cGMP pathway

Progress in Neurobiology Vol. 58, pp. 89±120, 1999 # 1999 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0301-0082/99/$ - see front matter

PII: S0301-0082(98)00077-X

IN VIVO STUDIES OF THE CEREBRAL GLUTAMATE RECEPTOR/NO/cGMP PATHWAY ERNESTO FEDELE* and MAURIZIO RAITERI Department of Experimental Medicine, Section of Pharmacology and Toxicology, University of Genova, Viale Cembrano 4, 16148 Genova, Italy (Received 20 July 1998) AbstractÐOverwhelming 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 ampli®ed 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 ®rst reports, appeared in the early 90's, have demonstrated that microdialysis monitoring of cGMP in the extracellular environment of the cerebellum and hippocampus exactly re¯ects 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 in¯uenced 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. # 1998 Elsevier Science Ltd. All rights reserved

CONTENTS 1. Introduction 2. Methodological considerations 3. In vivo studies of the NO/cGMP system in the adult rat brain 3.1. Cerebellum 3.1.1. Modulation by glutamate receptors 3.2. Hippocampus 3.2.1. Modulation by glutamate receptors and interactions with GABAergic circuits 3.2.2. Nicotine and cGMP 3.2.3. Galanin facilitation of cGMP synthesis 3.2.4. Hippocampal excitation: neurochemical and behavioural correlates 3.3. Other brain regions 4. The hippocampal and cerebellar NMDA/NO synthase/guanylyl cyclase pathway during ageing 5. cGMP production under ischemic conditions 6. Conclusions Acknowledgements References

ABBREVIATIONS [K+]o Extracellular K+ concentration 7Cl-KYNA7-Chloro-kynurenic acid 7-NINA 7-Nitroindazole monosodium salt AMPA a-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid ANF Atrial natriuretic factor cGMP Cyclic GMP, guanosine 3':5'-cyclic monophosphate CGP 52432 [3-[[(3,4-Dichlorophenyl)methyl]amino]propyl] (diethoxymethyl)phosphinic acid

CGS 19755 4-Phosphonomethyl-2-piperidinecarboxylic acid CO Carbon monoxide CTZ Cyclothiazide D-AP5 D(ÿ)-2-Amino-5-phosphonopentanoic acid DNQX 6,7-Dinitroquinoxaline-2,3-dione GABA g-Aminobutyric acid GluRs Glutamate receptors GTP Guanosine triphosphate IBMX 3-Isobutyl-1-methylxantin

* Corresponding author. 89

90 90 91 91 91 98 98 103 108 109 110 111 113 116 116 116

90 IPSP L-NAME L-NARG LTD LTP M-blue mEPSCs mGluR MK-801 nAChRs NADPH NBQX dione

E. Fedele and M. Raiteri Inhibitory post-synaptic potential NG-Nitro-L-arginine methyl ester NG-Nitro-L-arginine Long term depression Long term potentiation Methylen blue Miniature excitatory postsynaptic currents Metabotropic glutamate receptor Dizocilpine Nicotinic acetylcholine receptors Nicotinamide adenine dinucleotide phosphate 6-Nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-

1. INTRODUCTION Since its discovery, nitric oxide (NO) has attracted the attention of neuroscientists due to the pivotal roles that this gaseous transmitter plays in a variety of physiological and pathological processes of the brain. In neurons, NO is produced by either neuronal or endothelial isoforms of NO-synthase (NOS), Ca2+-calmodulin dependent, iron-heme containing ¯avoenzymes that require NADPH and tetrahydrobiopterin to convert L-arginine into L-citrulline and NO (Bredt and Snyder, 1990; Dinerman et al., 1994). As a gas, NO can easily di€use from the producing to neighbouring cells and this peculiar property has made it the most interesting and innovative molecule involved in cell to cell signalling during the last decade. In the intracellular compartment, the major target of NO, identi®ed so far, is represented by the heme group of soluble guanylyl cyclase (sGC) to which NO binds to promote the synthesis of the classical second messenger guanosine 3',5'cyclic monophosphate [cGMP; Miki et al. (1977); Palmer et al. (1987); Garthwaite et al. (1988)]. A milestone in this ®eld of research has been the demonstration that the NOS/sGC pathway is particularly coupled to the glutamatergic neurotransmission, triggering key events in synaptic plasticity phenomena involved in learning and memory, such as long term potentiation and depression [LTP and LTD, respectively; Crepel and Jaillard (1990); O'Dell et al. (1991); Shibuki and Okada (1991); Schuman and Madison (1991); Daniel et al. (1993); Zhuo et al. (1994a,b); Boulton et al. (1995); Boxall and Garthwaite (1996)]. Most of the knowledge regarding NO/cGMP synthesis, and its modulation by glutamate receptors (GluRs), has been gained essentially from in vitro and ex vivo experimental research but the development and consolidation of intracerebral microdialysis techniques has recently permitted the investigation in vivo of this neurochemical circuit. The present article will thus be focused on microdialysis studies in which cGMP extracellular levels and their changes have been monitored as index of the functioning of the GluR/NOS/guanylyl cyclase pathway in vivo. 2. METHODOLOGICAL CONSIDERATIONS Microdialysis is based on the in situ sampling of endogenous molecules by means of a microdialysis

NMDA N-Methyl-D-aspartate NO Nitric oxide NOS Nitric oxide synthase ODQ 1H-[1,2,4]Oxadiazolo[4,3-alpha]quinoxalin-1-one sGC Soluble guanylyl cyclase SNAP S-Nitroso-N-acetyl-D,L-penicillamine SZ Seizures trans-ACPDtrans-1-Aminocyclopentane-1,3-decarboxylic acid TTX Tetrodotoxin VDCCs Voltage dependent calcium channels WDS Wet dog shakes WR Wild running

probe, a specially designed cannula part of which consists of a semipermeable membrane, stereotaxically implanted into a precise brain region and continuously perfused with a physiological solution. The probe mimics a blood vessel: endogenous molecules are ®ltered through the membrane by di€usion from the extracellular environment into the perfusion ¯uid, which is collected and analyzed. Therefore, the presence in the extracellular compartment is the prerequisite for a molecule to be sampled by microdialysis. In this view, it may appear rather surprising that cGMP can be found in brain tissue dialysates since cyclic nucleotides are classical intracellular second messengers. However, in 1983 Tjornhammar and collaborators showed that stimulation of intracellular cGMP production in liver slices was paralleled by the e‚ux of the nucleotide into the incubation medium, an e€ect which could be inhibited by probenecid, thus indicating the involvement of a carrier-mediated mechanism (Tjornhammar et al., 1983). Later on, these results were reproduced using cerebellar slices stimulated with NO donors, glutamate or depolarized with high potassium and also demonstrated that neuronal cells were able to extrude cGMP from the cytosol into the extracellular space (Tjornhammar et al., 1986). Another point that is worth emphasizing regards the choice of measuring cGMP in the dialysates. Indeed, it might be argued that analysis of NO (gaseous NO, nitrite and nitrate, arginine and citrulline) would certainly represent a direct monitoring of NOS activity and its modulation by neurotransmitters' receptors. However, there are several aspects which, in our opinion, need to be taken into account: ®rst, determination of the cyclic nucleotide gives the possibility to investigate the whole pathway down to the end-product and to characterize each step involved using the selective pharmacological tools presently available. Second, it is clear that guanylyl cyclase ampli®es NO signalling thus permitting the detection of subtle changes which might be lost if looking at NO. For example, it has been shown that, under the same experimental conditions, 1 mM N-methyl-D-aspartate (NMDA) elicits a 20fold augmentation of extracellular cGMP whereas only a 300% increase of NO is observed in response to 10 mM of the GluR agonist (Luo et al., 1993). This means that it is possible to use rather low concentrations of drugs to evoke meaningful cGMP changes; in fact, concentrations as low as 100 mM

Cerebral GluR/NO/cGMP Pathway

for NMDA and 10 mM for AMPA are sucient to observe a statistically signi®cant elevation of cGMP dialysate levels (Vallebuona and Raiteri, 1993, 1994; Fedele and Raiteri, 1996). For those who may not be so familiar with microdialysis, these concentrations might still seem too high; however, it should be borne in mind that, when a drug is administered through the probe, the dialysis membrane limits the passage of the molecule from the perfusion ¯uid to the tissue (and vice versa) due to physico-chemical interactions. As a rough rule of thumb, it can be assumed that the amount of a low-molecular weight molecule reaching the tissue is ca 10±20% of the original concentration infused in the probe. Last but not least, the analytical techniques used for NO determination are quite laborious, time-consuming and often require expensive equipment whereas cGMP analysis is based on rather rapid radioimmuno assays with negligible cross-reactivity, very high sensitivity (1±2 fmol/100 ml) and that do not need special pre-treatment of the sample.

