Sustained Long Term Potentiation and Anxiety in Mice Lacking the Mas Protooncogene*

Thomas WaltherDagger §, Detlef Balschun, Jörg-Peter Voigtparallel , Heidrun Finkparallel , Werner Zuschratter, Carmen BirchmeierDagger , Detlev GantenDagger **, and Michael BaderDagger §Dagger Dagger

From the Dagger  Max-Delbrück-Center for Molecular Medicine (MDC), D-13122 Berlin-Buch, Germany, § Transgenics in Berlin-Buch GmbH, Berlin-Buch, Germany,  Institute for Neurobiology, D-39008 Magdeburg, Germany, parallel  Institute of Pharmacology and Toxicology, Medical Faculty (Charité) of the Humboldt University, D-10098 Berlin, Germany, and ** Department of Clinical Pharmacology, Free University, D-12200 Berlin, Germany

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The Mas protooncogene is a maternally imprinted gene encoding an orphan G protein-coupled receptor expressed mainly in forebrain and testis. Here, we provide evidence for a function of Mas in the central nervous system. Targeted disruption of the Mas protooncogene leads to an increased durability of long term potentiation in the dentate gyrus, without affecting hippocampal morphology, basal synaptic transmission, and presynaptic function. In addition, Mas-/- mice show alterations in the onset of depotentiation. The permissive influence of Mas ablation on hippocampal synaptic plasticity is paralleled by behavioral changes. While spatial learning in the Morris water maze is not significantly influenced, Mas-deficient animals display an increased anxiety as assessed in the elevated-plus maze. Thus, Mas is an important modulating factor in the electrophysiology of the hippocampus and is involved in behavioral pathways in the adult brain.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Imprinting, the transcriptional silencing of one allele of a gene depending on the sex of the transmitting parent, is an epigenetic regulatory mechanism in mammalian embryogenesis (reviewed by Barlow (1) and Hall (2)). It tightly controls the expression level of a gene, and imprinted genes mostly code for proteins with an indispensable function during ontogenesis. This has been shown by the targeted disruption of several imprinted genes, such as Igf2r, Mash-2, and WT-1, which led to an embryonic or perinatal lethal phenotype of the mutant animal (3-5).

The Mas protooncogene is a maternally imprinted gene in fetal mice located on chromosome 17 in a cluster of imprinted genes, including Igfr2 (6). However, it was recently shown that Mas is not imprinted in adult mice (7) and humans (8). Originally, it was detected by its tumorigenic properties in cell culture (9). It codes for a protein belonging to the class of G protein-coupled receptors with seven transmembrane domains. Angiotensin II and III have been suggested to be ligands for Mas as Mas-transfected cells react with an accumulation of inositol phosphates and calcium after stimulation with these peptides (10). Furthermore, angiotensin II can enhance [3H]thymidine incorporation in stably Mas-transfected cells after growth factor stimulation (11). However, binding of angiotensins to Mas-expressing cells was never shown and, therefore, a direct interaction of angiotensins with Mas has been doubted. It could be shown that Mas and the product of the Mas-related gene (Mrg) enhance the effects of angiotensins on cells expressing angiotensin receptors of the AT1 subtype (12, 13). This family of proteins, Mas, Mrg, and the third known homolog, rat thoracic aorta (RTA) (14), seems to exert a novel modulatory function at least on angiotensin receptors.

Mas is expressed predominantly in testis and forebrain but also in kidney and heart of rodents (15, 16). In the brain, Mas mRNA is localized mainly to neurons in the hippocampal formation, olfactory tubercle, olfactory bulb, piriform cortex and amygdala (16-18). Its expression in the brain is developmentally regulated, being high during embryogenesis and decreasing after birth (16, 18). The localization and the increased expression in hippocampus after brief seizure episodes suggests a role of Mas in synaptic plasticity and memory (19).

