Developmental Brain Research, 48 (1989) 187-195 187 Elsevier

BRESD 50933

Development of N-methyl-D-aspartate excitotoxicity in cultured hippocampal neurons

Christine Peterson, John H. Neal* and Carl W. Cotman Department of Psychobiology, University of California, Irvine, CA 92717 (U.S.A.)

(Accepted 7 February 1989)

Key words: Neurotoxicity; Glutamate; N-Methyl-D-aspartate; Hippocampus; Development; Receptor autoradiography; Excitatory amino acid; MK-801

Immature hippocampal neurons (E-18) were maintained in defined medium for up to 3 weeks and their susceptibility to N-methyl-D-aspartic acid (NMDA)-induced cell death was studied at various days in vitro. Upon acute exposure to NMDA (5 rain), hippocampal neurons in vitro (8-12 days after plating) showed cell body swelling and dendritic degeneration that preceded cell death 24 h later. NMDA-induced neurodegeneration could be prevented by MK-801 treatment but not by tetrodotoxin. In contrast, immature (5-7 days old) neurons were unaltered by exposure to 500 gM NMDA for either 5 min or 24 h. One explanation for the resistance of immature neurons to glutamate neurotoxieity may be related to maturation of the NMDA receptor complex. Glutamate binding to the NMDA receptor in vivo increased from 14.6 + 1.6% (0 day) to 55.2 + 4.5% (day 7), 79 + 4.9% (day 14), 93.8 + 2.8% (day 21) until it reached the adult Sprague-Dawley value of 100 _+ 0.8% (day 90).

INTRODUCTION

Excitatory amino acid receptor activation may

play a role in the pathogenesis of hypoxic- or ischemic-induced neuronal injury (for review see ref.

24). High glutamate concentrations which are found

throughout the CNS may accumulate in the extra-

cellular space I during hypoxia and ischemia. This

may reflect a combination of increased presynaptic glutamate release 11 and impaired glutamate uptake 6.

Excessive activation of glutamate receptors may rapidly damage neurons 8'13'19.

The mechanisms by which excess glutamate pro- duces neuronal injury are not well understood.

Pharmacological studies of glutamate neuroexcita- tion suggest that it may be mediated by 3 separate

receptor subclasses: N-methyl-D-aspartate (NM- D A ) , quisqualate and kainate 7'27. Blockade of

N M D A receptors with the selective N M D A antag- onist 2-amino-7-phosphonoheptanoic acid dimin- ishes the neuronal loss associated with ischemia 26,

anoxia 8 and hypoglycemia 28. Furthermore, MK-801

protects chick retinal neurons from N M D A toxicity 2°. It has been reported that immature neurons are more resistant to neuronal damage 23

than mature neurons.

Excess glutamate is a potent and rapidly acting

neurotoxin in vitro. Since the hippocampus contains a large proport ion of N M D A receptors and is

selectively vulnerable to a variety of metabolic

insults, it was chosen as a model system to test the

effects of maturation on in vitro N M D A neurotox- icity. Neuronal sensitivity to N M D A was determined

in vitro where defined drug concentrations could be

administered in a controlled environment and the

resultant morphological changes monitored serially. Methods were standardized to maintain hippo- campal neurons in serum-free media that were

relatively free of non-neuronal elements. The ap-

pearance of N M D A neurotoxicity in these cultures in vitro was compared to the development of

glutamate binding to the postsynaptic N M D A re- ceptor in vivo.

* Present address: Dept. Neurological Surgery, University of Southern California Sch. Med., Los Angeles, CA 90007, U.S.A. Correspondence: C. Peterson, Department of Psychobiology, University of California, Irvine, CA 92717, U.S.A.

0165-3806/89/$03.50 (~) 1989 Elsevier Science Publishers B.V. (Biomedical Division)

188

MATERIALS AND METHODS

Timed pregnant Sprague-Dawley rats that were obtained from Charles Rivers Breeding laboratories (Wilmington, MA) were housed individually and parturition was designated as day 1. [3H]Glutamate (50.9 Ci/mmol), protosol and aqueous counting scintillant were from New England Nuclear (Boston, MA). (+)-5-Methyl- 10,11-dihydro-[SH]dibenzo[a,d] - cyclohepten-5,10-imine maleate (MK-801) was kindly provided by Dr. L. lversen at Merck, Sharp and Dohme, England, U.K. Tissue culture supplies were from Falcon (N J) or Coming (Coming, N.Y.). Tissue culture media, trypan blue and fetal bovine serum was from Gibco (Long Island City, N.Y.). Defined media components were from Sigma Chemical Company (St. Louis, MO). All other chemicals were from Sigma Chemical Company.

