The effects of intra-dorsal hippocampus infusion of pregnenolone sulfate on memory function and hippocampal BDNF mRNA expression of biliary cirrhosis-induced memory impairment in rats

Accepted Manuscript The effects of intra-dorsal hippocampus infusion of pregnenolone sulfate on memory function and hippocampal BDNF mRNA expression of biliary cirrhosis-induced memory impairment in rats Mona Dastgheib, Ahmad Reza Dehpour, Mansour Heidari, Leila Moezi PII: DOI: Reference:

S0306-4522(15)00741-1 http://dx.doi.org/10.1016/j.neuroscience.2015.08.018 NSC 16504

To appear in:

Neuroscience

Accepted Date:

7 August 2015

Please cite this article as: M. Dastgheib, A.R. Dehpour, M. Heidari, L. Moezi, The effects of intra-dorsal hippocampus infusion of pregnenolone sulfate on memory function and hippocampal BDNF mRNA expression of biliary cirrhosis-induced memory impairment in rats, Neuroscience (2015), doi: http://dx.doi.org/10.1016/ j.neuroscience.2015.08.018

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The effects of intra-dorsal hippocampus infusion of pregnenolone sulfate on memory function and hippocampal BDNF mRNA expression of biliary cirrhosis-induced memory impairment in rats

Mona Dastgheiba, Ahmad Reza Dehpour b, Mansour Heidari c, Leila Moezia,d*

a. Department of Pharmacology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran

b. Experimental research center, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran

c. Department of Medical Genetics, Tehran University of Medical Sciences, Tehran, Iran. d. Nanomedicine and Nanobiology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

*Correspondence to: Dr Leila Moezi, Department of Pharmacology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran Tel/Fax: 0098-711-2307591 Email: [email protected]

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ABSTRACT Learning and memory impairment is one of the most challenging complications of cirrhosis and present treatments are unsatisfactory. The exact mechanism of cirrhosis cognitive dysfunction is unknown. Pregnenolone sulfate (PREGS) is an excitatory neurosteroid acts as a NMDA receptor agonist and GABAA receptor antagonist. In this study we evaluated the effect of intra CA1 infusion of PREGS on cirrhotic rats' memory function using Y-maze test. Hippocampal BDNF mRNA expression was also evaluated.Three weeks after bile duct ligation (BDL) surgery, rats were under stereotaxic surgery for insertion of 2 guide cannulas in CA1 region of the hippocampus. After one week recovery, PREGS administered through CA1 cannulas in cirrhotic rats, while control or sham groups received vehicle. For evaluation of NMDA receptor role in memory enhancing effects of PREGS, DL-2-Amino-5-phosphonopentanoic acid (AP5) which is a potent and competitive antagonist of NMDA receptor, co-administered with PREGS and for assessment of hippocampal BDNF mRNA expression, quantitative RT-PCR was used. Results showed that 28 days after bile duct ligation, cirrhotic animals' memory significantly decreased in comparison with control and sham groups, while PREGS infusion could restore memory impairment (P<0.05). PREGS effects on memory of cirrhotic rats were antagonized by DAP5. RT-PCR findings have shown that hippocampal relative BDNF mRNA expression was upregulated in PREGS-treated groups in comparison with BDL group (P<0.001). Our findings suggest that PREGS has memory enhancing effect in cirrhosis memory deficit in acute therapy and this effect may be through NMDA (glutamate) receptor involvement and BDNF mRNA expression.

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Key words: Liver cirrhosis, Pregnenolone sulfate, Learning and memory, NMDA receptor, Brain-derived neurotrophic factor INTRODUCTION Liver cirrhosis described as liver function impairment due to hepatocytes fibrosis which presented by various degrees of signs and symptoms(Garcia-Tsao and Lim, 2009).Among several etiology of liver cirrhosis, cholestasis is one of disease causes which may be induced by common bile duct ligation (BDL) in animal models (Kountouras et al., 1984). In bile duct ligated animal, hepatocellular excretion of bile constituents is impaired and bile salts accumulation in the body and their deficiency in the intestinal lumen causes cholestasis, which can induce liver cirrhosis at last(Gartung et al., 1996).Hepatic encephalopathy (HE) is one of the most important complications of liver cirrhosis, which presents with cognitive dysfunctions such as learning and memory impairment (Zarrindast et al., 2012), but some studies have shown that cognitive dysfunction might be induced by liver cirrhosis even in the absence of HE(Romero-Gomez, 2007).Learning and memory impairment is one of the most challenging complication of cirrhosis and there are no specific treatments for cognitive dysfunction in this disease and all treatments are unsatisfactory (Gonzalez-Usano et al., 2014). The exact mechanism of learning and memory dysfunction in cirrhosis is unknown but several neurotransmitter systems such as glutamatergic, GABAergic, opioidergic, cholinergicand serotonergic can be altered in cirrhosis (Butterworth, 1996, Cauli et al., 2009, Ebrahimi-ghiri et al., 2012, Zarrindast et al., 2012). Moreover,hippocampal brain derived neurotrophic factor 3

(BDNF) level which is a member of the nerve growth factor (NGF) family and has been shown to play an important role in normal peripheral and central nervous systemdevelopment, decreases in cirrhosis. BDNF along with glutamatergic, GABAergic, opioidergic, cholinergicand serotonergic systems are involved in learning and memory processes (Tapia-Arancibia et al., 2004, Khakpai et al., 2012, Kim et al., 2012, Hosseini et al., 2013, Nasehi et al., 2013, Seyedabadi et al., 2014). Glutamatergic neurotransmission is known to play an important role in the course of hippocampal modulation of learning and memory processes, long-term potentiation (LTP) and synaptic plasticity(Harris et al., 1984, Bashir et al., 1993, Jamali-Raeufy et al., 2011, Khakpai et al., 2012).NMDA receptors are among the glutamatergic subtype receptors, widely distributed in the mammalian CNS with high densities in cerebral cortex superficial layers like CA1 region of the hippocampus(Morris, 1989, Richter-Levin et al., 1994, Davis and Linn, 2003).Besides, compelling evidence has confirmed the essential involvement of hippocampus upon memory processes in animals and humans(Squire, 1992, Braak and Braak, 1993, Alvarez and Banzan, 1999).It has been shown acorrelation between the NMDA hippocampal receptors density and the learning ability in rats(Keller et al., 1992). NMDA antagonists are reported to block induction of LTP when administered into the dorsal hippocampus(Hrabetova and Sacktor, 1997, Pourmotabbed et al., 1998, Xu et al., 1998). As already stated, glutamatergic synaptic transmissionsare altered in patients with liver disease and cholestasis(Felipo, 2006, Monfort et al., 2007).The alterations in glutamatergic neurotransmission may then be partly responsible for the memory impairment seen in cirrhotic patients.There has been little fundamental research evaluating the glutamatergic system 4

