Overexpression of a Calcium-binding Protein, S 1 …learnmem.cshlp.org/content/2/1/26.full.pdftherefore affect neuronal and behavioral phenotypes. Previously, we generated transgenic

RESEARCH

Overexpression of a Calcium-binding Protein, S 1 O013, in Astrocytes Alters Synaptic Plasticity and Impairs Spatial Learning in Transgenic Mice R o b e r t G e r l a i , 1'4 J. M a r t i n W o j t o w i c z , 2 A l e x a n d e r M a r k s , 3 a n d J o h n R o d e r 1

~Division of Molecular Immunology and Seurobiology Samuel Lunenfeld Research Institute Mount Sinai Hospital Toronto, Ontario, Canada 2Department of Physiology Molecular Research Council (MRC) Group in Nerve Cells and Synapses University of Toronto Toronto, Ontario, Canada 3Banting and Best Medical Research Department University of Toronto Toronto, Ontario, Canada

A b s t r a c t

Recent evidence suggests that slowly propagating Ca 2÷ waves from astrocytes can modulate the funct ion of neurons . Altering astrocytic calcium processes in vivo may therefore affect neurona l and behavioral phenotypes. Previously, we generated transgenic mice that overexpress an astrocytic calcium-binding protein, S100[3. lmmunocy tochemis t ry and in situ hybridizat ion showed elevated expression in the astrocytes of the h ippocampus and other brain regions. Neurons in the h ippocampus were negative for $100[$. In this paper we analyze the hippocampal electrophysiology and learning propert ies of mice from two transgenic lines. Significant differences were found between the h ippocampal slices of normal and transgenic mice in their response to h igh f requency (100 Hz) stimulation. The overall distr ibution of post-tetanic excitatory postsynaptic potent ials (EPSP) of the slices from the transgenic mice was shifted significantly toward smaller values to a degree that 25% of slices exhibited

4Corresponding author.

depression. The altered hippocampal neurophysiology was accompanied by an impai rment in a h ippocampal -dependent learning task. Transgenic mice showed significant impai rment in a spatial version of the Morris water maze, however, they performed normal ly in non-spatial tasks. Probe trials showed that transgenic mice, though significantly impaired, also acquired spatial information. The results suggested that the impai rment was not due to motor dysfunction, impaired vision or motivation of the transgenic mice, findings compatible with a possible h ippocampal mechanism. We conclude that overexpression of S100~3 in astrocytes impairs, but does not abolish, the ability to solve a spatial task, and it leads to a significantly decreased post-tetanic potentiat ion in the h ippocampal slice. We hypothesize that the changes are due to calcium mediated processes. Our results support the not ion that astrocytes are involved in h igher brain functions.

I n t r o d u c t i o n

Several observations support the hypothesis that astrocytes play a role in modulating neural function. First, astrocytes ensheath many synapses

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ABNORMAL LEARNING AND LTD IN 5100~ TRANSGENIC MICE

and their membranes are closely apposed ( 10 nm) to the axolemma (reviewed in Barres 1991 ). Sec- ond, astrocytes can respond to neuronal activity (Dani et al. 1992; Murphy et al. 1993), or glutamate application (CorneU-Bell et al. 1990; Menn- erick and Zorumski 1994), by generating Ca 2+ waves which propagate slowly. Third, electrically evoked astrocyte Ca 2+ waves in cell culture crossed gap junctions into neurons in a unidirec- tional manner (Nedergaard 1994). Although these studies make a convincing case for neuron-astro- cy t e -neu ron signaling, the mechanisms whereby neurons are modulated are not known, but could involve ( 1 ) the control of K + fluxes in the narrow extracellular synaptic space by Ca 2 + dependent K channels in astrocytes (Laming 1989); (2 ) regulation of Ca 2 ÷ concentrat ions by voltage-activated Ca channels in astrocytes (Barres 1991); or (3) uptake of glutamate released from the presynaptic neuron (Barres 1991; Smith 1994; Mennerick and Zorumski 1994). A possible consequence of astro- cy t e -neu ron signaling could involve elevations of neuronal intracellular Ca 2 + concentrat ion leading to the modulat ion of a number of calcium-dependent neuronal processes including learning and memory (Smith 1994) and long-term potentiat ion (LTP), a calcium-dependent neurophysiological phenomenon that has been suggested as a synaptic mechanism underlying memory (Bliss and Collin- gridge 1993). One may therefore hypothesize that perturbat ion of astrocytic signaling in vivo could alter synaptic processes involved in learning and memory, a possibility that has not been investigated.

Recently, molecular genetics have been used to identify genes and their interactions that play key roles in the biological mechanisms underlying synaptic processes, such as LTP and long-term depression (LTD) and higher brain function, such as learning and memory. Creating mice with null mu- tations (gene knockout) in the gene for cx-calcium calmodulin-dependent kinase II (CAM kinase II; Silva et al. 1992a,b), fyn, a protein tyrosine kinase (Grant et al. 1992), PKC ~/ (Abeliovich et al. 1993), and mGluR1 (Aiba et al. 1994) have pro- vided evidence for the success of the molecular genetic approaches. In all of these cases, the mice exhibited reduced or altered hippocampal LTP.

We decided to manipulate the gene for S10013 for several reasons. S100[3 is a 20-kD calcium-binding protein expressed in the cytoplasm of astrocytes (Matus and Mughal 1975; Haan et al. 1982; Zimmer and Van Eldik 1987). It could act in glia to

alter calcium-dependent processes or it could also be released and stimulate neurons directly to increase the free intracellular Ca 2+ levels (Barger and Van Eldik 1992). Furthermore, we decided to overexpress the gene for S100~3 instead of elimi- nating expression because elevated levels of S10013 have been implicated in human neurode- generative diseases, including Down's syndrome and Alzheimer disease (Rabe et al. 1990; Griffin et al. 1989).

