Ž .Brain Research 765 1997 141–148

Research report

NMDA-glutamate receptors regulate phosphorylation of dendriticcytoskeletal proteins in the hippocampus

Carlos Sanchez a,1, Luis Ulloa1 a, Rafael J. Montoro b, Jose Lopez-Barneo b, Jesus Avila a,)´ ´ ´ ´a Centro de Biologıa Molecular, UniÕersidad Autonoma de Madrid, Cantoblanco, E-28049 Madrid, Spain´ ´

b Dept. Fisiologıa Medica y Biofısica, UniÕersidad de SeÕilla, SeÕilla, Spain´ ´ ´

Accepted 8 April 1997

Abstract

Ž .Most forms of synaptic potentiation need the activation of the N-methyl-D-aspartate NMDA subtype of glutamate receptors whichgenerate changes in dendritic morphology of postsynaptic neurons. Since microtubule proteins have an essential role in dendriticmorphology, they may be involved and regulated during the modifications of dendritic morphology associated with synaptic potentiation.

Ž .The phosphorylation of the microtubule-associated proteins MAPs has been analyzed in situ after activation or blockade ofNMDA-glutamate receptors in hippocampal slices. The phosphorylation of MAP1B and MAP2 has been studied by using severalantibodies raised against phosphorylation-sensitive epitopes. Whereas antibodies 125 and 305 recognize phosphorylated epitopes onMAP1B and MAP2, respectively, Ab 842 recognizes a phosphorylatable sequence on MAP1B only when it is dephosphorylated. NMDAtreatment decreased the phosphorylation state of the epitope recognized by the antibody 305 on MAP2 and caused a slight

Ždephosphorylation of MAP1B sequences recognized by Ab 125 and 842. Moreover, exposure to APV an antagonist of NMDA-glutamate.receptors counteracted the effect of NMDA and induced an increase in the phosphorylation state of these sequences in MAP2. Since

phosphorylation regulates the interaction of MAPs with cytoskeleton, the results suggest that the modulation of the phosphorylated stateof MAP2 by NMDA-glutamate receptors may be implicated in dendritic plasticity. q 1997 Elsevier Science B.V.

Keywords: Rat; Microtubule associated protein; Glutamate receptor; Synaptic plasticity; NMDA; APV

1. Introduction

In neural networks, the flow of information usuallyprogresses from the axon of a neuron to the somatoden-dritic tree of another neuron. Transmission of informationbetween neurons occurs at chemical synapses, which, uponrepeated stimulation, may undergo plastic changes that

w xdecrease or improve the efficiency of the transmission 5 .A structure that seems to be important for synaptic plastic-

Ž .ity is the dendritic postsynaptic density PSD , a regionwith the highest concentration of postsynaptic receptorsand a cytoskeletal specialization located under the post-

w xsynaptic membrane 1,9,10,22,38 . Cytoskeletal proteinscould then play a role in long-term synaptic modificationsoccurring in those neural regions like dendrites showing aparticularly high level of plasticity. Among the cyto-

) Ž . Ž .Corresponding author. Tel. q34 1 397-8440; Fax: q34 1 397-4799.

1 Authors who have contributed equally to this work.

skeletal proteins possibly involved in synaptic plasticityw xare MAP2, a specific dendritic protein 7,8,12,17,29,42 ,

and MAP1B which is also known to be present in den-w xdrites 6,66,67 . Two forms of MAP1B with distinct local-

izations have been shown in long-term cultured neurons: aŽ .MAP1B phosphoisoform modified by casein kinase 2 is

present in the soma and proximal dendrites, whereas un-phosphorylated MAP1B isoforms are present at more dis-

w xtal dendritic regions 66 .Ž .Activation of N-methyl-D-aspartate NMDA receptors

by either glutamate or NMDA is required in most cases ofw xsynaptic plasticity reported 5,15,20,35,40,41,49,58 . These

receptors mediate calcium influx into the postsynapticterminal, leading to the activation of Ca2q-dependent en-

Ž .zymes kinases, phosphatases or proteases which couldmodify cytoskeletal proteins and, as a consequence, den-dritic morphology. In fact, dephosphorylation of the cyto-skeletal protein MAP2 due to the activation of NMDA

w xreceptors has been reported 13,24,43,47,48 . This dephos-phorylation appears to result from the activation of cal-

w xcineurin, a calcium-dependent phosphatase 13,47 .

0006-8993r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved.Ž .PII S0006-8993 97 00563-5

( )C. Sanchez et al.rBrain Research 765 1997 141–148´142

MAP2 is a phosphoprotein that could be modified inŽ .vitro by several protein kinases such as: 1

w xcalciumrcalmodulin protein kinase 21,55,69 , a proteinthat is a major component of PSD and has been involved

w x Ž .in synaptic potentiation 25,33,34,39 ; 2 protein kinase Cw x27,61,62 , a protein that has an isotype, zPKC, present indendrites, which has been also involved in the mechanism

w x Ž .of synaptic potentiation 52 ; 3 proline-directed proteinkinases, including mitogen-activated protein kinasesŽ . Ž . w xMAPK and glycogen synthase kinase 3 GSK3 3,28,54 ;

Ž . w xand 4 cAMP-dependent protein kinase 57,69 . We havecharacterized an MAP2 sequence phosphorylated in vivo

w xin rat brain 53 that can be phosphorylated by MAPKs andGSK3 and dephosphorylated by PP1 and PP2A, in vitrow x54 . This sequence is located at a proline-rich region,close to the tubulin-binding motifs, of MAP2 which havebeen found to be very important in the regulation of the

w xinteraction between MAP2 and tubulin 16 . To follow thephosphorylation state of this MAP2 sequence in vivo, we

Ž .obtained an antibody Ab 305 which recognizes the phos-phorylated sequence.

