Plasticity of GABAA Receptors during Pregnancy and Postpartum Period: From Gene to Function.

Pregnancy needs complex pathways that together play a role in proper growth and protection of the fetus preventing its premature loss. Changes during pregnancy and postpartum period include the manifold machinery of neuroactive steroids that plays a crucial role in neuronal excitability by local modulation of specific inhibitory receptors: the GABAA receptors. Marked fluctuations in both blood and brain concentration of neuroactive steroids strongly contribute to GABAA receptor function and plasticity. In this review, we listed several interesting results regarding the regulation and plasticity of GABAA receptor function during pregnancy and postpartum period in rats. The increase in brain levels of neuroactive steroids during pregnancy and their sudden decrease immediately before delivery are causally related to changes in the expression/function of specific GABAA receptor subunits in the hippocampus. These data suggest that alterations in GABAA receptor expression and function may be related to neurological and psychiatric disorders associated with crucial periods in women. These findings could help to provide potential new treatments for these women's disabling syndromes.

Increased voluntary ethanol consumption and changes in hippocampal synaptic plasticity in isolated C57BL/6J mice.

Social isolation (SI) is a notable model of prolonged mild stress, characterized by multiple neurochemical and behavioral alterations, that appears particularly suitable for studying different aspects of the interplay between stress and ethanol (EtOH) consumption in order to characterize potential molecular mechanisms, including changes in the function of inhibitory GABAergic synapses, underlying such interaction. In C57BL/6J mice, SI is associated with an altered hippocampal concentration of the neuroactive steroids 3α-hydroxy-5α-pregnan-20-one (3α-5α-THP), an increased expression of the α4 and δ subunit of γ-aminobutyric acid type A receptors (GABAARs) in the dentate gyrus (DG), and a parallel enhancement of the stimulatory action of 4,5,6,7-tetrahydroisoxazolo[5,4-c] pyridin-3-ol (THIP) on GABAergic tonic currents recorded in voltage-clamped DG granule cells (DGGCs). In addition, SI in C57BL/6J mice determines an increase in voluntary EtOH consumption and EtOH preference when compared to group-housed (GH) control animals. Furthermore, in hippocampal slices of SI mice we also observed a marked reduction of both cellular excitability and long term potentiation (LTP) in pyramidal neurons of the CA1 hippocampal sub-region, effects that were prevented by the long term treatment of SI mice with the neuroactive steroid precursor progesterone. In this article, we summarize some of our recent findings on the effects of SI in C57BL/6J mice on voluntary EtOH intake, regulation of GABAARs gene expression and function and hippocampal long term synaptic plasticity.

An OFF-ON chemosensor for biological and environmental applications: sensing Cd2+ in water using catanionic vesicles and in living cells.

A new OFF-ON fluorescent chemosensor (L(1)) for Cd(2+) recognition based on a 5-chloro-8-hydroxyquinoline pendant arm derivative of 1,4,7-triazacyclononane ([9]aneN3) will be presented and its photochemical features in an MeCN-H2O 1 : 1 (v/v) mixture, in pure water, after inclusion within catanionic vesicles, and in living cells will be discussed. The coordination properties of L(1) both in solution and in the solid state were preliminarily studied and its selectivity towards Cd(2+)versus a set of different metal ions (Cu(2+), Zn(2+), Cd(2+), Pb(2+), Al(3+), Hg(2+), Co(2+), Ni(2+), Mn(2+), Mg(2+), K(+), Ca(2+), Ag(+), and Na(+)) was verified in MeCN-H2O 1 : 1 (v/v). In water, upon addition of increasing amounts of Cd(2+) to L(1) an enhancement of the fluorescence emission was detected. To overcome this serious drawback, L(1) was dissolved in an innovative catanionic vesicular solution based on sodium bis(2-ethylhexyl) sulfosuccinate, a traditional surfactant, and 1-dodecyl-3-methylimidazolium bromide, an ionic liquid. When enclosed within the vesicle bilayers in water, L(1) restored its fluorescence emission property upon addition of Cd(2+). Remarkably, L(1) enters the cellular membrane of living cells thus allowing the detection of intracellular Cd(2+). These findings encourage the application of this new fluorescent chemosensor in real samples for histological and environmental analyses.