We overexpressed S10013 in the astrocytes of transgenic mice using the human gene with its own endogenous promoter and locus controlling region, which led to expression levels that de- pended on gene copy number (Friend et al. 1992). Our transgenic mice expressed three- and eight- fold eIevated levels of human S100[3 mRNA and a two- to fivefold increase in protein levels in all brain regions including the hippocampus (Friend et al. 1992). We show that overexpression of S100[3 in vivo results in significantly altered synaptic processes characterized by smaller post-tetanic excitatory postsynaptic potentials and an increased incidence of synaptic depression in hippocampal slices and impaired spatial leafing in the transgenic mice.

Materials and Methods

ANIMALS AND HOUSING

Transgenic mice were generated from the CD1 strain as described previously (Friend et al. 1992). Mice from two transgenic lines (line 5 and line 8) carrying 8 and 70 copies of the human S10013 transgene (Friend et al. 1 9 9 2 ) w e r e behav- iorally tested at 12-13 weeks of age along with age-matched nontransgenic control CD1 mice. Both the transgenic and CD1 control mice were the second generation offspring from a segregating S10013 population. The mice were bred, raised, and kept simultaneously in the same room under identical conditions ( temperature, 20+-1°C; relative humidity, 45% ). They were housed in groups of three to four in standard plastic cages (25 × 18 × 12 cm) on sawdust bedding. Males and females were housed separately. Food and water were available ad lib. The photoperiodici ty was 12/12 hr with the light being turned on at 7 a.m. Electrophysiological experiments were carried out on 15- to 18-week-old line-8 transgenic and CD1 control mice. In one series of electrophysio-

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logical experiments, older ( 4 0 - 7 0 weeks old) mice were used.

THE MORRIS WATER MAZE

The hippocampus has been shown to be a crit- ical brain center involved in the regulation of ex- ploratory activities and in incorporating spatial information (O'Keefe and Nadel 1978). Mice and rats with dysfunctional hippocampus are impaired in tasks that require spatial learning (Morris 1990). One of the tests of spatial learning used most frequently is the Morris water maze (Morris 1984), in which mice or rats are required to locate a platform submerged under water. We employed two versions of this learning paradigm, the h idden platform with variable starting and the visible platform tests (Eichenbaum et al. 1990). The former tests for spatial learning, and the latter serves as a control, as it requires similar motivational, sen- sory, and motor abilities in a nonspatial context.

The Morris water maze used was a circular tank (diam., 120 cm, height of wall, 30 c m ) m a d e of white acrylic and filled with water that was made opaque with a nontoxic white color tem- pera. The temperature of the water was held con- stant at 23 + I°C. The water level was 5 cm below the edge of the tank so that the experimental mice could easily see visual cues surrounding the tank. A circular platform (20, 15, or 10 cm diam.) made of white acrylic was submerged 5-10 mm below the water level. The apparatus was placed in a well-lit room (4 × 4 m ) w i t h numerous visual cues. The exper imenter was not present in the room during the training and probe sessions and observed the mice from outside through a small win- dow.

BEHAVIORAL TESTING PROCEDURE

All mice were tested by the same experi- menter throughout the training and probe trials. The sequence of testing was randomized across genotypes so that possible environmental error variation or circadian activity rhythms could not bias the data. The experimental ly naive mice were first habituated to the water and the platform by placing them in the water tank and allowing them to swim for 30 sec and to practice cl imbing onto the platform three times. The mice were allowed to rest until the next day when the training tests

began. Particular attention was paid to handling the mice gently throughout the habituation and training sessions. For instance, mice were always picked up from the platform by gently making them climb onto the palm of the investigator and never by the tail, which could have caused distress to the mice (personal observation) and thus could have served as an unwanted punishment for finding the platform.

Two training paradigms were applied using the same mice. First, an 8-day hidden platform training was carried out, which, after a 3-day rest- ing period, was followed by a 5-day visible platform training. During the h idden platform training the mice were required to find the fixed location of a submerged platform in relation to extra tank visual cues, a task that requires spatial learning. The experimental mice were started singly from six predetermined locations from the wall of the tank each day to prevent them from associating the location of the platform with a single view. The sequence of starting was determined by a semirandom computer schedule. The amount of time, "escape latency," needed for the mice to locate and climb onto the platform was recorded. If the mouse did not find the platform within 60 sec, it was guided onto it gently. Mice were allowed to rest on the platform for 40 sec and then were returned to their home cage. The intertrial interval was 60 min. The training was carried out for 8 days continuously be tween 10 a.m. and 5 p.m. On the first and second days larger platforms (20 and 15 cm diam., respectively) were used to increase the chance for the mice to find the platform acciden- tally (Eichenbaum et al. 1990). From the third day onward the diameter of the platform was 10 cm. On the eighth day the mice were started from two novel locations to investigate the effect of the novel view of the same cue configuration on the performance of the mice.

On the ninth day, two probe trials were carried out in a random sequence each lasting 60 sec per mouse. In probe trial 1, by removing the hidden platform, we investigated the possibility that visual cues from the platform were perceived by and guided the experimental mice. In probe trial 2 we studied whether the mice were using extra- tank spatial cues to locate the hidden platform: We removed the platform, and in addition, we surrounded the tank with 150-cm-high cardboard paper and il luminated it wi th a 40 W red light bulb from above, thus preventing the experimental mice to use extra tank visual cues. In the two

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probe trials the experimental mice were allowed to search the water tank for the entire 60-sec session, and their swimming patterns were video recorded. The relative duration of time the mice spent in four equal quadrants of the tank was measured with an event recorder computer program (Gerlai and Hogan 1992), and in probe 2 the latency to cross the previous fixed location of the platform the first time (escape latency) was also recorded. The swimming patterns of the mice during probe 1 were analyzed further by measuring the total length the mice traveled and the number of quick turns. A quick turn was counted when the mouse changed its swimming direction more than 90 ° within a l O-cm 2 area.