involvement upon cholestasis-induced amnesiabut it has been demonstrated that prolonged exposure to ammonia increases extracellular glutamate in cultured rat astrocytes and deregulation of glutamate release from astrocyets may contribute to the dysfunction of glutamatergic neurons in patients with acute liver failure(Ohara et al., 2009). Pregnenolone sulfate (3β-hydroxy-5-pregnen-20–one sulfate, PREGS) is an excitatory neurosteroid.The term neurosteroid, first introduced by Baulieu and his colleagues in year 1981, refers to steroids that synthesized de novo in the central and peripheral nervous systems from cholesterol(Dubrovsky, 2005). Steroids are known to have genomic actions, but they can also have non-genomic effects by interacting with several types of neurotransmitter receptors and neuromodulatory proteins(Rupprecht, 2003). PREGS is a positive modulator of N-methyl-Daspartate (NMDA), sigma 1 receptor and negative modulator of γ-aminobutyric acid type A (GABAA) receptor activities(Mayo et al., 1993, Flood et al., 1995).It also increases hippocampal BDNF and acetylcholine content(Mathis et al., 1999, Naert et al., 2007). PREGS has been well described as a potent steroidal enhancer of learning and memory processes in rodents(Flood et al., 1995, Vallee et al., 1997, Darnaudery et al., 2000, Ladurelle et al., 2000), but there are little studies have evaluated the effect of PREGS on cirrhosis cognitive impairment(Gonzalez-Usano et al., 2014). For treatment of cirrhosis cognitive deficits, discovery of new agents with new mechanism of action are necessary to act on molecular targets which involved in neurological alteration(Gonzalez-Usano et al., 2014), therefore, the principle objective of present study was to determine the effect of intra CA1 infusion of PREGS on cirrhotic animals memory function. We also evaluated NMDA receptor involvement as a part of memory-enhancing effect of PREGS and BDNF mRNA expression in hippocampus. The result of this study will shed light of new treatment in cirrhotic patients’ amnesia. 5

EXPERIMENTAL PROCEDURE Chemicals The drugs used in the present study were pregnenolone sulfate sodium salt (3β-hydroxy-5pregnen 20one sulfate,PREGS) (a GABAA receptor antagonist and NMDA receptor agonist) and DL-2-Amino-5-phosphonopentanoic acid (AP5) (a selective NMDA receptor antagonist) provided from Sigma Chemical Co., USA. Other drugs which are xylazine hydrochloride 2% (an alpha-2 adrenergic receptor agonist) provided from Alfasan Co., Netherland and ketamine hydrochloride 10% (a NMDA receptor antagonist) which purchased from rotexmedica Co., Germany.PREGSand AP5were dissolved in0.3% salineby sonication for 10 min(Akwa et al., 2001). These drugs were injected in a volume of 2.5 µl in each hippocampus (5 µl/rat).Xylazine hydrochloride and ketamine hydrochloride were injected intraperitoneal (ip).

Animals Male wistar rats (220-250 g) were obtained from Tehran University of Medical Sciences, Iran. Animals were kept in the animal house, provided with food and water ad libitum having a 12:12 h light-dark cycle at a temperature controlled environment (22±2°C). Rats were allowed to adapt to the laboratory condition for at least 1 week before surgery. All experiments were performed between 9:00 AM and 12:00 AM and conducted in accordance with the international guideline for biomedical research involving animals, revised in 1985. The animal Research Ethic Committee of Tehran University of Medical Sciences approved the study. We used 10 animals in the each group.

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Bile duct ligation (BDL) and induction of cirrhosis Bile duct ligation (BDL) was performed using standard technique(Uchinami et al., 2006).According to this technique, rats were anesthetized with 60 mg/kgKetamine hydrochloride10% and 8 mg/kgxylazine hydrochloride 2%. After midline laparotomy, the common bile duct was exposed and twice ligated with 4-0 silk suture and dissected between the ligatures. Sham operation was performed by gently touching the bile duct. The abdomen was closed intwo layers, and the animals were allowed to recover on a heat pad.

Stereotaxic surgery Twenty one days after BDL,animals were anesthetized with an intraperitoneal (ip) injection of ketamine hydrochloride 10% (60 mg/kg) and xylazine hydrochloride 2% (8 mg/kg) and mounted in a stereotaxic apparatus (David Kopf Instruments). Guide cannulas (22 G) were bilaterally implanted into the CA1 (3.6 mm posterior to bregma, 3.6 mm lateral and 2.9 mm ventral) according to Watson and Paxinos atlas (Paxinos and Watson, 2007). These cannulaswere used for microinjection into the CA1 region of the hippocampus. Cannulas were secured to the skull with dental cement.After cannula implantation was performed, rats were given 1 week for recovery.

Neurosteroid administration After recovery from stereotaxic surgery, rats were injected with 2.5 µl PREGS (1, 2 and 5 ng) or the same volume of vehicle in each hippocampus on the day of behavioral testing. Drugs were 7

injected bilaterally (thus the total infusion volume were double as those mentioned above) into the hippocampus of freely moving rats via 28-gauge needles connected with polyethylene tubing to a micro syringe (Hamilton, USA).Solutions were infused for 1 min at a constant rate of 5µ l/min. Control rats received the vehicle at the same rate of infusion. Injection needles were removed from the guide cannula 2 min after infusions in order to prevent drug reflux. Neurosteroid was administered immediately beforethe Y-maze training session and 1 h before Ymaze testing session.

