Reduced anxiety‐like behavior and central neurochemical change in germ‐free mice

Background There is increasing interest in the gut‐brain axis and the role intestinal microbiota may play in communication between these two systems. Acquisition of intestinal microbiota in the immediate postnatal period has a defining impact on the development and function of the gastrointestinal, immune, neuroendocrine and metabolic systems. For example, the presence of gut microbiota regulates the set point for hypothalamic‐pituitary‐adrenal (HPA) axis activity. Methods We investigated basal behavior of adult germ‐free (GF), Swiss Webster female mice in the elevated plus maze (EPM) and compared this to conventionally reared specific pathogen free (SPF) mice. Additionally, we measured brain mRNA expression of genes implicated in anxiety and stress‐reactivity. Key Results Germ‐free mice, compared to SPF mice, exhibited basal behavior in the EPM that can be interpreted as anxiolytic. Altered GF behavior was accompanied by a decrease in the N‐methyl‐D‐aspartate receptor subunit NR2B mRNA expression in the central amygdala, increased brain‐derived neurotrophic factor expression and decreased serotonin receptor 1A (5HT1A) expression in the dentate granule layer of the hippocampus. Conclusions & Inferences We conclude that the presence or absence of conventional intestinal microbiota influences the development of behavior, and is accompanied by neurochemical changes in the brain.     

Essentiality of central GABAergic neuroactive steroid allopregnanolone for anticonvulsant action of fluoxetine against pentylenetetrazole-induced seizures in mice

Fluoxetine, a selective serotonin reuptake inhibitor, is known to increase the cortical content of allopregnanolone (ALLO) without altering the level of other neurosteroids. In contrast to the proconvulsant effect of many antidepressants, fluoxetine exhibits anticonvulsant effects. The present study was undertaken to examine the role of ALLO in the anticonvulsant action of fluoxetine against pentylenetetrazole (PTZ)-induced seizures in mice. Prior administration of GABA(A) receptor agonist muscimol or neurosteroid ALLO or progesterone, a precursor of ALLO or neurosteroidogenic drugs like FGIN 1-27, an agonist at the mitochondrial diazepam binding inhibitor receptor (MDR) or metyrapone, an 11beta-hydroxylase inhibitor, significantly potentiated the anticonvulsant effect of fluoxetine. In contrast, the effect of fluoxetine was counteracted by inhibition of the neurosteroid biosynthesis using drugs like 5alpha-reductase inhibitor, finasteride; 3beta-hydroxysteroid dehydrogenase inhibitor, trilostane; 3alpha-hydroxysteroid dehydrogenase inhibitor, indomethacin; MDR antagonist, PK 11195; or the GABA(A) receptor antagonist, bicuculline. Further, bilateral adrenalectomy had no significant effect on the anticonvulsant action of fluoxetine, suggesting negligible contribution from peripheral steroidogenesis. The anticonvulsant effect of fluoxetine was partially abolished in 5,7-DHT treated mice, indicating that the effect may also, in part, be dependent on serotonergic transmission. Thus, our data indicate that increased synthesis of ALLO in CNS is a major factor that ultimately leads to anticonvulsant effects of fluoxetine against PTZ-induced seizures.     

Anxiety and stress responses in female oxytocin deficient mice

Oxytocin is believed to attenuate the response of the hypothalamic-pituitary-adrenal axis to stress and to be anxiolytic. Stressors with a psychological component evoke both central and peripheral secretion of oxytocin in laboratory rodents. Oxytocin gene deletion mice provide a novel way to understand the role of oxytocin in stress and anxiety-related behaviours. We present our experience with female oxytocin deficient mice that were tested in an elevated plus maze (EPM), a behavioural test of anxiety, or exposed to psychogenic stressors (platform shaker or novel environment). Oxytocin-deficient mice not only displayed more anxiety-related behaviour, but also released more corticosterone after a psychogenic stressor and manifested greater stress-induced hyperthermia compared to wild-type mice. The diurnal variation of corticosterone and the response of corticosterone to corticotropin-releasing factor were not significantly different between genotypes. We also measured Fos-immunoreactive protein, an index of neuronal activation, in the medial amygdala of female mice after EPM testing. The medial amygdala is important for processing of psychogenic stress and anxiety and also contains oxytocin pathways and oxytocin receptors. The expression of Fos in the medial amygdala of mice not exposed to the EPM was not different between genotypes. Following EPM exposure, Fos expression was greater in oxytocin null compared to wild-type mice. Our findings support the hypothesis that central oxytocin is anxiolytic, and attenuates the stress response to psychogenic provocation in female mice.     