3. IN VIVO STUDIES OF THE NO/cGMP SYSTEM IN THE ADULT RAT BRAIN 3.1. Cerebellum 3.1.1. Modulation by Glutamate Receptors The ®rst evidence that monitoring in vivo cGMP extracellular levels by intracerebral microdialysis could be exploited to investigate the NOS/GC system appeared in the literature in 1993 (Vallebuona and Raiteri, 1993). This study was carried out in the cerebellum, a brain structure with a well-known laminar cytoarchitecture and de®ned neuronal circuitry, where cGMP tissue concentrations are the highest among the di€erent regions of the CNS. Furthermore, in this area GluRs, NO and the cyclic nucleotide have been implicated in LTD (Shibuki and Okada, 1991; Daniel et al., 1993; Boxall and Garthwaite, 1996), the proposed cellular mechanism for cerebellar motor learning (Ito, 1989). When cGMP was found in the cerebellar extracellular space, the question arose as to its origin under physiological, unstimulated conditions. The marked sensitivity to increasing concentrations of the NOS inhibitor NG-nitro-L-arginine (L-NARG) indicates that the basal levels of cGMP are almost completely maintained by endogenously produced NO which, in turn, stimulates sGC (Fig. 1). However, a small portion (20%) of cerebellar cGMP remains insensitive to the NOS inhibitor suggesting the existence of other routes for cGMP production. One possibility relies on the presence of low levels of carbon monoxide (CO), synthesized by heme oxygenase II, which is able to activate the soluble cyclase (Verma et al., 1993); alternatively, novel NARG-insensitive NOS isoforms might exist. It has to be reminded that conversion of GTP to cGMP can also be catalyzed by another member of the GC family, namely the particulate or membrane-bound GC. This latter hypothesis has recently found decisive support from the results obtained with 1H[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ), a new, selective and potent inhibitor of sGC

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with no e€ects on glutamate ionotropic receptors, constitutive or inducible NOS and particulate GC (Garthwaite et al., 1995). ODQ behaved exactly as L-NARG (Fig. 1): it greatly decreased extracellular cGMP in a concentration-dependent fashion but left 20% of the basal values una€ected (Fedele et al., 1996). Thus, it can be concluded that, in the cerebellum, the NO-stimulated cytosolic enzyme is responsible for the majority of cGMP extracellular levels, the membrane-bound form playing a minor role. Finally, since the phosphodiesterase inhibitor 3-isobutyl-1-methylxantin (IBMX) only doubled cGMP extracellular concentrations (Vallebuona and Raiteri, 1993), it would seem that the cerebellum does not possess an ecient phosphodiesterase degradative system, as reported previously (Greenberg et al., 1978). Overall, these ®ndings lead us to consider another point: since a `physiological' basal production of NO/cGMP takes place, then there may be an endogenous signal which activates the pathway. For the reasons mentioned above, the excitatory glutamatergic system is the most likely candidate. Indeed, administration of GluR antagonists has revealed that AMPA, but not NMDA, sites are activated by endogenous glutamate released during in vivo spontaneous ongoing synaptic activity; in fact, 6,7-dinitroquinoxaline-2,3-dione (DNQX), but not D(ÿ)-2-amino-5-phosphonopentanoic acid (D-AP5) or dizocilpine (MK-801), was able to diminish, on its own, extracellular cGMP when infused in the cerebellum (Fig. 2). This result is in keeping with the notion of the tonic and phasic nature of AMPA and NMDA receptors, respectively; however, the inhibition by DNQX was lower than that observed with L-NARG or ODQ, suggesting that other receptors [possibly metabotropic glutamate receptor (mGluR), see below] may be involved in the tonic control of NO/cGMP production. On the other hand, NMDA and AMPA receptors can be recruited by exogenous stimulation with selective agonists or by enhancing glutamate neurotransmission (Vallebuona and Raiteri, 1993; Luo et al., 1994; Fedele and Raiteri, 1996). As expected from in vitro (Bredt and Snyder, 1989; Garthwaite et al., 1989; Raiteri et al., 1991) and ex vivo (Wood, 1991; Wood et al., 1990) studies, marked cGMP elevations occur in vivo in response to infusion of NMDA, the e€ects being prevented by selective competitive or non-competitive receptor antagonists (Figs. 3 and 4). Also blockade of NOS or sGC activity by L-NARG and ODQ, respectively, results in the abrogation of the NMDA e€ects, demonstrating that NO synthesis is the primary event triggered by the GluR subtype, followed by stimulation of guanylyl cyclase. Several lines of investigation have shown that coactivation of glutamate and glycine sites is needed for NMDA receptors to function (Johnson and Ascher, 1987; Kleckner and Dingledine, 1988; Pittaluga and Raiteri, 1990). Microdialysis experiments in the cerebellum (Fedele et al., 1997a) have con®rmed that NMDA GluRs do need the presence of glycine in the extracellular space to respond to exogenous NMDA, in as much as co-infusion of the glycine site antagonist 7-chloro-kynurenic acid com-

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pletely abolished the increased cGMP e‚ux, in a competitive manner (Fig. 4). In accordance with in vitro experiments on cerebellar slices (Southam et

al., 1991), the strychnine-insensitive sites associated with the NMDA-receptor complex are not saturated by the endogenous ligand also in vivo since addition

Fig. 1. Time-course pro®le of the e€ects of NOS and sGC inhibition on the extracellular cGMP basal levels in the cerebellum of freely-moving rats. Animals were stereotaxically implanted with microdialysis probes (8 mm dialysis zone) transversally positioned in the cerebellum. Experiments have been carried out 24 hr after surgery by perfusing the probes with modi®ed Ringer's solution at a ¯ow rate of 5 ml minÿ1. L-NARG (upper panel) and ODQ (lower panel) were administered locally by retrodialysis for 80 min as indicated in the respective panels. Results are expressed as percent of the mean basal value which has been computed by averaging the cGMP content in the ®rst three fractions collected before drug treatments. The dramatic decrease of extracellular cGMP by either L-NARG or ODQ clearly indicates that the basal nucleotide concentrations result from an endogenous nitrinergic tone which stimulates the cyclase enzyme activity [data taken from Vallebuona and Raiteri (1993); Fedele et al. (1996)].

Cerebral GluR/NO/cGMP Pathway

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Fig. 2. E€ects of various GluR antagonists on the basal levels of cerebellar cGMP. All drugs have been applied locally in the cerebellum through the dialysis probe for the time indicated by the arrow. DNQX causes a signi®cant inhibition (60±70%) of the cyclic nucleotide basal levels whereas D-AP5 or MK-801 are ine€ective, thus indicating that cerebellar cGMP basal levels are almost completely maintained by endogenous glutamate mainly acting at AMPA, but not NMDA, receptors [data taken from Vallebuona and Raiteri (1993); Fedele and Raiteri (1996)]. Other receptors, however, might be involved (i.e. mGluRs; see text for explanation).

of the selective agonist D-serine to the dialysis stream greatly potentiated NMDA-induced e€ects. Therefore, administration of glycine-mimetic drugs may prove therapeutically useful to enhance

impaired cerebellar glutamate signalling in physiopathological conditions (see below). As discussed above, AMPA receptors are also coupled to cGMP production. Although these sites

Fig. 3. Increased cGMP production by exogenous NMDA: pharmacological characterization. A 20-min pulse of NMDA has been applied locally into the cerebellum whereas receptor antagonists or enzyme inhibitors were present into the perfusion stream one fraction before, together and after NMDA application. Each bar represents the time-point at which the maximal e€ect of NMDA was observed. Prevention of the NMDA-induced increase of cGMP by L-NARG con®rms that the e€ect is not direct on sGC but requires the intermediate production of NO by NOS [data taken from Vallebuona and Raiteri (1993); Fedele et al. (1996)].

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Fig. 4. In¯uence of the strychnine-insensitive glycine site on the NMDA-induced elevation of cerebellar cGMP. The e€ect of a 20-min pulse of NMDA is abolished by the glycine receptor antagonist 7-chlorokynurenic acid (7Cl-KYNA) indicating that endogenous glycine is needed for NMDA receptors to function. As expected, 7Cl-KYNA behaves as a competitive antagonist since its e€ect can be completely reversed by the selective glycinergic agonist D-serine (D-ser). The observation that, in the absence of the antagonist, D-serine is able to potentiate the cGMP response to exogenous NMDA suggests that the glycine sites associated to the NMDA receptor complex are not saturated by the endogenous ligand. Each bar represents the time-point at which the maximal e€ect of NMDA was observed [data taken from Fedele et al. (1997a)].

Fig. 5. The cerebellar NOS/sGC system is potently activated by AMPA receptors. Local infusion of AMPA (20 min) dose-dependently augments cGMP extracellular levels in a DNQX-sensitive manner. Also in this case, the e€ect is completely sensitive to L-NARG or ODQ, demonstrating that the glutamatergic receptor triggers the activation of NOS, production of NO and the subsequent stimulation of sGC. Each bar represents the time-point at which the maximal e€ect of AMPA was observed [data taken from Fedele and Raiteri (1996)].

Cerebral GluR/NO/cGMP Pathway Table 1. E€ect of cyclothiazide on the AMPA-induced increase of extracellular cGMP in the cerebellum Over¯ow (fmol/100 ml) S1 S2 S2/S1

AMPA

AMPA+Cyclothiazide

1047279 309 228 0.29520.06

12222 166 8692122 0.7112 0.10

Control rats received two consecutive pulses of AMPA (100 mM) and treated rats received cyclothiazide (100 mM) during the second pulse of AMPA. Over¯ow was calculated by subtracting the basal out¯ow from the total cGMP e‚ux induced by AMPA, S2/S1 ratios for the two animal groups were computed and compared [data taken from Fedele and Raiteri (1996)].

are tonically stimulated by endogenous glutamate, they are not fully activated in vivo as shown by the marked cGMP elevations that follow infusion of exogenous AMPA (Fig. 5). As in the case of the NMDA-induced response, L-NARG and ODQ antagonized the stimulatory e€ect of AMPA, demonstrating that it activates the NOS/sGC pathway in a conventional manner. Receptors of the AMPA type are known to desensitize and this property has been object of several electrophysiological and biochemical studies in vitro. Intriguing results have been obtained when in vivo AMPA receptor desensitization is precluded by cyclothiazide (Fedele and Raiteri, 1996). The fact that cyclothiazide is able to further enhance cGMP

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elevations induced by AMPA (Table 1) would be in line with its anti-desensitizing properties. The original ®nding that this drug elicits signi®cant, DNQXsensitive, cGMP responses when present alone in the perfusion ¯uid, reasonably suggests that the physiological tone exerted on AMPA receptors by endogenous glutamate keeps them in a partly desensitized state (Fig. 6). Surprisingly, however, this latter e€ect is also completely abrogated by the NMDA channel-blocker MK-801, indicating that cyclothiazide not only enhances the function of an ongoing AMPA receptor-mediated transmission by preventing its desensitization, but also triggers events leading to activation of NMDA receptors which are not under tonic stimulation (see Fig. 2). It might be conceivably hypothesized that the two receptors are co-localized onto NOS-positive neurons but the desensitized state of AMPA sites, caused by the endogenous glutamatergic tone, does not permit membrane depolarization sucient to relieve the voltage-dependent blockade of the NMDA receptor channel by Mg2+. In presence of cyclothiazide, AMPA-mediated depolarization reaches the threshold to activate adjacent NMDA receptors that, thus, participate in augmenting cGMP. As a matter of fact, functional relationships between colocalized ionotropic GluRs are well documented and, in particular, it has been shown that stimulation of AMPA sites appears to play a permissive

Fig. 6. Blockade of AMPA receptor desensitization by cyclothiazide (CTZ) triggers activation of NMDA receptors. The ®gure shows the time-course pattern of the e€ect of CTZ applied alone into the cerebellum for the time indicated by the arrow. The increased cGMP production is, as expected, prevented by co-perfusion of the AMPA receptor antagonist DNQX but, surprisingly, is also completely abolished by MK-801, suggesting the participation of NMDA receptors in the cGMP response to cyclothiazide [see text for a detailed discussion; data taken from Fedele and Raiteri (1996)].