DL-2-Amino-5-phosphonopentanoic acid (AP5) administration DL-2-Amino-5-phosphonopentanoic acid (AP5)is a selective NMDA receptor antagonistthat competitively inhibits the ligand (glutamate) binding site of NMDA receptors(Akwa et al.,

2001). AP5 (0.5 µg/rat) was administered 5 min before effective dose of PREGS through hippocampus (CA1) in cirrhotic rats. AP5 dose was selected according to dose response curve of AP5 in cirrhotic animals. Preliminary results in cirrhotic animals indicated that AP5 (0.5 µg/rat) did not affect memory performances and only blocked NMDA receptor, while the higher dose (1 µg/rat) decreased animal memory function. The selection of initial doses (0.5 and 1 µg/rat) was according to other similar studies on AP5(Akwa et al., 2001, Yamada et al., 2015).

Y-Maze test Behavioral study was conducted 4 weeks after BDL. Memory performances were evaluated by using a two-trial arm discrimination task in a Y-maze, a hippocampus relevant spatial task 8

(Conrad et al., 1996)based on the innate tendency of rodents to explore novelty(Dellu et al., 2000).The Y-maze apparatus in this study was made of wood, with three arms placedsymmetrically at a 120° angle from one another. The size of each arm was 40 cm long, 15 cm wide and 35 cm high. Each arm had special cues on the walls, so the animal could distinctdifferent arms from each other. This test has already been applied to steroid modulation of spatial memory performance in rats and mice(Petit et al., 2011). Briefly the test consisted of two trials (training and test) separated by an inter trial interval of 1 h.During the training trial, rats were allowed for 10 min to visit two arms (familiar arms 1 and 2) of the Y-maze, and during the test trial, they had free access to the three arms for 8 min, i.e. the familiar arms and the "novel arm" that was previously closed. The time spend in each arm was recorded and the percentage of time in the novel armduring the retention phase was reported as an index of memory performances. The apparatus was cleaned with 5% alcohol and allowed to drybetween sessions. The total number of arm entrances provided a measure of general motor activity.

Hippocampus removal After scarifying animal with head dislocation,its brain was removed and hippocampus isolated and kept in -80°C for future evaluation.

Measurement of BDNF mRNA expression

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sTotal RNA was extracted from ~ 200 µg of hippocampus using ONE STEP-RNA Reagent (Bio Basic, Canada inc.) according to the manufacturer’s instruction and the quantity and quality of RNA were analyzed using a nanodrop(ND-1000, Thermo Scientific Fisher, US) and gel electrophoresis. To eliminate any genomic contamination, RNA was treated with DNaseI (Qiagen, Hilden, Germany) as described by the manufacturer.Complementary DNA (cDNA) was synthesized using 1 µg of total RNA (Raoofian et al., 2013).The integrity and quality of cDNA were examined with β-Actinprimers as housekeeping (Figure 1). Real-time reverse transcriptase-PCR (RT-PCR) was carried out toevaluate the differences in expression patterns of BDNF gene among sample (Intact, Control, Sham, BDL and effective doses of PREGS (2 and 5ng/5µl) finding in behavioral study). The specific

primers

corresponding to the coding sequence including BDFF 5'-GGAGGCTAAGTGGAGCTGAC -3' ; BDFR 5'-GCTTCCGAGCCTTCCTTTAG-3' ; BACF 5'-TCCTGGGTATGGAATCCTG-3' and BACR 5'- CTTCTGCATCCTGTCAGCAA-3'were designed

for BDNF and β-Actin

respectively by Primer 3 software version 0.4 (frodo.wi.mit.edu) . Real time RT-PCR was performed in 20 µl reactions containing 1 µlcDNA target, 100 nM forward and reverse primers and 1× SYBR® Premix Ex Taq™ II (Takara, Tokyo, Japan). Experiments were carried out in triplicate using a CFX96™ Real-Time System (C1000TM Thermal Cycler) (Bio-Rad, Hercules, CA, USA). Amplification conditions were as follow: initial denaturation at 95°C for 10 min, followed by 40 cycles (denaturation at 95°C for 15second and annealingand extension at 60°C for 60 second). The relative value of the mRNA expression level of BDNF gene was calculated by comparing the cycle thresholds (CTs) of the target gene with that of housekeeping gene (β-Actin) using the 2−∆∆ct method and REST 2009 software.

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(Pfaffletal.2002). Serial dilutions of cDNAs were used for calculation of the efficiencies of the primer sets on real-time PCR. In this regard, it was found that the efficiencies of the various primer sets were similar.

Experimental design Experiment 1: Evaluation of the effect of intra-CA1 infusion of PREGS on memory function of cirrhotic animals Rats were divided into 6 groups (10 rats in each group): control group, sham group (was under manipulation of bile duct and stereotaxic surgery, received vehicle), BDL (was under bile duct ligation and stereotaxic surgery, received vehicle), and treatment groups (were under bile duct ligation and stereotaxic surgery, received PREGS at doses of 1, 2 and 5 ng/5µl 5 min before Ymaze training trial)

Experiment 2: Evaluation of NMDA receptor involvement in the PREGS function Bile duct ligated rats were divided into7 groups, 3 groups for finding ineffective dose of AP5 (received AP5 at doses of 0.5 and 1µg /rat or vehicle 5 min before Y-maze training trial) and4 treated groups (bile duct ligated animals received ineffective dose of AP5 or vehicle 5 min before the effective doses of PREGS).

Experiment 3: Quantitative measurement of BDNF mRNA expression 11

Intact (without any intervention), Control, Sham, BDL, and the effective doses of PREGS groups as explained previously, were evaluated for measurement of BDNF mRNA levels inhippocampus.

Statistical analysis Data are expressed as Mean ± SEM. The one way analysis of variance (ANOVA) followed by Tukey test was employed to analyze the data of Y-Maze test. Statistical software was SPSS (Ver16).Relative Expression Software Tool© (REST ©)(Ver2009) was used to analyze RT-PCR findings. P<0.05 was considered as a significance level.