As indicated above, the microtubule-associated protein,w xMAP1B, is also located in the dendrites 66,67 . MAP1B isw xa major cytoskeletal protein of neurons 6 and it is found

in the dendrites in two forms, unphosphorylated and phos-w xphorylated by casein kinase 2 14,63,64 , an enzyme that

w xhas also been involved in synaptic potentiation 11 . Thephosphorylated and dephosphorylated forms of MAP1B

Žcan be recognized by separate antibodies Ab 125 and Ab.842, respectively .

Since the interaction of MAPs with cytoskeletal pro-teins is regulated by phosphorylation, we have investigatedwhether changes in the phosphorylated state of dendriticcytoskeletal proteins MAP1B and MAP2 result from ma-noeuvres that alter the activity of NMDA-glutamate recep-tors. Our study was done in rat hippocampal slices incu-bated with NMDA or the selective glutamate-receptor

Ž .antagonist D- " -2-amino-phosphonopentanoic acidŽ .APV and the level of phosphorylation evaluated in situby immunohistochemistry using the specific antibodiesdescribed above. Our results indicate that both NMDA andAPV induce clear changes in the level of phosphorylationof MAP2. These results suggest that modifications of thephosphorylated state of cytoskeletal proteins located in thedendrites may participate in dendritic synaptic plasticity.

2. Materials and methods

2.1. Antibodies

Ž .Antibodies to MAP1B used in this study Fig. 1 in-Ž .clude mouse monoclonal IgM antibody 125 raised against

w xa phosphoepitope on MAP1B 63 and rabbit polyclonalAb 842 which recognizes the phosphorylatable sequence

ŽASTYSYETSD corresponding to residues 2050–2059 of

Fig. 1. Distribution of phosphorylation-sensitive epitopes on MAP1B andMAP2 molecules. Diagrams of the MAP1B and MAP2 molecules accord-

w x w xing to the sequences reported by Noble et al. 45 and Lewis et al. 37 ,respectively. TBD marks the position of the tubulin-binding domain asinferred from transfection studies, and CTR represents the carboxy-termi-nal repeated sequences described to MAP1B. MAP1B: antibodies raisedagainst MAP1B phosphorylation-sensitive sequences included mouse

Ž .monoclonal IgM Ab 125 which recognizes a CK2-phosphorylated epi-tope on N-terminal half of MAP1B, and rabbit polyclonal Ab 842 whichrecognizes a non-phosphorylated CK2-phosphorylatable sequencesŽ .ASTYSYETSD located inside the CTR of MAP1B whose in vivofunction is still unknown. MAP2: mouse monoclonal antibody 10 reactswith a region located between residues 160 and 1512 of MAP2 in aphosphorylation-independent way. Rabbit polyclonal antibody 305 recog-

) ) Žnizes the phosphorylated sequence RTPGT PGT PSY modified in.vitro by PDPKs and located at the proline-rich region of MAP2, close to

w xits C-terminal domain 54 .

w xMAP1B sequence according to Noble et al. 45 when it isdephosphorylated. These antibodies have been previously

w xcharacterized 63,64 and used to analyze different phos-phorylated isoforms of MAP1B both in primary cultures of

w x w xhippocampal neurons 66 and in brain sections 51 . Hy-bridoma supernatant of Ab 125 was diluted 1:15, whereasAb 842 was used at 1:250 dilution. The same dilutionswere used in Western blot to confirm the antibody speci-ficity and to quantify the phosphorylation changes. The

Ž .mouse monoclonal Ab AA6 Sigma, St. Louis, MO, USAis usually used as the most characterized antibody against

w xphosphorylation-independent epitope on MAP1B 51 , andit has been used as a control antibody to evaluate therelative amount of MAP1B from the different slices andaliquots.

Rabbit polyclonal antibody 305 was raised against aŽphosphorylated peptide w ith the sequence

.RTPGTPGTPSY containing residues 1616–1626 of mousew xMAP2 according to the sequence of Lewis et al. 37,54 . It

was used at a dilution 1:2000 for Western blotting and1:400 for immunocytochemistry. Mouse polyclonal anti-

( )C. Sanchez et al.rBrain Research 765 1997 141–148´ 143

body 10 was raised against the whole MAP2 molecule. Itonly recognizes high molecular weight MAP2 isoformsindependently of their phosphorylation. This antibody wasused at a dilution 1:30 000 for Western blotting and 1:200for immunocytochemistry. A localization scheme of allthese epitopes is shown in Fig. 1.

2.2. Preparation of hippocampal slices

Ž .Adult male Wistar rats 100–200 g were decapitatedunder ketolar anesthesia and the brains placed in a coldŽ .4–58C and oxygenated Krebs–Ringer bicarbonate bufferŽ . Ž .KRB, pH 7.4 , containing in mM : 126 NaCl, 5 KCl, 25NaHCO , 1.5 MgSO , 1.2 NaH PO , 0.5 CaCl , 10 glu-3 4 2 4 2

cose. The brain was quickly cut through the interhemi-spheric fissure and then cut at the base of one hemispherein a plane f108 inclined in relation to the horizontalplane. The hemisphere was then placed on the stage of avibratome where 300-mm-thick slices were selected per-pendicular to the main axis of the hippocampal gyrus.Slices were incubated at room temperature in the sameKRB solution for 60 min, and then the temperature wasraised to 348C for 30 min, with constant bubbling of 95%O and 5% CO .2 2

The effects of NMDA or APV were tested by immer-sion of slices in Krebs–Ringer buffer KRB at 348C con-taining 100 mM NMDA or 100 mM APV for 1 min.

