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Hyperammonemic rats show peripheral inflammation, increased GABAergic neurotransmission and neuroinflammation in cerebellum and hippocampus which induce motor incoordination and cognitive impairment. Neuroinflammation enhances GABAergic neurotransmission in cerebellum by enhancing the TNFR1-glutaminase-GAT3 and TNFR1-CCL2-TrkB-KCC2 pathways. Golexanolone reduces GABARats were treated with golexanolone and effects on peripheral inflammation, neuroinflammation, TNFR1-glutaminase-GAT3 and TNFR1-CCL2-TrkB-KCC2 pathways, and cognitive and motor function were analyzed.Hyperammonemic rats show increased TNFα and reduced IL-10 in plasma, microglia and astrocytes activation in cerebellum and hippocampus, and impaired motor coordination and spatial and short-term memories. Treating hyperammonemic rats with golexanolone reversed changes in peripheral inflammation, microglia and astrocytes activation and restored motor coordination and spatial and short-term memory. This was associated with reversal of the hyperammonemia-enhanced activation in cerebellum of the TNFR1-glutaminase-GAT3 and TNFR1-CCL2-TrkB-KCC2 pathways.Reducing GABA

Background Patients with liver cirrhosis may develop hepatic encephalopathy. Rats with chronic hyperammonemia exhibit neurological alterations mediated by peripheral inflammation and neuroinflammation. Motor incoordination is due to increased TNF-a levels and activation of its receptor TNFR1 in the cerebellum. The aims were to assess (a) whether peripheral inflammation is responsible for TNF-a induction in hyperammonemic rats, (b) the cell type(s) in which TNF-a is increased, (c) whether this increase is associated with increased nuclear NF-κB and TNFR1 activation, (d) the time course of TNF-a induction, and (e) if TNF-a is induced in the Purkinje neurons of patients who die with liver cirrhosis. Methods We analyzed the level of TNF-a mRNA and NF-κB in microglia, astrocytes, and Purkinje neurons in the cerebellum after 1, 2, and 4 weeks of hyperammonemia. We assessed whether preventing peripheral inflammation by administering an anti-TNF-a antibody prevents TNF-a induction. We tested whether TNF-a induction is reversed by R7050, which inhibits the TNFR1-NF-κB pathway, in ex vivo cerebellar slices. Results Hyperammonemia induced microglial and astrocyte activation at 1 week. This was followed by TNF-a induction in both glial cell types at 2 weeks and in Purkinje neurons at 4 weeks. The level of TNF-a mRNA increased in parallel with the TNF-a protein level, indicating that TNF-a was synthesized in Purkinje cells. This increase was associated with increased NF-κB nuclear translocation. The nuclear translocation of NF-κB and the increase in TNF-a were reversed by R7050, indicating that they were mediated by the activation of TNFR1. Preventing peripheral inflammation with an anti-TNF-a antibody prevents TNF-a induction. Conclusion Sustained (4 weeks) but not short-term hyperammonemia induces TNF-a in Purkinje neurons in rats. This is mediated by peripheral inflammation. TNF-a is also increased in the Purkinje neurons of patients who die with liver cirrhosis. The results suggest that hyperammonemia induces TNF-a in glial cells and that TNF-a released by glial cells activates TNFR1 in Purkinje neurons, leading to NF-κB nuclear translocation and the induction of TNF-a expression, which may contribute to the neurological alterations observed in hyperammonemia and hepatic encephalopathy.

Rats with chronic hyperammonemia reproduce the cognitive and motor impairment present in patients with hepatic encephalopathy. It has been proposed that enhanced GABAergic neurotransmission in hippocampus may contribute to impaired learning and memory in hyperammonemic rats. However, there are no direct evidences of the effects of hyperammonemia on GABAergic neurotransmission in hippocampus or on the underlying mechanisms. The aims of this work were to assess if chronic hyperammonemia enhances the function of GABA(A) receptors in hippocampus and to identify the underlying mechanisms. Activation of GABA(A) receptors is enhanced in hippocampus of hyperammonemic rats, as analyzed in a multielectrode array system. Hyperammonemia reduces membrane expression of the GABA transporters GAT1 and GAT3, which is associated with increased extracellular GABA concentration. Hyperammonemia also increases gephyrin levels and phosphorylation of the beta 3 subunit of GABA(A) receptor, which are associated with increased membrane expression of the GABA(A) receptor subunits alpha 1, alpha 2, gamma 2, beta 3, and delta. Enhanced levels of extracellular GABA and increased membrane expression of GABA(A) receptors would be responsible for the enhanced GABAergic neurotransmission in hippocampus of hyperammonemic rats. Increasing extracellular cGMP reverses the increase in GABA(A) receptors activation by normalizing the membrane expression of GABA transporters and GABA(A) receptors. The increased GABAergic neurotransmission in hippocampus would contribute to cognitive impairment in hyperammonemic rats. The results reported suggest that reducing GABAergic tone in hippocampus by increasing extracellular cGMP or by other means may be useful to improve cognitive function in hyperammonemia and in cirrhotic patients with minimal or clinical hepatic encephalopathy.

Aims Chronic hyperammonaemia and inflammation synergistically induce neurological impairment, including motor incoordination, in hepatic encephalopathy. Hyperammonaemic rats show neuroinflammation in the cerebellum which enhances GABAergic neurotransmission leading to motor incoordination. We aimed to identify underlying mechanisms. The aims were (1) to assess if S1PR2 is involved in microglial and astrocytic activation in the cerebellum of hyperammonaemic rats; (2) to identify pathways by which enhanced S1PR2 activation induces neuroinflammation and alters neurotransmission; (3) to assess if blocking S1PR2 reduces neuroinflammation and restores motor coordination in hyperammonaemic rats. Methods We performed ex vivo studies in cerebellar slices from control or hyperammonaemic rats to identify pathways by which neuroinflammation enhances GABAergic neurotransmission in hyperammonaemia. Neuroinflammation and neurotransmission were assessed by immunochemistry/immunofluorescence and western blot. S1PR2 was blocked by intracerebral treatment with JTE-013 using osmotic mini-pumps. Motor coordination was assessed by beam walking. Results Chronic hyperammonaemia enhances S1PR2 activation in the cerebellum by increasing its membrane expression. This increases CCL2, especially in Purkinje neurons. CCL2 activates CCR2 in microglia, leading to microglial activation, increased P2X4 membrane expression and BDNF in microglia. BDNF enhances TrkB activation in neurons, increasing KCC2 membrane expression. This enhances GABAergic neurotransmission, leading to motor incoordination in hyperammonaemic rats. Blocking S1PR2 in hyperammonaemic rats by intracerebral administration of JTE-013 normalises the S1PR2-CCL2-CCR2-BDNF-TrkB-KCC2 pathway, reduces glial activation and restores motor coordination in hyperammonaemic rats. Conclusions Enhanced S1PR2-CCL2-BDNF-TrkB pathway activation mediates neuroinflammation and incoordination in hyperammonaemia. The data raise a promising therapy for patients with hepatic encephalopathy using compounds targeting this pathway.