To clarify this role we generated and analyzed mice lacking the Mas protooncogene. The animals develop and breed normally and show no obvious morphological alterations in the brain. However, hippocampal long term potentiation and anxiety are markedly affected by the mutation. Thus, Mas seems to modulate the function of receptors in the brain thereby influencing synaptic plasticity and behavior.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Mas Gene Deletion-- The Mas gene was isolated from a genomic library of 129/Sv mice essentially as described for the BALB/c allele (16). A 950-base pair HindIII/BamHI and a 4.4-kilobase pair ScaI/XbaI fragment were used to construct the targeting vector with a neomycin resistance and herpes simplex virus-thymidine kinase gene. The construct was linearized with ClaI and transfected into E14-1 embryonic stem (ES)1 cells (20) by electroporation (single pulse of 0.5 mF, 240 V, in a 0.4-cm cuvette). The transfected cells were cultivated on confluent neomycin-resistant mitomycin-treated primary embryonal fibroblasts in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum (Life Technologies, Inc.) and 1300 units/ml leukemia-inhibiting factor (Life Technologies, Inc.) and selected by the addition of 0.4 mg/ml G418 (Life Technologies, Inc.) and 2 µM Gancyclovir (Syntex) for 8 days. Ninety-six double-resistant clones were isolated and screened for homologous recombinants by Southern blots. Southern blotting was performed according to standard protocols using a KpnI/PstI fragment of the Mas gene. Four targeted clones were obtained, and two of them were injected into C57/Bl6 blastocysts. After retransfer into the uterus of pseudopregnant C57/Bl6 mice, five chimeras were born, three of which produced offspring heterozygous for the targeted mutation. Mas-/- mice were established by mating of the heterozygotes. Mas and beta -actin expression was analyzed by RNase protection assay using a commercially available kit (Ambion), 20 µg of total RNA from testis, 40 µg from brain, and 50 µg from yeast as a control. As probes a 629-nucleotide RNA transcribed from a cloned EcoRV/ScaI fragment of the Mas gene and complementary to 531 nucleotides of the native Mas mRNA and a 304-nucleotide RNA complementary to 250 nucleotides of the beta -actin mRNA (Ambion) were used.

Immunocytochemical and Histochemical Methods-- For immunocytochemical staining all animals were anesthetized with Hypnodil and perfusion-fixed through the aorta with 4% paraformaldehyde in 0.1 M phosphate-buffered saline, pH 7.4. After postfixation overnight, 60-mm thick sections were cut with a vibratome, washed in phosphate-buffered saline (three times, 5 min each), and incubated for 15 min in 50% methanol, phosphate-buffered saline with 1% hydrogen peroxide to inactivate endogeneous peroxidase. Sections were then washed and incubated in 5% bovine serum albumin for 1 h to suppress nonspecific binding. Subsequently, sections were incubated in one of the following well characterized primary antibodies raised against parvalbumin (mouse monoclonal (Sigma); dilution, 1:5000), calbindin D-28K (mouse monoclonal (Sigma); dilution, 1:5000) calretinin (rabbit polyclonal (Swant); dilution, 1:4000), or glial fibrillary acidic protein (GFAP) (mouse monoclonal (Boehringer Mannheim); dilution, 1:500) for 36 h at room temperature. The sections were then thoroughly washed in phosphate-buffered saline and incubated with species-specific biotinylated second antibodies at a dilution of 1:200 for 1.5 h and subsequently with a Vectastain Elite kit (Vector Laboratories) at a dilution of 1:200 for 1.5 h. Visualization of the immunoreaction was performed with 3,3'-diaminobenzidine and 0.01% hydrogen peroxide in Tris-HCl buffer, pH 7.6, for 5-10 min. Finally, sections were mounted on glass slides, dehydrated, and coverslipped in D.P.X. (Aldrich).

