Age-related Changes in the NMDA Receptor/Nitric Oxide/cGMP Pathway in the Hippocampus and Cerebellum of Freely Moving Rats Subjected to Transcerebral Microdialysis

The N-methyl-d -aspartate (NMDA) receptor/nitric oxide synthase/guanylate cyclase pathway was studied during aging by monitoring extracellular cGMP in the rat hippocampus and cerebellum during in vivo microdialysis. In the hippocampus the basal cGMP efflux decreased by 50% from 3 to 12 months of age, whereas it remained constant with age in the cerebellum. Locally perfused NMDA (1 mM) evoked remarkable cGMP responses in 3-month-old rats; in the hippocampus the cGMP production was already dramatically reduced at 12 months, whereas in the cerebellum a similar impairment occurred much later (24 months). The nitric oxide donor S-nitroso-N-penicillamine (1 mM) elicited cGMP responses which slightly decreased from 3 to 12–24 months in the hippocampus, while no significant decrement with age could be seen in the cerebellum. Local perfusion of the phosphodiesterase inhibitor 3-isobutyl-1 methylxanthine (IBMX, 1 mM) produced large increases in hippocampal cGMP levels. The response decreased at 12 and 24 months, apparently in parallel with the fall in the basal level of cGMP. No significant differences across ages were observed following IBMX infusion in the cerebellum. The decreases in basal outflow and in the NMDA-evoked cGMP response seen in the aged hippocampus were not compensated for by supplying l -arginine. Infusion of d -serine (1 mM) enhanced (150–200%) extracellular cGMP in the cerebellum with no age-related differences. The activity in vitro, of hippocampal nitric oxide synthase at 24 months was 33% lower than at 3 months, whereas the cerebellar enzyme did not show any age-related decay. Aging seems therefore to affect differentially the NMDA receptor/nitric oxide synthase/cGMP pathway in the rat hippocampus versus the cerebellum. In the hippocampus the early fall in the NMDA-evoked cGMP response seems to originate from deficits in NMDA receptor function and nitric oxide synthase and guanylate cyclase activities; in the cerebellum, the decreased response to NMDA in the old animals seems essentially to be due to impairment of NMDA receptor function.     

Roles of neuroactive amino acids in ammonia neurotoxicity

Many neurologic disorders are related to congenital or acquired hyperammonemia (HA). Advanced symptoms of HA range from seizures in acute stages to stupor and coma in more chronic conditions, manifesting variable imbalance between the inhibitory and excitatory neurotransmission. Evidence obtained with the use of experimental HA models suggests that acute neurotoxic effects of ammonia are mediated by overactivation of ionotropic glutamate (GLU) receptors, mainly the N-methyl-D-aspartate (NMDA) receptors, and to a lesser degree the KA/AMPA receptors. NMDA receptor–mediated neurotoxicity may be potentiated by impaired control of their function by metabotropic GLU receptors, which are inactivated by ammonia. Prolonged overactivation of the NMDA receptors upon extended ammonia exposure causes their downregulation. The GLU receptor changes may be related to their excessive exposure to extrasynaptic GLU. Ammonia promotes GLU accumulation in the extrasynaptic space by enhancing its release from neurons, and/or by decreasing its reuptake to the nerve endings and astrocytes, where the effect results from inactivation (downregulation) of the astrocytic glutamate transporter GLT1. Excitotoxic effects of ammonia are augmented by increased synthesis of nitric oxide (NO), which is associated with NMDA receptor activation and/or increased synaptic transport of arginine (ARG). A shift toward neural inhibition is promoted by positive modulation of the γ-aminobutyric acid (GABA)ergic tone resulting from excessive accumulation in the brain of endogenous central benzodiazepine receptor agonists, and from upregulation of astrocytic peripheral benzodiazepine receptors leading to elevated levels of prognenelone-derived neurosteroids, which positively modulate the GABA(A) receptor complex. Inhibitory neurotransmission may also be favored by enhanced release from astrocytes of an inhibitory amino acid, taurine. J. Neurosci. Res. 51:133–138, 1998. © 1998 Wiley-Liss, Inc.     

