Neuroscience Letters, 154 (1993) 43-46 43 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/93/$ 06.00

NSL 09478

Developmental alterations in N-methyl-D-aspartate stimulated [3H]norepinephrine release in rat brain cortex and hippocampus

Laurie M. Brown

Department of Pharmacology, Northeastern Ohio Universities College of Medicine, Rootstown, OH 44272 (USA)

(Received 20 November 1992; Revised version received 30 January 1993; Accepted 1 February 1993)

Key words: N-Methyl-D-aspartate; [3H]Norepinephrine release; Slice; Development; Cortex; Hippocampus

Developmental alterations in N-methyl-D-aspartate (NMDA)-stimulated [3H]norepinephrine release from rat brain cortical and hippocampal slices were studied. NMDA (10-1000/IM) resulted in a concentration-dependent increase in [3H]norepinephrine efflux; maximal responses (% released~ in the cortex were: (1.53 + 0.12, 3.68 + 0.20, 2.94 + 0.20, 4.60 + 0.28 and 5.28 + 0.33) and the hippocampal responses were: (1.90 + 0.18, 3.84 + ~23, 3.60 + 0.28, 5.16 + 0.38 and 5.81 + 0.45) at varying postnatal ages (1, 7, 14, 21 and 90 days) respectively. Cortical tissue from 7-day-old pups exhibited a transient increase in maximal efflux and a decrease in ECs0. These results indicated that developmental alterations in the NMDA receptor appear to be translated into differences in NMDA stimulated [3H]norepinephrine release.

Excitatory amino acid receptors exhibit a great deal of postnatal development in the rat brain. The N-methyl-D- aspartate (NMDA) receptor is a subclass of excitatory amino acid receptor with many regulatory sites [10]. Many authors have repor~d that NMDA receptor bind- ing exhibits a transient inorca~e, with postnatal develop- ment [4, 12, 16]. Functional studies using inositol phos- phate turnover and long-term pQl~entation also demon- strate transient increases in NMDA stimulated activity during development [8, 9]. Studies examining [3H]D-as- partate uptake are controversial, some studies report a transient increase with development and'Others do not [5, 15]. ~i~,, .

These studies were performed t0'e~amine the postna- tal developmental changes in NMDA stimulated [3H]norepinephrine ([3H]NE) release. This system was chosen because it exhibits sensitivity to NMDA antago- nists and is enhanced by the co-agonist glycine [7, 11]. Recent studies indicate that NMDA stimulated efflux in slices is mediated by presynaptic receptors [6].

Combined groups of male and female Sprague-Da- wley rat pups up to 21 days of age (1, 7, 14 and 21 days) were used in these studies. Only male rats were used at 90 days (adult) of age. They were obtained from a colony established at Northeastern Ohio Universities from

Correspondence: L.M. Brown, Department of Pharmacology, North- eastern Ohio Universities, College of Medicine, 4209 State Route 44, P.O.Box 95, Rootstown, OH 44272-0095, USA.

Zivic-Miller Laboratories (Zelienople, PA), Animals were maintained on a 12 h light/dark cycle with food and water available ad libitum. L-[3H]Norepinephrine (31-37 Ci/mmol) was purchased from Dupont (New England Nuclear, Wilmington, Delaware). NMDA and other drugs were obtained from Sigma Chemical Co. (St. Louis, MO).

The assay to determine overflow of [3H]NE was per- formed according to reported methods [1, 11, 14]. Briefly, cortical and hippocampal brain slices were pre- pared and washed thoroughly in an oxygenated, 37°C, magnesium-free Krebs-Ringer bicarbonate buffer~ Slices were incubated with [3H]NE (15/tC1 in 4 ml) in the pres- ence of 25 ~tM pargyline and 1.25 mM ascorbid acid. After washing to remove excess [3H]NE, aliquots of the tissue were loaded onto individual nylon mesh bott0med baskets and ,moved at one minute intervals th r~gh a series of 5 vials containing buffer and the drugs to be tested. The first three vials contained no drug and served as measures of non-stimulated efflux. The last two vials (stimulated efflux) contained NMDA in varying concen- trations (10, 50, 100, 500 and 1000/IM). Percent efflux was calculated by net efflux (average stimulated- average unstimulated efflux) divided by total tissue radioactivity multiplied by 100. Under the experimental conditions, greater than 90% of the radioactivity recovered in the medium of a stimulated sample co-eluted with NE deter- mined by HPLC [2]. Logistic (minimum, ECs0, slope and maximum) parameters for NMDA concentration-re-

