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cyclic GMP pathway in rat striatum
Pergamon PII:
Neuroscience Vol. 91, No. 1, pp. 51–58, 1999 Copyright 䉷 1999 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/99 $20.00+0.00 S0306-4522(98)00601-0
THE PARAFASCICULAR THALAMIC NUCLEUS BUT NOT THE PREFRONTAL CORTEX FACILITATES THE NITRIC OXIDE/CYCLIC GMP PATHWAY IN RAT STRIATUM S. CONSOLO,* A. CASSETTI and M. C. UBOLDI Istituto di Ricerche Farmacologiche Mario Negri, 20157 Milan, Italy
Abstract—We investigated whether the parafascicular thalamic nucleus and the prefrontal cortex, the two major excitatory inputs to the striatum, modulate the nitric oxide/cyclic GMP pathway in rat striatum. Electrical stimulation (10 pulses of 0.5 ms, 10 V applied at 10 Hz, 140 mA) delivered bilaterally to the parafascicular thalamic nucleus for a total of 4, 10 and 20 min, time-dependently facilitated cyclic GMP output in the dorsal striatum of freely moving rats, assessed by trans-striatal microdialysis. Electrical stimulation to the prefrontal cortex for a total duration of 20 min did not affect striatal cyclic GMP levels. The facilitatory effect observed after electrical stimulation of the parafascicular thalamic nucleus was blocked by co-perfusion with tetrodotoxin, suggesting that the effect is mediated by neuronal process(es). The non-competitive N-methyl-d-aspartate receptor antagonist, dizocilpine maleate (30 mM infused into the dorsal striatum), and the competitive one, 3-[(R)-carboxypiperazin-4-yl]-propyl-phosphonic acid (50 mM infused), but not local perfusion of the a-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid antagonist, 6-nitro-7-sulphamoylbenzo(f)quinoxaline-2,3-dione (15 mM perfused locally), abolished the cyclic GMP response in the striatum. The nitric oxide synthase inhibitor, 7-nitroindazole, applied locally (1 mM), blocked the electrically evoked increase in striatal extracellular cyclic GMP. This increase was also prevented by local application (100 and 300 mM) of 1H-(1,2,4)-oxadiazolo-(4,3a)-quinoxalin-1one, a selective inhibitor of soluble guanylyl cyclase. The results provide direct functional evidence of selective thalamic facilitation of the nitric oxide/cyclic GMP pathway in the dorsal striatum, through activation of N-methyl-d-aspartate receptors. 䉷 1999 IBRO. Published by Elsevier Science Ltd. Key words: cGMP release in vivo, microdialysis in vivo, AMPA receptors, NMDA receptors, 7-NINA, ODQ.
The brain generates nitric oxide (NO) from larginine by a Ca 2⫹-calmodulin and NADPHdependent neuronal NO synthase (NOS). 8,27 Soluble guanylyl cyclase is a major target of the NO formed. 17 In the striatum, NOS is present in a population of aspiny interneurons which amount to 1–2% of the total striatal neurons. These cells show immunoreactivity for glutamate decarboxylase, the synthetic enzyme for GABA, 28,49 and contain two neuropeptides, somatostatin and neuropeptide Y. 12,16,46 The terminals of the NOS-positive cells make synapses on perikarya and dendrites of other striatal cells including the spiny GABAergic projection neurons, 50 which are rich in soluble guanylyl cyclase and cGMP, 1,2 and express mRNAs that encode soluble guanylyl cyclase. 35 The NO formed in striatal
NOS-containing neurons may diffuse and activate the soluble guanylyl cyclase present in the neighbouring spiny cells, thus causing an accumulation of cGMP. By increasing cGMP, NO can exert a variety of short- and long-term neuromodulatory effects. 7,21,22 The major activators of NO and cGMP formation in the brain are excitatory amino acids, mainly glutamate. 17 The excitatory input to the striatum is derived from the cortex or thalamus. The corticostriatal pathway utilizes l-glutamate as a neurotransmitter, which appears to be largely mediated by a-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) or kainate glutamatergic receptors. 9,23,26 Pharmacological studies 4,10,11,20 have strengthened the possibility that the thalamostriatal input is also mediated by glutamate, 40 operating mainly through the N-methyl-d-aspartate (NMDA) type of glutamatergic receptor, whose physiological properties differ from those of the AMPA or kainate receptors. 33 In addition, anatomical data showed that the cortico- and thalamostriatal inputs preferentially contact separate striatal efferent neurons, distinguished on the basis of the density of dendritic spines. 13 It would therefore seem that the cortico- and
*To whom correspondence should be addressed. Abbreviations: AMPA, a-amino-3-hydroxy-5-methylisoxazole4-propionic acid; MK-801, dizocilpine maleate; NBQX, 6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3 dione; 7-NINA, 7-nitroindazole monosodium salt; NMDA, N-methyl-daspartate; NO, nitric oxide; NOS, nitric oxide synthase; ODQ, 1H-(1,2,4)-oxadiazolo-(4,3a)-quinoxalin-1-one; R-CPP, 3-[(R)-carboxypiperazin-4-yl]-propyl-phosphonic acid; TTX, tetrodotoxin. 51
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thalamostriatal input may be processed by distinct neuronal populations in the striatum. Thus, knowledge of which excitatory afferents to the striatum are involved in the activation of the striatal NO/cGMP system could help us to better understand how cortical and thalamic information is integrated in the basal ganglia. In this study, with the aim of identifying the excitatory input(s) involved in the regulation of the striatal NO/cGMP system, we measured the extracellular cGMP concentration in the striatum of freely moving rats during electrical stimulation of the frontal cortex or the parafascicular nucleus. We also investigated whether the electrically evoked striatal cGMP efflux was associated with the stimulation of NO production after activation of NMDA or nonNMDA glutamatergic receptors. EXPERIMENTAL PROCEDURES
Animals Male CD/COBS rats (210–270 g; Charles River, Calco, Italy) were used. They were given free access to food and water and housed under standard conditions of humidity (60%), room temperature (22⬚C) and light/dark cycle (lights on at 7.00 a.m.). The release experiments were performed between 8.00 a.m and 1.00 p.m. Procedures involving animals and their care were conducted in conformity with the institutional guidelines, which are in compliance with national (D.L.n 116, G.U., suppl. 40, Febbraio 1992, Circolare No. 8, G.U., 14 luglio 1994) and international law and policies (EEC Council Directive 86/609, OJ L 358, 1, Dec. 12, 1987; Guide for the Care and Use of Laboratory Animals, U.S. National Research Council, 1996).
Radiochemical Centre, Buckinghamshire, U.K.). The sensitivity of the assay was about 1.0 fmol/sample. At the end of the release experiments, the placement of the dialysis probe was verified histologically. Animals with the probe outside the dorsal striata (3%) were discarded. cGMP in vitro recovery was 30.1 ^ 0.16% for a probe 7.0 mm long (three different fibre units) in our perfusion conditions (flow rate 5 ml/min; 100 ml/sample). Implantation of electrodes Bipolar electrodes were constructed of fine nichrome wire (each wire 62.5 mm in diameter; Plastic One, Roanoke, VA, U.S.A.) and their tips were 0.2 mm apart. Under Equithesin anaesthesia, rats were stereotaxically implanted bilaterally with the electrodes in the parafascicular nucleus or prefrontal cortex on the same day as probe implantation. The coordinates were: for the parafascicular nucleus nose bar ⫺2.5 mm; AP, ⫺3.9 mm; L, ^1.4 mm from bregma and V, ⫺6.8 mm from the occipital bone; for the prefrontal cortex nose bar ⫺2.5 mm; AP, ⫹3 mm; L, ^0.8 mm; V, ⫺3 mm from bregma. 36 The electrode leads were attached to a multipin socket and fixed to the skull with dental acrylic. All solder joints were tested for continuity by checking impedance. On the day on which the electrodes were implanted, a dialysis probe was inserted transversally through both dorsal striata as described above. After each experiment, the placement of the bipolar electrode was verified by standard histological techniques. Animals in which the electrode had not been properly placed (10%) were discarded. Electrical stimulation in vivo After collecting three consecutive 20 min samples (baseline), tetani (10 pulses, 0.5 ms 10 V, at 10 Hz, 140 mA) were applied bilaterally in the parafascicular nucleus over 10 s and repeated at 2 min intervals for 4, 10 and 20 min. Similar tetani were delivered bilaterally in the prefrontal cortex over 10 s for a total duration of 20 min. A 20 min fraction was collected for cGMP assay during and after electrical stimulation.