2.3. Protein purification from hippocampal slices

Slices were quickly resuspended and frozen in 50 mMMES, pH 6.4, 100 mM b-glycerophosphate, 5 mM EGTA,0.25 mM MgCl , 50 mM NaF, 1 mM sodium orthovana-2

date, 1 mM ATP, 1 mM phenylmethylsulfonyl fluorideŽ .PMSF , 20 mgrml leupeptin and 20 mgrml aprotinin.Slices were homogenized with a Teflon–glass homoge-nizer at 0–48C. The homogenates were then boiled for 5min in the presence of loading buffer and centrifuged at20 000=g for 10 min. Proteins were analyzed on 5%

w xSDS–PAGE according to Laemmli 36 and subsequentlytransferred to a nitrocellulose sheet as described by Tow-

w xbin et al. 60 . Bound antibodies were visualized with theŽ .ECL Amersham kit and exposure to Kodak X-Omat

films. Autoradiograms were quantified by densitometry ona Molecular Dynamics densitometer model 300, equippedwith Image Quant software version 3.0.

2.4. Immunocytochemistry on tissue slices

Slices were fixed in 4% paraformaldehyde in 50 mMPipes, pH 6.8, 5 mM MgCl , 2 mM EGTA for 12 h. They2

were embedded gradually in 10, 20 and 30% sucrose inŽphosphate-buffered saline PBS, 20 mM sodium phos-.phate, pH 7.4, 140 mM NaCl for 48 h and then included

Ž .in O.C.T. Tissue-Tek, Elkhart, USA compound for cryo-stat sectioning.

Sections of 10 mm were transferred onto gelatin-coatedslides and rinsed in PBS. Immunocytochemistry was per-formed using the peroxidase method, with diaminobenzi-

Ž .dine DAB as chromogen. Endogenous peroxidase wasinactivated by incubating sections in a solution of 0.03%hydrogen peroxidase in PBS for 10 min. Slices wereincubated in 1% calf fetal serum in PBS with 0.1% TritonX-100 for 1 h, followed by the incubation with primary

Ž .antiserum diluted in the above-mentioned buffer overnightat 48C. The slices were rinsed briefly with PBS and

Žincubated with the secondary antibody peroxidase-con-.jugated for 1 h. To minimize the non-specific staining, a

preabsorption of secondary antibodies was performed byincubating them for 30 min at room temperature withacetone powder obtained from a rat brain cell extract.Slices were rinsed several times with PBS and finally with100 mM Tris-HCl, pH 7.6. The color reaction was devel-oped with 0.5 mgrml DAB and 0.06% H O in 100 mM2 2

Tris-HCl, pH 7.6, 50 mM NaCl, 50 mM MgCl . The2

reaction was stopped with 0.1% sodium azide. Slices werethen dehydrated through graded ethanol and xylene andcoverslipped.

To perform double immunofluorescence analysis, slicesŽ .previously transferred onto gelatin-coated slides wereincubated with 1 mgrml sodium borohydride in PBS with

Ž .0.1% Triton X-100 PBS-TX for 5 min and blocked for 1h with 1% calf fetal serum in PBS-TX. Primary antibodieswere diluted in 3% BSA in PBS-TX. Slices were rinsedseveral times with PBS-TX and incubated for 1 h withTexas Red or fluorescein-conjugated secondary antibodiesŽ .previously preabsorbed, see above . They were rinsedwith PBS-TX and coverslipped in fluoromount. Sampleswere examined in a Zeiss epifluorescence microscope andphotographs of specific fields were taken by automaticmode exposure.

3. Results

We have studied the MAP1B and MAP2 phosphoryla-tion after NMDA-glutamate receptor activation or block-ade in hippocampal slices. The phosphorylation was ana-lyzed in situ by immunohistochemistry of hippocampalslices and quantified by Western blot using antibodiesraised against phosphorylation-sensitive epitopes on both

Ž . w xMAPs Fig. 1 54,63 .

3.1. MAP1B phosphorylation after NMDA-glutamate re-ceptor actiÕation or blockade

Although MAP1B expression decreases to low levels inadult brain, prominent expression persists in brain areas

w xwith unusually high regenerative activity 68 , suggesting arole in neurogenesis and neurite plasticity. Since phospho-rylation is the main mechanism that modulates the interac-tion of MAPs with other cytoskeletal proteins, we havefocused on MAPs phosphorylation.

( )C. Sanchez et al.rBrain Research 765 1997 141–148´144

Fig. 2. Analysis of MAP1B phosphorylation at 125 and 842 epitopes afterNMDA or APV treatment. Aliquots with the same amount of MAP1BŽ .corrected by immunoreactivity with Ab AA6 from control hippocampal

Ž . Ž . Ž .slices C or treated with NMDA N or APV A were analysed byŽ . Ž .Western blot with Ab 125 panel A or Ab 842 panel B . These

antibodies recognize different phosphorylated isoforms of MAP1B lo-w xcated in dendrites both in cultured hippocampal neurons 66 and cat

w xbrain sections 51 .

Among the different phosphorylation sites described inthe MAP1B molecule, epitopes 125 and 842 are present in

w xadulthood 63 and have been localized in the dendritesw xboth in cultured hippocampal neurons 66 as well as in

w xbrain sections 51 . The phosphorylation state of thesesequences can be analyzed in situ by using antibodiesagainst these two epitopes. Ab 125 recognizes a CK2-phosphorylated epitope that may be located near the tubu-lin-binding domain in the N-terminal half of MAP1B. Incontrast, Ab 842 recognizes a CK2-phosphorylatable se-quence only when it is dephosphorylated. In this regard, asynthetic peptide with this sequence is phosphorylated byCK2 in vitro and the phosphorylation is abolished by the

w xantibody 63,65 . The sequence recognized by Ab 842 isŽlocated at position 2050–2059 of MAP1B according to

w x.the sequence of Noble et al. 45 , just inside the character-Ž . Ž .istic carboxy-terminal repeated sequences CTR Fig. 1 .