In patients with liver cirrhosis, minimal hepatic encephalopathy (MHE) is triggered by a shift in peripheral inflammation, promoting lymphocyte infiltration into the brain. Rifaximin improves neurological function in MHE by normalizing peripheral inflammation. Patients who died with steatohepatitis showed T-lymphocyte infiltration and neuroinflammation in the cerebellum, suggesting that MHE may already occur in these patients. The aims of this work were to assess, in a rat model of mild liver damage similar to steatohepatitis, whether: (1) the rats show impaired motor coordination in the early phases of liver damage, (2) this is associated with changes in the immune system and infiltration of immune cells into the brain, and (3) rifaximin improves motor incoordination, associated with improved peripheral inflammation, reduced infiltration of immune cells and neuroinflammation in the cerebellum, and restoration of the alterations in neurotransmission. Liver damage was induced by carbon tetrachloride (CCl4) injection over four weeks. Peripheral inflammation, immune cell infiltration, neuroinflammation, and neurotransmission in the cerebellum and motor coordination were assessed. Mild liver damage induces neuroinflammation and altered neurotransmission in the cerebellum and motor incoordination. These alterations are associated with increased TNFa, CCL20, and CX3CL1 in plasma and cerebellum, IL-17 and IL-15 in plasma, and CCL2 in cerebellum. This promotes T-lymphocyte and macrophage infiltration in the cerebellum. Early treatment with rifaximin prevents the shift in peripheral inflammation, immune cell infiltration, neuroinflammation, and motor incoordination. This report provides new clues regarding the mechanisms of the beneficial effects of rifaximin, suggesting that early rifaximin treatment could prevent neurological impairment in patients with steatohepatitis.

Background: Hyperammonemia is a main contributor to minimal hepatic encephalopathy (MHE) in cirrhotic patients. Hyperammonemic rats reproduce the motor incoordination of MHE patients, which is due to enhanced GABAergic neurotransmission in the cerebellum as a consequence of neuroinflammation. In hyperammonemic rats, neuroinflammation increases BDNF by activating the TNFR1–S1PR2–CCR2 pathway. (1) Identify mechanisms enhancing GABAergic neurotransmission in hyperammonemia; (2) assess the role of enhanced activation of TrkB; and (3) assess the role of the TNFR1–S1PR2–CCR2–BDNF pathway. In the cerebellum of hyperammonemic rats, increased BDNF levels enhance TrkB activation in Purkinje neurons, leading to increased GAD65, GAD67 and GABA levels. Enhanced TrkB activation also increases the membrane expression of the γ2, α2 and β3 subunits of GABAA receptors and of KCC2. Moreover, enhanced TrkB activation in activated astrocytes increases the membrane expression of GAT3 and NKCC1. These changes are reversed by blocking TrkB or the TNFR1–SP1PR2–CCL2–CCR2–BDNF–TrkB pathway. Hyperammonemia-induced neuroinflammation increases BDNF and TrkB activation, leading to increased synthesis and extracellular GABA, and the amount of GABAA receptors in the membrane and chloride gradient. These factors enhance GABAergic neurotransmission in the cerebellum. Blocking TrkB or the TNFR1–SP1PR2–CCL2–CCR2–BDNF–TrkB pathway would improve motor function in patients with hepatic encephalopathy and likely with other pathologies associated with neuroinflammation.

Cognitive and motor impairment in minimal hepatic encephalopathy (MHE) are mediated by neuroinflammation, which is induced by hyperammonemia and peripheral inflammation. GABAergic neurotransmission in the cerebellum is altered in rats with chronic hyperammonemia. The mechanisms by which hyperammonemia induces neuroinflammation remain unknown. We hypothesized that GABAA receptors can modulate cerebellar neuroinflammation. The GABAA antagonist bicuculline was administrated daily (i.p.) for four weeks in control and hyperammonemic rats. Its effects on peripheral inflammation and on neuroinflammation as well as glutamate and GABA neurotransmission in the cerebellum were assessed. In hyperammonemic rats, bicuculline decreases IL-6 and TNFα and increases IL-10 in the plasma, reduces astrocyte activation, induces the microglia M2 phenotype, and reduces IL-1β and TNFα in the cerebellum. However, in control rats, bicuculline increases IL-6 and decreases IL-10 plasma levels and induces microglial activation. Bicuculline restores the membrane expression of some glutamate and GABA transporters restoring the extracellular levels of GABA in hyperammonemic rats. Blocking GABAA receptors improves peripheral inflammation and cerebellar neuroinflammation, restoring neurotransmission in hyperammonemic rats, whereas it induces inflammation and neuroinflammation in controls. This suggests a complex interaction between GABAergic and immune systems. The modulation of GABAA receptors could be a suitable target for improving neuroinflammation in MHE.

Patients with liver cirrhosis may develop covert or minimal hepatic encephalopathy (MHE). Hyperammonemia (HA) and peripheral inflammation play synergistic roles in inducing the cognitive and motor alterations in MHE. The cerebellum is one of the main cerebral regions affected in MHE. Rats with chronic HA show some motor and cognitive alterations reproducing neurological impairment in cirrhotic patients with MHE. Neuroinflammation and altered neurotransmission and signal transduction in the cerebellum from hyperammonemic (HA) rats are associated with motor and cognitive dysfunction, but underlying mechanisms are not completely known. The aim of this work was to use a multi-omic approach to study molecular alterations in the cerebellum from hyperammonemic rats to uncover new molecular mechanisms associated with hyperammonemia-induced cerebellar function impairment. We analyzed metabolomic, transcriptomic, and proteomic data from the same cerebellums from control and HA rats and performed a multi-omic integrative analysis of signaling pathway enrichment with the PaintOmics tool. The histaminergic system, corticotropin-releasing hormone, cyclic GMP-protein kinase G pathway, and intercellular communication in the cerebellar immune system were some of the most relevant enriched pathways in HA rats. In summary, this is a good approach to find altered pathways, which helps to describe the molecular mechanisms involved in the alteration of brain function in rats with chronic HA and to propose possible therapeutic targets to improve MHE symptoms.