Microdialysis and cyclic GMP assay
Drugs
Under Equithesin anaesthesia [1% pentobarbital/4% (v/v) chloral hydrate; 3.5 ml/kg, i.p.] the rats were placed in a stereotaxic frame. Dialysis tubing (AN 69 membrane; Hospal Dasco, Bologna, Italy) was inserted transversely through both dorsal striata. The dialysis probe was covered with super-epoxy glue along its whole length, except for the parts that corresponded to the areas of interest (two 3.5 mm sections separated by a glued central zone 2.5 mm long). The coordinates for implanting the probe were: nose bar ⫺2.5 mm; A, 1.5 mm from bregma; V, 5.3 mm from the temporal bone. 36 On the day after implantation, each rat was placed in a Plexiglas cage and the dialysis probe was perfused at a constant rate of 5 ml/min 32,45 with Ringer solution containing (in mM) 145 NaCl, 1.26 CaCl2, 1 MgCl2 and 3 KCl in distilled water. The solution was buffered to pH 7.4 with 2 mM sodium phosphate buffer. The perfusate during the first 45 min equilibration period was discarded and then collected at 20 min intervals in 3 ml polyethylene test tubes. After a 60 min period of perfusion to allow the cGMP output to reach a steady baseline, vehicle or drugs were applied in the dorsal striata by reverse dialysis up to the end of experiments, either alone or in combination with electrical stimulation of the parafascicular nucleus or the prefrontal cortex (described below). Experimental details are specified in the figure legends. The 20 min perfusate samples were immediately frozen on dry ice and stored at ⫺ 20⬚C until assayed for their cGMP content. cGMP was determined using a commercially available Amersham dual range radioimmunoassay kit (Amersham
All reagents were of analytical grade. Drugs used in this study and their sources were: 6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3 dione (NBQX, mol. wt 336.28), 1H-(1,2,4)oxadiazolo-(4,3a)-quinoxalin-1-one (ODQ, mol. wt 187.16), 7-nitro-indazole monosodium salt (7-NINA, mol. wt 197.13) and 3-[(R)-2-carboxypiperazin-4-y]-propyl-1phosphonic acid (R-CPP, mol. wt 270.22), all from Tocris Cookson (Bristol, U.K.) and dizocilpine maleate (MK-801, mol. wt 337.37) from RBI (Natick, MA, U.S.A.). R-CPP was dissolved in a few microlitres of 0.1 N NaOH and diluted to final volume in Ringer solution. None of the drugs used interfered with the cGMP assay. Statistical analysis Values calculated as fmol cGMP in a 20 min dialysate fraction, not corrected for the in vitro recovery of the microdialysis probe, were used for statistical analysis. A two-way split-plot ANOVA for repeated measures followed by a multiple comparison test (Tukey’s test for unconfounded means) were used, as specified in the figure legends. RESULTS
Cyclic GMP outflow in vivo from the dorsal striatum evoked by electrical stimulation of the parafascicular nucleus Electrical stimulation (10 pulses of 0.5 ms, 10 V
Parafascicular nucleus facilitates striatal NO/cGMP system
Fig. 1. Time-dependent effect of electrical stimulation of the parafascicular thalamic nucleus or the prefrontal cortex (see inset) on in vivo cGMP release in dorsal striata. Electrical stimuli (10 0.5 ms, 10 V pulses applied during 10 s, 140 mA, 10 Hz) were delivered bilaterally in the parafascicular nucleus for 10 and 20 min after 60 min baseline collection. The columns represent the peak increase (second 20 min period) in cGMP release after electrical stimulation. Data are means ^ S.E.M (vertical bars) of five animals, expressed as fmol of cGMP released/20 min in dialysate samples. Each bar represents *P ⬍ 0.01 vs the 0 min stimulation; aP ⬍ 0.01 vs the 10 and 20 min stimulation by two-way split-plot ANOVA followed by Tukey’s test. In the inset, electrical stimulation was applied bilaterally in the prefrontal cortex for 20 min at 10 Hz. The columns represent the average values of cGMP release after electrical stimulation expressed as fmol of cGMP released/ 20 min in dialysate samples and the data are means ^ S.E.M. (vertical bars) of five animals.
applied at 10 Hz, 140 mA) was delivered bilaterally in the parafascicular thalamic nucleus over 10 s for a total duration of 4, 10 and 20 min, and the dialysate in the dorsal striatum was collected every 20 min. In these conditions, the extracellular cGMP concentration increased in a time-dependent manner. Striatal cGMP outflow peaked 40 min after starting the tetani, and reached 44% above the basal efflux during 4 min of stimulation (P ⬍ 0.01) and about 70% (P ⬍ 0.01) during the 20 and 30 min periods of electrical stimulation (Fig. 1). No seizures or abnormal behavioural activation were noticed during electrical stimulation. Tetani similar to those applied in the parafascicular nucleus were delivered bilaterally in the prefrontal cortex over 10 s for a total duration of 20 min and the dialysate in the dorsal striatum was collected every 20 min (Fig. 1, inset). Electrical stimulation did not affect cGMP, the average outflow being 8.18 ^ 0.20 fmol/20 min in the electrically stimulated group and 8.29 ^ 0.10 fmol/20 min in the control group.
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Fig. 2. Effect of electrical stimulation (E.S.) of the parafascicular nucleus on cGMP output in rat dorsal striata and its sensitivity to tetrodotoxin (TTX). Perfusion with Ringer solution containing 5 mM TTX was started 40 min before electrical stimuli (10 Hz for 10 min) and continued until the end of the experiment. For each time-point data are means ^ S.E.M. (vertical bars) of five rats, expressed as fmol of cGMP released/ 20 min. Two-way split-plot ANOVA showed a significant interaction between E.S. group and the (TTX ⫹ E.S.) group; F3,21 11.79, aP ⬍ 0.0001. Tukey’s test showed significant differences in cGMP release compared with the control group (*P ⬍ 0.01 and **P ⬍ 0.05).
Effects of tetrodotoxin on in vivo cyclic GMP outflow from dorsal striatum evoked by electrical stimulation of the parafascicular nucleus The facilitatory effect observed after electrical stimulation of the parafascicular nucleus was markedly reduced by 5 mM tetrodotoxin (TTX) added to the perfusion solution from 40 min before electrical stimulation until the end of the experiment (Fig. 2). Thus, the evoked cGMP release it is likely to be neuronal, although a non-neuronal origin of cGMP cannot be excluded. By itself, the TTX did not modify the basal release of cGMP from dorsal striata. Effects of MK-801, R-CPP and NBQX on in vivo cyclic GMP outflow from dorsal striatum evoked by electrical stimulation of the parafascicular nucleus The effects of the glutamate receptor antagonists MK-801, R-CPP and NBQX were studied on striatal cGMP release evoked by electrical stimulation of the parafascicular nucleus (Fig. 3). Local application through the microdialysis probe of the non-competitive NMDA glutamatergic receptor antagonist, MK-801, at the concentration of 30 mM, 60 min before electrical stimulation prevented the rise in extracellular cGMP in the dorsal striatum (Fig. 3A) (F3,27 13.2; P ⬍ 0.01). The dose of MK-801 was chosen in the light of previous evidence that the
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Fig. 3. Effect of MK-801 (A), R-CPP (B) and NBQX (C) on in vivo striatal cGMP outflow evoked by electrical stimulation (E.S.) of the parafascicular thalamic nucleus. Electrical stimuli (10 Hz for 10 min) were delivered bilaterally in the parafascicular nucleus 60 min after MK-801 or R-CPP and 40 min after NBQX. MK-801 (30 mM), R-CPP (50 mM) and NBQX (30 mM) were infused by reverse dialysis after 60 min baseline collection up to the end of the experiment. For each time-point data are the mean ^ S.E.M. (vertical bars) of four or five rats, expressed as fmol of cGMP released/20 min. Two-way split-plot ANOVA showed in A and B the following interactions: E.S. group vs MK-801 ⫹ E.S. group (F3,27 13.2; aP ⬍ 0.01) and E.S. group vs R-CPP ⫹ E.S. group (F3,24 17.5; aP ⬍ 0.01); post hoc Tukey’s test showed in A, B and C significant differences in cGMP release compared with the control group (*P ⬍ 0.01).
