Polyamines modulate the neurotoxic effects of NMDA in vivo

163

Brain Research, 616 (1993) 163-170 Elsevier Science Publishers B.V.

BRES 18994

Polyamines modulate the neurotoxic effects of NMDA in vivo Muhammad Munir, Swaminathan Subramaniam * and Paul McGonigle Department of Pharmacology, Universityof Pennsylvania, School of Medicine, Philadelphia, PA 19104 (USA) (Accepted 2 February 1993)

Key words: NMDA receptor; Excitotoxicity; Dizocilpine; Neuroprotection

The ability of polyamines to alter NMDA-induced neurotoxicity in neonatal rats was examined to determine whether polyamines modulate NMDA receptor activity in vivo. Unilateral injections of NMDA a n d / o r polyamines were made into the striatum of 7-day-old rats. After 5 days, the brains were removed and 20/.~m thick coronal sections were cut and stained with Cresyl violet. A computer-based image analysis system was used to densitometrically measure the cross-sectional area of intact tissue in the control and injected hemispheres. Administration of NMDA (5-40 nmol) produced a dose-dependent tissue damage that ranged from 7 to 52% of the area of the uninjected hemisphere. The polyamine agonist spermine (10-500 nmol) dose-dependently exacerbated the toxicity of a 15 nmol dose of NMDA, increasing the size of the lesion by up to 50%. Administration of spermine alone produced dose-dependent tissue damage that ranged from 9 to 52%. The damage produced by both NMDA and spermine could be completely inhibited by co-administration of the NMDA antagonist MK-801. The polyamine inverse agonist 1,10-diaminodecane (DA-10, 50-400 nmol) inhibited the damage produced by NMDA in a dose-dependent manner, with a maximal inhibition of 50%. Administration of DA-10 alone produced limited damage at doses above 100 nmol. The weak partial agonist diethylenetriamine had no effect by itself or on NMDA-induced toxicity at the doses tested. These results indicate that polyamines can modulate the activity of NMDA receptors in vivo and suggest that polyamines or related compounds may have important therapeutic potential as neuroprotective agents.

INTRODUCTION

The N-methyl-o-aspartate (NMDA) subtype of the glutamate receptor is involved in a variety of physiological processes in the central nervous system including the generation of long term potentiation, neuronal development and neuronal plasticity 3'32'4°. This receptor has also been implicated in the excitotoxic damage associated with neurological disorders such as stroke and epilepsy 5'34'51. Antagonists selective for the NMDA receptor prevent the damage observed in various in vivo and in vitro models of ischemia suggesting that over-stimulation of NMDA receptors is responsible for this damage 14'45'51. Moreover, direct administration of NMDA into the neonatal rat striatum mimics the damage produced by occlusion of the carotid artery followed by a period of hypoxia16'28. Several investigators have demonstrated that NMDA antagonists can attenuate neuronal damage even when administered min-

utes to hours after an ischemic or excitotoxic insult 6'1°'28'46. This demonstration of delayed rescue of neurons has led to the suggestion that NMDA antagonists may have therapeutic utility as neuroprotective agents. The NMDA receptor is a ligand-gated ion channel complex that has multiple regulatory sites in addition to the glutamate recognition site. These include a high affinity site for glycine that is thought to be required for channel opening 17'19, a voltage dependent site for Mg 2+ that blocks ion flUX24'25'33, and a site associated with the ion channel where non-competitive antagonists such as MK-801 bind to produce an open channel block 15'23. There is also a site for Zn 2+ that inhibits agonist-induced channel opening in a voltage-independent manner 38'58. In addition, NMDA receptors are sensitive to the extracellular concentration of H + and to the redox state 1'56'57. Another putative regulatory site on the NMDA re-

Correspondence: P. McGonigle, Department of Pharmacology, University of Pennsylvania, School of Medicine, Philadelphia, PA 19104-6084, USA. Fax: (1) (215) 898-9982. * Present address: Bldg. 10, Room 5N-248, NIH/NINDS, 9000 Rockville Pike, Bethesda, MD 20892, USA.

