Effects of GABA A Receptors in Dorsal Hippocampus on Memory Retention of Cholestatic Rats

The molecular mechanisms that result in cognitive decits following cholestasis are mainly unknown. As there are many GABA A receptors in the hippocampus CA1 region and the crucial role for GABA in modulating memory, we evaluated the effects of GABA A receptor agents in the CA1 of cholestatic rats on memory retention. The interaction between GABAergic and opioidergic systems in the CA1 on memory was also investigated. The effects of administration of GABA A receptor agonist and antagonist, muscimol (60, 120 and 240 ng/ side) and bicuculline (100, 200 and 400 ng/ side), into CA1 on memory retention were studies using passive avoidance learning (PAL) task in bile duct ligated (BDL) rats. Naloxone (250 ng/ side), the mu-opioid receptor antagonist, was also co-administered alone or with bicuculline (400 ng/ side) to indicate the interaction between opioidergic and GABAergic system. Cholestasis inhibited memory retrieval is shown by the decrease in the step-through latency (STLr). Administration of muscimol or bicuculline alone after training potentiated or attenuated respectively amnesia in BDL rats dose-dependently. Naloxone (250 ng /side) alone increased STLr in BDL-treated rats. Bicuculline (100 ng/side) alone antagonized the amnesic effect of muscimol (120 ng/side). Coadministration of bicuculline and naloxone or muscimol and naloxone caused a signicant difference in STLr compared to only naloxone treated rats which show the interaction between two systems on memory retention in cholestssis. Bicuculline (100 ng/side) microinjection alone antagonized the amnesic effect of muscimol (120 ng/side). We indicated the contribution of intra-CA1 GABA A receptors on memory retention in cholestatic rats by the PAL test. Blockade of each GABA A or mu-opioid receptors alone could attenuate the amnesia in BDL rats. Furthermore, blockade of both GABA A or mu-opioid receptors reversed the memory decit in BDL-treated rats, which shows the interaction between GABAergic and opioidergic systems on memory retention in this test.


Introduction
Cholestasis arises from structural and functional impairments of the hepatobiliary system (Roma and Crocenzi 2008) that nally leads to elevation of concentrations of biliary constituents in plasma, jaundice, and liver injury. Bile duct ligation (BDL) is an experimental model for cholestatic liver disorders that induces jaundice and liver dysfunction Sheen et al., 2010). Neurocognitive de cits as the main complication of liver dysfunction have been indicated in both humans (Swain 2009) and animals (Magen et al., 2009).
The precise mechanism that results in cognitive de cits following cholestatic liver disease is mainly unknown and therefore, there is no effective treatment available (Bashiri et al., 2018). There are some reports indicate that dysfunctions in several neurotransmitter systems, including acetylcholine, opioids,gamma-aminobutyric acid, noradrenaline, serotonin, dopamine, and glutamate may be involved in the pathogenesis of behavioral and cognitive abnormalities caused by cholestatic liver disease ( For example, it has been shown that the endogenous opioids increase in the cholestatic liver disease, supporting the involvement of the opioid system in its pathophysiology (Ebrahimkhani et al., 2006;Yurdaydin, 2001). Furthermore, it has been shown that GABAergic system dysfunction contributes to the pathogenesis of the cholestatic liver disease (Cauli et al., 2009). GABA A receptors, as the major inhibitory receptors in the nervous system, play the main role in cognition (Chapouthier and Venault 2002;Reis et al., 2009;Torkaman-Boutorabi et al., 2013). They are found at high concentration in the hippocampus, which plays crucial roles in cognition processes including, acquisition, consolidation, and retention (Lorenzini et al., 1996). For example, application of GABAergic receptor agonists could impair memory, but the GABAergic receptor antagonists enhance memory processes in inhibitory avoidance tests (Castellano and McGaugh 1990;Rassouli et al., 2010;Sardari et al., 2014).
There is no integrative study about the effects of GABAergic system on cognition function in cholestatic liver disease. Therefore, we aimed to evaluate the involvement of GABA A receptors in the dorsal hippocampal on memory retention of cholestatic animals. In the rst series of experiments, we microinjected a selective GABA A receptor agonist, muscimol, and the GABA A receptor antagonist, bicuculline, at different doses into CA1 of healthy and BDL rats. In another series of experiments, naloxone, the µ-opioid receptor antagonist, was microinjected alone or with bicuculline and muscimol into CA1 for detecting the possible interaction between the two opioidergic and GABAergic systems. Memory retention was evaluated by a passive avoidance learning (PAL) task in different animal groups.