RESULTS The effect of intra-CA1 infusion of PREGS on memory function of cirrhotic animals PREGS effect on time duration which spent in the novel arm Figure 2A, shows the effect of PREGS (1, 2 and 5 ng/5µl) on time duration which spent in the novel arm. According to the results, time duration which spent in the novel arm in BDL group decreased compared with control or sham group (P<0.05). PREGS treatment in BDL group caused increase in time duration which spent in the novel arm (P>0.05 compared with control or sham group).

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PREGS effect on time percent which spent in the novel arm Figure 2B, shows the effect of PREGS(1, 2 and 5 ng/5µl) on time percent which spent in the novel arm. 28 days after BDL surgery, time percent which spent in the novel arm decreased significantly compared with control or sham group (P<0.01). Intra CA1infusion of PREGS at doses of 2 and 5 ng/5µl enhanced time percent which spent by cirrhotic animals in the novel arm compared with cirrhotic group (P<0.05) while the dose of 1 ng/5µl was ineffective.

PREGS effect on the number of novel arm visits Figure 2C, shows the effect of PREGS (1, 2 and 5 ng/5µl) on the number of novel arm visits. Data analysis has demonstrated that, the numbers of novel arm visits have not shown any differences among groups.

Evaluation of NMDA receptor involvement in the PREGS function of cirrhotic rats The effect of AP5 on time duration, time percent and the number of novel arm visits in BDL group Figure3A, shows the effects of AP5 (0.5 and 1 µg/rat) on time duration which spent in the novel arm in cirrhotic animals. According to the results, AP5 at dose of 1 µg/rat reduced time duration which spent by cirrhotic animals in novel arm compared with BDL group (P<0.05), but the dose 0.5 µg/rat was ineffective.

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Figure 3B, shows the effects of AP5 (0.5 and 1 µg/rat) on time percent which spent in the novel arm in cirrhotic animals. Results have shown that, AP5 at dose of 1 µg/rat reduced time percent which spent by cirrhotic animals in novel arm compared with BDL group (P<0.05), but the dose 0.5 µg/rat was ineffective Figure 3C, shows the effects of AP5 (0.5 and 1 µg/rat) on the number of novel arm visits in cirrhotic animals. Data analysis has demonstrated that, the numbers of novel arm visits have not shown any differences among groups.

The effect of PREGS and AP5 co-administration on time duration which spent in novel arm Figure 4A, shows the effects of PREGS (2 and 5ng/5µl) and AP5 (0.5 µg/rat) co-administration on time duration which spent in novel arm in cirrhotic animals. According to results, PREGS (2 and 5ng/5µl) and AP5 (0.5 µg/rat) co-administration have not shown any effects on time duration.

The effect of PREGS and AP5 co-administration on time percent which spent in novel arm Figure 4B, shows the effects of PREGS (2 and 5ng/5µl) and AP5 (0.5 µg/rat) co-administration on time percent which spent in novel arm in cirrhotic animals.Results have shown that, coadministration of PREGS (2 and 5ng/5µl) and AP5 (0.5 µg/rat) reduced time percent which spent by cirrhotic animals in novel arm compared with corresponding PREGS groups.

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The effect of PREGS and AP5 co-administration on number of novel arm visits Figure 4C, shows the effects of PREGS (2 and 5ng/5µl) and AP5 (0.5 µg/rat) co-administration on the number of novel arm visits in cirrhotic animals. Data analysis have demonstrated that, PREGS (2 and 5ng/5µl) and AP5 (0.5 µg/rat) co-administration have not shown any effects on the number of novel arm visits. Quantitative measurement of hippocampal BDNF mRNA expression The relative hippocampal BDNF mRNA expression of intact, sham and BDL groups compared with control Figure 5A shows the relative hippocampal BDNF mRNA expression of intact, sham and BDL groups compared with control. Hippocampal BDNF mRNA expression of intact and sham groups have not shown any differences compared with control but it is down-regulated in BDL group compared with control by a factor of 0.691 (P<0.001) The relative hippocampal BDNF mRNA expression of intact, control, sham and PREGS (2 and 5 ng/5µl) compared with BDL Figure 5B, shows the relative hippocampal BDNF mRNA expression of intact, control, sham and PREGS (2 and 5 ng/5µl) groups compared with BDL group. Hippocampal BDNF mRNA expression is up-regulated by intra CA1 infusion of PREGS (2 and 5 ng/5µl) compared with BDL group by a factor of 3.90and 1.95 respectively (P<0.001) DISCUSSION

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Around 40% of cirrhotic patients suffer from minimal hepatic encephalopathy (MHE), with mild cognitive dysfunction which impairs their quality and span of life(Gonzalez-Usano et al., 2014),and some studies have shown that cognitive dysfunction might be induced by liver cirrhosis even in the absence of HE(Romero-Gomez, 2007).According to our findings, 28 days after bile duct ligation, memory of animals significantly decreased in comparison with control and sham groups. We also previously demonstratedmemory impairment induced by cirrhosis in rats, using Y-maze test (Javadi-Paydar et al., 2013).Our result is also in direction with other studies which have shown spatial memory impairment in cholestatic rats (Huang et al., 2009, Huang et al., 2010, Hosseini et al., 2013). It also has been demonstrated that the discrimination ability between novel object and experienced sample were impaired in BDL animals (GarciaMoreno et al., 2005, Zarrindast et al., 2012) Treatment of MHE is unsatisfactory, and there are no specific and curative treatments for the neurological changes in MHE.For treatment of cirrhosis cognitive deficits, discovery of new agents with new mechanism of action are necessary to act on molecular targets which involved in neurological alteration (Gonzalez-Usano et al., 2014). Pregnenolone sulfate (PREGS) acts as an excitatory neuromodulator and has a variety of neuropharmacological actions like memory enhancement.PREGS increases neuronal activityby inhibiting GABAA receptors (Majewska et al., 1988, Park-Chung et al., 1999) and/or potentiating NMDA receptors (Wu et al., 1991, Park-Chung et al., 1997). It mayaffect brain functions by modulating the release of neurotransmittersfrom presynaptic terminals in addition to itsdirect actions on ligand-gated ion channels. It