Effects of Chronic Bifidobacteria Administration in Adult Male Rats on Plasma Metabolites: A Preliminary Metabolomic Study.

Probiotics are live microorganisms distributed in the gastrointestinal tract that confer health benefits to the host when administered in adequate amounts. Bifidobacteria have been widely tested as a therapeutic strategy in the prevention and treatment of a broad spectrum of gastrointestinal disorders as well as in the regulation of the "microbiota-gut-brain axis". Metabolomic techniques can provide details in the study of molecular metabolic mechanisms involved in Bifidobacteria function through the analysis of metabolites that positively contribute to human health. This study was focused on the effects of the chronic assumption of a mixture of Bifidobacteria in adult male rats using a metabolomic approach. Plasma samples were collected at the end of treatment and analyzed with a gas chromatography-mass spectrometry (GC-MS) platform. Partial least square discriminant analysis (PLS-DA) was performed to compare the metabolic pattern in control and probiotic-treated rats. Our results show, in probiotic-treated animals, an increase in metabolites involved in the energetic cycle, such as glucose, erythrose, creatinine, taurine and glycolic acid, as well as 3-hydroxybutyric acid. This is an important metabolite of short-chain fatty acids (SCFA) with multitasking roles in energy circuit balance, and it has also been proposed to have a key role in the prevention and treatment of neurodegenerative diseases.

Melatonin: From Neurobiology to Treatment.

Melatonin, the major regulator of the sleep/wake cycle, also plays important physiological and pharmacological roles in the control of neuronal plasticity and neuroprotection. Accordingly, the secretion of this hormone reaches the maximal extent during brain development (childhood-adolescence) while it is greatly reduced during aging, a condition associated to altered sleep pattern and reduced neuronal plasticity. Altogether, these properties of melatonin have allowed us to demonstrate in both experimental models and clinical studies the great chronobiotic efficacy and sleep promoting effects of exogenous melatonin. Thus, the prolonged release formulation of melatonin, present as a drug in the pharmaceutical market, has been recently recommended for the treatment of insomnia in over 55 years old subjects.

Changes in expression and function of extrasynaptic GABAA receptors in the rat hippocampus during pregnancy and after delivery.

Pregnancy is associated with changes in mood and anxiety level as well as with marked hormonal fluctuations. Increases in the brain concentrations of neuroactive steroids during pregnancy in rats are accompanied by changes in expression of subunits of the GABA type A receptor (GABA(A)-R) in the brain. Granule cells of the dentate gyrus (DGGCs) exhibit two components of inhibitory GABAergic transmission: a phasic component mediated by synaptic GABA(A)-Rs, and a tonic component mediated by extrasynaptic GABA(A)-Rs. Recordings of GABAergic currents were obtained from hippocampal slices prepared from rats in estrus, at pregnancy day 15 (P15) or P19, or at 2 d after delivery. Exogenous GABA or 3alpha,5alpha-THP induced an increase in tonic current in DGGCs that was significantly greater at P19 than in estrus. Neither tonic nor phasic currents were affected by pregnancy in CA1 pyramidal cells. Immunohistochemical analysis revealed a marked increase in the abundance of the delta subunit of the GABA(A)-R and a concomitant decrease in that of the gamma(2) subunit in the hippocampus at P19. Expression of the alpha(4) subunit did not change during pregnancy but was increased 2 d after delivery. Treatment of rats from P12 to P18 with the 5alpha-reductase inhibitor finasteride prevented the changes in tonic current and in delta and gamma(2) subunit expression normally apparent at P19. These data suggest that the number of extrasynaptic GABA(A)-Rs is increased in DGGCs during late pregnancy as a consequence of the associated marked fluctuations in the brain levels of neuroactive steroids.

Neonatal exposure to estradiol in rats influences neuroactive steroid concentrations, GABAA receptor expression, and behavioral sensitivity to anxiolytic drugs.