drug selectively antagonized the responses to NMDA up to 30 mM in rat cortical slice preparations. 52 By itself, MK-801 did not modify cGMP efflux in the rat dorsal striatum (Fig. 3A). Similarly (Fig. 3B), the selective and competitive antagonist of NMDA-type receptors, R-CPP which, unlike MK-801, does not interfere with the voltagesensitive sodium channels of excitable cells, 31 when applied into the dorsal striatum by reverse dialysis at the concentration of 50 mM, 60 min before the tetanus, completely prevented the increase in striatal cGMP induced by electrical stimulation (F3,24 17.5; P ⬍ 0.01 by two-way split-plot ANOVA). By itself, R-CPP did not modify striatal cGMP efflux (Fig. 3B). In contrast, NBQX, a highly selective nonNMDA receptor antagonist, 39 infused by reverse dialysis at the concentration of 30 mM (Fig. 3C) did not prevent the increase in striatal cGMP release in vivo evoked by electrical stimulation of the parafascicular nucleus, nor did it modify the basal striatal cGMP efflux. It should be noted that a lower dose of NBQX, 15 mM, infused in the dorsal striata completely prevented the increase in striatal acetylcholine release in vivo induced by electrical stimulation of the cortex. 10 In addition, NBQX antagonized the electrophysiological responses to AMPA in rat neocortex slices, 39 at lower concentrations, 0.1–1.0 mM and, when applied locally (12.5– 25 nmol) in either the rat dorsal hippocampus 30 or the striatum, 34 it prevented the neurodegeneration induced by the non-NMDA receptor agonist AMPA and kainate.
Effect of nitric oxide synthase inhibition on the cyclic GMP efflux from the dorsal striatum evoked by electrical stimulation of the parafascicular nucleus The selective neuronal NOS inhibitor, 7-NINA (1 mM), applied in the dorsal striatum through the microdialysis probe, 20 min before electrical stimulation delivered bilaterally in the parafascicular nucleus, prevented the increase in striatal cGMP (F3,21 9.7; P ⬍ 0.01 by two-way spit-plot ANOVA) (Fig. 4).
Effect of nitric oxide-sensitive guanylyl cyclase inhibition on the increase in cyclic GMP efflux from dorsal striatum evoked by electrical stimulation of the parafascicular nucleus The potent and selective inhibitor of NO-sensitive guanylyl cyclase, ODQ, was infused by reverse dialysis into the dorsal striata, at concentrations of 100 or 300 mM (Fig. 5A, B), from 20 min before tetanus until the end of the experiment. One hundred micromolar ODQ partially inhibited while 300 mM completely prevented cGMP generation in response to electrical stimulation of the parafascicular nucleus (F3,18 57.1, P ⬍ 0.01; F3,18 40.4, P ⬍ 0.01 by two-way split-plot ANOVA). By itself, ODQ at both doses reduced the efflux of cGMP, which reached its nadir (12 and 28%) 20 min after drug application and remained low until the end of the experiment (Fig. 5).
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Fig. 4. Effect of the NOS inhibitor 7-NINA on striatal cGMP increase evoked by electrical stimulation (E.S.) of the parafascicular thalamic nucleus. 7-NINA (1 mM) was infused into the dorsal striata after 60 min baseline collection up to the end of the experiment. Electrical stimuli (10 Hz for 10 min) were delivered bilaterally in the parafascicular nucleus 20 min after 7NINA. For each time-point data are the mean ^ S.E.M. (vertical bars) of four rats, expressed as fmol of cGMP released/20 min. Split-plot ANOVA indicated a significant interaction between the E.S. group and 7-NINA ⫹ E.S. group (F3,21 9.7; a P ⬍ 0.01). Post hoc Tukey’s test showed a significant increase in extracellular cGMP levels of the E.S. group compared with the control group (*P ⬍ 0.01). DISCUSSION
The present study provides in vivo evidence that, of the two major excitatory inputs to the striatum, it is the parafascicular thalomostriatal pathway, not the corticostriatal one, that facilitates the NO/cGMP system of the dorsal striatum. Thus, we found that electrical stimulation of the excitatory parafascicular nucleus trans-synaptically increased cGMP in the dorsal striatum of freely moving rats, whereas similar tetani applied in the frontal cortex did not alter the basal striatal cGMP outflow. The stimulus parameters used were chosen on the basis of their ability to maintain intracranial self-stimulation in rats 37 and to increase acetylcholine release in vivo in the dorsal striatum, 4,10,41 when applied in either the frontal cortex or the parafascicular nucleus. The cGMP release evoked by electrical stimulation of the parafascicular nucleus was time dependent and was a process dependent on neuronal activation since it was blocked by TTX. The increase in extracellular cGMP levels probably reflected an increase in the intracellular content of this nucleotide, as shown previously in a number of peripheral tissues (e.g., bovine aorta endothelial cells, 38 hepatocytes 6 and pancreatic acinar cells. 25) Studies in vivo, in accordance with an in vitro study
Fig. 5. Effect of the guanylyl cyclase inhibitor ODQ on the in vivo striatal cGMP outflow evoked by electrical stimulation (E.S.) of the parafascicular thalamic nucleus. ODQ 100 mM (A) and 300 mM (B) was infused locally after collection of three 20 min baseline fractions up to the end of the experiment. Electrical stimuli (10 Hz for 10 min) were delivered bilaterally in the parafascicular nucleus 20 min after ODQ. For each timepoint data are the mean ^ S.E.M. (vertical bars) of four rats, expressed as fmol of cGMP released/20 min. Two way splitplot ANOVA showed in A and B the following interactions: E.S. group vs ODQ 100 mM ⫹ E.S. (F3,18 57.1; aP ⬍ 0.01); E.S. group vs ODQ 300 mM ⫹ E.S. group (F3,18 40.4; a P ⬍ 0.01). Post hoc Tukey’s test showed in A and B significant differences in cGMP release compared with the control group (*P ⬍ 0.01).
