doi:10.1006/mcne.2002.1104

Molecular and Cellular Neuroscience 20, 330–342 (2002)MCN

Role of Heparin-Binding Growth-AssociatedMolecule (HB-GAM) in Hippocampal LTP andSpatial Learning Revealed by Studies onOverexpressing and Knockout Mice

Ivan Pavlov,* ,†,1 Vootele Voikar,* ,1 Marko Kaksonen,*Sari E. Lauri,* ,†,2 Anni Hienola,* Tomi Taira,* ,†

and Heikki Rauvala* ,3

*Laboratory of Molecular Neurobiology, Institute of Biotechnology and Department ofBiosciences, P.O. Box 56, 00014 University of Helsinki, Finland; and †Division of AnimalPhysiology, Department of Biosciences, P.O. Box 17, 00014 University of Helsinki, Finland

Heparin-binding growth-associated molecule (HB-GAM)is an extracellular matrix-associated protein with neuriteoutgrowth-promoting activity and which is suggested tobe implicated in hippocampal synaptic plasticity. To studythe functions of HB-GAM in adult brain we have producedHB-GAM overexpressing mice and compared phenotypicchanges in the transgenic mice to those in the HB-GAMnull mice. Both mutants were viable and displayed nogross morphological abnormalities. The basal synaptictransmission was normal in the area CA1 of hippocampalslices from the genetically modified mice. However, long-term potentiation (LTP) was attenuated in the mice over-expressing HB-GAM, whereas enhanced LTP was de-tected in the HB-GAM-deficient mice. Changes in LTPseen in vitro were paralleled by behavioral alterations invivo. The animals overexpressing HB-GAM displayedfaster learning in water maze and decreased anxiety inelevated plus-maze, while the HB-GAM knockouts dem-onstrated an opposite behavioral phenotype. These re-sults show that HB-GAM suppresses LTP in hippocampusand plays a role in regulation of learning-related behavior.

INTRODUCTION

Although the role of cell-to-cell adhesion moleculesin hippocampal plasticity and memory is well-recog-

1 These authors contributed equally to this work.2 Current address: Department of Anatomy, School of Medical

Sciences, University of Bristol, University walk, Bristol BS8 1TD, UK.

To whom correspondence and reprint requests should be ad-

dressed. Fax: �358 9 191 59068. E-mail: heikki.rauvala@helsinki.fi.

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nized (Schachner, 1997; Cotman et al., 1998; Murase andSchuman, 1999; Benson et al., 2000), surprisingly little isknown about cell-matrix interactions in control of syn-aptic function. Only few components of extracellularmatrix have been shown to contribute to neuronal plas-ticity in hippocampus (Nakic et al., 1998; Lauri et al.,1999; Saghatelyan et al., 2000, 2001).

Recent experiments suggest that the extracellularmatrix-associated protein called heparin-bindinggrowth-associated molecule (HB-GAM), also known aspleiotrophin (Li et al., 1990), is involved in the regula-tion of hippocampal long-term potentiation (LTP). HB-GAM is a secreted, developmentally regulated 18-kDaprotein, which is implicated in the regulation of neuriteoutgrowth, axonal guidance and synaptogenesis in vitro(for review, see Rauvala and Peng, 1997). This moleculebelongs to the family of proteins containing throm-bospondin type 1 repeat (TSR) sequence motifs. Othermembers of the family (e.g., F-spondin, midkine) areinvolved in various specific cell surface interactionssuggesting some common biological functions for pro-teins containing TSR domains (for review, see Adamsand Tucker, 2000; Kilpelainen et al., 2000). HB-GAMexpression is most prominent at embryonic and perina-tal stages of development (Rauvala, 1989) though it alsopersists into adulthood in some neuronal populations.In the adult brain HB-GAM expression is restricted tocertain regions which exhibit pronounced synapticplasticity including hippocampal area CA1 (Wanaka et

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al., 1993). In pyramidal neurons of hippocampus HB- GAM expression is activity-dependent and increases

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after high-frequency stimulation (Lauri et al., 1996). Inaddition, application of purified recombinant HB-GAMeffectively inhibits LTP in area CA1 of hippocampalslices (Lauri et al., 1998).

In order to directly study the role of endogenousHB-GAM in hippocampal functions we have producedmutant mice overexpressing HB-GAM and used knock-out mice lacking HB-GAM (Amet et al., 2001). We haveanalyzed synaptic function and plasticity in hippocam-pal slices from mutant animals in vitro. In parallel,hippocampus-associated forms of behavior were stud-ied to obtain information about hippocampal functionin transgenic and knockout animals in vivo. We dem-onstrate that opposite genetic manipulations producereverse phenotypes as revealed by electrophysiologicaland behavioral studies. The data presented herestrongly support the idea that HB-GAM is involved inthe regulation of hippocampal plasticity and behavior.

RESULTS

Transgenic Mice Overexpressing HB-GAM

HB-GAM transgenic mice were produced using aconstruct in which the coding region of HB-GAM (Me-renmies and Rauvala, 1990) is under the control ofhuman PDGF �-chain promoter (Fig. 1A), which pro-duces preferential expression in neurons (Sasahara etal., 1991). Northern blot analysis of adult hippocampusrevealed expression of the transgene-derived mRNA ofthe expected size in litters from two independentlyderived founders. The transgene was expressed ap-proximately at the same level as the endogenously oc-curring HB-GAM mRNA in both litters (Fig. 1B). Thetwo transgenic lines were similar in terms of the trans-gene expression level and in phenotype analysis. Inagreement with the Northern analysis, the heterozy-gous transgene-positive mice showed about twofoldoverexpression of the HB-GAM protein in the hip-pocampus in Western blots (not shown).