Hippocampal cultures

Hippocampi from embryonic rats (18-19 days old) were dissected in Hank's balanced salt solution without calcium or magnesium but with 4.2 mM sodium bicarbonate, 1 mM pyruvate, 20 mM HEPES and 3 mg/ml bovine serum albumin (BSA) 3. The tissue was mechanically dissociated in 2 ml of Hanks' dissecting buffer by trituration through a fire-polished siliconized glass pipette tip (0.5-1 mm diameter). An equal volume of Dulbecco's modified Eagles medium with 10% fetal bovine serum (final concentration), 20 mM HEPES, 26.2 mM sodium bicarbonate and 1 mM pyruvate was added and the cell suspension was mixed and non-triturated debris was allowed to settle for 3 min. The supernatant was transferred to a new tube and centrifuged at room temperature for 3 min at 400 g. The supernatant was discarded and the pellet resuspended in 4 mi of Dulbecco's modified Eagles medium that contained 2.4 mg/ml of BSA, 26.2 mM sodium bicarbonate, 1 mM sodium pyruvate and 20 mM HEPES (DMEM- BSA). The suspension was recentrifuged and the pellet resuspended in DMEM-BSA. An aliquot was removed for counting with a hemocytometer; viable cells excluded trypan blue. Cells were plated (day 0) onto 12 mm poly-D-iysine (50/ag/ml)-coated cover- slips at a density of 75 viable cells/mm 2. One hour after plating, the excess medium, that contained non-attached cells and debris, was aspirated. Three

coverslips were placed with the cell side down into 35-mm plastic tissue culture dishes that contained 2 ml of antibiotic-free DMEM-BSA that was supple- mented with 0.41 ktM biotin, 0.1 mg/ml BSA, 10~M carnitine-HCl, 10 ktM ethanolamine-HCI, 85 /~M galactose, 5/~g/ml insulin, 100/~M putrescine-HCI, 0.03 /~M selenite, 0.003 /~M triiodo-thyronine, 5 ~g/ml transferrin, 0.25 ~M vitamin B12, I).06 ktM corticosterone, 3.6 ~M linoleic acid, 3.6 ktM linolenic acid, 0.02/~M progesterone, 0.3 gM retinol-all trans, 0.35 ~tM retinyl acetate, 2.3/~M tocopherol, 2.1 ,uM tocopheroi acetate. Cells plated in this manner were generally viable for up to 3 weeks.

Different cell types were identified immunocyto- chemically. Briefly, the cells were fixed with 3.7% buffered formalin (30 min), rinsed with phosphate- buffered saline (PBS), incubated with 1.25% oval- bumin; 1% non-fat dry milk (15 min) to block non-specific binding, primary antibody (45 min), PBS (3 min), secondary antibody conjugated to peroxidase anti-peroxidase (45 rain), PBS (3 rain), 3,3-diaminobenzidine (8 min), PBS (3 rain), series of upgraded alcohols (3 min), xylene (3 min) and mounted. Rabbit antisera to glial fibrillary acidic protein 9 and mouse monoclonal to phosphorylated and non-phosphorylated neurofilament antibodies (Sternberger and Meyer, MD) were used.

In vitro treatment with N M D A and MK-801

Cultures were treated at various days after plating as follows: the media conditioned by the hippo- campal cells was removed and saved. Fresh medium that contained either NMDA and/or MK-801 was pre-equilibrated for at least 15 min at 37 °C before it was added to respective dishes. The coverslips were lifted up to expose the cells to the drug- containing media and the cells were then incubated at 37 °C for either 5 min or 24 h. To terminate the incubation the medium was aspirated and the dishes rinsed twice with pre-equilibrated DMEM-BSA. The original conditioned medium was returned to its respective dish and the coverslips were again lifted up and then placed down. Cell viability was esti- mated 24 h later by trypan blue exclusion. Five fields of cells were counted at a magnification of ×400 with an Olympus BH2 light microscope. Throughout the duration of the experiment, the total number of cells did not change (222.9 _+ 3.1 total cells). The number

of cells remained relatively constant since the dead

cells probably do not float away as they would in conventional cultures in which the cells are facing up.

