Nitric oxide does not mediate acute glutamate neurotoxicity, nor is it neuroprotective, in rat brain slices

Neuropharmarology

Vol. 33, No. II, pp. 1431-1438, 1994 Copyright e 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 002%3908/94 $7.00 + 0.00

0028-3908(94)00093-X

Nitric Oxide Does not Mediate Acute Glutamate Neurotoxicity, nor is it Neuroprotective, in Rat Brain Slices G. GARTHWAITE Wellcome

Research Laboratories,

Langley

and

J. GARTHWAITE*

Court, South Eden Park Road, Beckenham

BR3 3BS, U.K.

Summary-Nitric oxide (NO), generated upon glutamate receptor activation, elicits cyclic GMP accumulation through stimulation of guanylyl cyclase. NO is also a potential cytotoxin that has been suggested, on the basis of tissue culture experiments, to mediate neuronal damage associated with excessive activity of the /V-methyl-o-aspartate (NMDA) subtype of glutamate receptor. We have investigated the involvement of NO in the toxicity of glutamate receptor agonists in brain slice preparations. Slices of cerebellum and hippo~dmpus from the developing rat exhibited neuronal necrosis following exposure (5-30 mm) to NMDA (IO0 HIM or 1 mM). When the exposures were carried out in the presence of NO synthase inhibitors, at concentrations suppressing NMDA-induced NO formation (as judged by measurements of cyclic GMP accumulation), the extent of injury was unaffected. To determine if exogenous NO is able to replicate NMDA toxicity, the slices were exposed to high concentrations of NO donating compounds for up to 2 hr. No damage was detectable. NO donors, moreover, neither reduced NMDA toxicity, nor potentiated the degeneration caused by just suprathreshold NMDA concentrations. The toxicities of non-NMDA agonists, or of glutamate itself, were also unaltered by NO synthase inhibitors or NO donors. Similar results were obtained using hippocampal slices from more mature animals. We conclude that the acute neurodegeneration mediated by NMDA or non-NMDA receptors in the slice preparations is not mediated by NO, nor is NO neuroprotective under these conditions. Keywords-Nitrjc

oxide, glutamate, NMDA

receptors,

Neuronal death caused by excessive stimulation of excitatory amino acid (EAA) receptors may contribute to the

neurodegeneration taking place as a result of cerebral ischaemia since, in experimental animals, blockers of N-methyl-D-aspartate (NMDA) or a-amino-3-hydroxySmethyl-4-isoxazolepropionate (AMPA) subtypes of glutamate receptor confer neuroprotection; more speculatively, this mechanism may also participate in chronic types of neurodegenerative disorder, such as Alzheimer’s disease and Huntington’s chorea (Choi, 1988; Meldrum and Garthwaite, 1990). Studies in brain slices and cultured neurones have suggested that the cell death associated with both NMDA and AMPA receptor stimulation is dependent on Ca’+, but the downstream events culminating in irreversible injury remain unclear. A number of Ca*+activated enzymes could participate, one being the nitric oxide (NO) generating enzyme, NO synthase. NO is formed in neurones in response to increases in intracellular Ca’+ in the submicromolar range. Under physiologi-

*To whom

correspondence

should

be addressed. 1431

excitotoxicity,

neurodeg~neratjon.

cal conditions, it functions as an inter-cellular messenger molecule that raises the levels of cyclic GMP by stimulating guanylyl cyclase enzymatic activity (Garthwaite, 1991). Excessive production of NO, however, could cause damage through various mechanisms, including inhibition of metabolic pathways and, in the presence of superoxide anions, the formation of toxic free radicals (Moncada ef al., 1991; Nathan, 1992). In cultures of cerebral cortex and other brain regions (hippocampus, striatum), Dawson et al. (1991, 1993) reported that NMDA neurotoxicity, but not that of the non-NMDA receptor agonist, kainate, was partly mediated by NO as the neuronal loss could be reduced by NO synthase inhibitors and could be replicated by exposure to NO-donating chemicals. It was further proposed that the small numbers of NO synthase-containing neurones (l-2% of the total) were responsible for killing the larger population of neighbouring neurones whilst the NOgenerating cells themselves were resistant to NMDA and NO neurotoxicity. If generally valid, these findings could have important repercussions for the understanding and treatment of neurodegenerative disorders. Currently, however, the

