European Journal of Neuroscience, Vol. 8, pp. 2619-2634, 1996 0 European Neuroscience Association

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

Keith M. Kendrick, Rosalinda Guevara-Guzman4, Carlos de la Riva, Jakob Christensen', K. 0stergaard2 and Piers C. Emson3 Department of Neurobiology and 3MRC Molecular Neuroscience Group, The Babraham Institute, Babraham, Cambridge CB2 4AT, UK 'Institute of Neurobiology, University of Aarhus, DK-8000, Aarhus C, Denmark *PharmaBiotec, Department of Anatomy and Cell Biology, University of Odense, OK-5000 Odense, Denmark 4Present address: Department of Physiology, Faculty of Medicine, UNAM, Mexico

Keywords: acetylcholine, glutamate, dopamine, kainate, microdialysis, neuroprotection, nitric oxide, NMDA, striatum

Abstract

The effects of N-methybaspartate (NMDA), kainate, S-a-amino-3-hydroxyd-methyl-4-isoxazole propionate (AMPA) and KCI on striatal nitric oxide (NO), acetylcholine (ACh), dopamine (DA), serotonin (5-HT), aspartate (ASP), glutamate (GLU) and y-aminobutyric acid (GABA) release were measured in anaesthetized rats in vivo by microdialysis and in vitro in organotypic slice cultures. Local NMDA (1-1 00 pM) infusion by retrodialysis dose-dependently increased levels of classical transmitters, NO2-, NO3-, citrulline and arginine at similar thresholds (10 pM) Similar patterns of NMDA-evoked (50 pM) release were seen in striatal cultures. NMDA- evoked changes were all calcium-dependent and blocked by NMDA (APV or MK-801) but not AMPNkainate (DNQX) receptor antagonists, excepting DA which could be prevented by both. In vivo, kainate increased NO2-, NO3-, CIT and ARG levels at 50 and 100 pM but was less potent than NMDA. Kainate also evoked significant ACh, DA and GLU release dose-dependently starting at 1-10 pM whereas 5-HT, ASP and GABA required 50 or 100 pM doses. Kainate effects were inhibited by DNQX, but not by APV, and were calcium-dependent. AMPA failed to alter NOz-, NO3-, CIT or ARG levels at 50 or 100 pM doses but dose-dependently increased ACh and DA. Similar results were seen with kainate (50 pM) and AMPA (50 pM) in v i m KCI evoked NO2-, NO3-, CIT and ARG release as well as that of the classical transmitters in vivo and in vitro. In vivo administration of the NO synthase inhibitor L-nitroarginine (L-NARG; 100 pM) significantly reduced NO2-, NO3- and CIT levels and prevented NMDA, kainate or KCI-evoked increases. It also potentiated ACh, ASP, GLU and GABA release and reduced that of DA in response to 50 pM NMDA whereas treatment with an NO-donor (SNAP; 10 pM) significantly reduced evoked ACh, ASP and GLU release. The NO synthase inhibitor L-NARG potentiated kainate-evoked ACh release and reduced that of DA, although less potently than NMDA, but it had no effect on KCI-evoked transmitter release. Overall, these results show that both NMDA and kainate increase striatal NO release at similar dose-thresholds as for classical transmitter release suggesting that NO is dynamically released under physiological and not just pathological conditions. Reduction of striatal NO levels also potentiates calcium- dependent transmitter release in response to NMDA and, to a lesser extent, kainate, whereas increasing them reduces it. This is consistent with a role for NO as a neuroprotective agent in this region acting to desensitize NMDA receptors.

Introduction

The kee radical gas nitric oxide (NO) has been proposed to act as a messenger, or novel type of neurotransmitter in the brain (Garthwaite, 1991 ; Snyder and Bredt, 1991). In the striatum, nicotinamide-adenine dinucleotide phosphate (NADPH) diaphorase staining has identified a population of 1-2% of medium to large aspiny interneurons which contain the neuronal nitric oxide synthase enzyme (nNOS) which is

responsible for NO production. These cells also contain somatostatin and neuropeptide Y (Vincent and Johansson, 1983) and were, until recently, thought not to contain a classical transmitter. Histochemical staining after colchicine treatment has revealed the presence of GADb7 in NOS-containing cells (Kubota et al., 1993), however it is more likely that, given the large amounts of NOS in these cells, NO is

Correspondence to: Dr. K.M. Kendrick. as above

Received 10 April 1996, revised 5 August I996, accepted 12 August 1996

2620 NMDA and kainate-evoked NO and striatal transmitter release

their primary transmitter. Nitric oxide may play this 'transmitter' role by diffusing down a concentration gradient and influencing haem- containing molecules, such as guanylate cyclase and the cytochromes, and by nitrosylating a number of proteins (Garthwaite, 1991; Snyder and Bredt, 1991 ). In a previous study we have shown that retrodialysis infusions of NO-donors and NO alter striatal classical transmitter release and these effects can be mimicked by cyclic GMP agonists, blocked by NO-scavengers and are in most cases either partially or complctcly calcium-dependent (Guevara-(iuzman et al., 1994).

While there is strong evidence that at high concentrations NO is a highly neurotoxic, at lower ones it might play a neuroprotective role within the striatuni since it reduces NMDA or glutamate (GLU) evoked calcium entry into striatal cells (Manzoni et al., 1992; Manzoni and Bockaert, 1993; Emson et al., 1995). Further, in vitro studies on striatal slices have shown that NOS inhibitors potentiate NMDA- evoked y-aminobutyric acid (GABA) relcase (Moller et al., 1995). It has therefore been hypothesized that endogenous NO may act to reduce the sensitivity of NMDA receptors to glutamate challenges, although there is no in vivo evidence to support this.

