Ernesto Fedele

In vivo studies of the cerebral glutamate receptor/NO/cGMP pathway

Overwhelming evidence indicates that the glutamate/nitric oxide (NO) synthase/soluble guanylyl cyclase system is of primary importance in a variety of physiological and pathological processes of the brain. Most of our knowledge on this neurochemical pathway derives from in vitro and ex vivo studies but the recent improvement of microdialysis techniques combined with extremely sensitive measurements of the amplified end-product cyclic GMP (cGMP) has given new impulses to the investigation of this cascade of events, its modulation by neurotransmitters and its functional relevance, in a living brain. The first reports, appeared in the early 90s, have demonstrated that microdialysis monitoring of cGMP in the extracellular environment of the cerebellum and hippocampus exactly reflects what is expected to occur at the intracellular level; thus, in vivo extracellular cGMP is sensitive to NO-synthase and soluble guanylyl cyclase inhibitors, can be increased by NO-donors or phosphodiesterase blockers and is modulated by glutamate receptor stimulation in a NO-dependent fashion. Since then, other microdialysis studies have been reported showing that the brain NO synthase/guanylyl cyclase pathway is mainly controlled by NMDA, AMPA and metabotropic glutamate receptors but can be also influenced by other transmitters (GABA, acetylcholine, neuropeptides) through polysynaptic circuits interacting with the glutamatergic system. The available data indicate that this technique, applied to freely-moving animals and combined with behavioural tests, could be useful to get a better insight into the functional roles played by NO and cGMP in physiological and pathological situations such as learning, memory formation, epilepsy, cerebral ischemia and neurodegenerative diseases.

Subthalamic stimulation activates internal pallidus: Evidence from cGMP microdialysis in PD patients

Parkinsons disease patients benefit from deep brain stimulation (DBS) in subthalamic nucleus (STN), but the basis for this effect is still disputed. In this intraoperative microdialysis study, we found elevated cGMP extracellular concentrations in the internal segment of the globus pallidus, despite negligible changes in glutamate levels, during a clinically effective STN‐DBS. This supports the view that a clinically beneficial effect of STN‐DBS is paralleled by an augmentation (and not an inactivation) of the STN output onto the GPi. Ann Neurol 2005;57:448–452

Cyclo-oxygenase-1 and -2 differently contribute to prostaglandin E2 synthesis and lipid peroxidation after in vivo activation of N-methyl-D-aspartate receptors in rat hippocampus

Using intracerebral microdialysis, we reported previously that acute in vivo activation of NMDA glutamate receptors triggers rapid and transient releases of prostaglandin E2 (PGE2) and F2‐isoprostane 15‐F2t‐IsoP in the hippocampus of freely moving rats. The formation of the two metabolites – produced through cyclo‐oxygenase (COX) enzymatic activity and free radical‐mediated peroxidation of arachidonic acid (AA), respectively, – was prevented by the specific NMDA antagonist MK‐801, and was largely dependent on COX‐2 activity. Here, we demonstrate that besides COX‐2, which is the prominent COX isoform in the brain and particularly in the hippocampus, the constitutive isoform, COX‐1 also contributes to prostaglandin (PG) synthesis and oxidative damage following in vivo acute activation of hippocampal NMDA glutamate receptors. The relative contribution of the two isoforms is dynamically regulated, as the COX‐2 selective inhibitor NS398 immediately prevented PGE2 and 15‐F2t‐IsoP formation during the application of NMDA, whereas the COX‐1 selective inhibitor SC560 was effective only 1 h after agonist infusion. Our data suggest that, although COX‐2 is the prominent isoform, COX‐1 activity may significantly contribute to excitotoxicity, particularly when considering the amount of lipid peroxidation associated with its catalytic cycle. We suggest that both isoforms should be considered as possible therapeutic targets to prevent brain damage caused by excitotoxicity.