Environmental enrichment upregulates micro‐RNA‐183 and alters acetylcholinesterase splice variants to reduce anxiety‐like behavior in the little Indian field mouse (Mus booduga)

Environmental enrichment (EE) has an influential role in reducing behavioral reactivity to stress. We previously observed that EE reduces the anxiety‐like behavior in the field mouse Mus booduga accompanied by a reduction in the expression of molecules involved in the stress pathway. In this study, we demonstrate the effect of different housing condition on regulation of micro‐RNA‐183‐SC35‐mediated splicing of acetylcholinesterase (AChE). Adult male M. booduga were captured from an agricultural field and housed under nonenriched standard conditions (SC) for 7 days and considered as directly from the wild (DW). On day 8, individuals were randomly assigned to three groups; DW, SC, and EE. The DW group's anxiety‐like behavior was assessed in the elevated plus maze (EPM) and open field test (OFT). The SC and EE groups were transferred to their respective conditions and housed for another 30 days. The mice housed in EE showed less anxiety‐like behavior on EPM and in OFT compared with DW and SC mice. Interestingly, miR‐183 expression was increased following exposure to EPM in EE mice but not in SC mice. Subsequently, the upregulated miR‐183 expression suppresses the SC35 expression and shifting of splicing from AChE‐S (synaptic) to AChE‐R (read‐through) form, whereas standard housing condition downregulate miR‐183 and induces the splicing of AChE. The upregulated AChE‐R form possibly terminates ACh transmission, which is reflected in the level of anxiety‐like behavior. Overall, the present study suggests that EE effectively regulates the miR‐183 pathway to reduce anxiety‐like behavior. © 2012 Wiley Periodicals, Inc.     

Catamenial‐like seizure exacerbation in mice with targeted ablation of extrasynaptic δGABA‐a receptors in the brain

Neurosteroids play a key role in catamenial epilepsy, a menstrual cycle‐related seizure clustering in women with epilepsy. While neurosteroids act on all GABA‐A receptor isoforms, they cause greater effects on extrasynaptic δGABA‐A receptors that mediate tonic inhibition in the brain. Previously, we identified a potential GABA‐A receptor mechanism for catamenial epilepsy. However, the precise functional role of extrasynaptic δGABA‐A receptors in the pathophysiology of catamenial epilepsy remains unclear. In this study, we utilized mice lacking extrasynaptic δGABA‐A receptors (δKO) to investigate whether reduction of tonic inhibition affects catamenial seizure susceptibility or intensity. Intact female wildtype (WT) and δKO mice were subjected to hippocampus kindling until they exhibited stage 5 seizures. Elevated gonadal hormone‐based neurosteroid levels were induced by standard gonadotropin regimen and neurosteroid withdrawal (NSW) was triggered by finasteride. NSW increased susceptibility to, as well the intensity of evoked catamenial‐like seizures in WT and δKO mice. However, fully kindled δKO mice exhibited an accelerated and augmented response to NSW, with a more rapid increase in seizure susceptibility and intensity than WT mice undergoing the NSW paradigm. Moreover, δKO mice in NSW showed reduced benzodiazepine sensitivity, but in stark contrast to the increased neurosteroid sensitivity observed in WT animals, δKO mice displayed no change in neurosteroid sensitivity in response to NSW. The increased catamenial seizure exacerbation and alterations in antiseizure drug responses are consistent with NSW‐induced changes in the abundance of δGABA‐A receptors. Collectively, these findings provide evidence of a potential protective role for extrasynaptic δGABA‐A receptors in catamenial‐like seizures. © 2017 Wiley Periodicals, Inc.     

