Effect of phosphatidylserine on the binding properties of glutamate receptors in brain sections from adult and neonatal rats

BRAIN RESEARCH ELSEVIER

Brain Research 730 ( IY96) 337-135

Research report

Effect of phosphatidylserine on the binding properties of glutamate receptors in brain sections from adult and neonatal rats Joi;1 Gagnk ‘, Caroline Gig&r-e a, George Tocco b. Maurice Ohayon “, Richard F. Thompson Michel Baudry b, Guy Massicotte ‘L”

Accepted

I6 July

h.

IGM

Abstract The effects ol‘ phosphatidylserine (PS) on the binding properties of the AMPA (cy-amino-3-hydroxy-5-tnethylisoxazolepr~~p~~)nic acid) and NMDA ( N-methyl-D-aspartate) subtypes of glutamate receptors were analyzed by quantitative autoradiography of [ ‘HIAMPA. [3H]6-cyano-7-nitroquinoxaline-2.3-dione (CNQX) and [jH]glutamate binding on rat brain tissue sections. Preincubation of brain sections with PS produced an increase in [ ‘HIAMPA binding without modifying the binding properties of [3~l~~~~. an antagonist of AMPA receptors. Thij ef‘fect of PS appeared to be specific for the AMPA subtype of glutamate receptors as the \ame treatment did not modify [?H]glutamate binding to the NMDA receptors. Furthermore, the PS-induced increase in [jH]AMPA binding was different in various brain structures, being larger in the molecular layer of the cerebellum and almost absent in the striatum. Preincubation with calcium also augmented [ ‘H]AMPA binding, and the lack of additivity of the effects of calcium and PS on [‘H~AMPA binding strongly suggest& that both treatments share a common mechanism(s) for producing increased agonist binding. Finally. the effect of PS on AMPA receptolproperties wah markedly reduced in rat brain sections prepared from neonatal rats at a developmental stage that is normally characterized by the absence of LTP expression in certain brain regions. The present data are consistent with the hypothesis that alteration in the lipid composition of synaptic membranes may be an important mechanism for regulating AMPA receptor properties. which could be involved in producing long-lasting changes in synaptic operation. Krvrrorrk

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1. Introduction Long-term potentiation (LTP) is a long-lasting increase in synaptic efficacy that is generally considered to be a cellular device for information storage in various brain structures such as the hippocampus, the striatum and the cerebral cortex [ 10.16,34,38,45]. In area CA1 of the hippocampus. LTP induction requires sufficient postsynaptic depolarization to activate NMDA receptors [ 12,261, thereby resulting in a calcium influx into pyramidal cells, which in turn provides the intracellular trigger for LTP appearance [30,31]. Furthermore. LTP expression appears to be partly

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mediated by a modification of postsynaptic currents elicited by activation of the AMPA subtype of glutamate receptor [ 18,23,39]. Several groups have proposed that LTP expression may involve the uncovering of AMPA receptors which prior to LTP, are either not present in the postsynaptic membrane or are non-functional. i.e.. electrophysiologically silent [22,25]. This hypothesis, which assumes the appearance of new functional AMPA receptors during synaptic potentiation, is consistent with previous observations indicating that LTP expression in the rat hippocanpus is associated with increased binding of [‘H]AMPA to glutamate/AMPA receptors [8,32.48]. Thus. understanding the biochemical mechanisms participating in the regulation of AMPA receptors might provide important clues to the very basis of LTP expression and possibly memory. A growing body of evidence indicated that the binding

