Overexpression of urokinase-type plasminogen activator in transgenic mice is correlated with impaired learning. (spatal karnn/olfactor ear/ n/condltoned tase ...
Proc. Natl. Acad. Sci. USA Vol. 91, pp. 3196-3200, April 1994 Neurobiology
Overexpression of urokinase-type plasminogen activator in transgenic mice is correlated with impaired learning (spatal karnn/olfactor ear/ n/condltoned tase averson/neural plstity)
NOAM MEIRI*, TAMAR MASOSt, KOBI ROSENBLUM*, RUTH MISKINt, AND YADIN DUDAI* Departments of *Neurobiology and tBiochemistry, The Weizmann Institute of Science, Rehovot 76100, Israel
Communicated by Michael Sela, December 27, 1993 (received for review October 27, 1993)
cell migration and invasion, have been linked to activation of plasminogen (12-15). Two distinct molecular forms of PA are known in mammals, the urokinase-type (uPA) and the tissue-type (tPA), encoded by two different genes (13-15). PA genes, in particular the uPA gene, exhibit remarkable inducibility and can respond, usually at the level of transcription, to a variety of stimuli (13-15). For uPA, a specific high-affinity receptor found on the surface of several cell types appears to play an important role in the spatial control of PA-catalyzed proteolysis (14, 15). tPA displaying high affinity for fibrin appears to function in physiological thrombolysis in plasma, whereas uPA is considered to be more involved in tissue proteolysis (13-15). In neural cells and tissues, PA activity was correlated with cell migration (16-19), neurite outgrowth and growth cones (20, 21), development (19, 22-25), and regeneration (26, 27). Expression of the gene encoding tPA has been recently shown to be induced in the rat brain in a time course characteristic of immediate early genes in response to seizures, kindling, and long-term potentiation (11). It is not clear whether plasminogen, known to be synthesized in the liver, is available in the brain outside the circulation. That this may be possible has been suggested by the finding that cultured rat brain microglial cells synthesize plasminogen in a constitutive and induced fashion (28). We have now taken advantage of a line of transgenic mice overexpressing uPA in brain (29, 30) to analyze the role of uPA in learning and memory. We report that overexpression of murine uPA in the mouse brain is correlated with impaired behavior in several learning tasks, without significantly affecting sensory or motor capabilities required for successful performance in these tasks. Our data thus corroborate the assumption that uPA participates in neural mechanisms subserving behavioral plasticity.
ABSTRACT Transgenic mice desi ated aMUPA overproduce in the brain murine urokinase-type plainogen activator (uPA), an extracellular protease implicated in tissue remodeling. We have now lid, by in situ hybridization, extensive signal of uPA mRNA in the aMUPA cortex, hippocampus, and amygdala, sites that were not labeled in counterpart wild-type mice. Furthermore, biochemical measurements reveal a remarkably high level of enzymatic activity of uPA in the cortex and hippocampus of aMUPA compared with wildtype mice. We have used the aMUPA mice to exami whether the abnormal level of uPA in the cortex and the limbic system affects lrning ability. We report that aMUPA mice perform poorly in tasks of spatial, olfactory, and taste-aversion learning, while displaying normal sensory and motor capabiliti. Our results snuest that uPA is involved in neural processes subserving a variety of laing types.
The formation of a lasting memory trace in the brain passes through time phases (1-3). Short-term memory lasts for minutes to hours and is brought about by biophysical and posttranslational modifications in synaptic proteins (4, 5). Longterm memory lasts for days to years and is considered to be subserved by structural alterations in neuronal circuits (6). The prevailing molecular framework for realization of structural change subserving long-term memory proposes that modulation of gene expression is involved (2). Indeed, ample evidence indicates that memory consolidation requires de novo protein synthesis (1, 7) and is accompanied by gene activation (8, 9). The morphological and metabolic modifications correlated with the formation of long-term plasticity and memory in identified neuronal circuits suggest that widespread molecular mechanisms of tissue remodeling are recruited in these processes. Ultrastructural analysis revealed evidence for dynamic metabolism of synaptic membrane (6), and molecular analysis revealed modulation of genes whose products take part in cell-cell interactions, such as the neural cell adhesion molecule NCAM (10) and tissue-type plasminogen activator (tPA) (11). Plasminogen activators (PAs) are prime candidate enzymes to be involved in neuronal plasticity, due to their functional potential and versatile inducibility (reviewed in refs. 12-15). PAs are secreted serine proteases specifically converting the ubiquitous inactive zymogen plasminogen into plasmin, a trypsin-like protease of broad substrate specificity. Plasmin, in turn, dissolves the fibrin network ofthe blood clot and is capable of activating latent metalloproteases. The cascade of proteases initiated by PA can degrade most matrix and basement membrane components and interferes with cell-cell and cell-matrix interactions. Events requiring limited extracellular proteolysis, such as tissue remodeling and
MATERIALS AND METHODS Transgenic Mice. Transgenic mice designated aMUPA generated by Miskin et al. (29) were previously described. aMUPA mice carry the cDNA encoding murine uPA linked to the promoter of the murine aA-crystallin gene. The mice were generated from the NIH inbred mouse line FVB/N, which contains an rd mutation leading to photoreceptor degeneration and hence to visual impairment (31). The external phenotype of aMUPA mice did not differ from normal, except that the body weight of aMUPA mice was lower than normal by about 15%. Histological examination of the eye, brain, and other organs of the transgenic mice also did not reveal any abnormality. aMUPA mice produce transgenic uPA in the ocular lens and also in ectopic sites such as retina and brain (29, 30). Throughout the present study one line of aMUPA mice was used, and if not otherwise indicated the mice were in the homozygous state. The mice were housed
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Abbreviations: CTA, conditioned taste aversion; PA, plasminogen activator; tPA, tissue-type PA; uPA, urokinase-type PA; WT, wild type.