Glucocorticoid receptor deletion from the dorsal raphé nucleus of mice reduces dysphoria‐like behavior and impairs hypothalamic‐pituitary‐adrenocortical axis feedback inhibition

Glucocorticoids can cause depression and anxiety. Mechanisms for glucocorticoid effects on mood are largely undefined. The dorsal raphé nucleus (DRN) produces the majority of serotonin in the brain, and expresses glucocorticoid receptors (GR). Because we previously showed that antidepressants used to treat depression and anxiety decrease DRN GR expression, we hypothesized that deleting DRN GR would have anxiolytic‐ and antidepressant‐like effects. We also hypothesized that DRN GR deletion would disinhibit activity of the hypothalamic–pituitary–adrenal (HPA) axis. Adeno‐associated virus pseudotype AAV2/9 expressing either Cre recombinase (DRNGRKO mice) or GFP (DRN‐GFP mice) was injected into the DRN of floxed GR mice to test these hypotheses. Three weeks after injection, mice underwent 21 days of social defeat or control handling and were tested for anxiety‐like behavior (open‐field test, elevated‐plus maze), depression‐like behavior [sucrose preference, forced‐swim test (FST), tail‐suspension test (TST)], social interaction, and circadian and stress‐induced HPA activity. DRN GR deletion decreased anxiety‐like behavior in control but not in defeated mice. DRN GR deletion decreased FST and tended to decrease TST despair‐like behavior in both control and defeated mice, but did not affect sucrose preference. Exploration of social (a novel mouse) as well as neutral (an empty box) targets was increased in DRNGRKO mice, suggesting that DRN GR deletion also promotes active coping. DRN GR deletion increased stress‐induced HPA activity without strongly altering circadian HPA activity. We have shown a novel role for DRN GR to mediate anxiety‐ and despair‐like behavior and to regulate HPA negative feedback during acute stress.     

Long‐lasting marked inhibition of periaqueductal gray‐evoked defensive behaviors in inescapably‐shocked rats

Clinical evidence suggests that depression and trauma predispose the subject to panic. Accordingly, here we examined the late effects of uncontrollable stress, a presumptive model of depression and/or traumatic disorder, on panic‐like behaviors evoked by electrical stimulation of the dorsal periaqueductal gray (DPAG). Changes in anxiety and depression were also assessed in the elevated plus‐maze (EPM) and forced‐swimming test (FST), respectively. Rats with electrodes in the DPAG were subjected to a 7‐day shuttle‐box one‐way escape yoked training with foot‐shocks either escapable (ES) or inescapable (IS). The day after the end of one‐way escape training, rats were trained in a two‐way escape novel task (test‐session) to ascertain the effectiveness of uncontrollable stress. DPAG stimulations were carried out in an open field, both before the escape training and 2 and 7 days after it, and EPM and FST were performed on the 8th and 10th days afterwards, respectively. Controls were either trained with fictive shocks (FS) or subjected to intracranial stimulations only. Although the ES rats performed significantly better than the IS group in the two‐way escape task, groups did not differ with respect to either the anxiety or depression scores. Unexpectedly, however, IS rats showed a marked attenuation of DPAG‐evoked freezing and flight behaviors relative to both the ES and FS groups, 2 and 7 days after one‐way escape training. The conjoint inhibition of passive (freezing) and active (flight) defensive behaviors suggests that IS inhibits a DPAG in‐built motivational system that may be implicated in depressed patients' difficulties in coping with daily‐life stress.     

Isoallopregnanolone reduces tic‐like behaviours in the D1CT‐7 mouse model of Tourette syndrome

Tourette syndrome (TS) is a neuropsychiatric disorder characterised by multiple, persistent tics. These semi‐voluntary motor and phonic manifestations are typically aggravated by exposure to acute stress, yet the mechanisms underlying this exacerbation remain unclear. Using a well‐characterised animal model of TS, the D1CT‐7 mouse, we recently showed that acute stress increases tic‐like responses and causes sensorimotor gating deficits, as measured by the prepulse inhibition of the startle. We showed that these effects are promoted by the brain synthesis of the neurosteroid allopregnanolone (AP). In line with this idea, inhibition of AP synthesis by finasteride was found to suppress the tic‐exacerbating effects of stress; conversely, AP administration resulted in a marked enhancement of the number of tic‐like motor bursts. Given that the primary mechanism of AP is based on the positive allosteric modulation of GABA_A receptors, in the present study, we hypothesised that the enhancement in tic‐like behaviours induced by either stress or AP may be countered by isoallopregnanolone (isoAP), the natural 3β‐epimer of AP that acts as an antagonist to the AP‐binding site within GABA_A receptors. In agreement with our hypothesis, isoAP (5‐10 mg kg^‐1, s.c.) dose‐dependently reduced the number of tic‐like behaviours induced by stress in D1CT‐7 mice. These effects were comparable to those elicited by both the benchmark TS therapy haloperidol (0.3 mg kg^‐1, i.p.), as well as finasteride (25 mg kg^‐1, i.p.). IsoAP also countered the prepulse inhibition deficits secondary to stress in D1CT‐7 mice. Finally, isoAP opposed the enhancement of tic‐like behaviours induced by AP (15 mg kg^‐1, i.p.). Given that isoAP is well‐tolerated and has an optimal safety profile, these data suggest that this steroid may have therapeutic properties in TS.     