‘5Z 1996 Elsevier Science B.V. All rights reserved

properties of the AMPA subtype of glutamate receptor are regulated in part by their lipid environment and/or phospholipid metabolites generated by various phospholipases [4,1 1,171. In particular, treatment of synaptic membranes with phospholipase C or A, (PLA , ) results in an increased affinity of agonists for the receptor [33.35-37,501. lncreased AMPA binding is also observed following treatment of rat brain sections with PLA?; as the same treatment produces a decrease in antagonist binding to the receptor. it has been proposed that the PLA,-induced alteration in ligand binding reflects changes in AMPA receptor conformation rather than number [35]. Moreover, the effects of phospholipase on AMPA receptors are probably due to modifications of the lipid environment of the receptors, rather than to the formation of phospholipid metabolites. such as arachidonic acid [33]. In particular. incorporation of phosphatidylserine (PS) into synaptic membranes also results in an increased affinity of agonists for AMPA receptors [6]. Surprisingly, PLA 2 treatment of neonatal membranes does not augment the affinity of AMPA receptors but results in decreased AMPA binding [.5,35]. As this opposite effect of PLA, occurs, at a developmental stage during which LTP is not expressed, we have argued that PLA,-induced modifications in the lipid environment of synaptic membranes are critical factors for producing changes in AMPA receptor properties, as observed in synaptic potentiation. It is of interest to mention that LTP expression in the adult hippocampus is accompanied by an increase in [ 3H]AMPA binding without modifications in NMDA receptor binding properties [32,48]. If the selective changes in glutamate receptor properties observed in LTP reflect alterations in the lipid environment, it should be possible to differentially modify the properties of both subtypes of glutamate receptors (NMDA and AMPA) by manipulating the phospholipid composition of membranes. Here, we present the results of our experiments performed on brain tissue sections aimed at further characterizing phospholipid-induced alterations in glutamate receptor binding in both adult and neonatal rats. To investigate possible regional differences in the effects of phospholipids on AMPA receptors, exogenous phospholipids and cholesterol were applied to thin, thaw-mounted sections of frozen rat brains. The binding properties of AMPA receptors were determined by qualitative and quantitative autoradiography of [jH]AMPA and [jH]CNQX binding to the sections. We also analyzed [‘HIglutamate binding to the NMDA receptors to determine the specificity of phospholipid actions on AMPA receptor properties. Our data indicate that PS selectively modifies the conformation ol AMPA receptors in various brain structures of adult rats, an effect that is not present at a developmental stage characterized by an absence of LTP. The results therefore further support the idea that changes in the lipid environment of glutamate receptors participate in mechanisms of synaptic plasticity.

2. Materials

and methods

Sprague-Dawley rats (200-250 g) were kept on a 12-h day and night cycle with food and water ad libitum. Naive rats were sacrificed and their brains quickly removed and frozen in isopentane (methyl butane) at -20°C. then kept at -70°C until sectioned. Horizontal and coronal IO-pm thick sections were cut in a cryostat and thaw-mounted on chrome-alum gelatin-coated slides. Different amounts ot phospholipids (phosphatitylserine (PS), phosphatidylcholine (PC). phosphatidylethanolamine (PE). sphingomyelin (SP)) or cholesterol (Sigma, St. Louix. MO) in chloroform solution were evaporated to dryness in a Speed Vat centrifuge. The dry residues were resuspended in 2.0 ml of 100 mM Tris-acetate. pH 7.4. containing 100 PM EGTA, and the final suspensions were sonicated t’or 30 \. Adjacent sections were preincubated for 60 min in 50 ml of Tris-acetate buffer (100 mM, pH 7.4) containing 100 I_LM EGTA with or without different concentrations of phospholipids and cholesterol at 35°C. After a rapid wash in ice-cold Tris-acetate buffer, they were incubated with different radioactive ligands. 2.2. Binding cc.sstt~s 2.2. I. AMPA receptor properties For [3~]~~~~ binding, sections were incubated for 45 min at 0-4°C in Tris-acetate buffer (50 mM. pH 7.4; 100 PM EGTA) containing 50 mM potassium thiocyanate in the presence of 75 nM of [ 3H]AMPA (specific activity: 53 Ci mm01 ‘, NEN-DuPont, Wilmington, DE). For [‘H]CNQX binding, sections were incubated for 45 min at O-4°C in Tris-acetate buffer (50 mM. pH 7.4: SO PM EGTA) containing 1 mM glycine (to inhibit the glycine site of the NMDA receptor) in the presence of 50 nM [“H]CNQX (16.1 Ci mom’, NEN-DuPont). Nonspecific binding was defined for both ligands and the binding measured in the presence of I mM quisqualatc. The sections were then rinsed in cold incubation buffer twice l01 IO s. once for 5 s in SO%>of- the same buffer, followed by 3 dips in distilled water. They were then rapidly either wiped off the slides with a GF/C filter and the radioactivity in the filters was counted in a liquid scintillation counter, or they were dried under a stream of warm air and used for autoradiography.

