European Journal of Pharmacology 602 (2009) 343–347

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European Journal of Pharmacology

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Behavioural Pharmacology

Possible GABAergic mechanism in the protective effect of allopregnenolone againstimmobilization stress

Anil Kumar ⁎, Richa Goyal, Atish PrakashPharmacology Division, University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh-160014, India

⁎ Corresponding author. Reader in Pharmacology, Uceutical Sciences, Panjab University, Chandigarh-160014fax: +91 172 2541142.

E-mail address: kumaruips@yahoo.com (A. Kumar).

0014-2999/$ – see front matter © 2008 Elsevier B.V. Aldoi:10.1016/j.ejphar.2008.11.038

a b s t r a c t

a r t i c l e i n f o

Article history:

Acute stress may be experie Received 30 August 2007Received in revised form 10 November 2008Accepted 18 November 2008Available online 27 November 2008

Keywords:AllopregnenoloneImmobilization stressγ-amino butyric acidOxidative damage

nced in response to an immediate physical, emotional or psychological stimulus.Stress is known to promote long-term changes in multiple neural systems. Present study was conducted withan aim to elucidate the possible role of GABAergic system in the protective effect of allopregnenolone againstimmobilization stress induced behavioral and biochemical alterations. Mice were immobilized for periods of6 h. Animals were divided into different groups, consisting of six in each. Various behavioral tests (mirrorchamber, actophotometer) followed by oxidative parameters (malondialdehyde level, glutathione, catalase,nitrite and protein) were assessed in animals. 6 h immobilization significantly caused antinociceptive,locomotor activity impairment, anxiety-like behavior and oxidative damage as compared to unstressedanimals. Allopregnenolone (10 and 20 mg/kg, i.p.) treatment significantly reduced tail flick latency, improvedambulation, anti-anxiety like effect and attenuated oxidative damage as compared to stressed mice.Protective effect of allopregnenolone (10 mg/kg, i.p.) was further antagonized by picrotoxin (1.0 mg/kg) andpotentiated by muscimol (0.05 mg/kg) pretreatments (Pb0.05). Present study suggests that allopregneno-lone's protective effect could be due its interaction with γ-amino butyric acid receptor complex.

© 2008 Elsevier B.V. All rights reserved.

1. Introduction

Steroids were reported to influence brain excitability as early as1942. Allopregnenolone is an active metabolite of progesterone thatgenerates in the brain via de novo synthesis from cholesterol ormetabolism of peripheral sources of progesterone. Subsequent studiesdetermined that the progesterone metabolite (allopregnenolone), apotent positive modulator of γ-amino butyric acid type A receptorcomplex potentiates γ-amino butyric acid gated chloride conductance(Harrison and Simmonds, 1984). Allopregnenolone acts by a stereospecific interaction at a unique steroid recognition site associated withthe γ-amino butyric acid receptor complex (Lambert et al., 1995).

Although, a variety of neurotransmitter systems and ionic channelsare sensitive to neurosteroids modulation, the physiological andpharmacological significance of these widespread effects remainspoorly understood. Consistent with its ability to facilitate γ-aminobutyric acid (GABAergic) transmission, allopregnenolone producesanesthetic (Mok et al., 1991), hypnotic (Mendelson et al., 1987),anticonvulsant (Kokate et al., 1994) and anxiolytic (Weiland andOrchinik, 1995) effects. Allopregnenolone has been shown to be apotent antistress (Purdy et al., 1991), antiaggressive, neuroprotective(Frye, 1995) and hyperphagic activity (Chen et al., 1996). In addition,