1432

G. GARTHWAITEand J. GARTHWAITE

situation is confused as support has been received from some tissue culture studies (Tamura et al., 1992; Vige et al., 1993; Reif, 1993) but not others (Demerit-Pallardy et al., 1991; Pauwels and Leysen, 1992; Regan et al., 1993; Hewett et al., 1993; Zinkand et al., 1993; LafonCazal et al., 1993). Paradoxically, it has also been suggested that NO can be neuroprotective either by inhibiting NMDA receptor function (Manzoni et al., 1992; Lei et al., 1992) or by blocking free radical damage (Wink et al., 1993). Much of the confusion presumably arises because of subtle differences in tissue culture conditions; the composition of the cultures with respect to the numbers of NO-generating cells may also be critical. The use of freshly-prepared brain slices should circumvent such variables but, with one exception (Izumi et al., 1992), results on intact slices have not been reported. In the present study we have used slice preparations of two brain regions, cerebellum and hippocampus, to answer three specific questions: does NO mediate EAA neurotoxicity, can exogenous NO mimic the toxicity of EAAs, and is NO cytoprotective?

METHODS

Most of the experiments were carried out on brain slices from the developing (8 days postnatal) Wistar rat, using methods described in detail previously (Hajos et al., 1986). In brief, the slices were cut 0.4 mm thick and incubated at 37°C in a Krebs solution of the following composition (mM): NaCl (120), KC1 (2), CaCl, (2) NaHCO, (26), MgSO, (1.19), KH,PO, (1.18) and glucose (1 l), continuously gassed with 95% O,, 5% CO,. Following a preincubation period lasting at least 90 min, the experiment was started by addition of test compounds. After various exposure periods (see results), the slices were usually transferred to fresh incubation medium for 1.5-2 hr before being fixed and embedded. Unless otherwise stated, antagonists were added 15 min before the EAA receptor agonist and were present for the first 15 min of the recovery period. Semithin sections (1 pm) were stained with toluidine blue and examined by light microscopy. Quantitation was performed by direct cell counts, as described (Garthwaite and Garthwaite, 1986, 1991) or, in the case of glutamate toxicity, by measuring the depth of necrosis (Garthwaite et al., 1992). The results are given as means f SEM for n slices, each slice being from a different animal. For each experimental condition, no more than 2 slices were used on any one day. In other experiments, an attempt was made to reproduce the results described by Izumi et al. (1992) on young adult rat hippocampal slices and so we followed the methods for slice preparation and incubation exactly as described by these authors, including the use of animals (either sex) of the same age range (27-32 days).

Measurements of cyclic GMP levels were made using standard radioimmunoassay methods (Southam et al., 1991). S - nitroso -N - acetylpenicillamine, S - nitrosoglutathione and L-NC-monomethylargine were synthesised at the Wellcome Research Laboratories; AMPA was bought from Tocris, dizocilpine and 3-morpholinosydnonimine N-ethylcarbamide (SIN-l) were gifts from Merck, Sharpe and Dohme (Terlings Park, U.K.) and Cassella-Reidel Pharma (Frankfurt, Germany) respectively; all other special chemicals were from Sigma (St Louis, MO, U.S.A.). RESULTS

