Concurrent pharmacological MRI and in situ microdialysis of cocaine reveal a complex relationship between the central hemodynamic response and local dopamine concentration.

The mechanisms underlying the signal changes observed with pharmacological magnetic resonance imaging (phMRI) remain to be fully elucidated. In this study, we obtained microdialysis samples in situ at 5-min intervals during phMRI experiments using a blood pool contrast agent to correlate relative cerebral blood volume (rCBV) changes with changes in dopamine and cocaine concentrations following acute cocaine challenge (0.5 mg/kg iv) in the rat over a duration of 30 min. Three brain areas were investigated: the dorsal striatum (n = 8), the medial prefrontal cortex (mPFC; n = 5), and the primary motor cortex (n = 8). In the striatum and mPFC groups, cocaine and dopamine temporal profiles were tightly correlated, peaking during the first 5-min period postinjection, then rapidly decreasing. However, the local rCBV changes were uncorrelated and exhibited broader temporal profiles than those of cocaine and dopamine, attaining maximal response 5-10 min later. This demonstrates that direct vasoactivity of dopamine is not the dominant component of the hemodynamic response in these regions. In the motor cortex group, microdialysis revealed no local change in dopamine in any of the animals, despite large local cocaine increase and strong rCBV response, indicating that the central hemodynamic response following acute iv cocaine challenge is not driven directly by local dopamine changes in the motor cortex. The combination of phMRI and in situ microdialysis promises to be of great value in elucidating the relationship between the phMRI response to psychoactive drugs and underlying neurochemical changes.

Benzodiazepine-sensitive GABA(A) receptors limit the activity of the NMDA/NO/cyclic GMP pathway: a microdialysis study in the cerebellum of freely moving rats.

In the cerebellum, infusion of NMDA (200 microM) for 20 min evoked a marked (200%) increase of extracellular cyclic GMP (cGMP) levels. The selective GABA(A) receptor agonist muscimol (0.01-100 microM) was able to counteract the NMDA effect with an EC(50) of 0.65 microM; the inhibitory effect of muscimol (10 microM) was prevented by bicuculline (50 microM). Diazepam (10 microM) significantly potentiated the muscimol (1 microM) inhibition; furthermore, when coinfused with 0.1 microM muscimol (a concentration not affecting, on its own, the cGMP response to NMDA), diazepam (10 microM) reduced the NMDA effect. Similar results were obtained with zolpidem (0.1-1 microM). Finally, local infusion of the benzodiazepine site antagonist flumazenil (10 microM), together with muscimol and diazepam, almost completely restored the effect of NMDA on extracellular cGMP levels. It is concluded that GABA(A) receptors potently control the NMDA/nitric oxide/cGMP pathway in the cerebellum in vivo. In terms of the alpha subunit composition, we can deduce that the cerebellar GABA(A) receptor does not contain alpha(6) or beta(4) subunits because it is diazepam-sensitive. Moreover, the observation that zolpidem is active at a rather low concentration, in combination with localization studies present in the literature, tend to exclude the presence of alpha(5) subunits in the receptor composition and suggest the involvement of an alpha(1) subunit.

Intracerebral administration of L-kynurenine decreases N-methyl-D-aspartate receptor-mediated production of cGMP in the cerebellum and hippocampus of unanaesthetized rats subjected to transcerebral microdialysis.

The effects of intracerebral administration of L-kynurenine (L-KYN) on the N-methyl-D-aspartate (NMDA) receptor-mediated, nitric oxide (NO)-dependent cGMP responses have been studied in vivo in the cerebellum and hippocampus of freely-moving rats subjected to transcerebral microdialysis. Administration of exogenous NMDA in the cerebellum through the dialysis probe evoked a 3-fold increase of basal extracellular levels of cGMP that was concentration-dependently reduced by co-infusion of L-KYN. In the hippocampus, local administration of cyclothiazide caused a significant enhancement of the cyclic nucleotide dialysate concentrations that was accompanied by behavioural manifestations characteristic of preconvulsive states. Co-infusion of L-KYN largely decreased the neurochemical effects of cyclothiazide and completely prevented the appearance of the behavioural episodes. It is concluded that administration of L-KYN by increasing endogenous kynurenic acid concentrations might exert neuroprotective and anticonvulsive effects through blockade of the NMDA receptor/NO/cGMP pathway.

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 N(G)-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 gamma-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 GABA(A) and GABA(B) receptors modulating the glutamate receptor/NO/cyclic GMP pathway in the rat hippocampus.

Intrahippocampal perfusion of bicuculline (50 microM) in Mg2+-free medium caused elevation of extracellular cGMP and epileptic-like behaviour. Both effects were partially prevented by blocking NMDA receptors with MK-801 or Mg2+ ions. Similarly, the GABA(B) receptor antagonists CGP52432 (0.1-30 microM) and CGP35348 (0.3-1 mM) evoked increases of extracellular cGMP. CGP52432 also elicited behavioural responses ranging from wet dog shakes to convulsions. MK-801 or Mg2+ ions reduced the effects of CGP52432. Local application of muscimol (100-300 microM) or (-)baclofen (300 microM) caused inhibition of extracellular cGMP. Administration of the AMPA/kainate receptor antagonist NBQX (100 microM) caused cGMP elevation which was almost abolished by co-perfusion of muscimol and (-)baclofen. In the presence of physiological Mg2+, perfusion of AMPA (30 microM) failed to affect cGMP levels, although rats displayed wet dog shakes episodes. When AMPA was co-perfused with low concentrations of bicuculline or CGP52432, cGMP elevations were observed in 60% of the rats. Addition of both antagonists to AMPA resulted in 85% of rats displaying a cGMP response. To conclude: (a) extracellular hippocampal cGMP is controlled by inhibitory GABA(A) and GABA(B) receptors tonically activated through GABAergic interneurons receiving AMPA/kainate-mediated glutamatergic inputs; (b) the GABAergic receptors are not endogenously saturated and can be further stimulated by exogenous agonists; (c) blockade of the GABA-mediated inhibition causes increase of cGMP and epileptic-like behaviour, due largely to endogenous activation of NMDA receptors; (d) reproducible cGMP responses to AMPA can be observed when the inhibitory GABAergic inputs to the NO/guanylyl cyclase system are blocked, confirming the previously proposed existence of AMPA/kainate receptors able to increase the nucleotide synthesis.

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 microM) 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 microM 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 microM) or by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA; 100 microM) was largely attenuated by 100 microM 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 microM) 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 microM) 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 microM) 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 microM 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 microM 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.