Hyperammonemia contributes to altered neurotransmission and cognition in patients with hepatic encephalopathy. Hyperammonemia in rats affects differently high- and low-affinity AMPA receptors (AMPARs) in cerebellum. We hypothesized that hyperammonemia would alter differently membrane expression of AMPARs GluA1 and GluA2 subunits by altering its phosphorylation. This work aims were: 1) assess if hyperammonemia alters GluA1 and GluA2 subunits membrane expression in cerebellum and 2) analyze the underlying mechanisms. Hyperammonemia reduces membrane expression of GluA2 and enhances membrane expression of GluA1 in vivo. We show that changes in GluA2 and GluA1 membrane expression in hyperammonemia would be due to enhanced NMDA receptors activation which reduces cGMP levels and phosphodiesterase 2 (PDE2) activity, resulting in increased cAMP levels. This leads to increased protein kinase A (PKA) activity which activates phospholipase C (PLC) and protein kinase C (PKC) thus increasing phosphorylation of GluA2 in Ser880, which reduces GluA2 membrane expression, and phosphorylation of GluA1 in Ser831, which increases GluA1 membrane expression. Blocking NMDA receptors or inhibiting PKA, PLC or PKC normalizes GluA2 and GluA1 phosphorylation and membrane expression in hyperammonemic rats. Altered GluA2 and GluA1 membrane expression would alter signal transduction which may contribute to cognitive and motor alterations in hyperammonemia and hepatic encephalopathy.

Minimal hepatic encephalopathy (MHE) is a neuropsychiatric syndrome produced by central nervous system dysfunction subsequent to liver disease. Hyperammonemia and inflammation act synergistically to alter neurotransmission, leading to the cognitive and motor alterations in MHE, which are reproduced in rat models of chronic hyperammonemia. Patients with MHE show altered functional connectivity in different neural networks and a reduced response in the cognitive potential mismatch negativity (MMN), which correlates with attention deficits. The mechanisms by which MMN is altered in MHE remain unknown. The objectives of this work are as follows: To assess if rats with chronic hyperammonemia reproduce the reduced response in the MMN found in patients with MHE. Analyze the functional connectivity between the areas (CA1 area of the dorsal hippocampus, prelimbic cortex, primary auditory cortex, and central inferior colliculus) involved in the generation of the MMN and its possible alterations in hyperammonemia. Granger causality analysis has been applied to detect the net flow of information between the population neuronal activities recorded from a local field potential approach. Analyze if altered MMN response in hyperammonemia is associated with alterations in glutamatergic and GABAergic neurotransmission. Extracellular levels of the neurotransmitters and/or membrane expression of their receptors have been analyzed after the tissue isolation of the four target sites. The results show that rats with chronic hyperammonemia show reduced MMN response in hippocampus, mimicking the reduced MMN response of patients with MHE. This is associated with altered functional connectivity between the areas involved in the generation of the MMN. Hyperammonemia also alters membrane expression of glutamate and GABA receptors in hippocampus and reduces the changes in extracellular GABA and glutamate induced by the MMN paradigm of auditory stimulus in hippocampus of control rats. The changes in glutamatergic and GABAergic neurotransmission and in functional connectivity between the brain areas analyzed would contribute to the impairment of the MMN response in rats with hyperammonemia and, likely, also in patients with MHE.

Patients with hepatic encephalopathy (HE) show working memory and visuo-spatial orientation deficits. Hyperammonemia is a main contributor to cognitive impairment in HE. Hyperammonemic rats show impaired spatial learning and learning ability in the Y maze. Intracerebral administration of extracellular cGMP restores learning in the Y-maze. The underlying mechanisms remain unknown. It also remains unknown whether extracellular cGMP improves neuroinflammation or restores spatial learning in hyperammonemic rats and if it affects differently reference and working memory. The aims of this work were: Spatial working and reference memory were assessed using the radial and Morris water mazes and neuroinflammation by immunohistochemistry and Western blot. Membrane expression of NMDA and AMPA receptor subunits was analyzed using the BS3 crosslinker. Extracellular cGMP was administered intracerebrally using osmotic minipumps. Chronic hyperammonemia induces neuroinflammation in hippocampus, with astrocytes activation and increased IL-1β, which are associated with increased NMDA receptors membrane expression and impaired working memory. This process is not affected by extracellular cGMP. Hyperammonemia also activates microglia and increases TNF-α, alters membrane expression of AMPA receptor subunits (increased GluA1 and reduced GluA2) and impairs reference memory. All these changes are reversed by extracellular cGMP. These results show that extracellular cGMP modulates spatial reference memory but not working memory. This would be mediated by modulation of TNF-α levels and of membrane expression of GluA1 and GluA2 subunits of AMPA receptors.

Exposure to low levels of environmental contaminants, including pesticides, induces neurodevelopmental toxicity. Environmental and food contaminants can reach the brain of the fetus, affecting brain development and leading to neurological dysfunction. The pesticide endosulfan is a persistent pollutant, and significant levels still remain detectable in the environment although its use is banned in some countries. In rats, endosulfan exposure during brain development alters motor activity, coordination, learning, and memory, even several months after uptake, and does so in a sex-dependent way. However, the molecular mechanisms driving these effects have not been studied in detail. In this work, we performed a multiomics study in cerebellum from rats exposed to endosulfan during embryonic development. Pregnant rats were orally exposed to a low dose (0.5 mg/kg) of endosulfan, daily, from gestational day 7 to postnatal day 21. The progeny was evaluated for cognitive and motor functions at adulthood. Expression of messenger RNA and microRNA genes, as well as protein and metabolite levels, were measured on cerebellar samples from males and females. An integrative analysis was conducted to identify altered processes under endosulfan effect. Effects between males and females were compared. Pathways significantly altered by endosulfan exposure included the phosphatidylinositol signaling system, calcium signaling, the cGMP-PKG pathway, the inflammatory and immune system, protein processing in the endoplasmic reticulum, and GABA and taurine metabolism. Sex-dependent effects of endosulfan in the omics results that matched sex differences in cognitive and motor tests were found. These results shed light on the molecular basis of impaired neurodevelopment and contribute to the identification of new biomarkers of neurotoxicity.

Background & Aims Chronic hyperammonemia induces neuroinflammation which mediates cognitive impairment. How hyperammonemia induces neuroinflammation remains unclear. We aimed to assess whether: chronic hyperammonemia induces peripheral inflammation, and whether this then contributes to neuroinflammation, altered neurotransmission and impaired spatial learning — before assessing whether this neuroinflammation and impairment is reversible following hyperammonemia elimination or treatment of peripheral inflammation with anti-TNF-α. Methods Chronic hyperammonemia was induced by feeding rats an ammonia-containing diet. Peripheral inflammation was analyzed by measuring PGE2, TNF-α, IL-6 and IL-10. We tested whether chronic anti-TNF-α treatment improves peripheral inflammation, neuroinflammation, membrane expression of glutamate receptors in the hippocampus and spatial learning. Results Hyperammonemic rats show a rapid and reversible induction of peripheral inflammation, with increased pro-inflammatory PGE2, TNF-α and IL-6, followed at around 10 days by reduced anti-inflammatory IL-10. Peripheral anti-TNF-α treatment prevents peripheral inflammation induction and the increase in IL-1b and TNF-α and microglia activation in hippocampus of the rats, which remain hyperammonemic. This is associated with prevention of the altered membrane expression of glutamate receptors and of the impairment of spatial memory assessed in the radial and Morris water mazes. Conclusions This report unveils a new mechanism by which chronic hyperammonemia induces neurological alterations: induction of peripheral inflammation. This suggests that reducing peripheral inflammation by safe procedures would improve cognitive function in patients with minimal hepatic encephalopathy. Lay summary This article unveils a new mechanism by which chronic hyperammonemia induces cognitive impairment in rats: chronic hyperammonemia per se induces peripheral inflammation, which mediates many of its effects on the brain, including induction of neuroinflammation, which alters neurotransmission, leading to cognitive impairment. It is also shown that reducing peripheral inflammation by treating rats with anti-TNF-α, which does not cross the blood-brain barrier, prevents hyperammonemia-induced neuroinflammation, alterations in neurotransmission and cognitive impairment.