Increasing the function of the glutamate‐nitric oxide‐cyclic guanosine monophosphate pathway increases the ability to learn a Y‐maze task

N‐methyl‐D‐aspartate (NMDA) receptors play a crucial role in learning. However, the molecular mechanisms by which NMDA receptors contribute to learning processes are not known in detail. Activation of NMDA receptors leads to increased calcium in the postsynaptic neuron. Calcium binds to calmodulin and activates neuronal nitric oxide synthase, increasing nitric oxide (NO), which activates soluble guanylate cyclase, increasing cGMP. Part of this cGMP is released to the extracellular space. Several reports indicate that impairment of this glutamate‐NO‐cGMP pathway reduces the ability to learn a Y‐maze conditional discrimination task by rats. The aim of this work was to assess whether enhancing the function of this pathway increases the ability to learn this task. Prenatal exposure to the polybrominated diphenylether PBDE‐99 during embryonic days 2–9 or 11–19 enhances the function of the glutamate‐NO‐cGMP pathway in cerebellum in vivo as assessed by microdialysis in freely moving rats. This was associated with an increase in the ability to learn the Y‐maze task. Rats prenatally exposed to PBDE need fewer trials than control rats to learn the Y‐maze task. These results show that the function of the glutamate‐NO‐cGMP modulates the ability of rats to learn the Y‐maze task, that the function of the pathway under physiological conditions is not optimal for learning, and that performance in the Y‐maze task may be improved by enhancing slightly the function of the pathway and cGMP formation. © 2009 Wiley‐Liss, Inc.     

NMDA receptor-mediated cGMP synthesis in primary cultures of mouse cerebellar granule cells appears to involve neuron-astrocyte communication with NO operating as the intercellular messenger

The possibility that neuron-astrocyte communication may be responsible for glutamate (Glu)-stimulated cGMP formation even in relatively homogeneous primary cultures of mouse cerebellar granule cells (7 days in vitro) was investigated. Pharmacological analysis using selective excitatory amino acid (EAA) receptor antagonists showed that cGMP production, stimulated in these cultures by Glu and a variety of endogenous EAAs structurally-related to Glu (namely, L-aspartate, L-cysteine sulphinate, L-homocysteate, S-sulpho-L-cysteine), was mediated wholly by N-methyl-D-aspartate (NMDA) receptor activation. Moreover, EAA-induced responses were dependent on the presence of extracellular calcium but unaffected by addition of the L-type voltage-sensitive calcium channel blockers nifedipine (10 μM) or verapamil (5 μM). The mode of calcium entry was also shown to be important since the calcium ionophore, Λ23187 (10 μM), was unable to stimulate cGMP levels above basal. cGMP formation was blocked by the competitive nitric oxide synthase inhibitor, L-N^G-nitroarginine (100 μM), consistent with a role of nitric oxide (NO) in this signalling pathway. In the presence of added haemoglobin (1 μM), acting as a membrane-impermeable NO scavenger, Glu-stimulated cGMP formation was abolished implying that NO must act as an intercellular messenger. When the neuronal population was destroyed following a 24 hr exposure to the excitotoxin, S-sulpho-L-cysteine (200 μM), Glu-stimulated cGMP formation was abolished; whereas responses to the NO donor, sodium nitroprusside (SNP), although markedly reduced were still double that stimulated by Glu in the absence of the excitotoxin, suggesting the presence of non-neuronal cells that can generate cGMP if supplied directly with NO. Consistent with this suggestion, low levels of the glial specific enzyme, glutamine synthetase, were detected in granule cell cultures. Furthermore, omission or delayed addition of the antimitotic agent, cytosine arabinoside (20 μM), to the growth medium caused a significant increase in the level of Glu-stimulated cGMP formation. © 1996 Wiley-Liss, Inc.     