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recent studies have shown the neuroprotective effect of allopregne-nolone in several experimental animal models of neurodegeneration,including seizures (Lonsdale and Burnham 2007), Alzheimers (BrintonandWang 2006), Niemann-Pick C disease (Mellon et al. 2008), kainiteexcitotoxicity (Ciriza et al., 2004) and middle cerebral artery occlusion(MCAO) (Sayeed et al. 2006). However, their exact mechanism of theiraction in stress and related conditions is not fully understood. Most ofits characteristics are due to the release of the adenocorticotropichormone and corticosteroids (cortisone in rats and cortisol inhumans) into the blood stream as a result of activation ofhypothalamic pituitary adrenal axis. It is well reported that immobi-lization stress stimulates hypothalamic-pituitary-adrenal axis result-ing to an increased production of corticosterone (Kipp and Rivers,1987). Besides, exposure to stressful situations stimulates numerouspathways leading to increased production of free radicals (Liu et al.,1996). Excessive free radicals generation produces lipid peroxidation,deoxy ribonucleic acid damage, cell death and precipitated variousstress related pathological conditions such as anxiety, memory anddepression. Stress may also impair antioxidant defenses, leading tooxidative damage by altering oxidants and antioxidant balance(Matsumoto et al., 1999). Both immobilization and variable stressare followed by an increase in lipid peroxidation, measured in plasmaand in brain structures (Liu et al., 1994). In addition, decreasedactivities of the antioxidant defense enzymes have been observed inthe brain of the rats treated with glucocorticoids (steroid hormonesreleased by the adrenals in response to physical and psychologicalstressors) and exposure to physiological levels of these hormones

344 A. Kumar et al. / European Journal of Pharmacology 602 (2009) 343–347

exacerbates the induction of reactive oxygen species (Manolli et al.,2000). Present investigation was designed with the aim to elucidatethe possible role of γ-amino butyric acid (GABAergic) system in theprotective effect of allopregnenolone against immobilization stressinduced behavioral and biochemical alterations.

2. Materials and methods

2.1. Animals

Albinomice (Laca strain) of either sexweighing between 22 and 30 gbred in Central Animal House (CAH) facility of the Panjab University,Chandigarh, India were used. The animals were housed under standardlaboratory conditions and maintained on natural 12-h light and 12-hdark cycle and had free access to food and water. Animals wereacclimatized to laboratory conditionsbefore the experiment. Each groupconsists ofminimal 6 animals. Themanipulationswere carried out in thelight phase between 09.00AMand15.00 PM. The experimental protocolwas approved by the Institutional Animal Ethics Committee (IAEC) andconducted according to the IndianNational Science AcademyGuidelinesfor the use and care of experimental animals.

2.2. Drug and treatments

The following drugs were used in the present study — allopregne-nolone (10, 20 mg/kg), picrotoxin (1.0 mg/kg) andmuscimol (0.05 mg/kg). Animals (n=48) were divided into several groups, consisting of sixin each. First and second group was named as unstressed and stressedgroup (6 h immobilization) respectively. Allopregnenolone (10 mg/kgand 20 mg/kg, i.p.), picrotoxin (1.0 mg/kg, i.p.) and muscimol (0.05 mg/kg, i.p.) were treated as group 3–6 respectively. Pretreatment ofpicrotoxin (1.0 mg/kg, i.p.) or muscimol (0.05 mg/kg, i.p.) with lowerdose of allopregnenolone (10 mg/kg i.p.) is categorized as group 7 and8 respectively. Allopregnenolone was administered 30 min beforeimmobilization stress. However, picrotoxin (1.0 mg/kg) or muscimol(0.05 mg/kg) were administered 10 min before allopregnenolonetreatment in combination studies. Allopregnenolone and musimolwere dissolved in distilled water. However, picrotoxin was dissolved ina few drops of 0.25% dimethylsulfoxide (DMSO) and then made up withwater. All the drugs were administered intraperitoneally. Doses of thesedrugswere chosenbasedon thepublished reports of our own laboratory.

2.3. Immobilization stress

Animals were immobilized for 6 h by taping all the four limbs toboard after placing them on their backs using zinc oxide hospital tape.Release was affected by unraveling the tape after moistening withacetone in order to minimize pain or discomfort. In unstressed group,themicewere kept in animal cagewith soft bedding in the experimentalroom (Sur and Bhattacharya, 1997).

2.4. Behavioral assessments

Various behavioral parameters were assessed in mice after 6 himmobilization stress.

2.4.1. Antinociceptive testThe nociceptive threshold was determined as the tail flick latencies

elicited in response to radiant heat (D'Amour and Smith,1941). Baselinelatencies to tail flick withdrawal from the radiant heat source (3–5 s)were established. A cut-off time of 10 s was set to prevent any injury onthe tail (Kulkarni, 1999).