on cerebellar slices, showed that extracellular cGMP levels in the hippocampus 44 and cerebellum 32,45 of conscious rats changed in response to various drugs that activated or inhibited cGMP production. The thalamic modulation of cGMP occurred through selective activation of the NMDA type of glutamatergic receptors, as inferred from the findings that the non-competitive NMDA receptor antagonist, MK-801, and the competitive one,
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R-CPP, but not the AMPA/kainate antagonist NBQX, prevented the cGMP release evoked by electrical stimulation of the parafascicular nucleus. The NMDA receptors mediating the action of the excitatory thalamic afferents on cGMP appear to be located in the striatum itself, as both MK-801 and R-CPP were applied by reverse dialysis into the dorsal striatum. Thus, another study found no AMPA receptors on striatal somatostatin/NOScontaining neurons. 42 The overall results are consistent with the finding that NMDA receptor activation accounts for a large part of cGMP synthesis in the CNS 17 and support the pharmacological evidence that the thalamostriatal pathway is mediated selectively by NMDA receptors. 4,10,11,20 The finding that thalamic afferents operate preferentially through the NMDA receptor might explain why excitation of the cortical afferents, which operate preferentially through the AMPA/kainate type of glutamatergic receptors (see Introduction), did not modify cGMP output in the striatum even though NOS-positive cells receive direct cortical input. 48 Going a step further, the present study found that the NMDA-mediated cGMP response to electrical stimulation of the parafascicular nucleus was dependent on NO production in the striatum since 7-NINA, the selective neuronal NOS inhibitor, when applied locally prevented the rise in cGMP. This confirms observations in the hippocampus 14,17 and cerebellum cortex 18,32 that changes in cGMP levels induced by stimulation of NMDA receptors were mediated by NO synthesis. The observation that ODQ, a selective in vitro 7,19 and in vivo 15 inhibitor of soluble guanylyl cyclase targeted by NO, completely prevented the rise in striatal cGMP in response to stimulation of the parafascicular nucleus suggested that the NO formed increased cGMP accumulation by activating soluble guanylyl cyclase. The mechanism by which thalamostriatal afferents modulate the NO/cGMP system through NMDA receptor activation might be either direct or by activating a local polysynaptic loop. The latter is more likely since (i) the NOS/neuropeptide Y-positive cells are not targets for thalamic afferents fibres 24 but they are from the cortex; 48 and (ii) NOSneurons are not enriched in NMDA receptor 1 mRNA 3,29 (the subunit necessary for the functional activation of the NMDA receptor), in contrast to the majority of the other striatal neurons. Instead, the thalamic projections exert a synaptic facilitatory control over striatal cholinergic interneurons which innervate NOS-positive neurons 47 expressing
muscarinic cholinergic receptors. 5 In addition, the parafascicular nucleus modulates the activity of both of types of GABAergic neuron present in the striatum, 20 i.e. the efferent neurons, probably the less spiny neurons receiving innervation from the thalamic neurons, 13 and the interneurons. From the overall results, it may be argued that excitation of the parafascicular nucleus releases glutamate on to NMDA receptors, which leads to the activation of NOS and formation of NO, through a not yet clarified mechanism, possibly involving GABAergic and/or cholinergic interneurons. The NO formed binds to and activates striatal soluble guanylyl cyclase, present mainly in spiny output neurons, thereby increasing cGMP and activating cGMP-dependent pathways. A primary action of elevated cGMP levels is the stimulation of cGMPdependent protein kinase G, the major intracellular receptor protein for cGMP, which phosphorylates substrate proteins to exert its multiple actions. 51 One appropriate demonstration of this pathway has been described in striatonigral nerve terminals where NO, by raising cGMP levels, activates cGMPdependent protein kinase and phosphorylation of a dopamine- and cAMP-regulated phosphoprotein of mol. wt 32,000. 43 CONCLUSIONS
This study provides direct functional in vivo evidence that the parafascicular nucleus facilitates the NO/cGMP system in the striatum, through selective activation of the NMDA type of glutamatergic receptors. The cortex seems not to operate through the NO/cGMP pathway in regulating striatal activity. These findings have important implications for a better understanding of how thalamic information is integrated in the basal ganglia, suggesting that the NOS-positive neurons have an important part. Together with previous findings that the thalamus modulates the activity of GABAergic neurons 20 and the cholinergic interneurons, 4,10 this strongly suggests a close relation between the parafascicular thalamic nucleus and the striatum, which are presumably fundamental to the basic circuitry of the basal ganglia in physiological and pathological conditions.
Acknowledgements—This study was kindly supported by Comune di Cesano Boscone (to M.C.U). We thank Ms E. Colli for skilful technical assistance. The generous contribution of the Fondazione Angelo ed Angela Valenti, Milan, Italy, is gratefully acknowledged.
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(1991) Glutamate, nitric oxide and cell–cell signalling in the nervous system. Trends Neurosci. 14, 60–67. 18. Garthwaite J., Garthwaite G., Palmer R. M. J. and Moncada S. (1989) NMDA receptor activation induces nitric oxide synthesis from arginine in rat brain slices. Eur. J. Pharmac. 172, 413–416. 19. Garthwaite J., Southam E., Boulton C. L., Nielsen E. B., Schmidt K. and Mayer B. (1995) Potent and selective inhibition of nitric oxide-sensitive guanylyl cyclase by 1H-[1,2,4]-oxadiazolo-[4,3-a]-quinoxalin-1-one. Molec. Pharmac. 48, 184–188. 20. Giorgi S., Rimoldi M., Rossi A. and Consolo S. (1997) The parafascicular thalamic nucleus modulates messenger RNA encoding glutamate decarboxylase 67 in rat striatum. Neuroscience 80, 793–801. 21. Goy M. F. (1991) cGMP: the wayward child of the cyclic nucleotide family. Trends Neurosci. 14, 293–298. 22. Hawkins R. D. (1996) NO honey, I don’t remember. Neuron 16, 465–467. 23. Herrling P. L. (1985) Pharmacology of the corticocaudate excitatory postsynaptic potential in the cat: evidence for its mediation by quisqualate or kainate receptors. Neuroscience 14, 417–426. 24. Kachidian P., Vuillet J., Nieoullon A., Lafaille G. and Kerkerian-Le Goff L. (1996) Striatal neuropeptide Y neurones are not a target of thalamic afferent fibers. NeuroReport 7, 1665–1669. 25. Kapoor C. L. and Krishna G. (1977) Hormone-induced cyclic guanosine monophosphate secretion from guinea pig pancreatic lobules. Science 196, 1003–1005. 26. Kita H. (1996) Glutamatergic and GABAergic postsynaptic responses of striatal spiny neurons to intrastriatal and cortical stimulation recorded in slice preparations. Neuroscience 70, 925–940. 27. Knowles R. G., Palacios M., Palmer R. M. J. and Moncada S. (1989) Formation of nitric oxide from l-arginine in the central nervous system: a transduction mechanism for stimulation of the soluble guanylate cyclase. Proc. natn. Acad. Sci. U.S.A. 86, 5159–5162. 28. Kubota Y., Mikawa S. and Kawaguchi Y. (1993) Neostriatal GABAergic interneurones contain NOS, calretinin or parvalbumin. NeuroReport 5, 205–208. 29. Landwehrmeyer G. B., Standaert D. G., Testa C. M., Penney J. B. and Young A. B. (1995) NMDA receptor subunit mRNA expression by projection neurons and interneurons in rat striatum. J. Neurosci. 15, (7) 5297–5307. 30. Lees G. J. and Leong W. (1994) NBQX prevents contralateral but not ipsilateral seizure-induced cytotoxicity of kainate but not AMPA-reversal at high doses of NBQX. NeuroReport 5, 2153–2156. 31. Lehmann J., Schneider J., McPherson S., Murphy D. E., Bernard P., Tsai C., Bennet D. A., Pastor G., Steel D. J., Bohem C., Cheney D. L., Liebman J. M., Williams M. and Wood P. L. (1987) CPP, a selective N-methyl-d-aspartate (NMDA)-type receptor antagonist: characterization in vitro and in vivo. J. Pharmac. exp. Ther. 240, 737–746. 32. Luo D., Leung E. and Vincent S. R. (1994) Nitric oxide-dependent efflux of cGMP in rat cerebellar cortex: an in vivo microdialysis study. J. Neurosci. 14, 263–271. 33. MacDermott A. B. and Dale N. (1987) Receptors, ion channels and synaptic potentials underlying the integrative actions of excitatory amino acids. Trends Neurosci. 10, 280–284. 34. Massieu L. and Tapia R. (1994) 2,3-Dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline protects against both AMPA and kainate-induced lesions in rat striatum in vivo. Neuroscience 59, 931–938. 35. Matsuoka I., Giuili G., Poyard M., Stengel D., Parma J., Guellaen G. and Hanoune J. (1992) Localization of adenylyl and guanylyl cyclase in rat brain by in situ hybridization: comparison with calmodulin mRNA distribution. J. Neurosci. 12, 3350– 3360. 36. Paxinos G. and Watson C. (1982) The Rat Brain in Stereotaxic Coordinates. Academic, Sydney. 37. Phillips A. G. and Fibiger H. C. (1978) The role of dopamine in mantaining intracranial self-stimulation in tha ventral tegmentum nucleus accumbens, and medial prefrontal cortex. Can. J. Psychol. 32, 58–66. 38. Schini V., Schoeffter P. and Miller R. C. (1989) Effect of endothelium on basal and on stimulated accumulation and efflux of cyclic GMP in rat isolated aorta. Br. J. Pharmac. 97, 853–865.