U-69,593 microinjection in the infralimbic cortex reduces anxiety and enhances spontaneous alternation memory in mice

The present report investigated the contributions of the ventromedial prefrontal cortex to the control of spontaneous alternation/working memory and anxiety-related behaviour. In Experiment 1, we examined the effects of microinjections of the selective kappa(1) receptor agonist, U-69,593, in the infralimbic cortex (IL) of CD-1 mice on several ethologically-derived anxiety indices in the elevated plus-maze (EPM) and defensive/withdrawal (D/W) anxiety in the open field, as well as on memory in the EPM transfer-latency (T-L) test and implicit spontaneous alternation memory (SAP) in the Y-maze. In week 1, pretreatment with one injection of vehicle, 1, 10 or 25 nmol/1.0 microliter U-69,593 in the IL dose-dependently prolonged T-L and produced a dose-dependent anxiolytic behavioural profile in the first EPM trial. Following a 24-h delay, the same mice were given a drug-free second trial in the EPM tests of T-L memory and anxiety. Whereas T-L memory was not disturbed, small but detectable carry-over effects were observed in trial-2 EPM behaviour relative to vehicle-treated animals. In week 2, the same groups of mice were again pretreated with one injection of the same doses of U-69,593 in the IL and given a D/W test in an open field, followed immediately by an 8-min SAP trial in the Y-maze. The smallest U-69,593 dose was anxiolytic in the D/W test, and SAP/working memory was dose-dependently enhanced in the Y-maze. In Experiment 2, we evaluated whether 0.5 microliter volume microinjections would produce comparable behavioural and carry-over effects in the IL of three new groups of CD-1 mice, in the event that the 1.0 microl volume injections used in Experiment 1 diffused beyond the IL and therefore may have confounded some effects. Experiment 2 procedures were carried out in the same manner as in Experiment 1, except the animals were tested in reverse order. Thus in week 1, SAP memory was tested in the Y-maze followed by D/W anxiety in the open field for half of the animals in each group, and the other half was tested in reverse order. In week 2, T/L memory and anxiety were tested in the EPM in 2 trials as described in Experiment 1. Pretreatment with one injection of vehicle, 10 or 25 nmol/0.5 microliter U-69,593 in the IL reduced D/W anxiety and enhanced SAP memory regardless of testing order in week 1. In week 2, the same groups of mice were again pretreated with one injection of the same doses of U-69,593 in 0.5 microliter volumes in the IL and tested in the EPM. In a similar fashion to Experiment 1, U-69,593 dose-dependently prolonged T/L and produced an anxiolytic behavioural profile in the first EPM trial. Following a 24-h delay, T/L recall memory was again not significantly influenced, but a robust anxiolytic behavioural profile was observed in the second drug-free anxiety trial in the EPM relative to vehicle-treated animals. Results are discussed relative to a) injection volumes and testing order, b) the possible influence kappa receptors may exert on neurochemical responsivity to anxiety-provoking environments in the IL area of the mPFC, c) the possibility that kappa-mediated anxiolysis from the IL in CD-1 mice results from interactions with neurochemical systems involved in the blunting of incoming anxiety-provoking information, d) evidence that SAP memory may be an implicit subtype of working memory, and e) the possibility that IL implicit working memory processes may modulate the induction and expression of anxiety-related behaviour.     