Adult transgenic mice appeared to be healthy anddisplayed no apparent abnormalities in brain morphol-ogy and structure. Brain samples were sectioned incoronal and horizontal directions and stained with stan-dard hematoxylin-eosin method for histological analy-sis. The layered structures of the cerebral cortex andhippocampus of HB-GAM overexpressing mice werenot different from wild-type animals (Fig. 1C). Thedimensions of larger anatomical structures also ap-peared normal. Cell densities were measured morecarefully in cortex and hippocampus but no differences

FIG. 1. Generation of mice overexpressing HB-GAM. (A) Thetransgene construct. (B) Northern blot showing hippocampal ex-pression of HB-GAM in transgene-negative (lane 1) and transgene-positive (lanes 2 and 3) littermates (e.g., endogenous mRNA; tg,mRNA transcribed from the transgene) in three individuals (lanes1–3) from two litters that were progeny of two original founders.The level of expression of the transgene is similar to the level ofendogenous HB-GAM mRNA in both founders. (C) The overallhippocampal structure assessed by hematoxylin-eosin staining ap-pears normal in transgenic mice.

331HB-GAM in Hippocampal LTP and Learning and Memory

between genotypes were revealed. Neuronal density inmotor cortex of HB-GAM overexpressing mice was58042 � 5627 neurons/mm3 (n � 4) and 59297 � 3689neurons/mm3 in wild-type controls (n � 4). To take acloser look at the axonal projections of the mutant brain,we stained coronal sections with Bielschowsky-silverimpregnation method. Thalamical tracts to cortex andhippocampal connections appeared also normal withthis staining (data not shown).

Electrophysiological and behavioral characterizationwas carried out on mice produced from the twofounders in both FVB and hybrid FVB/129Sv geneticbackgrounds. Heterozygous transgene-positive andtheir transgene-negative littermates were used for theanalysis.

Electrophysiological Analysis of MiceOverexpressing HB-GAM

To study basal properties of synaptic transmission inhippocampal slices from HB-GAM overexpressing micewe tested single stimulus-evoked responses in CA1area. Input-output curves showing the ratio of presyn-aptic fiber volley amplitude and the slope of the fEPSPwere indistinguishable in mice overexpressing HB-GAM and wild-type littermate controls (Fig. 2A).Therefore, the baseline synaptic transmission in hip-pocampus seems to be intact in the mutants.

Further, we studied paired-pulse facilitation (PPF)induced by a pair of stimulus of afferent fibers at inter-vals between 20 to 200 ms. PPF is commonly consideredas the measure of presynaptic neurotransmitter releaseprobability (Manabe et al., 1993). No significant differ-ences in the PPF between transgenic mice and controlanimals were detected (Fig. 2B).

We have previously shown that application of recom-binant HB-GAM into stratum radiatum of CA1 area ofhippocampal slice blocks LTP (Lauri et al., 1998). Thus,it was of interest to study whether the enhanced HB-GAM expression in neurons of the transgenic micewould produce a similar effect. Indeed, high-frequencystimulation of Schaffer collaterals produced signifi-cantly lower level of potentiation in slices from miceoverexpressing HB-GAM (Fig. 2C, P � 0.01, Student’st test). The normalized fEPSP slope for the slices fromcontrol animals 1 h after LTP induction was 177 � 10%of the average slope before HFS. In slices obtained fromtransgenic animals the normalized fEPSP slope 1 h afterstimulation was 130 � 5% of the average slope beforetetanus.

LTP in CA1 region requires postsynaptic depolariza-tion to release voltage-dependent Mg2� block from

FIG. 2. Attenuated LTP in mice overexpressing HB-GAM. Datarepresents mean � SEM. (A) Input– output curves showing therelationship between presynaptic fiber volley (PSFV) amplitudeand fEPSP slope in wild-type (WT) and HB-GAM overexpressingtransgenic (TG) mice. The curves for the wild-type and transgenicmice are identical (WT: n � 17 slices/7 animals; TG: n � 19 slices/9animals). (B) PPF in slices from transgenic mice is slightly lower ascompared to that in slices from wild-type controls (TG: n � 19slices/9 animals; WT: n � 17 slices/7 mice). (C) Mice overexpress-ing HB-GAM produce lower level of LTP induced by HFS (100Hz/1 s) (n � 18 slices/9 mice) in comparison to wild-type controls(n � 15 slices/7 mice). Superimposed single fEPSPs from bothgenotypes before and 1 h after LTP induction are shown on theright. (D) The ability to follow HFS stimulation is identical inHB-GAM overexpressing (n � 10 slices/5 mice) and wild-type mice(n � 11 slices/5 mice). Averaged traces of field recordings duringLTP induction are shown superimposed. (E) Normalized fEPSPslopes during HFS stimulation are plotted against the number ofthe stimulus in the train (TG: n � 10 slices/5 mice; WT: n � 11slices/5 mice). (F) Paired-pulse facilitation remains similar intransgenic mice and control animals 60 min after LTP induction(WT: n � 15 slices/7 mice; TG: n � 18 slices/9 mice).

