Involvement of L1.1 in memory consolidation after active avoidance conditioning in zebrafish

Involvement of L1.1 in Memory Consolidation afterActive Avoidance Conditioning in Zebrafish

Gabriele Pradel,1 Rupert Schmidt,1 and Melitta Schachner2

1 Biotechnology Center, Justus-Liebig-University, Leihgesterner Weg 217, D-35392Giessen, Germany

2 Zentrum fur Molekulare Neurobiologie, Universitat Hamburg, Martinistrasse 52, D-20246Hamburg, Germany

Received 23 November 1999; accepted 9 March 2000

ABSTRACT: To investigate the involvement of thecell adhesion molecules L1.1, L1.2, NCAM, and tenas-cin-C in memory formation, zebrafish (Brachydanio re-rio) were trained in an active avoidance paradigm tocross a hurdle to avoid mild electric shocks after a lightsignal. Application of [14C]deoxyglucose prior to thetraining session revealed an increased energy demand inthe optic tectum during acquisition of the active avoid-ance response compared with untrained fish and withfish not learning the task (nonlearners).In situ hybrid-ization with digoxigenin-labeled cRNA probes directedagainst zebrafish L1.1, L1.2, NCAM, and tenascin-Crevealed an enhanced expression of L1.1 and NCAMmRNA in the optic tectum of learners 3 h after acquisi-tion of the task compared with untrained fish, nonlearn-ers, overtrained fish, and learners decapitated 1 or 6 hafter acquisition. Levels of L1.2 mRNA were not signif-icantly increased in the tectum 3 h after learning. Tena-

scin-C was neither expressed in the optic tectum ofuntrained fish nor in the tectum of learners. To test fora possible involvement of L1.1 in memory consolidation,antibodies were injected intracerebroventricularly 1 hafter the last training trial. Two days later, injectedzebrafish were tested for recall and evaluated by a re-tention score (RS), ranging from 1.0 for immediate re-call to 0.0 indicating no savings. The average retentionscore of L1.1 antibody-injected fish (RS5 0.29) wasdifferent from that of tenascin-C antibody-injected (RS5 0.71) or uninjected fish (RS 5 0.78), indicating apivotal function of L1.1 in long-term memory formationin zebrafish. © 2000 John Wiley & Sons, Inc. J Neurobiol 43:

389–403, 2000

Keywords: cell adhesion molecules; L1; learning andmemory; NCAM; optic tectum; synaptic plasticity; te-nascin-C; zebrafish

Learning events induce temporary modifications inthe efficacy of neurotransmission and may finallyresult in the formation of a long-term memory trace,depending on ultrastructural changes at synaptic con-nections. Several cell adhesion molecules involved inthe formation and maintenance of cell contacts duringontogenetic development of the nervous system have

been implicated in the molecular mechanisms of syn-aptic plasticity, learning, and memory formation(Schmidt, 1995, 1999; Fields and Itoh, 1996;Schachner, 1997). Among them are L1 and NCAM,two members of the immunoglobulin superfamily,and the extracellular matrix proteins ependymins andtenascin-C.

An increased number of spine synapses expressingNCAM-180 was detected in the rat perforant path24 h after induction of long-term potentiation (Schus-ter et al., 1998), and an increase in NCAM immuno-reactivity was observed after learning in chick (Solo-monia et al., 1998) and rat (Doyle et al., 1992b). In a

Correspondence to: M. Schachner ([email protected]).

Contract grant sponsor: Deutsche Forschungsgemeinschaft;contract grant number: Schm 478/10 (RS and MS).© 2000 John Wiley & Sons, Inc.

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one-trial passive avoidance training, immunoreactiv-ity for NCAM increased 5 to 6 h after learning atsynapses of the chick striatum (Skibo et al., 1998).Recombinant L1 fragments and antibodies against L1and NCAM inhibited long-term potentiationin vitro(Luthi et al., 1994; Ronn et al., 1995). Furthermore,intracerebral application of antibodies against L1 andNCAM inhibited memory consolidation after spatial(Doyle et al., 1992a; Arami et al., 1996) and appeti-tive (Roullet et al., 1997) learning in rats, and passiveavoidance conditioning in chicks (Scholey et al.,1993, 1995; Alexinsky et al., 1997). Polyclonal anti-bodies against ependymins and monoclonal antibod-ies against the HNK-1 cell recognition epitope pre-vented memory consolidation in fish (Shashoua andMoore, 1978; Schmidt, 1987; Piront and Schmidt,1988; Schmidt and Schachner, 1994; Pradel et al.,1999).

In the chick, Scholey et al. (1993, 1995) reportedtwo waves of glycoprotein synthesis after learning.The first one was terminated within 1 h, whereas thesecond one occurred between 5 and 8 h posttraining.Antibodies to NCAM-induced amnesia only whenadministered during the second phase. Recently, athird wave of protein synthesis was described inchicks 16 h after learning (Tiunova et al., 1998).Suppressed NCAM expression in transgenic mice im-paired learning and altered behavior (Cremer et al.,1994; Stork et al., 1997), whereas ectopic overexpres-sion of L1 in astrocytes enhanced learning of a watermaze task in mice (Wolfer et al., 1998). Furthermore,NCAM-180 mutant mice exhibited behavioralchanges in circadian clock functions (Shen et al.,1997).

A remarkable correlate of flexibility in recognitionmolecule expression in synaptic plasticity is the up-regulation of ependymins in meningeal fibroblasts(Rother et al., 1995) and of tenascin-C in glial cellsand subpopulations of neurons (for review, seeFaissner, 1997). Tenascin-C is highly expressed inearly phases of neural development, but it is down-regulated in the adult, with the exception of certainbrain areas, e.g., the pituitary, the hippocampus, andthe cerebellum (see e.g., U. Bartsch et al., 1992;Schachner, 1997). Tenascin-C is up-regulated in thelesioned optic nerve of mice, rats (S. Bartsch et al.,1992; Gocht and Lo¨hler, 1993; Ajemian et al., 1994)and goldfish (Battisti et al., 1995), but neither in theinjured optic nerve of amphibians (Becker et al.,1995) nor in zebrafish (Bernhardt et al., 1996). Nakicand colleagues (1998) observed reexpression of tena-scin-C mRNA in the rat hippocampus 4 h after long-term potentiation in adult rats, and antibodies to te-

nascin-C interfered with filial auditory imprinting inchicken (Metzger et al., 1995).