The roles of high- and low-affinity AMPA receptors in modulating extracellular glutamate in the cerebellum remain unclear. Altered glutamatergic neurotransmission is involved in neurological alterations in hyperammonemia, which differently affects high- and low-affinity AMPA receptors. The aims were to assess by in vivo microdialysis (a) the effects of high- and low-affinity AMPA receptor activation on extracellular glutamate in the cerebellum; (b) whether chronic hyperammonemia alters extracellular glutamate modulation by high- and/or low-affinity AMPA receptors; and (c) the contribution of NMDA receptors and EAAC1 transporter to AMPA-induced changes in extracellular glutamate. In control rats, high affinity receptor activation does not affect extracellular glutamate but increases glutamate if NMDA receptors are blocked. Low affinity AMPA receptor activation increases transiently extracellular glutamate followed by reduction below basal levels and return to basal values. The reduction is associated with transient increased membrane expression of EAAC1 and is prevented by blocking NMDA receptors. Blocking NMDA receptors with MK-801 induces a transient increase in extracellular glutamate which is associated with reduced membrane expression of EAAC1 followed by increased membrane expression of the glutamate transporter GLT-1. Chronic hyperammonemia does not affect responses to activation of low affinity AMPA receptors. Activation of high affinity AMPA receptors increases extracellular glutamate in hyperammonemic rats by an NMDA receptor-dependent mechanism. In conclusion, these results show that there is a tightly controlled interplay between AMPA and NMDA receptors and an EAAC1 transporter in controlling extracellular glutamate. Hyperammonemia alters high- but not low-affinity AMPA receptors.

Hyperammonemia is a main contributor to cognitive impairment and motor in-coordination in patients with hepatic encephalopathy. Hyperammonemia-induced neuroinflammation mediates the neurological alterations in hepatic encephalopathy. Intracerebral administration of extracellular cGMP restores some but not all types of cognitive impairment. Motor in-coordination, is mainly due to increased GABAergic tone in cerebellum. We hypothesized that extracellular cGMP would restore motor coordination in hyperammonemic rats by normalizing GABAergic tone in cerebellum and that this would be mediated by reduction of neuroinflammation. The aims of this work were to assess whether chronic intracerebral administration of cGMP to hyperammonemic rats: 1) restores motor coordination: 2) reduces neuroinflammation in cerebellum: 3) reduces extracellular GABA levels and GABAergic tone in cerebellum: and also 4) to provide some advance in the understanding on the molecular mechanisms involved. The results reported show that rats with chronic hyperammonemia show neuroinflammation in cerebellum, including microglia and astrocytes activation and increased levels of IL-1b and TNFa and increased membrane expression of the TNFa receptor. This is associated with increased glutaminase expression and extracellular glutamate, increased amount of the GABA transporter GAT-3 in activated astrocytes, increased extracellular GABA in cerebellum and motor in-coordination. Chronic intracerebral administration of extracellular cGMP to rats with chronic hyperammonemia reduces neuroinflammation, including microglia and astrocytes activation and membrane expression of the TNFa receptor. This is associated with reduced nuclear NF-kappa B, glutaminase expression and extracellular glutamate, reduced amount of the GABA transporter GAT-3 in activated astrocytes and reduced extracellular GABA in cerebellum and restoration of motor coordination. The data support that extracellular cGMP restores motor coordination in hyperammdnemic rats by reducing microglia activation and neuroinflammation, leading to normalization of extracellular glutamate and GABA levels in cerebellum and of motor coordination. (C) 2018 Elsevier Inc. All rights reserved.

γ-Secretase inhibitors (GSIs) are potential therapeutic agents for Alzheimer’s disease (AD); however, trials have proven disappointing. We addressed the possibility that γ-secretase inhibition can provoke a rebound effect, elevating the levels of the catalytic γ-secretase subunit, presenilin-1 (PS1). Acute treatment of SH-SY5Y cells with the GSI LY-374973 (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester, DAPT) augments PS1, in parallel with increases in other γ-secretase subunits nicastrin, presenilin enhancer 2, and anterior pharynx-defective 1, yet with no increase in messenger RNA expression. Over-expression of the C-terminal fragment (CTF) of APP, C99, also triggered an increase in PS1. Similar increases in PS1 were evident in primary neurons treated repeatedly (4 days) with DAPT or with the GSI BMS-708163 (avagacestat). Likewise, rats examined after 21 days administered with avagacestat (40 mg/kg/day) had more brain PS1. Sustained γ-secretase inhibition did not exert a long-term effect on PS1 activity, evident through the decrease in CTFs of APP and ApoER2. Prolonged avagacestat treatment of rats produced a subtle impairment in anxiety-like behavior. The rebound increase in PS1 in response to GSIs must be taken into consideration for future drug development., This study was funded in part by the EU BIOMARKAPD-Joint Programming on Neurodegenerative Diseases (JPND) project by the Instituto de Salud Carlos III (ISCIII grants PI11/03026 and PI15/00665 for JSV and PI14/00566 for MSGA), co-financed by the Fondo Europeo de Desarrollo Regional (FEDER) “Investing in your future,” and through CIBERNED (ISCIII). We also acknowledge financial support from the Spanish Ministerio de Economía y Competitividad, through the “Severo Ochoa” Programme for Centres of Excellence in R&D (SEV-2013-0317).

It has been proposed that developmental exposure to pesticides contributes to increasing prevalence of neurodevelopmental disorders in children, such as attention deficit with hyperactivity (ADHD) and to alterations in coordination skills. However, the mechanisms involved in these alterations remain unclear. We analyzed the effects on spontaneous motor activity and motor coordination of developmental exposure to a representative pesticide of each one of the four main chemical families: organophosphates (chlorpyrifos), carbamates (carbaryl), organochlorines (endosulfan), and pyrethroids (cypermethrin). Pesticides were administered once a day orally, in a sweet jelly, from gestational day 7 to post natal day 21. Spontaneous motor activity was assessed by an actimeter and motor coordination using the rotarod, when rats were adults. The effects were analyzed separately in males and females. Extracellular GABA in cerebellum and NMDA receptor subunits in hippocampus were assessed as possible underlying mechanisms of motor alterations. Motor coordination was impaired by developmental exposure to endosulfan, cypermethrin, and chlorpyrifos in females but not in males. The effect of endosulfan and cypermethrin would be due to increased extracellular GABA in cerebellum, which remains unaltered in male rats. Chlorpyrifos increased motor activity in males and females. Cypermethrin decreased motor activity mainly in males. In male rats, but not in females, expression of the NR2B subunit of NMDA receptor in hippocampus correlated with motor activity. These results show sex-specific effects of different pesticides on motor activity and coordination, associated with neurotransmission alterations. These data contribute to better understand the relationship between developmental exposure to the main pesticide families and motor disorders in children.

