Associative learning deficit in two experimental models of hepatic encephalopathy

Behavioural Brain Research 198 (2009) 346–351

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Associative learning deficit in two experimental models of hepatic encephalopathy Marta Méndez a , Magdalena Méndez-López a , Laudino López a,∗ , María Ángeles Aller b , Jaime Arias b , Jorge L. Arias a a b

Laboratorio de Neurociencias, Departamento de Psicología, Universidad de Oviedo, Plaza Feijoo s/n, 33003 Oviedo, Spain Departamento de Cirugía I, Facultad de Medicina, Universidad Complutense de Madrid, Ciudad Universitaria s/n, 28040 Madrid, Spain

a r t i c l e

i n f o

Article history: Received 26 May 2008 Received in revised form 4 November 2008 Accepted 9 November 2008 Available online 14 November 2008 Keywords: Hepatic encephalopathy Cirrhosis Active avoidance Passive avoidance Rat

a b s t r a c t People with hepatic insufficiency can develop hepatic encephalopathy (HE), a complex neuropsychological syndrome covering a wide range of neurological and cognitive and motor alterations. The cognitive deficits include disturbances in intellectual functions such as memory and learning. In spite of its high prevalence in western societies, the causes of HE have not yet been clearly established. For this reason, experimental models of HE are used to study this condition. In this work, two experimental models were used, one Type B HE (portacaval shunt) and the other Type C HE (cirrhosis by intoxication with thioacetamide), to evaluate its effect on two tasks of associative learning: two-way active avoidance and step-through passive avoidance. The results show an impediment both in acquisition and retention of active avoidance in both models of HE. However, in passive avoidance, only the rats with portacaval shunt presented a memory deficit for the aversive event. In our opinion, these results can be explained by alterations in the neurotransmission system presented by animals with hepatic insufficiency, which are mainly caused by a rise in cerebral histamine and a dysfunction of the glutamatergic system. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Hepatic encephalopathy (HE) is a neuropsychiatric disorder derived from hepatic insufficiency. However, in spite of significant progress in this area in recent years, there are still many unanswered questions about HE, in relation to its ethiopathogeny [9,25,38], treatment [7] and diagnosis [37,43]. It is, therefore, necessary to develop different experimental models of hepatic insufficiency and to recur to further animal experimentation [8,6,12]. The most accepted and widely used classification for HE is the one proposed by Ferenci et al. [19], which classifies it into three main types, depending on the origin or cause. The first is Type A, which refers to encephalopathy associated with acute liver damage. The next is Type B, which concerns HE related to portosystemic shunt, which does not require any hepatocytic alteration. The third, referred to as Type C, is associated with cirrhosis and portal hypertension with portosystemic shunt. Type C HE can be episodic, persistent, and minimal [19]. Persistent HE constitutes the most common expression of chronic HE and evolves with evident symptoms, both neurological and cognitive, which have a negative impact on the patients’ social and professional life. However, in min-

∗ Corresponding author. Tel.: +34 985104188; fax: +34 985104144. E-mail address: [email protected] (L. López). 0166-4328/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2008.11.015

imal HE, there is a noteworthy absence of clinical symptoms during routine neurological exploration. But if more specific neurophysiological and neuropsychological methods are used, cognitive deficits can be observed that affect performance of daily skills along with some psychomotor sluggishness that can particularly affect driving vehicles [50,51]. Minimal HE represents, therefore, the first stage in the spectrum of HE, and patients who manifest minimal HE have increased probability of suffering overt HE later. Moreover, as HE can be derived from three types of hepatic insufficiency, it is necessary to develop experimental models that can imitate, as closely as possible, the characteristics of type A, B or C HE. Experimental models of HE should produce alterations similar to those developed by human patients, although of different ethiology, as in the models, the alterations are produced artificially in the laboratory. Patients with hepatic insufficiency show cognitive deficits, although no clear signs of neurological dysfunction, as occurs in minimal HE. They have also shown alterations in attention and in resolving tasks that evaluate visuospatial and visuoconstructive orientation, and difficulties in learning and memory tasks [4,39,40,52–54]. Alterations in learning and memory capacity in models of chronic HE have been reported previously [16,35,46,55]. The goal of this work is, therefore, to follow on from these studies by evaluating the capacity for associative learning, with implication of emotional memory and, therefore, participation of the amygdala as a key structure, in two experimental models, one of Type B (the