164 ceptor mediates the effects of polyamines. Spermidine and spermine enhance the binding of [3H]MK-801 to the NMDA receptor in vitro 42 and structure-activity studies have led to the suggestion that polyamines act as allosteric modulators of channel opening through a distinct binding site 6°. Spermidine and spermine have been classified as agonists at this site based on their ability to enhance the binding of [3H]MK-801, whereas diethylenetriamine (DET) has been classified as a weak partial agonist based on its ability to competitively block the effects of agonists 55'6°. The polyamine 1,10diaminodecane (DA-10) inhibits the binding of [3H]MK-8013°'6°, but this effect is attenuated by DET suggesting that DA-10 may be an inverse agonist at the polyamine binding site 59. Polyamines retain their ability to modulate [3H]MK-801 binding in solubilized preparations of receptor indicating that they interact with a site intrinsic to the receptor molecule 43'44. Polyamines can also modulate the activity of the NMDA receptor by altering the affinity of the receptor for the co-transmitter glycine41'47. The ability of polyamines to modulate the NMDA receptor in vitro and the presence of micromolar concentrations of polyamines in the central nervous system of several species 5° are consistent with a role for polyamines as endogenous modulators of NMDA receptor function. There is some in vitro electrophysiological evidence that polyamines modulate NMDA receptor function. In hippocampal neurons, the agonist spermine increases NMDA-elicited currents and the inverse agonist DA-10 has the opposite effect 2'59. Spermine has also been shown to increase NMDA-elicited currents in Xenopus oocytes expressing NMDA receptors and this response can be blocked by the putative polyamine antagonist putrescine 3t'52. In cultured spinal cord neurons, spermine potentiates NMDA-induced currents by delaying the onset of desensitization22. Despite their ubiquitous presence in the CNS, there is very little direct evidence that polyamines modulate the NMDA receptor in vivo. In mice, it has been reported that intracerebroventricular administration of spermidine enhances the ability of N-methyl-DLaspartate to induce seizures 53. In the present study, the ability of polyamines to alter the excitotoxicity mediated by NMDA receptors was examined to provide further evidence that polyamines modulate NMDA receptor function in vivo. The effects of the full agonist spermine, the partial agonist DET and the inverse agonist DA-10 on the extent of damage produced by intracerebral administration of NMDA in the neonatal rat were measured to determine how these effects correlate with the modulatory effects of these agonists observed in vitro. The ability of some polyamines to

attenuate the excitotoxicity produced by NMDA in this model suggests that these or related compounds may have therapeutic potential as neuroprotective agents. MATERIALS

AND METHODS

Materials (+)-MK-801 was purchased from Research Biochemicals Inc. (Natick, MA). Spermidine, spermine, diethylenetriamine and 1,10-diaminodecane were obtained from Aldrich (Milwaukee, WI). All other compounds were purchased from Sigma Chemical Co. (St. Louis, MO).

Production of lesions 7-day-old male and female Sprague-Dawley rat pups were anaesthetized with diethyl ether for 3 min and the animals were positioned in a plaster of Paris mold of head and body. A Kopf small animal apparatus was used to determine stereotaxic coordinates and intrastriatal injections were administered with a 26 gauge, beveled tip, Hamilton syringe. The tip of the needle was positioned 2.5 m m lateral to bregma just anterior to the coronal suture at a depth of 5.5 m m from the skin and was left in place for 3 rain following injection to reduce leakage. These coordinates result in a lesion that simulates the damage produced by unilateral carotid artery ligation and timed exposure to hypoxia 1s'26'27. Stock solutions of N M D A , spermine, DA-10, D E T and MK-801 were prepared in 0.01 M Tris buffer and their pH was adjusted to 7.4. Required dilutions were made in 0.01 M Tris, pH 7.4. The total volume of injection was 0.5/xl for groups not receiving MK-801 and 1.0 p.l for groups receiving MK-801. Each treatment or control group consisted of 5 - 6 animals. There was no attempt to pair litter mates between groups. Body temperature was maintained by placing the pups under a warming lamp during a 3 h recovery period following injections. The warming lamp kept the ambient temperature at 37°C. The pups were then returned to their mothers for 5 days and housed in a climatecontrolled facility with 12 h light/dark cycles. O n post-natal day 12, the animals were decapitated and their brains were removed and frozen.