44

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4 -

3 "

2 "

1

~' o

CORTEX

. . . . . . . | . . . . . . . i . . . . . . . | . . . . . . . . 1 0 1 0 0 1 0 0 0 1 0 0 0 0

[ N M D A I ~.M

• day 1

• day 7

• day 14

0 day 21

day 90

HIPPOCAMPUS

w 7 n- 6. j ~ E w L Z . , =e • day 1

"~ 5 • • day 7

4 • day 14

0 day 21

E 3- "~ 2 -" a day 90

¢e 1

0 i . . . . . . . . i . . . . . . . . i . . . . . . . q . . . . . . . . I 0 'I O0 1000 10000

[ N M D A ] ~ M

Fig. 1. NMDA-stimulated [3H]norepinephrine release from the cortex and hippocampus of rats at varying ages. NMDA (10, 50, 100, 500 and 1000 pM) caused concentration-dependent increases in efflux. Rats of various ages (1, 7, 14, 21 and 90 days postnatal age) exhibited increases in maximal efflux and Ecso values with development. The data is pre- sented in tabular form in Table 1. The data represents the mean val-

ues ± S.E.M. of 3 to 5 experiments.

sponse curves and differences between curves were ana-

lyzed using A L L F I T , a computerized non l inea r curve fit-

ting rout ine which calculates the residual variance of

curves and performs an F-test to determine statistical sig-

nificance [3].

In the cortex and h ippocampus (Fig. 1 and Table 1)

N M D A induced concen t ra t ion-dependent responses at

all ages tested. The m i n i m u m values of the cortical data

set and h ippocampal data set were held cons tant to assist

in curve fitting. The slope values of the cortical concen-

t ra t ion- response curves were statistically equivalent and

the slope values of the h ippocampa | data set were also

equivalent. The maximal and ECs0 responses in both the

cortex and h ippocampus usually increased with develop-

ment as compared to tissues from one day old brain.

In cortical tissue, the ECs0 at day 7 was significantly

lower than all other ECs0 values. The ECs0 values at days

21 and 90 were statistically equivalent and were signifi-

cantly larger than the ECs0 values at days 1, 7 and 14.

The maximal percent [3H]NE released at day 7 was

higher than days 1 and 14. The maximal response at days

21 and 90 were no t different and were significantly larger

than the maximal responses at days 1, 7 and 14.

In h ippocampal tissue the ECs0 values were similar at

days 21 and 90 and significantly larger than the ECso

values at days 1, 7 and 14. The maximal responses at

days 21 and 90 were similar and larger than the responses

at 1, 7 and 14 days.

The specificity of the response was examined using the

N M D A antagonis t 2 -amino-5 -phosphonopen tano ic acid

(AP-5). The cortical response with N M D A ( t 0 0 / I M ) in

the absence or presence of AP-5 (200 p M ) at various

TABLE I

COMPUTER GENERATED CONCENTRATIONS-EFFECT CURVE PARAMETERS FOR NMDA-STIMULATED [3H]NOREPINEPH- RINE EFFLUX FROM THE CORTEX AND HIPPOCAMPUS OF RATS AT VARYING AGES

Parameters were estimated by fitting the data to a logistic concentration-effect curve as determined by the computer program ALLFIT. Min. values were constrained to the average response obtained using 10/.tM NMDA. A plot of the data is shown in Fig. 1. Values are the means ± S.E.M. (computer-generated).

Parameter 1 day 7 days 14 days 21 days 90 days

Cortex Min. (% relsd.) 0.22 +_ 0.07 0.22 + 0.07 0.22 ± 0.07 0.22 ± I).07 0.22 + 0.07 Slope 1.86 + 0.55 2.25 ± 0.76 1.53 + 0.28 2.11 ± 0.49 1.88 ± 0.38 ECs0 (pM) 34.89 + 9.46 18.20 ± 4.22* 38.72 + 8.10 79.08 ± 8.99* 85.69 ± 10.65" Max. (% relsd.) 1.53 _+ 0.12 3.68 ± 0.20* 2.94 ± 0.20* 4.60 ± 0.28* 5.28 4- 0.33*

Hippocampus Min. (% relsd.) 0.30 + 0.05 0.30 + 0.05 0.30 ± 0.05 0.30 + 0.05 0.30 ± 0.05 Slope 3.81 _+ 5.18 2.59 + 0.76 1.98 _+ 0.48 2.30 + 0.57 1.87 _+ 0.43 ECs0 (pM) 46.68 + 7.87 35.64 + 7.67 47.92 _+ 8.91 92.15 ± 13.03" 96.17 ± 16.36" Max. (% relsd.) 1.90 +_ 0.18 3.84 + 0.23* 3.60 + 0.28* 5.16 + 0.38* 5.81 _+ 0.45*

*Significantly different from the corresponding value in 1-day-old animals.