increases action potential-dependent

andspontaneous glutamate release by a variety of mechanisms(Zheng, 2009). 16

PREGS appears to be the most potent memory enhancer which is reported in different animal models even when injected in very low doses (Flood et al., 1995). Its memory enhancing effects have been shown in many different studies for example Alzheimer, scopolamine- induced learning deficits and age-related disorders of memory (Meziane et al., 1996, Maurice et al., 1999, Urani et al., 2004) In present study, we investigated the effect of PREGS (1, 2 and 5 ng/5µl) on memory impairment induced by cirrhosis in rats, using Y-maze test. Intra CA1 infusion of PREGS (2 and 5 ng/5µ l) could restore memory impairment in cirrhotic rats at doses of 2 and 5ng/5µl but not at dose of 1 ng/5µl. Number of total arm entrances in all groups have not shown any differences, which shows that locomotor activity did not have not any changes among groups. As mentioned previously, different neurotransmitter systems like GABAergic and glutamatergicsystems (which their role in learning and memory are well known) are involved in cirrhosis memory impairment. According to studies, GABAergic tone is increased in cirrhosis (Ahboucha et al., 2006). On the other hand hyperammonemia and liver failure also alter NMDA receptor function and its related signal transduction. The glutamate-nitric oxide-cGMP pathway is impaired in brain of cirrhotic animal model (Llansola et al., 2007). Our findings is in direction of Gonzales-Usano A and his colleagues study indicated that hyperammonemia affects glutamate-NO-cGMP pathway and recovery of learning ability by PREGS is due to restoration of this pathway and also it seems to be due to reduction of GABA receptors and enhancement of NMDA receptors activation (Gonzalez-Usano et al., 2014). It is known that glutamatergic neurotransmission plays an important role in the course of hippocampal modulation of cognitive processes, long term potentiation (LTP) and synaptic 17

plasticity (Harris et al., 1984, Bashir et al., 1993, Jamali-Raeufy et al., 2011, Khakpai et al., 2012). It also plays an essential role in information processing during spatial learning and memory. NMDA receptors, widely distributed in mammalian CNS with high densities in dorsal region (CA1) of hippocampus(Morris, 1989, Richter-Levin et al., 1994, Davis and Linn, 2003).The alterations in glutamatergic neurotransmission may then be partly responsible for the memory impairment seen in cirrhotic patients (Felipo, 2006, Monfort et al., 2007). In present study, we evaluated the role of NMDA receptor as a part of memory enhancing effects of PREGS by using AP5 as a most potent competitive antagonist of NMDA receptor, which a lot of studies have shown its effect on memory function(Davis et al., 1992, Morris et al., 2013). For this purpose, AP5 was administered at doses (0.5 and1 µg/rat) through CA1infusion in cirrhotic animals. Our results showed that AP5 at dose 0.5µg/rat, did not significantly affect memory performances. According to obtained dose-response curve, the highest ineffective dose of AP5 (which had no effect on memory but only blocked the NMDA receptor) was coadministered with the most effective dose of PREGS (2 and 5 ng/5µl) in cirrhotic animals.Our results showed that memory-enhancing effect of PREGS (2 ng/5µl) was antagonized by coadministration of PREGS (2 ng/5µl)and AP5 (0.5 µg/rat), which may demonstrate that NMDA receptor role as a part of PREGS memory-enhancing effect. On the other hand, we evaluated the relative hippocampal BDNF mRNA expression of intact, sham and BDL groups compared with control and also between PREGS-treated and non-treated groups, using Quantitative RT-PCR. Our results have indicated that hippocampal BDNF mRNA expression is down-regulated in bile duct-ligated animal compared with control which was in direction with Magen and his colleague which have shown that BDL reduced expression of the 18

BDNF mRNA(Magen et al., 2010). In our study we also illustrated for the first time that intra CA1 infusion of PREGS (2 and 5 ng/5µl) enhanced BDNF mRNA expression in BDL rats.

CONCLUSION Our findings suggest that PREGS has memory enhancing effect in cirrhosis memory deficit in acute therapy and this effect may be through NMDA receptor involvement and hippocampal BDNF mRNA expression enhancement.

Acknowledgments−The authors would like to thank Pharmacology Departments of Shiraz University of Medical Sciences (SUMS) and Tehran University of Medical Sciences (TUMS) and also Miss Shahrzad.M for their sincere cooperation and support during the study.

REFERENCES Ahboucha S, Pomier-Layrargues G, Mamer O, Butterworth RF (2006) Increased levels of pregnenolone and its neuroactive metabolite allopregnanolone in autopsied brain tissue from cirrhotic patients who died in hepatic coma. Neurochemistry international 49:372-378. Akwa Y, Ladurelle N, Covey DF, Baulieu EE (2001) The synthetic enantiomer of pregnenolone sulfate is very active on memory in rats and mice, even more so than its physiological neurosteroid

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counterpart: distinct mechanisms? Proceedings of the National Academy of Sciences of the United States of America 98:14033-14037. Alvarez EO, Banzan AM (1999) Ventral hippocampal glutamate receptors in the rat: possible involvement in learning mechanisms of an active avoidance response. J Neural Transm 106:9871001. Bashir ZI, Bortolotto ZA, Davies CH, Berretta N, Irving AJ, Seal AJ, Henley JM, Jane DE, Watkins JC, Collingridge GL (1993) Induction of LTP in the hippocampus needs synaptic activation of glutamate metabotropic receptors. Nature 363:347-350. Braak H, Braak E (1993) Entorhinal-hippocampal interaction in mnestic disorders. Hippocampus 3 Spec No:239-246. Butterworth RF (1996) The neurobiology of hepatic encephalopathy. Seminars in liver disease 16:235244. Cauli O, Rodrigo R, Llansola M, Montoliu C, Monfort P, Piedrafita B, El Mlili N, Boix J, Agusti A, Felipo V (2009) Glutamatergic and gabaergic neurotransmission and neuronal circuits in hepatic encephalopathy. Metabolic brain disease 24:69-80. Conrad CD, Galea LA, Kuroda Y, McEwen BS (1996) Chronic stress impairs rat spatial memory on the Y maze, and this effect is blocked by tianeptine pretreatment. Behavioral neuroscience 110:13211334. Darnaudery M, Koehl M, Piazza PV, Le Moal M, Mayo W (2000) Pregnenolone sulfate increases hippocampal acetylcholine release and spatial recognition. Brain research 852:173-179. Davis