To further characterize the potential roles for NO as an intercellular messenger andor neuroprotective agent in the striatum, under physio- logical conditions, we have used both in vivo (microdialysis) and in vitro (organotypic slice cultures) methods to assess whether NMDA, kainate or S-a-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-type receptors are important for mediating glutamate-induced striatal NO release and whether thresholds are similar to those for altered classical transmitter release. Secondly we have investigated whether reduced NO release in the striatum in response to glutamate receptor activation might alter evoked release of classical transmitters. In both in vivo and in vitro experiments NO release was measured through monitoring release of its co-product, citrulline (CIT), which is produced in btoichiometi-ic quantities following NOS catalysis of the oxygenation of arginine (ARG; Bredt and Snyder, 1989; Knowles et al.,1989). We have also measured the main breakdown products of NO, nitrite (NO2-) and nitratc (NO3-).

Materials and methods

Animals Adult male Wistar rats (240-340 g) were used. For microdialysis sampling animals were anaesthetized with urethane (1.2 gkg; i.p.), placed in a stereotaxic frame (Kopf Instruments) with the incisor bar set at 5 mm above the interaural line and probes (CMA Microdialysis, Stockholm, Sweden, CMA-12, 0.5 mm o.d., 3 mm membrane length) positioned in the medial striatum (1.0 mm rostral to bregma: 3.0 mm lateral to the midline and 5.0 mm from the surface of the brain). Body temperature was maintained at 37.5OC using a heated blanket. Placements of probes within the medial striatum were confirmed histologically after the animal was killed at the end of the experiment and with reference to a stereotaxic atlas (Pellegrino et al., 1979). All animal use procedures were in strict accordance with UK Home Office guidelines and specifically licensed under the Animals (Scientific Procedures) Act 1986.

In vivo microdialysis sampling experiments In all experiments a Krebs Ringer solution (138 mM NaC1, 11 mM KCI, 1.5 mM CaC12, 1 mM MgC12, 11 mM NaHC03 and 1 mM NaH2P04 adjusted to pH 7.4) containing 5 pM neostigmine methyl sulphate (Sigma) was filtered (0.22 pm), degassed and passed through the microdialysis probes at 1.5 pl/min using a syringe pump (CMA- 100, CMA Microdialysis). Sampling started 1.5 h after probe insertion

to allow major damage release to subside (as evidenced by the measurement of stable baseline transmitter concentrations). Samples were then collected at 15 min intervals. A liquid switch was placed between the syringe pump and the microdialysis probe to enable different drugs to be administered locally via the probe (retrodialysis).

In the first series of experiments five 15 min baseline samples were taken before 15 min retrodialysis infusions of NMDA (Tocris Cookson, UK) at 1, 10,50 and 100 pM or kainate (kainic acid, Tocris Cookson) at I, 10, 50 and 100 pM. or AMPA (Tocris Cookson) at 50 and 100 pM or KCl (100 mM). A further eight samples were taken after each pharmacological challenge. In furthcr experiments 50 pM NMDA was given in the presence of calcium-free Krebs Ringer (for 30 min before and during the NMDA challenge) or in the presence of the NMDA receptor antagonist ~,~-2-amino-5-phosphonopentanoic acid (APV 100 pM. Tocris Cookson), or an AMPAkainate receptor antagonist, 6,7-dinitroquinoxaline-2,3-dione (DNQX; 200 pM, Tocris Cookson) for 1 h prior to and during the NMDA challenge and for a further 30 min following it. Using the same protocol, kainate (50 pM) was also given in the presence of calcium-free Ringer, DNQX (200 pM) or APV (100 pM). Each treatment and each drug dose was given to six to ten animals. Changes in substance concentrations during drug challenges were expressed as a percentage of baseline (basclinc was dcrived from the two samples preceding the drug challenge) and analysed statistically using a Wilcoxon test. Compar- isons between doses of different drugs were carried out by analysis of variance followed by post hoc Mann-Whitney I/ tests.

In a second series of experiments five 15 min baseline samples were taken before a 15 min challenge with 50 pM NMDA, 50 pM kainate or 100 mM KC1. After a further four baseline samples the NOS inhibitor, r-nitroarginine (L-NARG- 100 pM, Sigma) was retrodialysed into the striatum for 2 h (or after eight baseline samples for 1 h) and then a further 15 min NMDA, kainate or KCI challenge was given in the presence of the L-NARG. After this a further four baseline samples were taken. As a control the NMDA, kainate and KC1 challenges were given at the same time points but without L-NARG treatment. Six different animals received each experimental and control treatment. In addition, two further groups of six animals received the two doses of NMDA but NOS inhibitors (L-NARG at 100 mgkg and 7-nitroindazole, a specific nNOS inhibitor, at 20 mgkg, i.p.; Alexis, Nottingham, UK) were administered peripher- ally 2 h prior to the start of the second NMDA challenge. Finally. in a further group of six animals two 50 pM NMDA challenges were given 3 h apart with the sccond challenge being given in the presence of a low dose (10 pM) of an NO donor, S-nitroSo-N-acetyl-D,L- penicillamine (SNAP; Alexis), which by itself does not influence classical transmitter release except for a small inhibition of dopamine (DA) release (Guevara-Guzman et al., 1994).