Chronic liver disease (e.g. cirrhosis) affects brain function. There is a high incidence of mild cognitive impairment and psychomotor slowing in patients with cirrhosis. This condition, known as minimal hepatic encephalopathy (MHE) affects more than 2 million people in the European Union and has serious health, social and economic consequences. There are no effective treatments for MHE. Rat models of MHE reproduce cognitive and motor alterations seen in patients, showing reduced performance in different types of cognitive tests, including learning a conditional discrimination task in a Y maze. Reduced ability to learn the Y maze task is due to reduced function of the glutamate -nitric oxide (NO)cGMP pathway in cerebellum, assessed in vivo by microclialysis. This results in reduced formation of cGMP in response to activation of NMDA receptors and impairment of learning ability. Both hyperammonemia and neuroinflammation contribute to impair this pathway. The effect is mediated by enhanced tonic activation of NMDA and GABAa receptors and of MAP-kinase p38. Based on these mechanistic studies new therapeutic strategies acting on specific targets in the brain have been designed and tested, which have successfully restored the function of the glutamate-NO-cGMP pathway in vivo and learning ability in rats with MHE. This can be achieved by therapeutic treatments using: a) phosphodiesterase 5 inhibitors (sildenafil, zaprinast), that increase cGMP levels by reducing its degradation b) extracellular cGMP c) antagonists of type A GABA receptors (bicuculline) d) neurosteroids that modulate GABAergic tone (pregnenolone sulfate) e) inhibitors of cyclooxygenase (ibuprofen) which reduce neuroinflammation f) inhibitors of MAP- kinase p38 (5[3239063), that reduce microglial activation and neuroinflammation Translation of some of these treatments to clinical practice would improve cognitive function, quality of life and life span of patients with cirrhosis and MHE and reduce health systems costs. Focal points: Benchside The mechanisms underlying cognitive and motor alterations in minimal hepatic encephalopathy (MHE) are beginning to be clarified in animal models. A number of therapeutic targets have been identified to improve cognitive and motor Function in MHE. Also, serum level of 3-nitrotyrosine is the first peripheral biomarker identified for diagnosis of MHE in cirrhotic patients, with high diagnostic accuracy, high sensitivity and specificity. Bedside In the European Union more than 2 million patients with liver cirrhosis show MHE with mild cognitive impairment. MHE is an important, until now underestimated, health, social and economic problem. Early diagnosis and treatment of MHE will significantly improve quality of life and life span of the patients and reduce costs of hospitalization and treatment Industry There are no specific treatments for the neurological alterations in MHE. A number of therapeutic targets have been identified in animal models to improve cognitive and motor function in MHE. This is a new market waiting for development of appropriate therapeutic treatments which would improve quality of life and survival of patients. Development of a kit for diagnosis of MHE in clinical practice is pending. Governments - regulatory agencies Early diagnosis and treatment of MHE will significantly improve quality of life and life span of the patients and reduce costs of hospitalization and treatment. Screening of the presence of MHE in patients with liver diseases will reduce costs. (C) 2014 European Society for Translational Medicine. Published by Elsevier Ltd. All rights reserved.

Several million patients with liver cirrhosis suffer minimal hepatic encephalopathy (MHE), with mild cognitive and coordination impairments that reduce their quality of life and life span. Hyperammonaemia and peripheral inflammation act synergistically to induce these neurological alterations. We propose that MHE appearance is because of the changes in peripheral immune system, which are transmitted to brain, leading to neuroinflammation that alters neurotransmission leading to cognitive and motor alterations. We summarize studies showing that MHE in cirrhotic patients is associated with alterations in the immune system and that patients died with HE show neuroinflammation in cerebellum, with microglial and astrocytic activation and Purkinje cell loss. We also summarize studies in animal models of MHE on the role of peripheral inflammation in neuroinflammation induction, how neuroinflammation alters neurotransmission and how this leads to cognitive and motor alterations. These studies identify therapeutic targets and treatments that improve cognitive and motor function. Rats with MHE show neuroinflammation in hippocampus and altered NMDA and AMPA receptor membrane expression, which impairs spatial learning and memory. Neuroinflammation in cerebellum is associated with altered GABA transporters and extracellular GABA, which impair motor coordination and learning in a Y maze. These alterations are reversed by treatments that reduce peripheral inflammation (anti-TNFα, ibuprofen), neuroinflammation (sulphoraphane, p38 inhibitors), GABAergic tone (bicuculline, pregnenolone sulphate) or increase extracellular cGMP (sildenafil or cGMP). The mechanisms identified would also occur in other chronic diseases associated with inflammation, aging and some mental and neurodegenerative diseases. Treatments that improve MHE may also be beneficial to treat these pathologies.

The use of pesticides has been associated with impaired neurodevelopment in children. The aims of this work were to assess: 1) the effects on spatial learning of developmental exposure to pesticides 2) if the effects are sex-dependent and 3) if hippocampal neuroinflammation is associated with the impairment of spatial learning. We analyzed the effects of developmental exposure to four pesticides: chlorpyrifos, carbaryl, endosulfan and cypermethrin. Exposure was from gestational day 7 to post-natal day 21 and spatial learning and memory was assessed when the rats were young adults. The effects of pesticides on spatial learning were pesticide and gender-dependent. Carbaryl did not affect spatial learning in males or females. Endosulfan and chlorpyrifos impaired learning in males but not in females. Cypermethrin improved spatial learning in the Morris water maze both in males and females while impaired learning in the radial maze only in males. Spatial learning ability was lower in control female rats than in males. All pesticides induced neuroinflammation, increasing IL-1b content in the hippocampus and there is a negative correlation between IL-1b levels in the hippocampus and spatial learning. Neuroinflammation would contribute to the effects of pesticides on spatial learning. (C) 2016 Elsevier Ltd. All rights reserved.