Quantification of brain injury 20 Izm thick frozen coronal sections were taken at intervals of 0.5 m m through the striatum. The sections were fixed in 4% paraformaldehyde dissolved in 0.1 M phosphate buffer and stained for Nissl substance with Cresyl violet. The damage was quantified by measuring and comparing the cross-sectional area of intact tissue in each hemisphere using the D U M A S video-based computerized image analysis system. An optical density threshold was selected that included all of the control hemisphere in the area calculation and excluded the damaged portions of the lesioned hemisphere. This approach minimizes error in the estimate of lesion size due to shrinkage associated with damaged tissue. For each animal, the extent of damage was determined by averaging the measured damage in four serial coronal sections showing maximum lesions at the level of the striatum.

Data analysis The data are expressed as percent damage produced by the injection and were determined using the following formula: % damage = 100x (C - I ) / C where I is the area of intact tissue remaining on the injected hemisphere and C is the area of the uninjected control hemisphere. Comparisons of two groups were m a d e using the Student's t-test. Comparisons of more than two groups were made using one-way A N O V A followed by a N e w m a n - K e u l ' s or D u n n e t t ' s test. All errors are expressed as S.E.M.

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RESULTS Unilateral injection of NMDA into the striatum produced deformity of the injected hemisphere and necrosis in the striatum which extended into adjacent areas such as the cortex and hippocampus when higher doses were administered (Fig. 1A). The tissue damage was usually confined to the injected hemisphere. Near the site of injection, there was a very small population of surviving cells. At the periphery of the lesion, there was also a clear decrease in the number of cells, most surviving cells appeared pyknotic and there was significant gliosis. Increasing doses of NMDA produced a graded, dose-related increase in the extend of damage measured as a decrease in cross-sectional area as described in Methods (Fig. 2). Over a dose range of 5 to 40 nmol, the tissue damage ranged from 7 _+ 1% to 52 + 4%. The effects of co-administration of polyamines were measured in the presence of a 15 nmol dose of NMDA, which produced a 30 +_4% lesion. Co-injection of the polyamine spermine resulted in dose-dependent alterations in the extent of damage produced by NMDA. At doses below 50 nmol, spermine appeared to attenuate the excitotoxic effects of NMDA but this effect was not statistically significant. However, at doses above 50 nmol, spermine increased the damage produced by NMDA in a dose-dependent fashion (Figs. 1C and 3). The highest dose of spermine

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DOSE OF NMDA (nmol) Fig. 2. Effect of N M D A on tissue damage in rat brain. Different doses of N M D A were injected into the striatum of 7-day-old rats and the animals were sacrificed 5 days later. The extent of damage was quantitated by m e a s u r e m e n t of the cross sectional area of normal and intact tissue in the injected and uninjected hemispheres, respectively. Brain damage was assessed by densitometric m e a s u r e m e n t of intact tissue area in Nissl-stained sections as described in Materials and Methods. The damages produced by all doses greater than 5 nmol were significantly greater than the damage produced by the lowest dose ( A N O V A , F = 36.9, P < 0.0001; N e w m a n - K e u l ' s , P < 0.01). Each point represents the m e a n + S.E.M. of at least 6 animals.

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DOSE OF SPERMINE (nmol) Fig. 3. Effect of spermine on tissue damage in the presence and absence of N M D A . Rats were injected with different doses of spermine with or without 15 nmol N M D A and the damage was assessed as described in Materials and Methods. Addition of the agonist spermine at higher doses increased the tissue damage produced by N M D A . The dashed line represents the percent damage produced by injection of 15 nmol N M D A alone which was 30.4+_3. The three highest doses of spermine co-administered with N M D A produced a significant increase in the percent damage when compared to injection of N M D A alone ( A N O V A , F = 20.9, P < 0.0001; Dunnett's, P < 0.01). T h e two lowest concentrations of spermine did not produce a significant decrease in damage when co-administered with N M D A . Injection of spermine alone also caused significant tissue damage. The damage produced by all doses of spermine greater than 50 nmol were significantly greater than the damage produced by the lowest dose (ANOVA, F = 55.3, P < 0.0001; Newm a n - K e u l ' s , P < 0.01). Each point represents the m e a n _+S.E.M. of 5 - 7 animals.