In vivo excitatory amino acid receptor binding Sprague-Dawley rats (aged 0, 7, 14, 21 or 90

days) were decapitated and the whole brain was

removed and frozen on powdered dry ice. The brains were stored at -80 °C until analyzed. Horizontal brain sections (6/~m) were prepared as described previously ~7. Briefly, the brain sections were thaw- mounted onto acid-washed gelatin-coated micro-

scope slides and stored overnight at -20 °C. Each brain was sectioned sequentially such that the next slide contained a slice that was adjacent to the slide

before it; slices were cut until all the slides contained at least 3 sections. Intermittent slices were saved for protein determinations 14.

On the following day, the slides were completely air-dried, incubated in 50 mM Tris acetate buffer pH 7.2 for 20 min at 2 °C and then 10 min at 30 °C. To measure glutamate binding to the NMDA receptor, the sections were incubated for 10 min at 2 °C in 0.9 ml of 50 mM "Iris acetate buffer, pH 7.2 that also contained 100 nM [3H]glutamate, 100 /~M 4-acet- amide- 4"- isothiocyanostilbene- 2, 2"- disulfonic acid and 5/~M quisqualate. The incubations were termi- nated by rinsing the slides at 2 °C for a total of 30 s

in 4 sequential containers filled with 50 mM Tris acetate buffer, pH 7.2. Some of the sections were briefly dried by an air stream, removed with a glass fiber filter, solubilized with protosol and counted by

liquid scintillation. Non-specific glutamate binding to the NMDA receptor was determined in adjacent sections that were incubated in the presence of 200 /~M NMDA. Counts per minute were converted to disintegrations per minute (dpm) by external stan- dardization on a Beckman LS3800. Non-specific binding was subtracted from total dpm bound by the brain section before the calculations were done. Proteins were determined by the modified Lowry technique ]4 and all values were expressed per/~g protein.

Autoradiograms were generated by opposing in- tact tissue sections to tritium-sensitive Ultrafilm (LKB products, Rockville, MD). After an appropri- ate exposure time of 3-4 wks at 4 °C, the films were

189

developed and optical densities were measured with

a video camera and computer-assisted microdensi-

tometry image analyzer (MCID system). Conversion to fmol/#g protein was estimated by comparison to

quenched brain paste sections. This method allows the density and distribution of receptor binding to be determined in multiple brain regions. Anatomical

regions were identified by comparison with a rat brain atlas.

Statistical analyses All statistical comparisons were done by one-way

analysis of variance (Statview 512+). Specific be- tween-group differences were determined with Scheffe's F-test (P < 0.05).

RESULTS

In vitro growth of hippocampal neurons Hippocampal neurons from embryonic rats that

are maintained in defined media remained viable for

at least 3 weeks. Neurite extension could be ob- served as early as 3 h after plating. By 3 days most

of the cells had neurites, displayed a characteristic neuronal morphology (e.g. pyramidal-shaped cell bodies and thin branched dendritic processes) and were immunologically positive for neurofilament proteins. Immediately after plating, about 76 +

90

8o

70

6o

50 (n

40

30

20

10

0 0

I I I

5 10 15

Days in culture

Fig. 1. Survival of hippocampal neurons in vitro. Suspensions of embryonic rat hippocampi were seeded at a density of 75 viable cells/cm 2. At various days after plating (day 0) the cells were treated with trypan blue and a minimum of 5 fields per coverslip were counted at a magnification of × 400. The values for each point are expressed as means + S.E.M. and were calculated from a minimum of 5 separate experiments. The values for each of the 5 experiments were determined in triplicate. Significant differences (P < 0.05) were determined by analysis of variance with a post-hoc Scheffe's F-test.

190

Fig. 2. Morphological appearance of hippocampal cultures treated with NMDA and MK-801. Cells from embryonic rat hippocampus were plated at a density of 75 viable cetls/cm e (day 0). At the indicated days the cells were treated with either nothing, 500/tM NMDA, 200 nM MK-801 or 500 ~tM NMDA + 200 nM MK-801 for 5 min. x288. A: untreated 7-day-old cultures; B: NMDA treated 7-day-old culture; C: untreated 10-day-old culture; D: NMDA treated 10-day-old culture; E: MK-801/NMDA treated 10-day-old culture.

