Jolrrnd o/il.‘enrochrmrstrv Raven Press, Ltd., New York 0 1994 International Society for Neurachemistry

Regional Profile of Developmental Changes in the Sensitivity of the N-Methyl-D-Aspartate Receptor to Polyamines

Swaminathan Subramaniam and Paul McGonigle

Department of Pharmacology, University of Pennsylvania School oj’Medicine, Philadelphia, Pennsylvania, U.S.A.

Abstract: The NMDA receptor exhibits increased sensitiv- ity to stimulation during early development compared with the adult. In this study, we examined modulation of the NMDA receptor by polyamines during development to see if it correlates with differences in the functional responsive- ness of the NMDA receptor. [3H]MK-801 binding was measured in discrete brain regions in the presence and absence of polyamines in 3-, 7-, 15-, 25-, and 60-day-old Sprague-Dawley rats. [3H]MK-801 binding increased be- tween postnatal days 3 and 15, with adult levels of binding being reached between days 15 and 25. Spermidine (75 pM) caused maximal stimulation of [3H]MK-801 binding during early development, ranging from 250% in the thala- mus to 450% in the caudate putamen at postnatal day 3. This effect gradually declined to levels seen in the adult by postnatal days 15-25. During all developmental stages, the stimulation seen was greater in the caudate putamen compared with the hippocampus. Diethylenetriamine (1 mM) exhibited similar developmental and regional hetero- geneity in its effects on [3H]MK-801 binding, producing substantial stimulation of binding in the neonate, but not in the adult. The EC,,, and Em,, values for the stimulatory effect of spermidine were significantly higher at day 7 compared with the adult. Unlike spermidine and diethylene- triamine, there was no regional variation in the effects of the putative “polyamine site” inverse agonist 1, l O-diami- nodecane at any age and only a slightly attenuated inhibi- tion at postnatal day 3 compared with the adult. This lack of complementarity in the regional and developmental pro- files of spermidine and diethylenetriamine, on the one hand, and 1 ,lo-diaminodecane, on the other, suggests that their effects on [3H]MK-801 binding are mediated at different sites. The altered sensitivity of the NMDA recep- tor to polyamines during development could reflect the ex- pression of molecular variants with different sensitivities to modulation by polyamines. Key Words: Development -Sperm idi ne- 1 , l o - Diaminodecane-Diethylenetria- mine-Quantitative autoradiography-Modulation. J. Neurochem. 62, 1408-1 41 5 (1 994).

The N-methyl-D-aspartate (NMDA) receptor is a ligand-gated ion channel that is permeable to Ca2+. Radioligand binding and electrophysiological studies have revealed that it can be modulated through a num- ber of distinct sites. These include, apart from the transmitter (glutamate) recognition site, a site for gly-

cine, activation of which is obligatory for channel opening (Kleckner and Dingledine, 1988), a voltage- dependent site for the divalent cation Mg2+, where this cation acts to block ion flux (Engberg et al., 1979; MacDonald et al., 1982; Nowak et al., 1984), and a distinct site at which the divalent cation Zn2+ acts to inhibit channel opening (Peters et al., 1987). In addi- tion, there is a site through which polyamines, such as spermine and spermidine, activate the NMDA recep- tor (Ransom and Stec, 1988). The polyamine diethyl- enetriamine (DET) has been classified as a weak par- tial agonist at this “polyamine site” based on its abil- ity to block the effects of spermine and spermidine while producing only a small effect of its own (Wil- liams et al., 1989; Subramaniam and McGonigle, 1991). 1,lO-Diaminodecane (DAIO) has been pro- posed to be an inverse agonist based on its ability to reduce [3H]MK-80 1 binding in a DET-sensitive rnan- ner (Williams et al., 1990).