Histochemical Timm staining was performed using a modification of the Timm method (21, 22) described by Danscher (23). Briefly, animals were anesthetized with Hypnodil and perfused transcardially with Na2S (0.1%) in 0.16 M Sörensen's phosphate buffer (pH 7.4) followed by buffered glutaraldehyde (3%). Horizontal vibratome sections (30 mm) were mounted on glass slides and incubated in Timm's developing solution for 90-120 min at 30 °C. Cytoarchitectonical details of immunostained and Timm-stained sections were analyzed under a Leica DMRXE microscope, and micrographs of wild-type and Mas-/- mice were taken with a 10× numerical aperture Fluotar or 40× numerical aperture 1.0 Plan Apo objective.

Electrophysiology-- Hippocampal slices were prepared from male mice (7-12 weeks old). After decapitation, the brain was rapidly removed and placed into cold oxygenated physiological solution (ACSF in mM: NaCl 124, KCl 4.9, MgSO4 1.3, CaCl2 2.5, KH2PO4 1.2, NaHCO3 25.6, D-glucose 10, adjusted with 95% O2 and 5% CO2 to pH 7.4). The hippocampi were dissected out, cut into 400-µm thick slices with a tissue chopper, and transferred to a submerged chamber. There, the slices were maintained at 33 °C, superfused with oxygenated ACSF and allowed to recover for at least 1 h. A monopolar, lacquer-coated, steel electrode was placed in the stratum moleculare of the dentate gyrus to stimulate the medial perforant path input. A glass electrode filled with ACSF was lowered to the same level, but about 200 µm apart to record field excitatory postsynaptic potentials (fEPSPs). The initial slope of the fEPSP was used as a measure of this potential, thereby avoiding errors due to a contamination by population spikes. The medial perforant path input was distinguished by its localization and the presence of paired-pulse depression at an interpulse interval of 40 ms. After input-output (I/O) curves were generated by recording fEPSP slopes at an increasing stimulation intensity, the stimulation voltage was adjusted to 40% of the maximum, and test stimuli were applied every 5 min. Once stable responses were obtained for 50 min, LTP was induced by a triple tetanization consisting of 15 bursts of 8 pulses, 200 Hz, interburst interval 200 ms, and applied three times every 10 min. The experiments with mutant mice were interleaved with experiments with normal controls.

For examination of depotentiation, LTP was generated by a single tetanization consisting of 15 bursts of 4 pulses, 100 Hz, interburst interval 200 ms. 5 min after tetanization, potentiation was reversed by a 2-min train of low frequency stimulation at 5 Hz.

To check paired-pulse depression (PPD), three paired pulses with interpulse intervals of 10, 40, 100, 200, and 500 ms were delivered at 60-s intervals at a stimulation voltage adjusted to 40% of the maximum. The three responses were averaged, and the degree of PPD being defined as percent PPD (fEPSP slope second pulse × 100/fEPSP slope first pulse).

Within group comparisons were performed with nonparametric analysis of variance (Friedman-test) and the Wilcoxon matched pairs signed rank test. Intergroup differences were analyzed by the Mann-Whitney U test.

Morris Water Maze Test-- Mice were kept under standardized conditions with an artificial 12-h dark-light cycle. They had free access to food (standard diet Altromin 1326) and water. All behavioral testing was performed between 0900 and 1200 h. The water maze was a square pool (80 cm × 80 cm, 60 cm high) filled with water (20 °C) to a depth of 40 cm. The platform (144 cm2) was submerged by 0.5 cm. For three consecutive days, each mouse was placed in the pool three times with a 5-min interval between trials. Mice were given a 120-s swim. After locating the platform, the mice were allowed to remain on it for 15 s before being returned to the home cage. Mice that did not locate the platform within 120 s were placed on it and scored as 120 s. On day 4 (probe trial), the platform was removed. The time the mice searched in the area where the platform was located during training serves as measure of memory. Escape latencies for individual mice were averaged from each daily trial. Between group comparisons were analyzed by nonparametric analysis of variance (Kruskal-Wallis test), and within group escape latencies were analyzed by nonparametric analysis of variance on repeated measures (Friedman test) with training days as repeated measure.