Stimulation with N-methyl-D-aspartate or kainic acid increases cyclic guanosine monophosphate-like immunoreactivity in turtle retina: Involvement of nitric oxide synthase

In brain and retina, stimulation with excitatory amino acids (EAA) can generate nitric oxide (NO) and increase levels of cyclic guanosine monophosphate (cGMP). Because nitric oxide synthase (NOS) has been found in retinas of all species examined to date, an NO signal-transduction pathway is likely to be present in all retinas. We tested the hypothesis that stimulation of ionotropic glutamate receptors in turtle retina would result in increases in cGMP through an NOS/NO/cGMP pathway. Following in vitro incubations of turtle eye cups with the glutamate receptor agonists, N-methyl-D-aspartate (NMDA) or kainic acid (KA), we quantified the increases in cGMP-like immunoreactivity (cGMP-LI) by using enzyme-linked immunosorbant assay (ELISA) and localized the increased cGMP-LI by using an antibody against cGMP. Stimulation with NMDA or KA increased cGMP-LI in bipolar and amacrine cells as well as in some somata in the ganglion cell layer. Either KA or NMDA produced statistically significant increases in total retinal cGMP-LI by ELISA. To test the involvement of NO, we used the NOS inhibitors 7-nitroindazole and L-nitroarginine. Both inhibitors blocked virtually all of the KA- or NMDA-stimulated increases in cGMP-LI. These results indicate that activation of ionotropic glutamate receptors can increase cGMP in select retinal neurons. Differences between the agonist-evoked increases of retinal cGMP-LI suggest that there can be specificity in the activation of the NOS/NO/cGMP signal-transduction pathway by glutamate. This suggests that, in addition to short-term electrical changes, activation of ionotropic glutamate receptors also may produce longer term modulatory or metabolic effects involving NO/cGMP. J. Comp. Neurol. 404:75–85, 1999. © 1999 Wiley-Liss, Inc.     

Role of nitric oxide and cyclic GMP in glutamate-induced neuronal death

Glutamate is the main excitatory neurotransmitter in mammals. However, excessive activation of glutamate receptors is neurotoxic, leading to neuronal degeneration and death. In many systems, including primary cultures of cerebellar neurons, glutamate neurotoxicity is mainly mediated by excessive activation of NMDA receptors, leading to increased intracellular calcium which binds to calmodulin and activates neuronal nitric oxide synthase (NOS), increasing nitric oxide (NO) which in turn activates guanylate cyclase and increases cGMP. Inhibition of NOS prevents glutamate neurotoxicity, indicating that NO mediates glutamate-induced neuronal death in this system. NO generating agents such as SNAP also induce neuronal death. Compounds that can act as “scavengers” of NO such as Croman 6 (CR-6) prevent glutamate neurotoxicity. The role of cGMP in the mediation of glutamate neurotoxicity remain controversial. Some reports indicate that cGMP mediates glutamate neurotoxicity while others indicate that cGMP is neuroprotective. We have studied the role of cGMP in the mediation of glutamate and NO neurotoxicity in cerebellar neurons. Inhibition of soluble guanylate cyclase prevents glutamate and NO neurotoxicity. There is a good correlation between inhibition of cGMP formation and neuroprotection. Moreover 8-Br-cGMP, a cell permeable analog of cGMP, induced neuronal death. These results indicate that increased intracellular cGMP is involved in the mechanism of neurotoxicity. Inhibitors of phosphodiesterase increased extracellular but not intracellular cGMP and prevented glutamate neurotoxicity. Addition of cGMP to the medium also prevented glutamate neurotoxicity. These results are compatible with a neurotoxic effect of increased intracellular cGMP and a neuroprotective effect of increased extracellular cGMP.     

The glial glutamate transporter in hyperammonemia and hepatic encephalopathy: Relation to energy metabolism and glutamatergic neurotransmission

Abnormalities in glutamate metabolism and glutamatergic neurotransmission appear to play a major role in the pathogenesis of hyperammonemia and hepatic encephalopathy. Astrocytes may be involved in these derangements as ammonia has been shown to impair the ability of these cells to take up glutamate. This study presents a northern blot analysis of the GLT-1 glutamate transporter in hyperammonemic rats, and in rats with thioacetamide-induced acute liver failure. Our findings demonstrate a downregulation of GLT-1 mRNA in both conditions. This article examines the potential impact of deficits in glutamate uptake on energy metabolism and glutamatergic neurotransmission in the context of abnormalities in glial-neuronal interactions. We propose that an ammonia-induced abnormality in astroglial glutamate uptake constitutes a critical aspect in the pathogenesis of hepatic encephalopathy and other hyperammonemic conditions. GLIA 21:124–133, 1997. © 1997 Wiley-Liss, Inc.     