Pruritus is a common symptom in chronic liver diseases, which may also alter thermal sensitivity. The underlying mechanisms remain unclear and treatments are not satisfactory. Portal-systemic shunting has been proposed to alter thermal sensitivity in cirrhotics. Inflamma-tion-induced enhanced activity of the Transient Receptor Potential Vanilloid 1 (TRPV1) may contribute to pruritus and thermal hyperalgesia. Sildenafil reduces neuroinflam-mation in portacaval shunt (PCS) rats. The aims were to assess whether: (1) PCS rats show enhanced scratching or thermal sensitivity; (2) TRPV1 activity is enhanced in PCS rats; (3) treatment with sildenafil reduces TRPV1 activation, scratching and thermal hyperalgesia. Rats were treated with sildenafil beginning 3 weeks after surgery. The number of scratches performed were counted. Thermal hyperalgesia was analyzed using the Hargreaves' Plantar Test. TRPV1 activation by measuring the increase in Ca2+ induced by capsaicin in dorsal root ganglia neurons. PCS rats show enhanced scratching behavior, reaching 66 +/- 5 scratches/ h (p < 0.01) at 21 days after surgery, while controls show 37 +/- 2 scratches/ h. PCS rats show thermal hyperalgesia. Paw withdrawal latency was reduced (p < 0.05) to 10 +/- 1 s compared to controls (21 +/- 2 s). Capsaicin-induced calcium increase was higher in dorsal root ganglia cultures from PCS rats, indicating TRPV1functional increase. PCS rats show enhanced scratching behavior and thermal sensitivity and are a good model to study these alterations in chronic liver diseases. Enhanced sensitivity and activity of TRPV1 channel underlies these alterations. Treatment with sildenafil reduces TRPV1 channel sensitivity and activity and normalizes scratching behavior and thermal sensitivity.

AimsPatients with liver disease may develop hepatic encephalopathy (HE), with cognitive impairment and motor in-coordination. Rats with HE due to portacaval shunts (PCS) show motor in-coordination. We hypothesized that in PCS rats: (i) Motor in-coordination would be due to enhanced GABAergic tone in cerebellum; (ii) increased GABAergic tone would be due to neuroinflammation; (iii) increasing cGMP would reduce neuroinflammation and GABAergic tone and restore motor coordination. To assess these hypotheses, we assessed if (i) treatment with sildenafil reduces neuroinflammation; (ii) reduced neuroinflammation is associated with reduced GABAergic tone and restored motor coordination. MethodsRats were treated with sildenafil to increase cGMP. Microglia and astrocytes activation were analyzed by immunohistochemistry, extracellular GABA by microdialysis, and motor coordination in the beam walking. ResultsPCS rats show neuroinflammation in cerebellum, with microglia and astrocytes activation, increased IL-1b and TNF-a and reduced YM-1 and IL-4. Membrane expression of the GABA transporter GAT1 is reduced, while GAT3 is increased. Extracellular GABA and motor in-coordination are increased. Sildenafil treatment eliminates neuroinflammation, microglia and astrocytes activation; changes in membrane expression of GABA transporters; and restores motor coordination. ConclusionsThis study supports an interplay between cGMP-neuroinflammation and GABAergic neurotransmission in impairing motor coordination in PCS rats.

We developed a simple analytical method for the simultaneous determination of representatives of various groups of neurotoxic insecticides (carbaryl, chlorpyrifos, cypermethrin, and alpha-endosulfan and beta-endosulfan and their metabolite endosulfan sulfate) in limited amounts of animal tissues containing different amounts of lipids. Selected tissues (rodent fat, liver, and brain) were extracted in a special in-house-designed mini-extractor constructed on the basis of the Soxhlet and Twisselmann extractors. A dried tissue sample placed in a small cartridge was extracted, while the nascent extract was simultaneously filtered through a layer of sodium sulfate. The extraction was followed by combined clean-up, including gel permeation chromatography (in case of high lipid content), ultrasonication, and solid-phase extraction chromatography using C-18 on silica and aluminum oxide. Gas chromatography coupled with high-resolution mass spectrometry was used for analyte separation, detection, and quantification. Average recoveries for individual insecticides ranged from 82 to 111%. Expanded measurement uncertainties were generally lower than 35%. The developed method was successfully applied to rat tissue samples obtained from an animal model dealing with insecticide exposure during brain development. This method may also be applied to the analytical treatment of small amounts of various types of animal and human tissue samples. A significant advantage achieved using this method is high sample throughput due to the simultaneous treatment of many samples.

Extracellular protein kinases, including cAMP-dependent protein kinase (PICA), modulate neuronal functions including N-methyl-D-aspartate (NMDA) receptor-dependent long-term potentiation. NMDA receptor activation increases calcium, which binds to calmodulin and activates nitric oxide synthase (NOS), increasing nitric oxide (NO), which activates guanylate cyclase, increasing cGMP, which is released to the extracellular fluid, allowing analysis of this glutamate-NO-cGMP pathway in vivo by microdialysis. The function of this pathway is impaired in hyperammonemic rats. The aims of this work were to assess (1) whether the glutamate-NO-cGMP pathway is modulated in cerebellum in vivo by an extracellular PKA, (2) the role of phosphorylation and activity of calcium/calmodulin-dependent protein kinase II (CaMKII) and NOS in the pathway modulation by extracellular PKA, and (3) whether the effects are different in hyperammonemic and control rats. The pathway was analyzed by in vivo microdialysis. The role of extracellular PKA was analyzed by inhibiting it with a membrane-impermeable inhibitor. The mechanisms involved were analyzed in freshly isolated cerebellar slices from control and hyperammonemic rats. In control rats, inhibiting extracellular PKA reduces the glutamate-NO-cGMP pathway function in vivo. This is due to reduction of CaMKII phosphorylation and activity, which reduces NOS phosphorylation at Ser1417 and NOS activity, resulting in reduced guanylate cyclase activation and cGMP formation. In hyperammonemic rats, under basal conditions, CaMKII phosphorylation and activity are increased, increasing NOS phosphorylation at Ser847, which reduces NOS activity, guanylate cyclase activation, and cGMP. Inhibiting extracellular PICA in hyperammonemic rats normalizes CaMKII phosphorylation and activity, NOS phosphorylation, NOS activity, and cGMP, restoring normal function of the pathway.