Organotypic striatal slice cultures and in vitro sampling ptutocol Organotypic striatal cultures were made using previously described methods (0stergaard et al., 1995). Cultures were sampled (5 min sample duration) during superfusion at 0.1 mYmin with Krebs Ringer (pH 7.4) containing 10 pM neostigmine. Samples were collected into Eppendorf tubes (0.5 ml of 2.0 M acetic acid was added) and freeze- dried. For HPLC analysis they were re-dissolved in 100 p1 of distilled water and levels were divided by five to reflect this concentration factor. An initial wash period of 1 h was given before sampling and seven control samples were taken before a 5 min stimulation with NMDA (50 pM). kainatc (50 pM), AMPA (50 pM) or KC1 (56 mM). In addition, NMDA stimulation was given when an NMDA receptor antagonist MK-801 (50 pM) was added for the entire sampling period

NMDA and kainate-evoked NO and striatal transmitter release 2621

or with calcium-tree ringer. For each treatment 4-12 different cultures were used and mean concentrations in the two samples prior to treatment were compared with those during it using a paired t test. Comparisons between the different agonists were made by calculating a mean percentage change in release followed by an analysis of variance and post hoc Mann-Whitney U tests.

HPLC methods for amino acid, monoamine and nitrite and nitrate measurements Concentrations of aspartate (ASP), GLU, GABA, ARG and CIT were measured by HPLC with fluorescence detection (Gilson, 157, Anachem, Luton, UK) following derivitization with o-phthaldial- dehyde (OPA). A binary gradient was used (A = 35 mM sodium acetate + 10% methanol, adjusted to pH 5.8 and B = 20% buffer A + 80% methanol adjusted to pH 6.7). The gradient started at 23% €3, rose to 45% B at 2 min, to 70% B at 7 rnin and to 97% B at 10 min and returned to 23% B at 18 min, with a flow rate of 0.52 ml/ min and a 12.5 cm X 3 mm C18 reversed phase cartridge column with 3 pm particle packing (S3 ODS2, part number 858528; Phase Separations, UK) and an integral guard column (part number 830053). In all cases, 5 p1 of sample was automatically derivatized for 2 min with 3 pl OPA (Sigma) buffered with NaOH (2.5 p1 of a saturated solution added to 40 p1 of OPA) to offset sample acidity and injected via a 10 pl loop using an autosampler (Gilson 231). This column and gradient elution profile reliably separates all the transmitter amino acids as well as CIT and ARG with detection sensitivities of 1-5 nM. Serotonin (5-HT) and DA were measured by microbore HPLC with electrochemical detection (Kendrick and Hinton, 1992) and a detection sensitivity of 100-200 pM for a 5 pl injection (i.e. 0.5-1 fmol injected). Acetylcholine (ACh) concentrations were detertnined using a microbore column and enzyme reactor coupled with electro- chemical detection with a platinum electrode at +0.5 V (Bioanalytical Systems, West Lafayette; Sepstik 530 mm X 1 mm analytical column and ACh/Ch IMER). The mobile phase used was 80 mM sodium phosphate with 5 ml/l of Kathon (Bioanalytical Systems) at pH 8.5. The flow rate used was 125 pl/min and detection sensitivity for ACh was 1 nM for a 3 p1 injection (i.e. 3 fmol). For striatal culture samples injection volumes were increased to 10 p1 with a resultant decreased detection limit.

Nitrite (NO2-) and nitrate (NO3-) levels were determined by anion exchange chromatography using an anion exchange column (IonPac AS12A, 4 mm X 25 cm; Uionex, Camberley, UK) with an AG12A guard column. The mobile phase was 25 mM KCI, pumped at 1.0 mumin using an HPLC pump with peek heads (CMA 250; CMA Microdialysis) and with peek tubing and connectors throughout. The N02- and N03- were detected by an absorbance detector at 215 nm (Dionex AD20) and samples injected via an autosampler with a 10 p1 peek loop (CMA 200/240, CMA Microdialysis). Using this protocol N02- eluted at 4.5 min and NO3- at 6.8 min with a detection sensitivity of 50 nM for a 10 pl injection (i.e. 0.5 pmol injected). For the slice cultures 20 pl of sample was injected giving a detection sensitivity of 25 nM. NaNOz and NaN03- were used as standards and linearity was seen in the range of 50 nM-20 pM. Interference from chloride ions was prevented by the column being saturated by chloride in the KCl mobile phase. The NO3- contamination levels were quantified in dialysate and drug solutions and concentrations given following subtraction of such contamination levels.

Results Effects of NMDA on NO and classical transmitter release In vivo microdialysis sampling showed that NMDA evoked dose- dependent increases in ACh, DA, 5-HT, ASP, GLU and GABA

release in the striatum (Fig. 1) with a 10 pM dose being the minimum one to evoke significant increases excepting GABA which was significantly increased at 1 FM. Release of ARG, CIT, NO*. and NO3- also showed similar dose-dependent increases and sensitivity to NMDA (Fig. I). The magnitudes of the NMDA-evoked changes in each of these substances were similar, with the main increase occurring during the challenge; although for N O z and N03- the return to baseline levels often took a further 15 min. The basal concentrations (mean t SEM) of NO2- (551 +. 36 nM) were close to those of CIT (296 ? 51 nM), although those of NO3- were much higher (1558 t 229 nM). Basal ARG levels were 945 5 123 nM. The effects of a 50 pM dose of NMDA on release of both classical transmitters, ARG, CIT, NO2- and NO3- could be significantly inhibited by APV (100 pM; Fig. 2) or by removal of calcium from the dialysis medium (Fig. 3). With APV itself, there were small but significant overall decreases in concentration of CIT (from 239 t 34 nM to 192.8 ? 26.8 nM, P < 0.01 two tailed paired t-test), N02- (from 594 ? 115.7 nM to 503 t 107 nM, P < 0.001), N03- (from 1563 i 236.5 nM to 1357 +. 214.1 nM, P < O.OOl), ARG (from 998 2 111.4 nM to 827 +- 107.2 nM, P < 0.01) and GABA (from 64.35 ? 13.1 nM to 45.1 ? 6.4 nM, P < 0.05). However, DA concentrations were significantly increased (from 1886.8 t 202.2 pM to 2300.7 2 256 pM, P < 0.05).