S,

Butcher

SP,

Morris

RG

(1992)

The

NMDA

receptor

antagonist

D-2-amino-5-

phosphonopentanoate (D-AP5) impairs spatial learning and LTP in vivo at intracerebral concentrations comparable to those that block LTP in vitro. The Journal of neuroscience : the official journal of the Society for Neuroscience 12:21-34. 20

Davis SF, Linn CL (2003) Activation of NMDA receptors linked to modulation of voltage-gated ion channels and functional implications. American journal of physiology Cell physiology 284:C757768. Dellu F, Contarino A, Simon H, Koob GF, Gold LH (2000) Genetic differences in response to novelty and spatial memory using a two-trial recognition task in mice. Neurobiology of learning and memory 73:31-48. Dubrovsky BO (2005) Steroids, neuroactive steroids and neurosteroids in psychopathology. Progress in neuro-psychopharmacology & biological psychiatry 29:169-192. Ebrahimi-ghiri M, Nasehi M, Rostami P, Mohseni-Kouchesfehani H, Zarrindast MR (2012) The effect of cholestasis on rewarding and exploratory behaviors induced by opioidergic and dopaminergic agents in mice. Archives of Iranian medicine 15:617-624. Felipo V (2006) Contribution of altered signal transduction associated to glutamate receptors in brain to the neurological alterations of hepatic encephalopathy. World journal of gastroenterology : WJG 12:7737-7743. Flood JF, Morley JE, Roberts E (1995) Pregnenolone sulfate enhances post-training memory processes when injected in very low doses into limbic system structures: the amygdala is by far the most sensitive. Proceedings of the National Academy of Sciences of the United States of America 92:10806-10810. Garcia-Moreno LM, Conejo NM, Gonzalez-Pardo H, Aller MA, Nava MP, Arias J, Arias JL (2005) Evaluation of two experimental models of hepatic encephalopathy in rats. Brazilian journal of medical and biological research = Revista brasileira de pesquisas medicas e biologicas / Sociedade Brasileira de Biofisica [et al] 38:127-132. Garcia-Tsao G, Lim JK (2009) Management and treatment of patients with cirrhosis and portal hypertension: recommendations from the Department of Veterans Affairs Hepatitis C Resource 21

Center Program and the National Hepatitis C Program. The American journal of gastroenterology 104:1802-1829. Gartung C, Ananthanarayanan M, Rahman MA, Schuele S, Nundy S, Soroka CJ, Stolz A, Suchy FJ, Boyer JL (1996) Down-regulation of expression and function of the rat liver Na+/bile acid cotransporter in extrahepatic cholestasis. Gastroenterology 110:199-209. Gonzalez-Usano A, Cauli O, Agusti A, Felipo V (2014) Pregnenolone sulfate restores the glutamate-nitricoxide-cGMP pathway and extracellular GABA in cerebellum and learning and motor coordination in hyperammonemic rats. ACS chemical neuroscience 5:100-105. Harris EW, Ganong AH, Cotman CW (1984) Long-term potentiation in the hippocampus involves activation of N-methyl-D-aspartate receptors. Brain research 323:132-137. Hosseini N, Nasehi M, Radahmadi M, Zarrindast MR (2013) Effects of CA1 glutamatergic systems upon memory impairments in cholestatic rats. Behavioural brain research 256:636-645. Hrabetova S, Sacktor TC (1997) Long-term potentiation and long-term depression are induced through pharmacologically distinct NMDA receptors. Neuroscience letters 226:107-110. Huang LT, Chen CC, Sheen JM, Chen YJ, Hsieh CS, Tain YL (2010) The interaction between high ammonia diet and bile duct ligation in developing rats: assessment by spatial memory and asymmetric dimethylarginine. International journal of developmental neuroscience : the official journal of the International Society for Developmental Neuroscience 28:169-174. Huang LT, Tiao MM, Tain YL, Chen CC, Hsieh CS (2009) Melatonin ameliorates bile duct ligation-induced systemic oxidative stress and spatial memory deficits in developing rats. Pediatric research 65:176-180. Jamali-Raeufy N, Nasehi M, Zarrindast MR (2011) Influence of N-methyl D-aspartate receptor mechanism on WIN55,212-2-induced amnesia in rat dorsal hippocampus. Behavioural pharmacology 22:645-654. 22

Javadi-Paydar M, Ghiassy B, Ebadian S, Rahimi N, Norouzi A, Dehpour AR (2013) Nitric oxide mediates the beneficial effect of chronic naltrexone on cholestasis-induced memory impairment in male rats. Behavioural pharmacology 24:195-206. Keller EA, Borghese CM, Carrer HF, Ramirez OA (1992) The learning capacity of high or low performance rats is related to the hippocampus NMDA receptors. Brain research 576:162-164. Khakpai F, Nasehi M, Haeri-Rohani A, Eidi A, Zarrindast MR (2012) Scopolamine induced memory impairment; possible involvement of NMDA receptor mechanisms of dorsal hippocampus and/or septum. Behavioural brain research 231:1-10. Kim DH, Kim JM, Park SJ, Cai M, Liu X, Lee S, Shin CY, Ryu JH (2012) GABA(A) receptor blockade enhances memory consolidation by increasing hippocampal BDNF levels. Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology 37:422-433. Kountouras J, Billing BH, Scheuer PJ (1984) Prolonged bile duct obstruction: a new experimental model for cirrhosis in the rat. Br J Exp Pathol 65:305-311. Ladurelle N, Eychenne B, Denton D, Blair-West J, Schumacher M, Robel P, Baulieu E (2000) Prolonged intracerebroventricular infusion of neurosteroids affects cognitive performances in the mouse. Brain research 858:371-379. Llansola M, Rodrigo R, Monfort P, Montoliu C, Kosenko E, Cauli O, Piedrafita B, El Mlili N, Felipo V (2007) NMDA receptors in hyperammonemia and hepatic encephalopathy. Metabolic brain disease 22:321-335. Magen I, Avraham Y, Ackerman Z, Vorobiev L, Mechoulam R, Berry EM (2010) Cannabidiol ameliorates cognitive and motor impairments in bile-duct ligated mice via 5-HT1A receptor activation. British journal of pharmacology 159:950-957. Majewska MD, Mienville JM, Vicini S (1988) Neurosteroid pregnenolone sulfate antagonizes electrophysiological responses to GABA in neurons. Neuroscience letters 90:279-284. 23