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39. Sheardown M. J., Nielsen E. O., Hansen A. J., Jacobsen P. and Honore´ T. (1990) 2,3-Dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline: a neuroprotectant for cerebral ischemia. Science 247, 571–574. 40. Streit P. (1980) Selective retrograde labelling indicating the transmitter of neuronal pathways. J. comp. Neurol. 191, 429–463. 41. Taber M. T. and Fibiger H. C. (1994) Cortical regulation of acetylcholine release in rat striatum. Brain Res. 639, 354–356. 42. Tallaksen-Greene S. J. and Albin R. L. (1994) Localization of AMPA-selective excitatory amino acid receptor subunits in identified populations of striatal neurons. Neuroscience 61, 509–519. 43. Tsou K., Snyder G. L. and Greengard P. (1993) Nitric oxide/cGMP pathway stimulates phosphorylation of DARPP-32, a dopamine- and cAMP-regulated phosphoprotein, in the substantia nigra. Proc. natn. Acad. Sci. U.S.A. 90, 3462–3465. 44. Vallebuona F. and Raiteri M. (1994) Extracellular cGMP in the hippocampus of freely moving rats as an index of nitric oxide (NO) synthase activity. J. Neurosci. 14, 134–139. 45. Vallebuona F. and Raiteri M. (1995) Age-related changes in the NMDA receptor/nitric oxide/cGMP pathway in the hippocampus and cerebellum of freely moving rats subjected to transcerebral microdialysis. Eur. J. Neurosci. 7, 694–701. 46. Vincent S. R., Johansson O., Hokfelt T., Skirboll L., Elde R. P., Terenius L., Kimmel J. and Goldstein M. (1983) NADPHdiaphorase: a selective histochemical marker for striatal neurons containing both somatostatin- and avian pancreatic polypeptide (APP)-like immunoreactivities. J. comp. Neurol. 217, 252–263. 47. Vuillet J., Dimova R., Nieoullon A. and Kerkerian-Le Goff L. (1992) Ultrastructural relationship between choline acetyltransferase and neuropeptide Y-containing neurons in the rat striatum. Neuroscience 46, 351–360. 48. Vuillet J., Kerkerian-Le Goff L., Kachidian P., Bosler O. and Nieoullon A. (1989) Ultrastructural correlates of functional relationships between nigral dopaminergic or cortical afferent fibers and neuropeptide Y-containing neurons in the rat striatum. Neurosci. Lett. 100, 99–104. 49. Vuillet J., Kerkerian-Le Goff L., Kachidian P., Dusticier G., Bosler O. and Nieoullon A. (1990) Striatal NPY-containing neurons receive GABAergic afferents and may also contain GABA: an electron microscopic study in the rat. Eur. J. Neurosci. 2, 672–681. 50. Vuillet J., Kerkerian-Le Goff L., Kachidian P., Salin P. and Nieoullon A. (1989) Ultrastructural features of NPY-containing neurons in the rat striatum. Brain Res. 477, 241–251. 51. Wang X. and Robinson P. J. (1997) Cyclic GMP-dependent protein kinase and cellular signaling in the nervous system. J. Neurochem. 68, 443–456. 52. Wong E. F. H., Kemp J. A., Priestley T., Knight A. R., Woodruff G. N. and Iversen L. L. (1986) The anticonvulsivant MK-801 is a potent N-methyl-d-aspartate antagonist. Proc. natn. Acad. Sci. U.S.A. 83, 7104–7108. (Accepted 6 October 1998)
Neuroscience Vol. 91, No. 1, pp. 51–58, 1999 Copyright 䉷 1999 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/99 $20.00+0.00 S0306-4522(98)00601-0
THE PARAFASCICULAR THALAMIC NUCLEUS BUT NOT THE PREFRONTAL CORTEX FACILITATES THE NITRIC OXIDE/CYCLIC GMP PATHWAY IN RAT STRIATUM S. CONSOLO,* A. CASSETTI and M. C. UBOLDI Istituto di Ricerche Farmacologiche Mario Negri, 20157 Milan, Italy
Abstract—We investigated whether the parafascicular thalamic nucleus and the prefrontal cortex, the two major excitatory inputs to the striatum, modulate the nitric oxide/cyclic GMP pathway in rat striatum. Electrical stimulation (10 pulses of 0.5 ms, 10 V applied at 10 Hz, 140 mA) delivered bilaterally to the parafascicular thalamic nucleus for a total of 4, 10 and 20 min, time-dependently facilitated cyclic GMP output in the dorsal striatum of freely moving rats, assessed by trans-striatal microdialysis. Electrical stimulation to the prefrontal cortex for a total duration of 20 min did not affect striatal cyclic GMP levels. The facilitatory effect observed after electrical stimulation of the parafascicular thalamic nucleus was blocked by co-perfusion with tetrodotoxin, suggesting that the effect is mediated by neuronal process(es). The non-competitive N-methyl-d-aspartate receptor antagonist, dizocilpine maleate (30 mM infused into the dorsal striatum), and the competitive one, 3-[(R)-carboxypiperazin-4-yl]-propyl-phosphonic acid (50 mM infused), but not local perfusion of the a-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid antagonist, 6-nitro-7-sulphamoylbenzo(f)quinoxaline-2,3-dione (15 mM perfused locally), abolished the cyclic GMP response in the striatum. The nitric oxide synthase inhibitor, 7-nitroindazole, applied locally (1 mM), blocked the electrically evoked increase in striatal extracellular cyclic GMP. This increase was also prevented by local application (100 and 300 mM) of 1H-(1,2,4)-oxadiazolo-(4,3a)-quinoxalin-1one, a selective inhibitor of soluble guanylyl cyclase. The results provide direct functional evidence of selective thalamic facilitation of the nitric oxide/cyclic GMP pathway in the dorsal striatum, through activation of N-methyl-d-aspartate receptors. 䉷 1999 IBRO. Published by Elsevier Science Ltd. Key words: cGMP release in vivo, microdialysis in vivo, AMPA receptors, NMDA receptors, 7-NINA, ODQ.