To investigate the learning performance of the mice in a nonspatial task, a 5-day visible platform test was carried out 3 days after the probe trials. The mice were required to find the variable location of the submerged platform by associating its location with a single visible cue, a 15-cm-high column (3 cm diam.) with yellow and red markings. To prevent a possible interference with spatial information learned previously, the tank was surrounded with 150-cm-high cardboard paper that blocked the view to external cues. The mice, started from a fixed location from the wall of the tank, were required to find the platform at each of six predetermined variable locations of a six-trial training each day. The platform locations and their sequence were determined by a semirandom computer schedule so that the minimum daily distance the mice were required to swim to the platform was equal in the hidden and visible platform tests. The size of the platform was decreased between days 1 and 3 as described in the hidden platform training. Other parameters of the test were also the same as in the hidden platform training.

Nonspatial learning was also studied in a slightly modified visible platform training that was designed to increase the difficulty of the nonspatial task and to make some aspects of the test more comparable to the hidden platform training. As in the hidden platform training, naive, previously untested, S10013-8 transgenic and normal control CD 1 mice were used. The 12- to 13-week-old mice were first habituated to the tank as explained above. The training procedure was also identical to the one described above, except that a small platform (10 cm diam.) was used throughout the training and the cue marking the platform was smaller than the one used before (a 5-cm-high and 1-cm-thick column with yellow and red markings).

ANALYSIS OF HIPPOCAMPAL LTP

The recently reported findings in CAM kinase II (Silva et al. 1992a; 1992b) andfyn (Grant et al. 1992) null mutant mice, together with the results of previous investigations applying pharmacologi- cal manipulations (for review, see Morris 1990), suggest that abnormal hippocampal LTP impairs the solving of spatial tasks such as the spatial version of the Morris tank test. Therefore, in addition to behavioral studies testing for hippocampal function, we measured LTP in the hippocampal slices (CA1 area) of normal and age-matched SIO0~ transgenic mice. Mice were anesthetized with halothane and decapitated, and their brains quickly removed. The hippocampus was separated and cut into 400-txm-thick, transverse slices, which were kept at room temperature in oxygen- ated chambers for at least 1 hr prior to electrophysiological experiments. Randomly selected slices were transferred to a separate perfusion chamber perfused continuously with an artificial cerebrospinal fluid (ACSF) at 32°C. The ACSF was composed of 124 mM NaCI, 3 mM KCI, 1.25 mM NaHPO4, 2 mM MgCI2, 2 mM CaCI 2, 26 mM NaHCO3, 10 mM dextrose. The ACSF was kept in equilibrium with 95% 02 and 5% CO2, and the pH was maintained at 7.4. The slicing procedure usually yields 10--15 slices from each mouse. How- ever, because of time constraints, only two to three slices were usually used for electrophysiological recordings.

The Schaffer collateral/commissural afferents to the CA1 area of the hippocampus were stimulated via tungsten electrodes with 1- to 1.5-mA current pulses of 0.01-msec duration. Field synaptic responses (excitatory postsynaptic potentials, EPSPs) were recorded in the stratum radiatum with glass pipettes filled with ACSF ( - - 3 - 5 ~ m tip diam.). The Axopatch-2 amplifier (Axon Instru- ments Inc.) was used. The data were transferred from the amplifier to a computer and averaged. The Schaffer collateral/commissural pathway was stimulated at a frequency of 0.2 Hz. The stimulation intensity was adjusted to give field EPSPs just subthreshold for generation of the population ac- tion potentials. The responses were averaged once per minute, and the initial slope of the field EPSPs was used as a measure of synaptic transmission. The averaged, baseline responses were recorded for at least 20 min and considered stable if their deviations from the mean were <15%. Unstable slices were rejected. In all experiments, LTP was

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induced with two 100-Hz trains, lasting 1 sec and repeated at a 5-sec interval. In a subset of slices the procedure was repeated after 20 min to induce a second LTP. The magnitude of LTP was expressed as a relative change (%) with respect to the overall average of responses during the 10-min period preceding the tetanus.

Generally, hippocampal slices from mice are more difficult to dissect and maintain in vitro than more standard rat slices. In this study - 4 0 % of slices were rejected from the analysis. The 40% rejection rate was the same in normal and transgenic animals. The two most common causes of rejection were (1) the slope of the field EPSP de- viated by > 15% of the overall baseline average, and (2) the field EPSPs were smaller than --0.5 mV and/or the ratio of presynaptic volley to the EPSP amplitude was >2.

In the majority of experiments only one animal was tested per day. However, in one series of 13 experiments, we tested control and transgenic animals in tandem, on the same day. In these experiments we tested slices from the two animals in alternate sequence to avoid bias attributable to possible deterioration of slices. We found no ob- vious difference between the results of this series and the other experiments.