Animals Husbandry
Locally produced male Wistar rats (250-280 g) were used in this study. Animals were housed at about 26 ± 2°C temperature and a 12/12°h light/dark cycle. Rats had free access to the taped water and standard laboratory chow. Rats were handled following the protocols approved by the institutional ethics committee of Shaanxi Provincial People's Hospital. Furthermore, the present experimental procedures were done in accordance with the guidelines for animal experimentation of the European Communities Council Directive (EU/63/2010) and reported according to the ARRIVE guidelines (Lilley et al., 2020).

Drugs
We used (+)-bicuculline (Sigma, St. Louis, USA), muscimol hydrochloride (Sigma, St Louis, MO) and naloxone hydrochloride (Tocris Cookson Inc., Bristol, UK). Naloxone and muscimol were dissolved in 0.9% saline. Bicuculline was dissolved in a drop of acetic acid glacial and then made up to 5 mL volume with 0.9% saline.

Bile duct ligation surgery
Laparotomy was done through the administration of sodium pentobarbital (50 mg/kg, i.p.) to cause general anesthesia. Surgery of BDL was performed by ligation of the main bile duct by two ligatures approximately 0.5 cm apart and transection at the midpoint between the two ligatures (Bergasa et al., 1994). To do postoperative care, rats were kept in separate cages to prevent wound dehiscence. About 4 h after surgery, rats were moved to their original cage (Rastegar et al., 2002). Operation-related mortality was less than 5%.

Cannula guide implantation
Animals were anaesthetized with xylazine/ketamine (5 and 50 mg/kg, i.p., respectively). To do stereotaxic surgery, two 23 gauges guide cannulas were moved until the tips were above the CA1 (about 2 mm). The stereotaxic coordinates used for the CA1 were: AP: -2 mm from bregma, V: -1.5 mm from the skull surface and L: ±1.6 from the sagittal suture (Paxinos and Watson 1986). The cannulas were secured to anchor screws with dental acrylic. After one week of recovery, rats were used for the experiments.

Intracerebral drug microinjections
The injection cannula (30 gauge) were used for bilateral microinjection of drugs into the CA1. Then, injection cannula was attached to a Hamilton syringe (1µL) by a PE-10 connection tube. The volume of injection was 0.5 µL solution on each side (1 µL/rat) during a 60-s period. The needles were not removed until 1 min after injection for complete diffusion.

PAL and memory test (step-through test)
The test was performed for all experimental groups on 14 days after BDL surgery. The procedure and apparatus were approximately the same as the previous reports (Hasanein and Shahidi 2010; Hasanein and Teimuri Far 2015). The step-through passive avoidance apparatus included of a transparent plastic lighted chamber (20 cm×20 cm×30 cm) and a dark chamber in dark opaque plastic with the same size of light chamber. The oor of both compartments was made of stainless steel rods (spaced 1 cm apart). A shock generator provides electricity in the oor of dark chamber. An opaque guillotine door closed the opening between the two chambers.

Training
All the rats received two trials for habituating to the apparatus at rst. To do these trials, the animals were placed in a lighted chamber facing away from the door and the guillotine door was raised 5 sec later. The rat prefers dark environment naturally. Once the animal enters the dark chamber, the door was closed and then after 30 sec the animal was moved to its home cage. The trial of habituation was repeated 30 min later and then followed by the rst acquisition trial after 30 min. When the rat had placed all 4 paws in the dark chamber, step-through latency or STLa was recorded as the latency to enter the dark chamber. If rat waited more than 100 sec to enter the dark chamber, it was eliminated from the experiments. A mild electrical shock (0.5 mA) was applied (3 sec) after entering the animal to the dark chamber. The animal was moved from the dark chamber into its cage after 30 sec. Then the procedure was repeated after 2 min. Every time the animal reentered the dark and had placed all 4 paws in the dark chamber, it received a foot-shock. The training was terminated if the rat remained 120 sec in the light compartment.

Retention test
Twenty-four hours after the acquisition trial, the retention trial was done. First, the animal was placed in the lighted chamber and after 5 sec, the guillotine door was raised. We recorded step-through latency (STLr) up to 300 sec. If the animal did not enter the dark chamber in 300 sec, the retention trial was terminated.

Experimental design
The following experiments were performed on each rat group consisting of seven animals and each rat was used only once. Experiment 1. Different doses of muscimol were microinjected into CA1 after training in BDL and non-BDL rats. Four groups of rats received intra-CA1 injections of saline or muscimol (60, 120 and 240 ng/ side) immediately after training. In the retention trial, all of these rats were microinjected with saline, 5 min before the retention trial. Experiment 2. Different doses of bicuculline were microinjected into CA1 after training in BDL and non-BDL rats. Four groups of rats were microinjected into CA1 with saline or bicuculline (100, 200 and 400 ng/side) immediately after training. On the retention trial, all of these rats were administered saline, 5 min before the retention trial. Experiment 3. The effects of microinjection of naloxone alone or bicuculline plus naloxone after training on memory retention in BDL and non-BDL rats were investigated. Four groups of animals received intra-CA1 injections of saline, naloxone alone (250 ng/side), naloxone (250 ng/side) plus bicuculline (200 ng/side) and naloxone (250 ng/side) plus muscimol (120 ng/side) after training. On the retention trial, all of these rats received saline into CA1, 5 min before the retention trial. Experiment 4. This experiment examined the effect of post-training microinjection of bicuculline with muscimol on memory retention in BDL rats. Four groups of BDL animals received intra-CA1 injections of saline, bicuculline (100 ng/side), muscimol (120 ng/side) and bicuculline (100 ng/side) with muscimol (120 ng/side) after training. On the test day, all of these animals received intra-CA1 injections of saline, 5 min before the test.