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Fig. 7. The response of extracellular cGMP to exogenous AMPA in the absence of cyclothiazide does not include a NMDA component. These experiments clearly show that, when desensitization is not precluded by cyclothiazide, NMDA receptors do not contribute to the augmentation of cerebellar cGMP induced by AMPA receptor stimulation. In fact, the increase of cGMP by either low or high concentrations of AMPA is not a€ected by co-perfusion of the NMDA channel blocker MK-801. Each bar represents the time-point at which the maximal e€ect of AMPA was observed [data taken from Fedele and Raiteri (1996)].

Fig. 8. Cyclothiazide potentiates the cGMP increase induced by exogenous AMPA: antagonism by MK801. Co-operativity between AMPA and NMDA receptors in the production of cerebellar cGMP occurs only in the absence of AMPA receptor desensitization. In fact, cyclothiazide (CTZ) potentiates the AMPA-induced increase of cGMP levels and MK-801 is able to revert this e€ect, leaving the AMPAmediated component of the response una€ected. Each bar represents the time-point at which the maximal e€ect of AMPA was observed [data taken from Fedele and Raiteri (1996)].

Cerebral GluR/NO/cGMP Pathway

role on the activation of NMDA receptors in the presence of otherwise inhibiting Mg2+ concentrations (Desce et al., 1992; Raiteri et al., 1992). Although this explanation seems certainly plausible, additional experiments have shown that the scenario is far more complex. If the level of membrane depolarization were the limiting event to activate NMDA receptors, one would expect to overcome the problem by stimulating AMPA receptors with high agonist concentrations, regardless of desensitization; in other words, also cGMP elevations induced by exogenous AMPA should be sensitive, at least in part, to MK-801. This is not the case, however, as the selective NMDA non-competitive antagonist did not signi®cantly a€ect the enhancement of the cyclic nucleotide elicited by either low or high AMPA concentrations (Fig. 7). Therefore, the presence of cyclothiazide appears to play a critical role to bring NMDA receptors into action. This conclusion has been con®rmed in subsequent experiments showing that the potentiation of the AMPAinduced increase of extracellular cGMP by cyclothiazide (at a concentration ine€ective per se) is completely reverted by MK-801 (Fig. 8). It is worth noting that the NMDA channel-blocker antagonizes only the cyclothiazide component of the response without a€ecting that of AMPA. Although peculiar, this ®nding is reminiscent of some in vitro results on striatal cultured neurons, demonstrating that cGMP accumulation induced by AMPA/kainate agonists, insensitive to MK-801, was partially blocked by the NMDA antagonist in the presence of concanavalin A, a lectin known to prevent desensitization at nonNMDA receptors (Marin et al., 1993). In a recent study, Barnes et al. (1994) have shown that glutamate release from synaptosomes can be increased by AMPA only if cyclothiazide is present. In the light of this ®nding, we can venture a fascinating theory: there are two AMPA receptors, which can in¯uence the NO/cGMP pathway; one is localized onto NOSproducing neurons and shows no or a very low degree of desensitization; the other site is present on glutamatergic nerve terminals facing the above mentioned cells and represents a positive-feedback autoreceptor which, on the contrary, is `silent' unless desensitization is prevented. Under normal conditions, cGMP is mainly produced by endogenous or exogenous stimulation of the former AMPA site without the participation of NMDA receptors. When cyclothiazide is present, alone or in combination with exogenous AMPA, the glutamate AMPA autoreceptor becomes operative, increasing glutamate release which, in turn, stimulates NMDA receptors. This would explain why MK-801 is active only when desensitization of AMPA receptors is precluded. Indeed, increased glutamate release has been observed in cerebellar dialysates following cyclothiazide administration (Fedele and Raiteri, unpublished observations). Alternatively, it can be speculated that the thiazide can induce AMPA receptor changes, which favour, by unknown mechanisms, a functional interaction with neighbouring NMDA receptors, leading to their activation. Of course, a more simple explanation would see cyclothiazide as a direct activator of NMDA receptors. At face value, this possibility seems rather unli-

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kely since data reported in the literature have shown that NMDA-activated currents or NMDA-mediated ion in¯ux are not potentiated by cyclothiazide (Yamada and Tang, 1993; Hoyt et al., 1995). In addition to NMDA and non-NMDA receptors, in vitro experiments with cerebellar slices have shown that mGluR stimulation also results in the increase of cGMP, an e€ect mediated by NOS activated following Ca2+ release from intracellular stores (Okada, 1992). This ®nding has been con®rmed in vivo by showing that local application of the mGluR agonist trans-1-aminocyclopentane-1,3decarboxylic acid (trans-ACPD) elicits a six-fold increase in the extracellular nucleotide levels (Luo et al., 1994). Finally, in vivo extracellular cGMP increments in the cerebellum can also be observed when endogenous glutamate release is enhanced by systemic injections of harmaline (Luo et al., 1994). This alkaloid is able to evoke a ®ve-fold augmentation of cGMP in a tetrodotoxin (TTX)- and Ca2+-dependent fashion, the e€ect being sensitive to either NOS or sGC inhibition. Moreover, local application of CNQX or D-AP5 diminishes the harmaline-induced potentiation, further con®rming that AMPA and NMDA receptors, targeted by the endogenous ligand, are deeply involved in the control of NO/ cGMP production in this brain region (Fig. 9). To conclude, we will try to organize all these neurochemical data into a conceivable picture for a better understanding of the anatomical/functional relationships between the cerebellar glutamatergic and the NO/cGMP systems (Fig. 10). Cerebellar granule cells, the target of mossy ®bers, possess NMDA, AMPA and metabotropic receptors (Nicoletti et al., 1986; Garthwaite and Brodbelt, 1989; Masu et al., 1991; Seeburg, 1993; Pin et al., 1994) and contain high levels of NOS (Bredt et al., 1990; Vincent and Kimura, 1992); a similar distribution occurs in inhibitory GABAergic interneurons (Chan-Palay, 1982; Hussain et al., 1991; Southam et al., 1992). On the other hand, sGC, cGMP-dependent protein kinase and the G-substrate have been mainly localized in Purkinje neurons (Schlichter et al., 1980; Lohmann et al., 1981; Zwiller et al., 1981; Ariano et al., 1982; Nakane et al., 1983; De Camilli et al., 1984; Matsuoka et al., 1992), which express glutamate ionotropic and metabotropic receptors, but seem to lack the machinery for NO production. Therefore, under physiological conditions in vivo, endogenous glutamate is released from mossy ®bers onto granule cells where it activates NOS mainly through AMPA receptors (possibly also mGluRs), whereas NMDA receptors are silent, probably due to the Mg2+ blockade of the receptor channel. NO produced in granules can di€use into Purkinje neurones where it binds to sGC, thus stimulating the production of cGMP. The cyclic nucleotide is then metabolized by phosphodiesterases but part of it is extruded into the extracellular space where it can be monitored by microdialysis. Alternatively, or in addition, NO can be formed in basket cells activated by parallel ®bres via AMPA receptors. NMDA receptors, however, can be recruited when AMPA receptor desensitization is prevented, by stimulation with the exogenous agonist or when endogenous

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Fig. 9. Harmaline-evoked cGMP elevation in the cerebellum of freely-moving rats. Local infusion of harmaline causes signi®cant, Ca2+- and TTX-dependent augmentation of extracellular cGMP, which depends upon NOS and sGC activities since it is greatly reduced by L-NARG or methylen blue (Mblue). The results showing a decreased cGMP response in the presence of D-AP5 or CNQX, indicates that the endogenous glutamate released by harmaline mainly acts onto NMDA and AMPA receptors to stimulate the NOS/sGC pathway. Each bar represents the time-point at which the maximal e€ect of harmaline was observed [data taken from Luo and Vincent (1994)].

glutamate release is enhanced by harmaline. The e€ect of harmaline deserves some comments. The alkaloid activates selectively cerebellar climbing ®bers (LaMarre et al., 1971; Llinas and Volkind, 1973) which directly contact Purkinje cells; but these neurons apparently do not contain NOS. Interestingly, it has been demonstrated that collaterals of the climbing ®bers excite the deep cerebellar nuclei where the mossy ®bers pathway originates (Llinas and Muhlethaler, 1988; Van der Want et al., 1989; Audinat et al., 1992), which, in turn, activates granule cells. 3.2. Hippocampus 3.2.1. Modulation by Glutamate Receptors and Interactions with GABAergic Circuits The hippocampus is the limbic region of the brain mostly involved in learning and in the early stages of memory formation. It is widely accepted that hippocampal LTP represents the cellular mechanism associated with cognitive functions and evidence has accumulated indicating that the GluR/NOS/sGC pathway is instrumental for the expression of this phenomenon of neuronal plasticity. As in the cerebellum, microdialysis experiments with L-NARG and ODQ (Vallebuona and Raiteri, 1994; Fedele et al., 1996) have shown that extracellular cGMP levels in the hippocampus depend on the activity of NOS and sGC but a substantial role is also played by particulate guanylyl cyclase, in as much as the two selective enzyme inhibitors diminish cGMP production only by 50% (Fig. 11).