332 Pavlov et al.

NMDA receptor channel complex. Thus, the ability ofsynapses to follow high-frequency stimulation is criticalfor the induction of LTP. We did not find any differencein synaptic responses evoked by 100Hz stimulation be-tween HB-GAM overexpressing mice and wild-typecontrols. The slow NMDA receptor-mediated compo-nents of field recordings during LTP induction werealso indistinguishable in both experimental groups(Figs. 2D and 2E). In addition, no changes were re-vealed between the genotypes in PPF measured 1 hafter LTP induction (Fig. 2F).

Behavioral Performance of HB-GAMOverexpressing Mice

Animals expressing the HB-GAM transgene devel-oped normally and we did not detect any difference intheir basic sensory abilities, motor coordination orspontaneous locomotor activity in comparison to thewild-type mice.

Both the transgenic mice and their wild-type litter-mate controls demonstrated very rapid spatial learning(Fig. 3A) in the water maze although the escape laten-cies did not differ between the genotypes (genotype F1,20 � 1.1, P � 0.30; training block F5, 100 � 32.9, P �0.01; genotype � training block F5, 100 � 0.7, P �0.58). However, significant differences were found inthe first transfer test (Fig. 3B). A repeated-measuresANOVA revealed significant interactions of genotypewith platform crossings (F2, 40 � 5.1, P � 0.01) andswimming time in zones (F2, 40 � 2.9, P � 0.05).Subsequent post hoc Newman–Keuls test confirmedthat the transgenic mice displayed a significant prefer-ence (P � 0.01) to the trained quadrant regarding bothcrossings and swimming time when compared withrespective values for remaining quadrants. In the wild-type group such preferences were not evident (P �0.1).Additionally, post hoc comparison revealed that thetransgenic mice swam longer in the trained zone (P �0.05) as compared with the wild-type mice.

The performance of the wild-type mice improvedwith further training and no differences were estab-lished in the second transfer test with regard to theannulus crossings—both groups crossed preferentially

FIG. 3. Facilitated learning in HB-GAM transgenic mice in the spa-tial water maze study. (A) Mean escape latency across training blocks(TG: open squares, n � 11; WT: filled squares, n � 11). (B–D) Time(percentage) spent inside the zone (6.25% of the water maze totalarea) around the platform and corresponding zones in the other

quadrants of the pool during the transfer tests (TT1, TT2, and TT3,respectively). (E) The percentage of time spent swimming in theoutermost ring of the water maze (30.6% of the maze area)—thigmo-taxis. Black bars represent the values for WT, gray bars for TG mice,dotted line marks the chance level. *P � 0.05 between the groups.

333HB-GAM in Hippocampal LTP and Learning and Memory

(P � 0.01) the annulus at the trained location. How-ever, the analysis of the time in zones (Fig. 3C) revealedsignificant effect of the genotype (F1, 20 � 5.0, P �0.05) and post hoc comparison confirmed that trans-genic mice spent slightly more time (P � 0.05) in thetrained zone than wild-type mice. However, the pref-erence to the trained quadrant as compared with re-maining quadrants was highly significant (P � 0.01) inboth groups.

Both groups learned very rapidly the task when theplatform was removed to the new, previously oppositequadrant of the water maze (Fig. 3A, reversal learning;genotype F1, 20 � 1.9, P � 0.18; training block F1, 20 �43.0, P � 0.01; genotype � training block F1, 20 � 0.2,P � 0.64). In the third transfer test (Fig. 3D) there wasno difference between wild-type and transgenic mice,both groups searched selectively in two quadrantswhere platform had been located during the wholetraining period. The analysis of thigmotaxis (Fig. 3E) byrepeated measures ANOVA revealed genotype � trans-fer test interaction (F2, 40 � 3.0, P � 0.05) and furtherpost hoc comparisons showed that thigmotaxis wassignificantly higher in wild-type mice during the sec-ond transfer test (P � 0.05). We did not see any dif-ference in the swimming distance during the transfertests. Spatial training was followed by cued version ofthe task and there was no difference between groups inescape latencies to the visible platform (data notshown).

In addition, the transgenic mice displayed a tendencytowards decreased anxiety in the elevated plus maze(Fig. 6A). Namely, they made more entries onto theopen arms (F1, 44 � 4.04, P � 0.05), stayed there for alonger time (percentage of time on open arms: F1, 44 �4.89, P � 0.05) and crossed more lines (F1, 44 � 4.7,P � 0.05). However, the number of closed arm entriesas a measure of locomotion in the elevated plus mazedid not differ between groups (F1, 44 � 0.00, P � 1.00).

In the classical fear conditioning paradigm (Fig. 6B),the transgenic mice displayed less freezing to the CStone than control mice (F1, 20 � 6.4, P � 0.05), but thecontext dependent freezing was not different (F1, 20 �2.0, P � 0.17).

Electrophysiological Analysis of HB-GAM-DeficientMice

Recently gene-targeted mice lacking HB-GAM havebeen produced in C57BL/6J�129/Ola hybrid back-ground (Amet et al., 2001). To reduce the possible vari-ation due to genetic background, mice studied in thecurrent work have been bred into the 129S2/SvHsd

strain to generate an inbred line. Like in the HB-GAMknockout mice mentioned above no differences in in-put-output curves were seen in the present work inknockout mice as compared to wild-type controls, sug-gesting normal basic synaptic transmission in the mu-tants (Fig. 4A). The PPF was also indistinguishable inthe slices from HB-GAM deficient and control mice(Fig. 4B).