Regulator of G protein signaling 2 differentially regulates nicotine‐induced anxiolytic‐ and antidepressant‐like effects in mice

This study assessed the role of regulator of G protein signaling 2 (RGS2) in nicotine‐induced anxiolytic‐ and antidepressant‐like effects using RGS2 wildtype (WT) and RGS2 knockout (KO) mice. RGS2 negatively regulates monoaminergic neurotransmission, which is implicated in the pathology of anxiety and depression. We hypothesized that deletion of RGS2 would enhance nicotine‐induced anxiolytic‐ and antidepressant‐like effects, which were assessed using the elevated plus maze and tail suspension tests, respectively. Anxiolytic‐like effects were observed in both RGS2 WT and KO mice after administration of low dose of nicotine (0.05 mg/kg, base) compared to respective saline controls. Additionally, administration of nicotine (0.1 mg/kg, base) compared to saline resulted in anxiolytic‐like effects in RGS2 KO mice, but not RGS2 WT mice, suggesting genetic deletion of RGS2 facilitated anxiolytic‐like effects of nicotine. Administration of nicotine (0.5 and 1 mg/kg, base) compared to saline resulted in antidepressant‐like effects in RGS2 WT mice. Antidepressant‐like effects were observed in RGS2 KO mice only at the highest tested dose of nicotine (1 mg/kg, base) compared to saline controls, suggesting that genetic deletion of RGS2 decreased sensitivity to antidepressant‐like effects of nicotine. Together, the data suggest that RGS2 differentially regulated nicotine‐induced affective behavioral responses. These data suggest that individuals with RGS2 polymorphisms may experience differential affective responses to tobacco smoking, which may make them vulnerable to developing nicotine addiction.     

Wfs1‐deficient animals have brain‐region‐specific changes of Na+, K+‐ATPase activity and mRNA expression of α1 and β1 subunits

Mutations in the WFS1 gene, which encodes the endoplasmic reticulum (ER) glycoprotein, cause Wolfram syndrome, a disease characterized by juvenile‐onset diabetes mellitus, optic atrophy, deafness, and different psychiatric abnormalities. Loss of neuronal cells and pancreatic β‐cells in Wolfram syndrome patients is probably related to the dysfunction of ER stress regulation, which leads to cell apoptosis. The present study shows that Wfs1‐deficient mice have brain‐region‐specific changes in Na⁺,K⁺‐ATPase activity and in the expression of the α₁ and β₁ subunits. We found a significant (1.6‐fold) increase of Na‐pump activity and β₁ subunit mRNA expression in mice lacking the Wfs1 gene in the temporal lobe compared with their wild‐type littermates. By contrast, exposure of mice to the elevated plus maze (EPM) model of anxiety decreased Na‐pump activity 1.3‐fold in the midbrain and dorsal striatum and 2.0‐fold in the ventral striatum of homozygous animals compared with the nonexposed group. Na‐pump α₁‐subunit mRNA was significantly decreased in the dorsal striatum and midbrain of Wfs1‐deficient homozygous animals compared with wild‐type littermates. In the temporal lobe, an increase in the activity of the Na‐pump is probably related to increased anxiety established in Wfs1‐deficient mice, whereas the blunted dopamine function in the forebrain of Wfs1‐deficient mice may be associated with a decrease of Na‐pump activity in the dorsal and ventral striatum and in the midbrain after exposure to the EPM. © 2014 Wiley Periodicals, Inc.     

Transgenic overexpression of interleukin-1 receptor antagonist in the CNS influences behaviour, serum corticosterone and brain monoamines

The effects of brain-directed overexpression of human soluble interleukin-1 receptor antagonist (hsIL-1ra) on behaviour, serum corticosterone (CST) levels and concentrations of dopamine (DA), 5-hydroxytryptamine (5-HT) and their metabolites in different brain regions, were investigated in six months old homozygotic transgenic male mice (Tg hsIL-1ra(+/+)). The transgenic and age-matched wild type (WT) mice were subjected to a battery of behavioural tests for analysis of open field (OF) behaviours, anxiety in elevated plus maze (EPM), and motor performance in rotarod. One week after the last behavioural test, half of the mice from each genotype were subjected to a mild stress, while the remaining mice served as controls for the determination of serum CST levels and monoamine concentrations in different brain regions. Tg hsIL-1ra(+/+) mice had higher locomotor scores and showed less habituation in the OF test, spent more time in the open arms of the EPM and had similar motor performance as compared to WT mice. The serum CST levels were comparable, both in basal conditions and upon stress, in the two genotypes. Tg hsIL-1ra(+/+) mice had lower concentrations of DA, 5-HT and their metabolites in several brain regions, with different effects on monoamine turnover upon stress. In conclusion, brain-directed overexpression of hsIL-1ra resulted in increased locomotion and decreased habituation, an anxiolytic effect, but did not influence motor performance. Finally, the activation of hypothalamo-pituitary-adrenal (HPA)-axis was comparable in the two genotypes, however Tg hsIL-1ra(+/+) mice had a modified metabolism of brain monoamines as compared to WT mice.     