Recently, we established an active avoidance par-adigm for zebrafish to investigate the influence of theHNK-1 carbohydrate epitope on long-term memoryformation (Pradel et al., 1999). Here, we studied thecontribution of the HNK-1-bearing molecules L1,NCAM, and tenascin-C in long-term memory forma-tion in zebrafish. First, we determined by the deoxy-glucose method which brain areas are primarily in-volved in learning of an active shock avoidanceresponse in zebrafish. In these regions we analyzedthe correlation of L1.1, L1.2 (two zebrafish isoformsrelated to mouse L1; Tongiorgi et al., 1995a), NCAM,and tenascin-C mRNA expression with long-termmemory formation after learning. Furthermore, anti-bodies against the respective cell adhesion moleculeswere intracerebroventricularly injected to test for apossible functional involvement of these cell adhesionmolecules in the memory consolidation process. Partsof our results have recently been published as ab-stracts (Pradel et al., 1997, 1998; Schmidt et al.,1998).

MATERIALS AND METHODS

Animals

Zebrafish (Brachydanio rerio), average body weight 6406 132 mg, were obtained from Tagis-Aquarium (Dreieich-Sprendlingen, Germany) and kept at 26°C at a day to nightcycle of 12h–12 h.

Active Avoidance Conditioning

Zebrafish were trained in an active avoidance paradigm asdescribed in detail by Pradel et al. (1999). The shuttle boxused for training was 25 cm long, 12 cm wide, and 12 cmhigh and was subdivided into two compartments by a hur-dle, reaching up to 2 cm beneath the water surface. Aconditioning light signal (red light, 3 W) was presentedalternatingly at either side of the box. Twelve seconds afteronset of the light signal, mild electric shocks (needle pulsesof 3 V and 1.25 mA at 40 Hz; unconditioned stimulus) wereadministered via stainless steel electrodes in the illuminatedcompartment of the shuttle box. The conditioning lightsignal was kept on during the whole period of each triallasting for 50 s. To avoid the electric shock, fish had to crossthe hurdle within 12 s after onset of the light. The condi-tioned and unconditioned stimuli were then applied at theother side of the shuttle box. Photo detectors above thehurdle inactivated the shock generator when the fish movedinto the dark compartment of the box and reactivated it, ifthe fish returned to the illuminated compartment beforetermination of the training cycle. Application of the stimuli

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was controlled by a computer. The whole training sessionconsisted of up to 40 trials, lasting 33 min without anyinterruption. The training was automatically terminatedwhen fish responded eight times correctly to the conditionedlight signal within 10 (or fewer) consecutive trials (learningcriterion). These fish were designated “learners.” Fish cross-ing the hurdle only after onset of the electric shock and fishnot changing the side of the shuttle box at all and, therefore,not reaching the learning criterion within 40 consecutivetrials were designated “nonlearners.” As a control group for2-deoxyglucose experiments and forin situ hybridizations,fish were placed into the inactivated shuttle box for the sametime period (“untrained control”). “Overtrained” fish servedas a further control group: To this purpose learners wereretrained twice a week for a period of 6 weeks. After 12training sessions overtrained animals make hardly any mis-takes in the avoidance trials.

[14C]2-Deoxyglucose Autoradiography

Zebrafish were injected intraperitoneally with [U-14C]2-deoxyglucose (2-DG; specific activity, 11 GBq/mmol; Am-ersham, Braunschweig, Germany) at an activity of 7.4 kBq/fish in 2 mL sterile 0.9% saline. After injection, fish werereturned to the tank for recovery (13 min). Subsequently,fish were placed into the shuttle box to become accustomedto the apparatus prior to the training (7 min). In the 2-DGexperiments, the active avoidance training lasted for 25 min,regardless of whether the fish reached the learning criterion(learners) or not (nonlearners). Immediately after comple-tion of training, fish were decapitated, and brains wererapidly frozen in isopentane at280°C. Untrained controlfish were treated identically except that they were placedinto the inactivated shuttle box. Frontal cryosections (10mm) were rapidly dried at 50°C and exposed to X-ray films(beta-max; Amersham) for 21 days at room temperature.

cRNA Probes for Cell AdhesionMolecules

Digoxigenin-labeled L1.1 and L1.2 cRNA probes were syn-thesized as described by Tongiorgi et al. (1995a). The L1.1probe (3392 bp) includes the third to sixth immunoglobulin(Ig)-like domains, the five fibronectin type III (FN) do-mains, the transmembrane, and the intracellular segments. Italso includes 526 noncoding bases of the 39-region withoutthe poly-A1 tail (Tongiorgi et al., 1995a). The L1.2 probe(1963 bp) spans the 59-half of the molecule and includes thefirst to fifth Ig domains, as well as 337 bases of 59-nontrans-lated sequence (Becker et al., 1998). The digoxigenin-la-beled tenascin-C probe (1152 bp) was synthesized as de-scribed by Tongiorgi et al. (1995b). It encompasses the FNtype III-like domains 7 and 8 and the fibrinogen domain.Synthesis of the digoxigenin-labeled NCAM probe (1067bp) was described by Bernhardt et al. (1996). The probecorresponds to the second to fifth Ig-like domain.