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postnatal days (1-3, 6, 15-16 and 20-23) was (2.53 + 0.42, 0.45 + 0.27; 4.57 +0.29, 1.00 + 0.13; 2.34 + 0.21, 0.08 + 0.03; 3.18 + 0.18, 0.33 + 0.05), re- spectively. The response elicited by NMDA was signifi- cantly (P < 0.05) inhibited by AP-5 in cortical and hip- pocampal tissues (data not shown) of varying ages.

NMDA caused a concentration-dependent increase in [3H]NE release in the cortex and hippocampus at all ages tested. In cortical tissue, a transient increase in maximal response and a decrease in ECs0 was observed at day 7. In hippocampal and cortical tissue a general develop- mental pattern of increase in ECs0 and maximal response was observed. Additionally, in both tissues the ECs0 and maximal values appear to have reached a plateau at their adult levels at 21 and 90 days of age. The brains of younger animals (1, 7 and 14 days) have ECs0 and maxi- mal values which were significantly lower than those ob- tained from 21- and 90-day-old rats.

The most interesting finding is the transient increase in maximal response and decrease in ECs0 observed in cor- tical tissue at day 7. Some investigators have reported transient increases in developmental parameters in corti- cal tissue while others have not. In rat brain cortex [3H]- D-aspartate uptake does not exhibit a transient increase with development but rather a gradual increase in uptake sites [5]. Cultured cortical tissue responds in a similar fashion, with a gradual increase in receptor sites with development [17]. Binding studies using the competitive NMDA ligand [3H]CPP in the visual cortex exhibit a transient increase in binding at 2 3 weeks and a differen- tial pattern of development for NMDA and non-NMDA receptors [4].

In the hippocampus, some radioligand binding studies have reported a gradual increase in binding with no ob- servable transient increase with development. [3H]D-as- partate binding autoradiography, a measure of presyn- aptic uptake sites in the hippocampus, does not exhibit a transient increase but rather a steady increase with in- creasing age [13]. Many investigators however, have ob- served a transient increase in hippocampal responses. In rat hippocampus, the density of NMDA receptor sites increases with age with a transient increase at day 8 [16]. Additionally, long-term potentiation appears to be greatest on day 15 in the hippocampus and is two to three times larger than the response elicited from adult animals [91.

In the present study, a transient increase in [3H]NE was observed in the cortex but not the hippocampus. There are a variety of possible explanations for these findings. Generally, there appear to be fewer examples of transient increases in presynaptic systems. Na+-depend - ent [3H]D-aspartate binding and excitatory amino acid uptake studies do not consistently demonstrate transient

increases. In the present studies, NMDA stimulated [3H]norepinephrine release was used as the test system and neurotransmitter release in this system is a presynap- tically based event [6].

Additionally, the release assay used in this study is a multistepped process and amplification at the receptor level with development may be differentially translated to the final outcome of a change in neurotransmitter re- lease. Increases in receptor number with development may not guarantee alterations in neurotransmitter re- lease. There also seems to be a further degree of incom- patibitily as changes in the ECs0 for NMDA with devel- opment were observed in the present study but, very few examples of alterations in affinity with radioligand bind- ing techniques have been reported. Affinity is an impor- tant component in determining the ECs0 value. These studies give some indication that other components im- portant in ECs0 determinations which occur after the binding of the ligand to the receptor may be altered dur- ing development.

These studies demonstrate developmental alterations in NMDA stimulated [3H]NE release in the cortex and the hippocampus. The cortical tissue exhibited a tran- sient increase of the response while the hippocampal tis- sue did not. These studies indicated that alterations in receptor binding that occur with development may be accompanied by alterations in NMDA stimulated neu- rotransmitter release.

This project was supported by Biomedical research support grant funds from the National Center for Re- search Resources 2S07 RR05806-13.

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