However, here we found that LTP was enhanced inmice lacking endogenous HB-GAM. The average slopeof fEPSPs 60 min after high-frequency stimulation was171 � 9.5% of the baseline in the knockouts and 141 �5.9% of the baseline in the wild-type controls (Fig. 4C).The ability to follow the tetanic stimulation appeared tobe identical in HB-GAM deficient mice and controls(Figs. 4D and 4E). The LTP level in wild-type animalsused as controls for HB-GAM overexpressing andknockout mice was considerably different. The mostprobable reason for that is a difference in genetic back-ground in animals used to produce transgenic andknockout mice. It is well documented that variousmouse strains vary in their ability to express synapticplasticity (Nguyen et al., 2000a,b). In addition, we didnot find any differences in LTP induced by lower stim-ulation frequency trains (10 Hz/1 s; data not shown).PPF was not affected one hour after HFS either inmutants or wild-type mice (Fig. 4F).

Behavioral Performance of HB-GAM-DeficientMice

All mutant mice were viable and healthy. They didnot display any defects related to the development andgeneral sensory abilities and were indistinguishablefrom wild-type mice in motor coordination, strengthand balance as assessed by rotarod and beam balancing.These findings were consistent with those reported byAmet et al., (2001).

The escape time in the water maze decreased rapidlyin both groups (Fig. 5A). However, in the knockoutgroup it was slightly delayed (block 2) and easily dis-turbed (block 6 after the first free swimming). The re-peated measures ANOVA revealed significant effect oftraining block (F8, 256 � 33.5, P � 0.01) and geno-type � training block interaction (F8, 256 � 2.3, P �0.05). The results of the first transfer test confirmedslightly better memory of wild-type mice (Fig. 5B). Theycrossed more frequently the correct platform location(genotype F1, 32 � 3.7, P � 0.05; number of crossingsF2, 64 � 26.2, P � 0.01; genotype � crossings F2, 64 �5.0, P � 0.01) and searched more preferentially in theplatform area (genotype F1, 32 � 4.3, P � 0.05; zone

334 Pavlov et al.

time F2, 64 � 41.2, P � 0.01; genotype � time F2, 64 �5.2, P � 0.01). However, both groups displayed signif-icant preference to the trained quadrant when com-pared with remaining three quadrants.

There was no difference in the second transfer test(Fig. 5C) performed after the 9th training block either inplatform crossings or time spent in the circle around theplatform. Both groups learned well to find the platformwhen it was removed to the opposite quadrant (Fig. 5A,reversal learning; genotype F1, 32 � 0.2, P � 0.66;training block F4, 128 � 13.6, P � 0.01; genotype �training block F4, 128 � 1.4, P � 0.36), but in thetransfer test the control mice showed a more selectiveswimming strategy (Fig. 5D). There was no differencein the platform crossings (genotype F1, 32 � 1.2, P �0.27, genotype � crossings F2, 64 � 1.5, P � 0.23) ortime in zones (genotype F1, 32 � 2.8, P � 0.1; geno-type � time F2, 64 � 2.3, P � 0.2). The time spent in thenew platform zone did not differ between genotypes.However, post hoc comparison showed that the wildtype mice made significantly more crossings at the oldlocation and spent significantly more time in the previ-ous target zone (P � 0.05) than the knockout mice.

The analysis of thigmotaxis (Fig. 5E) revealed signif-icant genotype � transfer test interaction and post hocNewman–Keuls test confirmed longer time in thigmo-taxis in the first (P � 0.01) and third (P � 0.05)transfer tests in the knockout mice. Again, the swim-ming distances did not differ between the groups in anyof the transfer tests and no difference was found invisible platform testing.

The knockout mice had a higher anxiety-like behav-ior than control mice in the elevated plus maze (Fig.6C). The latency to enter open arm was significantlylonger (F1, 52 � 9.61, P � 0.01), the number of openarm entries (F1, 52 � 4.95, P � 0.05), and the numberof crossed lines (F1, 52 � 5.1, P � 0.05) were lower inknockout mice with no difference in number of closedarm entries (F1, 52 � 2.62, P � 0.11).

Fear conditioning experiment (Fig. 6D) revealedlower context-dependent freezing in knockout mice (F1,49 � 3.9, P � 0.05). The freezing in the presence of theCS tone in novel context was similar in both groups (F1,49 � 0.01, P � 0.9).

DISCUSSION

We studied the role of HB-GAM in hippocampalsynaptic plasticity and learning and memory using mu-tant mice approach. To this end, we characterized trans-genic animals overexpressing HB-GAM and mice withtargeted deletion of the HB-GAM gene.