(500 nmol) increased the level of damage approximately 50% above the damage produced by NMDA alone. The damage produced by this combination did not exceed the extent of damage observed after the administration of the highest concentration of NMDA (40 nmol) alone. Injection of spermine alone also produced a significant loss of tissue (Fig. 1D). This lesion had slightly different morphological characteristics than the lesion produced by NMDA. At the site of injection, there was a more complete loss of cells and at the periphery of the lesion, there was a more pronounced gliosis. Increasing doses of spermine produced a graded, dose-related increase in the extent of tissue damage (Fig. 3). The dose-response curve for the excitotoxic effect of spermine alone was shifted to the right of the dose-response curve in the presence of NMDA, however, the damage produced by the highest dose of spermine (500 nmol) was the same in the presence and absence of NMDA. To insure that the toxicity produced by NMDA and spermine was mediated by the NMDA receptor, these drugs were co-injected with the non-competitive NMDA receptor antagonist MK-801. The antagonist inhibited the neurotoxic effects of both spermine and NMDA (Fig. 1B,F).

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Fig. 4. Effect of co-administration of MK-801 on tissue damage produced by spermine and N M D A . Rats were injected with spermine or N M D A with or without 15 nmol MK-801 and the damage was assessed as described previously. The percent protection produced by MK-801 was determined by comparing the tissue damage in the presence and absence of the antagonist. The selective N M D A antagonist MK-801 almost completely inhibited the neurotoxic effects of both N M D A and spermine at all doses (t-test, P < 0.001). The percent damages in the absence of MK-801 were as follows: spermine (50 n m o l ) = 1 0 . 3 + 2 ; spermine (100 n m o l ) = 3 5 + 2 ; spermine (200 n m o l ) = 3 3 + 3 ; N M D A (15 n m o l ) = 32-+3. Each bar represents the m e a n -+ S.E.M. of 5 - 6 determinations.

A 15 nmol dose of MK-801 provided greater than 90% protection against a 15 nmol dose of NMDA and doses of spermine up to 200 nmol (Fig. 4). To verify that the apparent protection produced by co-administration of MK-801 was not due to the in-

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DOSE OF DET (nmol) Fig. 6. Effect of D E T on tissue damage in the presence and absence of spermine. Rats were injected with different doses of D E T with or without 100 nmol spermine and the damage was assessed as described in Materials and Methods. T h e weak partial agonist D E T had no effect on the neurotoxic effect of spermine ( A N O V A , F = 1.5), nor did it exhibit any neurotoxic effect of its own ( A N O V A , F = 0.2). The solid line represents the percent damage produced by injection of 100 nmol spermine alone. Each point represents the m e a n + S.E.M. of 5 - 6 animals.

crease in injection volume necessitated by the relative insolubility of MK-801, the effect of injection volume on the damage produced by 15 nmol NMDA and 100 nmol spermine was measured. There was no significant difference between the damage produced by 0.5/zl and 1.0/xl injections (n = 6) for either NMDA or spermine. Co-administration of the inverse agonist DA-10 with 15 nmol NMDA resulted in an apparent dose-dependent decrease in the extent of damage produced by NMDA (Fig. 5). A dose of 200 nmol DA-10 reduced the tissue damage produced by NMDA alone by approximately 50% (Fig. 1E). Administration of DA-10 alone produced no tissue damage at doses up to 100 nmol, but at higher doses produced a small but significant loss of tissue. Administration of the weak partial agonist DET produced little or no loss of tissue at doses up to 200 nmol (Fig. 6). Co-administration of up to 200 nmol of DET with a dose of spermine that produces a 35% lesion did not inhibit the neurotoxic effect of spermine. Co-administration of up to 200 nmol of DET with a 15 nmol dose of NMDA produced damage of 32 + 4% (n = 6) and did not have a significant effect on the toxicity produced by NMDA. A dose of 2/zmol of DET was found to be invariably lethal. DISCUSSION The effects of intracerebral injection of NMDA into the neonatal rat striatum reported here are consistent with the effects originally described by McDonald et