Elevated-Plus Maze Test-- The plus maze was made of gray Plexiglas and consisted of two open arms (30 × 5 cm) and two enclosed arm (30 × 5 × 10 cm). The arms extended from a central platform (5 × 5 cm). A slight lip (0.3 cm) on the open arms helped to prevent animals falling from the maze. The apparatus was raised to a height of 43 cm by a single central support. The maze was indirectly illuminated by two standing lamps providing 300 and 200 lux at the ends of the open and closed arms, respectively. The experiments were conducted in a sound-attenuated experimental chamber. After adaptation to laboratory conditions for at least 1 h the mouse was placed on the central platform facing an open arm. The behavior of the mouse was recorded for 10 min by an overhead videocamera linked to a recorder in an adjacent laboratory. The data were calculated as percent of total for both frequency (open arm entries/total entries × 100) and duration (time on section/total × 100). Data were analyzed by nonparametric analysis of variance (Kruskal-Wallis test) followed by post hoc Dunn's test.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Gene Targeting-- The targeted disruption of the Mas protooncogene was achieved by transfecting ES cells with the construct shown in Fig. 1A. Homologous recombination led to the deletion of the region coding for the amino-terminal 253 amino acids of Mas including six transmembrane domains in homozygous mice derived from the ES cell clones (Fig. 1B) and to a loss of Mas expression in brain and testis (Fig. 1C).


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Fig. 1.   Generation of mice lacking the Mas protooncogene. A, targeting strategy for the disruption of Mas by homologous recombination in ES cells. The part of the Mas gene coding for the first 253 of 324 amino acids including the ATG codon is replaced by the neomycin resistance gene (neo) containing a transcriptional terminator. The thymidine kinase gene of herpes simplex virus (HSV-tk) is expressed from the 3'-end of the construct to allow the exclusion of clones with random integrations by Gancyclovir. RPA, ribonuclease protection assay; restriction sites: B, BamHI; C, ClaI; E, EcoRI; EV, EcoRV; H, HindIII; K, KpnI; N, NcoI, S, SalI; Sa, SacI; Sc, ScaI; X, XhoI; Xb, XbaI. B, Southern blot analysis of EcoRV/KpnI-cut genomic DNA from offspring of two Mas+/- mice. C, analysis of Mas expression. Total RNA from brain and testis of Mas+/+, Mas+/-, and Mas-/- mice was isolated and assayed for the expression of Mas and beta -actin by RPA. The protected fragment of 531 base pairs is absent in both organs of Mas-/- mice.

The Mas-/- animals are healthy, grow normally, and show no obvious developmental abnormalities. Despite the high expression of Mas in testis (16), the Mas-/- mice are fertile and, in males, the number and motility of spermatocytes as well as testis morphology is unchanged. Furthermore, there is no difference in drinking behavior and in intraarterially measured mean arterial pressure between wild-type and Mas-/- mice.2

Brain Anatomy-- The developmental regulation of Mas expression in the hippocampus suggests a role of Mas in the morphogenesis of this brain region. Therefore, we analyzed the fine structure of the hippocampus in Mas-/- and wild-type mice using immunocytochemical markers and histochemical staining techniques which are frequently employed to visualize distinct cytoarchitectonial distribution patterns within the hippocampal formation (24). As neuronal markers we used antibodies against the calcium-binding proteins parvalbumin, calretinin (Figs. 2, C-F, and 3, C-F), and calbindin D-28K (not shown), which characterize specific subpopulations of interneurons and principal cells (25-27). Moreover, the spatial distribution of astrocytes was mapped by antibodies against GFAP (not shown) and the distribution of zinc positive terminals (Figs. 2, A and B, and 3, A and B) especially of the mossy fibers, was analyzed by the Timm staining (21, 22, 28, 29). As shown in Figs. 2 and 3, no obvious alterations could be detected in the morphology of the hippocampus and its subregions indicating that the cytoarchitectonical distribution patterns and the fine wiring of neuronal subtypes is not affected by Mas ablation.


