in brain and plasma concentrations of neurosteroids and the plasticity of gamma-aminobutyric acid type A (GABA(A)) receptors in the brain during pregnancy and after delivery was investigated in rats. The concentrations in the cerebral cortex and plasma of pregnenolone as well as of progesterone and its neuroactive derivatives allopregnanolone (3alpha-hydroxy-5alpha-pregnan-20-one) and allotetrahydrodeoxycorticosterone (5alpha-hydroxy-3alpha,21-diol-20-one) increased during pregnancy, peaking around day 19, before returning to control (estrus) values immediately before delivery (day 21). In the postpartum period, steroid concentrations in plasma and brain did not differ from control values. The densities of [3H]GABA, [3H]flunitrazepam, and t-[35S]butylbicyclophosphorotionate (TBPS) binding sites in the cerebral cortex also increased during pregnancy, again peaking on day 19 and returning to control values on day 21; receptor density was decreased further 2 days after delivery and again returned to control values within 7 days. These changes were accompanied by a decrease in the apparent affinity of the binding sites for the corresponding ligand on day 19 of pregnancy. The amount of the gamma2L subunit mRNA decreased progressively during pregnancy, in the cerebral cortex and hippocampus, returned to control value around the time of delivery and did not change in the postpartum period. On the contrary, the amount of alpha4 subunit mRNA was not modified during pregnancy both in the cerebral cortex and hippocampus whereas significantly increased 7 days after delivery only in the hippocampus. No significant changes were apparent for alpha1, alpha2, alpha3, beta1, beta2, beta3 and gamma2S subunit mRNAs. Administration of finasteride, a specific 5alpha-reductase inhibitor, to pregnant rats from days 12 to 18 markedly reduced the increases in the plasma and brain concentrations of allopregnanolone and allotetrahydrodeoxycorticosterone as well as prevented both the increase in the densities of [3H]flunitrazepam and [35S]TBPS binding sites and the decrease of gamma2L mRNA normally observed during pregnancy. The results demonstrate that the changes in the plasticity of GABA(A) receptors that occur in rat brain during pregnancy and after delivery are related to the physiological changes in plasma and brain concentrations of neurosteroids.

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.

Role of brain allopregnanolone in the plasticity of gamma-aminobutyric acid type A receptor in rat brain during pregnancy and after delivery.

The relation between changes in brain and plasma concentrations of neurosteroids and the function and structure of gamma-aminobutyric acid type A (GABAA) receptors in the brain during pregnancy and after delivery was investigated in rats. In contrast with plasma, where all steroids increased in parallel, the kinetics of changes in the cerebrocortical concentrations of progesterone, allopregnanolone (AP), and allotetrahydrodeoxycorticosterone (THDOC) diverged during pregnancy. Progesterone was already maximally increased between days 10 and 15, whereas AP and allotetrahydrodeoxycorticosterone peaked around day 19. The stimulatory effect of muscimol on 36Cl- uptake by cerebrocortical membrane vesicles was decreased on days 15 and 19 of pregnancy and increased 2 days after delivery. Moreover, the expression in cerebral cortex and hippocampus of the mRNA encoding for gamma2L GABAA receptor subunit decreased during pregnancy and had returned to control values 2 days after delivery. Also alpha1, alpha2, alpha3, alpha4, beta1, beta2, beta3, and gamma2S mRNAs were measured and failed to change during pregnancy. Subchronic administration of finasteride, a 5alpha-reductase inhibitor, to pregnant rats reduced the concentrations of AP more in brain than in plasma as well as prevented the decreases in both the stimulatory effect of muscimol on 36Cl- uptake and the decrease of gamma2L mRNA observed during pregnancy. These results indicate that the plasticity of GABAA receptors during pregnancy and after delivery is functionally related to fluctuations in endogenous brain concentrations of AP whose rate of synthesis/metabolism appears to differ in the brain, compared with plasma, in pregnant rats.

Molecular and functional adaptation of the GABA(A) receptor complex during pregnancy and after delivery in the rat brain.

The abundance of gamma-aminobutyric acid receptor type A (GABAA receptor) subunit mRNAs and polypeptides as well as muscimol-stimulated 36Cl- uptake were measured in rat cerebral cortex or hippocampus at various times during pregnancy and after delivery. RNase protection assays revealed that the amount of the gamma2L subunit mRNA decreased progressively during pregnancy, in the cerebral cortex and hippocampus, and then returned to control values around the time of delivery. A similar pattern was observed for the alpha5 subunit mRNA in the cerebral cortex, whereas no significant changes were apparent for alpha1, alpha2, alpha3, alpha4, beta1, beta2, beta3 and gamma2S subunit mRNAs. The amounts of gamma2 and alpha1 proteins in the cerebral cortex were measured by immunoblot analysis; whereas the abundance of gamma2 protein decreased during pregnancy, no change was detected in the amount of alpha1 protein. Evaluation for functional significance of the down-regulated gamma2 and alpha5 subunit was made by determining the GABAA receptor function assessed by measurement of muscimol-stimulated 36Cl- uptake in cerebral cortical membrane vesicles. Muscimol-induced 36Cl- uptake was markedly reduced during of pregnancy compared with rats in oestrus. At this same time, the potentiating effects of diazepam and allopregnanolone on muscimol stimulation of 36Cl- uptake also were reduced. In contrast, the effects of muscimol, allopregnanolone and diazepam were significantly increased, relative to animals in oestrus, after delivery.

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.

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.