2.4.2. Measurement of locomotor activityAnimal was kept in actophotometer for the first 3 min and then

locomotor activity was recorded using actophotometer for a period

of 5 min. The apparatus was placed in a darkened, light-soundattenuated and ventilated testing room. Each animal was observedover a period of 5 min in a square (30 cm) closed arena equipped withinfrared light sensitive photocells using digital photoactometer andvalues expressed as counts per 5 min (Reddy and Kulkarni, 1998).

2.4.3. Measurement of anxiety: mirror chamber testThe mirror chamber consisted of a wooden chamber having a

mirror chamber enclosed within it. During the 5 min test session,following parameters were noted a) latency to enter the mirrorchamber, b) total time spent in mirror chamber, c) number of entriesin mirror chamber. Animals were placed individually at the distalcorner of the mirror chamber at the beginning of the test. Ananxiogenic response was defined as decreased number of entries andtime spent in the mirror chamber (Kulkarni and Reddy, 1996).

2.5. Biochemical assessments of oxidative damage

2.5.1. Tissue preparationAll the animals were sacrificed by decapitation on the same day

following behavioral assessments. The brains were removed, rinsed inisotonic saline and weighed. A 10% (w/v) tissue homogenate wasprepared with 0.1 M phosphate buffer (pH 7.4). The post nuclearfraction was obtained by centrifugation of the homogenate at12,000 ×g for 20 min at 4 °C.

2.5.2. Lipid peroxidation assayThe quantitative measurement of lipid peroxidation in the whole

brain was measured according to the method of Wills (1966). Theamount of malondialdehyde formed was measured by the reactionwith thiobarbituric acid at 532 nm using Perkin Elmer lambda 20spectrophotometer. The results were expressed as nanomole ofmalondialdehyde per milligram of protein using the molar extinctioncoefficient of chromophore (1.56×10 M−1 cm−1).

2.5.3. Estimation of reduced glutathioneReduced glutathione in the brain was estimated according to the

method of Ellman (1959). A 1.0ml of homogenatewas precipitatedwith1.0 ml of 4% sulfosalicylic acid by keeping themixture at 4 °C for 1 h andthe sampleswere immediately centrifuged at 1200 ×g for 15min at 4 °C.The assay mixture contains 0.1 ml of supernatant, 2.7 ml of phosphatebuffer of pH 8.0 and 0.2 ml of 0.01 M dithiobisnitrobenzoic acid (DTNB).The yellow color developed was read immediately at 412 nm usingPerkin Elmer lambda 20 spectrophotometer. The resultswere expressedas nanomole GSH per milligram of protein.

2.5.4. Nitrite estimationNitrite is the stable end product of nitric oxide (NO) in living

system. Accumulation of nitrite was measured in cell free super-natants from brain homogenates by spectrophotometer assay basedon Greiss reagent 15 (1% sulphanilamide 0.1% naphthylethylenedia-mine dihydrochloride 2.5% phosphoric acid) and incubated at roomtemperature for 10 min to yield a chromophore. Absorbance was readat 543 nm spectrophotometrically. The nitrite concentration wascalculated from a standard curve using sodium nitrite as standard andexpressed as micro molar nitrite per milliliter homogenate (Greenet al., 1982).

2.5.5. Catalase estimationCatalase activity was assayed by the method of Luck (1971)

wherein the breakdown of hydrogen peroxides (H2O2) is measured at240 nm. Briefly, assay mixture consisted of 3 ml of H2O2 phosphatebuffer and 0.05 ml of supernatant of tissue homogenate (10%), andchange in absorbance was recorded at 240 nm. The results wereexpressed as micromole H2O2 decomposed per milligram of protein/min.

Fig. 1. Effect of allopregnenolone on locomotor activity against 6 h immobilizationstress. Values are expressed as Mean±S.E.M. ⁎Pb0.05 as compared to Unstressed,⁎⁎Pb0.05 as compared to stressed (immobilized stress), ⁎⁎⁎Pb0.05 as compared to AP(10 mg/kg), +Pb0.05 as compared to MUS (0.05 mg/kg), (One-Way ANOVA followed byTukey's test) AP = Allopregnenolone, PTX = Picrotoxin, MUS = Muscimol.