Polychlorinated biphenyls (PCBs) are persistent organic pollutants that accumulate in the food chain and are present in human blood and milk. Children born to mothers exposed to PCBs show cognitive deficits, which are reproduced in rats perinatally exposed to PCBs. It has been proposed that PCB-induced cognitive impairment is due to impairment of the glutamate-nitric oxide (NO)-cGMP pathway. The aim of the present work was to assess whether chronic exposure to the nondioxin-like PCB52, PCB138, or PCB180 alters the function of this pathway in primary cultures of rat cerebellar neurons and to assess whether different PCBs have similar or different mechanisms of action. PCB180 and PCB138 impair the function of the glutamate-NO-cGMP pathway at nanomolar concentrations, and PCB52 impairs the function of the glutamate-NO-cGMP pathway at micromolar concentrations. The mechanisms by which different PCBs impair the function of the glutamate-NO-cGMP pathway are different. Each PCB affects the pathway at more than one step but with different potency and, for some steps, in opposite ways. Exposure to the PCBs alters the basal concentrations of intracellular calcium, NO, and cGMP. The three PCBs increase NO; however, PCB52 and PCB138 increase basal cGMP, while PCB180 decreases it. PCB52 and PCB138 decrease the activation of soluble guanylate cyclase by NO, and PCB180 increases it. Long-term exposure to PCB52, PCB180, or PCB138 reduces the activation of NO synthase and the whole glutamate-NO-cGMP pathway in response to activation of N-methyl-d-aspartate receptors. The EC(50) was 300 nM for PCB52 and 2 nM for PCB138 or PCB180. These results show that chronic exposure to nondioxin like PCBs impairs the function of the glutamate-NO-cGMP pathway in cerebellar neurons by different mechanisms and with different potencies. Impaired function of this pathway would contribute to the cognitive alterations induced by perinatal exposure to PCBs in humans.

Polychlorinated biphenyls (PCBs) are persistent organic pollutants present in human blood and milk. Exposure to PCBs during pregnancy and lactation leads to cognitive impairment in children. Perinatal exposure to PCB 153 or PCB 126 impairs the glutamate-nitric oxide-cGMP pathway in cerebellum in vivo and learning ability in adult rats. The aims of this work were: (1) to assess whether long-term exposure of primary cultures of cerebellar neurons to PCB 153 or PCB 126 reproduces the impairment in the function of the glutamate-nitric oxide-cGMP pathway found in rat cerebellum in vivo; (2) to provide some insight on the steps of the pathway affected by these PCBs; (3) to assess whether the mechanisms of interference of the pathway are different for PCB 126 and PCB 153. Both PCB 153 and PCB 126 increase basal levels of cGMP by different mechanisms. PCB 126 increases the amount of soluble guanylate cyclase while PCB 153 does not. PCB 153 reduces the amount of calmodulin while PCB 126 does not. Also both PCBs impair the function of the glutamate-nitric oxide-cGMP pathway by different mechanisms, PCB 153 impairs nitric oxide-induced activation of soluble guanylate cyclase and increase in cGMP while PCB 126 does not. PCB 126 reduces NMDA-induced increase in calcium while PCB 153 does not. When PCB 153 and PCB 126 exhibit the same effect, PCB 126 was more potent than PCB 153, as occurs in vivo.

Hepatic encephalopathy (HE) is a complex neuropsychiatric syndrome affecting patients with liver diseases, mainly those with liver cirrhosis. The mildest form of HE is minimal HE (MHE), with mild cognitive impairment, attention deficit, psychomotor slowing and impaired visuo-motor and bimanual coordination. MHE may progress to clinical HE with worsening of the neurological alterations which may lead to reduced consciousness and, in the worse cases, may progress to coma and death. HE affects several million people in the world and is a serious health, social and economic problem. There are no specific treatments for the neurological alterations in HE. The mechanisms underlying the cognitive and motor alterations in HE are beginning to be clarified in animal models. These studies have allowed to design and test in animal models of HE new therapeutic approaches which have successfully restored cognitive and motor function in rats with HE. In this article we review the evidences showing that: (C) 2015 Elsevier Ltd. All rights reserved.

Inflammation contributes to cognitive impairment in patients with hepatic encephalopathy (HE). However, the process by which peripheral inflammation results in cognitive impairment remains unclear. In animal models, neuroinflammation and altered neurotransmission mediate cognitive impairment. Taking into account these data, we hypothesized that in rats with HE: (1) peripheral inflammation is a main contributor to neuroinflammation; (2) neuroinflammation in hippocampus impairs spatial learning by altering AMPA and/or NMDA receptors membrane expression; (3) reducing peripheral inflammation with infliximab (anti-TNF-a) would improve spatial learning; (4) this would be associated with reduced neuroinflammation and normalization of the membrane expression of glutamate receptors. The aims of this work were to assess these hypotheses. We analyzed in rats with portacaval shunt (PCS) and control rats, treated or not with infliximab: (a) peripheral inflammation by measuring prostaglandin E2, IL10, IL-17, and IL-6; (b) neuroinflammation in hippocampus by analyzing microglial activation and the content of TNF-a and IL-1b; (c) AMPA and NMDA receptors membrane expression in hippocampus; and (d) spatial learning in the Radial and Morris water mazes. We assessed the effects of treatment with infliximab on peripheral inflammation, on neuroinflammation and AMPA and NMDA receptors membrane expression in hippocampus and on spatial learning and memory. PCS rats show increased serum prostaglandin E2, IL-17, and IL-6 and reduced IL-10 levels, indicating increased peripheral inflammation. PCS rats also show microglial activation and increased nuclear NF-kB and expression of TNF-a and IL-1b in hippocampus. This was associated with altered AMPA and NMDA receptors membrane expression in hippocampus and impaired spatial learning and memory in the radial and Morris water maze. Treatment with infliximab reduces peripheral inflammation in PCS rats, normalizing prostaglandin E2, IL-17, IL-6, and IL-10 levels in serum. Infliximab also prevents neuroinflammation, reduces microglial activation, translocates NF-kB into nucleoli and normalizes TNF-a and IL-1b content in hippocampus. This was associated with normalization of AMPA receptors membrane expression in hippocampus and of spatial learning and memory. The results suggest that peripheral inflammation contributes to spatial learning impairment in PCS rats. Treatment with anti-TNF-a could be a new therapeutic approach to improve cognitive function in patients with HE.

It has been proposed that extracellular cGMP modulates the ability to learn a Y maze task, but the underlying mechanisms remained unknown. Here we show that extracellular cGMP, at physiological concentrations, modulates learning in the Y maze in a biphasic way by modulating the glutamate-nitric oxide-cGMP pathway in cerebellum. Extracellular cGMP reduces glycine receptors activation inducing a voltage-dependent calcium-channels-mediated increase of calcium in Purkinje neurons. This calcium increase modulates CaMKII phosphorylation in a biphasic way. When basal calcium concentration is low extracellular cGMP reduces CaMKII phosphorylation, increasing nitric oxide synthase activity, the glutamate-NO-cGMP pathway function and learning ability. When basal calcium is normal extracellular cGMP increases CaMKII phosphorylation, reducing nitric oxide synthase activity, the pathway function and learning. These data unveil new mechanisms modulating learning in the Y maze and likely other learning types which may be therapeutic targets to improve learning in pathological situations associated with altered cGMP levels.