For [ ‘HIglutamate binding to NMDA receptors, scctions were incubated with 100 nM of [3H]glutamate (51 Ci.mmol ‘. NEN-DuPont) for 45 min at 0-4°C in Trisacetate buffer (SO mM, pH 7.4; SO PM EGTA) containing 5 FM AMPA. I FM kainic acid and IO PM quisqualate to eliminate glutamate binding to non-NMDA sites and 100 PM SITS (4-acetanido-4’-isothiocyanato-stilbenc-

3.7’-disulfonic acid) to block glutamate uptake sites. Nonspecific binding was defined as binding measured in the presence of I mM glutamate. The sections were rinsed in incubation bufter twice for IS s. once for 5 s in 50% of the same buffer. followed by 3 dips in distilled water. The sections were wiped off or dried as described above for AMPA binding.

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Dried sections as well as tritium standards (ARC. St. Louis. MO) were exposed to tritium-sensitive film (Amersham Hyperfilm) for 7 days ([‘H]AMPA and [ ‘H]CNQX) or 14 days ([ ‘H]glutamate). The films were developed in Sigma developer and fixer. Optical densities of different brain regions were converted to radioactive units, using tritium standards on the film after measurement with an image analysis system (Brain software running on the DLIMAS system from Drexel University). ANOVA was performed followed by Scheffe post-hoc analysis with the conventional criterion of statistical significance: P < 0.05.

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3. Results PE

Coronal sections at the level of the hippocampus were preincubated at 35°C [or 60 min in Tris-acetate buffer containing various phospholipids (PS, PC, PE, SP) and cholesterol concentrations. After washing in Tris-acetate buffer the sections were incubated with 7.5 nM [‘H]AMPA for 45 min at 0°C and specific [ ‘H]AMPA binding was determined by measuring radioactivity remaining in the sections following washing in ice-cold buffer. A signil‘icant increase in [ ‘H]AMPA binding was observed at IS nM (73%) PS and maximal elevation was obtained at PS concentrations between 500-800 nM (55%); other phospholipids such as PC, PE. SP. or cholesterol were not capable ot’ stimulating [-‘H]AMPA binding in brain sections (Fig. I A). A concentration of 400 nM of PS was used in subsequent experiments as it provided a near maximal increase in [ ’ H]AMPA binding. Considerable experimental evidence has shown that various treatments reported to increase [jH]AMPA binding in synaptic membranes often differentially modulate the binding properties of the antagonist [ ‘H]CNQX for the AMPA subtvpe of glutamate receptor [8,35.49,50]. To test the possibility that phospholipids differentially regulate the binding properties of agonist and antagonist ligands, we determined [ ‘H]AMPA and [ ’ H]CNQX binding after phospholipid treatment of brain sections. Coronal sections were preincubated with 400 nM of various phospholipids

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and cholesterol. [ ‘H]AMPA and [ ‘H]CNQX binding were then measured bv wiping off the sections and counting the amount of specifically bound radioacti\:ity. As shown in Fi g. IB. the PS-induced increase in [‘H]AMPA binding was not associated with significant changes in [ ‘H]CNQX no other phospholipids or cholesbinding. Furthermore, terol were shown to interfere with [ ‘H]CNQX binding to AMPA receptors. These results cont’irm that PS is the only phospholipid regulating AMPA receptor properties, and the observation that the PS-induced modulation of AMPA receptor properties is specific to the agonist strongly suggests that the changes involve alterations in receptor conformation rather than modifications in the maximal nuniber of binding sites.