During development, there is a critical period dur- ing which regions of the CNS, such as the visual and olfactory cortex, show increased plasticity that can be disrupted by NMDA antagonists (Cline et al., 1987; Kleinschmidt et al., 1987; Lincoln et al., 1988; Gu et al., 1989). There are several reports indicating that increased functional responsiveness of the NMDA re- ceptor contributes to this increased plasticity in the neonatal brain. For example, NMDA receptors on immature neurons exhibit decreased sensitivity to membrane potential and are less sensitive to blockade by Mg2+ (Ben-An et al., 1988; Bowe and Nadler, 1990; Morrisett et al., 1990), and NMDA currents in

Received June 23, 1993: revised manuscript received August 26, 1993; accepted August 27, 1993.

Address correspondence and reprint requests to Dr. P. McGoni- gle at Department of Pharmacology, University of Pennsylvania School of Medicine, 36th and Hamilton Walk, Philadelphia, PA

The present address of Dr. S. Subramaniam is Bldg. 10, Rm. 5N-254, National Institutes of Health, 9000 Rockville Pike, Be- thesda, M D 20892, U.S.A.

Abhreviuiions used: ANOVA, analysis of variance; CPu, caudate putamen; DA 10, 1,lO-diaminodecane; DET, diethylenetriamine; NMDA, N-methyl-D-aspartate.

19104-6084, U.S.A.

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ONTOGENY OF POLYAMINE EFFECTS ON THE NMDA RECEPTOR 1409

the neonate have been shown to have a duration that is several times longer than that in the adult (Hestrin, 1992). The immature brain is also more sensitive to the toxic effects of NMDA than the adult brain (McDonald et al., 1988).

The increased sensitivity of the NMDA receptor in the neonatal brain could be a result of altered allos- teric regulation at one of the several modulatory sites. A previous report that examined the ontogeny of mod- ulation of binding of the open-channel ligand [3H]- MK-80 1 ([3H]dizocilpine) by glycine concluded that there was no change in glycine modulation during de- velopment (Boje and Skolnick, 1992). On the other hand, there have been two reports showing that modu- lation of the NMDA receptor by polyamines changes during development (Williams et al., 199 1 ; Mishra and Delivoria-Papadopoulos, 1992). In one study carried out in rat forebrain membranes, the modula- tory properties of spermine on '*'I-MK-80 1 binding were shown to be different in the neonate compared with the adult (Williams et al., 1991). In the other study, carried out in guinea pig fetuses, the stimula- tion of [3H]ME;-80 1 binding by spermine was shown to decline with gestational age (Mishra and Delivoria- Papadopoulos, 1992). However, neither study ad- dresses the question of regional variations in the on- togeny of polyalmine effects. This is of interest in view of our previous finding that the effects of polyamines on the NMDA receptor exhibit regional heterogeneity (Subramaniam and McGonigle, 199 1, 1993). In this study, we haw examined regional effects of poly- amines on the NMDA receptor during development with a view to characterizing these differences further. Stimulation of [3H]MK-80 1 binding by agonist poly- amines was greatest in all regions during early develop- ment. The weak partial agonist DET produced marked stimulation of [3H]MK-801 binding in all re- gions in the neonate. There tended to be greater stimu- lation of [3H]hlK-80 1 binding by spermidine in the caudate putamen (CPu) as compared with the hippo- campal regions, in the neonate, which is consistent with the regional differences seen in the adult.

MATISRIALS AND METHODS ['H]MK-801 (20-25 Ci/mmol) was obtained from New

England Nuclear-DuPont (Wilmington, DE, U.S.A.). Ke- tamine hydrochloride was purchased from Research Bio- chemicals Inc. (IVatick, MA, U.S.A.). Spermidine, DET, and DAlO were obtained from Aldrich (Milwaukee, W1, U.S.A.). All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.).

Timed pregnant Sprague-Dawley rats were obtained on the 14th day of gestation (Charles River Breeding Labs., Newfield, NJ, U.S.A.). After birth ofthe litter, the pups were pooled and randomly reallocated to the mothers ( 10 pups per mother) such that the average weight and sex composi- tion of the litters were similar. At each postnatal time point, pups culled from each litter (three males and three females) were killed and their brains removed and frozen in isopen-

tane at -25°C prior to storage at -70°C. On postnatal day 25, the remaining pups were weaned and housed four per cage. All rats were housed in a climate-controlled facility with a 12-h light/dark cycle and fed Purina rat chow and water ad libitum.