Anxiolytic‐ and panicolytic‐like effects of Neuropeptide S in the mouse elevated T‐maze

Neuropeptide S (NPS) regulates various biological functions by selectively activating the NPS receptor (NPSR). Recently, epidemiological studies revealed an association between NPSR single nucleotide polymorphisms and susceptibility to panic disorders. Here we investigated the effects of NPS in mice subjected to the elevated T maze (ETM), an assay which has been proposed to model anxiety and panic. Diazepam [1 mg/kg, intraperitoneally (i.p.)] elicited clear anxiolytic effects reducing the latency to emerge from the closed to the open (CO) arm without modifying the latencies from the open to the closed (OC) arm. By contrast, chronic fluoxetine (10 mg/kg i.p., once a day for 21 days) selectively increased OC latency, suggesting a panicolytic‐like effect. NPS given intracerebroventricularly at 0.001–1 nmol elicited both anxiolytic‐ and panicolytic‐like effects. However, although the NPS anxiolytic dose–response curve displayed the classical sigmoidal shape, the dose–response curve of the putative panicolytic‐like effect was bell shaped with peak effect at 0.01 nmol. The behaviour of wild‐type [NPSR(+/+)] and receptor knock out [NPSR(?/?)] mice in the ETM task was superimposable. NPS at 0.01 nmol elicited anxiolytic‐ and panicolytic‐like effects in NPSR(+/+) but not in NPSR(?/?) mice. In conclusion, this study demonstrated that NPS, via selective activation of the NPSR, promotes both anxiolytic‐ and panicolytic‐like actions in the mouse ETM.     

Light/dark cycle manipulation influences mice behaviour in the elevated plus maze

The sensitization of animal models of anxiety is of great importance to detect potential anxiolytic drugs. Our goal was to evaluate the influence of manipulations of the light/dark cycle on the basal anxious behaviour of mice and the efficacy of two anxiolytic treatments in the mouse elevated plus maze (EPM). Male Swiss mice were exposed to different conditions of illumination for one week prior to testing. In the first experiment of the study, we evaluated the anxiolytic effects of diazepam, at the dose of 1 mg/kg, intraperitoneally (i.p.) administered 30 min before the test. In the second experiment, we examined the effects of WAY 100635, a 5-HT(1A) receptor antagonist, at the doses of 0.03 and 2 mg/kg, i.p. administered 30 min before the test. The locomotor activity of control mice and the anxiolytic efficacy of diazepam in the EPM were not affected by manipulation of the light/dark cycle. Conversely, the effects of WAY 100635, which were qualitatively different from those of diazepam, seemed to be influenced by the illumination conditions imposed before the test. We can conclude that diazepam's effect, which is characterized by a strong "disinhibition", was more robust than the 5-HT(1A) antagonist's effect, which was more anxioselective. Moreover, the light conditions imposed on mice before the test may be an important factor in the variability of the response to serotonergic but not to benzodiazepine treatments.     

Palmitoylethanolamide stimulation induces allopregnanolone synthesis in C6 Cells and primary astrocytes: involvement of peroxisome-proliferator activated receptor-α