1.5% of the a t tached cells excluded t rypan blue (Fig.

1). Be tween days 1 and 4, this percentage increased

non-significantly (to 84%) when compared to day 0.

Some of the cells may have sufficiently repai red their

membranes so as to exclude t rypan blue, thereby

producing an apparen t increase in cell survival. By

days 5 -8 the number of live cells decl ined to about

75% and from days 9 to 12 it fell f rom 70 to 66%.

Although the ratio of live versus dead cells declined,

the total number of cells (222.9 + 3.1; n = 70) within

the 5 fields, remained unchanged. By using cover- slips faced down the dead cells probably remained

attached longer which probably contributed to the

constant cell numbers through the course of the

experiment. Furthermore, manipulating the cover-

slips did not alter cell survival.

The cu l tureswere relatively free of non-neuronal

elements. Serum-free media did not support glial cell

proliferation, since the number of astrocytes did not

increase with time. The number of cells that were

immunologically positive for glial fibrillary acidic

protein was generally less than 5% of the live cells

plated on day 0. No obvious changes in astrocyte

morphology was observed during any of the treat- ments.

Resistance o f immature neuronal cultures to N M D A

treatment in vitro

N M D A treatment did not lead to neuronal de-

generation in immature hippocampal neurons. Five-

to seven-day-old cultures that were treated with 500 g M N M D A for 24 h (Fig. 2B) were similar in

appearance to control cultures (Fig. 2A). The cell

bodies were round and smooth and the dendritic trees were long and highly branched. This observa-

tion was also independent of cell density since

cultures plated at 75, 150, 500 or 1500 viable cells/mm 2 were similarly unaffected. The absence of

a neurotoxic action at 24 h was not due to the

presence of excess magnesium ions which can block the N M D A receptor. Incubating 5-7-day-old cells

for 24 h with 500 g M N M D A in the absence of

magnesium did not produce neuronal damage.

Susceptibility o f mature neuronal cultures to N M D A

excitoxicity

N M D A treatment decreased the number of viable

cells in mature cultures of hippocampal neurons. Increasing N M D A concentrations correlated posi- tively to the number of dead cells (e.g. trypan-blue stained cells). Ten-day-old cultures exposed to 5 min

exposure of 50 nM N M D A had the same morpho- logical appearance as the untreated controls. Treat- ment of 10-day-old cultures with 5 /zM N M D A decreased survival about 15% when compared to untreated controls. Survival declined to about 30%

191

when neurons were treated with 500 ktM N M D A

(Fig. 2D). Thus, this concentration of N M D A (500

#M) was used in all subsequent experiments. When 8-12-day-old hippocampal neurons were treated

with 500/~M N M D A for 5 min, severe swelling and

generation was observed 24 h later (Fig. 2D). The

cell bodies became shrunken and irregular while the

dendritic trees deteriorated throughout the culture.

In 8-, 9-, 10-, 11- or 12-day-old cultures, cell survival

declined 8.7%, 20.5%, 30.3%, 43.4% or 49.9%,

respectively, when compared to non-drug-treated

neurons (Table I). Some neurons survived the brief

N M D A exposure. This may be related to either

subpopulations of resistant neurons or that the time

of exposure to or the dosage of N M D A was

insufficient.

Reversal o f in vitro N M D A excitotoxicity with MK-

801

Treatment of neuronal cultures with an N M D A

antagonist prevented neuronal damage. MK-801

treatment alone increased cell survival 6.6% (day 6),

9.8% (day 7), 6.8% (day 8), 9.0% (day 9), 11.1% (day 10), 16.2% (day 11) and 15.8% (day 12) when

TABLE I

Treatment o f hip pocampal cultures with N M D A and M K-801

Cells from embryonic rat hippocampus were plated at a density of 75 viable cellsdcm 2 (day 0). At the indicated days the cells were treated with either nothing, 500/~M NMDA, 200 nM MK-801 or 500 #M NMDA + 200 nM MK-801 for 5 rain. Values are expressed as the percentage of live (e.g. trypan blue excluding cells) when compared to the total number of cells counted. Five fields of cells were counted at x40 with an Olympus BH2 microscope. The total number of cells (222.1 _+ 3.1) did not change during the time course of these experi- ments.