J. Gagn~et al. / Brain Research 740 (1996) 337-345

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3.2, Qualitative and quantitatit, e [ aH]AMPA and [-~H]glutamate binding autoradiography of horiz.ontal sections: effects of preincubation with PS

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Several groups h a v e p r o p o s e d that L T P expression may i n v o l v e the appearance o f n e w functional A M P A receptors [22,25]. Interestingly, L T P expression in the adult hipp o c a m p u s has been reported to be a c c o m p a n i e d by increased in [ 3 H ] A M P A binding without modifications in N M D A receptor properties [4,37]. W e also p r o p o s e d that

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Fig. 3. Effect of calcium on the PS-induced increase in [~H]AMPA binding in different areas of the rat brain. Adjacent horizontal sections were preincubated at 35°C for I h with 400 nM PS in the absence or presence of 2.0 mM calcium. They were then washed in buffer alone and processed fl~r [aH]AMPA binding as described in Section 2, Materials and methods. Autoradiographs (data not shown) were analyzed for various hippocampus regions and the molecular layer of the cerebellum as described in the legend to Fig. 2. The results represent PS-induced stimulation of ['H]AMPA binding (expressed as percentage increases over basal binding) under both control (open bars) or calcium (closed bars) conditions. The data represent the means ± S.E.M. of at least 8 different measurements per rat with 4 different rats.

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Fig. 2. Effect of PS treatment on [aH]AMPA binding in different areas of the rat brain. Adjacent horizontal sections were preincubated at 35°C for 1 h in Tris-acetate buffer (100 mM, pH 7.4, containing 100 /xM EGTA) without (A) or with (B) 400 nM of PS. They were then washed in buffer alone and processed for [3H]AMPA binding as described in Section 2, Materials and methods. Autoradiographs shown in A and B were analyzed with an image analysis system and the results are expressed in pmol/mg of protein obtained for different regions of the rat brain (C). The data represent the means±S.E.M, of at least 8 different measurements per rat with 4 different rats. * Statistical significance as compared to basal values. OR, stratum oriens; RAD, stratum radiatum: DG, molecular layer of the dentate gyms: INTCTX, inner layer (III-VI) of the parieto-temporal cortex; EXTCTX, outer layers (1-II) of the same cortex; STRIAT, striatum; GRAN, granular layer of the cerebellum; MOL, molecular layer of the cerebellum; THAL, thalamic nuclei.

modifications in the lipid e n v i r o n m e n t of synaptic m e m branes may be critical for generating changes in A M P A receptor properties during synaptic potentiation [4,6,33,37]. H o w e v e r , one important question is whether PS treatment specifically modulates A M P A receptors in various brain structures. To d e t e r m i n e this possibility, the effects of PS treatments on the binding properties o f the A M P A and N M D A subtypes of glutamate receptors were analyzed by quantitative autoradiography of [aH]AMPA and t a l l ] g l u t a m a t e binding on brain sections.

3.2.1. AMPA receptor binding Horizontal sections were preincubated at 35°C for 60 rain in the presence or absence of 400 n M PS. F o l l o w i n g washes, [ a H ] A M P A binding was p e r f o r m e d in the absence o f PS, and the sections were then processed for autoradiography. Visual e x a m i n a t i o n of autoradiography clearly indicated increased binding in the hippocampus, c e r e b e l l u m and other brain structures (Fig. 2B). Autoradiographs were quantitatively analyzed with an i m a g e analyzing system, and the data f r o m several sections were averaged. The results indicated that the increase in [ a H ] A M P A binding elicited by PS preincubation was u n i f o r m l y distributed in all regions of the h i p p o c a m p u s (Fig. 2C). PS treatment p r o d u c e d a 30 +_ 5% rise in [ 3 H ] A M P A binding in the C A 3 and CA1 strata radiatum and oriens. A similar increase was o b s e r v e d in the dentate gyrus and cerebral internal cortex; h o w e v e r , in the striatum, the elevation of [ a H ] A M P A binding was smaller and did not reach statistical significance (10 ___6%). Finally, the m o l e c u l a r layer of the c e r e b e l l u m exhibited the largest augmentation of