Gonadal steroids, in particular estradiol, exert important actions during pre- and perinatal periods in the regulation of sexual dimorphism and development of the nervous system. We have now examined the effects of neonatal estradiol administration in female rats on brain concentrations of the neuroactive steroids allopregnanolone and tetrahydrodeoxycorticosterone, expression of GABA(A) receptor subunits, and behavioral sensitivity to benzodiazepines and allopregnanolone. Administration of beta-estradiol 3-benzoate on the day of birth resulted in marked decreases in the concentrations of progesterone and allopregnanolone in the cerebral cortex at 21, 60, and 180 days after birth. The concentrations of tetrahydrodeoxycorticosterone, 17beta-estradiol, and dehydroepiandrosterone in the brain at 60 days were not affected by such treatment. Neonatal administration of beta-estradiol 3-benzoate also increased the cerebrocortical abundance of alpha(1), alpha(2), and gamma(2) subunits of the GABA(A) receptor without affecting that of alpha(3), alpha(4), alpha(5), or delta subunits. Diazepam induced a greater reduction in locomotor activity as well as a more pronounced anxiolytic-like effect in the elevated plus-maze test in rats subjected to neonatal treatment with beta-estradiol 3-benzoate than in vehicle-treated controls, while allopregnanolone induced a similar effect in both groups. These effects of estradiol suggest that it plays a major role in regulation both of GABAergic transmission and of the abundance of endogenous modulators of such transmission during development of the central nervous system.

Changes in neuroactive steroid content during social isolation stress modulate GABAA receptor plasticity and function.

Rats deprived of social contact with other rats at a young age experience a form of prolonged stress that leads to long-lasting alteration in their behavior profile. This chronic stress paradigm is thus thought to be anxiogenic for these normally gregarious animals and their abnormal reactivity to environmental stimuli, when reared under this condition, is thought to be a product of prolonged stress. Neurochemical, molecular, and electrophysiological evidences demonstrate that social isolation is associated with alteration in the structure and function of GABA(A) receptors and suggest that endogenous content of the progesterone metabolite 3alpha,5alpha-TH PROG may be an important determinant in regulating brain excitability and sensitivity to stimuli and point out its possible role in psychiatric and neurological disorder.

Neuroactive steroids and GABAA receptor plasticity in the brain of the WAG/Rij rat, a model of absence epilepsy.

The role of neuroactive steroids and GABA(A) receptors in the generation of spontaneous spike-and-wave discharges (SWDs) was investigated in the WAG/Rij rat model of absence epilepsy. The plasma, cerebrocortical, and thalamic concentrations of the progesterone metabolite 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-TH PROG) were increased in the WAG/Rij rat at 2 months of age compared with those in control (Wistar) rats. In contrast, the brain and peripheral levels of 3alpha,5alpha-tetrahydrodeoxycorticosterone (3alpha,5alpha-TH DOC) did not differ between the two rat strains at this age. At 6 months of age, when absence epilepsy worsens in WAG/Rij rats, the plasma concentration of 3alpha,5alpha-TH PROG remained high whereas that of 3alpha,5alpha-TH DOC had increased, the cerebrocortical levels of both 3alpha,5alpha-TH PROG and 3alpha,5alpha-TH DOC had increased, and the thalamic concentrations of these metabolites had decreased. At 6 months of age the expression of the alpha(4) and delta subunits of the GABA(A) receptor in relay nuclei was increased. Finally, chronic stress induced by social isolation elicited a reduction in the amount of 3alpha,5alpha-TH PROG in the thalamus of 2-month-old WAG/Rij rats that was associated with a reduction in the number and overall duration of SWDs at 6 months of age. Absence epilepsy in the WAG/Rij rat is thus associated with changes in the abundance of neuroactive steroids and in the expression of specific GABA(A) receptor subunits in the thalamus, a brain area key to the pathophysiology of this condition.

Plastic neuronal changes in GABA(A) receptor gene expression induced by progesterone metabolites: in vitro molecular and functional studies.