168

al. 26'27. N M D A produces a marked tissue necrosis in the striatum and adjacent areas and the extent of damage is dose-dependent. These investigators also demonstrated that the decrease in cross-sectional area was highly correlated with the loss of choline acetyltransferase activity. Since choline acetyltransferase is a specific marker for neurons, this observation supports the measurement of decreases in cross-sectional area as a quantitative measure of neuronal injury. Moreover, they demonstrated that the damage could be inhibited in a dose-dependent manner by a series of N M D A receptor antagonists, indicating that the damage is caused by stimulation of the N M D A receptor 29. The polyamine spermine increased the neurotoxicity of N M D A in a dose-dependent fashion. Spermine has been classified as an agonist based on its ability to increase the binding of [3H]MK-801 to the N M D A receptor 6°. MK-801 binds to a site on or within the ligand-gated ion channel of the N M D A receptor and spermine appears to have an allosteric effect on the channel to enhance access of MK-801 to its binding site 6°. In hippocampal neurons and oocytes, spermine increases NMDA-elicited currents 31'59. The in vivo effect reported here is consistent with the hypothesis that spermine increases NMDA-receptor channel opening and thereby enhances the ability of N M D A to produce neurotoxic damage. The ability of spermine to produce significant neurotoxicity on its own could be attributed to an increase in the excitation produced by endogenous glutamate in the presence of spermine. MK-801 completely inhibited the damage produced by a 15 nmol dose of N M D A indicating that this toxicity was mediated by the N M D A receptor. This is consistent with previous reports that MK-801 potently and dose-dependently inhibits N M D A neurotoxicity 29. MK-801 also inhibited the damage produced by spermine, suggesting that the neurotoxic effects of spermine are mediated by the N M D A receptor. Moreover, the damage produced by administration of N M D A and spermine individually were not additive when they were co-administered, consistent with a common mechanism of action. It has been reported that the neuroprotective effects of MK-801 in some models of excitotoxicity are due to the induction of hypothermia rather than blockade of the N M D A receptor 4. However, MK-801 has been shown to produce a mild hyperthermia rather than hypothermia in the neonatal rat 35. In contrast to higher doses, the lowest doses of spermine provided some measure of protection against the damage produced by NMDA. This biphasic response appears to correspond to the biphasic spermine dose-response curve for the stimulation of [3H]MK-801 binding observed in vitro. However, the inhibitory com-

ponent of this response, which would be expected to correspond to a protective effect in vivo, occurs at the highest concentrations of spermine 6°. Thus, it is unlikely that this protective effect involves modulation of the N M D A receptor. It is well established that polyamines have several other important actions in vivo, including a putative role in the synthesis of DNA and RNA 49. Moreover, the rapid induction of the parent enzyme ODC following injury or stress suggests that polyamines actively participate in the adaptive or protective response of neurons to traumatic stress. Indeed, an elevation in polyamine synthesis within injured peripheral neurons was found to be essential for neuronal survival after axonal injury H. Thus, it is likely that moderate increases in the concentration of spermine augment the in vivo adaptive response to toxic insult, but larger increases in spermine concentration enhance glutamate or N M D A toxicity by modulation of the N M D A receptor. The polyamine DA-10 inhibited the neurotoxicity produced by N M D A in a dose-dependent manner. DA-10 has been classified as an inverse agonist based on its ability to inhibit the binding of [3H]MK-801 in a D E T sensitive manner 59. Moreover, DA-10 has been shown to decrease NMDA-elicited currents in hippocampal neurons and oocytes 3x'59. The in vivo effect observed in this study is consistent with the hypothesis that DA-10 inhibits N M D A receptor channel opening. The ability of DA-10 to produce some tissue damage at higher doses suggests that DA-10 may also work by a mechanism unrelated to the N M D A receptor. This apparent non-NMDA receptor mediated effect at high doses of DA-10 appears to limit the extent of protection that DA-10 can provide against NMDA-induced neurotoxicity. The polyamine D E T did not inhibit the neurotoxicity produced by spermine. D E T has been classified as a weak partial agonist based on its ability to enhance the binding of [3H]MK-801 in vitro 55 and to inhibit the effect of spermidine in a dose-dependent manner 59. At the doses tested, D E T produced no significant neurotoxic effects when administered alone or in combination with NMDA, consistent with its classification as a weak partial agonist. The inability to inhibit the effect of spermine in vivo may be due to the relative lack of potency of this compound. In vitro, a concentration of 1 mM D E T is required to inhibit the effect of 75 ~ M spermine. In this study, the highest dose of D E T was only twice the dose of spermine. Ten-fold higher doses of D E T were found to be lethal and thus limited the range of doses available to test whether D E T can block the effects of spermine in vivo. This is the first demonstration that agonist and