In Situ Hybridization

After training (see above), fish were transferred into a smalltank for 1, 3, or 6 h. Untrained fish were also kept in a smalltank for the same time periods. Subsequently, fish weredecapitated and brains were rapidly frozen in isopentane at280°C. Frontal cryosections (12mm) were fixed in 4%paraformaldehyde in 10 mM sodium phosphate buffer (pH7.4, containing 120 mM NaCl) for 16 h at 4°C. Sectionswere then washed in sodium phosphate buffer, acetylated,dehydrated in a graded series of ethanol, and air-dried(Bernhardt et al., 1996). Sections were incubated with pre-hybridization buffer (100 mM tris-(hydroxymethyl)-amin-omethane (Tris), pH 7.5, 50 mM EDTA, 10 vol% Den-hardt’s solution (503), 40 mM NaCl, 0.5% baker’s yeasttRNA) for 3 h at37°C, and subsequently incubated for 16 hat 55°C with the respective cRNA probes (see above) di-luted in hybridization buffer (20 mM Tris, pH 7.5, 50 vol%formamide, 10% dextran sulfate, 1 mM EDTA, 2 vol%Denhardt’s solution (503), 340 mM NaCl, 100 mM dithio-threitol, 0.05% baker’s yeast tRNA, 0.01% poly-A1 RNA).The following day, sections were washed in buffer 1 (100mM Tris, pH 7.5, containing 150 mM NaCl) and incubatedin 1% blocking reagent (Boehringer, Mannheim, Germany)and 0.5% bovine serum albumin diluted in buffer 1. Digoxi-genin-labeled probes were detected using antidigoxigeninFab fragments (Boehringer) diluted in the blocking bufferand visualized by an alkaline phosphatase reaction (BCIP/NBT solution; Sigma, Deisenhofen, Germany). Linearity ofbinding and detection of thein situ hybridization protocolwas tested with a dot blot assay.

Western Blot Analysis

Zebrafish brains were homogenized in 25 mM Tris buffer[pH 7.4, containing 1 mM EDTA and 1 mM proteaseblocking reagent (Boehringer)] at a concentration of 200 mgof brain tissue per milliliter of buffer. The homogenate wasincubated in 0.1% Nonidet P-40 for 1 h at 4°C andseparatedby reducing polyacrylamide gel electrophoresis (8%crosslinked; Laemmli, 1970). Proteins were electrophoreti-cally transferred to nitrocellulose membranes (Towbin etal., 1979; Gershoni and Pallade, 1983), preincubated in 10mM Tris buffer, pH 7.4, containing 150 mM NaCl and 5%skim milk powder (TBSM) for 1 h atroom temperature andimmunostained (2 h at room temperature) with either anti-L1.1 or anti–tenascin-C antibody diluted in TBSM. Subse-quently, nitrocellulose membranes were washed in TBSMcontaining 0.05% Nonidet P-40 (Sigma) and incubated withperoxidase-conjugated goat anti-rabbit IgG (Sigma) dilutedin TBSM (90 min at room temperature). Nitrocellulosemembranes were finally washed in 10 mM Tris buffercontaining 150 mM NaCl and 0.1% Tween 20 (Sigma).Peroxidase activity was visualized by the enhanced chemi-luminescence protein detection system (Amersham).

L1.1 in Memory Formation 391

Antibody Injection

One hour after training, learners were anesthetized in ice-cold water and the respective antibody was injected into thetectal brain ventricle by means of a Hamilton syringe(Pradel et al., 1999). Polyclonal antibodies to bacteriallyexpressed fragments of zebrafish L1.1 and tenascin-C wereraised in rabbits. The His-tag vector system (Qiagen,Hilden, Germany) was used to express theSmaI-ClaI frag-ment of L1.1, which comprises Ig domains 1, 2, and 3, andhalf of FN type III domain 4. The same vector was used toexpress FN type III domains 7 and 8 and the fibrinogendomain of zebrafish tenascin-C (entire clone 9 as publishedby Tongiorgi et al., 1995b). Inclusion bodies were harvestedfrom transformedEscherichia coli(JM 101), and the re-combinant proteins were purified via their His-tag using aNi-NTA-agarose column according to the manufacturer’sinstructions (Qiagen). Rabbits were immunized by subcuta-neous injection of the respective recombinant protein (firstinjection, 500mg, in Freund’s complete adjuvant; secondand third injections, after 3 and 5 weeks, 300mg, inFreund’s incomplete adjuvant), and serum was collected 10

days after the third injection. Antisera were characterized byimmunoblotting of zebrafish brain homogenate. IgGs werepurified by chromatography on a DEAE affi-blue column(Biorad, Munchen, Germany) and concentrated by ultrafil-tration on Amicon PM10 filters (Millipore, Eschborn, Ger-many). Antibodies were administered at a concentration of2.6 mg in 1 mL 10 mM sodium phosphate buffer (pH 7.4,containing 120 mM NaCl and 2.5 mM KCl). After injection,fish were returned to the tank. They recovered within aminute, exhibiting no behavioral abnormalities. The methodof antibody injection into brain ventricles has been de-scribed earlier (Pradel et al., 1999). It has been controlled bystandard histochemical methods that the injection needlereached the ventricle and that it did not damage the braintissue beneath the ventricle. Furthermore, some zebrafishwere killed 8 or 18 h after injection of the L1.1 antibody andprocessed for indirect immunohistofluorescence. Eighthours after injection, the fluorescent secondary antibodylocalized the antigen-bound L1.1 antibody in the tectalparenchyma. L1.1 distribution was almost identical to thedistribution observed, when the primary antibody was ad-ministered on brain sections. In fish killed 18 h after intra-cerebroventricular injection, specific fluorescence staininghad largely faded, presumably because of clearance of theprimary L1.1 antibody from the brain extracellular fluid.

Quantitative Evaluation of Retention

Two days after the training session, learners were placedinto the shuttle box again to test for recall. In the testsession, the training procedure was repeated under identicalconditions, and, for quantitative evaluation, a retentionscore was calculated, using the algorithm of Piront andSchmidt (1988):

RS5

(number of trials to criterion in training2 8)2 ~number of trials to criterion in test2 8!

