132 Developmental Brain Research. 51 (1990) I32-i34 Elscvic~

BRESD 60338

Cha s in excitatory amino acid modulation of phosphoi itide metabolism during development

Elizabeth Palmer, Kurt Nangel-Taylor, Jeffrey D. Krause, Arnold Roxas and Carl W. Cotman

Department of Psychobiology, University of California, lrvine, CA 92717 (U.S.A.)

(Accepted 22 August 1989)

Key words: Quisqualate; N-Methyl-D-aspartate; Cortex; Cerebellum; Olfactory bulb; Hippocampus; Thalamus/hypothalamus; Phosphoinositide metabolism

Two different developmental patterns of stimulation of phosphoinositide (PI) metabolism by excitatory amino acid (EAA) receptors were observed during the postnatal maturation of various brain regions. A 'burst' in PI metabolism was seen at postnatal day 6 (PND6) in olfactory bulb and cerebellum and at PND9 in hippocampus. In cortex and thalamus/hypothalamus high levels of PI metabolism were observed initially, and then began to decline at PND15 and PND18, respectively. NMDA inhibition of PI metabolism was generally found to parallel the EAA activation but the persistence of inhibition varied in the different brain regions.

Excitatory amino acid (EAA) receptors may be di- vided into at least 4 subtypes based on pharmacological and electrophysiological investigations. These receptors are named according to their selective agonists; AMPA (a-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid) or QA (quisqualic acid), NMDA (N-methyl-D-aspartate), KA (kainic acid), and AP4 (2-amino-4-phosphono- butyrate). A fifth EAA receptor has been identified which stimulates phosphoinositide (PI) metabolism 15'~9-21. This receptor is activated by QA 18'2°'21, ibotenic acid 16"1s,

glutamate 16'18 and trans-l-amino-cyclopentyl-l,3-dicar-

boxylic acid (ACPD) ~5'19. We have recently shown that

EAA receptors exert a bidirectional control over phospho- inositide (PI) metabolism in the neonate rat hippocampuslS. In the neonate hippocampus 10/~M QA stimulates the formation of inositol phosphates by approximately 17- fold ~8. The co-application of 100 mM NMDA inhibits QA-induced stimulation by at least 50%. This inhibition is blocked by competitive and non-competitive NMDA an- tagonists such as 3-((+)-2-carboxypiperazin-4-yl)propyl-1- phosphonic acid and MK-80118. In addition, AMPA recep-

tor activation leads to a small increase in PI metabolism that is blocked by the AMPA antagonist, CNQX (6-cyano- 2,3-dihydroxy-7-nitro-quinoxaline) 5.

Recent evidence suggests that E A A s play an impor- tant role in synaptic growth and stabilization. In the rat hippocampus stimulation of PI metabolism by glutamate and a structural analog, ibotenate, has been shown to be most robust during the first week of postnatal develop- ment, dropping down to almost negligible levels by

PND2416. In addition, an increase in glutamate-induced PI metabolism is observed following deafferentation, during the time period in which neuronal regeneration is taking place and new synapses are being formed 17. Many

examples of a critical role for N M D A receptors in early learning and synaptic specificity exist. In the visual cortex of kittens NMDA antagonists can prevent the ocular dominance shift normally seen in response to a monoc- ular experience s . In the developing olfactory system NMDA antagonists can block the learning of a prefer- ence for an artificial odor that normally occurs during a specific developmental time when the odor is given concomitantly with tactile stimulation u'21.

The aim of this study was to characterize EAA receptor modulation of PI metabolism in different brain regions during postnatal development. That is, does the same receptor type stimulate PI metabolism in different brain regions, does the same developmental pattern for PI metabolism exist for all regions, and does NMDA modulate the EAA-induced stimulation to the same extent in different regions?

Brain regions were dissected from rats of the indicated ages and transverse 0.4 mm slices were prepared from tissue that had been cut to have a diameter of approx. 3 mm. The slices were immediately suspended in the following buffer: (in mM) NaCI 127, KCI 2, KH2PO4 1.25, NaHCO 3 26, CaCI 2 2, MgSO4 2 and D-glucose 10 equilibrated with 95% 02/5% CO 2 to raise the pH to 7.4. The slices were preincubated at 33 °C for 60 min under a constant stream of O2/CO 2 and then washed 3

Correspondence: C.W. Cotman, Department of Psychobiology, University of California, Irvine, CA 92717, U.S.A