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Fig. 2.   Comparison of Timm staining, parvalbumin, and calretinin immunostaining in the hippocampal formation of wild-type and Mas-/- mice. A and B, histochemical detection of zinc-containing boutons by the Timm-silver sulfide method reveals prominent staining of the mossy fiber tract through the hilus and CA4 and CA3 region of both wild-type (A) and Mas-/- mice (B). Moderate staining was present in stratum oriens and stratum moleculare of CA1 through CA4 and subiculum, while the molecular layers of the dentate gyrus (DG) are spared. C and D, the immunocytochemical distribution of parvalbumin containing cells in various areas of the hippocampal formation does not show any significant alterations between wild-type (C) and mice lacking the Mas protooncogene (D). E and F, calretinin immunoreactivity was found in a small number of nonpyramidal neurons in the hippocampal subfields CA1-CA4 and in the hilus of both wild-type (E) and mice lacking the Mas protooncogene (F). In addition, prominent calretinin immunoreactivity is present in the inner molecular layer of the dentate gyrus and the neuropil of the hilus. The scale bar indicates 100 µm.


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Fig. 3.   Comparison of Timm staining, parvalbumin, and calretinin immunostaining in the dentate gyrus of wild-type and Mas-/- mice at higher magnification. A and B, besides a prominent Timm staining in the hilus, fine zinc precipitates are seen in mossy fiber collaterals within the granular cell layer of both Mas+/+ (A) and Mas-/- mice (B). Note that the inner molecular layer is spared from Timm staining. C and D, fine structural analysis of parvalbumin immunoreactivity reveals similar distribution patterns in the granular cell layer and the molecular layers of the dentate gyrus of both Mas+/+ (C) and Mas-/- mice (D). E and F, strong calretinin immunoreactivity is found in cell bodies and neuropil within the hilus (HI) and in neuropil of the inner molecular layer of the dentate gyrus while the granular cell layer (GL) and the outer molecular layer (ML) shows only few bouton-like structures of both Mas+/+ (E) and Mas-/- mice (F). The scale bar indicates 20 µm.

Electrophysiology-- The strong expression of Mas in the dentate gyrus (17) led us to investigate LTP, a use-dependent form of synaptic plasticity, in this brain region. LTP, first discovered and most intensively studied in the mammalian hippocampus, has many features that make it a candidate mechanism underlying memory formation at the cellular level. Induction of LTP in the dentate gyrus and CA1 region critically depends on activation of the N-methyl-D-aspartate subtype of the glutamate receptor (30), but the subsequent biochemical events are apparently not completely identical in both regions (reviewed in Chen and Tonegawa (31)).

fEPSPs were recorded in the stratum moleculare of the dentate gyrus and the increase in the initial slope of fEPSPs after high frequency tetanic stimulation was taken as a measure for LTP. Mas-deficient mice exhibited a pronounced prolongation of the synaptic potentiation, becoming significantly different from the controls after 2 h (Fig. 4). In fact, there was no decrease in LTP detectable during the recording period of 6 h. This improved maintenance of LTP was not due to an altered basal synaptic function in Mas mutants as evidenced by input-output (I/O) curves (Fig. 5C). Although Mas-deficient mice displayed a trend to a higher excitability, this was not statistically significant. To the best of our knowledge Mas-deficient mice are the first genetically engineered animal model with an improved LTP.


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Fig. 4.   Long term potentiation in the dentate gyrus of Mas-/- and Mas+/+ mice determined by the initial slope of the fEPSPs. A and B, examples of individual experiments. A triple theta -burst stimulation was applied at the time indicated by the arrows. The insets represent analogue traces, taken during baseline recording (1), immediately after tetanization (2), and 6 h post-tetanus (3). Note the increased slope of the fEPSP after tetanization. The superimposed traces 1 + 3 indicate the remaining potentiation after 6 h. C, average values (means ± S.E.) calculated as percentage of base-line measures (control, n = 8, triangles; Mas-/-, n = 6, squares). Note the more robust synaptic potentiation in the Mas-deficient mice in comparison with the wild-type control. The difference became statistically significant after 3 h (Mann-Whitney U test; p < 0.05).