Previous studies have suggested that HB-GAM is aninducible molecule suppressing synaptic plasticity inthe CA1 region of hippocampus (Lauri et al., 1998; Amet

FIG. 4. HB-GAM deficient mice display an increased level of LTP.(A) Input–output curves are not different in HB-GAM knockout mice(KO: n � 8 slices/4 mice) and wild-type controls (WT: n � 10slices/5 mice). (B) No difference was seen in PPF obtained in slicesfrom KO (n � 8 slices/4 mice) and WT (n � 8 slices/4 mice). (C)Knockout mice (n � 6 slices/6 mice) exhibit a higher level of LTPinduced by 100 Hz train stimulation than wild-type controls (n � 6slices/6 mice). Superimposed single fEPSPs from both genotypestaken before and 1 h after HFS are shown on the right. (D) Pooled datashowing the ability of mice lacking HB-GAM (n � 6 slices/6 animals)and wild-type controls (n � 6 slices/6 mice) to follow HFS. Averagedtraces from knockout and wild-type mice are superimposed. (E) Thepercentage change in the slope of fEPSPs during HFS from the 1stfEPSP in the train is similar in both genotypes (WT: n � 6 slices/6mice; KO: n � 6 slices/6 mice). (F) PPF remains unchanged 1 h afterLTP induction both in HB-GAM deficient (n � 4 slices/4 mice) andwild-type animals (n � 4 slices/4 mice).

335HB-GAM in Hippocampal LTP and Learning and Memory

et al., 2001). Developmentally regulated expression ofHB-GAM in rat brain peaks during the early postnatalperiod, while in adults HB-GAM is expressed in hip-pocampal pyramidal neurons in an activity dependentmanner. In addition, it was shown that exogenous re-combinant HB-GAM applied by pressure injection intoCA1 region of hippocampal slice precludes the induc-tion of tetanus-induced LTP (Lauri et al., 1998). Never-theless, it was not clear whether this effect is merely dueto the inhibition of the function of endogenous proteinexpressed in the pyramidal neurons or is the result ofspecific inhibitory effect on LTP itself. These two pos-sibilities could imply opposite explanations for the roleof HB-GAM in regulation of synaptic plasticity. An-other caveat in experiments utilizing injection tech-nique is the difficulty to control the amount of proteinin the tissue at a recording site. Besides, injection per sedamages the tissue and is expected to activate path-ways involved in regulation of neuronal repair andplasticity.

Here we took an advantage of using transgenic micedisplaying about twofold increase in HB-GAM expres-sion level, which is within physiological range. Approx-imately the same level of increase in HB-GAM expres-sion in CA1 area of hippocampus is seen after HFS ofSchaffer collaterals in slices (Lauri et al., 1996). It wasfound here that mice with enhanced expression of HB-GAM show decreased LTP in area CA1. These datastrongly suggest that increased level of HB-GAM in thehippocampal tissue can restrict the ability of pyramidalneurons to undergo plastic changes in response to HFS.It is tempting to speculate that in vivo HB-GAM acts asa local extracellular signal necessary for maintainingnetwork stability in synaptic circuitry and preventingpossible adverse effects of intensive afferent activation.Consistent with this hypothesis HB-GAM expressionhas been shown to be induced by stimuli causing neu-ronal injury or seizures (Wanaka et al., 1993; Takeda etal., 1995).

Further, electrophysiological studies of mice lackingHB-GAM revealed an opposite phenotype as comparedto the animals with the increased protein expression.

FIG. 5. Reduced learning in HB-GAM knockout mice in the spatialwater maze study. (A) Mean escape latency across training blocks(KO: open squares, n � 14; WT: filled squares, n � 20). (B–D) Time

(percentage) spent inside the zone (6.25% of the water maze totalarea) around the platform and corresponding zones in the otherquadrants of the pool during the transfer tests (TT1, TT2, and TT3,respectively). (E) The percentage of time spent swimming in theoutermost ring of the water maze (30.6% of the maze area)—thigmo-taxis. Black bars represent the mean values for WT, gray bars for KOmice, dotted line marks the chance level. *P � 0.05; **P � 0.01between the groups.

336 Pavlov et al.

We found that slices from HB-GAM knockouts producea higher level of LTP than wild-type controls. Interest-ingly, in the hybrid genetic background, enhanced plas-ticity was only revealed when using a sub-maximalstimulus for LTP induction (100 Hz/20 pulses) (Amet etal., 2001) while the level of LTP in response to 100Hz/1 s stimulation was not altered. It has been welldocumented that different mouse strains not only per-form differently in the behavioral tests, but also showvarious degrees of LTP implying that plasticity of hip-pocampal synapses in various mouse strains may be“tuned” to different temporal patterns of synaptic ac-tivity (Nguyen et al., 2000a,b). It is known that evensubstrains might show considerable variability in thesynaptic plasticity (Simpson et al., 1997). Such pheno-typic variability, in fact, provides an additional tool tostudy the role of particular gene product in neuronalplasticity and behavior using different breeding condi-tions. Thus, the parallel electrophysiological phenotypeof mice lacking HB-GAM in the two different geneticbackgrounds strongly supports the idea that HB-GAMacts as an endogenous, activity-induced suppressor ofLTP.

To further elucidate the role of HB-GAM in vivo weperformed behavioral testing of HB-GAM overexpress-ing and knockout mice. In particular, since the mutantmice showed an LTP phenotype, we were mainly inter-ested in the spatial learning and memory, forms ofbehaviour known to be linked to hippocampal function.To the best of our knowledge, so far there have been nodata available on the role of HB-GAM in the regulationof behavior.