Expression of specific gamma-aminobutyric acid type A (GABA(A)) receptor subunit genes in neurons is affected by endogenous modulators of receptor function such as neuroactive steroids. Neuroactive steroids such as the progesterone metabolite allopregnanolone might thus exert differential effects on GABA(A) receptor plasticity in neurons, likely accounting for some of the physiological actions of these compounds. Here we summarise experimental data obtained in vitro that show how fluctuations in the concentration of progesterone regulate both the expression and function of GABA(A) receptors. The data described in this manuscript are in agreement with the notion that fluctuations in the concentrations of progesterone and its metabolite allopregnanolone play a major role in the temporal pattern of expression of various subunits of the GABA(A) receptor. Thus, rapid and long-lasting increases or decreases in the concentrations of these steroid derivatives observed in physiological and patho-physiological conditions, or induced by pharmacological treatments, might elicit selective changes in GABA(A) receptor gene expression and function in specific neuronal populations. Given both the importance of GABA(A) receptors in the regulation of neuronal excitability and the large fluctuations in the plasma and brain concentrations of neuroactive steroids associated with physiological conditions and the response to environmental stimuli, these compounds are likely among the most relevant endogenous modulators that could affect emotional and affective behaviors.

Enhanced Glutamatergic Synaptic Plasticity in the Hippocampal CA1 Field of Food-Restricted Rats: Involvement of CB1 Receptors.

The endogenous endocannabinoid system has a crucial role in regulating appetite and feeding behavior in mammals, as well as working memory and reward mechanisms. In order to elucidate the possible role of cannabinoid type-1 receptors (CB1Rs) in the regulation of hippocampal plasticity in animals exposed to food restriction (FR), we limited the availability of food to a 2-h daily period for 3 weeks in Sprague-Dawley rats. FR rats showed a higher long-term potentiation at hippocampal CA1 excitatory synapses with a parallel increase in glutamate release when compared with animals fed ad libitum. FR rats showed a significant increase in the long-term spatial memory determined by Barnes maze. FR was also associated with a decreased inhibitory effect of the CB1R agonist win55,212-2 on glutamatergic field excitatory postsynaptic potentials, together with a decrease in hippocampal CB1R protein expression. In addition, hippocampal brain-derived neurotrophic factor protein levels and mushroom dendritic spine density were significantly enhanced in FR rats. Altogether, our data suggest that alterations of hippocampal CB1R expression and function in FR rats are associated with dendritic spine remodeling and functional potentiation of CA1 excitatory synapses, and these findings are consistent with increasing evidence supporting the idea that FR may improve cognitive functions.

Changes in GABA(A) receptor gene expression associated with selective alterations in receptor function and pharmacology after ethanol withdrawal.

Changes in the expression of subunits of the GABA type A (GABA(A)) receptor are implicated in the development of ethanol tolerance and dependence as well as in the central hyperexcitability associated with ethanol withdrawal. The impact of such changes on GABA(A) receptor function and pharmacological sensitivity was investigated with cultured rat hippocampal neurons exposed to ethanol for 5 d and then subjected to ethanol withdrawal. Both ethanol treatment and withdrawal were associated with a marked decrease in the maximal density of GABA-evoked Cl- currents, whereas the potency of GABA was unaffected. Ethanol exposure also reduced the modulatory efficacy of the benzodiazepine receptor agonists lorazepam, zolpidem, and zaleplon as well as that of the inverse agonists Ro 15-4513 and FG 7142, effects that were associated with a reduced abundance of mRNAs encoding the receptor subunits alpha1, alpha3, gamma2L, and gamma2S. Ethanol withdrawal restored the efficacy of lorazepam, but not that of low concentrations of zolpidem or zaleplon, to control values. Flumazenil, which was ineffective in control neurons, and Ro 15-4513 each potentiated the GABA response after ethanol withdrawal. These effects of withdrawal were accompanied by upregulation of the alpha2, alpha3, and alpha4 subunit mRNAs as well as of the alpha4 protein. Diazepam or gamma-hydroxybutyrate, but not baclofen, prevented the changes in both GABA(A) receptor pharmacology and subunit mRNA levels induced by ethanol withdrawal. Changes in GABA(A) receptor gene expression induced by prolonged exposure to and withdrawal of ethanol are thus associated with altered GABA(A) receptor function and pharmacological sensitivity.