STATISTICAL ANALYSIS

The daily behavioral performance of each mouse was calculated by averaging the six escape latencies ( two escape latencies of day 8 of the hidden platform test) of each day. The daily performance was analyzed by three-way repeated measure analysis of variance (ANOVA) with factors Genotype (normal CD 1, line 5 transgenic, line 8 transgenic), Sex (female, male), and Day (1, 2, ..., 8 for the hidden platform test; and 1, 2, ..., 5 for the visible platform test). The different genotypes of mice were also compared by post hoc Student- Newman-Keuls (SNK) multiple range tests by day with P~>O.05 for nonsignificant ranges. The escape latencies measured at the very first trial and those measured in probe 2 were also analyzed by two- way repeated measure ANOVA with factors Gen- otype (as above) and Trial (first trial, probe 2 trial). In probes 1 and 2 the relative time the mice spent in the quadrant that previously contained the platform, was measured and analyzed by two- way ANOVA with factors Genotype and Sex and by post hoc SNK test. The relative durations were also

compared with chance level (25%) by t-test. The average swimming speed (cm/sec) of the mice during probe 1 was calculated by dividing the total distance the mice traveled by the length of the recording session. Swimming speed and the number of quick turns in probe 1 were analyzed by two-way ANOVA with factors Genotype and Sex. LTP in normal and transgenic mice was compared by ANOVA and t-tests, and the incidence of LTD was analyzed by Fisher's exact test. The effect of repeated Induction and Genotype on LTP was analyzed by two-way repeated measure ANOVA.

Results

SYNAPTIC PLASTICITY IN HIPPOCAMPUS

Modification of synaptic efficacy in the CA1 area of both normal control and transgenic slices was measured by averaging the relative change of synaptic responses 20 min after tetanization. The responses to the high-frequency stimulation were significantly lower in S100{3 transgenic than in normal control mice (normal, 79.3% increase compared with baseline, S.E. = 16.8, n = 20; transgenic, 35.7% increase compared with baseline, S.E.= 11.4, n = 2 7 ; t=2.21, P<0.03). The cumulative distribution histogram (Fig. 1 ) also shows that the synaptic responses of the transgenic mice were shifted generally toward smaller values compared with those of the normal control mice. Al- though the shape of the distribution appears to be normal in the transgenic mice, interestingly, this shift resulted in a situation where two different post-tetanic responses may be distinguished: LTP and LTD. In six slices from four S100[3 animals the stimulation produced responses persistently below baseline, that is, LTD instead of LTP. Such depression was not observed in normal, control slices (0 of 20). The difference between normal and transgenic mice in the frequency of hippocampal slices showing the depression was significant (P<0.03, Fisher's exact test). Analysis of the subset of the slices showing LTP revealed no significant differences between normal and transgenic animals despite the apparent reduction of the overall LTP response in the transgenic mice. Furthermore, the subset of LTP responses from those transgenic mice whose sister slices exhibited LTD appeared to be also nonsignificantly different from the LTP seen in normal animals (Fig. 2).

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Figure 1: Cumulative probability histogram illustrates the distribution of post-tetanic responses in control CD1 (thick line) and $10013-8 transgenic (thin line) hippocampal slices. Means of the field EPSP slopes (expressed in percent of baseline) in CA1 area were measured 20 min after tetanic stimulation in 20 control and 27 $10013 slices. Values above 100% (baseline level) represent potentiation; values below 100% represent depression. Data were binned at 10% increments to obtain equal number of samples in the transgenic and control groups. The comparison of the distributions indicate a significant difference between normal and transgenic responses (Kolmogorov-Smirnov, D = 0.33, P<0.01 ).

The time course of LTP in Figure 2A appears to show a small rising trend between 12 and 30 min in both groups of animals. These trends were found not statistically significant in either group (SIO0~ F = 0.245, P>0.99; CD1 control F = 0.210, P>0.99). Nevertheless, to establish whether the time course of LTP in our experiments includes possible slow rising N-methyl-n-aspartate (NMDA)- receptor-independent component of LTP, similar to the one reported by Grover and Teyler (1990), we applied a specific receptor blocker, n-amino-5- phosphonovalerate (D-APV), prior to the tetanic stimulation to a separate group of slices (see Fig. 2A). However, no significant LTP could be induced in the presence of D-APV. In the same slices, subsequent tetanization in normal ACSF produced an average LTP of 65% (S.E.). The results demonstrate that LTP observed in our experiments is pre- dominantly NMDA-receptor dependent.

Because LTP in the transgenic animals was found nonsignificantly different from that of the controls, one might hypothesize that the LTP

mechanism may have been saturated by the standard tetanizing procedure. Such saturation could obscure possible differences between transgenic and control mice. To determine whether LTP was saturated, it was recorded in another set of slices as described above; however, LTP was induced and measured twice. The mean of LTP1 (measured 15-20 min after the first tetanus) was 66.5% (S.E. = 23.1, n = 13) in normal control and 53.5% (S.E. = 11.9, n = 15) in SIO0~ transgenic mice. The mean of LTP2 (measured 15-20 min after the second tetanus) was 121.7% (S.E. = 50.9, n = 11) in normal controls and 126.2% (S.E. = 27.5, n = 13) in SIO0J3 transgenic mice. ANOVA revealed no difference between normal and transgenic mice (Ge- notype: FL26=0.02, P>O.05). The effect of repeated induction, that is, the increase in LTP2 compared with LTP 1 bordered the level of signif- icance (F~,2~ = 3.82, P = 0.06), suggesting that LTP was not saturated at the first induction period. The interaction between factors Genotype and Induc- tion was not significant (F1,2] --0.02, P>0.05).

Other features of synaptic plasticity in the transgenic mice were normal. For example, the paired pulse facilitation (PPF) was evoked by pair- ing two synaptic responses at equal stimulus strengths (insets in Fig. 2). Typically, PPF of 60% (S.E. = 8, n = 9) was observed in control animals when two pulses were paired at a 30-msec interval. In six transgenic slices from three animals PPF was 48% (S.E. = 9). The difference was not significant ( t = - 0.89, P>O.05).