Indeed, immunocytochemical and neurochemical studies have shown that the hippocampus possesses moderate levels of the membrane-bound GC, probably localized in glial cells, which can be selectively stimulated by the atrial natriuretic factor (ANF) (Gibson et al., 1986; de Vente et al., 1988; Friedl et al., 1989; de Vente et al., 1990a). Once produced, cGMP is almost completely metabolized by hippocampal phosphodiesterases since IBMX is able to increase dramatically (eight-fold) the cyclic nucleotide extracellular levels (Vallebuona and Raiteri, 1994), in keeping with in vitro data showing that this brain region has a very ecient degradative system (Greenberg et al., 1978). Also in this region, NMDA receptors seem not to be involved in the maintenance of basal concentrations of cGMP (MK-801 does not a€ect nucleotide extracellular values), whereas data obtained with DNQX, 6nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione (NBQX) and cyclothiazide have revealed a complex, double modulation of the NOS/sGC system by AMPA receptors [Fig. 12; Vallebuona and Raiteri (1994); Fedele et al. (1997b,c)]. The marked increase of extracellular cGMP observed following infusion of the competitive non-NMDA receptor antagonists clearly demonstrates that, under basal conditions, endogenous glutamate inhibits the activity of NOsensitive guanylyl cyclase by tonically stimulating AMPA receptors. Of course, these e€ects can be explained only by assuming that AMPA receptor activation elicits the release of inhibitory transmitters, which negatively modulate the NO/cGMP production. Several studies have demonstrated that excitatory glutamatergic circuits in the hippocampus

Fig. 10. Schematic drawing illustrating the GluRs/NOS/sGC pathway in the cerebellum of the rat. According to the in vivo data summarized in this article and taking into account the neuro-anatomical information available, it would seem that, under unstimulated conditions, most of the extracellular cGMP in the cerebellum derives from the stimulation by endogenous glutamate of granule cell AMPA receptors that, in turn, activate NOS present in these neurons (also mGluRs might, in part, be involved). The NO produced di€uses into Purkinje neurons where it binds to sGC, thus inducing the production of cGMP. The cyclic nucleotide is then degraded by phosphodiesterases and, in part, extruded from the cell into the extracellular environment. Alternatively, NO can be produced by basket cells, which express NOS, AMPA receptors and are contacted by granule cells. Endogenous glutamate, probably released during mossy ®bers ongoing synaptic activity, does not saturate these sites, however, since exogenous AMPA is able to potently enhance extracellular cGMP. NMDA receptors present on granule cells are normally `silent', probably due to the Mg2+ blockade of the channel, but can be recruited using exogenous NMDA or by enhancing endogenous glutamate release with harmaline. Interestingly, under basal conditions, NMDA receptors become operative in stimulating cGMP production when AMPA receptor desensitization is prevented by cyclothiazide, indicating some sort of functional cross-talk between the two glutamate sites (see text for further explanation).

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Fig. 11. Extracellular cGMP origin in the hippocampus: possible involvement of soluble and particulate guanylyl cyclases. As in the cerebellum, the basal extracellular levels of cGMP are sensitive to NOS or sGC inhibition. However, in this brain region, L-NARG (upper panel) and ODQ (lower panel) are able, at best, to halve extracellular cGMP even at supramaximal concentrations. Since ODQ is a selective inhibitor of sGC, the remaining 40±50% of extracellular cGMP measured under basal conditions can be only attributed to the activity of NO-insensitive particulate guanylyl cyclase, unless new, ODQ-resistant isoforms of the soluble enzyme exist [data taken from Vallebuona and Raiteri (1994); Fedele et al. (1996)].

are under strong inhibitory GABAergic control (Lacaille et al., 1989; Sloviter, 1991) and, vice versa, it has been shown that hippocampal GABAergic inhibitory post-synaptic potentials (IPSPs) are modulated by AMPA receptors (Andreasen et al., 1988).

Thus, DNQX or NBQX would disinhibit NOS-containing neurons by decreasing the AMPA-mediated activation of local GABAergic circuits. Accordingly, cGMP elevations should be observed also by limiting GABAergic synaptic inhibition with selective

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Fig. 12. E€ect of GluR antagonists on the basal levels of hippocampal cGMP: involvement of GABAergic circuits. Similarly to the cerebellum, local perfusion of MK-801 does not alter extracellular cGMP, indicating that NMDA receptors are not responsible of the basal production of cGMP in the hippocampus. Unexpectedly, the AMPA receptor antagonists DNQX and NBQX increase, rather then inhibit, the nucleotide extracellular concentrations, suggesting that an AMPA-driven inhibitory control is tonically exerted on the NOS/sGC system by endogenous glutamate under physiological conditions. Hippocampal GABAergic circuits seem to mediate this paradoxical e€ect since bicuculline or CGP52432, GABAA and GABAB selective antagonists, respectively, mimic the e€ect of AMPA antagonists on cGMP. Accordingly, the GABAA and GABAB receptor agonists, muscimol and (ÿ)baclofen can decrease the basal extracellular cGMP concentration. The hypothesis of an AMPA-driven GABAergic inhibitory circuit controlling the hippocampal NOS/sGC pathway has been further corroborated by the experiments showing that the potentiating e€ect of NBQX can be diminished by the co-perfusion of muscimol or baclofen. Local administration of a mixture of NBQX, baclofen and muscimol almost completely abolished the e€ect of the AMPA receptor antagonist. Each bar represents the time-point at which the maximal e€ect of the various drugs was observed [data taken from Fedele et al. (1997b,c)].

GABA receptor antagonists. In fact, either bicuculline or CGP52432 could enhance extracellular cGMP, although to a di€erent extent (80 versus 25%, respectively), further indicating that the inhibitory tone is exerted mainly through GABAA receptors, with GABAB sites giving a minor contribution. Moreover, it would seem that neither of these receptors is saturated by the endogenous ligand since both the GABAA receptor agonist muscimol and the GABAB agonist (ÿ)baclofen were able to signi®cantly decrease cGMP basal values. Finally, additional evidence in favour of this view comes from the results showing that the NBQX-induced potentiation of cGMP synthesis can be reduced by pharmacologically restoring GABAergic synaptic inhibition with exogenous GABAA and GABAB agonists (Fig. 12). These observations highlight in

vivo functional interactions between hippocampal glutamatergic and GABAergic systems but they do not clarify the neurochemical signals responsible for the NO-mediated production of basal extracellular cGMP. However, it would seem that endogenous glutamate is again involved by tonically stimulating other AMPA receptors, which excite NOS-containing cells. In fact, local application of cyclothiazide alone results in a signi®cant, though modest increase of cGMP (Fig. 13); if the AMPA receptors, driving GABA-mediated synaptic inhibition on the NOS/ sGC pathway, were the only ones involved, prevention of desensitization would be expected to cause inhibition, and not potentiation, of extracellular cGMP levels. Altogether, these ®ndings suggest that the spontaneous production of NO and cGMP in the hippocampus results from the physiological bal-

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Fig. 13. Cyclothiazide unmasks the presence of AMPA receptors increasing cGMP production in the hippocampus of freely-moving rats. The ®gure shows the temporal pro®le of the e€ect of cyclothiazide directly infused into the hippocampus for the time indicated by the arrow. The drug signi®cantly increases extracellular cGMP and demonstrates that the hippocampal NOS/sGC system is also under a tonic, AMPA-mediated excitatory in¯uence, which becomes evident when desensitization is prevented [data taken from Fedele et al. (1997b)].

ance between two AMPA-driven inputs which exert opposite, excitatory and inhibitory, regulations on the biochemical pathway leading to cGMP formation (Fig. 16). Moreover, the fact that cyclothiazide renders the excitatory tone prevailing over the inhibitory one, might also indicate that the two AMPA receptors are pharmacologically di€erent. Recently, native AMPA receptor subtypes have been pharmacologically characterized in in vitro functional studies on the basis of their di€erent desensitizing properties (Pittaluga et al., 1997). In the previous paragraph, we have seen that, in the cerebellum, blockade of receptor desensitization by cyclothiazide reveals functional links between AMPA and NMDA receptors. In the hippocampus in vivo, this phenomenon of receptor±receptor interaction can be unmasked by lowering extracellular Mg2+ concentration (Fedele et al., 1997b,c), an experimental procedure that mimics a physiological relief of the NMDA channel block and has been extensively used to bring to light NMDA receptor functions. The mere omission of Mg2+ ions from the dialysis stream, however, is not sucient to induce NMDA receptor activation by endogenous glutamate basal concentrations since application of MK-801 alone does not a€ect extracellular cGMP. On the contrary, the cGMP response to cyclothiazide is markedly enhanced under Mg2+-free conditions and such additional elevation can be completely reverted by competitive or non-competitive NMDA receptor antagonists. Similar results can be observed with bicuculline and CGP52432, the surplus of cGMP production caused by low

magnesium being abolished by NMDA receptor blockade (Fig. 14). Therefore, it can be assumed that NMDA receptor activation is favoured when AMPA-mediated fast excitatory neurotransmission is enhanced and/or GABAergic synaptic inhibition is reduced. As already discussed, stimulation of AMPA receptors has been reported to permit NMDA receptor activation (Desce et al., 1992; Raiteri et al., 1992) and it is well established that blockade of the GABAergic inhibitory control over the hippocampal glutamatergic circuit facilitates NMDA-mediated events (WigstroÈm and Gustafsson, 1983; Herron et al., 1985; Dingledine et al., 1986; Gustafsson et al., 1987; Collingridge et al., 1988; Aram et al., 1989; Sloviter, 1991). Although far from being completely elucidated (the reader is referred to the original studies for a detailed discussion), the above mentioned ®ndings uncover a complex endogenous modulation of the NOS/sGC cascade that deserves further attention in the light of its important roles in glutamate-mediated neuronal processes of the hippocampus. Moving on to the pharmacological modulation of the GluR/NOS/sGC pathway with exogenous GluR agonists, microdialysis studies have con®rmed that hippocampal cGMP formation can be accelerated by NMDA administration, in a concentration- and D-AP5-dependent manner; moreover, the fact that the potentiation is prevented by L-NARG shows that NMDA receptors do not directly activate soluble guanylyl cyclase but NO production is required (Fig. 15). In contrast, when AMPA is infused in the hippocampus, at concentrations able to evoke