The AMPAkainate receptor antagonist, DNQX (200 pM) had no effect on NMDA-evoked CIT, NO2-, NO3-, ARG or classical transmit- ter release excepting DA which it blocked (Fig. 2). Removal of calcium from the dialysate significantly reduced CIT (from 212.8 i: 8 to 188.3 5 4.5 nM, P < 0.05), NO2- (from 595.5 i 113 to 458.6 ? 95 nM, P < 0.01), N03-(from 1818.2 ? 298 to 1583.7 ? 273 nM, P < 0.011, ARG (from 1073.6 t 101 to 919.6 2 116 nM, P < 0.02), ACh (from 63.1 i: 10 to 53.4 -C 10 nM, P < 0.05), DA (from 3.8 ? 0.9 to 3.1 t 0.6 nM, P < 0.05), ASP (from 290.8 2 43 to 258 t 45 nM; P < 0.05), GLU (from 1977 +- 597 to 1784 2 538 nM, P < 0.05) and GABA (from 56.2 i 6.9 to 46.8 ? 6.7 nM, P < 0.05).

It was found that NMDA (50 ,uM) evoked significantly increased levels of ACh, GABA, ARG, CIT, NO2- and N03- in in vitro culture experiments too (Fig. 4). However, DA concentrations were not detectable in the cultures and, in contrast to in vivo experiments, ASP and GLU levels were not significantly altered (Fig. 4). These effects of NMDA on neurochemical release were significantly inhibited by MK-801 or by removing calcium from the perfusion medium (Fig. 4). MK-801 also decreased basal levels of CIT (from 60.2 t 1.2 to 2.1 i: 0.8 nM, P < 0.05 t test), NO2- (from 394 ? 42 to 166 ? 78 nM, P < 0.05) and N03- (from 1602 ? 290 to 768 i: 105 nM, P < 0.05). However, ARG concentrations were not significantly altered (45.6 ? 10 versus 47.6 ? 8 nM, P>0.05). None of the classical transmitter concentrations were significantly affected by MK-801. Removal of calcium from the perlusion medium significantly reduced levels of CIT (to 40.2 i: 1.4 nM, P < 0.05) and NO3- (to 1012 2 56 nM, P < 0.05). Reductions in NO2- did not quite achieve significance (to 221 2 48 nM, P = 0.08) and ARG levels were unaffected (44.8 ? 10 nM). Of the classical transmitters only GABA concentrations were reduced (from 2.6 ? 0.5 to 1.8 2 0.3 nM, P < 0.05).

Effects of kainate on striatal NO and classical transmitter release In vivo, kainate evoked dose-dependent increases in ACh, DA and GLU in the 1-50 pM range (Fig. 1). 5-HT was only significantly increased by a 100 pM and ASP by a 50 pM kainate dose. For ASP and ARG a 100 pM dose was significantly less potent than a 50 pM

2622 NMDA and kakte-evoked NO and striatal transmitter release

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NMDA and kainate-evoked NO and striatal transmitter release 2623

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FIG. 2. Mean t SEM striatal concentrations of transmitters, CIT, NO,, NO,- and ARG, in in vivo microdialysis samples (15 min) taken during two 50 pM NMDA challenges with either DNQX (200 pM; open squares) or APV (100 pM, filled squares) being given by retrodialysis for 1 h prior to the second NhIDA challenge and for 30 min following it. * P < 0.05 compared with control samples. The NMDA-evoked 5-HT release was also blocked by APV but not by DNQX (data not shown). Six animals were used in each treatment group.

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2624 NMDA and k a k t e - e v o k e d NO and striatal transmitter release

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FIG. 3. Mean t SEM striatal levels of transmitters, CIT, NOz, NO3- and ARG, in in vivo microdialysis samples (15min) taken during wccessive 50 pM NMDA or kainate challenges with calcium removed from the dialysate for 1 h prior to the second NMDA or kainate challenge and for 30 min after if. *P < 0.05 compared with control samples. The NMDA-evoked 5-HT release was also calcium dependent (data not shown). Data are from six animals in each case.

NMDA and kainate-evoked NO and striatal transmitter release 2625

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FIG. 4. Mean t SEM percentage change in concentrations of classical transmitters, CIT, N 0 1 , N03- and ARG, in striatal cultures during a 5 min exposure to NMDA (50 pM), NMDA + 50 pM MK-801, NMDA in Ca2+-free Ringer, kainate (50 pM) and AMPA (SO pM) or KCl (56 mM). *P < 0.05, **P 4 0.01 compared with basal concentrations; N, K and A show that the level of altered release was significantly ( P < 0.05) different from that with NMDA, kainate or AMPA respectively. Data are from four to twelve different cultures for each treatment. Baseline levels are loo%, indicated by a horizontal line.

2626 NMDA and kainate-evoked NO and striatal transmitter release

5) , and by removal of calcium from the dialysate (Fig. 3). The effects

of APV on basal ARG, CIT, NO2-, NO3- and DA, ASP, GLU and GABA were the same as those seen in the NMDA challenge experiment and DNQX itself also failed to influence NO or classical transmitter levels. In v i tm culture experiments showed that 50 pM kainate evoked significant release of ACh, NOz- and NO3- but not of CIT and ARG (Fig. 4).