To measure tonic levels of GABA-ergic inhibition in slices we applied l O-mi bicuculline, a GABAA-receptor blocker, to the perfusate. In both groups the intial slopes of field EPSPs were increased by about 50% with respect to baseline levels. However, PPF was consistently reduced from about 60% to about 25%. These results suggest the presence of tonic, presynaptic inhibition in slices from control and transgenic animals. However, there was no significant difference in the level of inhibition between the two groups of slices ( t = 1.59, P>0.05).

HIDDEN PLATFORM TEST

A significant behavioral impairment of transgenic mice was observable (see Fig. 3) in the hidden platform test (ANOVA: Genotype F2,51- 27.52, P<O.O001; Day F7,357 = 32.27, P<O.O001; Genotype×Day interaction F14,357=1.84, P = 0.0318). No significant sex difference was found

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Figure 2: LTP and LTD in S100[~ transgenic mice. (A) Time course of LTP in 19 slices from eight $1001~ transgenic (O) and 20 slices from eight normal control A CD1 (O) mice. Each point is an average, initial slope of field EPSPs in the CA1 area of the hippocampus. Samples of field EPSPs recorded before (1) and after (2) induction of LTP are illustrated in the inset. (#) The average of four experiments (one CD1 and three S100p) in which LTP was blocked by 50 tJ, M APV. Consequently, only a brief, post-tetanic potentiation, lasting up to 3 min was observed, and no significant LTP is seen. In this and subsequent plots, standard errors of averaged measurements are indicated. Note that LTP was measured up to 40 min after tetanization in a subset of six slices from three animals. (For the results of statistical analyses, see Results.) (B) Time course of LTP (A) and LTD (&) obtained from sister slices of the same four transgenic mice. Eight slices showed LTP, and six slices showed LTD. Note the strong, initial depression lasting 1-2 min, followed by sustained depression lasting at least 20 min. The magnitude of depression ( - 35%, S.E. = 16) was statistically significant (Wilcoxon signed rank test, P=0.031). Also note that LTPs in the transgenic mice of ,A and B are not significantly different from that of normal control (F2,2o=0.99, P>0.05). Paired pulse facilitation illustrated in the insets in A and B was not different from that of normal control in either group of transgenic slices (see Results). In all cases, "tetanus" indicates time of tetanic stimulation applied to the Schaffer collateral/ commissural afferents (11 min).

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in any of the genotype group. SNK tests (sexes pooled) revealed that from the third day onward the daily performance of normal mice was significantly (P<0.05) better (smaller escape latencies) than that of the two transgenic lines, whose performance was not significantly different from each other. The rate of learning after day 3, however, was not different in normal and transgenic mice: The regression coefficients of the partial curves (days 3 through 8) of transgenic and normal mice were not significantly different (ANOVA F2, 54 = 1.70, P>0.05). These results suggest that

the impairment of the transgenic mice arose at the beginning of the hidden platform training.

Comparison of the escape latencies (Fig. 3) measured at the first trial, prior to the training period, with those measured at probe 2 where extra tank visual cues were obscured, showed that all mice performed at the same level at both trials (two-way ANOVA all F values are <0.90, all P values are nonsignificant). These results suggest that normal and transgenic mice were not different at the beginning of the training and that both groups of mice relied on extra tank visual cues when






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Figure3: Learning performance of normal CD1 (n female s = 13,nmale s = 11), $10013 line 5 (nfernale s = 8 ,

n m a l e s = 1 0 ) and $10013 line 8 (nfemales=8, n m a l e s = 7 )

transgenic mice in the hidden platform test in the water maze (sexes pooled). The points of the curves (ffi, normal CD1; C), $10013 line 5; A, $10013 line 8) represent the mean of the daily average escape latencies. Error bars represent standard error. The escape latencies at the first trial and at the probe 2 trial (no extra tank visual cues present) are indicated by separate points. Note that on the first and second days larger platforms were used (20 and 15 cm diam., respectively). All mice improved with training; however, from day 3 the escape latencies of the normal control CD1 mice are significantly smaller than those of the two transgenic lines, whose values are not different from each other (SNK test with P> = 0.05 for nonsignificant ranges). The latencies measured at the first and probe 2 trials are not different from each other (two-way ANOVA). (For details about the training procedure and the results of the statistical analyses, see Materials and Methods and Results).

searching for the platform: Without these cues they performed at the level of the first trial. This suggestion is confirmed by the results of the two probe trials.

PROBE TRIALS

Figure 4 shows the relative duration the mice spent during probe 1 (extra tank visual cues present; Fig. 4a) and probe 2 (extra tank visual cues obscured, Fig. 4b) in the quadrant that previously contained the platform. In probe 1 normal mice spent significantly more time in the quadrant previously containing the hidden platform than

their transgenic counterparts (see Fig. 4). Never- theless, mice of all genotypes spent significantly more time in the correct quadrant than chance (25%) level. This latter result suggests that per- ception of visual cues from the platform was not necessary for mice of any genotype to find the

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Figure 4: Probe trials 1 (A) and 2 (B). The trials were carried out in a random order after the 8-day-long training in the hidden platform test. Both trials lasted for 60 sec. Relative duration of time (%) is shown spent by CD1, $10013 line 5, and line 8 transgenic mice in the quadrant of the water maze that previously contained the hidden platform. Chance level (25%) is indicated by the broken line. Error bars represent standard error. Nonsignificant ranges of SNK test (P> =0.05) are indicated by the thick bars below the graphs. Columns be- longing to the same range, i.e., unbroken line, are not significantly different from each other. Sample sizes are as in Fig. 1. (A) Probe trial 1 : The platform was removed from the tank. Note that the relative duration of time spent by transgenic mice is significantly below the level of that of the normal mice and that transgenic mice are not different from each other. All values are significantly above chance (25%) level (t= 5.992, P<0.001, dr= 17 for the comparison of the smallest value with chance level). (B) Probe trial 2: The platform was removed from the tank, and the view to extra tank visual cues was blocked. Note that the three genotypes of mice are not different from each other and also that their values are not significantly different form chance (25%) level either (t= 1.439, P>0.05, dr= 17, for the comparison of the largest difference between a genotype, line 5, and chance level).