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Fig. 5.   Depotentiation, basal synaptic transmission and PPD in the dentate gyrus of Mas-/- and Mas+/+ mice as determined by the initial slope of the fEPSPs. A, 5 min after generating LTP by a single theta  burst stimulation (TBS) at the time indicated by an arrow, the fEPSPs were depotentiated by a 2-min train of low frequency stimulation (LFS) at 5 Hz (p < 0.001, Friedman test). Average values (means ± S.E.) calculated as percentage of baseline measures (control, n = 7, triangles; Mas-/-, n = 8, squares). Note the significant increase of fEPSP slope of Mas-/- mice during the first 10 min after cessation of LFS (p < 0.05, Wilcoxon test). B, analogue traces of mutant (upper row) and control mice (lower traces), taken during base-line recording (1), immediately after tetanization (2), immediately after depotentiation (3), and 10 min thereafter (4). The superimposed traces 3 + 4 (broken line, trace 3) indicate the rebound of potentiation in Mas deficient animals after cessation of LFS. C, input/output-curves Mas-/- and Mas+/+ mice. fEPSP slopes were recorded at increasing stimulation intensities until a maximum was attained. There were no significant differences between both groups. Means ± S.E. are given. Mas+/+, n = 14; Mas-/-, n = 15. D, PPD calculated from the ratio of the second fEPSP slope to the first fEPSP slope. At all interpulse intervals, no significant differences were observed between Mas-deficient mice and the wild-type controls. Means ± S.E. are given (Mas+/+, n = 8; Mas-/-, n = 9).

The results of LTP experiments prompted us to test whether deletion of Mas may affect also other forms of synaptic plasticity. Since homosynaptic long term depression, the physiological counterpart of LTP, cannot be induced in the medial perforant path of the adult rat (32), we took advantage of depotentiation, the reversal of potentiation evoked by low frequency stimulation shortly after induction of LTP (33-35). Depotentiation was shown to share several biochemical characteristics with long term depression (35). Although in the Mas mutants tetanic stimulation resulted in a slightly higher magnitude of potentiation, application of a 2-min train of low frequency stimulation, 5 min after induction of LTP, resulted in both groups in a clear depotentiation of an fEPSP slope to intermediate levels significantly different from potentiation and base line (Fig. 5, p < 0.001). Thereafter, the mutants showed a significant rebound of potentiation during the following 10 min (p < 0.05), whereas the controls remained at the same low level.

Next, we examined whether paired-pulse depression was affected in mutant mice. This presynaptically governed form of short term plasticity is observed when two identical stimuli are delivered to homosynaptic afferent fibers in rapid succession (36, 37). In the medial perforant path, the response to the second stimulus is usually depressed by an extent which depends on the interpulse interval (38). Over the whole range of interpulse intervals (10-500 ms) virtually no difference was observed in paired-pulse depression between Mas-deficient mice and the normal controls (Fig. 5D).

Behavior-- The marked effects on LTP suggest a role of Mas in hippocampus-dependent learning behavior. Therefore, the spatial memory of Mas-/-, Mas+/-, and wild-type mice was tested in the Morris water maze task (39). All experimental groups showed a significant decrease in escape latencies onto a hidden platform over the 3 training days, reaching an asymptotic level of performance on day 3. There were no significant group differences in escape latencies on any day of training (Fig. 6A) There was a slight, nonsignificant trend toward a better performance of the Mas-/- and Mas+/- animals in the memory retention test on day 4 (probe trial; Fig. 6B). By measuring the distance swum on the search for the removed platform during probe trial, we observed a significantly increased swimming speed of the Mas-/- mice (Fig. 6C), which might be attributed to different causes including changes in sensomotor function and an increased anxiety.