~number of trials to criterion in training2 8!1 ~number of trials to criterion in test2 8!

The RS relates the number of trials to reach the criterion inthe test to the number of trials in the training session (eachminus the 8 correct responses). It assigns a positive RS toanimals exhibiting recall and a negative RS to animalsexhibiting more failures in the test than in the training.Animals that needed the same number of trials to criterionin the test as in the training session and, therefore, had noretention of the task, obtained an RS5 0.0. In fish exhib-iting immediate recall, the term (number of trials to criterionin test2 8) comes to 0, and, therefore, they obtain an RS5 1.0.

Quantitative Evaluation of 2-DGAutoradiography

Glucose utilization in the brains of learners, nonlearners,untrained fish, and overtrained fish was compared by quan-

Figure 1 Schematic drawing of brain regions investi-gated. (a) For quantitative evaluation of 2-DG-6-phosphateaccumulation, autoradiographs of frontal sections throughthe caudal part of the telencephalon, the rostral part of themesencephalon, the optic tectum, and the rostral part of thecerebellum were analyzed. (b)In situ hybridization signalsof L1.1, L1.2, NCAM and tenascin-C mRNAs were quan-titatively determined in the optic tectum from the stratummarginale to the stratum album centrale of a dorsal, adorsolateral and a lateral segment (frames) on one section ofeach fish. In order to compensate for differences betweenindividual hybridization experiments, a section of an un-trained control fish was assigned to each section of anexperimental fish (learner, nonlearner, overtrained and un-trained fish, respectively). BO, bulbus olfactorius; CC, cor-pus cerebelli; MS, medulla spinalis; Tel, telencephalon; Tg,tegmentum; TL, torus longitudinalis; TO, tectum opticum;VC, valvula cerebelli.

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Figure 2 Autoradiographs of brain regions investigated and corresponding phase contrast micro-graphs. Accumulation of the radioactive metabolite 2-DG-6-phosphate was slightly higher in themesencephalon (c) compared with the telencephalon (a) and the cerebellum (e) [see Fig. 1(a) forlocalization of frontal sections]. Accumulation of 2-DG-6-phosphate was strong in the valvulacerebelli, the ventral tegmentum, the torus longitudinalis, the torus semicircularis, and the tectumopticum. (b,d,f) Corresponding phase contrast micrographs. Arrows indicate the stratum periven-triculare and the stratum fibrosum et griseum superficiale. Bars5 180mm. CC, corpus cerebelli; Dc,central zone of the dorsal telencephalon; Dl, lateral zone of the dorsal telencephalon; Dm, medialzone of the dorsal telencephalon; EG, eminentia granularis; LI, lobus inferior; Tg, tegmentum; TL,torus longitudinalis; TO, tectum opticum; TS, torus semicircularis; VC, valvula cerebelli.


titative autoradiography of the accumulated radioactive me-tabolite 2-DG-6-phosphate (Sokoloff et al., 1977). Autora-diographs of frontal sections derived from three differentregions, thath is, the caudal part of the telencephalon, therostral mesencephalon, and the cerebellum [Fig. 1 (a)], wereanalyzed by means of an image analysis system (Quantimet;Zeiss, Jena, Germany). The mean optical density of therespective brain regions, that is, the extinction per unit areacaused by the silver grains on the autoradiographs, wasdetermined for 5 (telencephalon, mesencephalon, cerebel-lum) or 10 (tectum opticum) sections of one hemisphere

from each fish. To compensate for labeling differencesbetween individual animals resulting from the individualoverall metabolism or differences caused by the injectionprocedure, all values were standardized to the optical den-sity of that part of the brain exhibiting the smallest variation,thath is, the caudal telencephalon (set to 1), and expressedas relative optical density of accumulated 2-DG-6-phos-phate.

Quantitative Evaluation of In SituHybridization

In situ hybridization signals for L1.1, L1.2, and NCAMmRNA were investigated on one frontal section of therostral mesencephalon of each fish. All layers of the optictectum, ranging from the stratum album centrale to thestratum marginale were analyzed by means of an imageanalysis program (Soft Imaging System, Mu¨nster, Germa-ny). The stratum periventriculare was excluded from eval-uation, because its very intense staining in control sectionsprevented any reliable quantification of possible changesafter learning. The accumulated area of all labeled cells wasdetermined separately for a dorsal, a dorsolateral, and alateral segment of the optic tectum and divided by the totalarea of that respective segment [Fig. 1(b)]. Finally, theaverage was calculated from these three quotients to deter-mine the mean portion of a tectal square millimeter coveredby labeled cells. To compensate for differences in labelingintensity between individual hybridization experiments, afrontal section of an untrained control fish was assigned toeach frontal section derived from an experimental fish[learner, nonlearner, untrained fish, overtrained fish; Fig.1(b)]. Brains of experimental fish and corresponding controlfish were frozen simultaneously and treated identically. Forstandardization, the degree of labeling in the optic tectum ofeach experimental fish was divided by the degree of labelingin the optic tectum of the corresponding control fish. There-fore, the relative degree of labeling (rDL) was calculated asfollows:

rDL 5

average area of all labeled cells/square millimeter textum in experimental fish

average area of all labeled cells/square millimeter textum in control fish

Figure 3 Comparison of the relative optical density ofaccumulated 2-DG-6-phosphate. Accumulation of 2-DG-6-phosphate was significantly increased in the tectum opticumof learners (2a 5 .01, Wilcoxon test) and overtrained fish(2a 5 .05) compared with nonlearners and untrained fish(all values were standardized to the optical density of thetelencephalon, see Methods section). The whole mesen-cephalon exhibited a significant increase of accumulated2-DG-6-phosphate (2a 5 .02) compared with nonlearnersand untrained fish. There was no significant difference be-tween the relative optical density in the optic tectum oflearners and overtrained fish nor between the relative opticaldensity of nonlearners and untrained fish. For the telenceph-alon S.E.M. values refer to the optical density, and for thetectum opticum and cerebellum S.E.M. values refer to therelative optical density. Numbers in histograms indicate thenumber of independent fish analyzed. White asterisk, 2a5 .01; black asterisks, 2a 5 .05.