Mathis C, Meziane H, Ungerer A (1999) [Models for the study of memory and neurosteroids]. J Soc Biol 193:299-306. Maurice T, Phan VL, Urani A, Kamei H, Noda Y, Nabeshima T (1999) Neuroactive neurosteroids as endogenous effectors for the sigma1 (sigma1) receptor: pharmacological evidence and therapeutic opportunities. Japanese journal of pharmacology 81:125-155. Mayo W, Dellu F, Robel P, Cherkaoui J, Le Moal M, Baulieu EE, Simon H (1993) Infusion of neurosteroids into the nucleus basalis magnocellularis affects cognitive processes in the rat. Brain research 607:324-328. Meziane H, Mathis C, Paul SM, Ungerer A (1996) The neurosteroid pregnenolone sulfate reduces learning deficits induced by scopolamine and has promnestic effects in mice performing an appetitive learning task. Psychopharmacology 126:323-330. Monfort P, Erceg S, Piedrafita B, Llansola M, Felipo V (2007) Chronic liver failure in rats impairs glutamatergic synaptic transmission and long-term potentiation in hippocampus and learning ability. The European journal of neuroscience 25:2103-2111. Morris RG (1989) Synaptic plasticity and learning: selective impairment of learning rats and blockade of long-term potentiation in vivo by the N-methyl-D-aspartate receptor antagonist AP5. The Journal of neuroscience : the official journal of the Society for Neuroscience 9:3040-3057. Morris RG, Steele RJ, Bell JE, Martin SJ (2013) N-methyl-d-aspartate receptors, learning and memory: chronic intraventricular infusion of the NMDA receptor antagonist d-AP5 interacts directly with the neural mechanisms of spatial learning. The European journal of neuroscience 37:700-717. Naert G, Maurice T, Tapia-Arancibia L, Givalois L (2007) Neuroactive steroids modulate HPA axis activity and cerebral brain-derived neurotrophic factor (BDNF) protein levels in adult male rats. Psychoneuroendocrinology 32:1062-1078.

24

Nasehi M, Piri M, Abbolhasani K, Zarrindast MR (2013) Involvement of opioidergic and nitrergic systems in memory acquisition and exploratory behaviors in cholestatic mice. Behavioural pharmacology 24:180-194. Ohara K, Aoyama M, Fujita M, Sobue K, Asai K (2009) Prolonged exposure to ammonia increases extracellular glutamate in cultured rat astrocytes. Neuroscience letters 462:109-112. Park-Chung M, Malayev A, Purdy RH, Gibbs TT, Farb DH (1999) Sulfated and unsulfated steroids modulate gamma-aminobutyric acidA receptor function through distinct sites. Brain research 830:72-87. Park-Chung M, Wu FS, Purdy RH, Malayev AA, Gibbs TT, Farb DH (1997) Distinct sites for inverse modulation of N-methyl-D-aspartate receptors by sulfated steroids. Molecular pharmacology 52:1113-1123. Paxinos G, Watson C (2007) The rat brain in stereotaxic coordinates. Amsterdam ; Boston ;: Academic Press/Elsevier. Petit GH, Tobin C, Krishnan K, Moricard Y, Covey DF, Rondi-Reig L, Akwa Y (2011) Pregnenolone sulfate and its enantiomer: differential modulation of memory in a spatial discrimination task using forebrain NMDA receptor deficient mice. European neuropsychopharmacology : the journal of the European College of Neuropsychopharmacology 21:211-215. Pourmotabbed A, Motamedi F, Fathollahi Y, Mansouri FA, Semnanian S (1998) Involvement of NMDA receptors and voltage-dependent calcium channels on augmentation of long-term potentiation in hippocampal CA1 area of morphine dependent rats. Brain research 804:125-134. Raoofian R, Noori-Daloii MR, Saee-Rad S, Modarresi MH, Ghaffari SH, Mojarrad M, Abolhasani F, Heidari M (2013) Differential expression of human homeodomain TGIFLX in brain tumor cell lines. Acta medica Iranica 51:834-841.

25

Richter-Levin G, Errington ML, Maegawa H, Bliss TV (1994) Activation of metabotropic glutamate receptors is necessary for long-term potentiation in the dentate gyrus and for spatial learning. Neuropharmacology 33:853-857. Romero-Gomez M (2007) Critical flicker frequency: it is time to break down barriers surrounding minimal hepatic encephalopathy. Journal of hepatology 47:10-11. Rupprecht R (2003) Neuroactive steroids: mechanisms of action and neuropsychopharmacological properties. Psychoneuroendocrinology 28:139-168. Seyedabadi M, Fakhfouri G, Ramezani V, Mehr SE, Rahimian R (2014) The role of serotonin in memory: interactions with neurotransmitters and downstream signaling. Experimental brain research 232:723-738. Squire LR (1992) Memory and the hippocampus: a synthesis from findings with rats, monkeys, and humans. Psychological review 99:195-231. Tapia-Arancibia L, Rage F, Givalois L, Arancibia S (2004) Physiology of BDNF: focus on hypothalamic function. Frontiers in neuroendocrinology 25:77-107. Uchinami H, Seki E, Brenner DA, D'Armiento J (2006) Loss of MMP 13 attenuates murine hepatic injury and fibrosis during cholestasis. Hepatology 44:420-429. Urani A, Romieu P, Roman FJ, Yamada K, Noda Y, Kamei H, Manh Tran H, Nagai T, Nabeshima T, Maurice T (2004) Enhanced antidepressant efficacy of sigma1 receptor agonists in rats after chronic intracerebroventricular infusion of beta-amyloid-(1-40) protein. European journal of pharmacology 486:151-161. Vallee M, Mayo W, Darnaudery M, Corpechot C, Young J, Koehl M, Le Moal M, Baulieu EE, Robel P, Simon H (1997) Neurosteroids: deficient cognitive performance in aged rats depends on low pregnenolone sulfate levels in the hippocampus. Proceedings of the National Academy of Sciences of the United States of America 94:14865-14870. 26