Statistical analysis
All data are expressed as mean ± S.E.M. The SPSS statistical software package (version 21.5; SPSS, Chicago, IL, USA) was used to do the analysis. One-way analysis of variance (ANOVA) with Tukey as the post hoc test was applied for showing differences among groups. Probability values less than 0.05 were considered signi cant. Two-way ANOVA identi ed a signi cant interaction between STLr of rats who received different doses of drugs in BDL and non-BDL groups. Post hoc analysis (Tukey test) was applied following a signi cant F value, for assessing the speci c inter-group comparisons.

Effects of different doses of bicuculline microinjected into CA1 on memory retention in the PAL test
Muscimol effects microinjected into CA1 at doses 100, 200 and 400 ng/side on STLr in different control and BDL rats are indicated in Figure 2. Two-way ANOVA results showed a signi cant interaction between bicuculline effects administration at different doses on STLr in BDL and non-BDL animals (F (3, 48) = 10.96, P < 0.001). Tukey test revealed that post-training injection of bicuculline (100, 200 and 400 ng/side) in non-BDL rats could not alter STLr as shown in the left panel of Figure 2 (P's > 0.05). We found a signi cant difference in STLr of BDL rats, between post-training administered bicuculline (100 ng/side) (229.71± 3.63 sec) compared to vehicle-treated BDL group (201.71 ± 2.69 sec) (P < 0.01) (Fig. 2 right   panel). Moreover, intra-CA1 injection of bicuclline 200 and 400 ng increased STLr (245 ± 4.1 sec, 262.85± 6.26 sec, respectively) than vehicle-received BDL rats (201.71 ± 2.69 sec) (P < 0.001, P < 0.001, respectively) ( Fig. 2 right panel). Further analysis showed bicuculline increased STLr in BDL rats dose dependently, in this regard, we found a signi cant difference in STLr between bicuculline 100 ng and bicuculline 400 ng-treated BDL animals (P < 0.001). A signi cant difference in STLr between bicuculline 200 ng and bicuculline 400 ng-treated BDL animals (P < 0.05) were also found (right panel of Figure 2).
Effects of administration of bicuculline or muscimol with naloxone on memory retention in the PAL task Figure 3 indicates the effects of naloxone alone and bicuculline or muscimol plus naloxone on memory retention in BDL and non-BDL rats. The results of two-way ANOVA showed a signi cant interaction between the effects of naloxone alone and bicuculline or muscimol plus naloxone on STLr in BDL and non-BDL rats (F (3, 48) = 20.78, P < 0.001). As shown in the left panel of gure 3, administration of naloxone alone and bicuculline or muscimol plus naloxone could not change STLr of non-BDL rats (243.85 ± 1.86 sec, 246.85 ± 6.61 sec, 249.14 ± 5.47 sec, respectively) compared to saline-treated non-BDL animals (244.14 ± 4.66 sec) (P's > 0.05).

Effects of administration of bicuculline to BDL rats on the response induced by administration of muscimol in the PAL task
Tukey post test revealed that microinjection of the minimum dose of bicuculline (100 ng/side) could not alter STLr of BDL rats compared to saline treated BDL animals as indicated in Figure 4 (P > 0.05). Administration of the medium dose of muscimol (120 ng/side) induces a hyperalgesic effect in BDL rats (156.85 ± 3.39 sec) compared to saline treated BDL group (225.42 ± 3.46 sec) (P < 0.001). However, coadministration of bicuculline (100 ng/side) with muscimol (120 ng/side) antagonized the hyperalgesic effect of muscimol (120 ng/side) by increasing STLr in bicuculline plus muscimol injected cholestatic animals (220.71 ± 2.92 sec) compared to only muscimol-treated cholestatic animals (156.85 ± 3.39 sec) (P < 0.001).