The MAP1B phosphorylation was studied by Western-blotof control, NMDA or APV-treated hippocampal slicesŽ .Fig. 2 . Although no drastic changes in the levels ofphosphorylation were observed, the quantification of theautoradiograms by densitometry indicates that NMDA orAPV treatments modified the basal levels by 91"7%Ž . Ž .NMDA and 115"7% APV in the reaction with Ab

Ž . Ž .125 and 125"9% NMDA and 71"8% APV in theŽ .reaction with Ab 842 mean"S.D., ns5 . Therefore, it

seems that NMDA treatment induces only a slight dephos-phorylation of both sequences of MAP1B, whereas theblockade of NMDA receptors with APV causes the oppo-site, an increase of the phosphorylation state in the samesequences. The fact that the APV-induced changes arelarger than those produced by NMDA, is compatible withsome NMDA endogenous activity present in control slices.However these are slight and probably not significantchanges.

The immunohistochemistry analysis done in thin hip-pocampal slices reveals that the slight changes induced by

NMDA or APV on MAP1B occurs in the CA1 pyramidalcells and some minor modifications are observed in gran-

Ž .ule cells of the dentate gyrus data not shown . Bothantibodies react with the somas and dendrites of granulecells from dentate gyrus. In accordance with Westernblots, no main variations are found with Ab 125 after drugtreatment, while some changes are observed with Ab 842where NMDA treatment increases dendrite staining incontrast to APV treatment which causes a decreased reac-tion in dendrites. Similar results can be observed in pyra-midal CA1 region.

3.2. Alterations in the phosphorylation state of MAP2 afterNMDA-glutamate receptor actiÕation or blockade

MAP2 phosphorylation was more sensitive than MAP1Bto both NMDA and APV treatment. In this set of experi-ments, we first tested the reaction of the antibodies 305and 10 in untreated hippocampal slices. Fig. 3A illustratesthat CA1 pyramidal cells dendrites are immunostainedwith both antibodies, 10 and 305, raised against MAP2.Western blot analyses of homogenates obtained from hip-pocampal slices, untreated or treated with NMDA or APV,

Ž .were done Fig. 3B . Staining with Ab 10 shows thatŽsimilar amounts of MAP2 were loaded in the gel Fig. 3B,

.left . NMDA treatment induced a marked decrease in theŽ .reaction of hippocampal MAP2 dephosphorylation with

antibody 305. On the other hand, a dramatic increase in the

Fig. 3. The MAP2 sequence recognized by the antibody 305, in neuronalcell bodies and dendrites, is dephosphorylated after NMDA treatment ofhippocampal slices. A: immunocytochemistry of the CA1 region ofhippocampal slices with MAP2 antibodies was developed by peroxidase

Ž .reaction see Section 2: Materials and methods . Both antibodies recog-nize MAP2 present in cell bodies and dendrites of pyramidal neurons.Scale bars, 35 mm. B: Western blots of samples obtained from hippocam-

Ž . Ž . Ž .pal slices untreated C or treated with NMDA N or APV APV areŽ .shown see also Section 2 . The phosphorylation state of the sequence

recognized by antibody 305 decrease after treatment with NMDA. Thelevel of phosphorylation after treatment with APV increases even overthat of control hippocampal slices.

( )C. Sanchez et al.rBrain Research 765 1997 141–148´ 145

Table 1Dephosphorylation of the MAP2 sequence recognized by antibody 305after NMDA treatment of hippocampal slices

Antibody 10 Antibody 305

Control 1 1NMDA 0.61"0.2 0.06"0.04APV 0.72"0.2 25"2

These data corresponds to the mean values and standard deviations offive different experiments after densitometry of the autoradiograms shownin Fig. 5B. The values were obtained in arbitrary units and referred to

Ž .those obtained for controls considered as 1 . Whereas no main differ-ences were observed in the reaction of the antibody 10, dramatic changeswere detected in the reaction of the antibody 305 after NMDA or APVtreatments.

Žreaction of hippocampal MAP2 with Ab 305 phosphoryla-.tion was found after incubation of the slices with APV

Ž .Fig. 3B, right . Quantification of the antibody reactionwas done by densitometry of the autoradiograms. Valuesobtained are shown in Table 1. The phosphorylation state

Fig. 4. Dephosphorylation of MAP2 after NMDA treatment in dentateŽ .gyrus. Hippocampal slices were untreated CONTROL or treated with

NMDA or APV. Double immunofluorescence with antibodies 10 and 305Ž .of dentate gyrus are shown see also Section 2: Materials and methods .

The reaction of the antibody 305 is lost in granule cells after treatmentwith NMDA and it is observed after treatment with APV. Scale bar, 30mm. Times of exposure differed in each case.

Fig. 5. Dephosphorylation of MAP2 after NMDA treatment in CA1.Double immunofluorescence with MAP2 antibodies of hippocampal slices

Ž .untreated CONTROL and treated with NMDA or APV were obtained.In this case, CA1 regions are shown. No reaction with antibody 305 wasfound in pyramidal neurons after treatment with NMDA. The reactionwas again observed after treatment with APV. Scale bar, 30 mm. Timesof exposure differed in each case.

of the MAP2 sequence recognized by the antibody 305decreased about 10 times upon NMDA treatment, whereasan increase of nearly 50 times was observed in the pres-ence of APV. The result with antibody 10, which recog-nizes MAP2 independently of its phosphorylation state,shows a decrease around 40"20% in the MAP2 isoformwith a slower electrophoretic mobility in the presence ofNMDA and a decrease around 30"20% in the presenceof APV. Since no protein fragments were observed in

Žimmunoblots, we think that this slight probably not signif-.icant decrease of MAP2 is not due to proteolysis. Further-

more, similar changes in the MAP2 electrophoretic mobil-ity have been previously observed after MAP2 phosphory-

Žlation by proline-directed protein kinases mitogen-activated protein kinases, glycogen-synthase kinase 3, cy-

.clin-dependent kinases and it has been shown that thesequence recognized by the antibody 305 can be phospho-

w xrylated in vitro by proline-directed protein kinases 54 ,NMDA may be affecting the level of MAP2 phosphoryla-tion by those kinases.