Effects of AMPA on striatal NO and classical transmitter release In vivo microdialysis showed that AMPA produced significant dose- dependent increases in ACh and DA but not in any other substances (Fig. 1). In the in vitro culture experiments 50 pM AMPA only increased ACh concentrations significantly (Fig. 4).

Comparison of NMDA, kainate and AMPA actions on striatal NO and transmitter release Figure 1 shows that, in vivn, NMDA and kainate were significantly more potent than AMPA at increasing ACh concentrations at a 50 p M dose. Kainate was significantly more effective at increasing DA levels than NMDA at both 10 and 50 pM doses and AMPA was significantly more potent than NMDA at a 50 pM dose. For 5-HT, NMDA was significantly more potent than AMPA at a 50 pM dose and kainate more than AMPA at a 100 pM dose. For both ASP and GLU, NMDA was significantly more potent than AMPA at either 50 or 100 pM doses and than kainate at a 100 pM dose. Kainate evoked significantly more ASP and GLU release than AMPA at a 50 yM dose. For GABA, NMDA was significantly more potent than kainate at 1, 10, 50 and 100 pM doses and than AMPA at 50 and 100 pM. For ARG, NMDA was significantly more effective than kainate at 10 and 100 pM doses and than AMPA at 50 and 100 pM doses. For CIT and NO2-, NMDA was significantly more potent than kainate at 10 and 100 pM doses and than AMPA at 50 and 100 pM doses. Kainate was significantly more potent than AMPA at both 50 and 100 pM doses. NMDA and kainate were significantly more effective in increasing N03- levels than AMPA-at 50 and 100 pM doses.

For the in vitro culture studies, which only used 50 pM doses, NMDA and kainate evoked significantly more N03- release than AMPA and although there was a similar trend for NO; this did not quite achieve significance. The NMDA challenge evoked significantly more GABA release than kainate and more ARG release than kainate or AMPA (Fig. 4).

Effects of KCI challenges on striatal nitric oxide and classical transmitter release In viva experiments showed that a 100 mM KC1 challenge stimulated significant release of all the classical transmitters measured as well as ARG (149.7 ? 9.9% of baseline, P < 0.05), CIT (128 ? 4.996, P < 0.05), NO?- (146.7 5 12.896, P < 0.05) and NO3- (175.5 ? 31.4%, P < 0.05). In vitro culture experiments showed the same effects but with a 56 mM KCI challenge (Fig. 4).

Effects of NO synthase inhibitors on striatal NO release and NMDA, kainate and KCI-evoked transmitter release In vivn microdialysis showed when NMDA (50 pM), kainate (50 pM) and KCl (100 mM) were administered by retrodialysis twice, 3 h

apart, classical transmitter release in response to the second

challenge was the same or slightly but significantly reduced (Figs 6-8): excepting DA where significantly more release was evoked by the second challenge than by the first. Retrodialysis administration of the NOS inhibitor L-NARG for 2 h prior to the second NMDA challenge significantly potentiated release of ACh, ASP, GLU and GABA and reduced that of D.4 (Figs 6 and S). Infusions of 1.-NARG for 1 h instead of 2 h prior to the second NMDA challenge failed to produce this effect (data not shown). Peripheral treatment with L-NARG or with the specific nNOS inhibitor, 7-nitroindazole, significantly potentiated ACh, ASP, GLU and GABA release and reduced that of DA in response to NMDA (Figs 6 and 8).

Retrodialysis administration of L-NARG also potentiated the release of ACh and reduced that of DA in response to kainate 50 pM but had no significant effect on KCI (100 mM) evoked transmitter release (Fig. 7). The L-NARG potentiation of NMDA-evoked ACh release was significantly greater than that of kainate (523.5 -C 120% versus 51 L 11%, P 0.05 two-tailed Mann-Whitney U test, comparisons made between the percentage change with the second NMDA or kainate challenge (S2) under control conditions as opposed to a second challenge (S2) after 2 h of L-NARG treatment). Similarly the effect of L-NARG in reducing evoked DA release was significantly greater for NMDA than for kainate (209 t 25% versus 110 ? 28%, P < 0.05).

The 2 h retrodialysis treatments with L-NARG produced significant reductions in CIT (from 328.2 5 29.3 to 238.5 f 26.6 nM, paired t test, P < 0.01 two-tailed, n = 18 animals), NO2- (from 874.7 2 206.8 to 672.5 ? 163.4 nM, P < 0.01) and N03- (from 2209.7 L 198.1 to 1610.7 ? 139.5 nM, P < 0.001) and inhibited the ability of the second NMDA (Fig. 8), kainate and KC1 challenges to evoke significant increases in these three indicators of NO release. ARG levels could not be measured reliably since L-NARG itself produced large ARG concentrations. The 2 h period of L-NARG treatment also significantly reduced basal concentrations of ACh (from 187.3 f 27 to 102.1 ? 13.5 nM, P < 0.001) and ASP (from 328.5 5 24.4 to 237.7 i 12.9 nM, P < 0.001) but no other substances were affected (see Fig. 8).

Effects of an NO donor on NMDA-evoked striatal transmitter release Retrodialysis administration of SNAP ( 1 0 pM) significantly inhibited NMDA-evoked release of ACh, ASP and GLU (Fig. 6). It was confirmed in four animals that this dose of SNAP (10 pM) produced no significant effect on transmitter release apart from a small (10%) reduction in DA concentrations (data not shown) The carrier compound for SNAP. penicillamine, was shown to have no effect on neurotransmitter release at 10 pM (data not shown).

