From the 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,
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
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ABSTRACT |
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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.
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INTRODUCTION |
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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.
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EXPERIMENTAL PROCEDURES |
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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
-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
-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 MasElectrophysiology-- 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.
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RESULTS |
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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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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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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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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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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* 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.
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.
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REFERENCES |
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