Figure 4 Expression of NCAM, L1.1, L1.2, and tenascin-C mRNA in the optic tectum of learnersand untrained fish. Labeling of NCAM (a,b) and L1.1 (c,d) was predominant in cells of the stratumperiventriculare and the stratum fibrosum et griseum superficiale. Further expression was detectablein the stratum opticum, stratum griseum centrale and the stratum album centrale. No labeling wasdetectable in the stratum marginale. L 1.2 (e,f) was predominantly expressed by cells of the stratumperiventriculare, stratum griseum centrale and the stratum album centrale. Tenascin-C mRNA wasneither expressed in the optic tectum of untrained fish (h) nor in the optic tectum of learners (g).Hybridization with sense-mRNA (NCAM) revealed no labeling (i). (a,c,e,g) Optic tectum oflearners 3 h after training; (b,d,f,h) Optic tectum of untrained fish. Bars5 5 mm. Em, endomeninx;SAC, stratum album centrale; SFGS, stratum fibrosum et griseum superficiale; SGC, stratumgriseum centrale; SM, stratum marginale; SO, stratum opticum; SPV, stratum periventriculare.

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In addition, the numbers of labeled cells per square milli-meter of the tectum in experimental and in control fish werecompared.

RESULTS

To localize the regions in zebrafish brain that areactivated during learning of an active avoidance task,[U-14C]2-DG was injected intraperitoneally beforetraining, and deposition of 2-DG-6-phosphate wasdetected autoradiographically. The optical density ofthe radioactive metabolite was measured in the caudalpart of the telencephalon, in the mesencephalon, inparticular in the optic tectum, and in the cerebellum[Fig. 1(a)]. The brains of learners, nonlearners, un-trained fish, and overtrained fish were compared.

Accumulation of 2-DG-6-phosphate was slightlylower in the telencephalon (25%) and in the cerebel-lum (23%) compared with the mesencephalon (seeFig. 2). Within the telencephalon [Fig. 2(a,b)] signalintensities were rather homogeneous. In the mesen-cephalon [Fig. 2(c,d)], regions of high 2-DG-6-phos-phate accumulation were the optic tectum, the torussemicircularis, and the ventral tegmentum. The rostralpart of the cerebellum [Fig. 2(e,f)] exhibited much2-DG-6-phosphate in the corpus cerebelli, but less inthe eminentia granularis. Intense labeling was alsodetected in the granular cell layer of the valvulacerebelli [Fig. 2(c,d)].

2-DG-6-phosphate accumulation was quantified,and all values were standardized to the optical densityof the telencephalon to compensate for labeling dif-ferences between individual experiments. No differ-ences were observed between the relative optical den-sity of the telencephalon or the cerebellum ofzebrafish that performed the active avoidance task(learners and overtrained fish; Fig. 3) compared tononlearners and untrained fish. The optic tectum oflearners and overtrained fish, however, exhibited anincreased accumulation of 2-DG-6-phosphate. Here,the relative optical density was significantly higher inlearners (111%; 2a 5 .01, Wilcoxon test;n 5 7) andovertrained fish (17%; 2a 5 .05; n 5 7) comparedwith the relative optical density of nonlearners (n 5 7)and untrained fish (n 5 7). The increase of the relativeoptical density of the whole mesencephalon (learnersvs. nonlearners) was smaller (14%; 2a 5 .05;n 5 7),indicative of a pivotal role of the tectum and not of thetotal mesencephalon for learning of the avoidanceresponse. No significant differences were detectablebetween the relative optical density of learners andovertrained fish nor between nonlearners and un-trained fish.

Experiments concerning the expression of cell ad-hesion molecules after training concentrated on theoptic tectum because of its increased energy demandin learners.In situhybridization was used to detect themRNAs corresponding to NCAM, L1.1, L1.2, andtenascin-C (Fig. 4). Hybridization signals were alsomeasured quantitatively. In order to compensate forlabeling differences between individual hybridizationexperiments, the values obtained from each experi-mental fish (learner, nonlearner, untrained, or over-trained fish) were compared with the values obtainedfrom sections of a corresponding untrained controlfish on the same slide.

In situ hybridization exhibited predominant label-ing of NCAM mRNA [Fig. 4(a,b)] and L1.1 mRNA[Fig. 4(c,d)] in cells of the stratum periventriculareand the stratum fibrosum et griseum superficiale.NCAM and L1.1 were also expressed by some cells ofthe stratum opticum. Although L1.1 was clearly ex-pressed by cells of the stratum griseum centrale andthe stratum album centrale, expression of NCAM wasless obvious in these layers. No labeling was detect-able in the stratum marginale. L1.2 [Fig. 4(e,f)] waspredominantly expressed by cells of the stratumperiventriculare, the stratum griseum centrale, and thestratum album centrale.

The number of cells expressing NCAM, L1.1, andL1.2 was increased in the tectum of learners decapi-tated 3 h after avoidance conditioning [Fig. 4(a,c,e)]compared with untrained fish [Fig. 4(b,d,f)]. Increasedlabeling for NCAM and L1.1 mRNA apparently com-prised those cell populations defined in goldfish astype I neurons of the stratum fibrosum et griseumsuperficiale by the nomenclature of Meek (1981,1990). Identification of other labeled cell populationswill have to await further functional, that is, physio-logical and morphological, analysis of the zebrafishbrain. Tenascin-C was neither expressed in the optictectum of untrained fish [Fig. 4(h)] nor in the optictectum of learners [Fig. 4(g)] at any of the three timepoints examined (1, 3, or 6 h after the avoidanceconditioning). Tenascin-C mRNA was not detected inany other brain region except of cell layers in theimmediate proximity of the ventricles, some Purkinjecells, and—strongly expressed—in cells of the cor-pora mamillaria, but the physiological significance ofthis finding is not yet known (data not shown).