169 inverse agonist polyamines can modulate NMDA receptor function in vivo in a manner analogous to the modulation that has been observed in vitro. A major unresolved question is whether endogenous polyamines play a role in the modulation of NMDA receptor function under normal a n d / o r pathologic conditions. In models of cerebral ischemia, there is an increase in both ornithine decarboxylase (ODC) activity and putrescine concentration within hours of the initial insult 7'8'2°'37. Ornithine decarboxylase converts ornithine to the polyamine putrescine, which is subsequently metabolized to spermidine and spermine 48. These increases in ODC activity and putrescine concentration can be prevented by prior administration of the NMDA antagonist MK-8012t indicating that stimulation of the NMDA receptor is necessary for these changes to occur. Direct administration of excitotoxic doses of NMDA into the CNS also produces an increase in ODC activity and putrescine concentration 39. Moreover, even intrastriatal infusion of NMDA via a dialysis cannulae has been reported to produce a rapid increase in the extracellular concentration of spermine and spermidine 9. The finding that polyamines can modulate NMDA receptors in vivo suggests that these changes in polyamine concentration may significantly affect NMDA receptor function. Another unresolved question is whether specific polyamines or related compounds can provide protection against excitotoxic insults. Systemic administration of polyamines has been shown to inhibit the retinal damage and loss of body weight produced by monosodium glutamate, which is presumably producing some of its effects by activation of NMDA receptors 12. Polyamines have also been reported to inhibit the loss of neurons associated with cerebral ischemia in the guinea pig brain t3. Other investigators36'39 have demonstrated that the concentration of putrescine increases and the concentration of spermine decreases following transient ischemia or administration of toxic doses of NMDA. Moreover, the pattern of change in putrescine concentration correlates closely with the extent of ischemic cell injury 36. It remains to be determined whether these changes in polyamine concentration are part of a neuroprotective response that attenuates the toxic effects of NMDA receptor stimulation. The model of NMDA-induced brain injury used in this study has many features in common with neonatal rat models of hypoxic-ischemic injury. The cytopathology produced by the administration of NMDA is very similar to that produced by hypoxia-ischemia18'26 and hypobaric ischemiat6. The extent of brain injury produced by administration of NMDA peaks during development at around post-natal day 7 28. A similar devel-

opmental profile for hypobaric-ischemic brain injury has also been reported t6. Moreover, the pharmacological profile of drugs that reduce brain damage induced by direct administration of NMDA corresponds to the profile of neuroprotective activity exhibited by these compounds in neonatal models of hypoxic-ischemia16"29. Thus, the activity of compounds in this model of NMDA-induced excitotoxicity is likely to predict their effectiveness against hypoxial-ischemic neuronal injury. The activity of DA-10 described in this study indicates that polyamines may have important therapeutic potential as neuroprotective agents. Acknowledgements.

This work was supported by USPHS GM34781, NS 08803, and a grant from the Pew Foundation. We would like to thank Dr. Marie-Francoise Chesselet for her helpful suggestions.

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