Low tolerance and dependence liabilities of etizolam: molecular, functional, and pharmacological correlates.

The effects of prolonged exposure to and subsequent withdrawal of the thienotriazolobenzodiazepine etizolam on gamma-aminobutyric acid (GABA) type A receptor gene expression and function were compared with those of the benzodiazepine lorazepam. Exposure of rat hippocampal neurons in culture to 10 microM etizolam for 5 days reduced the amounts of alpha5 and gamma2S receptor subunit mRNAs, whereas etizolam withdrawal was associated with a persistent reduction in gamma2S mRNA and an increase in alpha2 and alpha3 mRNAs. Neither chronic exposure to nor withdrawal of etizolam affected the acute modulatory effects of etizolam or lorazepam on GABA-evoked Cl- current. Treatment with 10 microM lorazepam for 5 days reduced the amounts of alpha1 and gamma2S subunit mRNAs and increased that of alpha3 mRNA, whereas lorazepam withdrawal was associated with persistence of the changes in alpha3 and gamma2S mRNAs and an increase in alpha2 and alpha4 mRNAs. Parallel changes in the abundance of alpha1 and alpha4 subunit proteins induced by chronic exposure to and withdrawal of lorazepam, but not etizolam, were detected by immunocytofluorescence analysis. Chronic lorazepam treatment resulted in a reversible reduction in the modulatory efficacy of this drug and conferred on flumazenil the ability to potentiate GABA-evoked Cl- current. The anticonvulsant action of etizolam was not altered in mice chronically treated with this drug, whereas lorazepam-treated animals became tolerant to the acute anticonvulsant effect of this benzodiazepine. These data suggest that etizolam is endowed with a reduced liability to induce tolerance and dependence compared with classical benzodiazepines.

Inhibition by miltirone of up-regulation of GABAA receptor alpha4 subunit mRNA by ethanol withdrawal in hippocampal neurons.

Miltirone, a tanshinone isolated from the root of Salvia miltiorrhiza, has been characterized as a low-affinity ligand for central benzodiazepine receptors. We have now shown that this compound bound with low affinity (micromolar range) to central benzodiazepine recognition sites but did not interact with peripheral benzodiazepine receptors. It failed to potentiate Cl(-) currents induced by gamma-aminobutyric acid (GABA) both in Xenopus oocytes expressing recombinant human GABA(A) receptors and in cultured rat hippocampal pyramidal cells, but it inhibited the ability of diazepam to potentiate the effect of GABA in these systems. Miltirone (1-10 microM) also partially inhibited the increase in the abundance of the mRNA for the alpha(4) subunit of the GABA(A) receptor induced by ethanol withdrawal in cultured hippocampal neurons. These results suggest that miltirone might ameliorate the symptoms associated with discontinuation of long-term administration of ethanol or of other positive modulators of the GABA(A) receptor.

Molecular mechanisms of tolerance to and withdrawal of GABA(A) receptor modulators.

Here, we summarize recent data pertaining to the effects of GABA(A) receptor modulators on the receptor gene expression in order to elucidate the molecular mechanisms behind tolerance and dependence induced by these drugs. Drug selectivity and intrinsic activity seems to be important to evidence at the molecular level the GABA(A) receptor tolerance. On the contrary, we suggested that all drug tested are equally potentially prone to induce dependence. Our results demonstrate that long-lasting exposure of GABA(A) receptors to endogenous steroids, benzodiazepines and ethanol, as well as their withdrawal, induce marked effects on receptor structure and function. These results suggest the possible synergic action between endogenous steroids and these drugs in modulating the functional activity of specific neuronal populations. We report here that endogenous steroids may play a crucial role in the action of ethanol on dopaminergic neurons.

Involvement of the cannabinoid CB1 receptor in modulation of dopamine output in the prefrontal cortex associated with food restriction in rats.