The relative degree of labeling (rDL) was quanti-fied for NCAM, L1.1, and L1.2 mRNA in the ze-brafish optic tectum between the stratum marginaleand the stratum album centrale. The stratum periven-triculare had to be excluded from quantitative evalu-ation, because this layer is very tightly packed withcell bodies already exhibiting intense labeling in con-

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trol fish. Learners, nonlearners, untrained fish, andovertrained fish were compared 1, 3, and 6 h after theavoidance conditioning (Fig. 5). Quantitative evalua-tion revealed a significantly increased rDL for NCAMmRNA and L1.1 mRNA in learners 3 h after training(2a 5 .01, Wilcoxon test;n 5 6) compared withuntrained fish and nonlearners 1, 3, and 6 h aftertraining, with overtrained fish 3 h after training, andwith learners 1 and 6 h after training (each control,n5 5 or 6). Furthermore, for NCAM, a significantincrease in the rDL was detectable in the tectum oflearners 1 h after training (2a 5 .02,n 5 5) comparedwith the rDL of the corresponding control groups(each control,n 5 5). Almost identical results wereobtained, when the numbers of all labeled cells persquare millimeter of the optic tectum in learners andcontrol fish were compared instead of the labeled area(rDL). The increase in the expression of L1.2 in theoptic tectum of learners was not significant 1, 3, and6 h after training (each experimental group,n 5 4 or5). Further, there were no significant differences be-tween the rDLs of nonlearners, untrained fish, andovertrained fish (Fig. 5).

Expression of L1.1 and tenascin-C was character-

ized by Western blot analysis at the protein level.Polyclonal antibodies against L1.1 and tenascin-Cwere raised in rabbits and purified by affinity chro-matography. It was not possible to characterizeNCAM by this method because production of anti-bodies against zebrafish NCAM has so far failed. Fordetection of proteins immunopositive for L1.1 andtenascin-C, homogenates of zebrafish brain were sep-arated by reducing polyacrylamide gel electrophoresisand transferred to nitrocellulose membranes. Electro-phoretic blots stained with L1.1 antibodies (Fig. 6)revealed immunopositive protein bands at 200, 140,90, 55, and 50 kD. Blots stained with tenascin-Cantibodies revealed immunopositive proteins at 190,170, and 150 kD. These molecular weights are similarto those known for Ng-CAM and L1 of avian andmammalian brain (Sadoul et al., 1988; Burgoon et al.,1991) and tenascin-C (see, for instance, Faissner etal., 1988; S. Bartsch et al., 1992; Mitrovic andSchachner, 1995).

To test for a direct involvement of L1.1 in memoryconsolidation in zebrafish, L1.1 antibodies were in-jected intracerebroventricularly into learners 1 h afteracquisition of the active avoidance behavior. Two

Figure 5 Comparison of the relative degree of labeling (rDL) for NCAM, L1.1, and L1.2 mRNAs.Quantitative evaluation of labeling revealed a significantly enhanced rDL for NCAM and L1.1mRNAs in learners 3 h after training (2a 5 .01, Wilcoxon-test) compared with untrained fish andnonlearners 1, 3, and 6 h after training, to overtrained fish 3 h after training and to learners 1 and6 h after training. Furthermore, an increased rDL was detectable for NCAM mRNA in learners 1 hafter training (2a 5 .02) compared with untrained fish and nonlearners 1 h after training. Expressionof L1.2 was insignificantly increased in the optic tectum of learners 3 h after training. There was nosignificant difference between the rDL of untrained fish, nonlearners, and overtrained fish 1, 3, and6 h after training for either of the three mRNAs tested. Numbers in histograms correspond to thenumber of experimental fish. White asterisks, 2a 5 .01; gray asterisk, 2a 5 .02.


days later, injected learners were placed into the shut-tle box again and tested for recall. For quantitativeevaluation, an RS was calculated. Fish injected withL1.1 antibodies exhibited amnesia with an RS of 0.296 0.067 (mean6 S.E.M., n 5 20; Fig. 7). Fishinjected with tenascin-C antibodies achieved an RS of0.71 6 0.038 (n 5 20), and fish that had not beeninjected with any antibody had an RS of 0.786 0.043(n 5 20). The RS of anti-L1.1injected fish was sig-nificantly different from the RS of anti-tenascin-C-injected fish and of uninjected fish (p , .001, Stu-dent’s t test).

DISCUSSION

Recent experiments revealed the involvement of theHNK-1 carbohydrate epitope expressed by severalcell adhesion molecules (for a review, see Schachnerand Martini, 1995) in long-term memory formationafter an active avoidance conditioning in adult ze-brafish (Pradel et al., 1999). In this study, we inves-tigated changes in mRNA expression of the cell ad-hesion molecules L1.1, L1.2, NCAM, and tenascin-Cin zebrafish after active avoidance conditioning andtheir role in memory consolidation. In particular,learning-induced increases in L1.1 and NCAMmRNA expression were observed in the mesence-phalic optic tectum, where an enhanced energy de-mand during performance of the avoidance behavior

was measured by the 2-DG method. Increased celladhesion molecule expression was seen only during adistinct time window, in particular 3 h after the ac-quisition of the avoidance response. Furthermore, in-jection of L1.1 antibodies into the tectal brain ventri-cle 1 h after active avoidance conditioning impairedretention of the learned response.