( )C. Sanchez et al.rBrain Research 765 1997 141–148´146

Similar results were obtained by double immunofluo-rescence when the antibodies 10 and 305 were tested on

Ž . Ž .dentate gyrus Fig. 4 and CA1 Fig. 5 regions of hip-pocampal slices. Fig. 5 clearly shows that the reaction ofthe antibody 305 in granule cell dendrites was lost aftertreatment of hippocampal slices with NMDA, but it wasincreased after treatment with APV. No main changeswere observed when antibody 10 was tested. Since pyrami-dal cells have more visible dendrites than granule cells,clearer effects of NMDA and APV were observed in the

Ž . Ž .CA1 region Fig. 5 than in granule cells see Fig. 4 . Analmost complete absence of immunostaining with antibody305 was observed in pyramidal cell dendrites after treat-ment with NMDA. However, a marked reaction of theantibody with phospho-MAP2 was detected after treatmentwith APV.

4. Discussion

The interaction of glutamate with its receptors, andparticularly with the calcium permeable subtype selec-tively activated by NMDA, has been proposed to promoteseveral morphological changes at the postsynaptic regioninduced by calcium-dependent or independent enzymesw x5,38 . These modifications may be regulated by the den-dritic cytoskeleton. In this work, we have used an im-munocytochemical approach to test if two dendritic micro-tubule-associated proteins, MAP2 and MAP1B, are modi-fied in situ after activation of the NMDA subtype ofglutamate receptors, which is essential for most forms ofsynaptic potentiation. We have used two antibodies whichrecognize dendritic MAP2 and MAP1B sequences in theirphosphorylated states, which have been previously charac-

w xterized in vitro 53,54,63,64 , to follow modifications inthe phosphorylation of these specific MAP sequences afteractivation or blockade of NMDA receptors. The reactionof the antibodies was tested in dentate gyrus and CA1neurons, since these cells are known to contain a largenumber of NMDA receptors which have been correlatedwith morphological changes that may occur at the post-synaptic region during processes of synaptic potentiation

w xand plasticity 5,38 .NMDA treatment induced only slight and possibly in-

significant changes in the level of phosphorylation ofMAP1B. However, a dramatic decrease in the MAP2phosphorylation level at a specific proline-rich sequencewas observed in the presence of NMDA. A close relation-ship has recently been found between activation of aNMDA subtype of glutamate receptors and changes in

w xMAP2 expression and phosphorylation state 32,43,47,54 .Furthermore, reorganization of the neuronal cytoskeletonoccurs after activation of glutamate receptors, possibly

w xacting through MAP2 4 . Previous studies have indicatedthat NMDA stimulates the overall dephosphorylation of

w xthe dendritic MAP2 43 and only 10–25% of the MAP2

w xphosphorylation sites remain modified 13,24 . Quinlanw xand Halpain 47,48 have shown that glutamate produces a

rapid transient increase in whole MAP2 phosphorylation,possibly mediated by activation of calciumrcalmodulin-dependent protein kinases, protein kinase C and mitogen-activated protein kinases through activation of metabotropicreceptors, followed by a persistent whole MAP2 dephos-

Žphorylation after activation of the NMDA receptors possi-.bly mediated by activation of the protein phosphatase 2B

w x13,47 . Furthermore, the regulation of MAP2 phosphoryla-tion by glutamate release can differ in adults and neonatesw x48 . It may be suggested that only some of the MAP2

w xphosphorylated sites 62 can be modified by glutamatetreatment. In this work, it has been shown that a specific

Ž .MAP2 sequence and in a lower proportion, MAP1B ,recognized by the antibody 305, is one of those sequencesthat is dephosphorylated after activation of NMDA recep-tors in hippocampal slices.

In the presence of NMDA, a decrease in the presence ofphospho-MAP2 isoforms, reacting with antibody 305, wasfound in dendrites of neurons from dentate gyrus or CA1hippocampus. Those phosphoisoforms were modified at asequence present in the proline-rich region of MAP2, closeto the tubulin-binding domains, which is phosphorylatedby MAPKs and GSK3 and dephosphorylated by PP1, and

w xto a lesser extent by PP2A, in vitro 54 . The proline-richŽregion of MAP2 and tau another MAP-related protein

.with a similar sequence may be very important in regulat-w xing the interaction MAP2-tubulin 16,23 . Thus, the changes

in the phosphorylation state of the proline-rich region ofMAP2 could lead to modifications of its interaction withmicrotubules, or with other components of the cyto-skeleton, such as microfilaments, present in dendrites.

Since the MAP1B and MAP2 sequences recognized bythe antibodies 125, 842 and 305 are good substrates of PP1and no dephosphorylation was found in vitro with cal-

w xcineurin 54,65 , their dephosphorylation after activation ofNMDA receptors in hippocampal slices may be mediatedby activation of PP1. In the model proposed by Malenkaw x40 , calcium entry into dendritic spines, via NMDA recep-tors, would activate PP2B which could dephosphorylate

Ž .inhibitor 1 I1 . Dephosphorylation of I1 would lead to theactivation of PP1. Additionally, PP1 and PP2A are highly

w xconcentrated in dendrites and dendritic spines 26,46 .Whether the phosphorylation state of the MAP2 sequencestudied could be modified in a different way by activation

Žof other type of glutamate receptors perhaps by the activa-.tion of PDPKs like MAPKs , remains to be elucidated.

w xMAPKs have a high affinity for microtubules 44,50 andw x w xMAP2 59 , colocalize with MAP2 in dendrites 19 and

w xmight be involved in synaptic potentiation 18 . Thus,MAP2 phosphorylation by MAPKs could be regulated byneurotransmitter receptor activity.

The correct regulation of MAP2 phosphorylation statemay be important for synaptic plasticity and lead to synap-tic potentiation. Accordingly, defects in the expression

( )C. Sanchez et al.rBrain Research 765 1997 141–148´ 147

andror phosphorylation of MAP2 may underlie the alter-ation in synaptic connections and contribute to some of the

w xabnormalities described in schizophrenia 2 . Thus, certainpathological neuronal abnormalities may result from thederegulation of MAP2 phosphorylation and synaptic poten-tiation.