Increase in dopamine output on corticolimbic structures, such as medial prefrontal cortex (mPFC) and nucleus accumbens, has been related to reward effects associated with palatable food or food presentation after a fasting period. The endocannabinoid system regulates feeding behavior through a modulatory action on different neurotransmitter systems, including the dopaminergic system. To elucidate the involvement of type 1 cannabinoid receptors in the regulation of dopamine output in the mPFC associated with feeding in hungry rats, we restricted the food availability to a 2-h period daily for 3 weeks. In food-restricted rats the extracellular dopamine concentration in the mPFC increased starting 80 min before food presentation and returned to baseline after food removal. These changes were attenuated in animals treated with the CB1 receptor antagonist SR141716. To better understand how food restriction can change the response of mesocortical dopaminergic neurons, we studied several components of the neuronal circuit that regulates dopamine output in the mPFC. Patch-clamp experiments revealed that the inhibitory effect of the CB1 receptor agonist WIN 55,212-2 on GABAergic sIPSC frequency was diminished in mPFC neurons of FR compared to fed ad libitum rats. The basal sIPSC frequency resulted reduced in mPFC neurons of food-restricted rats, suggestive of an altered regulation of presynaptic GABA release; these changes were accompanied by an enhanced excitability of mPFC and ventral tegmental area neurons. Finally, type 1 cannabinoid receptor expression in the mPFC was reduced in food-restricted rats. Together, our data support an involvement of the endocannabinoid system in regulation of dopamine release in the mPFC through changes in GABA inhibitory synapses and suggest that the emphasized feeding-associated increase in dopamine output in the mPFC of food-restricted rats might be correlated with an altered expression and function of type 1 cannabinoid receptor in this brain region.

Mucuna pruriens (Velvet bean) rescues motor, olfactory, mitochondrial and synaptic impairment in PINK1B9 Drosophila melanogaster genetic model of Parkinson's disease.

The fruit fly Drosophila melanogaster (Dm) mutant for PTEN-induced putative kinase 1 (PINK1B9) gene is a powerful tool to investigate physiopathology of Parkinson's disease (PD). Using PINK1B9 mutant Dm we sought to explore the effects of Mucuna pruriens methanolic extract (Mpe), a L-Dopa-containing herbal remedy of PD. The effects of Mpe on PINK1B9 mutants, supplied with standard diet to larvae and adults, were assayed on 3-6 (I), 10-15 (II) and 20-25 (III) days old flies. Mpe 0.1% significantly extended lifespan of PINK1B9 and fully rescued olfactory response to 1-hexanol and improved climbing behavior of PINK1B9 of all ages; in contrast, L-Dopa (0.01%, percentage at which it is present in Mpe 0.1%) ameliorated climbing of only PINK1B9 flies of age step II. Transmission electron microscopy analysis of antennal lobes and thoracic ganglia of PINK1B9 revealed that Mpe restored to wild type (WT) levels both T-bars and damaged mitochondria. Western blot analysis of whole brain showed that Mpe, but not L-Dopa on its own, restored bruchpilot (BRP) and tyrosine hydroxylase (TH) expression to age-matched WT control levels. These results highlight multiple sites of action of Mpe, suggesting that its effects cannot only depend upon its L-Dopa content and support the clinical observation of Mpe as an effective medication with intrinsic ability of delaying the onset of chronic L-Dopa-induced long-term motor complications. Overall, this study strengthens the relevance of using PINK1B9 Dm as a translational model to study the properties of Mucuna pruriens for PD treatment.

Long-term administration with levonorgestrel decreases allopregnanolone levels and alters GABA(A) receptor subunit expression and anxiety-like behavior.