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Fig. 6.   Behavior of Mas-/-, Mas+/-, and Mas+/+ mice in the Morris water maze. A, escape latency to a hidden platform. Male mice (Mas+/+, n = 8; Mas+/-, n = 9; and Mas-/-, n = 7; age, 14-15 weeks) were trained on 3 consecutive days to swim to a hidden platform (escape). Escape latencies decreased significantly during training in all experimental groups (means ± S.E., p < 0.05). No overall group differences between Mas-/-, Mas+/-, and Mas+/+ mice were found. B, probe trial. The platform was removed and the time was measured that mice spent searching in the quadrant were the platform had been located during training (means ± S.E.). The dotted line represents the chance level. Mas-/- and Mas+/- mice spent more time in the trained quadrant than did Mas+/+ animals. The difference was, however, not statistically significant. C, swimming speed. The distance swum during 120 s in the probe trial was measured. Mas-/- mice swam significantly faster than the wild-type controls (means ± S.E., p < 0.05). Mas+/- mice showed an intermediate phenotype but the differences to the two other groups did not reach statistical significance. Key: black-square, Mas+/+; , Mas+/-; and square , Mas-/-.

To check on changes in anxiety, all groups were examined in the elevated-plus maze (40). The test is based upon the natural aversion of rodents for open spaces. Thus, mice, exposed to the maze, usually prefer the enclosed arms over the open arms. Mas-/- mice entered significantly less often the open arms of the maze and spent less time on this section than did the controls (Fig. 7). Mas+/- mice showed an intermediate behavior, but were not significantly different from either of the two other groups. Anxiety may be confounded with a reduced locomotor activity on the plus maze (41); however, no significant overall group differences were found in total entries on all arms. In conclusion, the behavioral data show a significantly higher level of anxiety in Mas-deficient mice. Impairments of sensomotor function, particularly of visuomotor function, which could interfere with the performance in the tests used, were not observed during the experiments. Therefore, the present data suggest a specific behavioral modification in Mas-/- mice.


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Fig. 7.   Behavior Mas-/-, Mas+/-, and Mas+/+ mice in the elevated-plus maze. A, open arm entries. Mas-/- mice (n = 9) entered less often the open arms of the maze compared with the wild-type controls (n = 9; means ± S.E., p < 0.05). Mas+/- mice (n = 11) showed an intermediate number of entries into the open arms, but the differences were not significant compared either to wild-type or to the Mas-/- mice. B, time in open arms. Mas-/- spent less time in the open arms compared with the wild-type control (means ± S.E., p < 0.05) and, again, Mas+/- mice showed an intermediate but not significantly different behavior. Key: black-square, Mas+/+; , Mas+/-; and square , Mas-/-.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

Mas and the closely related product of the Mrg gene have been shown to specifically stimulate intracellular signaling of angiotensin II (10, 12, 13). In areas where LTP has been observed, such as the hippocampal formation and the piriform cortex, Mas is colocalized with angiotensin II and the angiotensin II receptor AT1 (17, 18, 42). Angiotensin II was reported to reduce the performance in certain kinds of learning paradigms depending on an intact hippocampal formation and to inhibit LTP via AT1 receptors in the dentate gyrus (43-46). Here we show that in the same region, Mas ablation leads to an improved maintenance of LTP. These results shed new light on the functions of central angiotensin II and suggest that Mas may interact with the AT1 receptor mediating or enhancing the inhibition of LTP by this peptide. From our data, we can, however, not exclude that the effects of Mas on LTP are caused by a direct interaction with an hitherto unknown ligand or by the interaction with a receptor for a molecule different from angiotensin II. The more robust LTP in mutant mice is seemingly not a consequence of an altered excitability or a changed presynaptic function since both the I/O curve and PPD were identical in mutants and controls. The consistently strong depression observed over the whole range of interpulse intervals corroborates previous findings on PPD in the medial perforant path of the rat (38).