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Fig. i. Stimulation of [3HI inositol phosphate accumulation in slices from different brain regions. Slices were incubated in the presence of 10 pM QA (ll), 10 pM QA and 20 pM CNQX ([3), or 10 pM OA and 100 uM NMDA ([]). Values given are means + S.E.M. from 3 experiments measured in triplicate.

times with freshly oxygenated buffer. The slices were then labelled with [3H]myoinositol (14.0 C.mmo1-1, 5 mCi.ml -~) for 1 h at 33 °C4. After being washed 3 times

with freshly oxygenated buffer, 3-4 slices were distrib- uted to each test tube. LiCl (5 mM) and tetrodotoxin (500 nM) were added to the incubation cocktail (final volume 0.25 ml), followed by the antagonists and 5 min later by the agonists. In the experiments described in this paper QA (10 pM) was used as an agonist and CNQX (20 pM) was used as an antagonist in order to distinguish between responses due to the stimulation of the CNQX-sensitive (AMPA) and CNQX-insensitive receptors. NMDA (100 pM) was used as an antagonist to study the modulation of QA stimulation of PI metabolism. The reaction was allowed to proceed for 60 min under a constant stream of O2/CO 2. Levels of [3H]inositol phosphate produced were determined as described previously is.

In general, PI metabolism is relatively low at PND3 in all regions analyzed. The highest levels of inositol phosphate production at PND3 in response to stimulation by 10 pM QA were observed in thalamus/hypothalamus

133

(9-fold stimulation) while the least active region was

olfactory bulb where only a 2-fold stimulation was detected. In all regions except thalamus/hypothalamus the QA stimulation of PI metabolism was sensitive to CNQX with an approximate 25% decrease in OA- induced stimulation being observed, suggesting that the AMPA receptor is linked to PI metabolism very early in development. NMDA modulation of QA-induced stim- ulation was observed only in hippocampus and cerebel- lum at PND3.

At PND6 both olfactory bulb and cerebellum demon- strated maximal QA-induced IP stimulation. This time period corresponds to the sensitive period for the development of both neural and behavioral responses to olfactory preference training 9"~1"2~ and also corresponds to the time in which climbing fibers and parallel fibers begin to make contacts with Purkinje cells on PND3 and PND7, respectively in the cerebellum 724, A concomitant

strong inhibition of the QA stimulation by NMDA was also seen at this time.

In the hippocampus maximal stimulation was observed at PND9. In the hippocampus, between PND6 and PND9, there is a dramatic increase in the number of Na-independent [3H]glutamate binding sites 3 and an increase in NMDA binding sites 22. This time period

corresponds to the time in which the hippocampai pyramidal cells form synapses with invading commis- sures, Schaffer collaterals and perforant path projections, and also to an intense period of synaptogenesis when the cells in the dentate gyrus are making contacts with the major intrinsic and extrinsic fiber systems <12

By PND9 the only region in which QA-induced stimulation is still sensitive to CNQX is cortex, indicating that only in cortex does the AMPA receptor maintain a significant influence on PI metabolism. In olfactory bulb and cerebellum the levels of stimulation by QA have already begun to decline by PND9 and a concomitant decrease in NMDA inhibition is also observed.

The stimulation of PI metabolism by QA in both thalamus/hypothalamus and cortex does not exhibit the 'bursting' pattern seen in olfactory bulb and cerebellum at PND6 and hippocampus at PND9. Rather, the levels of stimulation are high at PND3 and remain high until PND12 in the cortex and PND15 in the thalamus/ hypothalamus. In both of these regions synaptogenesis begins very early in postnatal development and continues for 2-3 weeks l'2.m1314. It may be that in these regions PI

metabolism is important for activating processes that occur prior to synaptogenesis as the levels of IP stimu- lation are quite high when synaptogenesis is just begin- ning. The decrease in PI metabolism seems to start once the majority of synapses have been made ~.

By PND30 levels of IP stimulation are very much

134

reduced in all regions. N M D A modula t ion of the

Q A - i n d u c e d IP st imulat ion remains minimal in olfactory

bulb and cerebel lum and is notably present in hippocam-

pus. Al though present in cortex and thalamus/hypothal-

amus its relat ive impor tance is quest ionable as the levels

of s t imulat ion are so low.