Nicotine administration stimulates the in vivo N‐methyl‐D‐aspartate receptor/nitric oxide/cyclic GMP pathway in rat hippocampus through glutamate release

1 The in vivo effects of nicotine on the nitric oxide (NO) synthase/cyclic GMP pathway of the adult rat hippocampus have been investigated by monitoring the levels of extracellular cyclic GMP during microdialysis in conscious unrestrained animals. 2 Intraperitoneal (i.p.) administration of nicotine caused elevation of cyclic GMP levels which was prevented by mecamylamine. The effect of nicotine was abolished by local infusion of the NO synthase inhibitor NG‐nitro‐L‐arginine (L‐NOARG) or by the soluble guanylyl cyclase blocker 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxaline‐1‐one (ODQ). 3 Local administration of the NMDA receptor antagonists cis‐4‐(phosphonomethyl)‐2‐piperidinecarboxylic acid (CGS19755) and dizocilpine (MK‐801) inhibited by about 60% the nicotine‐induced elevation of cyclic GMP. Nicotine was able to stimulate cyclic GMP outflow also when administered directly into the hippocampus; the effect was sensitive to mecamylamine, L‐NOARG, ODQ or MK‐801. 4 Nicotine, either administered i.p. or infused locally, produced augmentation of glutamate and aspartate extracellular levels, whereas the outflows of γ‐aminobutyric acid (GABA) and glycine remained unaffected. Following local administration of high concentrations of nicotine, animals displayed symptoms of mild excitation (sniffing, increased motor and exploratory activity) during the first 20–40 min of infusion, followed by wet dog shake episodes; these behavioural effects were prevented by mecamylamine or MK‐801, but not by L‐NOARG or by ODQ. 5 It is concluded that (a) nicotine stimulates the production of NO and cyclic GMP in the hippocampus; (b) this occurs, at least in part, through release of glutamate/aspartate and activation of NMDA receptors. Modulation of the NMDA receptor/NO synthase/cyclic GMP pathway may be involved in the cognitive activities of nicotine.

In vivo microdialysis study of a specific inhibitor of soluble guanylyl cyclase on the glutamate receptor/nitric oxide/cyclic GMP pathway

1 Nitric oxide (NO) is known to stimulate soluble guanylyl cyclase, thereby eliciting an elevation of guanosine 3′:5′‐cyclic monophosphate (cyclic GMP) in target cells. Recently, a selective inhibitor of soluble guanylyl cyclase, 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ), has been identified and characterized in vitro. We have investigated the in vivo effects of ODQ on the glutamate receptor/NO/cyclic GMP pathway by monitoring extracellular cyclic GMP during microdialysis of the cerebellum or the hippocampus of freely‐moving adult rats. 2 Intracerebellar administration of ODQ (1–100 μm) via the microdialysis probe inhibited, in a concentration‐dependent manner, the basal extracellular level of cyclic GMP. The maximal inhibition, measured after a 20 min perfusion with 100 μm ODQ, amounted to 80% and persisted unchanged as long as ODQ was perfused. When ODQ was removed from the perfusion stream after 20 min, the levels of cyclic GMP started to recover, suggesting reversibility of guanylyl cyclase inhibition by ODQ. 3 The cyclic GMP response evoked in the cerebellum by NMDA (200 μm) or by α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA; 100 μm) was largely attenuated by 100 μm ODQ. The pattern of the inhibition curves suggests competition for guanylyl cyclase between ODQ and the NO generated by NMDA or AMPA receptor activation. 4 ODQ (100 μm) prevented the elevation of extracellular cyclic GMP levels provoked by intracerebellar infusion of the NO generator S‐nitroso‐N‐acetylpenicillamine (SNAP; 1 mM). The inhibition of the SNAP effect was rapidly relieved when ODQ was removed from the perfusion fluid. However, ODQ (100 μm) was unable to affect the cyclic GMP response elicited by 5 mM SNAP, in keeping with the proposed idea that ODQ binds to the ‘NO receptor’ in a reversible and competitive manner. 5 Infusion of ODQ (10, 100 or 300 μm) into the hippocampus of freely‐moving rats diminished the basal extracellular level of cyclic GMP. The maximal inhibition amounted to 50% and was produced by 100 μm ODQ. 6 The cyclic GMP response observed when 1 mM SNAP was perfused in the hippocampus, similar in percentage terms to that seen in cerebellum, was dramatically reduced during co‐infusion of 100 μm ODQ. 7 ODQ appears to act in vivo as a selective, reversible and possibly competitive inhibitor of the soluble guanylyl cyclase targeted by NO. This enzyme may generate most (about 80%) of the cyclic GMP found under basal conditions in the extracellular space of the cerebellum. In the hippocampus, about 50% of the basal cyclic GMP does not seem to originate from the ODQ‐sensitive soluble guanylyl cyclase.