Twenty-micrometer-thick brain sections were cut at - 14°C in a cryostat and thaw-mounted onto gelatin-coated slides. Sections were desiccated at 4°C for 3 h, kept under vacuum at -20°C overnight, and then stored at -70°C (Rainbow et al., 1984). Radiolabeling of sections was carried out as described previously (Subramaniam and McGonigle, 1991). Briefly, sections were thawed at room temperature for 30-45 min, and incubation was initiated by covering each section with a drop of buffer (300 pl) contain- ing [3H]MK-801 (6-8 nM), glutamate (100 pM), glycine (100 pM), and appropriate concentrations of polyamines in 30 mM N-(2-hydroxyethyl)piperazine-N-3-propanesul- fonic acid containing 1 mM EDTA buffer at pH 7.45. Non- specific binding was defined in the presence of 200 pMketa- mine. Incubations were carried out under humid conditions at 22°C and terminated after 2.5 h by draining the incuba- tion droplet and immersing the section in ice-cold buffer. This wash was carried out for 40 min with one change of buffer solution at 20 min. The sections were then dried at 50-60°C on a slide warmer and apposed to LKB-Ultrofilm in slide cassettes with appropriate plastic tritium standards for 15-21 days. The film was developed and fixed using Kodak GBX developer and fixer and allowed to dry. The autoradiograms were analyzed using an IBM-AT-based image processing system and the BRAIN software (Drexel University, Philadelphia, PA, U.S.A.). This system converts optical density into nanocuries of radioligand bound per milligram using a standard curve as reference. The plastic tritium standards (American Radiochemical Co., Bowling Green, OH, U.S.A.) were calibrated against tissue standards for tritium (Rainbow et al., 1984; Geary et al., 1985; Arty- myshyn et al., 1990). The demarcation of regions for the purpose of analysis was based on cresyl violet-stained sec- tions.

Data analysis The data were expressed as percent change in binding

measured in the absence of the polyamine. Concentration- response curves were fit to the data points using the equa- tion E = Em,, X L/(L + EC,o), where L is the concentration of polyamine, Em,, is the maximum response produced by the polyamine, and ECS0 is the concentration at which a half-maximal response is elicited. Analysis of variance (AN- OVA), followed by Dunnett's test or the t test, was used, as appropriate, to determine the significance of differences be- tween groups.

RESULTS

The density of [3H]MK-80 1-labeled binding sites was measured over the course of development by la- beling coronal sections at the level of the CPu and dorsal hippocampus with a high concentration of t3H]- MK-801 (25 nA4) in the presence of maximally effec- tive concentrations of glutamate, glycine, and spermi- dine (Fig. 1). Under these conditions, greater than 95% of binding sites should be labeled based on the affinity of [3H]MK-801 for its binding site on the NMDA receptor (Subramaniam and McGonigle,

J. Neurochem.. Vol. 62, No. 4, 1994

1410 S. SUBRAMANIAM AND P. McGONIGLE

P3

P7

PI 5

P25

P60

199 1 ) . In the CPu, there was a progressive increase in binding between postnatal days 3 and 25 (Fig. 2C). The rate of increase in binding was maximal between days 3 and 7, followed by a plateau between days 15 and 25. This profile was similar in the dorsolateral and ventromedial CPu. After day 25, there was a small decrease in binding in both regions to the level at day 60 (adult). A similar profile was seen in the cortex (Fig. 2B), except that in the outer layers of the cortex (layers 1-2) the density of binding sites re- mained high from day 15 onwards until day 60. Thus, the difference in the binding between the outer and inner layers of the cortex increased with developmen- tal age. The density of [3H]MK-801 binding sites in the hippocampal regions (Fig. 2A; dentate gyrus and

FIG. 1. Autoradiograms of [3H]MK- 801 -labeled brain sections during development. Sections at the level of the CPu and the dorsal hippo- campus were labeled with [3H]MK- 801 (25 nM) in the presence of glu- tarnate (1 00 pM), glycine (1 00 pM), and sperrnidine (75 pM) as de- scribed in Materials and Methods. “P3-60” refers to the postnatal age in days.