The mice overexpressing HB-GAM and having atten-uated LTP in CA1 in vitro appeared to learn faster thanthe wild-type controls in the spatial water maze task. Incontrast, HB-GAM knockout mice with enhanced LTPdisplayed significant reduction in their spatial memory.The enhancement of LTP in mutant mice has beenshown to be accompanied by improved (Manabe et al.,1998; Tang et al., 1999; Malleret et al., 2001), unalterd(Jun et al., 1998; Manabe et al., 2000), or impaired learn-ing (Gerlai et al., 1998; Migaud et al., 1998). On the otherhand, impaired (Tsien et al., 1996) or unaffected (Nos-ten-Bertrand et al., 1996; Zamanillo et al., 1999) learningFIG. 6. Alterations in the anxiety-like behavior and fear-condition-

ing in mice with different expression of HB-GAM. (A) Reducedanxiety-like behavior in HB-GAM transgenic mice in the elevatedplus maze (TG: n � 23; WT: n � 23). The percentage of time on openarms, the percentage of open arm entries, number of closed armentries. (B) Decreased cue-dependent fear conditioning in HB-GAMtransgenic mice (TG: n � 11; WT: n � 11). The percentage of freezingin the conditioning context (3 min) and during the CS tone (2 min) innovel context. (C) Enhanced anxiety-like behavior in HB-GAM knockout mice in the elevated plus maze (KO: n � 25; WT: n � 29). (D)

Decreased context-dependent fear conditioning in HB-GAM knock-out mice (KO: n � 24; WT: n � 27). The percentage of freezing in theconditioning context (3 min) and during the CS tone (2 min) in novelcontext. *P � 0.05 between the groups.

337HB-GAM in Hippocampal LTP and Learning and Memory

performance has been described in the cases where LTPwas inhibited.

As shown in this study mice overexpressing HB-GAM displayed intact context-dependent freezing.However, these mice froze significantly less than con-trol animals when CS was applied in a novel context.This combination along with a decreased anxiety in theelevated plus maze suggests a possible alteration of fearpathways in HB-GAM overexpressing mice. HB-GAMknockout animals on the contrary showed increasedanxiety-like behavior. Besides, they had decreased con-text-dependent freezing. This along with water mazeresults suggests impairment of hippocampus-depen-dent learning and memory. It is known that antianxietydrugs, such as benzodiazepine anxiolytics, produce sig-nificant impairment of memory (Korneyev, 1997). Thus,the phenotype with decreased anxiety and improvedlearning and memory as displayed by HB-GAM trans-genic mice is very intriguing considering possible phar-macological applications.

Increasing amount of evidence exists on the involve-ment of cell adhesion molecules in synaptic plasticity(Schachner, 1997; Murase and Schuman, 1999). Interest-ing parallels with our results can be found from thestudies on mouse mutants of the neural cell adhesionmolecules L1 and NCAM. The transgenic expression ofL1 in astrocytes inhibited hippocampal LTP (Luthi et al.,1996), but the mice displayed increased flexibility andselectivity in spatial learning (Wolfer et al., 1998). On theother hand, these mice showed weaker spatial retentionwhen tested in reversal learning. Inactivation of NCAMleads to the impairment of hippocampal LTP (Luthi etal., 1994), deficits in spatial learning (Cremer et al.,1994), increased anxiety (Stork et al., 1999), and aggres-sion (Stork et al., 1997). Furthermore, the loss of cad-herin-11 adhesion receptor in mice has been shown toenhance LTP and decrease anxiety (Manabe et al., 2000),whereas disruption of midkine (homologous protein toHB-GAM) was associated with increased anxiety injuvenile mice (Nakamura et al., 1998). These examplesstrongly suggest a modulation of memory processes aswell as fear and anxiety by molecules that control cell-to-cell and cell-to-extracellular matrix interactions.

Although not directly addressed in this study, thereare at least two plausible candidates for mediating theeffects of HB-GAM on synaptic plasticity: receptor-likeprotein tyrosine phosphatase � (PTP�/RPTP�) and syn-decan-3 (N-syndecan). HB-GAM is a functional ligandfor PTP�/RPTP� (Maeda and Noda, 1996) and has beendemonstrated to inactivate the catalytic activity of thisenzyme upon binding (Meng et al., 2000). During post-natal development PTP�/RPTP� has been shown to

modulate sodium channel function (Ratcliffe et al.,2000), which could affect Na� spikes and Ca2� entryinto the dendrites contributing to the mechanism ofLTP induction (Jaffe et al., 1992). It is postulated thatdynamic regulation of the balance between phospha-tase and kinase activity of specific substrates representsthe site for the control of synaptic plasticity and mem-ory (Soderling and Derkach, 2000; Malleret et al., 2001;Sweatt, 2001).

Another potential candidate to transduce HB-GAMsignal into the cell is the transmembrane heparan-sul-fate proteoglycan syndecan-3, which binds HB-GAM(Raulo et al., 1994) and contributes to LTP possibly viatyrosine kinase fyn signaling pathway (Lauri et al.,1999). Syndecan-3 expression in hippocampus is regu-lated upon changes in neuronal activity (Lauri et al.,1999). Mice lacking syndecan-3 display enhanced levelof LTP and defective learning thus resembling HB-GAM knockouts (M. Kaksonen, I. Pavlov, V. Voikar, S.Lauri, T. Taira, and H. Rauvala, manuscript in prepa-ration). Binding of HB-GAM to syndecan-3 leads tophosphorylation of a kinase-active protein complexcontaining src-family kinases c-Src and Fyn and theSrc-substrate cortactin (Kinnunen et al., 1998). Membersof the Src family tyrosine kinases are known to beinvolved in LTP induction (Lu et al., 1998; Salter, 1998;Lauri et al., 2000). It is noteworthy that Fyn-kinase de-ficient mice have been shown to display inhibited hip-pocampal LTP, impaired learning (Grant et al., 1992)and increased fear response (Miyakawa et al., 1994;Miyakawa et al., 1996). It is interesting to note that inaddition to its possible role in learning and memory,syndecan-3 plays a role in feeding behavior in hypo-thalamus where it is also expressed in an activity-de-pendent manner (Reizes et al., 2001).