The brain generates nitric oxide (NO) from larginine by a Ca 2⫹-calmodulin and NADPHdependent neuronal NO synthase (NOS). 8,27 Soluble guanylyl cyclase is a major target of the NO formed. 17 In the striatum, NOS is present in a population of aspiny interneurons which amount to 1–2% of the total striatal neurons. These cells show immunoreactivity for glutamate decarboxylase, the synthetic enzyme for GABA, 28,49 and contain two neuropeptides, somatostatin and neuropeptide Y. 12,16,46 The terminals of the NOS-positive cells make synapses on perikarya and dendrites of other striatal cells including the spiny GABAergic projection neurons, 50 which are rich in soluble guanylyl cyclase and cGMP, 1,2 and express mRNAs that encode soluble guanylyl cyclase. 35 The NO formed in striatal
NOS-containing neurons may diffuse and activate the soluble guanylyl cyclase present in the neighbouring spiny cells, thus causing an accumulation of cGMP. By increasing cGMP, NO can exert a variety of short- and long-term neuromodulatory effects. 7,21,22 The major activators of NO and cGMP formation in the brain are excitatory amino acids, mainly glutamate. 17 The excitatory input to the striatum is derived from the cortex or thalamus. The corticostriatal pathway utilizes l-glutamate as a neurotransmitter, which appears to be largely mediated by a-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) or kainate glutamatergic receptors. 9,23,26 Pharmacological studies 4,10,11,20 have strengthened the possibility that the thalamostriatal input is also mediated by glutamate, 40 operating mainly through the N-methyl-d-aspartate (NMDA) type of glutamatergic receptor, whose physiological properties differ from those of the AMPA or kainate receptors. 33 In addition, anatomical data showed that the cortico- and thalamostriatal inputs preferentially contact separate striatal efferent neurons, distinguished on the basis of the density of dendritic spines. 13 It would therefore seem that the cortico- and
*To whom correspondence should be addressed. Abbreviations: AMPA, a-amino-3-hydroxy-5-methylisoxazole4-propionic acid; MK-801, dizocilpine maleate; NBQX, 6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3 dione; 7-NINA, 7-nitroindazole monosodium salt; NMDA, N-methyl-daspartate; NO, nitric oxide; NOS, nitric oxide synthase; ODQ, 1H-(1,2,4)-oxadiazolo-(4,3a)-quinoxalin-1-one; R-CPP, 3-[(R)-carboxypiperazin-4-yl]-propyl-phosphonic acid; TTX, tetrodotoxin. 51
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thalamostriatal input may be processed by distinct neuronal populations in the striatum. Thus, knowledge of which excitatory afferents to the striatum are involved in the activation of the striatal NO/cGMP system could help us to better understand how cortical and thalamic information is integrated in the basal ganglia. In this study, with the aim of identifying the excitatory input(s) involved in the regulation of the striatal NO/cGMP system, we measured the extracellular cGMP concentration in the striatum of freely moving rats during electrical stimulation of the frontal cortex or the parafascicular nucleus. We also investigated whether the electrically evoked striatal cGMP efflux was associated with the stimulation of NO production after activation of NMDA or nonNMDA glutamatergic receptors. EXPERIMENTAL PROCEDURES
Animals Male CD/COBS rats (210–270 g; Charles River, Calco, Italy) were used. They were given free access to food and water and housed under standard conditions of humidity (60%), room temperature (22⬚C) and light/dark cycle (lights on at 7.00 a.m.). The release experiments were performed between 8.00 a.m and 1.00 p.m. Procedures involving animals and their care were conducted in conformity with the institutional guidelines, which are in compliance with national (D.L.n 116, G.U., suppl. 40, Febbraio 1992, Circolare No. 8, G.U., 14 luglio 1994) and international law and policies (EEC Council Directive 86/609, OJ L 358, 1, Dec. 12, 1987; Guide for the Care and Use of Laboratory Animals, U.S. National Research Council, 1996).
Radiochemical Centre, Buckinghamshire, U.K.). The sensitivity of the assay was about 1.0 fmol/sample. At the end of the release experiments, the placement of the dialysis probe was verified histologically. Animals with the probe outside the dorsal striata (3%) were discarded. cGMP in vitro recovery was 30.1 ^ 0.16% for a probe 7.0 mm long (three different fibre units) in our perfusion conditions (flow rate 5 ml/min; 100 ml/sample). Implantation of electrodes Bipolar electrodes were constructed of fine nichrome wire (each wire 62.5 mm in diameter; Plastic One, Roanoke, VA, U.S.A.) and their tips were 0.2 mm apart. Under Equithesin anaesthesia, rats were stereotaxically implanted bilaterally with the electrodes in the parafascicular nucleus or prefrontal cortex on the same day as probe implantation. The coordinates were: for the parafascicular nucleus nose bar ⫺2.5 mm; AP, ⫺3.9 mm; L, ^1.4 mm from bregma and V, ⫺6.8 mm from the occipital bone; for the prefrontal cortex nose bar ⫺2.5 mm; AP, ⫹3 mm; L, ^0.8 mm; V, ⫺3 mm from bregma. 36 The electrode leads were attached to a multipin socket and fixed to the skull with dental acrylic. All solder joints were tested for continuity by checking impedance. On the day on which the electrodes were implanted, a dialysis probe was inserted transversally through both dorsal striata as described above. After each experiment, the placement of the bipolar electrode was verified by standard histological techniques. Animals in which the electrode had not been properly placed (10%) were discarded. Electrical stimulation in vivo After collecting three consecutive 20 min samples (baseline), tetani (10 pulses, 0.5 ms 10 V, at 10 Hz, 140 mA) were applied bilaterally in the parafascicular nucleus over 10 s and repeated at 2 min intervals for 4, 10 and 20 min. Similar tetani were delivered bilaterally in the prefrontal cortex over 10 s for a total duration of 20 min. A 20 min fraction was collected for cGMP assay during and after electrical stimulation.
Microdialysis and cyclic GMP assay
Drugs
Under Equithesin anaesthesia [1% pentobarbital/4% (v/v) chloral hydrate; 3.5 ml/kg, i.p.] the rats were placed in a stereotaxic frame. Dialysis tubing (AN 69 membrane; Hospal Dasco, Bologna, Italy) was inserted transversely through both dorsal striata. The dialysis probe was covered with super-epoxy glue along its whole length, except for the parts that corresponded to the areas of interest (two 3.5 mm sections separated by a glued central zone 2.5 mm long). The coordinates for implanting the probe were: nose bar ⫺2.5 mm; A, 1.5 mm from bregma; V, 5.3 mm from the temporal bone. 36 On the day after implantation, each rat was placed in a Plexiglas cage and the dialysis probe was perfused at a constant rate of 5 ml/min 32,45 with Ringer solution containing (in mM) 145 NaCl, 1.26 CaCl2, 1 MgCl2 and 3 KCl in distilled water. The solution was buffered to pH 7.4 with 2 mM sodium phosphate buffer. The perfusate during the first 45 min equilibration period was discarded and then collected at 20 min intervals in 3 ml polyethylene test tubes. After a 60 min period of perfusion to allow the cGMP output to reach a steady baseline, vehicle or drugs were applied in the dorsal striata by reverse dialysis up to the end of experiments, either alone or in combination with electrical stimulation of the parafascicular nucleus or the prefrontal cortex (described below). Experimental details are specified in the figure legends. The 20 min perfusate samples were immediately frozen on dry ice and stored at ⫺ 20⬚C until assayed for their cGMP content. cGMP was determined using a commercially available Amersham dual range radioimmunoassay kit (Amersham
All reagents were of analytical grade. Drugs used in this study and their sources were: 6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3 dione (NBQX, mol. wt 336.28), 1H-(1,2,4)oxadiazolo-(4,3a)-quinoxalin-1-one (ODQ, mol. wt 187.16), 7-nitro-indazole monosodium salt (7-NINA, mol. wt 197.13) and 3-[(R)-2-carboxypiperazin-4-y]-propyl-1phosphonic acid (R-CPP, mol. wt 270.22), all from Tocris Cookson (Bristol, U.K.) and dizocilpine maleate (MK-801, mol. wt 337.37) from RBI (Natick, MA, U.S.A.). R-CPP was dissolved in a few microlitres of 0.1 N NaOH and diluted to final volume in Ringer solution. None of the drugs used interfered with the cGMP assay. Statistical analysis Values calculated as fmol cGMP in a 20 min dialysate fraction, not corrected for the in vitro recovery of the microdialysis probe, were used for statistical analysis. A two-way split-plot ANOVA for repeated measures followed by a multiple comparison test (Tukey’s test for unconfounded means) were used, as specified in the figure legends. RESULTS
Cyclic GMP outflow in vivo from the dorsal striatum evoked by electrical stimulation of the parafascicular nucleus Electrical stimulation (10 pulses of 0.5 ms, 10 V
Parafascicular nucleus facilitates striatal NO/cGMP system
Fig. 1. Time-dependent effect of electrical stimulation of the parafascicular thalamic nucleus or the prefrontal cortex (see inset) on in vivo cGMP release in dorsal striata. Electrical stimuli (10 0.5 ms, 10 V pulses applied during 10 s, 140 mA, 10 Hz) were delivered bilaterally in the parafascicular nucleus for 10 and 20 min after 60 min baseline collection. The columns represent the peak increase (second 20 min period) in cGMP release after electrical stimulation. Data are means ^ S.E.M (vertical bars) of five animals, expressed as fmol of cGMP released/20 min in dialysate samples. Each bar represents *P ⬍ 0.01 vs the 0 min stimulation; aP ⬍ 0.01 vs the 10 and 20 min stimulation by two-way split-plot ANOVA followed by Tukey’s test. In the inset, electrical stimulation was applied bilaterally in the prefrontal cortex for 20 min at 10 Hz. The columns represent the average values of cGMP release after electrical stimulation expressed as fmol of cGMP released/ 20 min in dialysate samples and the data are means ^ S.E.M. (vertical bars) of five animals.