NMDA and Kainate-evoked Release of Nitric Oxide and Classical Transmitters in the Rat Striatum: In Vivo Evidence that Nitric Oxide May Play a Neuroprotective Role

The effects of N-methy-d -aspartate (NMDA), kainate, S-α-amino-3-hydroxyd-5-methyl-4-isoxazole propionate (AMPA) and KCI on striatal nitric oxide (NO), acetylcholine (ACh), dopamine (DA), serotonin (5-HT), aspartate (ASP), glutamate (GLU) and γ-aminobutyric acid (GABA) release were measured in anaesthetized rats in vivo by microdialysis and in vitro in organotypic slice cultures. Local NMDA (1–100 μM) infusion by retrodialysis dose-dependently increased levels of classical transmitters, NO₂-, NO₃-, ctrulline and arginine at similar thresholds (10 γM) Similar patterns of NMDA-evoked (50 μM) release were seen in striatal cultures. NMDA-evoked changes were all calcium-dependent and blocked by NMDA (APV or MK-801) but not AMPN/kainate (DNQX) receptor antagonists, excepting DA which could be prevented by both. In vivo, kainate increased NO₂^-, NO₃^-, CIT and ARG levels at 50 and 100 μM but was less potent than NMDA. Kainate also evoked significant Ach₁ DA and GLU release dose-dependently starting at 1–10 μM whereas 5-HT, ASP and GABA required 50 or 100 μM doses. Kainate effects were inhibited by DNQX, but not by APV, and were calcium-dependent. AMPA failed to alter NO₂^-, NO₃^-, CIT or ARG levels at 50 or 100 μM doses but dose-dependently increased ACh and DA. Similar results were seen with kainate (50 μM) and AMPA (50 μM) in vitro KCI evoked NO₂^-, NO₃^-, CIT and ARG release as well as that of the classical transmitters in vivo and in vitro. In vivo administration of the NO synthase inhibitor L-nitroarginine (L-NARG; 100 μM) significantly reduced NO₂^-, NO₃^- and CIT levels and prevented NMDA, kainate or KCI-evoked increases. It also potentiated ACh, ASP, GLU and GABA release and reduced that of DA in response to 50 μM NMDA whereas treatment with an NO-donor (SNAP; 10 μM) significantly reduced evoked ACh, ASP and GLU release. The NO synthase inhibitor L-NARG potentiated kainate-evoked ACh release and reduced that of DA, although less potently than NMDA, but it had no effect on KCI-evoked transmitter release. Overall, these results show that both NMDA and kainate increase striatal NO release at similar dose-thresholds as for classical transmitter release suggesting that NO is dynamically released under physiological and not just pathological conditions. Reduction of striatal NO levels also potentiates calcium-dependent transmitter release in response to NMDA and, to a lesser extent, kainate, whereas increasing them reduces it. This is consistent with a role for NO as a neuroprotective agent in this region acting to desensitize NMDA receptors.     

Polychlorinated Biphenyls PCB 153 and PCB 126 Impair the Glutamate–Nitric Oxide–cGMP Pathway in Cerebellar Neurons in Culture by Different Mechanisms

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.     

Inhibition of nitric oxide formation does not protect murine cortical cell cultures from N-methyl-d-aspartate neurotoxicity

We examined the role of nitric oxide in N-methyl-d-aspartate (NMDA) receptor-mediated neurotoxicity in rat and mouse primary cortical cell cultures. In rat and mouse cultures, the NO synthase inhibitor, N^G-Nitro-l-arginine, blocked cGMP formation but not neuronal cell death following a 5–10 min exposure to 300–500 μM NMDA. N^G-Monomethyl-l-arginine was also unable to prevent neuronal death. In contrast, the non-competitive NMDA receptor antagonist, dextrophan, prevented both cGMP formation and cell death. While other data suggest that the synthesis of nitric oxide can mediate NMDA receptor-mediated neurotoxicity, present results suggest that such synthesis is not necessarily required.     