Cerebellar slices Two different exposure protocols were used for studying NMDA toxicity. In the first, the slices were incubated with 100 PM NMDA for 30 min. This results in the selective necrosis of differentiating granule cells, but not of Purkinje or Golgi cells, in the cerebellar cortex (Hajos et al., 1986). With 2 mM Ca2+ in the bathing medium (as used here) the extent of granule cell death is submaximal, as it increases by about 30% on raising to 2.5 mM (Garthwaite and the Ca2+ concentration Garthwaite, 1986). When the exposure was carried out in the presence of the NO synthase inhibitors, L-N~methylarginine (L-MeArg) or L-NC-nitroarginine (LNOArg) the number of necrotic cells was unchanged [Fig. l(a)], even though the inhibitor concentrations used (100 ~1M and 10 p M, respectively) completely suppressed NO formation, as evidenced by measurements of the cGMP response to NMDA [Fig. l(a)]. Haemoglobin (1 &IO0 p M, with 30 min preincubation), which binds and inactivates NO and hence also inhibits NMDAmediated cGMP accumulation (Southam and GarthWaite, 199la), also had no effect on NMDA toxicity; the values at the highest concentration tested (100 PM) were: 32 + 2 necrotic granule cells per lo4 pm* compared with the control value (NMDA alone) of 30 +_ 1 (n = 4). The second protocol employed brief (5 min) exposures to a higher NMDA concentration (1 mM), to match the paradigm used by Dawson et al. (1993) on cultured neurones. The numbers of granule cells killed using this method was about 25% of that observed using the first protocol, and was not significantly affected by either of the NO synthase inhibitors [Fig. l(b)]. We considered the possibility that, since the incubation medium did not contain any added L-arginine, the precursor of NO, the cells may be deficient in the amino acid and therefore be unable to sustain NO synthesis at the rates needed for it to contribute to degeneration. However, when the medium was supplemented with L-arginine at a concentration (300 PM) 10 times higher than its reported ECso for enhancing cGMP responses to NMDA in cerebellar slices (Garthwaite et al., 1989) and for promoting NMDA neurotoxicity in cortical cultures (Dawson et al.,

1433

NO and excitotoxicity

60 160

0-d

NMDA 1 mM for 5 mins

+ L-Arg 300 FM

+ L-NOArg 10pM

+ L-MeArg 1OOpM

NMDA 100 pM

r1 + L-MeArg 100~M

‘0

Necrosis

m

cGMP

+ L-NOArg 10pM

Fig. 1. NO synthase inhibitors abolish NMDA-mediated cGMP accumulation (filled columns) but fail, as does additional L-arginine, to modify NMDA neurotoxicity (open columns) in immature rat cerebellar slices. In (a), the concentration of NMDA used was 100 pM and the exposure period for the toxicity data was 30 min and for cGMP measurements, 2 min. In (b), slices were exposed to 1 mM NMDA for 5 min. In all cases, slices were preincubated for 15 min with the NO synthase inhibitors or L-arginine, before addition of NMDA. Values are means + SEM (n = 6-12); *P < 0.0001 (by Student’s t-test). 1993), no increase in the amount of damage could be detected [Fig. 1(b)]. Next, we evaluated the effects of exogenous sources of NO, using the NO-donors, S-nitroso-N-acetylpenicillamine (SNAP) and S-nitrosoglutathione (SNOG) as well as the compound, SIN- 1, which yields both NO and superoxide anions. At high concentrations (300 PM SNAP and SNOG, 3 mM SIN- 1; selected on the basis of their potencies at raising cyclic GMP levels in cerebellar slices: Southam and Garthwaite, 1991b) and with exposures for up to 2 hr, none of these compounds was toxic on its own (fz = 4; results not shown). It could be argued that in order to observe NO toxicity, simultaneous stimulation of NMDA receptors may be necessary. Therefore the NO donors were applied at the same time as a concentration of NMDA that was justsuprathreshold for causing injury (30pM for 30 min). No additional damage ensued (Fig. 2). In addition, the NO donors neither enhanced nor reduced the toxicity of a higher NMDA concentration (100 PM; Fig. 2). Experiments were also carried out using glutamate. The formidable properties of the glutamate transporter, coupled with the three-dimensional nature of the tissue, greatly restricts the neurotoxic potential of glutamate in brain slices and, except with very high concentrations, the damage is restricted to a band of tissue immediately adjacent to the bathing medium, and is blockable by