J. GagnF et al. / Brain Research 740 ( 1996~ 337 345

[~H]AMPA

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crease was present in the g r a n u l a r layer (15 4- 5%). T r e a t m e n t o f rat b r a i n s e c t i o n s w i t h c a l c i u m h a s p r e v i o u s l y b e e n s h o w n to h e i g h t e n [ 3 H ] A M P A b i n d i n g [49]. A d j a c e n t h o r i z o n t a l s e c t i o n s w e r e p r e i n c u b a t e d w i t h 2.0 m M c a l c i u m acetate at 35°C for 60 rain in the p r e s e n c e or a b s e n c e o f 4 0 0 n M PS a n d t h e n s u b j e c t e d to [ 3 H ] A M P A ligand b i n d i n g a u t o r a d i o g r a p h y . W e c o n f i r m e d that calc i u m t r e a t m e n t resulted in i n c r e a s e d [ ~ H ] A M P A b i n d i n g , but the c o m b i n a t i o n o f c a l c i u m a n d PS still e v o k e d h i g h e r b i n d i n g than e i t h e r t r e a t m e n t a l o n e ( d a t a not s h o w n ) . H o w e v e r , q u a n t i t a t i v e a n a l y s i s i n d i c a t e d t h a t c a l c i u m treatm e n t s u b s t a n t i a l l y r e d u c e d the m a g n i t u d e o f the P S - i n -

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Fig. 5. Effect of PS treatment on [~H]glutamate binding to the NMDA site in different areas of the rat brain. Adjacent horizontal sections were preincubated at 35°C for I h in Tris-acetate bufler (100 raM, pH 7.4, containing 1()() # M EGTA) without (A) or with (B) 400 nM of PS. The', were then washed in buffer alone and processed for [~Hlglutanlate binding to the NMDA subtype of glutamate receptor as descrihed in Section 2, Materials and methods. Autoradiographs ;',,ere analyzed as described m the legend to Fig. 2 and values were expressed in pmol/m~ of protein obtained for different regions ol the rat brahl (('). The data represent the means + S.E.M. of at least 8 different meast~rements per rat with 4 dift~zrent rats.

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Fig. 4. Effect of PS treatment on [~ H]AMPA binding in different areas of the neonatal rat Brain. Adjacent horizontal sections prepared from young ,mimals (PND ]()) were preincubated at 35°C for 1 h in Tris-acetate buffer (100 raM. pH 7.4, containing I00 #M EGTA) without (A) or with iB) 400 nM of PS. They were then washed in buffer alone and processed (or [~ H]AMPA binding as described in Section 2, Materials and methods. kutoradiographs were analyzed as described in the legend to Fig. 2, and values were expressed in pmol/mg of protein obtained for different ;egions of the rat brain (C). The data represent the means + S.E.M. of at !east g difl~.'rent measurements per rat vdth 4 different rats,

d u c e d c h a n g e in [ ~ H ] A M P A b i n d i n g in the various brain r e g i o n s a n a l y z e d (Fig. 3). Several g r o u p s h a v e d e m o n s t r a t e d that L T P e x p r e s s i o n is m a r k e d l y r e d u c e d in very y o u n g a n i m a l s [3,20]. W e e v a l u a t e d the effect o f PS t r e a t m e n t on [:~H]AMPA binding to b r a i n s e c t i o n s p r e p a r e d from p o s t n a t a l day l 0 ( P N D - 1 0 ) a n i m a l s . H o r i z o n t a l s e c t i o n s were p r e i n c u b a t e d w i t h 4 0 0 n M o f PS a n d [ ~ H ] A M P A b i n d i n g was then m e a s u r e d by q u a n t i t a t i v e a u t o r a d i o g r a p h y . W h e r e a s PS t r e a t m e n t o f adult rat s e c t i o n s p r o d u c e d an i n c r e a s e o f [ 3 H ] A M P A b i n d i n g , the s a m e t r e a t m e n t in y o u n g a n i m a l s