Involvement of the Optic Tectum inLearning

Glucose consumption can be used as an index of brainenergy demand. Therefore, the 2-DG technique pro-vides a tool for mapping the neuronal activity indifferent brain areas in relation to the presentation ofsensory stimuli and motor activity (Sokoloff et al.,1977). Our 2-DG analysis revealed a significantlyincreased metabolism in the optic tectum of zebrafishduring learning of the active avoidance response,compared with nonlearners and untrained fish. Asmaller increase in glucose consumption was mea-sured in the optic tectum of overtrained fish that hadalready learned the avoidance response in severalpreceding sessions. Apparently, the tectum is reacti-vated during retrieval of the stored information and/orduring performance of the task. The increased depo-sition of 2-DG-6-phosphate cannot be assigned to themotor activity itself, because untrained fish placedinto the inactivated shuttle box crossed the hurdle asfrequently as learners, that is, one to three times per

Figure 7 Comparison of retention scores (RS) of experi-mental fish groups. On an average, the RS of fish injectedwith L1.1 antibodies was significantly decreased comparedwith the RS of anti-tenascin-C–injected fish or uninjectedfish, respectively (p , .001, Student’st test; asterisk). Therewas no significant difference between the RS of fish injectedwith tenascin-C antibodies and uninjected fish. Numbers inhistograms correspond to the number of experimental fish.Bars5 S.E.M.

Figure 6 Western blot analysis of L1.1- and tenascin-C-immunopositive protein bands. Brain homogenate was sep-arated by reducing polyacrylamide gel electrophoresis (8%gels), blotted on nitrocellulose membranes, and immuno-stained with L1.1 and tenascin-C antibodies. Protein bandsimmunopositive for L1.1 were detectable at 200, 140, 90,55, and 50 kD. Tenascin-C-immunopositive protein bandsof the zebrafish homogenate exhibited apparent molecularweights of 190, 170, and 150 kD.

398 Pradel et al.

50-s cycle. Furthermore, the conditioned stimulus orthe physiological stress response resulting from thetraining procedure does not provide an explanation forthe increased glucose consumption, because nonlearn-ers were also exposed to the light and electric shockstimuli, but exhibited no increase in glucose con-sumption in the optic tectum. Thus, our data giveevidence for an activation of the tectum during learn-ing and recall of the active avoidance task and for aninvolvement of the teleostean tectum in associativelearning and memory.

The “optic” tectum of teleosts receives input fromthe retina and secondary visual centers, such as thedorsal and ventral thalamus, the prectectum and thenuclei isthmi (Northcutt and Wullimann, 1988; Meek,1990; Wullimann, 1998). It also receives nonvisualfibers from the torus semicircularis (Grover andSharma, 1981; Luiten, 1981; Northcutt, 1982) and thetelencephalon (Ito and Kishida, 1977; Airhart andKribel, 1985). These different fibers are segregatedinto separate tangential bands that subdivide the neu-rons and neuropil of the tectum into distinct layers(Vanegas et al., 1984; Meek, 1981, 1990). Majorefferent projections reach the telencephalon and thenuclei isthmi (Wullimann, 1998). In view of thisinput–output characteristic the teleostean optic tec-tum is a visual center, analyzing movement, form, andcolor. The topological representation of its multimo-dal input may, however, also provide the cytoarchi-tectonic organization for integrative functions of ori-entation in learning and retention of visuallystimulated tasks. The distinct contributions of theteleostean telencephalon, the optic tectum and dien-cephalic pretectal nuclei to learning and storage oflearned information have long been analyzed in avariety of training situations (Salas et al., 1996;Ohnishi, 1997; for reviews see Friedlander, 1983;Overmier and Hollis, 1983). There is growing evi-dence that the optic tectum is also strongly engaged inthe integration of other sensory modalities in additionto vision in fish (Sharma and Garcia-Valenzuela,1998), reptiles (Kardong and Berkhoudt, 1999), birds(Feldman et al., 1996) and possibly amphibians(Claas, 1994), in particular for the sake of prey-catching and avoidance responses.

In this study, the telencephalon exhibited nochanges in accumulation of 2-DG-6-phosphate afterconditioning. The teleostean telencephalon receivesinputs from many sensory systems (reviewed in Wul-limann et al., 1996). We chose its caudal part, whichreceives mainly olfactoric projections (Scalia andEbbesson, 1971; Wullimann, 1998) as a referenceregion to standardize the optical density of the auto-radiographs with regard to differences caused by the

general metabolic turnover of individual fish and theinjection of the radioactive tracer. Also the cerebel-lum exhibited no obvious changes in glucose con-sumption after learning of the avoidance response,although it is engaged in coordination of motor activ-ity. Other brain regions that may be involved in theavoidance learning were not analyzed in this study.

Learning-Induced Expression of CellAdhesion Molecules

Possible changes in the expression of L1.1, L1.2,NCAM, and tenascin-C mRNA after active avoidanceconditioning were then analyzed in the optic tectum,thath is, in the region of highest energy demandduring learning. Labeling was determined in the lay-ers of the tectum from the stratum album centrale tothe stratum marginale at different time points afterlearning and compared with the degree of labeling incontrol fish. Quantitative evaluation revealed an in-creased expression of L1.1 and NCAM mRNA in theoptic tectum of learners 3 h after learning. NCAMmRNA levels were already slightly increased 1 h afterlearning. Six hours after the avoidance conditioning,L1.1 and NCAM mRNA expression had returned tothe control level. The temporary increase in L1.1 andNCAM mRNA expression suggests an involvementof the translated cell adhesion molecules in memoryformation. In forthcoming studies, the antisense inter-vention approach will be used to elucidate, whetherdenovobiosynthesis of L1.1 and NCAM is a prerequi-site for memory consolidation in zebrafish, as sug-gested by earlier work on extracellular matrix glyco-proteins of the goldfish central nervous system: Here,ependymin mRNA was induced 20 min after activeavoidance learning and remained elevated for 6 h(Rother et al., 1995). Phosphorothioate derivatives ofoligodeoxynucleotide probes against ependyminmRNA inhibited long-term memory formation(Schmidt et al., 1995). Furthermore, it has to be con-sidered that the observed increase in mRNA expres-sion may reflect a physiological compensation for adecreased availability of the respective cell adhesionproteins that might have been bound to membranes orincorporated into cells in the course of learning andmemory formation.