Background: Patients with liver cirrhosis and minimal hepatic encephalopathy (MHE) show mild cognitive impairment and spatial learning dysfunction. Hyperammonemia acts synergistically with inflammation to induce cognitive impairment in MHE. Hyperammonemia-induced neuroinflammation in hippocampus could contribute to spatial learning impairment in MHE. Two main aims of this work were: (1) to assess whether chronic hyperammonemia increases inflammatory factors in the hippocampus and if this is associated with microglia and/or astrocytes activation and (2) to assess whether hyperammonemia-induced neuroinflammation in the hippocampus is associated with altered membrane expression of glutamate and GABA receptors and spatial learning impairment. There are no specific treatments for cognitive alterations in patients with MHE. A third aim was to assess whether treatment with sulforaphane enhances endogenous the anti-inflammatory system, reduces neuroinflammation in the hippocampus of hyperammonemic rats, and restores spatial learning and if normalization of receptor membrane expression is associated with learning improvement. Methods: We analyzed the following in control and hyperammonemic rats, treated or not with sulforaphane: (1) microglia and astrocytes activation by immunohistochemistry, (2) markers of pro-inflammatory (M1) (IL-1 beta, IL-6) and anti-inflammatory (M2) microglia (Arg1, YM-1) by Western blot, (3) membrane expression of GABA, AMPA, and NMDA receptors using the BS3 cross-linker, and (4) spatial learning using the radial maze. Results: The results reported show that hyperammonemia induces astrocytes and microglia activation in the hippocampus, increasing pro-inflammatory cytokines IL-1 beta and IL-6. This is associated with altered membrane expression of AMPA, NMDA, and GABA receptors which would be responsible for altered neurotransmission and impairment of spatial learning in the radial maze. Treatment with sulforaphane promotes microglia differentiation from pro-inflammatory M1 to anti-inflammatory M2 phenotype and reduces activation of astrocytes in hyperammonemic rats. This reduces neuroinflammation, normalizes membrane expression of glutamate and GABA receptors, and restores spatial learning in hyperammonemic rats. Conclusions: Hyperammonemia-induced neuroinflammation impairs glutamatergic and GABAergic neurotransmission by altering membrane expression of glutamate and GABA receptors, resulting in impaired spatial learning. Sulforaphane reverses all these effects. Treatment with sulforaphane could be useful to improve cognitive function in cirrhotic patients with minimal or clinical hepatic encephalopathy.

Cognitive function is impaired in patients with liver disease by unknown mechanisms. Long-term potentiation (LTP) in the hippocampus is considered the basis of some forms of learning and memory. The aims of this work were to assess (i) whether chronic liver failure impairs hippocampal LTP; (ii) if this impairment may be due to alterations in glutamatergic neurotransmission, and (iii) if impairment of LTP is associated with reduced learning ability. It is shown that liver failure in Wistar rats induces the following alterations in the hippocampus; (i) alters the phosphorylation of NMDA and AMPA receptors; (ii) reduces the expression of NMDA and AMPA receptors in membranes, (iii) reduces the magnitude of excitatory postsynaptic potentials (EPSPs) induced by activation of NMDA or AMPA receptors, and (iv) impairs NMDA receptor-dependent LTP. Liver failure also impairs learning of the Morris water maze task. Impairment of glutamatergic synaptic transmission and NMDA receptor-mediated responses may be involved in the alterations of cognitive function in patients with liver disease.

Polybrominated diphenylethers (PBDEs) are widely used as flame retardants. Significant amounts of PBDEs are present in the milk of lactating women. The possible neurotoxic effects of PBDEs are not well known. Perinatal exposure to PBDEs affects both motor and cognitive functions by mechanisms that remain unclear. Some types of learning depend on N-methyl-D-aspartate receptor activation, which increases intracellular calcium that binds to calmodulin and activates nitric oxide synthase, increasing nitric oxide formation that activates guanylate cyclase, increasing cGMP formation. Part of this cGMP is released to the extracellular fluid. We studied whether prenatal exposure of rats to PBDE99 alters the function of this glutamate-nitric oxide-cGMP pathway in rat brain in vivo. At 10 weeks of age, rats treated with PBDE99 showed increased function of the glutamate-nitric oxide-cGMP pathway in brain in vivo, as assessed by microdialysis in freely moving rats. The increased function of the pathway was reproduced in primary cultures of cerebellar neurons prepared from rats prenatally exposed to PBDE99 as well as in neurons cultured from normal rats and treated in vitro with PBDE99. Increased calmodulin content and activation of soluble guanylate cyclase by nitric oxide contributed to the increased function of the pathway.

In tests of spatial ability, males outperform females both in rats and in humans. The mechanism underlying this gender differential learning ability and memory in spatial tasks remains unknown. Long-term potentiation (LTP) in the hippocampus is considered the basis for spatial learning and memory. The aims of this work were (a) to assess spatial learning and memory in male and female rats in the radial and Morris mazes; (b) to assess whether basal synaptic activity and LTP in the hippocampus are different in male and female rats; and (c) to identify the molecular mechanisms responsible for the gender differences in LTP. We analyzed in young male and female rats (a) performance in spatial tasks in the radial and Morris water mazes; (b) basal synaptic activity in hippocampal slices; and (c) LTP and some mechanisms modulating its magnitude. The results reported allow us to conclude that female rats show larger AMPA receptor-mediate synaptic responses under basal conditions, likely due to enhanced phosphorylation of GluR2 in Ser880 and increased amounts of GluR2-containing AMPA receptors in postsynaptic densities. In contrast, the magnitude of tetanus-induced LTP was lower in females than in males. This is due to reduced activation of soluble guanylate cyclase and the formation of cGMP, leading to lower activation of cGMP-dependent protein kinase and phosphorylation of GluR1 in Ser845, which results in lower insertion of AMPA receptors in the synaptic membrane and a lower magnitude of LTP. These mechanisms may contribute to the reduced performance of females in the radial and Morris water mazes.

Background/Aims Patients with hepatic encephalopathy show altered motor function, psychomotor slowing and hypokinesia. The underlying mechanisms remain unclear. This work's aims were: (1) to analyse in rats with chronic liver failure due to portacaval shunt (PCS) the neurochemical alterations in the basal ganglia–thalamus–cortex circuits; (2) to correlate these alterations with those in motor function and (3) to normalize motor activity of PCS rats by pharmacological means. Methods Extracellular neurotransmitters levels were analysed by in vivo brain microdialysys. Motor activity was determined by counting crossings in open field. Results Extracellular glutamate is increased in substantia nigra pars reticulata (SNr) of PCS rats. Blocking metabotropic receptor 1 (mGluR1) in SNr normalizes motor activity in PCS rats. In ventro-medial thalamus of PCS rats GABA is increased and it is normalized by blocking mGluR1 in SNr. Blocking mGluR1 in SNr increases and mGluR1 activation reduces glutamate in motor cortex and motor activity. Conclusions Increased extracellular glutamate and activation of mGluR1 in SNr are responsible for reduced motor activity in rats with chronic liver failure. Blocking mGluR1 in SNr normalizes motor activity in PCS rats, suggesting that, under appropriate conditions, similar treatments could be useful to treat the psychomotor slowing and hypokinesia in patients with hepatic encephalopathy.