HB-GAM belongs to the TSR superfamily (Kilpel-ainen et al., 2000), which also includes F-spondin (Klaret al., 1992) and midkine (Matsubara et al., 1990; forreview see Adams and Tucker, 2000). These proteinspossess similar TSR sequence motifs as compared toHB-GAM and are involved in the mechanisms of neu-rite outgrowth and guidance in a TSR domain-depen-dent manner. This suggests that TSR domains may actas universal functional units to mediate common effectsof these molecules and probably are implicated in mod-ulation of synaptic plasticity. For example, F-spondinthat contains similar TSR domains as compared to HB-GAM and interacts through these domains with hip-pocampal neurons is also highly expressed in pyrami-dal neurons of the adult hippocampus (Feinstein et al.,1999). Further, recombinant TSR fragments of HB-GAMhave been recently shown to inhibit LTP when injected

338 Pavlov et al.

into hippocampal slices (I. Pavlov, E. Raulo, T. Taira,and H. Rauvala, unpublished results).

In conclusion, using mutant mice approach thepresent study demonstrates that manipulation of thelevel of HB-GAM in vivo affects hippocampal synapticplasticity and learning behavior, thus suggesting animportant role for this protein in brain function inadults. Notably, genetic manipulations increasing ordecreasing the in vivo expression of HB-GAM resultedin opposing modifications of both synaptic strength andbehavioral performance. Thus, HB-GAM transgenicand knockout mice provide an intriguing model forstudies of activity-dependent synaptic modification andbehavior.

EXPERIMENTAL METHODS

Generation of HB-GAM transgenic and knockoutmice. HB-GAM transgene was constructed by cloningPDGF-� promoter from pSP65-sis-CAT vector (Sasa-hara et al., 1991) in front of rabbit beta-globin intron andSV40 polyadenylation signal. HB-GAM cDNA (Meren-mies and Rauvala, 1990) was then inserted after theintron (Fig. 1A). The expression cassette was removedfrom the plasmid backbone by digestion, separated byagarose gel electrophoresis, purified and microinjectedinto FVB/NHsd zygotes. Founder animals and theirprogeny were screened by Southern blotting from tailbiopsies. PCR was used for routine genotyping. Hemi-zygous transgenic animals and their wild type litter-mates in inbred FVB/NHsd or in F1 FVB/NHsd �129S2/SvHsd hybrid backgrounds were used for exper-iments.

Gene targeted mice lacking HB-GAM in C57BL/6 �129 hybrid background have been described previously(Amet et al., 2001). Chimeric male animals were matedwith 129S2/SvHsd females and the inbred mutantmouse line was established by heterozygote siblingbreeding for 12 generations.

Histological analysis. Brains were dissected outfrom sacrificed animals and rinsed in PBS. They were cutin two parts from the level of bregma. Pieces were thenfixed overnight in 4% PFA and 0.5% glutaraldehyde (inPBS) and embedded in paraffin after dehydrating in in-creasing alcohol concentration and clearing in xylene. Thewhole samples were cut in 10-�m-thick sections andplaced on object glasses. Every other object glass from oneseries was stained with hematoxylin and eosin and everyother with Bielschowsky-silver staining method.

To study possible cell density differences betweenHB-GAM overexpressing and normal brains, we pho-

tographed a random sample from the coronal sectionsand calculated the cell density with selector-method(McMillan and Sorensen, 1992; Everall et al., 1997) inour volume of interest (1-mm-thick segment startingfrom the frontal tip of hippocampus and proceedingcaudally).

Electrophysiological analysis. For in vitro electro-physiology adult mice (1.5–2 months old) were decap-itated under deep pentobarbital anesthesia and trans-verse hippocampal slices 400 �m thick were cut onvibratome. The slices were allowed to recover at roomtemperature for at least 60 min before experiments.Recordings were carried out in an interface-type cham-ber at �32°C as described previously (Lauri et al., 1998).Perfusion solution was equilibrated with the mixture of5% CO2 and 95% O2 and applied with the constant rateof 1 ml/min.

Extracellular recordings from CA1 stratum radiatumwere obtained with glass capillary microelectrodesfilled with 150 mM NaCl (2–10 M�) using Axoclamp2A amplifier (Axon Instruments, Burlingame, CA).Field excitatory postsynaptic potentials (fEPSPs) wereelicited with a bipolar stimulation electrode placed inSchaffer collateral pathway of the hippocampal slice.Stimulus intensity was adjusted to gain half-maximalfEPSP amplitude, except for the collection of the datafor input–output curves. Baseline transmission wasmonitored at 1/20 Hz, pulse length 0.1 ms. After at least10 min of stable baseline LTP was induced by high-frequency stimulation (HFS, 100 Hz for 1 s), duringwhich the pulse length was doubled. For paired-pulsestimulation interpulse intervals of 20, 40, 60, 80, 100,150, and 200 ms were used. The LTP program (www.ltp-program.com) (Anderson and Collingridge, 2001)was used for data acquisition and analysis. The slope offEPSP was used as an indicator of synaptic efficacy andwas calculated between 20 and 80% of the maximalamplitude.