NMDA antagonists (Garthwaite et al., 1992). In agreement with our previous results, 300 FM glutamate killed granule cells only within the outer 30% of the crosssectional area (Fig. 3). This depth of necrosis was unaffected by L-NOArg (10 ,uM), SIN- 1 (3 mM) or SNAP (300pM) (Fig. 3). Non-N~DA agonists, such as AMPA and kainate, stimulate NO formation in cerebellar slices (Southam et al., 1991) and induce neurodegeneration in the same tissues, although with patterns that are distinct from each other and from that produced by NMDA: AMPA elicits a gradually-evolving, delayed, dark type of degeneration of Purkinje cells and an acute oedematous necrosis of some Golgi celts, but does not kill granule cells, whereas kainate induces acute necrosis of Golgi cells, without affecting Purkinje or granule cells (Hajos et al., 1986; Garthwaite and Garthwaite, 1991). Two exposure protocols for AMPA were tested: 30 min exposure to a 30 PM concentration, which produces degeneration of about 80% of Purkinje cells, and a longer (2 hr) incubation with a lower concentration (10 p M), where the corresponding damage is less (about 20%). In neither case did L-NOArg reduce the degeneration [Fig. 4(a, b)]. Moreover, AMPA neurotoxicity (10 PM, 2 hr) was unaffected by SIN-l, SNAP and L-arginine [Fig. 4(b)]. Likewise, kainate toxicity was

1434

G. GARTHWAITE and J. GARTHWAITE m

0

T

NMDA 100 pM NMDA 30 pM

T

ONMDA 30 mins

+ SIN-1 3mM

+ SNAP 300 pM

+ SNOG 300 pM

Fig. 2. NO donors do not enhance or diminish NMDA toxicity in cerebellar slices induced by a just suprathreshold NMDA concentrations (30pM: open bars) or by a higher, but submaximal, concentration (100 PM; hatched bars). Exposures to NMDA were for 30 min; NO donors were added 5 min before the NMDA. Data represent the means f SEM of 6 slices.

unchanged

additional

in the presence of NO synthase L-arginine [Fig. 4(c)].

inhibitors

or

Hippocampal slices To determine if the above negative findings were the result of using cerebellum, rather than one of the brain regions studied by Dawson et al. (1993), several key experiments were carried out on slices of hippocampus. Exposure of hippocampal slices from 8 day old rats to 100 PM NMDA for 20 min leads to total loss of pyrami-

da1 neurones in the CA1 and CA3 sectors together with necrosis of about 50% of granule cells in the dentate gyrus (Garthwaite and Garthwaite, 1989). No reduction in cell death was observed when the exposure was performed in the presence of the NO synthase inhibitors, L-MeArg (100 PM and 1 mM, n = 4 each) or L-NOArg (10 and 30 PM, n = 4 each). These tests were done using 15 min preincubation with the inhibitors, a protocol previously shown to block cyclic GMP responses to NMDA in hippocampal slices (East and Garthwaite,

35l30-

I

-r

T

l-

8 25.% 8 5 20E 5 15Jz z $ ‘@ 5-

Glutamate 300 pM for 30 min Fig. 3. Glutamate

+ L-NOArg 1OpM

+ SIN-1 3mM

+ SNAP 300 pM

toxicity (300 PM; 30 min exposure) in cerebellar slices is unaffected by L-NOArg NO donors, SIN-l and SNAP. Values are means f SEM of 6 slices.

or by the

NO and excitotoxicity

1435 DISCUSSION

[AMPA (3O PM), 30 mins]

NO and EAA

+ L-NOArg (10PM) [AMPA (lo r~). 2 h0uffi

+L-NOArg +L-Arg +SIN-1 (10~M) (3OOpM) (3mM)

+SNAP (300pM)

+L-NOArg +L-MeArg +L-Arg (10 PM) (100 pM) (300 FM)

Fig. 4. Lack of involvement of NO in non-NMDA neurotoxicity elicited by (a) short exposure to AMPA (30 PM for 30 min), (b) longer exposure to AMPA (I 0 p M for 2 hr) or (c) kainate (30 PM for 30 min) in immature rat cerebellar slices. Values are means k SEM of 6-12 slices. In each case, controls are shown on the left and the experimental conditions of the tests are indicated under each bar.

