Chronic hyperammonemia induces neuroinflammation in cerebellum, with glial activation and enhanced activation of the TNFR1-NF-kB-glutaminase-glutamate-GABA pathway. Hyperammonemia also increases glycinergic neurotransmission. These alterations contribute to cognitive and motor impairment. Activation of glycine receptors is reduced by extracellular cGMP, which levels are reduced in cerebellum of hyperammonemic rats in vivo. We hypothesized that enhanced glycinergic neurotransmission in hyperammonemic rats (1) contributes to induce neuroinflammation and glutamatergic and GABAergic neurotransmission alterations; (2) is a consequence of the reduced extracellular cGMP levels. The aims were to assess, in cerebellum of hyperammonemic rats, (a) whether blocking glycine receptors with the antagonist strychnine reduces neuroinflammation; (b) the cellular localization of glycine receptor; (c) the effects of blocking glycine receptors on the TNFR1-NF-kB-glutaminase-glutamate-GABA pathway and microglia activation; (d) whether adding extracellular cGMP reproduces the effects of strychnine.

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.

There is increasing evidence that extracellular cGMP modulates glutamatergic neurotransmission and some forms of learning. However, the underlying mechanisms remain unknown. We proposed the hypotheses that extracellular cGMP may regulate membrane expression of AMPA receptors. To do this extracellular cGMP should act on a membrane protein and activate signal transduction pathways modulating phosphorylation of the GluA1 and/or GluA2 subunits. It has been shown that extracellular cGMP modulates glycine receptors. The aims of this work were to assess: 1) whether extracellular cGMP modulates membrane expression of GluA1 and GluA2 subunits of AMPA receptors in cerebellum in vivo; 2) whether this is mediated by glycine receptors; 3) the role of GluA1 and GluA2 phosphorylation and 4) identify steps of the intracellular pathways involved. We show that extracellular cGMP modulates membrane expression of GluA1 and GluA2 in cerebellum in vivo and unveil the mechanisms involved. Extracellular cGMP reduced glycine receptor activation, modulating cAMP, protein kinases and phosphatases, and GluA1 and GluA2 phosphorylation, resulting in increased GluA1 and reduced GluA2 membrane expression. Extracellular cGMP therefore modulates membrane expression of AMPA receptors and glutamatergic neurotransmission. The steps identified may be therapeutic targets to improve neurotransmission and neurological function in pathological situations with abnormal glutamatergic neurotransmission.

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'.