L1.2 mRNA levels were only insignificantly in-creased in the zebrafish tectum, 3 h after learning. Incontrast to several observations in birds and mammals(Daniloff et al., 1989; S. Bartsch et al., 1992; Gochtand Lohler, 1993; Ajemian et al., 1994; Martini et al.,1994; Nakic et al., 1998), tenascin-C mRNA levelsremained undetectable in the tectum of trained ze-brafish as well as in untrained controls. Thus, species-


specific differences are likely to exist for tenascin-Cexpression and function.

Increased labeling of L1.1 and NCAM mRNAcannot be caused by handling of the fish or by motoractivity, because untrained control fish were treatedidentically and exhibited the same overall motor ac-tivity. Furthermore, exposure to the light stimuli andthe stress reaction elicited can be excluded as possiblereasons for the increased mRNA expression as non-learners were exposed to the same conditions, butexhibited no changes in mRNA expression. It is wellknown, however, that stress hormones may affectlearning and memory (Sandi and Rose, 1994;Schmidt, 1997). Because overtrained fish familiarwith the avoidance response exhibited control levelsof L1.1 and NCAM mRNA, increased expression ofthese molecules in learners cannot be assigned to theperformance of the avoidance task as such. Althoughcells transcribing higher levels of L1.1 and NCAM inthe superficial gray and plexiform layer of the optictectum after learning resemble type I neurons in shapeand position, unambiguous identification of all la-beled cell populations has to be pursued in view of thepossibility that both, neurons and glia, may be in-volved in remodeling of neuronal connections in syn-aptic plasticity and regeneration (Martini et al., 1994;Schwalb et al., 1995; Bernhardt et al., 1996; Schmidtand Schachner, 1998).

L1.1 in Memory Formation

Antibodies were intracerebroventricularly injected 1 hafter conditioning in order to test for a possible directinvolvement of L1.1 in memory consolidation. Twodays after the active avoidance conditioning, whenuninjected learners displayed good retention of thetask, animals injected with L1.1 antibodies were am-nesic. The effect of the polyclonal antibody againstL1.1 was very similar to the effects of monoclonalHNK-1 and polyclonal ependymin antibodies in ze-brafish (Pradel et al., 1999) and goldfish (Shashouaand Moore, 1978; Schmidt, 1987; Piront and Schmidt,1988; Schmidt and Schachner, 1994). Injection ofantibodies against tenascin-C, however, had no influ-ence on the recall of the avoidance response. Thisfinding is noteworthy, because tenascin-C protein ispresent in adult zebrafish brain tissue as shown byWestern blot analysis. Nevertheless, tenascin-CmRNA levels are too low to be detectable byin situhybridization in the tectum and most other brain areasof untrained and trained animals. IgG fractions fromnonimmune serum and the monoclonal antibodyC183, directed against a neuronal cell surface glyco-protein in teleosts (Bastmeyer et al., 1995), also do

not influence memory consolidation after the avoid-ance conditioning in zebrafish (Pradel et al., 1999). Inthe context of these controls, it is evident that inhibi-tion of memory consolidation in zebrafish was neithercaused by anesthesia, nor by the injection procedureas such, nor by the presence of any given IgG mole-cules injected into the cerebrospinal fluid or bound toneuronal surfaces. Accordingly, inhibition of memoryconsolidation in zebrafish by L1.1 antibodies appearsto depend on a specific modification of L1.1 function.L1.1 may be involved in interactions with moleculesof the extracellular matrix (e.g., laminin) or in bindingto other cell adhesion molecules (e.g., L1.1, NCAM)that were prevented by the injected anti-L1.1 antibod-ies circulating in the extracellular brain fluid.

In conclusion, our data give evidence for an im-portant role of L1.1 and possibly also of NCAM inlong-term memory formation in zebrafish. In thisstudy, we showed learning-induced expression ofL1.1 and NCAM in the zebrafish optic tectum 3 hafter training. Newly synthesized L1.1 and NCAMglycoproteins may, therefore, be available at synapsesat a time described as the second wave of glycoproteinsynthesis after learning in chicken (Scholey et al.,1993, 1995). It is generally accepted that memoryformation proceeds in several consecutive timephases, and L1 and NCAM may be synthesized toform new or to stabilize newly formed or preexistingsynapses. Our experiments revealed an inhibition ofmemory consolidation by inactivation of L1.1 withantibodies that were detected in the optic tectum atleast up to 8 h after injection. The time window for theamnestic effect of anti-L1.1 has yet to be determined.It will be of particular interest to compare, whetheranti-L1.1 interferes with memory formation in ze-brafish during the first and second phase of glycopro-tein synthesis after learning, like the anti-L1 antibodyafter passive avoidance learning in chick (Scholey etal., 1995) or only during the second phase, like anti-NCAM in the same paradigm (Scholey et al., 1993).The antibodies may exert their amnestic effects bybinding to specific domains of cell adhesion mole-cules, thereby preventing them from homophilic orheterophilic interactions with molecules in synapticmembranes or in the synaptic cleft. Antibodies mayalso interfere with specific steps of the signal trans-duction cascades necessary to transform synaptic con-nectivities that were activated during learning into apermanent, consolidated form during the memoryconsolidation process; e.g., Kandel and colleagues(Bailey et al., 1992) have considered rapid endocyto-sis and reexpression of apCAM observed inAplysiaasan important step in learning and memory formation.Similar to their well-established role in the develop-

400 Pradel et al.

ment and regeneration of the central nervous system,cell adhesion molecules, such as L1.1 and NCAM, arelikely to be involved in synaptic remodeling, stabili-zation, and possibly path finding of axonal sproutsduring those molecular mechanisms subserving theneural representation of behavioral plasticity.

The authors thank Dr. R. R. Bernhardt for helpful dis-cussion, to Dr. E. Tongiorgi for antibodies, to O. Heller forexpert advice and help with the image analysis, and to A.Kolar for technical assistance.

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Involvement of L1.1 in memory consolidation after active avoidance conditioning in zebrafish