CAI stratum radiatum) and the thalamus reached maximum levels at day 25 and then declined slightly to the level at postnatal day 60, as seen in the CPu. The relative proportion of binding sites in the various regions resembled that in the adult from day 25 on- wards. Also, the density of binding sites at day 60 was comparable to previously reported densities of the NMDA receptor in the adult rat measured by analysis of saturation binding data for [3H]MK-801 (Subra- maniam and McGonigle, I99 1).

The effect of spermidine changed dramatically dur- ing the course of development. At the earlier ages, spermidine had a much greater effect than in the adult in all of the regions examined (Fig. 3) . The stimula- tory effect was maximal at day 3 and varied signifi-

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1411 ONTOGENY OF POLYAMINE EFFECTS ON THE NMDA RECEPTOR

6000

m

- 4500 cotiex (layer 1.2)

1 ! 30001 - $ 15DOl- Cortex (layer 3-5)

8 3000 ventromedlal CPu

2 2 1500/p””’--- I dorsolatsral CPu

0- 0 10 20 30 40 50 60 70

Postnatal day

FIG. 2. Ontogeny of [3H]MK-801 binding sites in coronal sections at the level of the CPu and hippocampus. Brain sections from rats at different postnatal ages were labeled with 25 nM [3H]MK-801 in the presence of 100 pM glutamate, 100 pM glycine, and 75 pM spermidine. Cortical measurements were made at the level of the CPu. Each data pclint represents the mean & SE of quadruplicate determinations in !six rats. s.r., stratum radiatum.

cantly from region to region, ranging from 450% in the dorsolateral CPu to 250% in the thalamus. The relative level of spermidine stimulation across regions was not correlated with the density of NMDA recep- tors measured ,with [3H]MK-801. The effect of sper- midine steadily declined from day 3 to day 25, at which time it seemed to reach a plateau. At 60 days, the stimulatory effect of spermidine was much smaller, but remained significant ( p < 0.05) in the CPu and the outer layers of the cortex (layers 1-2).

The effect of DET also changed significantly during the course of development. Coincident with the devel- opmental profile of spermidine effects, DET pro- duced the greatest stimulation at the earliest ages, and this effect declined with age (Fig. 4). At day 3, stimula- tion ranged from 260% in the dorsolateral CPu to 180% in the thalamus. At all ages and in all regions, the stimulatory effect of DET was smaller than the effect of spermidine. Unlike spermidine, DET pro- duced an inhibition of [3H]MK-801 binding in the hippocampal regions, the thalamus, and the cortex at 25 and 60 days ( p < 0.05). In most regions, the adult level of inhibition was reached by day 25; however, in the cortex and thalamus, adult levels were reached by day 15.

In contrast to spermidine and DET, the putative inverse agonist IDA 10 exhibited a very different devel- opmental profile. DA 10 produced the smallest inhibi-

tion of [3HlMK-801 binding at day 3, and in most regions the effect of DA 10 was maximal by day 7 (Fig. 5). Adult levels of inhibition were observed by day 15 in the CPu and day 7 in the cortex. There was no significant change in the effect of DA 10 in the hippo- campus or thalamus during development. The rela- tive levels of inhibition by DAlO did not correlate with the density of NMDA receptors or the stimula- tory effects produced by the other polyamines. In ad- dition, there was no evidence of regional heterogene- ity once adult levels of inhibition were reached.