Table 1Effect of allopregnenolone on mirror chamber test against 6 h immobilization stress

Treatment Latency to entermirror chamber(s)

No. of entries inmirror chamber

Time spent inmirror chamber(s)

Unstressed 39.0±8.81 3.0±0.71 120±10.21Stressed 82.0±5.09a 0.5±0.029a 1.25±0.75a

AP (10) 38.76±2.76b 2.56±0.23b 37±3.13b

AP (20) 29.24±2.13c 3.37±0.33c 78±6.42c

PTX (1.0) 97.0±3.88 0.5±0.012 0.98±0.04PTX (1.0)+AP (10) 57.63±3.83c 1.07±0.78c 26.32±3.75c

MUS (0.05) 48.76±3.19b 1.56±0.36b 43.26±3.18b

MUS(0.05)+AP (10) 21.56±2.79c,d 3.24±0.64c,d 72.0±6.28c,d

Values are expressed as Mean±S.E.M. aPb0.05 as compared to naïve, bPb0.05 ascompared to control (immobilized stress), cPb0.05 as compared to AP (10 mg/kg),dPb0.05 as compared to MUS (0.05 mg/kg), (One-Way ANOVA followed by Tukey's test)AP = Allopregnenolone, PTX = Picrotoxin, MUS = Muscimol.

Table 2Effect of allopregnenolone on immobilization-induced oxidative damage

Treatment(mg/kg)

LPO (moles ofMDA/mg ofprotein)

Red. GSH(µM of GSH/mgof protein)

Catalase (µMoleof H2O2/min/mgof protein)

Nitrite (µg/ml)

(% of control) (% of control) (% of control) (% of control)

345A. Kumar et al. / European Journal of Pharmacology 602 (2009) 343–347

2.5.6. Protein estimationThe protein content was measured according to the method of

Lowry (Lowry et al., 1951) using bovine serum albumin as standard.

2.5.7. Statistical analysisAll the values are expressed as mean±S.E.M. The data were

analyzed by using one way analysis of variance (ANOVA) followed byTukey's test. Pb0.05 was considered statistically significant. Allstatistical procedures were carried out using sigma stat version 2.0.

3. Results

3.1. Effect allopregnenolone on behavior and its modification bymuscimol and antagonized by picrotoxin

6 h acute immobilization stress significantly caused antinociception,reduced locomotors, anxiety-like behavior (as indicated by delayedlatency to enter in mirror chamber, decreased number of entries andtime spent in the mirror chamber) as compared to unstressed mice.These effects were significant as compared to unstressed animals(Pb0.05). Pretreatmentwith allopregnenolone (10mg/kg and20mg/kg)significantly reduced tail flick latency (Fig. 1), improved ambulatorymovements (Fig. 2), anti-anxiety (Table 1), as compared to stressed(immobilized) mice (Pb0.05).

Picrotoxin (1.0 mg/kg, per se) did not produce any significant onbehavioral alterations as compared to stressed animals. However,picrotoxin (1.0 mg/kg) pretreatment with allopregnenolone (10 mg/kg)caused reversal of their protective effects. Animals showed significantantinociception (Fig. 1), impairment in ambulatory activity (Fig. 2),anxiety like behavior (Table 1) (Pb0.05). Further, pretreatment ofmuscimol (0.05 mg/kg) with lower dose of allopregnenolone (10 mg/kg)potentiated the protective effect of allopregnenolone which was sig-

Fig. 2. Effect of allopregnenolone on tail flick test against 6 h immobilization stress.Values are expressed as Mean±S.E.M. ⁎Pb0.05 as compared to Unstressed, ⁎⁎Pb0.05 ascompared to stressed (immobilized stress), ⁎⁎⁎Pb0.05 as compared to AP (10 mg/kg),+Pb0.05 as compared to MUS (0.05 mg/kg), (One-Way ANOVA followed by Tukey's test)AP = Allopregnenolone, PTX = Picrotoxin, MUS = Muscimol.

nificant as compared their effect per se (Pb0.05) (Figs. 1 and 2; Table 1).Further, muscimol (0.05 mg/kg, per se) did not produce any significanteffect on behavioral alterations as compared to stressed animals.

3.2. Effect allopregnenolone on oxidative damage and its modification bymuscimol and antagonized by picrotoxin

6 h acute immobilization stress significantly increased malondial-dehyde level, nitrite concentration and depletion of reduced glu-tathione levels and catalase activity that was significant as comparedto unstressed animals (Pb0.05). Pretreatment with allopregnenolone(10 mg/kg, and 20 mg/kg) significantly attenuated rise in malondial-dehyde, nitrite concentration and restored depleted reduced glu-tathione and catalase activity as compared to stressed animals(immobilized) (Pb0.05) (Table 2).