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VISIBLE PLATFORM TEST

platform location. In the absence of extra tank visual cues (probe 2), unlike in probe 1, there was no difference between normal and transgenic mice: All mice performed at chance level (see Fig. 4). This finding demonstrates that without the extra tank visual cues neither the normal nor the transgenic mice were able to find the previous location of the hidden platform.

The swimming patterns during probe 1 were analyzed, and motor abilities of normal control and S10013 transgenic mice were not found different by ANOVA. The frequency of quick turns and the swimming speed did not differ in normal and transgenic mice.

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The learning performance of the transgenic mice was not different from that of the normal in the visible platform test (Fig. 5): Both normal and transgenic mice showed significant improvement;

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Figure 5:

Gerlai et al.

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1 2 5

Days of training Learning performance of CD1 normal and

$10013 transgenic mice in the visible platform test in the water maze (sample sizes as in Fig. 1). The points (11, normal CD1; C), $10013 line 5; A, $10013 line 8) represent the mean of the daily average escape latencies. Error bars indicate standard error. Note that on the first and second days larger platforms were used (20 and 15 cm diam., respectively). All genotypes showed significant decrease in the escape latency with training. Mice of different genotypes are not different significantly from each other on any day. (For details about the training procedure and the results of the statistical analyses see Materials and Methods and Results.)

however, they were statistically indistinguishable from each other on every day of the training (all F values are <3.00; all P values are nonsignificant). These findings suggest that both the normal and the transgenic mice were able to see the visible cue and they were motivated to escape from the water.

MODIFIED VISIBLE PLATFORM WITH NAIVE MICE

One may argue that the visible platform training may be too insensitive to detect impairments in vision or motivation if it constitutes a relatively easy task. Because our mice could find the platform very quickly (Fig. 5) minor impairments af- fecting the performance in the hidden platform training might not have been detected by the visible platform test. Furthermore, because the visible platform test was applied after several days of training in the hidden platform test, one may argue that the level of habituation in the two tests is different. These potential problems are addressed by the modified visible platform training.

The results (Fig. 6) demonstrate that despite the initially high escape latencies, normal and S10013-8 transgenic mice were not different from each other in this test, and that both groups of mice improved significantly with the training. No difference between transgenic and normal mice was revealed by one-way ANOVAs for any day (all F values are <2.54; all P values are nonsignificant). When the visual cue was removed after the training, all mice performed significantly worse and no genotype difference was seen (ANOVA: Geno- type F1,22=0.22, P>0.05; Trim F1,22=33.12, P<0.0001; Genotype Trim F1, 2z = 1.01, P>O.05), suggesting that both normal and transgenic mice were able to see the small visible cue and used it to determine the platform location. The long ( 5 0 - 35 sec) escape latencies at the beginning of training, which are comparable to those obtained in the hidden platform test, demonstrate that the mice found the small platform, marked by a small visible cue, with greater difficulty than the larger platform in the previous visible platform test. Conse- quently, one may consider this test to be suffi- ciently sensitive to pick up motivational or visual impairment. However, the lack of difference found between the normal and transgenic mice, and the significant improvement of both, confirms the previous suggestion that the motivation and vision of transgenic mice are not different from those of the

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Figure 6:

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1 2 3 4 5 No cue marking

Days of training the platform

Learning performance of CD1 normal (nfema,es----6 , nrnales----6) and $10013-8 transgenic mice (nfemales = 7, nmales = 5) in the modified visible platform test. The diameter of the platform was 10 cm throughout the training; a small visible cue was applied to mark the platform location, and naive mice were measured in the test. The points of the curves (11, normal CD1 ; A, $100[3 line 8) represent the mean of the daily average escape latencies. The separate points show the results of a probe trial during which the visible cue was removed. Error bars indicate standard error. Both normal and Sl00lg-8 transgenic mice showed significant improvement with training. Normal and transgenic mice are not significantly different from each other on any day. Both genotypes of mice exhibited a significantly impaired performance when the visible cue was removed. (For details about the training procedure and the results of the statistical analyses see Materials and Methods and Results.)

normal mice. Furthermore, the results also rule out the possibility that habituation could play a role in the difference found between normal and transgenic mice in the hidden platform training. If habituation had been the cause of the difference, a similar effect should have been detected in the modified visible platform test, in which naive mice were measured.

D i s c u s s i o n

The results demonstrate that transgenic mice overexpressing S 10013 in astrocytes in the hippocampus exhibit decreased excitatory postsynaptic potentials in response to high-frequency stimula-

tion. This decrease in the response is also characterized by a higher incidence of LTD. These hippocampal neurophysiological abnormalit ies of the transgenic mice were accompanied by an impaired performance in the spatial task of the Mor- ris water maze. This lends support to the hypothesis that astrocytes are involved in information processing (Smith 1994). Because slow Ca 2+ waves are propagated from astrocytes to neurons through one-way gap junctions (Nedergaard 1994), it is tempting to speculate that S10013, which binds two Ca 2 + cat ions/molecule (Kligman and Hilt 1988; Calissano 1969), may attenuate the spread of these Ca 2+ waves. Alternatively, S10013 could be released from astrocytes (Shashoua et al. 1984; Van Eldik and Zimmer 1987) and bind di- rectl;¢ to unidentified receptors on neurons, induc- ing Ca 2 + responses within the neuron (Barger and Van Eldik 1992).