The stimulatory effect of spermidine was character- ized further by comparing concentration-response curves for the stimulation of [3H]MK-801 binding in serial sections at the level of the CPu in 7-day-old neonates and in 60-day-old adults. Data for the dorso- lateral CPu and the cortex (layers 3-5) are shown in Fig. 6. The concentration-response curves from sec-

500

400

- e

z

c 300

0

200

100

4oa

- 2

z

c 300

0

200

1 oa

400

- 2 300 c

8 $ 200

100

0

--C ventrornedial CPu a dorsolateral CPu

--t cortex (layer 1-2) --#- cortex (layer 3-5)

k, & dentate gyrus + CAI s. r. -&- thalamus

10 20 30 40 50 60 70

FIG. 3. Ontogeny of the effect of spermidine on [3H]MK-801 bind- ing in coronal sections at the level of the CPu and hippocampus. Brain sections from rats at different postnatal ages were labeled with 6 nM [3H]MK-801 in the presence of 100 pM glutamate and 100 pM glycine and in the presence or absence of 75 pM spermi- dine. Data are expressed as percentage of binding in the absence of spermidine. Each data point represents the mean f SE of qua- druplicate determinations in six rats. Open symbols represent a significant difference at p < 0.05 compared with the effect in 60-day-old rats by two-way ANOVA, followed by Dunnett’s test. s.r.. stratum radiatum.

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1412 S. SUBRAMANIAM AND P. McGONIGLE

400

--f ventromedial CPu --C dorsolateral CPu

-

+ coriex (layer 3-5) 400

125

f- CA1 s. r. 400 I thalamus

--O- ventromedial CPu + dorsolateral CPu -

0 1

postnatal day

FIG. 4. Ontogeny of the effect of DET on [3H]MK-801 binding in coronal sections at the level of the CPu and hippocampus. Brain sections from rats at different postnatal ages were labeled with 6 nM [3H]MK-801 in the presence of 100 pM glutamate and 100 pM glycine and in the presence or absence of 1 mM DET. Data are expressed as percentage of binding in the absence of spermidine. Each data point represents the mean t SE of quadruplicate deter- minations in six rats. Open symbols represent a significant differ- ence at p < 0.05 compared with the effect in 60-day-old rats by two-way ANOVA, followed by Dunnett’s test. s.r., stratum radia- turn.

tions of adult brain had a distinct inhibitory phase at the highest concentrations (not shown), whereas the curves from the neonate did not. Only the monotoni- cally increasing points of the concentration-response curves from the adult were used for the curve-fitting procedure. The EC,, and Emax values were signifi- cantly higher ( t test, p < 0.05) in both regions at day 7 than at day 6 0 . In the neonate, the Emax was 560 ? 57% in the dorsolateral CPu and 399 ? 71% in the cortex (layers 3-5). The corresponding figures for the adult were 8 1 & 18% and 6 2 & 7.4%. The ECso in the neonate was 36 f 3.4 and 22 k 6 pM in the dorsolat- era1 CPu and the cortex (layers 3-54 respectively. The corresponding figures for the adult were 8.5 -+ 2.9 and 8.9 k 3.5 yM, respectively.

DISCUSSION The densities of binding sites at 60 days in the dif-

ferent regions were comparable to B,,, values for [3H]-

MK-80 1 binding in these regions determined using saturation analysis (Subramaniam and McGonigle, 1991). This confirms that, in the presence of 100 FM glutamate, 100 pM glycine, and 75 pM spermidine, 25 nM [3H]MK-801 labels most of the available bind- ing sites. The development of [3H]MK-80 1 binding sites in different brain regions was similar, with a grad- ual increase in binding that reached adult levels in most regions between postnatal days 15 and 25. There was only a slight overshoot above adult levels of bind- ing in the CPu that was not seen in any of the other regions. This is in contrast with previous reports showing marked overexpression of NMDA-displace- able ~-[~H]glutamate binding during development (Tremblay et al., 1988; Insel et al., 1990; McDonald et al., 1990). However, this finding is consistent with the pattern of development of [3H]TCP ([3H]- tenocyclidine) binding sites (McDonald et al., 1990).