342

,1. Gagn~ et al. / Brain Research 740 (1996J 337 345

had no such effect. Ligand binding autoradiography confirmed this result, both qualitatively (Fig. 4B) and quantitatively (Fig. 4C). 3.2.2. N M D A r e c e p t o r b i n d i n g

Binding of 100 nM [3H]glutamate to NMDA receptors was determined in the presence of 1 /xM kainate, 5 /zM AMPA, 10 /xM quisqualate and 100 /xM SITS (4acetanido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid) to eliminate the non-NMDA receptor binding component and to block glutamate uptake sites. Under these conditions, [~H]glutamate binding represents binding to the NMDA receptor. In contrast to [3H]AMPA binding, preincubation of horizontal sections with 400 nM PS slightly decrease [3H]glutamate binding to NMDA receptors (Fig. 5B). Quantification and averaging of data obtained from several sections confirmed the visual examination and indicated no significant changes in [3H]glutamate binding in various brain regions following PS treatment (Fig, 5C).

4. Discussion

Our results confirm and extend the previously reported observation that incorporation of PS into membranes enhances [3H]AMPA binding [6]. In the present study, we also demonstrated that preincubation of frozen-thawed brain sections with PS resulted in an increase of[ 3H]AMPA binding, while the same treatment had no effect on [3H]glutamate binding to NMDA sites. No other phospholipids (PE, PC, and SP) and cholesterol tested were capable of producing a similar increase of [3H]AMPA binding in the various brain regions analyzed. Moreover, the different effects of PS treatment on [3H]AMPA and [3H]CNQX (antagonist) binding strongly support previous observations indicating that PS-induced changes in AMPA receptor properties are not due to alterations in the number of sites, but probably reflect modifications in receptor conformation [6]. Although the increase in [SH]AMPA binding elicited by PS in adult rats was relatively uniform across brain structures, two regions appeared to be differentially affected. Thus, quantitative autoradiography revealed that the molecular layer of the cerebellum exhibited the largest increase in [3H]AMPA binding, whereas the striatum showed the smallest. The mechanism(s) underlying these regional differences are unknown, although it is noteworthy that AMPA binding in the striatum has previously been shown to be less sensitive to the effect of sulfhydryl reagents [7] and PLA 2 treatment [50]. Finally, the PS-induced alteration in AMPA receptor properties was markedly reduced in rat brain sections prepared from young animals at a developmental period characterized by LTP impairment [3,20]. The transient rise in calcium concentration in postsynaptic elements resulting from NMDA receptor activation is likely to stimulate a number of biochemical pro-

cesses which could produce changes in AMPA receptor properties and thereby participate in LTP. in this regard, much experimental evidence indicates that the lipid environment of the receptor is an important factor in determining the binding properties of AMPA receptors, as both the incorporation of lipids and modification of phospholipids produced by phospholipase treatment of membranes or tissue sections produce similar changes in binding properties [6,33,35-37]. We previously found that calcium treatment of rat brain sections also evoked increased [ 3H]AMPA binding in various brain areas. This effect was time- and concentration-dependent and partly blocked by leupeptin, an inhibitor of calcium-dependent protease [49]. The lack of additivity in the effects of calcium and PS reported in the present study suggests that the same biochemical mechanism(s) is(are) involved in producing the increased in agonist binding to AMPA receptors. Several possibilities could account for the lack of additivity in the effects of calcium and PS. For instance, the asymmetric distribution of phospholipids in biological membranes is well established with most PS being localized in the inner leaflet of the lipid bilayer. This asymmetric distribution of PS is probably regulated by several factors, including (i) strong interaction between cytoskeletal proteins (such as spectrin) and the phospholipid, and (ii) constant translocation of PS from the outer leaflet to the inner leaflet of the cell membrane by an ATP-dependent aminophospholipid translocase [13,14,54]; under our experimental conditions, it is unlikely that the latter mechanism operates because of the lack of ATP-regenerating systems. However, as spectrin is a preferred substrate of calcium-dependent proteases, it is conceivable that calcium treatment releases PS/spectrin interactions, thereby allowing phosphatidylserine translocation to the outer leaflet. This could account fl)r the lack of additivity of calcium and PS treatment inasnmch as the effect of PS is the result of phospholipid insertion in the outer leaflet. The reduced effect of PS in young animals is consistent with this interpretation since a higher level of calpain-mediated proteolysis of spectrin is observed in the neonalal brain [41]. However. the lack of effect of PS treatment on neonatal brain sections may also reflect differential properties of synaptic membranes in neonates. In particular, the lipid composition of neuronal membranes changes during brain development [27] as does the expression of various subunits of AMPA receptors [5,43,44]. Further experiments are required to determine the relative contributions of these types of modifications to the differential regulation of AMPA receptor binding produced by PS treatment during the postnatal period. Independently of the intimate details of mechanisms involved in the effects of phosphatidylserine on [ ~H]AMPA binding in adult rats, as well as the absence of PS actions in young animals, these results may have potentially interesting implications regarding the biochemical processes underlying long-lasting changes in synaptic efficacy. Re-