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portocaval shunt) and one of Type C HE (thioacetamide-induced cirrhosis). 2. Material and methods 2.1. Animals A total of 54 male Wistar rats were used (220–255 g at the start of the experiments) from the animalarium of the Oviedo University. All the animals had ad libitum access to food and tap water and were maintained at constant room temperature (22 ± 1 ◦ C), with a relative humidity of 65 ± 10% and artificial light–dark cycle of 12 h (08:00–20:00/20:00–08:00 h). The procedures and manipulation of the animals used in this study were carried out according to the Directive 86/609/EEC of the European Communities Committee and Royal Decree 223/1988 of the Ministry of Agriculture, Food and Fisheries relating to the protection of the animals used for experimentation and other scientific purposes, and the study was approved by the local committee for animal studies (Oviedo University). 2.2. Procurement of experimental models The animals were randomly distributed into four groups: end-to-side portacaval shunt (PCS group, n = 9), sham-operated (SHAM group, n = 9) animals with cirrhosis by administration of thioacetamide (TAA group, n = 10), and controls (CO group, n = 10). The experimental models that require surgery were carried out under induction of anaesthesia by i.m. injection of ketamine (75 mg/kg) and diazepan (5 mg/kg). With respect to postsurgical care, the rats were maintained close to a source of heat until they recovered consciousness (10–15 min) to avoid postoperative hypothermia. Afterwards, they were introduced into individual polycarbonate cages for 15 days and then grouped into cages of five animals until their behavioural evaluation. The surgical procedures and protocols used for the different experimental models are described below. 2.2.1. Portacaval shunt The end-to-side portacaval shunt operation was performed according to the technique described by Lee and Fisher [28]. This was modified slightly by total clamping of the inferior vena cava during portacaval shunt performance [29]. The total time in which the portal vein and inferior vena cava were clamped for anastomosis was less than 15 min. The abdominal incision was closed on two layers with catgut and 2-0 silk. The postoperative period started immediately after the intervention and lasted until the behavioural evaluation 45 days later. 2.2.2. Sham operation A bilateral subcostal laparotomy with prolongation to the xyphoid apophysis, followed by isolation of the portal vein with later clamping for 5 min, was performed. The operative field was irrigated with saline solution during the intervention. Finally, the laparotomies were closed by continuous suture on the two layers with catgut and 2-0 silk. The postoperative period started immediately after the intervention and lasted until the behavioural evaluation 45 days later. 2.2.3. Thioacetamide The method used to produce cirrhosis was weekly administration of thioacetamide (Sigma, Germany) in drinking water as described by Xiangnong et al. [56]. The thioacetamide (TAA) was administered for 12 weeks and its concentration was modified weekly depending on the animals’ weight gain or weight loss. The initial concentration of TAA was 0.03%. After 12 weeks of treatment, in an attempt to minimise the influence of isolating the animals, they were placed in groups of five rats per cage and behavioural tests were started 2 weeks later. During this period and until the end of the experiments, the animals received TAA at 0.04%. 2.2.4. Control The control group was isolated in the same way as the TAA group during 12 weeks and received normal tap water. After this period, the animals were placed in groups of five rats per cage and behavioural tests were started 2 weeks later. 2.3. Determination of ammonia levels and liver morphology and histopathology in rats with TAA Eight TAA rats and eight control rats were sacrificed by exsanguination 14 weeks after starting TAA administration. Plasma ammonia was immediately measured by glutamate dehydrogenase enzyme assay on a clinical analyzer (COBAS MIRA autoanalyzer; Products: Biolabo SA, Maizy, France). Hepatic tissue was excised and samples of middle hepatic lobe were fixed in phosphate-buffered neutral formalin (10%) and embedded in paraffin. Sections (5 ␮m) were stained with haematoxylin eosin and Masson’s trichromic dye and assessed with light microscopy. The Knodell histological activity index (HAI) was used to evaluate the severity of the necroinflammatory process and fibrosis [27]. Other parameters of hepatic lesion were also studied, such as cholangiofibrosis, apoptosis and proliferation of the bile duct epithelial cell [26].