-0- cortex (layer 1-2) + cortex (layer 3-5)

25 t --t dentate gyrus

CAI s r. I thalamus

- 1001

0 1 0 10 20 30 40 50 60 70

postnatal day

FIG. 5. Ontogeny of the effect of DAlO on r3H]MK-801 binding in coronal sections at the level of the CPu and hippocampus. Brain sections from rats at different postnatal ages were labeled with 6 nM [3H]MK-801 in the presence of 100 pcM glutamate and 100 pM glycine and in the presence or absence of 100 pM DA10. Data are expressed as percentage of binding in the absence of spermidine. Each data point represents the mean -t SE of quadruplicate deter- minations in six rats. Open symbols represent a significant differ- ence at p < 0.05 compared with the effect in 60-day-old rats by two-way ANOVA, followed by Dunnett’s test. s.r., stratum radia- turn.

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ONTOGENY OF POLYAMINE

600 dorsolateral CPu

neonate

150

?

1 10 1000

500 7 I I

spermidine &A)

FIG. 6. Concentrai:ion-effect curves of the effect of spermidine on [3H]MK-801 binding at postnatal days 7 and 60. Coronal sec- tions at the level of the CPu were labeled with 6 nM [3H]MK-801 in the presence of 100 pM glutamate and 100 pM glycine and in the presence of increasing concentrations of sperrnidine. The binding at each concentrati,on of sperrnidine is represented as a percent- age of binding in the absence of sperrnidine. Each data point represents the mea,n -c SE from quadruplicate determinations in each region in six rat brains.

As suggested previously, it is possible that the discrep- ancy in the stoichiometry of the transmitter recogni- tion site and the PCP (phencyclidine) site is a result of alterations in the subunit composition of the NMDA receptor occur13 ng during development (McDonald et al., 1990).

Transient ovcrexpression of NMDA receptors in the neonate (Trt:mblay et al., 1988; Insel et al., 1990; McDonald et al., 1990) may account for the increased sensitivity of tbe neonatal brain to stimulation of NMDA receptors. Apart from this, however, there ap- pears to be increased functional responsiveness of the NMDA receptor in the neonate (Ben-An et al., 1988; Bowe and Nadler, 1990; Momsett et al., 1990; Hes- trin, 1992). Changes in stoichiometry of different com- ponents of the IVMDA receptor resulting in altered modulation ma.y account for the developmental change in NMDA-receptor sensitivity (McDonald et al., 1990). In the absence of selective, high-affinity ligands for the polyamine site on the NMDA receptor, it is impossible to determine directly if the polyamine site changes during development. However, poly- amine-stimulated [3H]MK-80 1 binding is an index of the ability of polyamines to modulate the receptor

1413 EFFECTS ON THE NMDA RECEPTOR

and can be used to follow changes in this allosteric modulatory mechanism resulting from either a change in the molecular configuration of the site itself or an alteration in its coupling to the NMDA recep- tor. The temporal profile of the effects of spermidine suggests greater efficacy, but lower potency during early development that gradually becomes adult-like between postnatal days 15 and 25. The weak partial agonist DET, which has small effects in the adult, was a strong stimulant of [3H]MK-80 1 binding during early development. This correlated with the strong stimulant effect of spermidine during early develop- ment consistent with both compounds acting at a common site. Unlike spermidine and DET, the ef- fects of the putative inverse agonist DA 10 had a dif- ferent developmental profile with smaller inhibitory effects seen during the early stages of development. In accord with these results, a previous study carried out using rat forebrain membranes reported that the po- tency of spermine to stimulate the NMDA receptor is lower in the neonate compared with the adult, al- though the potency of DAlO to inhibit the NMDA receptor is unchanged (Williams et al., 1991). These observations, along with our previous report showing lack of complementarity in the regional effects of DA 10 and spermidine (Subramaniam and McGoni- gle, 1991), suggest that the inhibitory effect of DAlO and the stimulatory effects of the agonist polyamines are mediated through different sites. Support for this notion has been provided by recent electrophysiologi- cal studies in hippocampal neurons showing that the inhibitory effect of DA10, unlike the stimulatory ef- fect of spermine, decreases at positive holding poten- tials, implying that it is acting as an open-channel blocker (Rock and MacDonald, 1992; Subramaniam et al., 1992). Consistent with this proposal, the block produced by DA 10 in single-channel recordings of the NMDA receptor was flickery in nature similar to Mg2+.