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Fig. 14. E€ects of cyclothiazide, bicuculline or CGP52342 on cGMP hippocampal levels under low extracellular magnesium concentration: role of NMDA receptors. The removal of MgCl2 from the infusion ¯uid does not a€ect cGMP basal values on its own, but results in the potentiation of cGMP responses to cyclothiazide, bicuculline or CGP52432, an e€ect that is completely reversed by the selective NMDA receptor antagonists MK-801 or CGS19755. These results show that NMDA receptors, stimulated by endogenous glutamate, can participate to cGMP formation when decreased GABAergic inhibition or AMPA receptor desensitization coincides with conditions that facilitate NMDA channel opening. Each bar represents the time-point at which the maximal e€ect of the various drugs was observed [data taken from Fedele et al. (1997b,c)].

marked e€ects in the cerebellum, no changes in extracellular cGMP can be observed. According to the picture proposed above, this may be due to exogenous activation of the two AMPA-driven circuits resulting in excitatory and inhibitory e€ects on NOS/sGC system, which annul each other. Supporting this idea, reproducible, robust cGMP elevations can be obtained with exogenous AMPA by reducing GABAA and GABAB mediated synaptic inhibition (Fig. 15) or by preventing AMPA receptor desensitization (Fedele and Raiteri, unpublished results). In the hippocampus, di€erent studies have shown that NMDA and AMPA receptors are mainly localized on pyramidal and granule cells [see Hollmann and Heinemann (1994)]; these neurones also express GABAA and GABAB receptors (Bowery et al., 1987; Chu et al., 1990; Wisden et al., 1992). On the other hand, NOS has been found in polymorphic neurons of the hilus, interneurons scattered in stratum oriens and radiatum, in the pyramidal and granule cell layers (Bredt et al., 1990, 1991; Leigh et al., 1990; Schmidt et al., 1992; Vincent and Kimura, 1992; Valtschano€ et al., 1993). Recent improvement of NADPH-diaphorase immunocytochemistry and the use of cNOS in situ hybridization have demonstrated that also CA1 pyramidal neurons express the NO synthesizing enzyme (Dinerman et al., 1994; Endoh et al., 1994; Wendland et al., 1994). As for soluble guanylyl cyclase, the `NO receptor' seems to be present in the pyramidal layer of the CA1±3 regions and in the granule cell layer of the dentate gyrus (Matsuoka et al., 1992; Burgunder and Cheung, 1994) where intense cGMP immunos-

taining can be detected (Southam and Garthwaite, 1993). Such an intricate scenario, with di€erent neuronal populations possessing multiple receptors and able to produce NO and cGMP, makes almost impossible the anatomical localization of the in vivo biochemical events here described. However, it is worth recalling that functional interplay between the hippocampal glutamatergic and GABAergic systems are instrumental for the occurrence of LTP (WigstroÈm and Gustafsson, 1983; Gustafsson et al., 1987; Collingridge and Singer, 1991), an electrophysiological process linked to memory formation that requires, at least under certain conditions, the activation of the NOS/sGC system (Schuman, 1995; Boulton et al., 1995). 3.2.2. Nicotine and cGMP Among the central neurotransmitter systems involved in learning and memory formation, a prominent role is held by glutamatergic and cholinergic neuronal circuits (Squire and Davis, 1981; Morris et al., 1986; Hawkins et al., 1993) especially in the hippocampus, a brain region that, since the late 50's (Scoville and Milner, 1957), is known to be of fundamental importance for information storage in mammals, including humans (Squire, 1992). Recent results have shown that stimulation of nicotinic acetylcholine receptors (nAChRs) improves memory performance in a wide variety of tasks both in experimental animals (Levin, 1992; Brioni and Arneric, 1993; Decker et al., 1993; Buccafusco et al., 1995; Zarrindast et al., 1996) and in healthy human volunteers (Levin, 1992; Warburton, 1992); in addition, nicotine has

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Fig. 15. Modulation of hippocampal cGMP levels by exogenous NMDA and AMPA. The upper panel shows that a 20-min local application of NMDA into the hippocampus evokes concentration-dependent increases of cGMP that are abolished by the selective receptor antagonist D-AP5. The observation that co-perfusion of L-NARG also precludes the cGMP response to exogenous NMDA, con®rms that NOS activation is an intermediate and crucial step for the e€ect to occur. The lower panel reports the e€ects of exogenous AMPA on cGMP extracellular levels. When the glutamatergic agonist is infused alone into the hippocampus, no signi®cant changes can be detected. On the contrary, exogenous AMPA evokes reproducible cGMP elevations in the presence of low concentrations of bicuculline and CGP52432. Each bar represents the time-point at which the maximal e€ect of the various drugs was observed [data taken from Vallebuona and Raiteri (1994); Fedele et al. (1997c)].

been found to ameliorate impaired cognitive functions in Alzheimer patients (Levin, 1992; Warburton, 1992; Lawrence and Sahakian, 1995;

Whitehouse and Kalaria, 1995; Wilson et al., 1995). Despite these unquestionable central e€ects, it is strikingly surprising that documentation of

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Fig. 16. Glutamatergic regulation of the hippocampal NOS/sGC pathway. The observation that hippocampal cGMP increases when AMPA receptors are either blocked (DNQX, NBQX) or stimulated (cyclothiazide can be seen as an AMPA receptor activator) clearly indicates the existence of a double, inhibitory and excitatory control by endogenous glutamate on the NOS/sGC pathway. The inhibitory tone seems to be mediated by GABA since the e€ect of AMPA receptor antagonists on cGMP can be abolished by GABAA and GABAB agonists. Thus, it can be concluded that one AMPA receptor is present on GABAergic interneurons and drives the release of GABA, which potently mediates synaptic inhibition onto the NOS-containing cells through both GABAA and GABAB receptors. The other AMPA receptor, whose potentiating e€ects on extracellular cGMP can be revealed by preventing its desensitization, is probably directly linked to the NOS/sGC system. In keeping with this view, exogenous AMPA does not elicit signi®cant changes of the basal cGMP levels, probably because the two AMPA-mediated excitatory and inhibitory e€ects annul each other. On the contrary, cGMP elevations in response to exogenous AMPA can be reproducibly observed when GABAA- and GABAB-mediated inhibition is prevented with selective antagonists or when desensitization is precluded by cyclothiazide. NMDA receptors are not operative under physiological conditions but their involvement in the enhancement of hippocampal cGMP synthesis can be demonstrated by stimulating the receptors with exogenous NMDA or by combining low Mg2+ with blockade of AMPA receptor desensitization or reduction of GABAA and GABAB synaptic inhibition. Whatever pharmacological manipulation is considered, NMDA activation leads to the appearance of epileptic-like episodes that, however, seem not to be related to the ability of the receptor in augmenting NO and cGMP formation. Moreover, endogenous glutamate release can be enhanced by presynaptic nicotinic acetylcholine receptors, resulting in the activation of the hippocampal NO/cGMP cascade mainly through NMDA receptors, although a contribution by AMPA sites can not be ruled out. For the sake of clarity, the NOS/sGC system has been depicted as a single compartment but it is well known that the two enzymes are localized also in di€erent hippocampal populations.

synaptic transmission mediated by nicotinic receptors in the brain is rather scanty; thus, in these last few years, the idea has emerged that nicotine may essentially modulate, rather than mediate,

transmission [see Wonnacott (1997) and references therein]. Since excitatory glutamate transmission in the hippocampus is intimately involved in cognitive processes, it can represent a likely target for nic-

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Fig. 17. E€ect of systemic nicotine on the hippocampal NO/cGMP cascade: relevance of NMDA receptors. The ®gure reports the time-course pattern of the e€ect of nicotine, administered intraperitoneally (arrow), on hippocampal cGMP. Upper panel, nicotine (Nic) enhances extracellular cGMP is a dosedependent fashion, the e€ect being abolished by the selective nAChR antagonist mecamylamine (Mec) delivered locally into the hippocampus by retrodialysis. The cGMP response depends upon NOS and sGC activities since the e€ect of nicotine is prevented by intrahippocampal administration of L-NARG or ODQ. Lower panel, the cGMP elevation by i.p. nicotine is greatly diminished by local infusion of MK-801 or CGS19755, suggesting the involvement of NMDA receptors [data taken from Fedele et al. 1988].

otinic receptor modulation. Actually, electrophysiological experiments on hippocampal slices or cultured neurons have found that nicotine is able to enhance the frequency of spontaneous miniature

excitatory postsynaptic currents (mEPSCs) without altering their amplitude, a ®nding consistent with a presynaptic facilitation of glutamate release (McGehee et al., 1995; Gray et al., 1996).

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Fig. 18. E€ect of local administration of nicotine on hippocampal cGMP e‚ux. In these experiments, nicotine has been delivered into the hippocampus by retrodialysis for the time indicated by the arrow. Also in this case, nicotine (Nic) is able to evoke mecamylamine (Mec)-sensitive elevations of extracellular cGMP in a NOS/sGC-dependent manner. In fact, either L-NARG or ODQ completely prevented the e€ect of nicotine. The result, showing that MK-801 signi®cantly decreased the e€ect of nicotine, further con®rms that hippocampal NMDA receptors mediate the cGMP increment induced by the alkaloid [data taken from Fedele et al. 1998].