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2.4. Apparatus A shuttle box (Ugo Basile 7552, Milan, Italy) was used to train the animals following a step-through passive avoidance paradigm. The box was made up of two Plexiglas compartments: an illuminated compartment (23 cm × 22 cm × 22 cm) lit by a 24 V 5 W lamp, and a dark compartment (23 cm × 22 cm × 22 cm), connected via a sliding door (7 cm × 3.5 cm × 7 cm). The grid floor of the apparatus consisted of stainless-steel bars 0.3 cm in diameter at 1 cm intervals, connected to a shock scrambler (Controller 7551, Milan, Italy). The front panel of this generator displays the function of the latency time, door, and shock indicators and the control door opening delay (10 s), duration and intensity of shock (3 s, 0.8 mA), and cut off time (5 min). Two-way active avoidance conditioning was conducted in a 50 cm × 24 cm × 23 cm automated shuttle box (Letica LE 916, Panlab,SL, Barcelona, Spain) constructed of plexiglas. The apparatus consisted of two equally sized compartments (25 cm × 23 cm × 24 cm) connected by an opening (10 cm × 14 cm). The floor was composed of stainless-steel bars wired to the stimulus generator. The shuttle box was enclosed in a sound-attenuating box (Letica LE 26, Panlab,SL, Barcelona, Spain) ventilated by an extractor fan and was illuminated by a fluorescent light located on the sound-attenuating box. The conditioned stimulus (CS) was an 80-dB and 60-Hz tone of 4 s duration. The unconditioned stimulus (US) was a 0.5-mA electrical footshock, presented immediately after the end of the CS, for 4 s at maximum. An intertrial interval of 30 s was used. The shuttle box was connected to a shock generator (Letica, LE 2706, Panlab SL, Barcelona, Spain) that controlled the training schedule and scored avoidances (the animal crosses over to the other compartment during the 4 s of the CS, thus avoiding the US), escapes (the animal crosses over to the other compartment during the 4-s US) and non-responses (the animal does not cross over to the other compartment during the US). 2.5. Experimental procedures Step-through passive avoidance training consisted of habituation, acquisition and retention phases. During habituation, rats were placed in the illuminated compartment and after 10 s the guillotine door was raised. The latency to cross to the dark compartment was recorded. The rat was then returned to the vivarium. One hour later, the acquisition phase began. The rat was placed in the illuminated compartment and after 10 s, the door was raised and the latency to cross the compartment was measured. Immediately afterwards, the door was closed and a 3 s unavoidable electric shock (0.8 mA) was delivered. The animal was kept in the dark compartment for 60 s and returned to the vivarium. Twenty-four hours later, retrieval was tested. The procedure was exactly the same as for the acquisition phase except that there was no shock. Latency to cross to dark compartment was measured. All behavioural tasks were performed between 10.00 and 13.00 h. A week after the retention trial of the passive avoidance task, each animal was placed singly in the two-way shuttle-box. The animals were trained in one daily session of 50 trials during 5 consecutive days. The first trial of the first session (day 1) started after 10 min of habituation to the apparatus, an interval in which the rat can explore the box freely and go from one compartment to the other. Each one of the 50 trials of each session consists of the presentation of a tone 4-s signal (CS) followed immediately by the appearance of a 0.5 mA (US) shock, of 4 s maximal duration. The 50 trials of each session were separated by a 30-s intertrial interval. For each trial, the rat could avoid the shock by crossing to the other compartment before the end of tone signal or terminate the shock by escaping into the other compartment. In each of these five sessions, the number of avoidances and escapes were recorded. All behavioural tasks were performed between 9.00 and 13.00 h. 2.6. Statistics All results are expressed as the mean ± SEM. PCS and TAA groups were compared with their respective control groups SHAM and CO (PCS vs. SHAM and TAA vs. CO). To compare groups during habituation, T-tests for independent samples were performed. The acquisition and retrieval phases of the passive avoidance task were analyzed by a two-way repeated measures ANOVA (between-factor: groups; within-factor: session). Significant interactions of session were followed by paired T-tests. To analyze the evolution between sessions in the number of avoidances, changes and percentage of escapes from shocks received in the active avoidance task, a two-way repeated measures ANOVA was conducted (between-factor: groups; within-factor: training day). Significant effect of factor training day was followed by one-way repeated measures ANOVA. Post-hoc Tukey’s tests were used to compare pairs of results.