There is evidence to suggest that modulators of poly- amine function are present in the CNS, and some of the regional differences in the modulatory effects of polyamines in the adult brain could be accounted for by washable endogenous factors (Subramaniam and McGonigle, 1991, 1993). However, it is unlikely that differences in the levels of endogenous polyamines or modulators of polyamine function account for the differences in the effects of agonist polyamines during development, because similar differences have been found in well-washed membrane preparations (Wil- liams et al., 1991).

Several subunits of the NMDA receptor have been cloned successfully, and different subunit combina- tions have distinctly different functional characteris- tics, such as affinities for agonists, sensitivities to com- petitive antagonists, and Mg2+ block (Moriyoshi et al., 199 1; Kutsuwada et al., 1992; Monyer et al., 1992). In situ hybridization in the adult brain has shown that these subunits are expressed differentially in different parts of the CNS (Kutsuwada et al., 1992; Monyer et

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1414 S. SUBRAMANIAM AND P. McGONIGLE

al., 1992). It is, therefore, possible that the heterogene- ity in NMDA-receptor function during development is a result of expression of molecular variants of the NMDA receptor at different times. In this connec- tion, a recent report examining splice variants of the NMDAR1 receptor is noteworthy (Durand et al., 1992). One of them, designated NRlb, exhibited re- duced spermine potentiation when injected into oo- cytes. Conceivably, predominant expression of the homomeric NR 1 b-type NMDA receptor in the adult could account for the reduced potentiation by spermi- dine that we observe in the adult. However, the exis- tence of multiple subunits of the NMDA receptor, whose properties have not been fully characterized, suggests that this may only be one of several possibili- ties. Apart from the temporal heterogeneity, there also appears to be a distinct pattern of heterogeneity that is seen between regions, such as the CPu and the hippocampus. This pattern of regional heterogeneity, which we have observed previously in the adult (Su- bramaniam and McGonigle, 1991, 1993), is not ac- companied by any difference in the potency of sper- midine and may be accounted for by a completely different mechanism.

The present results support the notion that stimula- tion of the NMDA receptor by polyamines changes during development. Some of the reported differences in NMDA-receptor function during development could arise from such heterogeneity. NMDA receptor heterogeneity has important implications for the de- velopment of therapeutic strategies. It is clear that the NMDA receptor in the developing brain is involved in the mediation of excitotoxic damage, especially be- cause it shows increased sensitivity to stimulation. This has been borne out in clinical studies showing that the consequences of status epilepticus on mental function are more severe in children less than 3 years of age (Aicardi and Chevrie, 1970; Fujiwara et al., 1979) and pathologic evidence showing that brain damage is more common following status epilepticus in children (Corsellis and Bruton, 1983). Potential therapeutic strategies involving the use of agents, such as MK-801, acting at the NMDA receptor channel site may not be useful in such situations because of their ability to sensitize the developing brain to excit- atory amino acid-mediated brain injury (McDonald et al., 1988). The present results demonstrate that the polyamine site coupled to the NMDA receptor in the developing brain is functional and can produce greater stimulation of the NMDA receptor. Consis- tent with this observation, it has been shown recently that the polyamine site antagonist ifenprodil preferen- tially protects neurons of an earlier developmental stage from NMDA-induced neurotoxicity (Zeevalk and Nicklas, 1992). Thus, agents acting at the polya- mine site can provide an alternative strategy for the development of therapeutic agents for the treatment of excitotoxic damage in the newborn.

Acknowledgment: This work was supported by grants from the Pew Foundation and the National Institutes of Health (GM-3478 1 and NS-08803).

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