In summary, it is proposed that the MAP2 phosphoryla-tion state can be regulated by the NMDA subtype ofglutamate receptors, whereas only small changes are foundin other dendritic cytoskeletal protein like MAP1B. Post-transductional modifications of MAPs can alter their inter-

w xaction with microtubules 30,31 andror other componentsw xof the cytoskeleton 56 present in dendrites and dendritic

spines, yielding changes in the cytoskeleton stability. Thesecytoskeletal alterations may lead to changes in the mor-phology of dendrites which, in turn, could produce func-tional modifications of synaptic transmission.

Acknowledgements

This work was supported by the ‘Comision Interminis-´Ž .terial de Ciencia y Tecnologıa’ CICYT , ‘Direccion Gen-´ ´

Ž .eral de Investigacion Cientıfica y Tecnica’ DGICYT´ ´ ´Spain, E.U. grant, and an institutional grant of Fundacion´Ramon Areces. C.S. was supported by a fellowship of´Comunidad de Madrid.

References

w x1 C. Aoki, P. Siekevitz, Ontogenetic changes in the cyclic adenosine3X-5X-monophosphate-stimulatable phosphorylation of cat visual cor-tex proteins, particularly of microtubule-associated-protein 2Ž .MAP2 : effects of normal and dark rearing and the exposure to

Ž .light, J. Neurosci. 5 1985 2465–2483.w x2 S.E. Arnold, V.M. Lee, R.E. Gur, J.Q. Trojanowski, Abnormal

Žexpression of two microtubule-associated proteins MAP2 and.MAP5 in specific subfields of the hippocampal formation in

Ž .schizophrenia, Proc. Natl. Acad. Sci. USA 88 1991 10850–10854.w x3 B. Berling, H. Wille, B. Roll, E.-M. Mandelkow, C. Garner, E.¨

Mandelkow, Phosphorylation of microtubule-associated proteinsMAP2a,b and MAP2c at Ser136 by proline-directed kinases in vivo

Ž .and in vitro, Eur. J. Cell Biol. 64 1994 120–130.w x4 D. Bigot, A. Matus, S.P. Hunt, Reorganization of the cytoskeleton in

rat neurons following stimulation with excitatory amino acids inŽ .vitro, Eur. J. Neurosci. 3 1991 551–558.

w x5 T.V.P. Bliss, G.L. Collingridge, Synaptic model of memory: long-Ž .term potentiation in the hippocampus, Nature 361 1993 31–39.

w x6 G.S. Bloom, F.C. Luca, R.B. Vallee, Microtubule-associated protein1B: identification of a major component of the neuronal cyto-

Ž .skeleton, Proc. Natl. Acad. Sci. USA 82 1985 5404–5408.w x7 A. Caceres, G.A. Banker, O. Steward, L.I. Binder, M.R. Payne,´

MAP2 is localized in the dendrites of hippocampal neurons whichŽ .develop in culture, Dev. Brain Res. 13 1984 314–321.

w x8 A. Caceres, M.R. Payne, L.I. Binder, O. Steward, Immunocytochem-´ical localization of actin and microtubule-associated protein MAP2

Ž .in dendritic spines, Proc. Natl. Acad. Sci. USA 80 1983 1738–1742.w x9 R.H.S. Calverley, D.G. Jones, Contributions of dendritic spines and

Ž .perforated synapses to synaptic plasticity, Brain Res. Dev. 15 1990215–249.

w x10 F.L.F. Chang, W.T. Greenough, Transient and enduring morphologi-cal correlates of synaptic activity and efficacy change in the rat

Ž .hippocampal slice, Brain Res. 309 1984 34–46.w x11 S. Charriat-Marlangue, S. Otani, C. Creuzet, Y. Ben Ari, J. Loeb,

Rapid activation of hippocampal casein kinase II during long termŽ .potentiation, Proc. Natl. Acad. Sci. USA 88 1991 10232–10236.

w x12 P. De Camilli, P.E. Miller, F. Navone, W.E. Theurkauf, R.B. Vallee,Distribution of microtubule-associated protein 2 in the nervoussystem of the rat studied by immunofluorescence, Neuroscience 11Ž .1984 817–846.

w x13 J. Dıaz Nido, R.J. Montoro, J.L. Lopez Barneo, J. Avila, High´ ´external potassium induces an increase in the phosphorylation of thecytoskeletal protein MAP2 in rat hippocampal slices, Eur. J. Neu-

Ž .rosci. 5 1993 818–824.w x14 J. Dıaz Nido, L. Serrano, E. Mendez, J. Avila, A casein kinase´ ´

II-related activity is involved in phosphorylation of microtubule-as-sociated protein MAP1B during neuroblastoma cell differentiation,

Ž .J. Cell Biol. 106 1988 2057–2060.w x15 F.A. Edwards, LTP a structural model to explain the inconsistencies,

Ž .Trends Neurosci. 18 1995 250–255.w x16 J. Ferralli, T. Doll, A. Matus, Sequence analysis of MAP2 function

Ž .in living cells, J. Cell Sci. 107 1994 3115–3125.w x17 E. Fifkova, M. Morales, Actin matrix of dendritic spines, synaptic

Ž .plasticity, and long-term potentiation, Int. Rev. Cytol. 139 1992267–307.

w x 2q18 S. Finkbeiner, M. Greenberg, Ca -dependent routes to Ras: mecha-nisms for neuronal survival, differenciation, and plasticity?, Neuron

Ž .16 1996 233–236.w x19 R.S. Fiore, V.E. Bayer, S.L. Pelech, J.M. Baraban, p42 mitogen-

activated protein kinase in brain: prominent localization in neuronalŽ .cell bodies and dendrites, Neuroscience 55 1993 463–472.

w x20 K. Fox, N.W. Daw, Do NMDA receptors have a critical function inŽ .visual cortical plasticity, Trends Neurosci. 55 1993 463–472.