3. Results 3.1. Ammonia levels, liver histopathology and body weights Ammonia serum level was higher in TAA rats compared to CO rats (TAA: 338.5 ± 48.4 ␮M; CO: 205.3 ± 33.9 ␮M; p = 0.041). Numerous macro and micronodules (72%) were seen on the surface

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of most of the TAA rats’ livers. The Knodell HAI was 7.83 ± 0.93. The TAA group presented chronic hepatitis in 69% of the animals; ductular proliferation was observed in 49% of the animals and cholangitis in 31%. Finally, a higher degree of apoptosis was observed in TAA rats. Animals from the CO group did not present significant morphological changes in hepatic tissue. Rats with cirrhosis (TAA) showed a final significant lower (p < 0.001) body weight (BW) than control animals (TAA: 296 ± 4.6 g; CO: 442.7 ± 11.7 g). Liver weight (LW) is also inferior (p < 0.001) in the TAA group in relation to CO group (TAA: 9.9 ± 0.3; CO: 13.1 ± 0.6 g). The ratio of LW to BW (LW/BW × 100) underwent a statistically significant increase (p = 0.034) in TAA rats compared to the CO animals (TAA: 3.35 ± 0.1; CO: 2.97 ± 0.1). However, there were no differences between the animals with PCS and the SHAM group regarding their body weight after the 45 days postsurgery (initial BW of the PCS group 238 ± 13.7 g, final BW 205 ± 15; initial BW of the SHAM group 221 ± 5.5 g, final BW 224 ± 18 g). In contrast, the ratio of LW to BW (LW/BW × 100) underwent a statistically significant increase in SHAM rats (p < 0.001) compared to the PCS animals (SHAM: 2.99 ± 0.2; PCS: 1.87 ± 0.1). La ratio of LW to BW (LW/BW × 100) between the SHAM group and the control animals (CO group) revealed no statistically significant differences (p = 0.391). 3.2. Active avoidance The two-way repeated measures ANOVA showed differences in the number of avoidances between PCS and SHAM (F(1,16) = 4.67, p = 0.046), differences between training days (F(4,64) = 25.192, p < 0.001), and a statistically significant interaction of group and training days (F(4,64) = 3.742, p = 0.008). More avoidances were found in SHAM compared to PCS on day 4 (t(16) = −3.438, p = 0.003) and on day 5 (t(16) = −3.364, p = 0.004) (Fig. 1A). SHAM showed an increase in avoidances over days, more avoidances were found on days 5 and 4 compared to days 1, 2 and 3 (p < 0.01), and on day 3 compared to day 1 (p = 0.008), whereas PCS showed more avoidances on days 5, 4 and 3 compared to day 1 (p < 0.008). In the percentage of escapes, ANOVA revealed no differences between PCS and SHAM (F(1,16) = 0.231, p = 0.637) and also no differences between training days (F(4,64) = 1.491, p = 0.215) (Fig. 1B). The ANOVA revealed differences in the number of avoidances shown by the CO and TAA groups (F(1,18) = 6.556, p = 0.02) and differences between learning days (F(4,72) = 24.96, p < 0.001). There was a statistically significant interaction of group and learning days (F(4,72) = 4.949, p = 0.001). Tukey’s test for independent samples showed differences between the CO and TAA groups in the number of avoidances on day 3 (t(18) = −2.135, p = 0.047), day 4 (t(18) = −3.385, p = 0.003) and day 5 (t(18) = −4.434, p < 0.001) (Fig. 1A). A further repeated measures ANOVA revealed that both the CO and TAA groups showed an improvement over days of training (CO: F(4,36) = 33.867, p = 0.001; TAA: F(4,36) = 3.184, F = 0.024). CO showed more avoidances on days 5, 4 and 3 compared to days 1 (p < 0.001) and 2 (p < 0.028), on day 3 compared to day 2 (p = 0.028), and on day 2 compared to day 1 (p = 0.003). TAA showed more avoidances only on day 5 compared to day 1 (p = 0.022). In the number of escapes, ANOVA revealed no differences between TAA and CO (F(1,18) = 0.635, p = 0.436) and no differences between training days (F(4,72) = 1.877, p = 0.124) (Fig. 1B). 3.3. Passive avoidance Analysis of the data showed that there were no differences between groups in the tendency to cross to the dark chamber during the habituation phase (PCS vs. SHAM t(16) = −0.258, p = 0.8; TAA vs. CO t(18) = (1.897, p = 0.074). The two-way repeated measures ANOVA of acquisition and retrieval phases showed that there