DECREASED POST-TETANIC POTENTIATION IS CHARACTERIZED BY INCREASED INCIDENCE OF ABNORMAL SYNAPTIC DEPRESSION IN THE HIPPOCAMPUS OF $100~ TRANSGENIC MICE

Our findings demonstate an overall decrease in the size of post-tetanic potentiation elicited by the 100 Hz tetani. This result, however, may be viewed in two different ways. One may stress that LTP and LTD are qualitatively different phenom- ena. In this view, the synaptic responses obtained as a result of high-frequency stimulation should be separately analyzed for potentiation and depression values. Such analysis indicated, not significantly reduced LTP, but significantly high incidence of LTD in the transgenic mice. One may therefore conclude that a mechanism underlying the observed behavioral impairment is not altered LTP but abnormal LTD. Although LTD is a normal feature of hippocampal synaptic physiology, it is usually induced by low frequency prolonged stimulation (Dudek and Bear 1992) and not by the high frequency tetani used in the present study. Because LTD was observed in --25% of the transgenic slices tested, assuming random sampling of the hippocampus with the tissue slice technique, we propose that approximately one-fourth of the CA1 area is incapable of producing normal LTP with the applied stimulation technique, but pro- duces LTD instead. Data showing LTP and LTD in neighboring slices from the same transgenic mice indicate that LTD is not a symptom seen only in

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Gerlai et al.

some, perhaps severely affected individuals, but rather a typical phenomenon present in all transgenic mice.

However, one may view LTP and LTD as processes that share certain common molecular mechanisms (Artola and Singer 1993; Lisman 1994). According to this view, and also because our LTP and LTD responses were elicited by the same stimulation technique, one may pool all data points and analyze them together. The results of such an analysis indicate an overall shift of the distribution curve of post-tetanic potentiation to- wards smaller values in the transgenic mice. One mechanism that commonly underlies LTP and LTD is the involvement of Ca 2 +. Ca 2 ÷ participates in the induction and maintenance of both LTP and LTD (for review, see Artola and Singer 1993). Be- cause exogenously added S10013 has been shown to increase Ca 2 ÷ influx and mobilization of intra- cellularly stored Ca 2 + in cultured glial and neuronal cells (Barger and Van Eldik 1992), chronically elevated S10013 levels in the transgenic mice could alter the calcium balance and a multitude of calcium dependent processes that influence LTP and LTD. One such process may be synaptic fa- tigue. The rapid depression of synaptic responses seen in a portion of transgenic slices immediately after the tetanic stimulation suggests a deficit in post-tetanic potentiation, usually attributed to ac- cumulation of calcium ions in the axonal termi- nals. It is possible that this early response leads to the subsequent, slower LTD. Another process re- suiting in LTD may be attributable to a chronic intracellular Ca 2 + level increase in the hippocampal neurons of the transgenic mice. S100~3 has been shown to be secreted by astrocytes (Shash- oua et al. 1984; Van Eldik and Zimmer 1987), a finding consistent with the increased extracellular immunoperoxidase staining of S 10013 observed in the hippocampus of S100~3 transgenic mice (Friend et al. 1992). The elevated extracellular S10013 level may increase the influx of calcium into neurons in an analogous manner observed in cultured glial and neural cells. A chronically elevated S10013 level may therefore lead to an increased intracellular calcium concentration in the neurons of the S10013 transgenic mice. Several observations suggest that induction of both LTP and LTD requires an increase of intracellular calcium relative to baseline level; however, the threshold of this relative increase is lower for LTD than for LTP (Artola and Singer 1993). One may speculate that such relative increase may be more difficult in

neurons with elevated intracellular calcium levels. Furthermore, a permanently increased calcium level during ontogenesis may induce such com- pensatory mechanisms in the neurons that desen- sitize them against high calcium concentrations. A desensitized neuron may respond less strongly to calcium increase during stimulation. As a result, the stimulation threshold required to induce LTD and LTP in the SIO013 transgenic slice may be shifted upward and the electrophysiological stimulation that would normally induce LTP may induce LTD. This hypothesis is supported by our preliminary observations indicating a lack of LTD in SIO013 slices in response to low frequency stimulation (data not shown) and also by the shift of the S10013 synaptic response distribution curve (Fig. 1). Interestingly, a similar shift toward synaptic depression was observed in transgenic mice harboring a point mutated gene for CAM kinase II (M. Mayford, J. Wang, E.R. Kandell, and T. O'Dell, unpubl.). These mice express a mutated form of CaMKII that autophosphorylates itself indepen- dently of Ca z +, therefore, working as if Ca z + levels were chronicaly elevated. Elevated S 10013 levels may lead to an analogous situation through in- creasing Ca 2+ and thus keeping the otherwise normally functioning CaMKII constitutively switched on.

In view of the many possible effects of S10013 protein on calcium levels in astrocytes and neurons we will direct future studies toward a better understanding of the actual function of this protein in the regulation of calcium levels in cells.

SPATIAL LEARNING TASK-SPECIFIC IMPAIRMENT IN $100~3 TRANSGENIC MICE CORRELATES WITH ABNORMAL HIPPOCAMPAL NEUROPHYSIOLOGY

S10013 has been implicated in the modulation of learning and memory. Reduction of its levels by intracerebral administration of anti-S100 protein antiserum in rats impaired learning processes such as maze performance (Karpiak et al. 1976) and reversal of handedness (Hyden and Lange 1970); and when the antiserum was applied to the hippocampus either in vitro or in vivo, it inhibited LTP (Lewis and Teyler 1986; Fazeli et al. 1990). Increased S10013 protein levels have also been shown to perturb behavior: S10013 transgenic mice displayed decreased T-maze spontaneous alternation rate (Gerlai et al. 1994) and novelty-induced female-specific hyperactivity (Gerlai et al. 1993;

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Gerlai and Roder 1993), behavioral abnormalities compatible with hippocampal dysfunction. Our present results demonstrate a hippocampal-dependent learning deficit as a result of overexpression of S10013 in transgenic mice.