Wu FS, Gibbs TT, Farb DH (1991) Pregnenolone sulfate: a positive allosteric modulator at the N-methylD-aspartate receptor. Molecular pharmacology 40:333-336. Xu X, Boshoven W, Lombardo B, Spranger J (1998) Comparison of the amnestic effects of NMDA receptor antagonist MK-801 and nitric oxide synthase inhibitors: L-NAME and L-NOARG in goldfish. Behavioral neuroscience 112:892-899. Yamada K, Shimizu M, Kawabe K, Ichitani Y (2015) Hippocampal AP5 treatment impairs both spatial working and reference memory in radial maze performance in rats. European journal of pharmacology 758:137-141. Zarrindast MR, Hoseindoost S, Nasehi M (2012) Possible interaction between opioidergic and cholinergic systems of CA1 in cholestasis-induced amnesia in mice. Behavioural brain research 228:116-124. Zheng P (2009) Neuroactive steroid regulation of neurotransmitter release in the CNS: action, mechanism and possible significance. Progress in neurobiology 89:134-152.

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Legends:

Fig.1.Gel electrophoresis of cDNA, examined with β-Actinprimers as housekeeping control gene.

Fig.2. The effects of PREGS (1, 2 and 5 ng/5µl) on time duration (a), time percent (b) and number of novel arm visits (c) in 28-days BDL rats. PREGS was administered through CA1 region of the hippocampus immediately before Y-maze training session and 1 h before Ymaze testing session. Data are presented as mean ±S.E.M. *P<0.05 and **P<0.01 compared with BDL-solvent group and ## P<0.01 compared with BDL group received PREGS (1 ng/5µ l).

Fig.3. The effect of AP5, a potent and competitive antagonist of NMDA receptor (0.5 and 1µg /rat) on time duration (a), time percent (b) and number of novel arm visits (c) in 28-days BDL rats.AP5 was administered through CA1 region of the hippocampus immediately before Y-maze training session and 1 h before Y-maze testing session.Data are presented as mean ±S.E.M. * P<0.05 compared with solvent-solvent group.

28

Fig.4. The effect of PREGS (2 and 5 ng/5µl ) and AP5 (0.5 µg/rat) co-administration on time duration (a), time percent (b) and number of novel arm visits (c) in 28-days BDL rats.AP5 was administered through CA1 region of the hippocampus 5 min before PREGS (2 and 5 ng/5µl ) administration. Data are presented as mean ±S.E.M. *, P<0.05 compared with solvent-solvent group and # P<0.05 compared with PREGS 2- solvent group.

Fig.5. The relative BDNF mRNA expression of intact, sham and BDL groups compared with control (a) and the relative BDNF mRNA expression of intact, control, sham and PREGS (2 and 5 ng/5µl ) compared with BDL group. Data are presented as mean ±S.E.M. *** P<0.001 compared with control and ### P<0.001 compared with BDL group.

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β-Actin

Fig.1

PREGS 5ng

PREGS 5ng

PREGS 2ng

PREGS 2ng

BDL

BDL

BDL

Sham

Sham

Control

Intact

Figure

Duration of novel arm visits (s)

Figure

300

*

250

* 200 150 100 50 0 Control

Sham Solvent

BDL Solvent

BDL PREGS 1 ng

Fig.2A

BDL PREGS 2 ng

BDL PREGS 5ng

% of time spent in novel arm

Figure

70 ##

50

##

**

60

*

**

##

*

40 30 20 10 0 Control

Sham Solvent

BDL Solvent

BDL PREGS 1 ng

Fig.2B

BDL PREGS 2 ng

BDL PREGS 5ng

Figure

9

Number of novel arm visits

8 7 6 5 4 3 2 1 0 Control

Sham Solvent

BDL Solvent

BDL PREGS 1 ng

Fig.2C

BDL PREGS 2 ng

BDL PREGS 5ng

Duration of novel arm visits (s)

120 100 80 60 40

20 0 Solvent

DAP5 0.5

BDL

Fig.3A

DAP5 1

% of time spent in novel arm

30 25 20 15 10

5 0 Solvent

DAP5 0.5

BDL

Fig.3B

DAP5 1

Number of novel arm visits

6 5 4 3 2

1 0 Solvent

DAP5 0.5

BDL

Fig.3C

DAP5 1

Duration of novel arm visits (s) 0 Solvent AP5 0.5 PREGS 2 PREGS 5

BDL

Fig.4A PREGS 2

AP5 0.5

AP5 0.5

Solvent

Solvent

Solvent

50 Solvent

Figure

250

200

150

100

PREGS 5

Figure

60

*

*

40 30

#

AP5 0.5

AP5 0.5

AP5 0.5

Solvent

Solvent

0

Solvent

10

Solvent

20 Solvent

% of time spent in novel arm

50

PREGS 2

PREGS 5

PREGS 2

PREGS 5

BDL

Fig.4B

Number of novel arm visits

AP5 0.5

AP5 0.5

0 Solvent

1 Solvent

2 Solvent

3

Solvent

Figure

8

7

6

5

4

Solvent AP5 0.5 PREGS 2 PREGS 5 PREGS 2 PREGS 5

BDL

Fig.4C

Relative BDNF mRNA expression from control 1.5

1

***

0.5

0 Control Intact Sham

Fig.5A BDL

5

Relative BDNF mRNA expression from BDL

###

4

3 ###

### 2

1

0 BDL

Intact

Control

Fig.5B

Sham

PREGS 2

PREGS 5

Research highlights •

Memory function is impaired in cirrhotic animals

•

BDNF expression decreased in cirrhotic rats

•

PREGS has memory enhancing effect in cirrhosis memory impairmentof rats

•

PREGSenhances BDNF mRNA expression in cirrhotic rats

•

Memory enhancing effect of PREGSin cirrhosis may be through NMDA receptor

30