Fig. 1. Bar charts represent avoidances (A) and escapes (B) in the active avoidance task during 5 training days of SHAM, PCS, CO and TAA groups (mean ± SEM). The SHAM group presented a better acquisition with more avoidances than PCS. SHAM vs. PCS on day 4 (*t(16) = −3.438, p = 0.003) and on day 5 (**t(16) = −3.364, p = 0.004). Differences between CO and TAA groups in the number of avoidances were on day 3 (# t(18) = −2.135, p = 0.047), day 4 (## t(18) = −3.385, p = 0.003) and day 5 (### t(18) = −4.434, p < 0.001).

were differences between PCS and SHAM groups (F(1,16) = 16.866, p < 0.001). Differences between acquisition and retrieval were found (F(1,16) = 49.92, p < 0.001) with a significant interaction effect between groups and session (F(1,16) = 10.869, p = 0.005). The differences between groups were present in the retrieval session. The PCS group, in contrast to the SHAM, showed similar latencies during retrieval and acquisition trials (SHAM: t(8) = (9.146, p < 0.001; PCS: t(8) = -2.287, p = 0.052) (Fig. 2). CO and TAA were similar in the task (F(1,18) = 0.228, p = 0.638). However, differences between acquisition and retrieval were found (F(1,18) = 109.105, p < 0.001), as both CO and TAA showed longer latencies during the retrieval trial compared with acquisition (CO t(9) = −7.774, p < 0.001; TAA t(9) = −7.005, p < 0.001) (Fig. 2). 4. Discussion The procedure used for two-way active avoidance includes multiple trials and sessions, which enable us to evaluate the typical avoidance response and constitutes an excellent learning and memory task. In this procedure, the animal may experience both anxiety and fear because it must initiate a response towards a location where it has previously experienced a noxious stimulus. The response may also be affected by an altered locomotor activity or by shock sensitivity. Increases in the number of avoidances on the different acquisition days can be attributed to processes of consol-

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Fig. 2. Bar charts represent latencies of crossing to the dark chamber during acquisition and retrieval in the step-through passive avoidance task of SHAM, PCS, TAA and CO groups (mean ± SEM). SHAM, CO and TAA displayed longer latencies during the retrieval trial compared to acquisition (*t(8) = −6.987, p < 0.001; # t(9) = −7.774, p < 0.0001 and t(9) = −7.005, p < 0.001, respectively).

idation and recall, and both experimental HE groups, PCS and TAA, showed a clear deficit compared to control groups. The Sham group showed a clear improvement in two-way active avoidance over the training period, and this was significantly greater on days 4 and 5 compared to the first three days, and on day 3 compared to the first day. However, the PCS animals showed a correct acquisition in the first 3 days but after this, they did not show any progress in learning. In fact, on days 4 and 5, the Sham group performed much better than the PCS group in relation to the number of avoidances it made (Fig. 1A). It is interesting to note that there were no differences in the number of escapes made by the Sham group and the PCS group on any of the five days of the test (Fig. 1B). This verifies that the deficit in acquisition of this task is not due to a freezing effect or to a greater tolerance to shock by the PCS group, as some authors have reported a lower reactivity of PCS animals to harmful stimuli [5]. Nor can be attributed to locomotor difficulties in the PCS group, as the hypoactivity described in these animals is more closely related to circadian rhythms than to motor impediments, or differences in anxiety or fear [35,46]. On the other hand, the TAA group showed a similar performance in the two-way active avoidance task to the PCS group, which was much poorer than that of the control group, as can be seen from the learning curve. In fact, the CO group presented a significantly different number of avoidances on days 3, 4 and 5 compared to the TAA group (Fig. 1A). On the other hand, TAA animals did not present any differences compared to the CO group and, as occurred with the PCS group, here also, we rule out differences in sensitivity to harmful stimuli or in the emotional reactivity of the groups, and motor alterations, as already demonstrated in a previous work [35]. Step-through passive avoidance learning is believed to be based on contextual memory, which is associated with the place and the event of being given the electric shock in the dark box. The hippocampus plays an important role in contextual memory; injuries of the hippocampus decrease performance in passive avoidance learning. Step-through passive avoidance learning involves not only contextual memory but also amygdala-dependent emotional memory, fear of the dark box. The performance of passive avoidance learning decreases by defects of either contextual memory or emotional memory. In the step-through passive avoidance task, the PCS group showed poorer recall latencies than the control group, suggesting a deficit in memory processes related to this task (Fig. 2). In contrast, the TAA group had similar latencies to the control group and showed no deficit in this task (Fig. 2). This correct performance of the TAA group in passive avoidance coincides with the data