applied at 10 Hz, 140 mA) was delivered bilaterally in the parafascicular thalamic nucleus over 10 s for a total duration of 4, 10 and 20 min, and the dialysate in the dorsal striatum was collected every 20 min. In these conditions, the extracellular cGMP concentration increased in a time-dependent manner. Striatal cGMP outflow peaked 40 min after starting the tetani, and reached 44% above the basal efflux during 4 min of stimulation (P ⬍ 0.01) and about 70% (P ⬍ 0.01) during the 20 and 30 min periods of electrical stimulation (Fig. 1). No seizures or abnormal behavioural activation were noticed during electrical stimulation. Tetani similar to those applied in the parafascicular nucleus were delivered bilaterally in the prefrontal cortex over 10 s for a total duration of 20 min and the dialysate in the dorsal striatum was collected every 20 min (Fig. 1, inset). Electrical stimulation did not affect cGMP, the average outflow being 8.18 ^ 0.20 fmol/20 min in the electrically stimulated group and 8.29 ^ 0.10 fmol/20 min in the control group.
53
Fig. 2. Effect of electrical stimulation (E.S.) of the parafascicular nucleus on cGMP output in rat dorsal striata and its sensitivity to tetrodotoxin (TTX). Perfusion with Ringer solution containing 5 mM TTX was started 40 min before electrical stimuli (10 Hz for 10 min) and continued until the end of the experiment. For each time-point data are means ^ S.E.M. (vertical bars) of five rats, expressed as fmol of cGMP released/ 20 min. Two-way split-plot ANOVA showed a significant interaction between E.S. group and the (TTX ⫹ E.S.) group; F3,21 11.79, aP ⬍ 0.0001. Tukey’s test showed significant differences in cGMP release compared with the control group (*P ⬍ 0.01 and **P ⬍ 0.05).
Effects of tetrodotoxin on in vivo cyclic GMP outflow from dorsal striatum evoked by electrical stimulation of the parafascicular nucleus The facilitatory effect observed after electrical stimulation of the parafascicular nucleus was markedly reduced by 5 mM tetrodotoxin (TTX) added to the perfusion solution from 40 min before electrical stimulation until the end of the experiment (Fig. 2). Thus, the evoked cGMP release it is likely to be neuronal, although a non-neuronal origin of cGMP cannot be excluded. By itself, the TTX did not modify the basal release of cGMP from dorsal striata. Effects of MK-801, R-CPP and NBQX on in vivo cyclic GMP outflow from dorsal striatum evoked by electrical stimulation of the parafascicular nucleus The effects of the glutamate receptor antagonists MK-801, R-CPP and NBQX were studied on striatal cGMP release evoked by electrical stimulation of the parafascicular nucleus (Fig. 3). Local application through the microdialysis probe of the non-competitive NMDA glutamatergic receptor antagonist, MK-801, at the concentration of 30 mM, 60 min before electrical stimulation prevented the rise in extracellular cGMP in the dorsal striatum (Fig. 3A) (F3,27 13.2; P ⬍ 0.01). The dose of MK-801 was chosen in the light of previous evidence that the
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Fig. 3. Effect of MK-801 (A), R-CPP (B) and NBQX (C) on in vivo striatal cGMP outflow evoked by electrical stimulation (E.S.) of the parafascicular thalamic nucleus. Electrical stimuli (10 Hz for 10 min) were delivered bilaterally in the parafascicular nucleus 60 min after MK-801 or R-CPP and 40 min after NBQX. MK-801 (30 mM), R-CPP (50 mM) and NBQX (30 mM) were infused by reverse dialysis after 60 min baseline collection up to the end of the experiment. For each time-point data are the mean ^ S.E.M. (vertical bars) of four or five rats, expressed as fmol of cGMP released/20 min. Two-way split-plot ANOVA showed in A and B the following interactions: E.S. group vs MK-801 ⫹ E.S. group (F3,27 13.2; aP ⬍ 0.01) and E.S. group vs R-CPP ⫹ E.S. group (F3,24 17.5; aP ⬍ 0.01); post hoc Tukey’s test showed in A, B and C significant differences in cGMP release compared with the control group (*P ⬍ 0.01).
drug selectively antagonized the responses to NMDA up to 30 mM in rat cortical slice preparations. 52 By itself, MK-801 did not modify cGMP efflux in the rat dorsal striatum (Fig. 3A). Similarly (Fig. 3B), the selective and competitive antagonist of NMDA-type receptors, R-CPP which, unlike MK-801, does not interfere with the voltagesensitive sodium channels of excitable cells, 31 when applied into the dorsal striatum by reverse dialysis at the concentration of 50 mM, 60 min before the tetanus, completely prevented the increase in striatal cGMP induced by electrical stimulation (F3,24 17.5; P ⬍ 0.01 by two-way split-plot ANOVA). By itself, R-CPP did not modify striatal cGMP efflux (Fig. 3B). In contrast, NBQX, a highly selective nonNMDA receptor antagonist, 39 infused by reverse dialysis at the concentration of 30 mM (Fig. 3C) did not prevent the increase in striatal cGMP release in vivo evoked by electrical stimulation of the parafascicular nucleus, nor did it modify the basal striatal cGMP efflux. It should be noted that a lower dose of NBQX, 15 mM, infused in the dorsal striata completely prevented the increase in striatal acetylcholine release in vivo induced by electrical stimulation of the cortex. 10 In addition, NBQX antagonized the electrophysiological responses to AMPA in rat neocortex slices, 39 at lower concentrations, 0.1–1.0 mM and, when applied locally (12.5– 25 nmol) in either the rat dorsal hippocampus 30 or the striatum, 34 it prevented the neurodegeneration induced by the non-NMDA receptor agonist AMPA and kainate.
Effect of nitric oxide synthase inhibition on the cyclic GMP efflux from the dorsal striatum evoked by electrical stimulation of the parafascicular nucleus The selective neuronal NOS inhibitor, 7-NINA (1 mM), applied in the dorsal striatum through the microdialysis probe, 20 min before electrical stimulation delivered bilaterally in the parafascicular nucleus, prevented the increase in striatal cGMP (F3,21 9.7; P ⬍ 0.01 by two-way spit-plot ANOVA) (Fig. 4).
Effect of nitric oxide-sensitive guanylyl cyclase inhibition on the increase in cyclic GMP efflux from dorsal striatum evoked by electrical stimulation of the parafascicular nucleus The potent and selective inhibitor of NO-sensitive guanylyl cyclase, ODQ, was infused by reverse dialysis into the dorsal striata, at concentrations of 100 or 300 mM (Fig. 5A, B), from 20 min before tetanus until the end of the experiment. One hundred micromolar ODQ partially inhibited while 300 mM completely prevented cGMP generation in response to electrical stimulation of the parafascicular nucleus (F3,18 57.1, P ⬍ 0.01; F3,18 40.4, P ⬍ 0.01 by two-way split-plot ANOVA). By itself, ODQ at both doses reduced the efflux of cGMP, which reached its nadir (12 and 28%) 20 min after drug application and remained low until the end of the experiment (Fig. 5).