Is this increase of excitatory synaptic transmission able to elicit neurochemical responses known to be linked to activation of GluRs? Indeed, systemic administration of nicotine causes dose-dependent, mecamylamine-sensitive augmentations of extracellular cGMP in the rat hippocampus as assessed by transcerebral microdialysis [Fig. 17, Fedele et al. 1998]. Of course, this route of administration does not help to de®ne whether extra- or intrahippocampal nACh receptors are responsible of the e€ect; however, it should be noted that in the experiments with mecamylamine, the nicotinic receptor antagonist has been infused locally into the hippocampus indicating that nAChRs localized inside this brain region are involved. Such view has been convincingly con®rmed by the ®nding that retrodialysis delivery of nicotine directly into the hippocampus also results in the elevation of cGMP extracellular levels (Fig. 18). In both cases, the increased rate of cGMP production induced by nicotine requires the intermediate activation of NOS, synthesis of NO and subsequent stimulation of soluble GC (Figs. 17 and 18). If nicotine acts presynaptically onto hippocampal glutamatergic nerve terminals stimulating transmitter release, the cGMP response should be mediated by postsynaptic GluRs. The ®ndings that competitive as well as non-competitive NMDA receptor antagonists greatly reduced the e€ect of nicotine ®t with this premise (Figs. 17 and 18). Neither drug, however, completely abolished the response to nicotine, suggesting that, besides NMDA, other receptors can contribute to the e€ect. Naturally, AMPA sites are another likely target for glutamate and, as considered above, they are able to activate the

NOS/sGC system. Unfortunately, the fact that nonNMDA receptor antagonists (i.e. DNQX or NBQX) markedly enhance, on their own, the cyclic nucleotide basal levels, thus masking their possible antagonistic activity on the nicotine-induced e€ect, has precluded testing this possibility. Alternatively, Ca2+-permeable postsynaptic nicotine receptors (Albuquerque et al., 1997), located onto NOS-positive neurons, might be directly implicated. McGehee et al. (1995) have, in fact, found that high concentrations of nicotine can activate postsynaptic nAChRs leading to potentiation of macroscopic currents in innervated lumbar sympathetic ganglion neurons. Although all these data are suggestive of a presynaptic action of nicotine that sets in motion the NOS/sGC machinery, still a direct demonstration of enhanced glutamate release represents the missing link in the whole story. Many in vitro studies on brain synaptosomes have documented that nicotine is able to enhance dopamine (Grady et al., 1992; Whiteaker et al., 1995; Clarke and Reuben, 1996), noradrenaline (Clarke and Reuben, 1996) and acetylcholine (Marchi and Raiteri, 1996; Wilkie et al., 1996) release, but evidence that it does so on glutamatergic nerve terminals is still lacking. However, microdialysis investigations have revealed (Fig. 19) that nicotine can potently stimulate endogenous glutamate/aspartate release in the hippocampus in vivo (Toth, 1996; Fedele et al., 1998), even if it is dicult to understand whether this represents a direct or indirect action. Since there is now overwhelming evidence from electrophysiological, neurochemical and behavioural studies that GluRs, NO and cGMP are essential for

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Fig. 19. Systemic or local administration of nicotine augments the release of endogenous excitatory amino acids in the hippocampus. The ®gure shows that nicotine, administered i.p. (upper panel) or continuously infused into the hippocampus (lower panel), potentiates the e‚ux of endogenous aspartate and glutamate, leaving that of GABA and glycine una€ected [data taken from Fedele et al. 1998].

learning and memory formation, the in vivo results summarized above (see also Fig. 16) might provide the basis for a better understanding of the cognitiveenhancing e€ects of nicotine; moreover, this type of study can be extended to other nootropic drugs to evaluate whether activation of the hippocampal NOS/sGC system represents a common mechanism of action for ameliorating cognitive de®cits.

3.2.3. Galanin Facilitation of cGMP Synthesis In a recent microdialysis study (Consolo et al., 1998), it has been found that hippocampal cGMP levels present in the extracellular milieu can be signi®cantly increased by galanin which shows a bellshaped dose±response curve typical of many neuropeptides. The augmentation induced by galanin was

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Fig. 20. Galanin increases cGMP levels in the ventral hippocampus by activating extrahippocampal receptors. The neuropeptide galanin (Gal) increases extracellular cGMP in the ventral, but not in the dorsal, hippocampus when administered i.c.v. The selective peptide receptor antagonist M40 completely antagonizes the galanin-induced elevation of cGMP. On the contrary, no e€ects can be observed if the peptide is delivered locally into the hippocampus, suggesting that galaninergic receptors located outside this brain region are implicated. In as much as hippocampal infusion of L-NAME, 7-NINA or ODQ abrogates the cGMP response to galanin, it can be concluded that the peptide activates the hippocampal NOS/sGC pathway in a conventional manner. The ®nding that MK-801, but not NBQX, prevents the stimulatory action of galanin indicates that the peptide exerts its e€ects on cGMP through the activation of NMDA receptors. Each bar represents the time-point at which the maximal e€ect of the various drugs was observed [data taken from Consolo et al. (1998)].

mediated by speci®c galaninergic receptors, being almost completely abolished by the selective antagonist M40 (Bartfai et al., 1993). In as much as NGnitro-L-arginine methyl ester (L-NAME), 7-NINA and ODQ were all able to prevent these e€ects, it can be concluded that galanin does not act on particulate GC but, instead, triggers the activation of NOS, which, in turn, stimulates the soluble form of the cyclase enzyme (Fig. 20). Interestingly, the potentiation of extracellular cGMP by galanin shows some sort of region selectivity since the e€ect can be observed in the ventral but not in the dorsal hippocampus. Although it is well documented that, within the hippocampus, galanin is localized in cholinergic a€erents originating from the septum and is released in vivo in relation to the activity of the septo-hippocampal pathway (Fisone et al., 1987; Consolo et al., 1994), the peptide e€ects on the hippocampal NOS/ sGC system do not seem to be mediated by intrahippocampal galanin receptors. In fact, the dialysate levels of the cyclic nucleotide are enhanced if galanin is administered i.c.v. whereas no changes can be observed by direct, intrahippocampal injection of the peptide. Whatever the situation might be, extrahippocampal galaninergic receptor stimulation results in the enhancement of NO/cGMP production via activation of hippocampal NMDA, but not AMPA, receptors (Fig. 20), indicating the involvement of endogenous glutamate release. Unfortunately, this parameter has not been monitored in the study by Consolo et al. (1998) and

therefore the question remains open. Surely, the e€ects of galanin can not be direct but need to be explained by assuming the involvement of polysynaptic circuits which cause excitation of glutamatergic inputs to the hippocampus leading to incremented activity of the NOS/sGC system; this reasoning appears justi®ed because di€erent studies have characterized galanin as an inhibitory, hyperpolarizing neuropeptide (Dutar et al., 1989; Lindskog et al., 1992; Zini et al., 1993). However, the existence of other types of galaninergic receptors able to induce cell excitation, as proposed in the case of galanin-induced stimulation of growth hormone release (Gabriel et al., 1993; Wynick et al., 1993), can not be excluded.

3.2.4. Hippocampal Excitation: Neurochemical and Behavioural Correlates There is no doubt that excessive glutamate transmission in the hippocampus leads to pre-epileptic/ epileptic states that, in the rat, are characterized by di€erent behavioural manifestations including increased motor and exploratory activity, wet dog shakes (WDS), wild running (WR) and seizures (SZ). Since most of the in vivo studies reviewed in this article have been carried out on awake, freelymoving animals and behavioural modi®cations, induced by di€erent treatments, have been reported (Vallebuona and Raiteri, 1994; Fedele et al., 1997b,c; Fedele et al., 1998), we can now attempt to draw some conclusions regarding the role of hippo-

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Table 2. Correlations between the rats' behaviour and the cGMP extracellular changes induced by various drugs acting at the GluR/NOS/sGC pathway Drugs (mM) NMDA (125) NMDA (250) NMDA (500) NMDA (1000) NMDA (500) D-AP5 (500) NMDA (500) L-NARG (10) NMDA (500) ODQ (100) Cyclothiazide (300)* Cyclothiazide (300)*+MK-801 (30) Cyclothiazide (300)*+L-NARG (100) Cyclothiazide (300)*+ODQ (100) Bicuculline (50)* Bicuculline (50)*+MK-801 (30) CGP52432 (30)* CGP52432 (30)*+MK-801 (30) Nicotine (20 mM) Nicotine (20 mM)+Mecamylamine (2 mM) Nicotine (20 mM)+MK-801 (30) Nicotine (20 mM)+L-NARG (100) Nicotine (20 mM)+ODQ (100) SNAP (1 mM) IBMX (1 mM)

WDS

WR

SZ

cGMP (% of basal)

+ ++ ++ ++ Ð ++ ++ ++ Ð ++ ++ +++ + ++ + ++ Ð Ð ++ ++ Ð Ð

Ð Ð + + Ð + + Ð Ð Ð Ð +++ + ++ Ð Ð Ð Ð Ð Ð Ð Ð

Ð Ð Ð + Ð Ð Ð Ð Ð Ð Ð ++ Ð Ð Ð Ð Ð Ð Ð Ð Ð Ð

155 275 300 310 120 110 107 274 154 82 102 420 200 175 115 325 160 150 102 90 250 800

See text for explanation. WDS, wet dog shakes; WR, wild running; SZ, seizures.+, Crosses represent the severity of the behavioural episodes. Asterisks indicate experiments carried out with Mg2+-free medium (data taken from Vallebuona and Raiteri, 1994; Fedele et al., 1997b,c; Fedele et al., 1998).

campal NO and cGMP in the genesis of limbic seizures. As can be seen in Table 2, hippocampal excitation coupled to behavioural responses can be achieved in di€erent ways: direct stimulation of NMDA receptors; blockade of AMPA receptor desensitization (cyclothiazide); decrease of GABAergic synaptic inhibition (bicuculline, CGP52432) or increase of endogenous glutamate release (nicotine). In all cases, as seen so far, these e€ects are paralleled by NOmediated elevations of extracellular cGMP levels. A ®rst interesting observation is that, whatever agent is used, the evoked behavioural and biochemical responses are greatly sensitive to blockade of NMDA receptors thus highlighting the primary role that these sites play in the activation of the NOS/sGC pathway and in transducing excessive hippocampal excitation into epileptic-like behavioural responses. Furthermore, the apparent strict correlation between altered behaviour and cGMP extracellular changes might indicate that NO and the cyclic nucleotide are implicated in the genesis of preconvulsive/convulsive phenomena, as already proposed by others (Mao et al., 1974; Ho€er et al., 1977; Wood et al., 1982; McCaslin and Morgan, 1986; Mollace et al., 1991; Bagetta et al., 1993; De Sarro et al., 1993). However, while this may occur in di€erent experimental conditions, the NOS/sGC pathway does not seem to be instrumental in inducing epileptic states when excitation is initiated primarily in the hippocampus, as can be inferred from the following observations. First of all, the behavioural alterations induced by NMDA, cyclothiazide or nicotine are not a€ected by L-NARG or ODQ, despite the ecacy of the latter drugs in abolishing the elevations of extracellular cGMP caused by the formers.