Discussion

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FIG. 5. Mean i- SEM smatal concentrations of transmitters, CIT, NO*-, N03- and ARG in in vivo microdialysis samples (15 min) taken during successive SO pM kainate challenges with either DNQX (200 kM; open squares) or APV (100 pM; filled squares) being given by retrodialysis for I h prior to the second kainate challenge and for 30 min following it. * P < 0.05 compared with control samples. Six animals were used in each treatment group.

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2628 NMDA and k a k t e - e v o k e d NO and striatal transmitter release

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NMDA and kainate-evoked NO and striatal transmitter release 2629

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2630 NMDA and kainate-evoked NO and striatal transmitter release

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NMDA and kainate-evoked NO and striatal transmitter release 263 1

transmitters. We have also provided the first in vivo evidence for a possible neuroprotective role for NO release in the striatum. Both NMDA- and to a lesser extent kainate-evoked transmitter release is potentiated by reducing NO levels, through inhibition of NOS activity. Conversely, enhancing NO levels during NMDA receptor activation using an NO donor reduces evoked transmitter release.

Our results showed parallel changes in the co-product of NO, CIT, and in its main breakdown products ( N O 1 and N03-) in response to NMDA, kainate and KCl and reductions in their levels after direct treatment with an NMDA receptor antagonist or direct or peripheral treatment with a total NOS inhibitor (L-NARG) or peripheral treatment with a specific nNOS inhibitor (7-nitroindazole). This suggests that changes in their levels reflect accurately altered NO release from striatal nNOS cells. Similar results for L-NARG inhibition of NOf and CIT in the striatum have been reported in vivo in a recent microdialysis study (Ohta et al.; 1994a) and in N02- and N03- ex vivo (Salter et al,, 1996). Our findings that striatal NO3- levels are three- fold higher in vivo and 4-fold higher in vitro than N O 1 also agree with previous work (Ohta et al., 1994a) and this ratio is similar to that in human saliva (Green et al., 1982).

The novel use of anion exchange chromatography to measure NO*- and NO3- concentrations in the brain directly is simple, and at least 40 times more sensitive (0.5 pmol versus 20 pmol) than the indirect Greiss reaction method used in other studies (Luo et a1.,1993; Luo and Vincent, 1994), although this method has been adapted using a diazotization reaction with spectrophotometric detection to produce sensitivities closer to ours (Ohta et al., 1994a).

The stimulatory effects of NMDA, kainate and KCI on concentra- tions of the NO precursor, ARG, were seen both in vivo and in vitro and closely paralleled similar increases in CIT. Citrulline can be converted to ARG by the enzymes arginosuccinate synthetase and arginosuccinate lysase which are both present in NOS-containing cells (Shuttleworth et al., 19953, although at relatively low levels in the striatum (Arnt-Ramos et al., 1992). It is therefore possible that the increased ARG concentrations we have seen during pharmacological challenges that also increase CIT may be due to the rapid conversion of CIT to ARG.

It is likely that NMDA effects on striatal NO release are mediated directly through NMDA receptors on nNOS-containing neurons since they are blocked by NMDA receptor antagonists (APV or MK-801), but not by an AMPA/kainate receptor antagonist (DNQX). The sensitivity of striatal nNOS cells to NMDA is surprising given that they only express very low levels of NMDARl mRNA (the subunit necessary for the functional activation of the NMDA receptor; Augood et a1.,1994; Landwehrmeyer et al., 1995). However, the same is true with striatal cholinergic neurons which express low levels of NMDARl (Landwehrmeyer et al., 1995) and which we have also found to be very sensitive to NMDA. Striatal nNOS/diaphorase and, to a lesser extent, cholinergic cells are also relatively resistant to excitotoxic damage produced by NMDA receptor agonists (Koh et 4 1 9 8 6 ; Davies and Roberts, 1988; Dawson et al., 1996) although some authors have failed to reproduce this (Mackenzie et al., 1995).

Cortical NOS-containing cells have calcium permeable kainate receptor-gated channels (Weiss et al., 1994) and our findings that kainate both evokes NO release and that this effect is blocked by DNQX, but not APV, suggests that this is also the case for striatal NOS cells. Striatal nNOS-containing neurons are also susceptible to kainic acid-induced toxicity and NOS inhibitors significantly reduce this effect (Dawson et al., 1993).

Our failure to find effects of AMPA on striatal NO release at doses of up to 100 pM does not rule out the possibility that GLU can evoke NO release through AMPA receptors although they would be much

less sensitive than NMDA or kainate. While a recent study has failed to find AMPA receptors on striatal somatostatin-containing cells (Tallaksen-Greene and Albin, 1994) there is evidence for calcium permeable AMPA receptor channels on NOS cells in the cortex (Weiss et al., 1994) and the AMPAlmetabotropic receptor agonist, quisqualate, has been shown to preferentially destroy striatal nNOS cells (Dawson et al., 1993, 1996).

Under physiological conditions, therefore, GLU may evoke NO release from striatal nNOS-containing cells through an action on both NMDA and kainate but probably not AMPA receptors. The NMDA receptors are nevertheless more sensitive than those for kainate in this respect since NMDA evokes NO release at a lower dose- threshold. Also, whereas NMDA receptor antagonists significantly reduce basal NO release AMPAkainate antagonists do not. This may be due to a greater efficacy for NMDA receptor activation to cause calcium entry into cells and thereby increase NO production. However, it is also possible that striatal DA release increases responsiveness to NMDA while reducing that to AMPA and kainate. Some stnatal nNOS cells express DI but none express D2 receptors (Le Moine et al., 1991) and while DA potentiates NMDA-evoked depolarization of cells through the D1 receptor it attenuates the effect of AMPA and kainate on them (Cepeda et al., 1993). This contrasts with DA actions via the D2 receptor which are inhibitory i n this respect for all three receptor types.