1991). In another set of experiments, a 1 hr preincubation (and 15 min post-incubation) period was used with t,-NOArg (3 and 10 PM, n = 4 each), but this still proved ineffective, as did haemoglobin (1, 10 and 100 PM, n = 4 each) applied in the same way. Finally, hippocampal slices were exposed to the NO donors, SNAP (300 PM), SNOG (300 PM) and SIN-l (3 mM) for 2 or 3 hr (with and without 90 min recovery), but no damage was detectable (n = 4 in each case). These findings are at odds with those reported for hippocampal slices from young adult (30 day old) rats by Izumi et al. (1992) who claimed that NMDA toxicity was substantially reduced by t-NOArg, L-MeArg and haemoglobin. We attempted, therefore, to reproduce the results of these authors, using identical methods. In our hands, none of these treatments (in all cases, 1 hr preincubation but no postincubation) was neuroprotective, whereas the damage could be completely prevented by the non-competitive NMDA antagonist, dizocilpine (Fig. 5; e = 4 for each condition).

neurotoxicity

Our results provide no support for the idea that NO mediates EAA neurotoxicity, either in immature cerebellar slices or in immature and young adult hippocampal slices as neither inhibition of NO synthase nor scavenging of NO with haemoglobin afforded any protection. Moreover, high concentrations of NO donating drugs were incapable of replicating the lethal effects of EAA receptor agonists. We conclude that, in the slice preparations we have studied, NO is neither necessary nor sufficient for EAA neurotoxicity. Our findings concur with those of several studies carried out on cultured neurones, for example of whole brain (Demerle-Pallardy et al., 1991) hippocampus (Pauwels and Leysen, 1992), cerebral cortex (Regan et al., 1993; Hewett et al., 1993; Zinkand et al., 1993) and of cerebellar granule cells (Lafon-Cazal et al., 1993). To the extent that non-NMDA toxicity in the slices did not appear to involve NO, our results also agree with those of Dawson et al. (1993). The major discrepancy between our results and previously-published data concerns NMDA toxicity in the young adult hippocampal slices where Izumi et al. (1992) found that L-MeArg, L-NOArg and haemoglobin protected against NMDA-induced necrosis whereas we, using the same concentrations and apparently identical methods, failed to see any such effects. We cannot find any obvious explanation for this discrepancy. It should be added that, in contrast to the lack of toxicity of NO donors in our experiments, Wallis et al. (1993) have reported that, in adult rat hippocampal slices, authentic NO has toxic actions (assessed by electrophysiological recording of synaptic transmission in the CA1 region) but it is difficult to relate the concentration used (150 ,u M) and the conditions under which it was applied (10 min in oxygen-free medium) to those in our experiments, or to those that might occur following NMDA receptor activation or in pathological conditions in vivo. In this regard, it might be anticipated that studies of EAA neurotoxicity in vivo should help clarify matters. Unfortunately not. Moncada ef al. (1992) reported that L-NOArg methyl ester (L-NAME) decreased NMDAinduced hippocampal lesions by up to 40% without affecting those caused by AMPA and Fujisawa et al. (1993) found similar (30%) protection by L-NAME against glutamate toxicity in the cerebral cortex. On the other hand, Lerner-Natoli et al. (1992) found no neuroprotection was afforded by chronic NO synthase inhibition on intrahippocampal NMDA lesions and in the experiments of Haberny et al. (1992), inhibition of NO synthesis potentiated the neurotoxicity of the NMDA receptor agonist, quinolinate. Under in vivo conditions, the problems imposed by the non-selectivity of the inhibitors on different isoforms of NO synthase (resulting in, for example, changes in blood flow) may complicate the outcome, in the same way that they appear to

1436

G. GARTHWAITE and J. GARTHWAITE

Fig. 5. NO synthase inhibitors and haemoglobin do not protect against NMDA toxicity in young adult rat hippocampal slices. All photomicrographs depict the CA1 region. A control (untreated) slice is shown in (a). In (b) the slice was treated with 100 /*M NMDA for 20 min and was then given 90 min recovery; all neurones are destroyed. The degeneration was unaffected by 100 PM L-MeArg (c), 10 PM L-NOArg (d) or 1 PM haemoglobin (e) but was abolished by 1 PM dizocilpine (f). The pictures are representative of 4-6 slices examined under each condition. Scale bar (f) = 20 pm.