Fluctuations in the concentrations of the neuroactive steroid allopregnanolone are thought to influence γ-amino-butyric acid type A (GABA(A)) receptor gene expression and function. Long-term treatment with ethinyl estradiol (EE) plus levonorgestrel (LNG), two of the most widely used steroids in the hormonal contraceptive pill, decreases allopregnanolone levels in rat cerebral cortex and plasma, alters GABA(A) receptor expression and induces anxiety-like behavior. We evaluated which component of the hormonal contraceptive pill is responsible for the aforementioned changes. Female rats were injected subcutaneously (s.c.) with EE (0.030 mg) or LNG (0.125 mg) once a day for 4 weeks. Compared to the respective vehicle-treated control groups, EE decreased cerebral cortical levels of allopregnanolone, progesterone and pregnenolone by 76, 72 and 33%, respectively and hippocampal levels by 52, 56 and 50%, respectively. Likewise, LNG decreased cerebral cortical levels of allopregnanolone, progesterone and pregnenolone by 75, 68 and 33%, respectively, and hippocampal levels by 55, 65 and 60%, respectively. Administration of LNG, but not EE, increased the abundance of the γ2 subunit peptide in cerebral cortex and hippocampus by 38 and 59%, respectively. Further, LNG, but not EE, decreased the time spent and the number of entries into the open arms of the elevated plus maze by 56 and 43%, respectively, an index of anxiety-like behavior. These results suggest that alterations in GABA(A) receptor subunit expression and anxiety-like behavior induced by long-term treatment with combined EE/LNG appear to be caused by LNG. Given that both EE and LNG decrease allopregnanolone levels in a similar manner, these results further suggest that changes in allopregnanolone levels are not associated with GABA(A) receptor expression.

Ex vivo skin delivery of diclofenac by transcutol containing liposomes and suggested mechanism of vesicle-skin interaction.

Recently, we described a novel family of liposomes, the Penetration Enhancer-containing Vesicles (PEVs), as carriers for enhanced (trans)dermal drug delivery. In this study, to go deeply into the potential of these new vesicles and suggest the possible mechanism of vesicle-skin interaction, we investigated transcutol containing PEVs as carriers for diclofenac, in the form of either acid or sodium salt. PEVs, prepared with soy phosphatidylcholine and aqueous solutions containing different concentrations of transcutol, were characterized by size distribution, zeta potential, incorporation efficiency, thermotropic behavior, and stability. (Trans)dermal diclofenac delivery from PEVs was investigated ex vivo through new born pig skin using conventional liposomes and a commercial gel as controls. The mode of action of the vesicles was also studied by performing a pre-treatment test and confocal laser scanning microscopy (CLSM) analyses. Results of the all skin permeation experiments showed an improved diclofenac (both acid and sodium salt) delivery to and through the skin when PEVs were used (especially in comparison with the commercial gel) thus suggesting intact PEVs' penetration through the pig skin. Images of the qualitative CLSM analyses support this conclusion. Thus, this work shows the superior ability of the PEVs to enhance ex vivo drug transport of both hydrophilic and lipophilic diclofenac forms.

Effects of voluntary ethanol consumption on emotional state and stress responsiveness in socially isolated rats.

Social isolation of rats immediately after weaning is thought to represent an animal model of anxiety-like disorders. This mildly stressful condition reduces the cerebrocortical and plasma concentrations of 3α-hydroxy-5α-pregnan-20-one (3α,5α-TH PROG) as well as increases the sensitivity of rats to the effects of acute ethanol administration on the concentrations of this neuroactive steroid. We further investigated the effects of voluntary consumption of ethanol at concentrations increasing from 2.5 to 10% over 4 weeks of isolation. Isolated rats showed a reduced ethanol preference compared with group-housed animals. Ethanol consumption did not affect the isolation-induced down-regulation of BDNF or Arc, but it attenuated the increase in the cerebrocortical concentration of 3α,5α-TH PROG induced by foot-shock stress in both isolated and group-housed animals as well as increased the percentage of number of entries made by socially isolated rats into the open arms in the elevated plus-maze test. Ethanol consumption did not affect expression of the α4 subunit of the GABA(A) receptor in the hippocampus of group-housed or isolated rats, whereas it up-regulated the δ subunit throughout the hippocampus under both conditions. The results suggest that low consumption of ethanol may ameliorate some negative effects of social isolation on stress sensitivity and behavior.