Days in Control MK-801 MK-801 + NMDA culture NMDA

6 80.8_+2.8 86.1+2.4 a 85.4+3.5 a 82.6-+3.8 7 77.3 -+ 1.7 84.9 -+ 1.8 a 83.9 _+ 2.4 ~ 75.5 _+ 3.9 b 8 73.6_+2.6 78.6+3.7 a 78.6+2.9 ~ 67.1+1.1 a'b'c 9 72.4 _+ 2.2 79.1 _+ 2.0 ~ 79.1 _+ 2.2 ~ 57.6-+ 9.3 a'b'c

10 70.8 + 1.5 78.8_+ 2.3 a 80.4_+ 2.6 a 49.3 _+ 8.7 a'b'c 11 59.0 + 3.3 68.6-+ 1.5 a 68.6 _+ 1.9 ~ 33.8_+ 3.1 ~'b'c 12 60.4_+ 3.6 69.9 _+ 5.2 a 70.8 _+ 3.5 ~ 30.2_+ 3.4 ~'b'c 13 55.9 _+ 3.9 67.4 _+ 1.7 a 66.4 _+ 2.6 a 29.4 _+ 3.3 ~'bx

a Differs from control (P < 0.05); b Differs from MK-801 (P < 0.05); c Differs from MK-801 + NMDA (P < 0.05).

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TABLE II

Increased glutamate binding during development

The values (fmol//~g protein) represent the binding of [3H]glutamate (100 nM) that can be displaced by 100 pM NMDA as determined by quantitative autoradiography.

Age (days)

Cortex Outer 209.5 + 3.& Inner 202.3 + 9.4 ~ Entorhinal 153.7 + 4.5 ~

Hippocampus CA1 stratum radiatum CA2 CA3 stratum radiatum CA4 Subiculum 153.7 -+ 4.5" Hilus Dentate

Cerebellum Granule cell layer Molecular layer Striatum 125.6 -+ 15.6 ~ Medial septum Lateral septum

0 7 14 21 90

374.2 + 19.1" 702.6 + 39.4 ~ 932.5 + 11.6 d 1066.9 + 85.4 ~ 265.6 + 7.0 b 549.0 + 17.U 700.9 + 16.3 d 679.9 _+ 66.8 242.2+ 10.1 b 561.2 + 6.2 c 713.8 + 27.0 d 712.4_+ 12.8

589.8 + 6.4" 994.2 + 58.9 c 1589.6 + 52.7 d 1619.2 + 69.4 568.8 _+ 27.5 I' 772.8 _+ 58.9 c 1171.4 + 35.7 d 1134.8 _+ 133 547.4 + 4.18 798.3 + 32.2 ~ 1224.3 + 20.9 d 1106.5 + 88.1 534.2 + 16.8 b 873.8 + 39.6 c 1168.9 _+ 53.2 d 1310.1 _+ 12.3 e 242.2 _+ 10.1 b 346.4 + 4.3 ~ 405.4 _+ 7.7 d 495.1 + 47.3 ~ 362.6 + 12.8 u 477.2 + 3.8 ~ 660.4 + 2913 d 593.5 _+ 69.3 ~ 428.3 + 41.3 b 801.9 + 14.7 c 1217.5 + 46.8 ~ 1543.6 _+ 112 ~

194.7 _+ 3.6 b 224.6 + 6.9 c 227.2 + 12.1 a 367.6 _+ 21.3 e 79.3 + 3.6 82.9 + 6.2 c 98.5 + 3.5 d 100.9 + 19.6

260.3 + 11.0 t' 433.8 + 17.1 ~ 613.6 + 20.2 d 632.2 + 65.4 216.7 + 10.0 b 328.6 + 19.9 d 431.8 + 20.9 512.7 + 47.1 c 251.9 _+ 11.6 b 382.0 + 23.0 ~ 501.9 + 24.3 d 596.0 _+ 19.6 ~

a Differs from postnatal days 7, 14, 21 and 90 (P < 0.05). b Differs from postnatal days 0, 14, 21 and 90 (P < 0.05). c Differs from postnatal days 0, 7, 21 and 90 (P < 0.05). d Differs from postnatal days 0, 7, 14 and 90 (P < 0.05).

Differs from postnatal days 0, 7, 14 and 21 (P < 0.05).

c o m p a r e d to n o n - d r u g t r e a t ed cont ro ls (Table I).