w x21 J. Goldenring, M. Vallano, R. DeLorenzo, Phosphorylation of micro-tubule-associated protein 2 at distinct sites by calmodulin-dependent

Ž .and cyclic-AMP-dependent kinases, J. Neurochem. 45 1985 900–905.

w x22 W.T. Greenough, C.H. Bailey, The anatomy of a memory: conver-gence of results across a diversity of tests, Trends Neurosci. 11Ž .1988 142–146.

w x23 N. Gustke, B. Trinazek, J. Biernat, E.M. Mandelkow, E. Man-delkow, Domain of protein t and interactions with microtubules,

Ž .Biochemistry 33 1994 9511–9522.w x24 S. Halpain, P. Greengard, Activation of NMDA receptors induces

rapid dephosphorylation of the cytoskeletal protein MAP2, Neuron 5Ž .1990 237–246.

w x 2q25 P.I. Hanson, H. Schulman, Neuronal Ca rcalmodulin-dependentŽ .protein kinases, Annu. Rev. Biochem. 61 1992 559–601.

w x26 T. Hashikawa, K. Nakazawa, S. Mikawa, H. Shima, M. Nagao,Immunohistochemical localization of protein phosphatase isoforms

Ž .in the rat cerebellum, Neurosci. Res. 22 1995 133–136.w x27 M. Hoshi, T. Akiyama, Y. Shinohara, Y. Hiyata, H. Ogawara, E.

Nishida, H. Sakai, Protein kinase C-catalyzed phosphorylation of themicrotubule-binding domain of MAP2 inhibits its ability to induce

Ž .tubulin polymerization, Eur. J. Biochem. 174 1988 225–230.w x28 M. Hoshi, K. Ohta, Y. Gotoh, A. Mori, H. Murofushi, H. Sakai, E.

Nishida, Mitogen-activated-protein-kinase-catalyzed phosphorylationof microtubule-associated proteins, microtubule-associated protein 2and microtubule-associated protein 4, induces an alteration in their

Ž .function, Eur. J. Biochem. 203 1992 43–52.w x29 G. Huber, A. Matus, Differences in the cellular distributions of two

microtubule-associated proteins, MAP1 and MAP2, in rat brain, J.Ž .Neurosci. 4 1984 151–160.

w x30 L. Jameson, T. Frey, B. Zeeberg, F. Dalldorf, M. Caplow, Inhibitionof microtubule assembly by phosphorylation of microtubule-associ-

Ž .ated proteins, Biochemistry 19 1980 2472–2479.w x31 G.V.W. Johnson, R.S. Jope, The role of microtubule-associated

( )C. Sanchez et al.rBrain Research 765 1997 141–148´148

Ž .protein 2 MAP-2 in neuronal growth, plasticity, and degeneration,Ž .J. Neurosci. Res. 33 1992 505–512.

w x32 H.M. Johnston, B.J. Morris, NMDA and nitric oxide increase micro-tubule-associated protein 2 gene expression in hippocampal granule

Ž .cells, J. Neurochem. 63 1994 379–382.w x33 P.T. Kelly, T.L. MacGuiness, P. Greengard, Evidence that the major

postsynaptic density protein is a component of a Ca2qrcalmodulin-Ž .dependent protein kinase, Proc. Natl. Acad. Sci. USA 81 1984

945–949.w x34 M.B. Kennedy, M.K. Benner, N.E. Erondu, Biochemical and im-

munochemical evidence that the ‘major postsynaptic density protein’is a subunit of a calmodulin-dependent protein kinase, Proc. Natl.

Ž .Acad. Sci. USA 80 1983 7357–7361.w x35 K. Kuba, E. Kumamoto, Long term potentiations in vertebrate

synapses, a variety of cascades with common subprocesses, Prog.Ž .Neurobiol. 34 1990 197–269.

w x36 U.K. Laemli, Cleavage of structural proteins during the assembly ofŽ .the head of bacteriophage T4, Nature 227 1970 680–685.

w x37 S.A. Lewis, D. Wang, N.J. Cowan, Microtubule-associated proteinMAP2 shares a microtubule-binding motif with tau protein, Science

Ž .242 1988 936–939.w x38 G.S. Lynch, M. Baudry, The biochemistry of memory: a new and

Ž .specific hypothesis, Science 224 1984 1057–1063.w x39 R.C. Malenka, J.A. Kauer, D.J. Perkel, M.D. Mauk, P.T. Kelly,

R.A. Nicoll, M.N. Waxham, An essential role for postynapticcalmodulin and protein kinase activity in long-term potentiation,

Ž .Nature 340 1989 554–557.w x40 R.C. Malenka, Synaptic plasticity in the hippocampus: LTP and

Ž .LTD, Cell 78 1994 535–538.w x41 R.C. Malenka, R.A. Nicoll, NMDA-receptor-dependent synaptic

plasticity: multiple forms and mechanisms, Trends Neurosci. 16Ž .1993 521–527.

w x Ž .42 A. Matus, Microtubule associated proteins, Ann. Neurosci. 11 198929–44.

w x43 R.J. Montoro, J. Dıaz Nido, J. Avila, J. Lopez Barneo, N-Methyl-D-´ ´aspartate stimulates the dephosphorylation of the microtubule-associ-ated protein 2 and potentiates excitatory synaptic pathways in the rat

Ž .hippocampus, Neuroscience 54 1993 858–871.w x44 M. Morishima-Kawashima, K.S. Kosik, The pool of MAP kinase

associated with microtubules is small but constitutively active, Mol.Ž .Biol. Cell 6 1996 893–905.

w x45 M. Noble, S.A. Lewis, N.J. Cowan, The microtubule binding do-main of microtubule associated protein MAP1B contains a repeatedsequence motif unrelated to that of MAP2 and tau, J. Cell Biol. 109Ž .1989 3367–3376.

w x46 C. Ouimet, E. Da Cruz e Silva, P. Greengard, The a and g1isoforms of protein phosphatase 1 are highly and specifically con-