J. GagnF et al./ Brain Research 740 ( 1990J 337 345

cent studies have suggested that a significant proportion of excitatory synapses on hippocampal CA I neurons express NMDA receptors but no AMPA receptors, thus making these synapses silent under conditions of low degree postsynaptic membrane depolarization. It has been proposed that when these silent synapses are subjected to highfrequency stimulation under conditions leading to LTP, they acquire AMPA receptor-type responses due to the functional addition of AMPA receptors in LTP [22,25]. The exact mechanisms by which synapses devoid of detectable AMPA receptors become responsive during LTP remain to be determined. Possible mechanisms may include the appearance of new functional AMPA receptors in postsynaptic elements, uncovering of receptors already present in the membranes and changes in the affinity of preexisting receptors. It is of interest that similar to PS treatment, LTP has been reported to be accompanied by an increase in AMPA receptor binding in adult hippocampus [32,49] with little change in [3H]glutamate binding to NMDA receptors [132]. Interestingly, in vivo administration of PS has been shown to increase LTP expression in the rat hippocampus [9] and the same treatment has been demonstrated to improve acquisition and retention of a variety of learned behaviors in aged rats [15,19,53], suggesting that the PS-induced change in AMPA receptors is an important step lk)r controlling synaptic function. The effect ~f PS treatment on AMPA binding was lkmnd to be different in various brain structures of adult rats. Whether this is due to receptor heterogeneity, to different levels of PS incorporation into synaptic membranes or to a regional variation in the ability of the tissue to generate the PS effect is not clear at present. For instance, phosphatidylserine has been shown to increase protein kmase C (PKC) activity and, since this type of kinase was reported to be critical for modulating AMPA receptor function in synaplic plasticity, it is tempting to propose that PS-induced changes in receptor binding could possibly be due to differential activation of PKC in various brain structures. In this respect, localization of PKC isozymes in rat brain was found to be very similar to the PS-induced increase in AMPA binding [21]. However, tile characteristics of the PS-induced enhancement in AMPA binding seen in the present study were not always consistent with the clectrophysiological properties of LTP in various brain regions. Although there is much evidence supporting the idea that alterations in AMPA receptor properties by phospholipids are associated with LTP lk)rmarion m area CA1 of the hippocampus [8,9,34,37], other investigations havc shown that LTP in area CA~ of the hippocampus possibly requires an increase in transmitter release rather than changes in receptor properties at the postsynaptic lexel [46,52]. Thus, it seems unlikely that the PS-induccd change in AMPA receptor properties observed in area CA3 of lhe hippocampus contributes to LTP formation in this hippocampal structure. Similarly, modifications in AMPA receptor properties by phospholipid would not