do in models of cerebral ischaemia (reviewed by Iadecola et al., 1994). Even in the tissue culture experiments where protective effects of NO synthase inhibitors have been observed, several discrepancies exist. For example, whereas Dawson et al. (1993) reported that, to be effective, NO synthase inhibitors needed to be present only during the exposure period, Vigt: et al. (1993) and Reif (1993) found insignificant effects using this protocol; but if they were administered during the 20-24 hr post-exposure period, neuroprotection was observed. The most effective regimen was for the inhibitors to be present both during the exposure and afterwards (VigC et al., 1993). This suggests that delayed NO formation participates importantly in the cell killing in this model. Curiously, in the experiments of Dawson et al. (1993) who did not deliberately postincubate with NO synthase inhibitors, cell death

(assessed by trypan blue staining) was still delayed as it did not occur until between 4 and 12 hr after the exposure to NMDA. The presence of residual amounts of NO synthase inhibitors in their cultures, after the (apparently) single washout of the exposing solution, may explain the protective effects observed. A critical factor, therefore, may be the time-course of cell death being studied in different paradigms. As discussed above, NO synthase inhibitors appear to confer protection under conditions where the neurones succumb in a delayed manner, possibly several hours after the NMDA insult. In our more acute slice experiments, on the other hand, the transition to irreversible injury in response to NMDA takes place rapidly as, based on conventional histopathological criteria, the vulnerable neurones progress to frank necrosis by the end of a 90min recovery period (Hajos et al., 1986;

1437

NO and excitotoxicity Garthwaite and Garthwaite, 1989). It remains possible, conversely, that neurones that survive acute degeneration in our experimental design, may succumb several hours later; of possible relevance is the observation that cerebellar granule cells, at least in tissue culture, can be killed by long (24-48 hr) exposure to NO generated from activated microglia by the inducible NO synthase (Boje and Arora, 1992). Consequently, neurones in vitro appear to die in response to NMDA receptor activation in at least two ways: acutely and in a Ca*+-dependent but NO-independent manner, and in a delayed fashion through mechanisms that may involve NO. Which, if any of these, is the more relevant to pathological conditions in vivo remains to be determined. NO and neuroprotection Given the possibility that NO could protect neurones by inhibiting NMDA receptor function (Manzoni et al., 1992; Lei et al., 1992) our experiments were also designed to reveal any neuroprotective effects of endogenous or exogenous NO. Should endogenous NO perform this function, NMDA toxicity should have been increased in the presence of NO synthase inhibitors, but this was not observed. Further, we could not detect any diminution in damage with high concentrations of NO donors. These findings are in keeping with the observation that SIN-l and SNAP do not reduce NMDAmediated depolarizations in cerebellar slices (East et al., 1991). NO and selective

vulnerability

The small percentage of neurones in the striatum and cortex that stain histochemically for NADPH diaphorase, now known to represent NO synthase, appear to be more resistant than the general population to experimental excitotoxic insults and also to survive preferentially in disease states such as Huntington’s chorea (reviewed by Vincent, 1994). A causal link between expression of NO synthase and low vulnerability to NMDA-mediated injury has been considered (Dawson et al., 1993). In the cerebellum, however, the neurones selectively vulnerable to NMDA, the granule cells, are also among the richest in NO synthase of any neurone in the brain and are overwhelmingly the major source of NO following NMDA receptor stimulation (Garthwaite and Garthwaite, 1987; Garthwaite et al., 1988). Further, in the dentate gyrus in vivo, NADPH diaphorase-containing neurones have been reported to be selectively vulnerable to transient ischaemia (Hong el al., 1993). In other regions, neurones deficient or rich in NO synthase can be equally resistant to pathological insult (Vincent, 1994). Therefore, there does not appear to be any simple causal relationship between expression of NO synthase and either resistance or vulnerability to pathological challenge. In the case of the cerebellum, at least, the neurones that are vulnerable to NMDA also express high levels of NMDA receptors whereas those that are not (Purkinje cells), are deficient (Garthwaite

et al.,

pression

1986), suggesting that the level of receptor exmay be the more important determining factor.

work was supported by a project grant from the Medical Research Council (U.K.). We thank Merck, Sharpe & Dohme (Terlings Park, U.K.) and CassellaReidel Pharma (Frankfurt, Germany) for the gifts of dizocilpine and SIN-I respectively. Acknowledgements-This

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