Although the knockout of the Mas gene resulted only in slight changes of depotentiation, the significant rebound of potentiation in mutant mice after low frequency stimulation supports the hypothesis that Mas is involved in dentate synaptic plasticity. Taken together the data suggest that in Mas-deficient mice the crossover point between LTP and long term depression/depotentiation (47) is shifted in favor of potentiation.

In contrast to its significant effects on LTP, which would classify Mas as a memory suppressor gene (48), its ablation did not result in clear changes of spatial learning in the Morris water maze. To resolve this contradiction, several explanations have to be considered. First, it appears reasonable to assume that learning depends on both, the magnitude and durability of potentiation (49), and only the latter was significantly improved by the Mas ablation. Therefore, only subtle effects, if any, on spatial learning can be anticipated which may be not detectable by the maze paradigm. It is also conceivable that not LTP itself covaries with learning but rather an associated parameter (49). This parameter could be under additional influence of factors that are not experimentally controlled. As a third explanation, learning in the water maze is not exclusively under hippocampal control, although the hippocampus plays an important role. Thus, effects of Mas ablation in other brain regions involved in the spatial learning (e.g. prefrontal and perirhinal cortex) may confound the correlation between hippocampal LTP and water maze learning. In line with these explanations, recent findings indicate that the inducibility and robustness of dentate gyrus LTP is not an ultimate condition for spatial learning and that saturation of LTP in this region does not disrupt spatial learning as might be postulated from theoretical considerations (31, 49-52).

The marked effect of Mas deficiency on anxiety was not expected from its role as positive modulator of central angiotensin II actions. We and others have shown that transgenic rats overexpressing angiotensin II in the brain exhibit an increased anxiety and that the specific antagonist for the AT1 receptor losartan has anxiolytic effects in the elevated-plus maze (53, 54). Mas is expressed in the rat and mouse amygdala; however, there are only low amounts of AT1 receptors suggesting that Mas may have an angiotensin-independent function in this brain region important for anxiety.

The Mas protooncogene has been shown to be imprinted in mouse embryos but not in adult mice and humans (6-8). Recent results obtained in our laboratory with Mas+/- mice confirm the imprinting in the mouse and demonstrate its restriction to distinct embryonic stages and to one single transcript of the Mas protooncogene.2 This may explain that Mas disruption does not lead to a phenotype as severe as was shown for the ablation of other imprinted genes like Igf2r, Mash-2, and WT-1 (3-5). Imprinting in the case of the murine Mas protooncogene may be necessary to constrain its expression in early developmental stages to limit its growth-promoting effects. However, despite the strong developmental regulation of its expression, Mas is not essential for the proper development of the brain and other organs but is a negative regulator of LTP and involved in the control of anxiety.

    ACKNOWLEDGEMENTS

We thank A. Hansson and U. Habenicht for help in the phenotypical analysis of brain and testis, respectively, and R. Brown and U. Frey for critical suggestions. The technical assistance of S. Hartmann, Ch. Sprang and M. Nitz is gratefully acknowledged.

    FOOTNOTES

* This work was supported by the Deutsche Forschungsgemeinschaft Grant Ba1374/1 (to M. B.) and by an "Innovationskolleg" (INK21/A1-1) (to H. F. and M. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Dagger To whom correspondence should be addressed: Max-Delbrück-Center for Molecular Medicine (MDC), Robert-Rössle-Str. 10, D-13122 Berlin-Buch, Germany. Tel.: 49-30-94062193; Fax: 49-30-96062110; E-mail: mbader{at}mdc-berlin.de.

1 The abbreviations used are: ES, embryonic stem; LTP, long term potentiation; fEPSP, field excitatory postsynaptic potential; PPD, paired-pulse depression; ACSF, artificial cerebrospinal fluid.

2 T. Walther and M. Bader, unpublished data.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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