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reported by Fedosiewicz-Wasiluk et al. [18], who did not observe any alterations in the passive avoidance latency of rats treated with intraperitoneal injections of thioacetamide. The contrast in the performance of the step-through passive avoidance task between rats with PCS and rats with TAA is no novelty, as differential effects have also been observed in the performance of spatial memory tasks in both experimental models of HE [35]. These discrepancies in the capacities of memory and learning between both experimental groups may be due to the pathophysiological differences generated in the two HE models. TAA rats suffer a liver failure secondary to inflammation related to oxygen reactive species, fibrosis and regenerating nodule development. On the contrary, in PCS rats the portal blood deprivation induces a liver atrophy, but the histological alterations shown by the liver of the rats administered TAA are not produced. Secondly, these two experimental models are also different in regards to the portal–systemic collateral circulation that is developed: PCS rats have a total portocaval shunt secondary to the performance of a surgical portacaval anastomosis, so that all the splanchnic blood drains into the systemic circulation. On the contrary, in TAA rats, portalsystemic shunts through the destructured liver parenchyma, that is, intrahepatic portalsystemic shunts, are progressively formed. Therefore, the shunt degree in PCS rats is the highest, extrahepatic and it is suddenly installed, whereas in TAA rats, it is lower, intrahepatic, and progressively developed. For this reason, TAA rats show liver and splanchnic involvement and hyperdynamic circulation whereas in PCS rats, the splanchnic hyperdynamic circulation is established as a consequence of the decreased resistance to the portal blood flow. These contrasts of the hepatic insufficiency generated in both models can produce a different effect on cerebral function and, thus, differentially affect the systems and structures involved in memory and learning. On the other hand, and in contrast to our findings, Bengtsson et al. [5] did not find any deficit in the step-through passive avoidance task by PCS animals. There could be two reasons for this: the evaluation of the performance of PCS animals in the work mentioned was done at a shorter postoperative time (behaviourally evaluated at 15 days postsurgery, compared to the 45 days in our PCS group) and, secondly, the maximum latency allowed to estimate recall in the task was much shorter than ours, because the maximum latency time allowed for the retrieval test was 120 s compared to the 300 s of our behavioural protocol (if we observe Fig. 2, we can see that a maximum latency of 120 s does not allow us to determine the differences between the PCS group and the SHAM group in our experiment). As mentioned previously, the specific causes of cerebral dysfunction in HE have not been clearly established. The increase in ammonia levels in the brain and its toxicity, whether in cirrhotic patients or PCS rats [10,14,48], is one of the most studied hypotheses. Also, the animals treated with TAA show an increased concentration of blood ammonia, as confirmed by our data and that of other authors [33]. However, postnatal treatment with a diet rich in ammonia did not seem to affect the correct performance of the avoidance task or the step-through passive avoidance [1]. It, therefore, seems that the rise in cerebral ammonia alone cannot explain the deficit in associative learning in TAA or PCS animals. Nevertheless, ammonia can collaborate in the alteration of the cerebral neurotransmission systems and thus, contribute to the development of the neurological and cognitive symptoms observed in HE. Thus, it is well known that cerebral histamine is highly increased both in rats with PCS and in patients with hepatic cirrhosis [20,30,31]. This activation of the histaminergic system could, therefore, contribute to the development of the neuropsychiatric symptoms observed in HE, such as sleep disturbances and altered circadian rhythms [31]. In fact, the use of pyrilamine, an H1 receptor antagonist, restores circadian rhythmicity and feeding in rats with