GEBR-7b, a novel PDE4D selective inhibitor that improves memory in rodents at non-emetic doses.

BACKGROUND AND PURPOSE Strategies designed to enhance cerebral cAMP have been proposed as symptomatic treatments to counteract cognitive deficits. However, pharmacological therapies aimed at reducing PDE4, the main class of cAMP catabolizing enzymes in the brain, produce severe emetic side effects. We have recently synthesized a 3‐cyclopentyloxy‐4‐methoxybenzaldehyde derivative, structurally related to rolipram, and endowed with selective PDE4D inhibitory activity. The aim of the present study was to investigate the effect of the new drug, namely GEBR‐7b, on memory performance, nausea, hippocampal cAMP and amyloid‐β (Aβ) levels.

In vivo activation of N-methyl-d-aspartate receptors in the rat hippocampus increases prostaglandin E2 extracellular levels and triggers lipid peroxidation through cyclooxygenase-mediated mechanisms

Cyclooxygenases (COX) are a family of enzymes involved in the biosynthesis of prostaglandin (PG) and thromboxanes. The inducible enzyme cyclooxygenase‐2 (COX‐2) is the major isoform found in normal brain, where it is constitutively expressed in neurons and is further up‐regulated during several pathological events, including seizures and ischaemia. Emerging evidence suggests that COX‐2 is implicated in excitotoxic neurodegenerative phenomena. It remains unclear whether PGs or other products associated to COX activity take part in these processes. Indeed, it has been suggested that reactive oxygen species, produced by COX, could mediate neuronal damage. In order to obtain direct evidence of free radical production during COX activity, we undertook an in vivo microdialysis study to monitor the levels of PGE2 and 8‐epi‐PGF2α following infusion of N‐methyl‐d‐aspartate (NMDA). A 20‐min application of 1 mm NMDA caused an immediate, MK‐801‐sensitive increase of both PGE2 and 8‐epi‐PGF2α basal levels. These effects were largely prevented by the specific cytosolic phospholipase A2 (cPLA2) inhibitor arachidonyl trifluoromethyl ketone (ATK), by non‐ selective COX inhibitors indomethacin and flurbiprofen or by the COX‐2 selective inhibitor NS‐398, suggesting that the NMDA‐evoked prostaglandin synthesis and free radical‐mediated lipid peroxidation are largely dependent on COX‐2 activity. As several lines of evidence suggest that prostaglandins may be potentially neuroprotective, our findings support the hypothesis that free radicals, rather than prostaglandins, mediate the toxicity associated to COX‐2 activity.

Acetylcholine release from rat hippocampal slices is modulated by 5-hydroxytryptamine