W h e t h e r this effect o f MK-801 was due to p ro tec t ion

o f the cells f r o m g l u t a m a t e that a c c u m u l a t e d in the

c o n d i t i o n e d m e d i a o r t h rough s o m e o t h e r n e u r o t r o -

phic ef fec t is unknown . MK-801 (100 nM) ef fec t ive ly

b locked N M D A toxici ty in h i p p o c a m p a l cul tures .

M o r p h o l o g i c a l l y the re were no a p p a r e n t d i f fe rences

w h e n c o m p a r e d to con t ro l cul tures (Fig. 2C ,E) .

MK-801 i m p r o v e d the survival of N M D A - t r e a t e d

cells so tha t the dec rease due to N M D A was only

5 . 8 % (6 days) , 8 . 6 % (7 days) , 6 . 7 % (8 days) , 9 .2%

(9 days) , 13 .4% (10 days) , 16 .9% (11 days) and

17 .2% (12 days) w h e n c o m p a r e d to u n t r e a t e d

con t ro l cul tures .

Increased synaptic activity is not a factor in NMDA- induced damage

N M D A toxici ty a p p e a r e d to be p r imary and not

s econda ry due to a d e v e l o p m e n t a l increase in syn-

apt ic activity. T w e n t y - f o u r h o u r t r e a t m e n t of h ippo-

c a m p a l cu l tures (13 days old) wi th t e t r odo tox in (500

/~M) did not a l ter cell survival (99.9 + 0 . 6 % ; n = 6)

w h e n c o m p a r e d to n o n - d r u g - t r e a t e d contro ls . Fur-

t h e r m o r e in s imilar ly aged cul tures , N M D A - i n d u c e d

neuro tox ic i ty was u n a l t e r e d by s i m u l t a n e o u s admin-

is t ra t ion with t e t r o d o t o x i n (98.8 + 0.8; n = 6).

Increased glutamate binding to the N M D A receptor during in vivo development

O n e hypothes i s to exp la in the res i s tance of young

h i p p o c a m p a l neu rons cul tures to exc i to tox ic d a m a g e

m a y be the i m m a t u r e d e v e l o p m e n t o f the N M D A

recep to r . To address this ques t i on the b ind ing of

g lu t ama te to the N M D A r e c e p t o r was e x a m i n e d in

brain slices f rom roden t s of d i f fe ren t ages.

B ind ing to the N M D A r e c e p t o r increases as the

animals a p p r o a c h matur i ty . T h e d e v e l o p m e n t of

N M D A - d i s p l a c e a b l e g l u t a m a t e b inding was esti-

m a t e d in rats f rom e m b r y o n i c day 20 (age 0) to

pos tna ta l day 90 (Table I I , Fig. 3). Sod ium- and

c a l c i u m - i n d e p e n d e n t g l u t a m a t e b ind ing to 6 -gm

thick t issue sect ions in Tr i s -ace ta te buf fe r inc reased

193

as the rats developed. Binding could be detected by day 0 although it was only 14.6 + 1.6% of the adult (90 day) value. Binding increased to 55.2 + 4.5% by day 7, 79.0 + 4.9% by day 14, 93.8 + 2.8% by day 21 until it reached the adult value of 100 + 0.8% at day 90.

Regional in vivo glutamate binding to the NMDA receptor

Tissue sections of rat brain at various ages were examined by quantitative receptor autoradiography (Table II) for possible developmental differences. These studies focussed on the developing rodent hippocampus for comparison to the in vitro studies although other regions were also examined. Most of

the glutamate binding in the brain regions reached

adult levels by day 21 postnatal. At this develop- mental age the regional distribution of NMDA receptors corresponds to that in the mature rodent brain 16. Cerebral cortex, hippocampus and striatum account for a large proportion of glutamate binding to the NMDA receptor, whereas brainstem, mid- brain and cerebellum had relatively low levels (Fig. 3). In the hippocampus at day 0 regional binding sites could be resolved; however, individual sub- fields could not be readily discerned. The combined value for the embryonic hippocampus was 20.9 + 1% when compared to the adult values. At later times it can be shown that receptors on hippocampal pyramidal cells (e.g. CA1, CA2, CA3 and CA4)

d

Fig. 3. In vivo glutamate binding to the NMDA receptor during development. Brain sections (6 #m) were prepared from 0-, 7-, 14-, 21- and 90-day-old rats. Binding of [3H]glutamate was performed as described in Methods. The intact sections were exposed to [3H]sensitive film for 4 weeks.