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PCS [32], and the use of a blocker of H1 receptors, hydroxyzine, improves the sleeping-waking cycle in patients with minimal HE [47]. However, these alterations in the histaminergic system could also contribute to the cognitive alterations observed in HE. The deficit in associative learning observed in PCS rats may be due to alterations in the histaminergic system [46], whereas there are no known data about the dysfunctions of cerebral neurotransmission in animals with TAA. Rubio et al. [44] inhibited the release and synthesis of histamine by H3 receptor agonists and observed a rise in two-way active avoidance. Also, the administration of l-histidine hindered a correct acquisition in the avoidance task. Similarly, other authors [21,45] observed an improved performance of the active avoidance task in the long-term after damage to the tuberomammillary nucleus, the only producer of histamine in the brain [24]. An increase in recall latencies was also observed when H1 and H2 receptor antagonists were used [15,22,57] or a reduction in these as cerebral histamine rises [58]. Hence, one could deduce that the deficit showed by PCS animals in the step-through passive avoidance could be facilitated by the action of histamine on the basolateral amygdala. This action consists of modulating the release of acetylcholine and thus participating in emotional memory [41]. The blockage of H3 histaminergic receptors increases the release of histamine, which acts on postsynaptic H2 receptors, inhibiting the release of acetylcholine from basolateral amygdala and impairing retention of emotional memory [11]. However, it has been noted that the disturbance of the amygdala or of the ventral hippocampus-amygdala pathway interferes with learning an avoidance task [34]. This might explain the deficit observed in the PCS animals, because the histamine receptors located in the ventral hippocampus modulate learning and memory processes. The administration of histamine in the ventral hippocampus 15 min after the acquisition of a conditioned avoidance task interferes with the process of consolidation, affecting the recall of the conditioned response on the following day [3]. In contrast, it is also known that hyperammonemia and liver failure alter different steps of the glutamatergic neurotransmission, and that glutamatergic neurotransmission plays an important role modulating learning and memory and motor function. In this sense, a dysfunction of the glutamatergic neurotransmission could contribute to the deficit in emotional memory involved in active avoidance. Increases in this neurotransmitter have been observed in the extracellular space, and also alterations related to its reuptake and its receptors [25,36]. More specifically, a reduction in NMDA and non-NMDA receptors was observed both in animals with PCS and in rat models of acute liver failure [13,42]. Moreover, it is also known that the conditioned stimulus information from the thalamus, hippocampus, and several cortical regions reaches the basolateral amygdala via glutamatergic projections, and that the administration of NMDA antagonists to the amygdala prevents the acquisition of fear conditioning [17]. Along these lines, Alvarez and Banzan [2] have observed that the administration of glutamic acid in the ventral hippocampus inhibits active avoidance acquisition, and that the effect was antagonized by AP7, the NMDA-glutamate receptor antagonist, and increased by AP3, the metabotropic glutamate receptor antagonist, which suggests that the NMDA-glutamate receptor inhibits the learning of active avoidance. Our data show that both portocaval shunt and thioacetamideinduced cirrhosis impair performance in associative learning tasks with emotional contents, possibly mediated by alterations in neurotransmission systems observed in HE. In previous works, it was reported that animals with PCS or TAA showed deficit in the acquisition of a spatial reference memory task in the round pool, a test frequently used to evaluate the hippocampus-dependent declarative memory [35]. In this work, we have shown a deficit