Experiments were performed with slices of rat hippocampus in order to investigate whether the release of acetylcholine in this area is modulated through 5-hydroxytryptamine (5-HT) receptors. The slices were prelabeled with [3H]choline then stimulated electrically twice for 4 min each at a frequency of 3 Hz. The overflow of tritium evoked was inhibited by exogenous 5-HT in a concentration-dependent manner. The 5-HT2 receptor agonist, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HC1 ((+/-)-DOI), did not mimic 5-HT. The effect of 5-HT was antagonized by methiothepin but not by the 5-HT2 antagonist, ketanserin. The 5-HT1 agonist, 5-methoxy-3-[1,2,3,6-tetrahydropyridin-4-yl]-1H-indole (RU 24969), inhibited the electrically evoked overflow of tritium, whereas the 5-HT1A-selective agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), was ineffective. Methiothepin itself, but not ketanserin, increased the evoked overflow of tritium. In contrast, the overflow was inhibited by the 5-HT uptake blocker, 6-nitroquipazine. The evoked overflow was also reduced by d-fenfluramine, a serotonin releaser. The concentration-inhibition curve for d-fenfluramine was shifted to the right by methiothepin. It is concluded that the release of ACh in rat hippocampus may be tonically inhibited by 5-HT through the activation of receptors, possibly belonging to the 5-HT1B subtype.

Age-related decrease of the NMDA receptor-mediated noradrenaline release in rat hippocampus and partial restoration by D-cycloserine

The release of [3H]noradrenaline ([3H]NA) evoked by N-methyl-D-aspartate (NMDA) from superfused rat hippocampus synaptosomes was monitored during aging. The maximal effects of NMDA decreased with age from 50% (1.5 months) to 10% enhancement (24 months). Quisqualic acid (100 microM) also enhanced [3H]NA release. Its effect decreased with age with a pattern partly different from that of NMDA. Glycine (1 microM) potentiated the [3H]NA releasing effect of 100 microM NMDA. Unexpectedly, the potentiation which amounted to 50% at 1.5 months, reached almost 200% and 300% in the 18- and 24-month-old rats, respectively, thus compensating in part for the age-related loss of the NMDA-induced effect. Concentration-response relationships for glycine at 3 vs. 24 months suggest that the glycine receptor is superresponsive in the aged brain. This may be due to more efficient glycine removal or/and to impaired release since uptake of the amino acid was increased by 350% in 24- vs. 3-month-old rats, while the K(+)-evoked tritium release from synaptosomes prelabeled with [3H]glycine was decreased. D-Cycloserine, although about 10 times less potent than glycine, strongly enhanced the NMDA-evoked [3H]NA release and may prove useful in cognitive deficits associated with aging and dementia.

Improvement of spatial memory function in APPswe/PS1dE9 mice after chronic inhibition of phosphodiesterase type 4D

Phosphodiesterase type 4 inhibitors (PDE4-Is) have received increasing attention as cognition-enhancers and putative treatment strategies for Alzheimers disease (AD). By preventing cAMP breakdown, PDE4-Is can enhance intracellular signal transduction and increase the phosphorylation of cAMP response element-binding protein (CREB) and transcription of proteins related to synaptic plasticity and associated memory formation. Unfortunately, clinical development of PDE4-Is has been seriously hampered by emetic side effects. The new isoform-specific PDE4D-I, GEBR-7b, has shown to have beneficial effects on memory at non-emetic doses. The aim of the current study was to investigate chronic cognition-enhancing effects of GEBR-7b in a mouse model of AD. To this extent, 5-month-old (5M) APPswe/PS1dE9 mice received daily subcutaneous injections with GEBR-7b (0.001 mg/kg) or vehicle for a period of 3 weeks, and were tested on affective and cognitive behavior at 7M. We demonstrated a cognition-enhancing potential in APPswe/PS1dE9 mice as their spatial memory function at 7M in the object location test was improved by prior GEBR-7b treatment. APPswe/PS1dE9 mice displayed lower levels of CREB phosphorylation, which remained unaltered after chronic GEBR-7b treatment, and higher levels of tau in the hippocampus. Hippocampal brain-derived neurotrophic factor levels and synaptic densities were not different between experimental groups and no effects were observed on hippocampal GSK3β and tau phosphorylation or Aβ levels. In conclusion, GEBR-7b can enhance spatial memory function in the APPswe/PS1dE9 mouse model of AD. Although the underlying mechanisms of its cognition-enhancing potential remain to be elucidated, PDE4D inhibition appears an interesting novel therapeutic option for cognitive deficits in AD.