Increasing extracellular cGMP in cerebellum in vivo reduces neuroinflammation, GABAergic tone and motor in-coordination in hyperammonemic rats.

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-κB, 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 hyperammonemic rats by reducing microglia activation and neuroinflammation, leading to normalization of extracellular glutamate and GABA levels in cerebellum and of motor coordination.

In vivo administration of extracellular cGMP normalizes TNF-α and membrane expression of AMPA receptors in hippocampus and spatial reference memory but not IL-1ß, NMDA receptors in membrane and working memory in hyperammonemic rats.

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-1ß, 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.

Intracellular and extracelluar cyclic GMP in the brain and the hippocampus.

Cyclic Guanosine-Monophosphate (cGMP) is implicated as second messenger in a plethora of pathways and its effects are executed mainly by cGMP-dependent protein kinases (PKG). It is involved in both peripheral (cardiovascular regulation, intestinal secretion, phototransduction, etc.) and brain (hippocampal synaptic plasticity, neuroinflammation, cognitive function, etc.) processes. Stimulation of hippocampal cGMP signaling have been proved to be beneficial in animal models of aging, Alzheimer's disease or hepatic encephalopathy, restoring different cognitive functions such as passive avoidance, object recognition or spatial memory. However, even when some inhibitors of cGMP-degrading enzymes (PDEs) are already used against peripheral pathologies, their utility as neurological treatments is still under clinical investigation. Additionally, it has been demonstrated a list of cGMP roles as not second but first messenger. The role of extracellular cGMP has been specially studied in hippocampal function and cognitive impairment in animal models and it has emerged as an important modulator of neuroinflammation-mediated cognitive alterations and hippocampal synaptic plasticity malfunction. Specifically, it has been demonstrated that extracellular cGMP decreases hippocampal IL-1ß levels restoring membrane expression of glutamate receptors in the hippocampus and cognitive function in hyperammonemic rats. The mechanisms implicated are still unclear and might involve complex interactions between hippocampal neurons, astrocytes and microglia. Membrane targets for extracellular cGMP are still poorly understood and must be addressed in future studies.

Chronic hyperammonemia alters extracellular glutamate, glutamine and GABA and membrane expression of their transporters in rat cerebellum. Modulation by extracellular cGMP.

Trafficking of glutamate, glutamine and GABA between astrocytes and neurons is essential to maintain proper neurotransmission. Chronic hyperammonemia alters neurotransmission and cognitive function. The aims of this work were to analyze in cerebellum of rats the effects of chronic hyperammonemia on: a) extracellular glutamate, glutamine and GABA concentrations; b) membrane expression of glutamate, glutamine and GABA transporters; c) how they are modulated by extracellular cGMP. Hyperammonemic rats show increased levels of extracellular glutamate, glutamine, GABA and citrulline in cerebellum in vivo. Hyperammonemic rats show: a) increased membrane expression of the astrocytic glutamine transporter SNAT3 and reduced membrane expression of the neuronal transporter SNAT1; b) reduced membrane expression of the neuronal GABA transporter GAT1 and increased membrane expression of the astrocytic GAT3 transporter; c) reduced membrane expression of the astrocytic glutamate transporters GLAST and GLT-1 and of the neuronal transporter EAAC1. Increasing extracellular cGMP normalizes membrane expression of SNAT3, GAT3, GAT1 and GLAST and extracellular glutamate, glutamine, GABA and citrulline hyperammonemic rats. Extracellular cGMP also modulates membrane expression of most transporters in control rats, reducing membrane expression of SNAT1, GLT-1 and EAAC1 and increasing that of GAT1 and GAT3. Modulation of SNAT3, SNAT1, GLT-1 and EAAC1 by extracellular cGMP would be mediated by inhibition of glycine receptors. These data suggest that, in pathological situations such as hyperammonemia, hepatic encephalopathy or Alzheimer's disease, reduced levels of extracellular cGMP contribute to alterations in membrane expression of glutamine, glutamate and GABA transporters, in the extracellular levels of glutamine, glutamate and GABA and in neurotransmission. This article is part of the issue entitled 'Special Issue on Neurotransmitter Transporters'.