Ž .centrated in dendritic spines, Proc. Natl. Acad. Sci. USA 92 19953396–3400.

w x47 E. Quinlan, S. Halpain, Postsynaptic Mechanisms for bidirectionalcontrol of MAP2 phosphorylation by glutamate receptors, Neuron 16Ž .1996 357–368.

w x48 E. Quinlan, S. Halpain, Emergence of activity-dependent bidirec-tional control of microtubule-associated protein MAP2 phosphoryla-

Ž .tion during postnatal development, J. Neurosci. 16 1996 7627–7637.

w x49 M. Recasens, Putative molecular mechanisms underlying long termŽ .potentiation LTP : the key role of exitatory amino acid receptors,Ž .Therapie 50 1995 19–34.

w x50 A. Reszka, R. Seger, C. Diltz, E. Krebs, E. Fischer, Association ofmitogen-activated protein kinase with the microtubule cytoskeleton,

Ž .Proc. Natl. Acad. Sci. USA 92 1995 8881–8885.w x51 B. Riederer, Differential phosphorylation of MAP1B during postna-

Ž .tal development of the cat brain, J. Neurocytol. 24 1995 45–54.w x52 T.C. Sacktov, P. Osten, H. Valsimis, X. Jiang, M.L. Naik, E.

Sublette, Persistent activation of the z isoform of protein kinase C inthe maintenance of long term potentiation, Proc. Natl. Acad. Sci.

Ž .USA 90 1993 8342–8348.w x53 C. Sanchez, J. Dıaz-Nido, J. Avila, Variation in in vivo phosphoryla-´ ´

tion at the proline rich domain of the microtubule associated proteinŽ . Ž .2 MAP2 during rat brain development, Biochem. J. 306 1995

481–482.w x54 C. Sanchez, P. Tompa, K. Szucs, P. Friedrich, J. Avila, Phosphoryla-´ ¨

tion and dephosphorylation in the proline-rich C-terminal domain ofŽ .microtubule-associated protein 2 MAP2 , Eur. J. Biochem. 241

Ž .1996 765–771.w x55 H. Schulman, Differential polymerization of MAP2 stimulated by

Ž .calcium-calmodulin and cyclic AMP, Mol. Cell. Biol. 4 19841175–1178.

w x56 S. Selden, T.D. Pollard, Phosphorylation of microtubule-associatedprotein regulates their interaction with actin filaments, J. Biol.

Ž .Chem. 258 1983 7064–7071.w x57 R.D. Sloboda, S.A. Rudolph, J.L. Rosenbaum, P. Greengard, Cyclic

AMP-dependent endogenous phosphorylation of a microtubule asso-Ž .ciated protein, Proc. Natl. Acad. Sci. USA 72 1975 177–181.

w x58 T.J. Teyler, Y. Cavus, C. Coussens, P. DiScenna, L. Grover, Y.P.Lee, Z. Little, Multideterminant role of calcium in hippocampal

Ž .plasticity, Hippocampus 4 1994 623–634.w x59 K.L. Thomas, S.P. Hunt, The regional distribution of extracellularly

regulated kinase-1 and -2 messenger RNA in the adult rat centralŽ .nervous system, Neuroscience 56 1993 741–757.

w x60 H. Towbin, T. Staehelin, J. Gordon, Electrophoretic transfer ofprotein from polyacrylamide gels to nitrocellulose sheets: procedure

Ž .and some application, Proc. Natl. Acad. Sci. USA 76 1979 4350–4354.

w x61 S. Tsuyama, G.T. Bramblett, K.P. Huang, M. Flavin,Calciumrphospholipid-dependent protein kinase recognizes sites inmicrotubule-associated protein 2 which are phosphorylated in livingbrain and are not accesible to other kinases, J. Biol. Chem. 261Ž .1986 4110–4116.

w x62 S. Tsuyama, Y. Terayama, S. Matsuyama, Numerous phosphates ofmicrotubule-associated protein 2 in living rat brain, J. Biol. Chem.

Ž .262 1987 10886–10892.w x63 L. Ulloa, J. Avila, J. Dıaz-Nido, Heterogeneity in the phosphoryla-´

tion of microtubule-associated protein MAP1B during rat brainŽ .development, J. Neurochem. 61 1993 961–972.

w x64 L. Ulloa, J. Dıaz-Nido, J. Avila, Depletion of casein kinase II by´antisense olibonucleotide prevents neuritogenesis in neuroblastoma

Ž .cells, EMBO J. 12 1993 1633–1640.w x65 L. Ulloa, V. Dombrad, J. Dıaz-Nido, K. Szucks, P. Gergerly, P.´

Friedrich, J. Avila, Dephosphorylation of distinct sits on microtubuleassociated protein MAP1 by protein phosphatases 1, 2A and 2B,

Ž .FEBS Lett. 330 1993 85–89.w x66 L. Ulloa, J. Dıez-Guerra, J. Avila, J. Dıaz-Nido, Localization of´ ´

differentially phosphorylated isoforms of microtubule associatedprotein 1B in cultured rat hippocampal neurons, Neuroscience 61Ž .1994 211–223.

w x67 R.B. Vallee, G.S. Bloom, F.C. Luca, Differential structure anddistribution of the high molecular weight brain microtubule associ-ated proteins MAP1 and MAP2, Ann. New York Acad. Sci. 466Ž .1986 134–144.

w x68 C. Viereck, R.P. Tucker, A. Matus, The adult rat olfactory systemexpresses microtubule-associated proteins found in the developing

Ž .brain, J. Neurosci. 9 1989 3547–3557.w x69 S.I. Walaas, A.C. Nairn, Multisite phosphorylation of microtubule-

Ž .associated protein 2 MAP2 in rat brain: peptide mapping distin-guishes between cyclic AMP, calciumrcalmodulin-, andcalciumrphospholipid-regulated phosphorylation mechanisms, J.

Ž .Mol. Neurosci. 1 1989 117–127.