Discussion
The present work indicated the in uences of GABA A receptor antagonist and agonist on memory retention in cholestasis, which is an animal model with increased tone of endogenous opioid system. We used PAL task which is a standard task for evaluating learning and memory based on the previous reports ( To exclude the hypomotility in inducing the observed effects, muscimol and bicuculiine doses in this study were selected based on the previous published report (Ebrahimi-Ghiri et al., 2018) and our own pilot studies. The drugs were administered immediately after training, therefore, the observed effects are attributed to consolidation of memory, which performs just after the training trial and lasts some time (Ghiasvand et al., 2011;McGaugh 1989).
We have shown in a previous study bicuculline and muscimol effects in cholestatic animals on thermal pain modulation, which also suggests the pharmacological interactions between GABA and opioid systems ).
In the current study, there was an impairment in memory retention of cholestatic animals in the PAL task, which is compatible with a previous study showing the negative effect of post-training microinjection of morphine on memory retention in PAL test (Hosseini et al., 2013). Furthermore, this report con rms previous behavioral studies about the inhibitory effect of morphine on learning and memory formation in the animal model of Y-maze (Ma et al., 2007). We have also shown that the amnesia induced in BDL rats was blocked by naloxone microinjection which veri es the role of endogenous opioids in the amnesia caused by cholestasis (Nasehi et al., 2013a, b). The in uence of endogenous opioids in the amnesia caused by cholestasis in the current work was further con rmed by indicating the effect of a mu-opioid receptor antagonist, naloxone, on blocking memory de cits in BDL rats. Consistent with our ndings it has been reported that muscimol and bicuculline could potentiate and attenuated respectively the amnesia caused by intra-CA1 microinjection of morphine in animals (Zarrindast et al., 2008).
The effects of bicuculline and muscimol on memory retention in non-BDL rats are in agreement with a previous study reported that microinjection of bicuculline and imuscimol into central amygdala, at similar doses with this work, could not alter memory retention in the PAL test (Rassouli et al., 2010). However, injection of muscimol intro amygdala has a negative in uence on memory performance, the discrepancy is possibly due to rats strain used or the low doses used in the current study. We used muscimol at doses 60, 120 and 240 ng /side less than 1 µg/rat used in that report.
These experiments revealed that administration of naloxone alone reversed although not completely the amnesia induced by cholestasis in treated animals. However, there was still signi cant difference compared to saline-received healthy animals. This recon rms the in uence of opioid system is in pathophysiology of amnesia induced by cholestasis and could suggest opioid antagonists as a probable therapeutic strategy for treatment of cholestatic amnesia (Ebrahimkhani et al., 2006;Yurdaydin, 2001).
In the third series of the experiments, we showed that coadministration of bicuculline and naloxone could completely antagonize the amnesic effect of cholestasis in treated rats. Furthermore, there was not any signi cant difference in STLr than saline-received healthy animals. However, there was signi cant difference in STLr between naloxone and bicuculline received BDL rats and only naloxone received BDL animals. Furthermore, coadministration of muscimol and naloxone induced signi cant change in STLr than only naloxone administartion in BDL groups. These observations show the reciprocal interactions between GABAergic and opioidergic systems in the CA1 on retention memory in BDL rats and lend credence to previous studies on modulation of memory by the opioidergic and GABAergic systems Our current experiments showed that microinjection of bicuculline (100 ng/side) that alone has no signi cant impact on STLr could antagonize the amnesic effect of muscimol (120 ng/side). Therefore, the observation that the amnesic effect of muscimol was antagonized by co-administration of the GABA A receptor antagonist, provides evidence that the effect was not a non-speci c effect of the injection procedure.

Conclusions
These experiments showed the in uence of intra-CA1 GABA A receptors in controlling memory retention in cholestatic animals by the PAL test. Our results indicate that decreasing and increasing GABAergic neurotransmission by bicuculline and muscimol respectively attenuates and potentiates amnesia caused by cholestasis. These effect were speci c and could not be attributed to the effects of the injection procedure. Therefore, the present report reveals that the GABA A receptors have participated in the regulation of memory retention in cholestatic animals. Furthermore, we showed a possible interaction between GABAergic and opioidergic systems in the CA1 of cholestatic rats on memory retention using PAL test by showing the effects of coadministartions of pharmacological opiodergic and GABAergic agents on STLr in BDL animals.

Declarations ACKNOWLEDGEMENTS AND FUNDING
This work was nancially funded by Hakim Sabzevari University.

Compliance with Ethical Standards
Con ict of interest: The authors have no competing interests to declare that are relevant to the content of this article. The authors declare that the research was conducted in the absence of any commercial or nancial relationships that could be construed as a potential con ict of interest.
Consent to publish: Not applicable.
Consent to Participate: Not applicable.
Ethical approval: Ethical approval was waived by the local Ethics Committee of Shaanxi Provincial People's Hospital. Hakim Sabzevari University in view of the retrospective nature of the study and all the procedures being performed were part of the routine care.

Availability of data and material
All data generated or analyzed during this study are included in this published article. The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any quali ed researcher.
Each bar and column represents mean ± S.E.M.