Palmitoylethanolamide (PEA) regulates many pathophysiological processes in the central nervous system, including pain perception, convulsions and neurotoxicity, and increasing evidence points to its neuroprotective action. In the present study, we report that PEA, acting as a ligand of peroxisome-proliferator activated receptor (PPAR)-α, might regulate neurosteroidogenesis in astrocytes, which, similar to other glial cells and neurones, have the enzymatic machinery for neurosteroid de novo synthesis. Accordingly, we used the C6 glioma cell line and primary murine astrocytes. In the mitochondrial fraction from cells stimulated with PEA, we demonstrated an increase in steroidogenic acute regulatory protein (StAR) and cytochrome P450 enzyme (P450scc) expression, both comprising proteins considered to be involved in crucial steps of neurosteroid formation. The effects of PEA were completely blunted by GW6471, a selective PPAR-α antagonist, or by PPAR-α silencing by RNA interference. Accordingly, allopregnanolone (ALLO) levels were increased in supernatant of PEA-treated astrocytes, as revealed by gas chromatography-mass spectrometry, and this effect was inhibited by GW6471. Moreover, PEA showed a protective effect, reducing malondialdehyde formation in cells treated with l-buthionine-(S,R)-sulfoximine, a glutathione depletor and, interestingly, the effect of PEA was partially inhibited by finasteride, a 5α-reductase inhibitor. A similar profile of activity was demonstrated by ALLO and the lack of an additive effect with PEA suggests that the reduction of oxidative stress by PEA is mediated through ALLO synthesis. The present study provides evidence indicating the involvement of the saturated acylethanolamide PEA in ALLO synthesis through PPAR-α in astrocytes and explores the antioxidative activity of this molecule, confirming its homeostatic and protective role both under physiological and pathological conditions.     

Increased elevated plus maze open-arm time in mice during spontaneous morphine withdrawal

Rats undergoing both naloxone-precipitated and spontaneous opioid withdrawal exhibit anxiogenic behaviors in the elevated plus maze (EPM). Recently, we observed an unexpected result, namely mice exhibited increased EPM open-arm time during naloxone-precipitated morphine withdrawal. This was surprising since this behavioral outcome is usually associated with an anxiolytic profile. This study demonstrates that mice exhibit an increase in both EPM open-arm time and % open-arm entries also during spontaneous opioid withdrawal.     

“Rapid actions of the neurosteroid allopregnanolone on ovarian and hypothalamic steroidogenesis: Central and peripheral modulation”

The present study aimed to determine whether an i.c.v. administration of allopregnanolone (ALLO) rapidly modifies the hypothalamic and ovarian 3β‐hydroxysteroid dehydrogenase (3β‐HSD) enzymatic activity and gene expression in in vivo and ex vivo systems in pro‐oestrus (PE) and dioestrus I (DI) rats. Animals were injected with vehicle, ALLO, bicuculline or bicuculline plus ALLO and were then killed. In the in vivo experiment, the hypothalamus, ovaries and serum were extracted and analysed. In the ex vivo experiment, the superior mesenteric ganglion ‐ ovarian nerve plexus ‐ ovary system was extracted and incubated during 120 minutes at 37 ºC. The serum and ovarian compartment fluids were used to determine progesterone by radioimmunoanalysis. In the in vivo experiments, ALLO caused a decrease in hypothalamic and ovarian 3β‐HSD enzymatic activity during PE. During DI, ALLO increased hypothalamic and ovarian 3β‐HSD activity and gene expression. The ovarian 3β‐HSD activity increased in both stages in the ex vivo system; gene expression increased only during DI. ALLO induced an increase in serum progesterone only in D1 and in the ovarian incubation liquids in both stages. All findings were reversed by an injection of bicuculline before ALLO. Ovarian steroidogenic changes could be attributed to signals coming from ganglion neurones, which are affected by the acute central neurosteroid stimulation. The i.c.v. administration of ALLO via the GABAergic system altered 3β‐HSD activity and gene expression, modulating the neuroendocrine axis. The present study reveals the action that ALLO exerts on the GABAA receptor in both the central and peripheral nervous system and its relationship with hormonal variations. ALLO is involved in the “fine tuning” of neurosecretory functions as a potent modulator of reproductive processes in female rats.     