The results p resented in this paper indicate that there

are regional variat ions in the deve lopmenta l pat terns of

PI metabol i sm in response to the st imulation of E A A

receptors . Al though it seems that in all regions the

recep tor subtype which elicits the strongest s t imulat ion of

PI metabol i sm is the recep tor which is s t imulated by Q A

but is insensitive to C N Q X , the CNQX-sensi t ive receptor

(the A M P A receptor ) does par t ic ipate in the modula t ion

of PI metabol i sm at PND3 and during the first month of

deve lopment in cerebra l cortex. The CNQX-insensi t ive

r ecep to r p robab ly corresponds to the A C P D receptor

which we have recent ly character ized in h ippocampus 19.

The deve lopmenta l pat terns of PI metabol ism ob-

served in cerebel lum, olfactory bulb and hippocampus

1 Aghajanian, G.K. and Bloom, EE., The formation of synaptic junctions in developing rat brain: a quantitative electron microscopic study, Brain Res., 6 (1967) 716-727.

2 Asanuma, C., Ohkawa, R., Stanfield, B.B. and Cowan, W.M., Observations on the development of certain ascending inputs to the thalamus in rats. I. Postnatal development, Dev. Brain Res., 41 (1988) 159-170.

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8 Kleinschmidt, A., Bear, M.E and Singer, W., Blockade of 'NMDA' receptors disrupts experience-dependent plasticity of kitten striate cortex, Science, 238 (1986) 355-358.

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seem to correspond to other deve lopmenta l critical periodsg.11.23 or intense per iods of synaptogenesis ~'7w24

in such a manner that during these important time

periods a 'burs t ' in QA- induced IP format ion is observed.

During these 'burs t ' per iods the QA- induced stimulation

is also very sensitive to inhibit ion by N M D A , suggesting

that the N M D A receptor activation may provide a

mechanism for modulat ing Ca 2+ levels in such a way that

Ca 2+ entering through the N M D A receptor would reduce

the need for Ca 2+ levels to he mainta ined by the release

of Ca 2+ from intracel lular pools through the inositol

1,4,5-tr isphosphate receptor . The fact that N M D A in-

hibit ion of QA- induced accumulat ion of inositol phos-

phates remains present in the h ippocampus even at

PND30 suggests that this modula t ion by N M D A may be

impor tant for maintaining plasticity in the hippocampus.

This project was supported by NINCD Grant 1 RO1 NS27320-01 and NINCH Grant PO1 HD24236.

14 Miller, M.W., Maturation of rat visual cortex. III. Postnatal morphogenesis and synaptogenesis of local circuit neurons, Dev. Brain Res., 25 (1986) 271-285.

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16 Nicoletti, F., Iadarola, M.J., Wroblewski, J.T. and Costa, E., Excitatory amino acid recognition sites coupled with inositol phospholipid metabolism: developmental changes and interac- tion with ax-adrenoreceptors, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 1931-1935.

17 Nicoletti, F., Wroblewski, J.T., Alho, H., Eva, C., Fadda, E. and Costa, E., Lesions of putative glutamatergic pathways potentiate the increase of inositol phospholipid hydrolysis elicited by excitatory amino acids, Brain Res., 436 (1987) 103--112.

18 Palmer, E., Monaghan, D.T. and Cotman, C.W:, Glutamate receptors and phosphoinositide metabolism: stimulation via quisqualate receptors is inhibited by N-methyt-o-aspartate re- ceptor activation, Mol. Brain Res., 4 (1988) 161-165.

19 Palmer, E., Monaghan, D.T. and Cotman, C.W., Trans-ACPD, a selective agonist to the Pf-coupled excitatory amino acid receptor, Eur. J. Pharm., 166 (1989) 585-587.

20 Recasens, M., Guiramand, J., Nourigat, A. Sassetti, I. and Devilliers, G., A new quisqualate receptor subtype (sAA2) responsible for the glutamate-induced inositol phosphate forma- tion in rat brain synaptoneurosomes, Neurochem. Int., 13 (1988) 463-467.

21 Sugiyama, H., Ito, I. and Hirono, C., A new type of glutamate receptor linked to inositol phospholipid metabolism, Nature (Lond.), 325 (1987) 531-533.

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23 Woo, C.C. and Leon, M., Sensitive period for neural and behavioral response development to learned odors, Dev. Brain Res., 36 ~1987) 309-313.

24 Woodward, D.J., Hoffer, B.J., Siggins, G.R. and Bloom, F.E., The ontogenetic development of synaptic junctions, synaptic activation and responsiveness to neurotransmitter substance in rat cerebellar Purkinje cells, Brain Res.. 34 (1971) 73-97.