It would thus appear that there is a paradox in the striatum where nNOS-containing cells are more sensitive to NMDA than kainate or AMPA but are more resistant to neurotoxic damage resulting from NMDA receptor agonists compared with those for kainate or AMPA receptors. While the increased sensitivity to NMDA may be explained by increased efficacy of calcium entry and/or by DA facilitation, how can the converse situation be explained where the nNOS cells are more resistant to calcium dependent neurotoxic actions of NMDA receptor agonists compared with those for kainate and AMPA? For this to occur, NMDA receptors on nNOS cells would have to become rapidly desensitized in response to toxic concentrations of GLU or NMDA receptor agonists. This desensitization is unlikely to involve NO since in striatal cultures from mice lacking the nNOS gene cells which would normally contain nNOS (i.e. somatostatin positive) are still resistant to NMDA toxicity (Dawson et al.. 1996). The cause of this desensitization therefore remains to be shown although it need not necessarily involve differential effccts on NMDA receptors on nNOS and non-nNOS containing cells since the paucity of these receptors on the NOS, and for that matter cholinergic cells, could still be invoked to explain their protection relative to other cell types.

In vitro experiments have previously shown that striatal NO-release may play a neuroprotective role by acting to reduce the sensitivity of the NMDA receptor and thereby reducing calcium entry into cells (Manzoni et al., 1992; Manzoni and Bockaert, 1993; Emson et al., 1995). We have now provided extensive in vivo support for this putative neuroprotective role in cholinergic and GABAergic striatal cells and excitatory amino acid terminals. Both peripheral and/or striatal application of NOS inhibitors significantly increases NMDA- evoked striatal release of ACh, ASP. GLU and GABA. This effect is most marked with ACh suggesting that cholinergic cells may be the most sensitive. Since these NMDA effects are calcium dependent reduction in NO levels must lead to increased calcium entry into cells following NMDA receptor activation. Conversely, we have shown that increasing NO release prior to an NMDA challenge by administration of an NO-donor, SNAP, significantly inhibits NMDA- evoked release of ACh, ASP and GLU suggesting that reduced calcium entry has occurred. While we cannot rule out a potential contribution to the modulation of NMDA-evoked transmitter release

2632 NMDA and kainate-evoked NO and striatal transmitter release

by other cellular actions of NO donors or inhibitors, these are unlikely to be of major importance. Our current in vivo data showing that NO-sensitive neurotransmitter effects of NMDA are calcium dependent, together with the direct in vitro evidence that NO donors can decrease NMDA-evoked calcium entry and GABA release (Emson et a/ . , 1995) in cultured striatal cells, strongly suggest that the primary effects of NO on NMDA-evoked transmitter release are mediated by modulating calcium entry. Thus NO could be acting functionally as a neuroprotective agent by reducing potentially damaging glutamatc- evoked calcium entry. Similar potentiating effects of NOS inhibitors on NMDA evoked ASP in cerebellar slices (Dickie et al., 1992) and GABA release in striatal slices (Moller et al., 1995) have been reported.

The specificity of the desensitizing action of NO on the NMDA receptor is confirmed by the absence of an effect of NOS inhibitors on KCI-evoked neurotransmitter release. While our results do indicate that kainate-evoked transmitter release is also influenced by NOS inhibition it is minor compared with NMDA; it potentiated ACh release by 50% compared with a 520% potentiation by NMDA. Also, whereas the NOS inhibitor increased NMDA-evoked GLU, ASP and GABA rclcasc. kainate did not. Further, we have recently shown in four animals that similar retrodialysis administration of L-NARG, as used in the present study, had no effect on AMPA (50 pM) induced ACh release, suggesting that the AMPA receptor in the striatum is not desensitized by NO either.

The neuroprotective effects that NO desensitization of the NMDA receptor might produce are obviously not absolute since large doses of NMDA receptor agonists are neurotoxic for both striatal cholinergic and GABAergic cells. Our findings must also be contrasted with those showing that cultures from mice lacking the nNOS gene or trcatcd with NOS inhibitors show increased resistance to acute excitotoxic damage (Dawson et a/. , 1996). However, these results are not contradictory since, under conditions where acute excitotoxic challcngcs arc given, cell death is rapidly caused by peroxynitrite formation following massive NO release. On the other hand the neuroprotective action we havc obscrved with non-toxic levels of NMDA receptor activation suggests that it could function to reduce the likelihood of cell damage following repeated but physiological activation of striatal neurons by excitatory glutamatergic inputs. It might also protect against the chronic neurodegenerative effects of ageing, for example and it is interesting to note that decreased NOS and guanylate cyclase activities have been reported in the hippocampus of agcd rats (Vallebuona and Raiteri. 1995).

Dopamine release showed the opposite pattern to other transmitters following NMDA and kainate challenges after L-NARG treatment. This parallels our findings in a previous study examining the effects of NO and NO-donors on striatal transmitter release (Guevara- Guzman et al.? 1994). A similar effect of NOS inhibitors in reducing NMDA-evoked DA release has been reported in striatal slices by two other groups (Hanbauer et al., 1992; Sandor et al., 1995). Other groups have reported the opposite effects in vivo (Lin st al.,1995; Shibata et al., 1996) using much higher and normally neurotoxic levels of NMDA (1 mM). Our previous work has shown that in vivo application of NO to the striatum indirectly inhibits DA release (Guevara-Guzman et ul., 1994) and this inhibitory action is confirmed by our present findings that NOS inhibitors significantly increase DA concentrations. Thus the toxic doses of NMDA used in these in viva studies would have released large amounts of NO and may have diminished apparent NMDA-evoked DA release under control condi- tions. NOS inhibitors would then have removed this inhibitory action of NO on DA release, resulting in an apparent potentiation.