Regulation by calcium of the nitric oxide/cyclic GMP system in cerebellar granule cells and astroglia in culture

Ca²⁺ entry induced by N-methyl-D-aspartate (NMDA) in neurons and by noradrenaline (NA) in astrocytes is known to increase intracellular cyclic GMP (cGMP) levels through stimulation of the Ca²⁺-dependent nitric oxide synthase type I (NOS-I). The possibility that Ca²⁺ entry could also down-regulate intracellular cGMP by activating a Ca²⁺/calmodulin-dependent phosphodiesterase (CaM-PDE) has been investigated here in primary cultures enriched in granule neurons or in astroglia from rat cerebellum. We show that the same agonists that stimulate nitric oxide (NO) formation (NMDA and NA at 100 μM) and the Ca²⁺ ionophore A23187 (10 μM) decrease cGMP generated in response to direct stimulation of soluble guanylyl cyclase (sGC) by NO donors in both cell types. This effect requires extracellular Ca²⁺ and is prevented by the calmodulin inhibitor W7 (100 μM). Membrane depolarization, manipulations of the Na⁺ gradient, and intracellular Ca²⁺ mobilization also decrease NO donor-induced cGMP formation in granule cells. In astroglia Ca²⁺ entry additionally down-regulates cGMP generated by stimulation of the particulate GC by atrial natriuretic peptide (ANF). Decreases in cGMP produced by A23187 were more pronounced in the absence than in the presence of the PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX; 1 mM), indicating that a CaM-PDE was involved. We also show that astroglial cells can accumulate similar amounts of cGMP than neurons in response to NO donors when IBMX is present but much lower levels in its absence. This may result from a lower ratio of sGC to PDE activities in astroglia. J. Neurosci. Res. 49:333–341, 1997. © 1997 Wiley-Liss, Inc.     

The NO-cGMP Pathway in Neonatal Rat Dorsal Horn

Incubation of slices of neonatal rat spinal cord with nitric oxide donor compounds produced marked elevations in cyclic guanosine 3',5'monophosphate (cGMP) levels. The excitatory amino acid receptor agonists N -methyl- d -aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) produced smaller increases, which were blocked by the nitric oxide synthase (NOS) inhibitor M l - N ^G-nitroarginine (NOArg), indicating that these cGMP responses were mediated by nitric oxide. Immunocytochemistry revealed that, in response to NMDA, cGMP accumulated in a population of small cells and neuropil in laminae II and III of the dorsal horn. This area was also shown, by reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry, to contain NOS. These observations suggest that, in the rat spinal cord, NMDA receptor activation is linked to the formation of NO and, hence, of cGMP. This pathway is located selectively in the superficial dorsal horn, consistent with a role in the processing of nociceptive signals.     

Ammonia triggers exocytotic release of L‐glutamate from cultured rat astrocytes

Ammonia toxicity to the brain involves NMDA receptor overactivation and glutamate excitotoxicity. The mechanisms underlying glutamate release from astrocytes in response to ammonia were addressed in this study. In cultured rat astrocytes, glutamate immunoreactivity (IR) was punctate and partly colocalized with transfected VAMP2‐YFP. NH₄Cl (5 mmol/L) and hypoosmotic exposure (205 mosmol/L) induced a rapid colchicine‐sensitive loss of cellular glutamate and glutamate appearance in the extracellular space. The NH₄Cl‐induced glutamate loss from astrocytes was strongly blunted after transfection of the cells with VAMP2 siRNA. Ammonia‐induced exocytosis of VAMP2‐YFP expressing vesicles was shown by total internal reflection fluorescence microscopy (TIRF‐M). Glutamate exocytosis in response to ammonia was sensitive to chelation of Ca²⁺, cyclooxygenase inhibition by indomethacin and colchicine. Ammonia triggered the rapid formation of prostanoids, which were identified as upstream events in ammonia‐induced glutamate exocytosis. Also, addition of prostaglandin E₂ or of tumor necrosis factor (TNF)‐α triggered glutamate exocytosis. Inhibition of ammonia‐induced glutamate exocytosis after transfection of VAMP2 siRNA inhibited ammonia‐induced RNA oxidation. It is concluded that ammonia triggers a prostanoid‐ and Ca²⁺‐dependent glutamate exocytosis, which is essential for induction of ammonia‐induced RNA oxidation. © 2009 Wiley‐Liss, Inc.     