Second, infusion of NO-donors, such as S-nitrosoN-acetyl-D,L-penicillamine (SNAP), does indeed augment cGMP levels but does not induce WDS, WR or SZ. Third, the dramatic increase of extracellular cGMP, due to blockade of phosphodiesterase degradation by IBMX, is not accompanied by any of the above behavioural manifestations. Therefore, it can be concluded that NMDA receptors are essential to initiate excitatory events that, by recruiting other neuronal networks, lead to epileptic attacks, whereas NO/cGMP production seems to be only a neurochemical consequence of the receptor activation. 3.3. Other Brain Regions The NOS/sGC pathway has been investigated in vivo also in brain regions other than cerebellum and hippocampus, always by monitoring extracellular cGMP levels by intracerebral microdialysis. In the frontal cortex, some very preliminary results have shown that basal extracellular cGMP apparently is not derived from endogenous NO production since inhibition of NOS by L-NAME does not alter the levels of the cyclic nucleotide (Laitinen et al., 1994). The observation that zinc protoporphyrin-IX signi®cantly reduces cortical cGMP (Laitinen et al., 1997) would suggest that CO, rather than NO, is involved. However, the use of metalloporphyrins to evoke the participation of CO may not be appropriate in as much as these chemicals are more potent inhibitors of sGC (Ignarro et al., 1984) than they are of heme oxygenase (Drummond and Kappas, 1981) and can inhibit NO-induced activation of the cyclase enzyme in vivo (Luo and Vincent, 1994). Indeed, a more detailed analysis has

Cerebral GluR/NO/cGMP Pathway

revealed that cortical cGMP production becomes markedly sensitive to blockade of NOS activity when phosphodiesterase metabolism is prevented by IBMX (Laitinen et al., 1997). This pharmacological manipulation results in a dramatic, six-fold increase of extracellular cGMP and indicates that, as already reported in the hippocampus (Vallebuona and Raiteri, 1994), a very ecient degradation of cyclic nucleotides takes place in the frontal cortex; thus, such a high rate of cGMP breakdown might have masked the inhibitory e€ects of L-NAME under physiological conditions (Laitinen et al., 1997). These results, however, point out that functional di€erences may exist between the cortex and the hippocampus: in this region a consistent decrease of cGMP can be reproducibly observed following perfusion of NOS inhibitors alone, despite the high activity of hippocampal phosphodiesterases (eight-fold cGMP increase by IBMX). As for the modulation by GluRs in the cortex, no data are unfortunately available to de®ne whether and to what extent NMDA and non-NMDA receptors control the NOS/sGC pathway in this region. There is now a large body of evidence suggesting that the GluR/NO/cGMP signalling system is implicated in the formation of olfactory memory (Breer and Shepherd, 1993 and references therein), but only recently this functional relationship has been proved by conducting microdialysis measurements in the olfactory bulb of the sheep during odour recognition (Kendrick et al., 1997). It is known that sheep learn to recognize the odour of their own lambs within 2 hr after delivery; during the ®rst 30 min post partum, microdialysis sampling of extracellular ¯uid in the olfactory bulb has detected marked elevations of glutamate, GABA, noradrenaline, NO and cGMP which are paralleled by olfactory memory formation in the maternal ewes as assessed by acceptance and rejection behavioural tests towards own and strange lambs, respectively. When sheep are treated with NOS inhibitors or ionotropic GluR antagonists, post partum augmentation of endogenous amino acids, NO and cGMP is completely prevented. On the other hand, blockade of sGC does not a€ect NO formation but abolishes cGMP responses; interestingly, prevention of cGMP synthesis also blocks glutamate and GABA elevations. Under these conditions, sheep do not recognize their own lambs and accept also strange lambs equally, indicating that a correct functioning of the GluR/NOS/sGC pathway is crucial for the development of olfactory memory. Conversely, if sheep receive these treatments after odour learning has taken place, they selectively take care of their own lambs thus demonstrating that memory recall does not need activation of the NO/ cGMP cascade. Apart from their original contribution to the understanding of NO and cGMP roles in olfactory memory, these results clearly indicate that microdialysis monitoring of extracellular cGMP in freelymoving animals during learning tasks might be exploited to gain a better insight into the mechanisms of cognitive processes.

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4. THE HIPPOCAMPAL AND CEREBELLAR NMDA/NO SYNTHASE/GUANYLYL CYCLASE PATHWAY DURING AGEING It is well accepted that cognitive functions decline during physiological ageing and neurodegenerative disorders are known to dramatically accelerate this process. Although many neuronal networks are certainly involved, evidence is now emerging indicating that impaired glutamate neurotransmission may have a central role. For instance, binding experiments have demonstrated an age-related loss of NMDA receptors in di€erent brain regions of rodents and primates (Peterson and Cotman, 1989; Tamaru et al., 1991; Wenk et al., 1991; Cimino et al., 1993; Magnusson, 1995), whereas functional experiments have revealed that the ability of NMDA receptors to stimulate noradrenaline release from hippocampal nerve endings signi®cantly decreases during senescence (Pittaluga et al., 1993). As for cGMP, it has been found that its basal as well as kainate-stimulated tissue content in the cerebellum are lower in old than in adult rats (Schmidt and Thornberry, 1978). Moreover, in the aged cerebellum, while the distribution pattern of the cyclic nucleotide is qualitatively similar to that in the adult cerebellum, the total area of immuno¯uorescence is reduced (de Vente et al., 1990b). The ®rst result of microdialysis experiments in the hippocampus of aged (12, 24 months) freely-moving rats was that extracellular levels of cGMP are almost halved in comparison to those measured in adult controls, whereas no signi®cant changes can be detected in the cerebellum [Table 3; Vallebuona and Raiteri (1995)]. It is clear that the basal level of extracellular cGMP is maintained by an endogenous nitrinergic tone; thus, one possible explanation is that hippocampal NOS activity is a€ected by senescence. Indeed, in vitro measurement of the enzyme activity showed a 30% reduction of the ability to convert L-arginine into L-citrulline in the hippocampus, but not in the cerebellum, of 24-month-old rats [Table 3; Mollace et al. (1995); Vallebuona and Raiteri (1995)]. Of course, a functional damage might also occur at the level of the receptors tonically stimulated by endogenous glutamate and responsible for the NO/cGMP basal production. In this case, NMDA receptors can be certainly excluded since we have seen that they do not participate in the sustenance of cGMP physiological levels (see Fig. 12). On the other hand, impairment of AMPA receptors can not be ruled out but, at the same time, can not be con®rmed experimentally due to the complex, excitatory and inhibitory controls that this receptor exerts on the hippocampal NOS/ sGC pathway (see above). A more dramatic decrement in the functioning of the NOS/sGC system can be seen when the production of cGMP is enhanced by activating NMDA receptors. In this case, both in the hippocampus and cerebellum of old animals the e€ect of exogenous NMDA, either calculated as absolute amounts of cGMP produced or as a percent increase over the corresponding basal values, is strikingly diminished (Fig. 21). Interestingly, it would seem that the cerebellum is more resistant than hippocampus to age-

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Table 3. cGMP dialysate levels and NOS activity in the hippocampus and cerebellum of adult and aged rats Age (months) Hippocampus 3 12 24 Cerebellum 3 12 18 24

cGMP levels (fmol/20 min)

NOS activity (dpm/mg protein)

18.8524.83 9.9222.03 11.8322.15

51 5442781 ND 34 58622113

235230 187228 180227 220228

71 09521066 ND ND 68 38521233

The in vivo basal levels of cGMP have been computed by averaging the nucleotide content of the ®rst three fractions collected in the absence of any pharmacological treatment. NOS activity has been evaluated in vitro by measuring the conversion of [3H]arginine into [3H]citrulline [data taken from Vallebuona and Raiteri (1995)].

related changes since in the former region a signi®cant drop in the NMDA-induced production is observed only at 18 and 24 months of age, while in the hippocampus this phenomenon occurs as early as at 12 months. It has been reported that strychnine-insensitive glycine sites associated with NMDA receptors are decreased during ageing (Kito et al., 1990; Miyoshi et al., 1990; Tamaru et al., 1991). In the cerebellum of old animals, the data obtained with D-serine have shown, however, that the glycine receptors on the

NMDA complex remain fully responsive to the exogenous agonist (Table 4). It is worth noting that administration of D-serine in the aged cerebellum results in a cGMP response to NMDA equal to that evoked in the adult cerebellum in the absence of the amino acid. As a consequence, the possibility exists that administration of glycinomimetic drugs ameliorates functional de®cits related to decreased activity of the NMDA/NOS/sGC circuit. Going a step further, it would appear that hippocampal sGC shows some sort of diminished activity during ageing, whereas the cerebellar enzyme seems normally functioning. In fact, hippocampal soluble guanylyl cyclase is signi®cantly (30%) less responsive to exogenous NO in old than in adult rats, when looking at the absolute quantity of cGMP produced following SNAP administration (Fig. 22). However, if the e€ects of SNAP on cGMP at di€erent ages are expressed as a percent increase over the corresponding basal levels, no di€erences between adult and old animals can be observed. In the absence of an accurate kinetic analysis, these results might be indicative of a reduction in the number of sGC enzymes in the aged hippocampus with the spared ones remaining fully responsive to NO. In this case, guanylate cyclase would contribute moderately, if not at all, to the dramatic loss of cGMP response to exogenous NMDA seen in elder animals. Finally, it might be argued that an increased activity of phosphodiesterase-mediated degradation of cGMP during the life-time underlies the changes observed in aged animals. If this were the case, one would expect blockade of phosphodiesterases to

Fig. 21. Ageing decreases the NMDA-induced elevation of extracellular cGMP in the hippocampus and cerebellum of freely-moving rats. NMDA has been infused locally by retrodialysis for 20 min in the hippocampus (1 mM) and cerebellum (500 mM) of rats of di€erent ages; 3-month old rats have been considered as controls. In the hippocampus, a dramatic loss of cGMP response occurs as early as at 12 months of age, whereas in the cerebellum a signi®cant decrease of the NMDA e€ect is observed at 18 and 24 months. Each bar represents the time-point at which the maximal e€ect of NMDA was observed [data taken from Vallebuona and Raiteri (1995)].