194

increased similarly; 44.2 + 2.9% (7 days), 67.1 + 1.9% (day 14), 96.8 + 2.5% (day 21) and 100 + 0% (day 90). The dentate, however, matured more slowly since it was only 27.7% (day 7), 51.9% (day 14), 78.9% (day 21) before it reached 100% (day 90). Binding in the hilus, however, developed more rapidly since it reached 61.1% by day 7, 80.4% (day 14), 112.8 (day 21) and 100% (day 90).

DISCUSSION

Immature hippocampal neurons appear to be relatively resistant to NMDA treatment but upon in vitro maturation there is an increased vulnerability to excitotoxic damage. This damage, which occurs after an acute exposure to NMDA (5 min), can be ameliorated by co-administration of the NMDA antagonist MK-801. This reversal implies that NM- DA's neurotoxic action may be triggered by activa- tion of the NMDA receptor. Treatment of hippo- campal cultures with cyanide, a form of histotoxic hypoxia, is ineffective at two days after plating; however, it causes marked neuronal loss by two weeks 23. Furthermore the neuronal loss at two weeks could be prevented by treatment of the cultures with magnesium which is known to block the NMDA-associated channel. This developmental increase in susceptibility to excitotoxic damage due to NMDA is similar to the effects of excess gluta- mate that were previously reported. In cultured hippocampal neurons the L-glutamate-induced cat- ion change in membrane permeability did not appear until 7 days after plating 21. At 5 days in vitro 500 ~tM glutamate did not alter the survival of cortical neurons; however, by 13 days in vitro there was a 44% loss in neuron survival 5. Hippocampal neurons treated with glutamate also show a decline in cell survival as the cultures mature 15. A decrease is seen by two days which is several days earlier than the effect reported here for NMDA treatment however. It is possible that glutamate may be acting at non-NMDA receptors or that the culture conditions are significantly different.

There are several hypotheses that may underlie the delayed neurotoxicity in the present study. NMDA induced-damage may be mediated by a calcium-dependent process (for discussion see ref. 24). NMDA treatment increases calcium uptake 2

and cytosolic-free calcium 12 in cultured neurons. Furthermore, manipulations that reduce calcium influx prevent glutamate-induced damage in cortical neurons 4, neuroblastoma cells TM and hippocampal pyramidal cells 15. During the second postnatal week, NMDA treatment produces a larger calcium influx in the apical dendrites of the CA1 pyramidal neurons 1°. Thus, the postnatal development of vul- nerability to excitotoxic damage in the hippocampus in vitro may be related to increased calcium fluxes which correlate to maturation of glutamate recep- tors. Furthermore anoxia affects older cultures more adversely than younger ones 23.

Another possibility is that the NMDA-induced excitoxicity may be increased due to the postnatal activation of the NMDA receptor which eventually leads to extracellular calcium uptake. In vivo studies with whole rat brain demonstrate a positive corre- lation between increasing postnatal age and gluta- mate binding to the NMDA receptor as well as glutamate uptake 25. When the appearance of in vitro neurotoxicity is compared to the development of in vivo binding~ however, an interesting correlation emerges. The estimated rate at which neurons acquire susceptibility to NMDA toxicity in vitro (5.4% increase in neuronal loss/day) closely parallels the increased binding of glutamate to the NMDA receptor in vivo (4.5% increase in binding/day). While there are differences between in vitro and in vivo development, this parallel suggests that recep- tor appearance and/or subsequent intraceltular pro- cessing (e.g. increase in intracellular calcium) may have similar time courses under both conditions: Clearly, however, a direct comparison of NMDA receptors and excitotoxicity in vivo and in vitro still needs to be directly assessed in parallel.

ACKNOWLEDGEMENTS

This work was supported in part by AG00538 (C.W.C.), the MacArthur Foundation Program on Successful Aging (C.W.C.), the French Foundation (C.P.), AG07855 (C.P.), the Alzheimer's Disease and Related Disorders Association (C.P,)~ CIP. is a recipient of an Allied Signal Corp. /ADRDA Faculty Scholar Award and an NIH First Investigator Re- search Service and Transition Award.

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