Learning deficits following hippocampal dam- age can be observed when the task demands a representation based on relations among multiple cues but not when the task encourages adaptation to an individual stimulus (Eichenbaum' et al. 1990). Therefore, our results showing impairment of S10013 transgenic mice in the hidden platform test with variable start locations (a spatial task that requires learning multiple extra tank cues) and normal performance in the visible platform tests (a nonspatial task that requires single cue learning) are compatible with the assumption of hippocampal dysfunction.

Nevertheless, transgenic mice could have used alternative, that is, nonspatial, strategies and improved without learning spatial cues. Such strategies, however, would not have allowed the mice to exhibit a preference for the quadrant of the tank with the platform, a result clearly demonstrated by the probe trials. These trials showed that in the presence of external visual cues, all mice were able to find the previous location of the platform; however, in the absence of these cues, they were not. These results show that transgenic mice, though significantly impaired compared with control mice, also learned the location of the hidden platform by using spatial information.

Despite the clear indication of impaired spatial learning performance, one may argue that the impairment of the transgenic mice could be attributable to several factors, other than learning. These factors may include impaired vision, motor dysfunction and/or decreased level of motivation. Previously, we found no sign of motor dysfunction in S10013 transgenic mice in dry open fields and in a bar cross apparatus (Gerlai and Roder 1993; Ger- lai et al. 1993. Although female-specific hyperactivity was noted in the transgenic mice in dry open fields, such abnormality was not observed in the water maze. This apparent discrepancy may be attributable to different time courses of testing in the two test situations. S10013 female-specific hyperactivity in the open field was associated with decreased habituation to novelty during a very short (9 min) time interval (Gerlai and Roder 1993). The habituation procedure used before and the repeated exposure applied during training over days in the water maze probably eliminated

the novelty of the situation together with the novelty-induced S10013 female-specific hyperactivity. This speculation is supported by our results, which show no difference between transgenic and normal mice, and between sexes, at the first trial and also show normal swimming speed and turn- ing frequency in transgenic mice at the probe 1 trial. Furthermore, normal and transgenic mice showed similar performance in the visible platform tests, suggesting that they were equally motivated to escape from the water and could see the visible cue. One may therefore conclude that the difference observed between the normal and transgenic mice in the hidden platform test was because of an impairment in the transgenic mice that compromised their abilities to learn and/or remember multiple spatial cues, a task associated with hippocampal function.

OVEREXPRESSION OF SIOOI3 IMPAIRS BUT DOES NOT ABOLISH SPATIAL LEARNING

After the third day of training in the hidden platform test, the level of performance was significantly worse in the $10013 transgenic mice. How- ever, the slopes of their learning curves were not different from control. Because we can exclude motor dysfunction, impaired vision, or difference in motivation to escape from the water, this tra- jectory of the learning curves suggests that the difference in the performance arose during the first 3 days of the testing.

The dynamics of learning in a spatial task may depend on several factors including the amount of spatial information needed to solve the task, the complexity of the environment, and its transient nature. At the beginning of the test the experimental animal encounters several novel cues to learn. As the training proceeds, however, the number of unlearned cues and the possible number of con- figural combinations between them decrease. One may therefore argue that the initially more de- manding spatial task becomes gradually easier and less sensitive in detecting a small impairment. This argument is compatible with our findings, and it implies that the transgenic mice were able to ac- cumulate spatial information to a degree that, after several days of training, allowed them to improve with a rate indistinguishable from normal. Our findings demonstrate that overexpression of $100J3, unlike the complete lack of a protein such as or-CAM kinase II (Silva et al. 1992a,b) or fyn

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Ger la i e t al.

(Grant et al. 1992), does not result in an abolish- ment of spatial learning; it only causes a certain degree of quantitative impairment.

It is of interest that S100~3 and other genes located on human chromosome 21 (e.g., superox- ide dismutase, 13-amyloid) are overexpressed in Down's syndrome, which involves trisomy of chromosome 21 (Epstein 1987). Overexpression of one or more of these genes could lead to mental retardation (Rabe et al. 1990). It is noteworthy that S10013 has also been shown to be expressed in elevated amounts in Alzheimer disease (Griffin et al. 1989), and this expression could conceivably influence higher brain functions.

Our present findings, demonstrating significant hippocampal neurophysiological alterations and learning impairment as a result of overexpression of S10013, suggest that the protein is involved in higher brain function. Because S100~3 is synthe- sized by and expressed in glial cells, our results also provide a strong support for the recent asser- tion that glial cells are actively involved in information processing in the brain.

Acknowledgments This work was supported by grants to J.R. from the

Network of Centres of Excellence on Neuroscience and the Ontario Mental Health Fundation, and by grants to J.M.W. and A.M. from the Medical Research Council of Canada (MRC). R.G is an MRC-Ciba-Geigy Postdoctoral Fellow and J.R. is an MRC Scientist.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.

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Received May 23, 1994; accepted in revised form February 15, 1995.





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Overexpression of a Calcium-binding Protein, S 1 …learnmem.cshlp.org/content/2/1/26.full.pdftherefore affect neuronal and behavioral phenotypes. Previously, we generated transgenic