Impairment of the nitric oxide/cyclic GMP pathway in cerebellar slices prepared from thehph-1 mouse

In this study, the effect of tetrahydrobiopterin deficiency on the nitric oxide/cGMP pathway has been investigated in cerebellar slices derived from thehph-1 mouse. This animal displays a partial deficiency of tetrahydrobiopterin. Basal levels of cGMP were significantly reduced (−29.5%) in thehph-1 mouse cerebellum compared to controls. Following kainate stimulation (500 μM) cGMP levels increased in both control andhph-1 preparations but were again significantly lower (−29.1 %) in thehph-1 mouse. Exposure of slices to the nitric oxide donors,S-nitroso-N-acetylpenicillamine andS-nitroso-glutathione, revealed no difference in cGMP accumulation between the two groups. These findings suggest that the cerebellar nitric oxide/cGMP pathway may be impaired in partial tetrahydrobiopterin deficiency states due to diminished nitric oxide formation.     

Pregnenolone Sulfate Restores the Glutamate-Nitric-Oxide-cGMP Pathway and Extracellular GABA in Cerebellum and Learning and Motor Coordination in Hyperammonemic Rats

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A presynaptic N‐methyl‐d‐aspartate autoreceptor in rat hippocampus modulating amino acid release from a cytoplasmic pool

A possible role of the N-methyl-d -aspartate receptor (NMDA-R) as a presynaptic autoreceptor was investigated using Percoll-purified hippocampus nerve terminals (synaptosomes). This preparation contained only a neglectable amount of postsynaptic structures. Two main effects of NMDA were observed. First, NMDA dose-dependently (10–100 μm ) and in the absence of Mg²⁺, stimulated basal release of aspartate and glutamate, but not of GABA. MK801 (10 μm ), an open NMDA-R-channel blocker, reduced this effect even below control levels, indicating endogenous NMDA-R activation. By superfusing synaptosomes, which prevents a tonic receptor occupation, also basal GABA release was stimulated by NMDA. The NMDA-induced potentiation of amino acid superfusate levels was blocked both by MK801 and Mg²⁺ (1 m m ), was slow in onset and returned to baseline after NMDA-removal. The NMDA-effect was also found in the absence of extracellular Ca²⁺, suggesting that amino acids were released from a non-vesicular (cytoplasmic) pool. Secondly, in KCl-depolarized synaptosomes exposed to 1 m m Mg²⁺, NMDA did not affect the release of the amino acids. MK801, however, reduced the KCl-evoked Ca²⁺-independent release of aspartate and glutamate, but not of GABA. l -trans-PDC, the selective inhibitor of the glutamate/aspartate transporter, prevented this MK801-effect, suggesting a coupling between NMDA-Rs and these transporters. These data provide evidence for a presynaptic NMDA autoreceptor in rat hippocampus. We speculate on the role of this NMDA-R to depolarize the presynaptic membrane by Na⁺-entry, which may induce reversal of amino acid transporters and thereby releasing amino acids from a cytoplasmic pool.     