The anxiolytic effect of acute ethanol or diazepam exposure is unaltered in μ-opioid receptor knockout mice

Previous researchers demonstrate an opioidergic involvement in the anxiolytic and rewarding actions of ethanol and diazepam. Therefore, to further characterize the role of the opioid system in the anxiolytic action of ethanol and diazepam, normal (C57BL/6J), hybrid (B6129F1) and μ-opioid receptor knockout mice were given i.p. ethanol (0, 1.0 or 1.6 g/kg) or diazepam (1.5 mg/kg). The anxiolytic properties of these agents were then tested in the elevated plus-maze. Additional ethanol-treated μ-opioid receptor knockout mice (1 g/kg) were pretreated with the κ-opioid receptor antagonist nor-BNI (0 or 3 mg/kg) to assess the involvement of κ-opioid activity in ethanol’s anxiolytic actions. The anxiolytic action of ethanol and diazepam in the μ-opioid receptor knockout mouse did not differ from the effects obtained in normal mice and pretreatment with nor-BNI did not significantly attenuate ethanol’s actions in μ-opioid receptor knockout mice. Thus, the anxiolytic actions of ethanol and diazepam appear to be independent of opioid system activity in the μ-opioid receptor knockout mouse.     

Prenatal and postnatal exposure to bisphenol a induces anxiolytic behaviors and cognitive deficits in mice

Bisphenol A (BPA), an environmental endocrine‐disrupting chemical, has been extensively evaluated for reproductive toxicity and carcinogenicity. However, little is known about the behavioral and neurochemical effects of BPA exposure. This study examined whether chronic daily exposure to an environmental endocrine‐disrupting chemical, bisphenol A [(BPA); 100 μg/kg/day or 500 μg/kg/day, p.o.], from prenatal Day 7 to postnatal Day 36 would lead to changes in anxiety and memory in mice. First, we observed the behavioral alterations of BPA‐treated mice using two anxiety‐related models, the open field test and elevated plus maze (EPM) test. In the open field test, BPA treatment (100 μg/kg/day) increased movement in the central zone. BPA treatment (500 μg/kg/day) also increased the time spent in the open arms in the EPM test. Second, we measured cognitive ability in the Y‐maze test and novel object test. BPA‐treated mice showed decreased alternation behavior in the Y‐maze at both of doses, indicating working memory impairment. BPA‐treated mice (100 μg/kg/day) also showed decreased novel object recognition as expressed by central locomotion and frequency in the central zone, showing recognition memory impairment. Finally, to measure changes in the dopaminergic and NMDAergic systems in the brain, we performed autoradiographic receptor binding assays for dopamine D₁ and D₂ receptors, the NMDA receptor, and the dopamine transporter. BPA treatment increased D₂ receptor binding in the caudate putamen (CPu) but decreased DAT binding. BPA treatment also decreased NMDA receptor binding in the frontal cortex and CA1, CA3, and DG of the hippocampus. Taken together, our results suggest that long‐term BPA exposure in mice can induce anxiolytic behaviors, cognitive deficits and changes in the dopaminergic and NMDAergic systems. Synapse 64:432–439, 2010. © 2010 Wiley‐Liss, Inc.     

Anxiolytic-like actions of reboxetine, venlafaxine and endurance swimming in stressed male rats

Despite being potent anxiolytic agents, benzodiazepines (BDZ) sometimes show reduced therapeutic efficacy in stressed rodents. However, the effectiveness of norepinephrine reuptake inhibitors (NRI) and serotonin-norepinephrine reuptake inhibitors (SNRI) or other anxiolytic interventions, e.g., exercise, remained elusive. Here, we demonstrated that male rats subjected to restraint stress for 4 weeks showed decreases in percent open arm time and open arm entry, as determined by elevated plus-maze test (EPM). Increases in inhibitory avoidance trial 2 and outer zone time were also observed in elevated T-maze (ETM) and open field test (OFT), respectively. To evaluate the anxiolytic-like actions of exercise and anxiolytic drugs, stressed rats were subjected for 4 weeks to swimming or daily gavage with 2mg/kg diazepam (BDZ), or 10mg/kg fluoxetine (selective serotonin reuptake inhibitor), reboxetine (NRI), or venlafaxine (SNRI). In EPM, the open arm activity was higher in the swimming, reboxetine-treated and venlafaxine-treated groups as compared to age-matched controls, while diazepam and fluoxetine were without effect. In ETM, a reduction in avoidance latency was observed only in swimming and venlafaxine-treated groups. However, the combined swimming and pharmacological treatment showed no additive anxiolytic-like effect. It could be concluded that restraint stress induced anxiety-like behaviors, which were not responsive to diazepam or fluoxetine, whereas reboxetine, venlafaxine and swimming showed anxiolytic-like actions in stressed rats.