Picrotoxin (1.0 mg/kg) per se treatment did not produce anysignificant effect on oxidative damage as compared to their stressedanimals (Table 2). Further, picrotoxin (1.0 mg/kg) pretreatment withlower dose of allopregnenolone (10 mg/kg) caused blocked of itsprotective effect as compared to their effect per se (Pb0.05). Muscimol(0.05 mg/kg) treatment caused significant antioxidant like effect ascompared to stressed animals. Further, muscimol (0.05 mg/kg)pretreatment potentiated the protective effect of allopregnenolone(10 mg/kg). These effects were significant as compared to their effectper se (Pb0.05) (Table 2).

Unstressed 0.168±0.031(100)

0.065±0.0018(100)

0.705±0.033(100)

318±2.94(100)

Stressed 0.611±0.033a

(363.6)0.014±0.0014a

(21.54)0.128±0.024a

(18.16)649±3.86a

(204.08)AP (10) 0.308±0.029b

(44.61)0.029±0.002b

(52.19)0.368±0.022b

(140.57)447±4.4b

(183.33)AP (20) 0.242±0.017c

(144.05)0.042±0.002c

(64.61)0.507±0.047c

(71.91)412±3.9c

(129.56)PTX (1.0) 0.686±0.002

(408.33)0.010±0.0023(15.38)

0.116±0.009(16.45)

678±4.56(213.21)

PTX (1.0)+AP(10)

0.502±0.041c

(298.80)0.022±0.0019c

(33.85)0.238±0.021c

(33.76)584±4.98c

(183.65)MUS (0.05) 0.378±0.0291b

(225)0.019±0.0011b

(29.23)0.317±0.024b

(44.96)378±2.88b

(118.87)MUS(0.05)+AP (10)

0.187±0.015c,d

(111.31)0.034±0.0031c,d

(52.31)0.428±0.032c,d

(60.71)427±3.92c,d

(134.28)

Values are expressed as Mean±S.E.M. aPb0.05 as compared to unstressed, bPb0.05 ascompared to stressed (immobilized stress), cPb0.05 as compared to AP (10 mg/kg),dPb0.05 as compared to MUS (0.05 mg/kg) (One-Way ANOVA followed by Tukey's test)AP = Allopregnenolone, PTX = Picrotoxin, MUS = Muscimol.

346 A. Kumar et al. / European Journal of Pharmacology 602 (2009) 343–347

4. Discussion

Stress activates hypothalamus–pituitary–adrenal axis (HPA) axisand influences several biological effects at both central and peripherallevel. Besides, neurotransmitters and neuropeptides also influenceHPA axis activity by acting at the hypothalamic or suprahypothalamiclevel. Role of neurosteroids in stress and related effects has been wellexplored (Purdy et al., 1991). Neurosteroid brings about a wide rangeof biochemical and behavioral changes augmenting adenohypophysialaxis (Shukla et al., 1989). Acute stress induces intense antinociception(Kulkarni 1980) accompanied by a massive secretion of adrenalsteroids and a fall in the adrenal ascorbic acid content. Gamma-amino-butyric-acid (GABA), an inhibitory neurotransmitter has beenreported to play an important role in many physiological andpsychological processes in stress and related pathologies (Mombereauet al., 2004). Allopregnenolone is a positive allosteric modulator of γ-amino butyric acid type A (GABAA) receptor, i.e., it increases theeffectiveness of inhibition signals relayed among γ-amino butyric acidchannels in nerves. Immobilization stress causes a significant increasein γ-amino butyric acid contents as well as an elevation of L-glutamicacid decarboxylase activity (Yoneda et al., 1983). Like most othersteroid hormones, neurosteroids are derived from cholesterol, a lipidthat plays an important role in the formation of cell membranes.Neurosteroids are known to effect neuronal growth and differentia-tion and to modulate various moods and reactions via neurotrans-mitter receptors, including the γ-amino butyric acid.