34~

appear to mediate LTP-like mechanisms m the striatum [10,51] as PS treatment did not increase [~H]AMPA binding in this area. In addition, electrophysiological studies have indicated that long-term depression (LTD), tile major mechanism of synaptic plasticity in the cerebellum, takes place in the molecular layer as a restllt of the simultaneous activation of climbing and parallel fibers, and that LTD could be due to a decreased responsiveness of AMPA receptors [28]. As PS treatment was reported to produce a maximal increase in [~H]AMPA binding in the molecular layer of cerebellum, we have to propose that other inmicellular processes possibly modulatc AMPA receptor function during LTD in this structure. Accordingly, pharmacological studies have recently shown that arachidonic acid or its lipoxygenase metabolites reduced AMPA receptor function [24,42] and PLA2 inhibitors have been reported Io reduce LTD lkwmation in cerebellar Purkinje neurons in culture [29]. In any event, the possibility of obtaining detailed maps of the biochemical processes rcgulatmg the binding properties of AMPA receptors should provide interesting information concerning tile nlechanisms of synaptic plasticity in different brain structures. The present results finally suggest that the reduced I,TP magnitude observed during the deveh~pmental period in certain brain regions may reflect the inability of PS to modulate AMPA receptor properties. Tile lack of modulation of AMPA receptors by PS at postnatal days (PND) 5 - 1 0 con'elates well with the capacity of tile hippocampus to elicit electrical LTP at this developmental period. In particular, synaptic potentiation was shown to bc virluall 5 absent in the first 8 PND in area CA 1 of the hippocampus and appears only after days 10-11 [3,20,40]; siinilarly, the rat visual cortex has been reported to elicit stable LTP only after PND 6 [47]. However, not all forms of synaptic potentiation associated with the developinental period seem to parallel the PS-induced changes in AMPA receptor properties. For instance, thalamocortical synapses of thc rat somatosensory corlex were found to generate inaxinla] L T P at PND 3-7 [16], a developmental stage characterized by a lack of PS effects on AMPA receptor properties. Whether this form of LTP at thalamocortical synapses is necessarily mediated by changes :it the postsynaptic level remains to be determined. For instance, i! is possible thai LTP processes observed during the developmental period require specific modifications in transmitter release instead of changes in AMPA receptor propertics. Independent of this question, tile present data further confirm the role of the lipid environment of AMPA receptors in the determination of receptor properties. It has been stressed previously that a simple modification of AMPA receptor number could not account for the qualitative and quantitative changes in wavelk)rm properties observed after LTP induction [1,2]. Our resuhs indicate that changes in membrane lipid composition, which are likely to take place as a restllt of the cascade of calcium-dependenl processes acliwited by stimuli leading to I.TP. produce modificatiol>

344

J. Gagng et al. / Brain Research 740 (1996) 337 345

in r e c e p t o r p r o p e r t i e s , r e f l e c t e d b y i n c r e a s e d b i n d i n g affinity for a g o n i s t s w i t h o u t a l t e r i n g affinity for a n t a g o n i s t s . M o r e o v e r , the d i f f e r e n t i a l e f f e c t o f m e m b r a n e lipid c o m p o s i t i o n o n A M P A r e c e p t o r p r o p e r t i e s d u r i n g the d e v e l o p m e n t a l p e r i o d c o u l d not o n l y a c c o u n t for t h e lack o f L T P d u r i n g this p e r i o d b u t also for d e v e l o p m e n t a l l y r e g u l a t e d m o d i f i c a t i o n s o f g l u t a m a t e r g i c s y n a p s e properties. F u r t h e r i n v e s t i g a t i o n s o f the m e c h a n i s m s u n d e r l y i n g P S - i n d u c e d c h a n g e s in A M P A r e c e p t o r s m i g h t t h u s p r o v i d e i n t e r e s t i n g i n f o r m a t i o n c o n c e r n i n g the v e r y b a s i s o f s y n a p t i c plasticity and therefore of learning and memory.

Acknowledgements T h i s w o r k w a s s u p p o r t e d b y a g r a n t f r o m the N a t u r a l S c i e n c e s a n d E n g i n e e r i n g R e s e a r c h C o u n c i l o f C a n a d a to G . M . a n d a g r a n t f r o m S a n k y o Co. Ltd. to M.B.

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