of both experimental HE groups in two-way active avoidance, a nondeclarative memory task independent of the hippocampus, as demonstrated by diverse studies [23,49], but a task that does require activation of the amygdala to modulate the consolidation of the memory that takes place in other brain regions [34]. In conclusion, these deficits confirm the validity of both models to study the cognitive alterations observed in human HE and complement the alterations found in other memory tests [16,39,55]. Likewise, performance of the step-through passive avoidance task, in which only the PCS group showed deficits, seems to indicate that the hepatic insufficiency produced in both models of HE has differential effects on the cerebral function, which may of great interest for future studies in the field of experimental HE. Acknowledgements ˜ Valdes for We would like to thank Piedad Burgos and Begona their technical assistance and Virginia Navascués Howard and Caroline Coope for revising the English text of this manuscript. This research was supported by grant SEJ2005-05067 from the Ministry of Science and Technology of Spain. References ˜ [1] Aguilar MA, Minarro J, Felipo V. Chronic moderate hyperammonemia impairs active and passive avoidance behavior and conditional discrimination learning in rats. Exp Neurol 2000;161:704–13. [2] Alvarez EO, Banzan AM. Ventral hippocampal glutamate receptors in the rat: possible involvement in learning mechanisms of an active avoidance response. J Neural Transm 1999;106:987–1001. [3] Alvarez EO, Banzan AM. The activation of histamine-sensitive sites of the ventral hippocampus modulates the consolidation of a learned active avoidance response in rats. Behav Brain Res 2008;189:92–9. [4] Amodio P, Schiff S, Del Piccolo F, Mapelli D, Gatta A, Umilta C. Attention dysfunction in cirrhotic patients: an inquiry on the role of executive control, attention orienting and focusing. Metab Brain Dis 2005;20:115–27. [5] Bengtsson F, Nobin A, Falck B, Gage FH, Jeppsson B. Portacaval shunt in the rat: selective alterations in behavior and brain serotonin. Pharmacol Biochem Behav 1986;24:1611–6. [6] Bhatnagar A, Majumdar S. Animal models of hepatic encephalopathy. Indian J Gastroenterol 2003;22:S33–6. [7] Blei AT, Córdoba J. Hepatic encephalopathy. Am J Gastroenterol 2001;96:1968–76. [8] Blei AT, Omary R, Butterworth RF. Animal models of hepatic encephalopathies. In: Bouton A, Baker B, Butterworth RF, editors. Animal models of neurological disease. II. Neuromethods, vol. 2. Clifton, New Jersey: The Humana Press Clifton; 1992. p. 183–222. [9] Butterworth RF. Pathogenesis of hepatic encephalopathy: new insights from neuroimaging and molecular studies. J Hepatol 2003;39:278–85. [10] Butterworth RF. The neurobiology of hepatic encephalopathy. Semin Liver Dis 1996;16:235–44. [11] Cangioli I, Baldi E, Mannaioni PF, Bucherelli C, Blandina P, Passani MB. Activation of histaminergic H3 receptors in the rat basolateral amygdala improves expression of fear memory and enhances acetylcholine release. Eur J Neurosci 2002;16:521–8. [12] Chamuleau RA. Animal models of hepatic encephalopathy. Semin Liver Dis 1996;16:265–70. [13] Chan H, Butterworth RF. Cell-selective effects of ammonia on glutamate transporter and receptor function in the mammalian brain. Neurochem Int 2003;43:525–32. [14] Ehrlich M, Plum F, Duffy TE. Blood and brain ammonia concentrations after portacaval anastomosis. Effects of acute ammonia loading. J Neurochem 1980;34:1538–42. [15] Eidi M, Zarrindast MR, Eidi A, Oryan S, Parivar K. Effects of histamine and cholinergic systems on memory retention of passive avoidance learning in rats. Eur J Pharmacol 2003;465:91–6. [16] Erceg S, Monfort P, Hernandez-Viadel M, Rodrigo R, Montoliu C, Felipo V. Oral administration of sildenafil restores learning ability in rats with hyperammonemia and with portacaval shunts. Hepatology 2005;41:299–306. [17] Fanselow MS, Kim JJ. Acquisition of contextual Pavlovian fear conditioning is blocked by application of an NMDA receptor antagonist d,l-2-amino5-phosphonovaleric acid to the basolateral amygdale. Behav Neurosci 1994;108:210–2. [18] Fedosiewicz-Wasiluk M, Holy ZZ, Wisniewska RJ, Wisniewski K. The influence of NMDA, a potent agonist of glutamate receptor, on behavioral activity of rats with experimental hyperammonemia evoked by liver failure. Amino Acids 2005;28:111–7. [19] Ferenci P, Lockwood A, Mullen K, Tarter R, Weissenborn K, Blei AT. Hepatic encephalopathy. Definition, nomenclature, diagnosis and quantification. Final

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