Hyperammonemia Increases GABAergic Tone in the Cerebellum but Decreases It in the Rat Cortex  Omar Cauli, Mohammad T. Mansouri, Ana Agusti, Vicente Felipo  Gastroenterology  Volume 136, Issue 4, Pages 1359-1367.e2 (April 2009) DOI: 10.1053/j.gastro.2008.12.057 Copyright © 2009 AGA Institute Terms and Conditions

Figure 1 Differential effects of activation or blockade of GABAA receptors on extracellular cGMP in cerebellum in vivo in control and hyperammonemic rats. Microdialysis probes were inserted in the cerebellum of control or hyperammonemic rats, perfused at 3 μL/min, and samples were taken every 30 minutes. After taking 5 samples to determine basal levels of cGMP, muscimol 2 or 10 μmol/L or bicuculline 50 μmol/L was administered in the perfusion stream, and cGMP was determined in the following 2 samples. Values are the mean ± SEM from 7 rats per group. Values significantly different (P < .05) from basal cGMP are indicated by asterisks. Values that are significantly different (P < .05) from control rats are indicated by “a.” Gastroenterology 2009 136, 1359-1367.e2DOI: (10.1053/j.gastro.2008.12.057) Copyright © 2009 AGA Institute Terms and Conditions

Figure 2 Differential effects of activation of GABAA receptors with muscimol on the function of the glutamate-NO-cGMP pathway in cerebellum in vivo in control and hyperammonemic rats. Microdialysis probes were inserted in the cerebellum of control or hyperammonemic rats, perfused at 3 μL/min, and samples were taken every 30 minutes. After taking 5 samples to determine basal levels of cGMP, muscimol 2 μmol/L (B) or 10 μmol/L (C) was administered in the perfusion stream as indicated by the horizontal bars. At the time indicated by the short horizontal bar, NMDA (0.3 mmol/L) was administered for 30 minutes to activate the glutamate-NO-cGMP pathway. cGMP was determined. (A) The effects of NMDA without pretreatment with muscimol. Values are given as percentage of basal and are the mean ± SEM from 7 rats per group. Values significantly different (P < .05) from basal cGMP are indicated by asterisks. Values significantly different (P < .05) from control rats are indicated by “a.” Gastroenterology 2009 136, 1359-1367.e2DOI: (10.1053/j.gastro.2008.12.057) Copyright © 2009 AGA Institute Terms and Conditions

Figure 3 Blocking GABAA receptors with bicuculline increases extracellular cGMP in cerebellum in vivo in hyperammonemic but not in control rats. Experiments were performed as in Figure 2, but bicuculline 50 μmol/L was administered instead of muscimol. Values are given as percentage of basal and are the mean ± SEM from 7 rats per group. Values significantly different (P < .05) from basal cGMP are indicated by asterisks. Values significantly different (P < .05) from control rats are indicated by “a.” Gastroenterology 2009 136, 1359-1367.e2DOI: (10.1053/j.gastro.2008.12.057) Copyright © 2009 AGA Institute Terms and Conditions

Figure 4 Differential effects of activation or blockade of GABAA receptors on extracellular cGMP in cerebral cortex in vivo in control and hyperammonemic rats. Microdialysis probes were inserted in the cerebral cortex of control or hyperammonemic rats, perfused at 3 μL/min, and samples were taken every 30 minutes. After taking 5 samples to determine basal levels of cGMP, muscimol 10 μmol/L or bicuculline 50 μmol/L was administered in the perfusion stream, and cGMP was determined in the following 2 samples. Values are the mean ± SEM from 7 rats per group. Values significantly different (P < .05) from basal cGMP are indicated by asterisks. Values significantly different (P < .05) from control rats are indicated by “a.” Gastroenterology 2009 136, 1359-1367.e2DOI: (10.1053/j.gastro.2008.12.057) Copyright © 2009 AGA Institute Terms and Conditions

Figure 5 Chronic treatment with bicuculline increases extracellular cGMP in cerebellum of hyperammonemic rats. Control or hyperammonemic rats were treated chronically with bicuculline (IP, 1 mg/kg per day) or with saline. After 18 days, extracellular cGMP was determined in cerebellum by microdialysis. Values are the mean ± SEM from 8 rats per group. Values significantly different (P < .05) from control rats are indicated by “a.” Values significantly different (P < .05) from rats not treated with bicuculline are indicated by asterisks. Gastroenterology 2009 136, 1359-1367.e2DOI: (10.1053/j.gastro.2008.12.057) Copyright © 2009 AGA Institute Terms and Conditions

Figure 6 Chronic treatment with bicuculline restores learning ability in hyperammonemic rats. Control and hyperammonemic rats treated chronically with bicuculline (IP; 0.01, 0.1, 0.3, or 1 mg/kg per day) or with saline were subjected to the conditional discrimination learning test in the Y-maze. The tests were initiated after 10 days of treatment. Values are the mean ± SEM of 7 rats per group for rats treated with bicuculline and 20 rats per group for control or hyperammonemic rats injected with saline. Values are given as the number of trials needed to learn. Values significantly different from control rats injected with saline are indicated by asterisks. Values significantly different from hyperammonemic rats injected with saline are indicated by “#.” Values significantly different in control and hyperammonemic rats injected with the same dose of bicuculline are indicated by “a.” *P < .05, **P < .01, #P < .05, aP < .05. Gastroenterology 2009 136, 1359-1367.e2DOI: (10.1053/j.gastro.2008.12.057) Copyright © 2009 AGA Institute Terms and Conditions

Supplementary Figure 1 Effects of hyperammonemia on the content of GABAA receptor subunits in cerebellum and cerebral cortex. The contents of GABAA receptor subunits α1, α4, α6, and γ2 were determined in cerebellum and cerebral cortex of control (C) and hyperammonemic (HA) rats by immunoblotting as indicated in methods. α4 Was not detected in cerebellum, and α6 was not detected in cortex. Representative immunoblottings are shown. The intensities of the bands were quantified and expressed as percentage of controls. Values are given in Supplementary Table 1. Gastroenterology 2009 136, 1359-1367.e2DOI: (10.1053/j.gastro.2008.12.057) Copyright © 2009 AGA Institute Terms and Conditions