The fact that NO may facilitate DA release in response to NMDA

challenges raises a number of potential functional consequences relating to the initiation of movement and also to the phenomenon of behavioural sensitization. In the former case one outcome of NOS inhibition could be to reduce striatal DA release in response to excitatory cortical and thalamic inputs resulting in a failure or delay in initiating movemenl. Indeed, NOS inhibitors can reduce NMDA- evoked locomotor responses in mice (Starr and Starr, 1995). Similarly, behavioural sensitization may involve a potentiation of NMDA receptor mediated DA release and altered NO levels could play a role in this. This is supported by the linding that NOS inhibitors block behavioural sensitization to methamphetamine in mice (Ohno and Watanabe, 1995).

Our results showing the respective actions of NMDA, kainate and AMPA on in vivo DA release confirm previous reports that all three can increase striatal DA concentrations with kainate and AMPA being more potent than NMDA (Westerink et al., 1992; Ohta et al., 1994b). Whereas NMDA effects on DA release could be blocked hy the NMDA receptor antagonist, APV, they could also be blocked by the AMPNkainate receptor antagonist. DNQX. This suggests that although there are NMDA receptors on striatal DA terminals (Chowdhury and Filenz, 1991; Desce et a/ . , 1992) the stimulatory effects of a 50 pM dose of NMDA on DA levels are mainly mediated indirectly by NMDA increasing GLU release which in turn acts on AMPA/kainate receptors on DA terminals to promote DA release. Indeed, this may also explain the lower potency for NMDA-evoked DA release compared w-ith AMPA and kainate.

Our finding that the NMDA receptor antagonist APV, but not the AMPNkainate receptor antagonist. DNQX: tonically increases striatal DA release is in agreement with previous studies (Imperato et al., 1990; Richard and Bennett, 1995) which have suggested that GLU tonically inhibits DA release via the NMDA receptor. It is generally thought that this is an indirect effect and there is some evidence that it may be mediatcd through disinhibition of local GABA neurons since it can be blocked by the GABAA receptor antagonist bicuculline (Leviel et ul., 1990). Our results support this hypothesis since APV, but not DNQX, also caused a significant reduction in basal striatal GABA release. The effect of APV might have been caused by reduced NO release, since NO can inhibit DA release in vivo (Guevara- Guzman et ul., 1994). However, this is unlikely since NMDA receptor antagonist-evoked DA release cannot be blocked by NOS inhibitors (Richard and Bennett, 1995).

Our results on the relative effects of NMDA, kainate and AMPA on striatal classical transmitter release other than DA are in broad agreement with some previous studies (Scatton and Lehmann. 1982; Young and Bradford, 1991; Ohta et al., 1994b) although they are more extensive. In vivo, NMDA and kainate were equipotent in stimulating ACh release and more effective than AMPA. The in vitro results showed this trend as well. However, this is in slight disagrcc- ment with a previous in vitro slice study which reported that NMDA was more potent than kainatc in evoking [3H]-ACh release (Scatton and Lehman, 1982). Similarly. NMDA and kainate were more potent in stimulating 5-HT release than AMPA in vivo which differs from a previous report claiming that they were all equipotent (Ohta et a]., 1994b).

Our results comparing the relative abilities of NMDA, kainate and AMPA for inducing ASP, GLU and GABA release are novel; previous studies have concentrated on NMDA (Young and Bradford, 1991). NMDA dose-dependently increased GABA release and at the lowest threshold for any of the transmitters (1 FM). It was also more potent than kainate both in vivo and in vitro. However, AMPA had no significant effect up to a 100 pM dose. This agrees with the finding that striatal GABAergic cells express high levels of NMDARl

NMDA and kainate-evoked NO and striatal transmitter release 2633

GAHAergic mRNA (Landwehrmeyer et nl., 1995). Similarly in vivo, NMDA dose-dependently increased A S P and GLU release and was more potent than kainate. Again, AMPA was without effect. Kainate also seemed to be showing a bell shaped response curve with the 100 FM dose for ASP being less effective than the 50 pM one. This was also the case with a nuinber of other substances and may reflect saturation effects of kainate a t 100 FM. In vitro none of the GLU agonists released ASP or GLU significantly, although this may be a reflection of the fact that the cortical and thalamic projection pathways to the striatum arc not intact in the slice cultures.

Acknowledgement R. G.-G. was supported by a scholarship from Consejo Nacional de Ciencia y Tecnologia y Direccion General dcl Personal Academico, Grant number "200594.

Abbreviations ACh AMPA ARC APV ASP CIT DA DNQX GABA GLU 5-HT L-NARC NMDA nNOS NO SNAP

acetylcholine (RS)-cl-anlino-3-hydroxy-5-methyl-4-isoxazolepropionic acid arginine ~,~-2-amino-S-phosphopentanoic acid aspartate citrulline dopamine 6,7-dinitroquinoxalinc-2,3-dione y-aminobutyric acid glutamate 5-hydrox ytryptamine L-nitroarginine N-methyl-o-aspartic acid neuronal nitric oxide spthase enzyme nitric oxide S-nitroso-N-acetyl-o,L-penicihminc

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