Student’s t test was used for statistical analysis of thedata. Difference was considered significant when P �0.05.

Behavioral analysis. Sex and age matched youngadult littermates were used. The test battery applied inthe present study was essentially the same as previ-ously described (Voikar et al., 2001). General observa-tional profile involved the evaluation of gross health,basic sensory and motor abilities (body weight, pos-tural, righting and visual placing reflexes, pain sensi-tivity, motor coordination and spontaneous locomotoractivity). In addition, tests assessing anxiety-like behav-ior and learning and memory were conducted. The

339HB-GAM in Hippocampal LTP and Learning and Memory

person performing the behavioural analysis was un-aware of the genotypes of the animals.

Elevated plus-maze. The maze consisted of two openarms (30 � 5 cm), two enclosed arms (30 � 5 cm with15-cm-high transparent side- and end-walls) and a con-necting central platform (5 � 5 cm). The open arms weredivided into six squares by lines drawn on the floor. Amouse was placed in the center of the maze facing one ofthe enclosed arms and during a 5-min observation periodthe following parameters were recorded: latency to the 1stopen arm entry, number of open and closed arm entries,number of lines crossed (only forward movements) on theopen arms, and the time spent in different parts of themaze (open and closed arms, central platform). An armentry was defined as a mouse having entered an arm withall four legs. Subsequently the percentage of the open armentries and the percentage of time on the open arms werecalculated.

Water maze. The system consisted of a black circu-lar water tank and computer-interfaced camera trackingsystem (Columbus Instruments, OH) as described pre-viously (Voikar et al., 2001).

The experiment with the transgenic mice was carriedout in 6 daily training sessions each consisting of 4 trialsseparated by 60-s intertrial interval. The transfer tests(TT, 60 s free swimming, platform unavailable) wereperformed 24 h after the 3rd and 6th training sessions.Thereafter the training was continued with the platformin the opposite quadrant for 2 days and TT was per-formed on the 3rd day.

The training of knockout mice consisted of 9 trainingsessions. On the first day, four trials were given withintertrial interval of 60 s. The training was continued for 5days with two daily sessions of 2 trials (5 h betweensessions). The TTs were performed on the 4th and 6th day.The training was continued with the platform in the op-posite quadrant for 3 days (5 sessions of two trials).

The animals were released to swim in random posi-tions facing the wall and the time to reach the escapeplatform was measured in every trial. In transfer teststhe swimming paths were recorded and analyzed bythe water maze software (Columbus Instr.). The spatialmemory was estimated by two parameters: (1) the num-ber of annulus crossings at the platform position andthe corresponding annuli in left, right and oppositequadrant; (2) the time spent in the zone around theplatform (covering 6.25% of the total area of the watertank) and in corresponding zones of the three otherquadrants. In addition, the swimming distances and thethigmotaxis were measured. Thigmotaxis was definedas time spent swimming within the outermost ring ofthe water maze covering 30.6% of the water maze area.

After completing the spatial version of water mazethe experiment with visible platform was carried out.Mice were tested in two blocks of four trials and theposition of the platform made visible by attaching yel-low flag on top of it was changed for every trial.

Fear conditioning. The fear conditioning experi-ments were carried out employing a computer-con-trolled fear conditioning system (TSE, Bad Homburg,Germany). Training was performed in a clear acryliccage (35 � 20 � 20 cm) within a constantly illuminated(�550 1x) fear conditioning box. A loudspeaker pro-vided a constant background noise (70 dB) for 120 sfollowed by 10 kHz tone (CS, 75 dB, pulsed 5 Hz) for 30s. The tone was terminated by a footshock (US, 0.8 mA,2 s, constant current) delivered through a stainless steelfloor grid (A 4 mm, distance 9 mm). Two CS-US pair-ings were separated by 30-s pause.

Contextual memory was tested 24 h after training inthe fear conditioning box for 180 s without tone stimu-lation. Freezing was scored as a behavioral parameter.Total time of freezing (absence of any movements formore than 3 s) was measured by infrared light barriersscanned continuously with a frequency of 10 Hz.

Tone (CS)-dependent memory was tested 2 h afterthe contextual memory test in a novel context. Newcontext was a similarly sized acrylic box. The lightintensity was reduced to 100 1x, the floor was plane(without shock grid) and the background color wasblack (as opposed to white color in training context).After 120 s of free exploration in novel context the CSwas applied for 120 s and freezing was measured again.

Statistical analysis. Data were analyzed by meansof one-way or repeated-measures analysis variance(ANOVA) with genotype as an independent variable.Post hoc comparisons were performed by Newman-Keuls test. Data on figures are expressed as groupmeans � standard error of mean (SEM).

ACKNOWLEDGMENTS

We thank the members of our laboratories and the animal care stafffor assistance. This study has been supported by the Academy ofFinland (Program of Molecular Neurobiology) and Sigrid JuseliusFoundation.

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Received October 12, 2001Revised January 18, 2002

Accepted January 22, 2002

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