Restoration of learning ability in hyperammonemic rats by increasing extracellular cGMP in brain

Intellectual function is impaired in patients with hyperammonemia and hepatic encephalopathy. Chronic hyperammonemia with or without liver failure impairs the glutamate-nitric oxide-cGMP pathway function in brain in vivo and reduces extracellular cGMP in brain as well as the ability of rats to learn a Y maze conditional discrimination task. We hypothesized that the decrease in extracellular cGMP may be responsible for the impairment in learning ability and intellectual function and that pharmacological modulation of the levels of cGMP may restore learning ability. The aim of this work was to try to reverse the impairment in learning ability of hyperammonemic rats by pharmacologically increasing extracellular cGMP in brain. We assessed whether learning ability may be restored by increasing extracellular cGMP in brain by continuous intracerebral administration of: (1) zaprinast, an inhibitor of the phosphodiesterase that degrades cGMP or (2) cGMP. We carried out tests of conditional discrimination learning in a Y maze with control and hyperammonemic rats treated or not with zaprinast or cGMP. Learning ability was reduced in hyperammonemic rats, which needed more trials than control rats to learn the task. Continuous intracerebral administration of zaprinast or cGMP restored the ability of hyperammonemic rats to learn this task. Pharmacological modulation of extracellular cGMP levels in brain may be a useful therapeutic approach to improve learning and memory performance in individuals in whom cognitive abilities are impaired by different reasons, for example in patients with liver disease who present hyperammonemia and decreased intellectual function.     

Region-specific causal mechanism in the effects of ammonia on cerebral glucose metabolism in the rat brain

Ammonia, which is considered to be the main agent responsible for hepatic encephalopathy, inhibits oxidative glucose metabolism in the brain. However, the effects of ammonia on cerebral glucose metabolism in different brain regions remains unclear. To clarify this issue, we added ammonia directly to fresh rat brain slices and measured its effects on glucose metabolism. Dynamic positron autoradiography with [¹⁸F]2-fluoro-2-deoxy-d-glucose and 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1) colorimetric assay revealed that ammonia significantly increased the cerebral glucose metabolic rate and depressed mitochondrial function, as compared to the unloaded control in each of the brain regions examined (cerebral cortex, striatum, and cerebellum), reflecting increased glycolysis that compensates for the decrease in aerobic metabolism. Pre-treatment with (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801), a N-methyl-d-aspartate (NMDA) receptor antagonist, significantly attenuated these changes induced by ammonia in cerebellum, but not in cerebral cortex or striatum. The addition of ammonia induced an increase in cyclic guanosine monophosphate (cGMP) levels in cerebellum, but not in cerebral cortex or striatum, reflecting the activation of the NMDA receptor-nitric oxide-cGMP pathway. These results suggested that NMDA receptor activation is responsible for the impairment of glucose metabolism induced by ammonia specifically in cerebellum.     

Evidence for a striatal NMDA receptor modulation of nigral glutamate release. A dual probe microdialysis study in the awake freely moving rat

Dual probe microdialysis was employed to characterize dialysate glutamate levels from the substantia nigra pars reticulata of awake freely moving rats, and to test its sensitivity to alterations in striatal neurotransmission including striatal N-methyl-d -aspartic acid (NMDA) receptor stimulation and blockade. Intranigral perfusion with low (0.1 mm ) Ca²⁺ medium (60 min) did not affect nigral glutamate levels, whereas intranigral perfusion with tetrodotoxin (10 μm , 60 min) increased nigral glutamate levels. Perfusion of KCl (100 mm , 10 min) in the dorsolateral striatum transiently stimulated nigral glutamate levels (maximal increase + 60%), whereas intrastriatal perfusion (60 min) with low Ca²⁺ medium and tetrodotoxin gradually increased nigral glutamate levels. Intrastriatal perfusion with NMDA (0.1–100 μm , 10 min) dose-dependently stimulated glutamate levels in the substantia nigra pars reticulata. The NMDA (1 μm )-induced increase in nigral glutamate release was transient and maximal (+60% within 20 min), whereas that for NMDA (10 μm ) had a slow onset but was long lasting (+35% after 60 min). Lower (0.1 μm ) and higher (100 μm ) NMDA concentrations were ineffective. The effect of intrastriatal NMDA (1 μm ) was prevented by coperfusion with MK-801 (1 μm ). Intrastriatal MK-801 (10 μm ) alone gradually increased glutamate levels up to +50% after 60 min of perfusion. The present results suggest that glutamate levels in the substantia nigra pars reticulata are sensitive to changes in neuronal transmission in the dorsolateral striatum, and that striatal NMDA receptors regulate nigral glutamate release in both a tonic and phasic fashion.