Parafascicular nucleus facilitates striatal NO/cGMP system
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Fig. 4. Effect of the NOS inhibitor 7-NINA on striatal cGMP increase evoked by electrical stimulation (E.S.) of the parafascicular thalamic nucleus. 7-NINA (1 mM) was infused into the dorsal striata after 60 min baseline collection up to the end of the experiment. Electrical stimuli (10 Hz for 10 min) were delivered bilaterally in the parafascicular nucleus 20 min after 7NINA. For each time-point data are the mean ^ S.E.M. (vertical bars) of four rats, expressed as fmol of cGMP released/20 min. Split-plot ANOVA indicated a significant interaction between the E.S. group and 7-NINA ⫹ E.S. group (F3,21 9.7; a P ⬍ 0.01). Post hoc Tukey’s test showed a significant increase in extracellular cGMP levels of the E.S. group compared with the control group (*P ⬍ 0.01). DISCUSSION
The present study provides in vivo evidence that, of the two major excitatory inputs to the striatum, it is the parafascicular thalomostriatal pathway, not the corticostriatal one, that facilitates the NO/cGMP system of the dorsal striatum. Thus, we found that electrical stimulation of the excitatory parafascicular nucleus trans-synaptically increased cGMP in the dorsal striatum of freely moving rats, whereas similar tetani applied in the frontal cortex did not alter the basal striatal cGMP outflow. The stimulus parameters used were chosen on the basis of their ability to maintain intracranial self-stimulation in rats 37 and to increase acetylcholine release in vivo in the dorsal striatum, 4,10,41 when applied in either the frontal cortex or the parafascicular nucleus. The cGMP release evoked by electrical stimulation of the parafascicular nucleus was time dependent and was a process dependent on neuronal activation since it was blocked by TTX. The increase in extracellular cGMP levels probably reflected an increase in the intracellular content of this nucleotide, as shown previously in a number of peripheral tissues (e.g., bovine aorta endothelial cells, 38 hepatocytes 6 and pancreatic acinar cells. 25) Studies in vivo, in accordance with an in vitro study
Fig. 5. Effect of the guanylyl cyclase inhibitor ODQ on the in vivo striatal cGMP outflow evoked by electrical stimulation (E.S.) of the parafascicular thalamic nucleus. ODQ 100 mM (A) and 300 mM (B) was infused locally after collection of three 20 min baseline fractions up to the end of the experiment. Electrical stimuli (10 Hz for 10 min) were delivered bilaterally in the parafascicular nucleus 20 min after ODQ. For each timepoint data are the mean ^ S.E.M. (vertical bars) of four rats, expressed as fmol of cGMP released/20 min. Two way splitplot ANOVA showed in A and B the following interactions: E.S. group vs ODQ 100 mM ⫹ E.S. (F3,18 57.1; aP ⬍ 0.01); E.S. group vs ODQ 300 mM ⫹ E.S. group (F3,18 40.4; a P ⬍ 0.01). Post hoc Tukey’s test showed in A and B significant differences in cGMP release compared with the control group (*P ⬍ 0.01).
on cerebellar slices, showed that extracellular cGMP levels in the hippocampus 44 and cerebellum 32,45 of conscious rats changed in response to various drugs that activated or inhibited cGMP production. The thalamic modulation of cGMP occurred through selective activation of the NMDA type of glutamatergic receptors, as inferred from the findings that the non-competitive NMDA receptor antagonist, MK-801, and the competitive one,
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R-CPP, but not the AMPA/kainate antagonist NBQX, prevented the cGMP release evoked by electrical stimulation of the parafascicular nucleus. The NMDA receptors mediating the action of the excitatory thalamic afferents on cGMP appear to be located in the striatum itself, as both MK-801 and R-CPP were applied by reverse dialysis into the dorsal striatum. Thus, another study found no AMPA receptors on striatal somatostatin/NOScontaining neurons. 42 The overall results are consistent with the finding that NMDA receptor activation accounts for a large part of cGMP synthesis in the CNS 17 and support the pharmacological evidence that the thalamostriatal pathway is mediated selectively by NMDA receptors. 4,10,11,20 The finding that thalamic afferents operate preferentially through the NMDA receptor might explain why excitation of the cortical afferents, which operate preferentially through the AMPA/kainate type of glutamatergic receptors (see Introduction), did not modify cGMP output in the striatum even though NOS-positive cells receive direct cortical input. 48 Going a step further, the present study found that the NMDA-mediated cGMP response to electrical stimulation of the parafascicular nucleus was dependent on NO production in the striatum since 7-NINA, the selective neuronal NOS inhibitor, when applied locally prevented the rise in cGMP. This confirms observations in the hippocampus 14,17 and cerebellum cortex 18,32 that changes in cGMP levels induced by stimulation of NMDA receptors were mediated by NO synthesis. The observation that ODQ, a selective in vitro 7,19 and in vivo 15 inhibitor of soluble guanylyl cyclase targeted by NO, completely prevented the rise in striatal cGMP in response to stimulation of the parafascicular nucleus suggested that the NO formed increased cGMP accumulation by activating soluble guanylyl cyclase. The mechanism by which thalamostriatal afferents modulate the NO/cGMP system through NMDA receptor activation might be either direct or by activating a local polysynaptic loop. The latter is more likely since (i) the NOS/neuropeptide Y-positive cells are not targets for thalamic afferents fibres 24 but they are from the cortex; 48 and (ii) NOSneurons are not enriched in NMDA receptor 1 mRNA 3,29 (the subunit necessary for the functional activation of the NMDA receptor), in contrast to the majority of the other striatal neurons. Instead, the thalamic projections exert a synaptic facilitatory control over striatal cholinergic interneurons which innervate NOS-positive neurons 47 expressing
muscarinic cholinergic receptors. 5 In addition, the parafascicular nucleus modulates the activity of both of types of GABAergic neuron present in the striatum, 20 i.e. the efferent neurons, probably the less spiny neurons receiving innervation from the thalamic neurons, 13 and the interneurons. From the overall results, it may be argued that excitation of the parafascicular nucleus releases glutamate on to NMDA receptors, which leads to the activation of NOS and formation of NO, through a not yet clarified mechanism, possibly involving GABAergic and/or cholinergic interneurons. The NO formed binds to and activates striatal soluble guanylyl cyclase, present mainly in spiny output neurons, thereby increasing cGMP and activating cGMP-dependent pathways. A primary action of elevated cGMP levels is the stimulation of cGMPdependent protein kinase G, the major intracellular receptor protein for cGMP, which phosphorylates substrate proteins to exert its multiple actions. 51 One appropriate demonstration of this pathway has been described in striatonigral nerve terminals where NO, by raising cGMP levels, activates cGMPdependent protein kinase and phosphorylation of a dopamine- and cAMP-regulated phosphoprotein of mol. wt 32,000. 43 CONCLUSIONS
This study provides direct functional in vivo evidence that the parafascicular nucleus facilitates the NO/cGMP system in the striatum, through selective activation of the NMDA type of glutamatergic receptors. The cortex seems not to operate through the NO/cGMP pathway in regulating striatal activity. These findings have important implications for a better understanding of how thalamic information is integrated in the basal ganglia, suggesting that the NOS-positive neurons have an important part. Together with previous findings that the thalamus modulates the activity of GABAergic neurons 20 and the cholinergic interneurons, 4,10 this strongly suggests a close relation between the parafascicular thalamic nucleus and the striatum, which are presumably fundamental to the basic circuitry of the basal ganglia in physiological and pathological conditions.
Acknowledgements—This study was kindly supported by Comune di Cesano Boscone (to M.C.U). We thank Ms E. Colli for skilful technical assistance. The generous contribution of the Fondazione Angelo ed Angela Valenti, Milan, Italy, is gratefully acknowledged.
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