Cerebral GluR/NO/cGMP Pathway

113

Table 4. E€ect of D-serine on the NMDA-induced elevation of cGMP in the cerebellum of rats of di€erent ages Age (months) 3 12 24

NMDA S1 4972 33 4032 42 1122 11

(fmol)

NMDA+D-serine S2

S2/S1

S1

3232 30 2252 32 772 12

0.65 0.56 0.69

541235 363248 151218

(fmol)

S2

S2/S1

936288 595255 320228

1.73 1.64 2.12

% Increase vs NMDA 166 236 193 232 207 259

Control animals received two consecutive pulses of NMDA (500 mM) and treated rats received D-serine (1 mM) during the second pulse of NMDA. S2/S1 ratios in aged animals were compared with those obtained in 3 month-old rats [data taken from Vallebuona and Raiteri (1995)].

result in an increase of cGMP much more pronounced in old than in adult animals. This possibility, however, can be de®nitely excluded since the e€ect of IBMX in the hippocampus was reduced, and not enhanced, during senescence, whereas it was not a€ected in the cerebellum (Fig. 23). Although many other processes can be involved, it is unequivocal that the NMDA/NOS/sGC pathway in the CNS is greatly a€ected by ageing, with marked regional di€erences. In the cerebellum, the age-related decline in cGMP production can be largely, if not exclusively, attributed to functional damages of the NMDA receptors themselves or to alterations of the intracellular transduction mechanisms which, following the receptors activation, convey the signal to NOS. This conclusion is supported by the observations that only the NMDA-induced cGMP accumulation is diminished in old rats whereas basal cGMP levels, NOS activity and sGC responsiveness to exogenous NO are not signi®-

cantly varied. In the old hippocampus, the situation is multifaceted and many factors, including de®cits of NMDA receptors, NOS and sGC, seem to contribute to the dramatic impairment of the biochemical cascade leading to cGMP synthesis.

5. cGMP PRODUCTION UNDER ISCHEMIC CONDITIONS The data collected during the past few years have shown that the e€ects of NO depend on the stage of evolution of the ischemic damage. During the acute phase, the gas is thought to have neuroprotective e€ects by promoting vasodilatation, by reducing vessels plugging and, probably, by limiting NMDA receptor functioning; soon after, however, the large amounts of NO produced by NOS neurons in response to exaggerate glutamate release or by inducible NOS, synthesized de novo in non-neuronal

Fig. 22. Age-related changes in the response of hippocampal and cerebellar sGC to the NO-donor SNAP. The increase of extracellular cGMP induced by local administration of SNAP (1 mM) is greatly (43%) reduced in the hippocampus of aged rats compared to control animals. However, if data are expressed as percent increase over the corresponding basal levels, no changes can be detected among the di€erent age groups (see text for explanation). No signi®cant alterations are observed in the cerebellum. Each bar represents the time-point at which the maximal e€ect of SNAP was observed [data taken from Vallebuona and Raiteri (1995)].

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Fig. 23. Ageing di€erentially a€ects phosphodiesterase activity in the hippocampus and cerebellum of freely-moving rats. In the hippocampus of 12- and 24-month old rats, the phosphodiesterase inhibitor IBMX, administered locally, shows a decreased (50%) ecacy in elevating extracellular cGMP whereas no changes can be observed in the cerebellum at all ages. Each bar represents the time-point at which the maximal e€ect of IBMX was observed [data taken from Vallebuona and Raiteri (1995)].

Fig. 24. Temporal pro®le of cGMP changes in the striatum of rats subjected to transient global ischemia. The ®gure shows that the extracellular levels of cGMP in the striatum increase during a 20-min period of global forebrain ischemia and remain higher than controls for up to 4 hr following recirculation. When rats are pre-treated with the NOS-inhibitor L-NAME, the cGMP peak during ischemia is markedly reduced and the cyclic nucleotide returns to baseline in ca 1 hr [data taken from Globus et al. (1995)].

Cerebral GluR/NO/cGMP Pathway

elements, appear to mediate neurotoxic events [for review, see Dawson (1994) and Iadecola (1997)]. Intracerebral microdialysis coupled to highly sensitive measurements of extracellular cGMP certainly o€ers a unique technique to characterize in vivo the neurochemical changes (and their temporal pro®le) related to glutamate release and NO production, which occur during experimental ischaemia. Indeed, in a preliminary study, Globus et al. (1995) have shown that, in the striatum of rats subjected to global forebrain ischemia, transiently induced by carotid legation combined with systemic hypotension, extracellular cGMP undergoes a two-fold augmentation and remains higher than controls up to 4 hr after recirculation has been restored. Administration of L-NAME greatly diminishes the elevation observed during and immediately after the insult and completely abolished the long lasting enhancement occurring in the post-ischemic period (Fig. 24). In our laboratory, we have investigated cGMP responses, and their pharmacological modulation, to elevated extracellular K+ concentrations ([K+]o), an event which characterizes a number of pathological conditions including ischaemia. In fact, it is known that a sudden, dramatic elevation of brain [K+]o (up to 60 mM) occurs within few minutes of ischaemia; in this situation neuronal cell membrane potential is virtually clamped at depolarized values of ca ÿ20 mV (anoxic depolarization) leading to electrical silence in the part of the brain interested by the insult [see for review, Szatkowski and Attwell (1994)]. In the cerebellum and hippocampus of freely-moving rats, local infusion of high KCl-con-

115

taining medium causes a seven- and four-fold augmentation of cGMP dialysate levels, respectively (Fig. 25; Fedele and Raiteri, unpublished data) which are paralleled by elevations of endogenous glutamate release. When Ca2+ ions are omitted, cGMP responses to high K+ are greatly reduced; similar results can be obtained by blocking NOS activity with L-NARG. Thus, it might be concluded that K+-induced depolarization leads to enhanced e‚ux of glutamate that, in turn, activates the NOS/ sGC system by stimulating GluRs. Surprisingly, however, experiments carried out with NMDA and non-NMDA receptor antagonists have revealed that GluRs play only a minor role, if any, in these neurochemical events. Indeed, MK-801, DNQX or the combination of the two antagonists does not a€ect at all the increase of extracellular cGMP elicited by high K+ depolarization (Fig. 25). These results are in keeping with those recently reported in the literature showing that the K+-induced increase of NOS activity, measured in vitro in rat and human cortical slices, is not altered by GluR antagonists, whereas it is sensitive to selective blockers of voltage dependent calcium channels [VDCCs; Alagarsamy et al. (1994); Fontana et al. (1997)]. Therefore, it appears that the in vivo calcium-dependent, NO-mediated cGMP rise during sustained depolarizations are due to activation of NOS by calcium ions entering the neurons mainly through VDCCs rather than through ion channels associated to GluRs. If these results are con®rmed also in models of experimental transient ischaemia, it will be of great interest to evaluate whether a more bene®cial neuroprotective therapy

Fig. 25. E€ect of KCl depolarization on cGMP extracellular levels in the cerebellum and hippocampus of rats. Microdialysis probes have been infused with a high-K+ containing medium (50 and 100 mM for the cerebellum and hippocampus, respectively) for 20 min. L-NARG, MK-801 and DNQX have been infused before, during and after the K+-depolarization. In the experiments assessing calcium dependency of the cGMP response, a Ca2+-free medium has been used from the beginning of the perfusion. Each bar represents the time-point at which the maximal e€ect of KCl was observed (Fedele and Raiteri, unpublished observations).

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can be achieved by simultaneously limiting excessive GluR activation and NO production, since the two events do not seem to be linked by a causal relationship. 6. CONCLUSIONS The evidence provided in this article indicate that monitoring of extracellular cGMP in freely-moving animals during intracerebral microdialysis represents a suitable in vivo experimental model to investigate the NOS/sGC pathway and its functional relationships with di€erent neurotransmitter systems as well as to de®ne its role in physiological and pathological processes. Moreover, this model could be useful also to test newly designed drugs able to act selectively at the di€erent steps of the pathway, from GluRs, to NOS, to soluble guanylyl cyclase, to phosphodiesterases. At this point, the reader would probably like to know why the classical intracellular second messenger cGMP should be present in the extracellular environment. Very little is known on this aspect of the nucleotide metabolism which would certainly deserve further investigation. The e‚ux of cGMP from cells may simply represent, together with degradation by phosphodiesterases, a means to regulate its intracellular levels, as proposed for cAMP (Rosenberg, 1992). However, there are reports in the literature showing that extracellular application of cGMP (which is not membrane permeable) is able to a€ect electrophysiological properties of neurons (Ho€er et al., 1971; Phillis et al., 1974; Stone et al., 1975; Siggins et al., 1976). More recently, Linden et al. (1995) have demonstrated that cGMP, bath-applied to patch-clamped Purkinje neurons, produces a large, though transient, attenuation of glutamate-evoked currents which seems to be due to an interaction of the cyclic nucleotide with the membrane surface. Similarly, extracellular e€ects have been reported also for cAMP which decreased chloride currents in hippocampal neurons by acting at GABAA receptors (Lambert and Harrison, 1990). Thus, although more extensive experimental evidence is needed, it might turn out that cGMP can also act as an intercellular messenger in cell to cell signalling. AcknowledgementsÐThis work was supported by grants from Italian MURST and from CNR Target Project on `Biotechnology'. The authors thank Dr Michela Bisaglia, Dr Giorgia Varnier and Miss Maria Antonia Ansaldo for their invaluable technical support and Mrs Maura Agate for the precious help in preparing the manuscript.

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