Acute stress has been reported to influence motor activity, anxietylike behavior and produce antinociceptive effect (Sevgi et al., 2006).Present study reconfirmed that acute immobilization stress (6 h)significantly caused antinociception, impaired locomotor activity,anxiety like behavior which was reversed by allopregnenolonepretreatment suggesting that γ-amino butyric acid is likely to play arole in the ability of allopregnenolone to alter the effects ofimmobilization stress. Antianxiety effect of allopregnenolone hasalso been cited in the literature (Rodgers and Johnson, 1998).

γ-Amino butyric acid receptor is a valuable in-vivo tool because itcan indicate whether a drug is acting in a complex site or not in orderto produce a pharmacological response.

It has also been seen that GABAergic system influences significantlyduring stress; particularly the benzodiazepine receptor binding (Brem-mer et al., 2000; Miller et al., 1987). Besides, GABAergic modulators arerecommended for the treatment of acute response to stress, episodicanxiety and fluctuations in generalized anxiety (Molodavkin et al.,2004).

Further, picrotoxin being a GABA antagonist decreases locomotoractivity and causes anxiety like behavior in animals. It is known thatpicrotoxin blocks the chloride channel coupled to the GABAA receptorand subsequently decreases the GABA inhibitory action and as aconsequence, induces neuronal over excitation resulting neuronalstress. In the present study, picrotoxin (γ-amino butyric acidantagonist) pretreatment blocked the protective effect of allopregne-nolone. Picrotoxin pretreatment with allopregnenolone significantlydecreases ambulatory activity, anxiety like behavior and increased tailflick latency. This suggests that GABAergic modulation could beinvolved in the protective effect of allopregnenolone. Further,muscimol (γ-amino butyric acid selective agonist) pretreatmentcaused potentiation of allopregnenolone's protective effects on bothbehavioral as well as oxidative damage. These results indicate the γ-amino butyric acid (GABAergic) mechanism of action of allopregne-nolone. Current evidence supports that the concentration of GABA hasmain role in allopregnenolone effects on GABAA receptors in rathypothalamic cultured neurons (Wang et al., 2007; Verleye et al.,2008).

It is generally well accepted that stress induces a wide range ofbiochemical and behavior changes (Natelson, 1983). Many of bio-chemical effects are thought to be mediated by stress-induced

neurochemical and hormonal abnormalities that are often associatedwith oxidative stress. Oxidative stress has been implicated in thepathophysiology of many neurological disorders. Antioxidant defensemechanisms include removal of oxygen, scavenging of reactiveoxygen/nitrogen species or their precursors, inhibition of reactiveoxygen species formation, binding of metal ions needed for thecatalysis of reactive oxygen species generation and up-regulation ofendogenous antioxidant defenses (Sherki et al., 2001). Oxidative stresscan cause cellular damage and neuro-degeneration by inducing thereactive oxygen species that oxidize vital cellular components such aslipids, proteins and deoxybric nucleic acid (DNA). Stress has beenknown to increase the malondialdehyde (MDA) levels and decreasesthe reduced glutathione activity (Molodavkin et al., 2004; Mombereauet al., 2004). It has also been seen that γ-amino butyric acid(GABAergic) system influences significantly in stress; particularlythe benzodiazepine receptor-binding (Bremmer et al., 2000). Also,application of immobilization stress to animals induced a significantalteration in the metabolism and function of various neurotransmit-ters in the central nervous system (CNS). In the present study, 6 himmobilized stress significantly increased lipid peroxidation, nitriteactivity and depleted reduced glutathione and catalase activity,suggesting oxidative damage which was significantly attenuated byallopregnenolone treatment. This indicates that allopregnenoloneproduces antistress and antioxidant like effect. However, cellular andreceptor basedmechanism in its protection is still far from elucidation.In the present study, picrotoxin pretreatment with allopregnenolonesignificantly reversed the protective action of allopregnenolone.Further, allopregnenolone pretreatment with muscimol potentiatedthe protective effect of allopregnenolone. Further, blockade ofallopregnenolone's protective effect by picrotoxin and potentiationbymuscimol, suggests the involvement of GABAergicmechanism in itsneuroprotection.

In summary, the present study suggests that γ-amino butyric acid(GABAergic) mechanism might be implicated in the protective effectof allopregnenolone in ameliorating immobilization stress-inducedbehavioral alterations and oxidative stress. Present findings furthersupport the therapeutic potential of allopregnenolone as neuropro-tectant in the treatment of stress-related disorders.

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