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Neurobiology: Meiri et aL five in a cage at 23TC under a 12 hr/12 hr light/dark cycle with water and food ad libitum, unless otherwise indicated. All the behavioral testing was carried out in the light phase. Female mice (60-90 days old, 20-30 g) were routinely used. The major findings reported below were replicated with males, yielding similar results. In Situ Hybrldizton. Mice were deeply anestetized with ly removed, frozen over dry ether and the brain was im ice, and kept at -70TC for 2 days until sliced on a cryostat. Sectioning, fixation, prehybridization, and hybridization were performed according to Hogan et al (32), except that 0.7 M NaCl was used in the hybridization buffer. RNA probes for hybridization were derived from the 1.1-kb EcoRl-Bgl II fragment of the murine uPA cDNA (33) subcloned into the pBS vector (Stratagene). For synthesis of the RNA probe in the antisense orientation, the plasmid was linearized by EcoRl and transcribed with T3 polymerase (Stratagene). For the sense RNA probe, the plasmid was linearized by Xba I and transcribed with T7 polymerase (Stratagene). All polymerase reactions included [-435S]thio]UTP (1000 Ci/mmol; Amersham; 1 Ci = 37 GBq). For hybridization reactions, labeled RNA probes (2-5 x 106 cpm) of sense or antisense orientation were applied to each slide in 25 p; of hybridization mixture. Posthybridization washing at high stringency, RNase treatment, exposure to emulsion (for 10 days), and photography were performed according to Orr-Urtreger et al. (34). PA Activity. Zymographic determination of PA activity was performed as described (29, 35). PA activity was visualized in the gels as clear bands on the darkly stained casein background. Counterpart gels from which plasminogen was omitted did not show any active bands. Water Maze Lrning. The procedures were modified from Morris (36). The mice were trained in a circular swimming pool 90 cm or 140 cm in diameter, depending on the experiment (see below), and 60 cm in height, containing water at 260C. A Plexiglas platform with surface area of 10 cm2 was either submerged 1 cm below water level or protruded 0.5 cm above water, depending on the version of the experiment. Two versions of the task were used, a spatial version and an olfactory version. In the spatial version, the island was hidden under water and the mice had to use extramaze cues to locate it. Extramaze sensory cues were open containers of menthol and almond extract, mice cages, the experimenter, a functioning tissue-culture hood, a dripping water tap, and an operating personal computer, all at fixed locations around the pool. In this version ofthe task, the island was kept in the same location throughout the experiment. In the olfactory version of the water maze task, the platform was protruding above water and marked by almond extract in a small perforated-wall Petri dish taped to the bottom of the platform above the water level. The island was shifted from one location of the pool to another in each session, so that the odor cue, rather than extramaze spatial cues, had to be used by the mouse. In both versions of the water maze experiments, the first day of the experiment was dedicated to swimming training for 90 sec in the absence of platform. In the following days the mice were given, unless otherwise indicated, three consecutive trials to locate the platform, each trial lasting up to 90 sec. If the mouse did not climb the platform within 90 sec, it was placed on it by hand. The mice were required to spend 15 sec of an intertrial interval on the platform. The escape-to-the-platform latency was measured with either a stopwatch or a computerized video tracking system (Lyon Electronique, Lyon, France). To determine whether the mice used spatial cues in locating the platform in the hidden platform version of the task, they were trained in the 140-cm pool for 9 days, two trials per day with an intertrial interval of 2 hr. On the 10th day the platform was removed and the time the mice spent in each
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quadrant of the pool was measured with the computerized video tracking system. Co 1nd Taste Aversion (CTA). CTA was performed similarly to the protocol used in our laboratory for rats (37). On days 1-3 of the experiment, the mice were trained to get their daily liquid ration during 15 min from two pipettes each containing 1 ml of water. Conditioning was performed either once or twice, on day 4 or on days 4 and 5. Saccharin (0.1%) was used as a novel taste, and LiCl as the malaise-inducing agent (0.3 M, 2% of body weight, injected i.p. 20 min after saccharin tasting). For two days following the conditioning, the mice were agivnallowed to drink water. Testing was then performed for three consecutive days on which the mice were presented simulusly with six pipettes, three containing 0.5 ml of 0.1% saccharin and three containing 0.5 ml of water. The aversion index was defined as [ml of water/(ml of water + ml of saccharin)] X 100. I some experiments the effect of LiCl injection was also quantified, on the conditioning day, by monitoring the time from the injection to (i) first occurrence of a "laying on the belly" posture, (ii) first food sniffing, and (iii) first food tasting (38). Activity and Motor Tests. The exploratory and motor behavior was determined in either a water open field or a dry open field. The water open field consisted of the water pool in the absence of the platform. The dry open field was a square cardboard box of dimensions 50 cm x 100 cm x 40 cm (width x length x height) divided graphically into nine equal areas. In the water open field, the swimming patternincluding path, rate, and location-was determined for a total of 90 sec with the computerized video tracking system. In the dry open field, the location of the mice was visually monitored and recorded every 5 sec for a period of 5 min. Ano Test. Since odor was used as a cue in the water maze experiment, and since gustatory behavior influences CTA, we determined the olfactory ability and gustatory motivation of the mice by using a buried-food retrieval test (39). Mice were deprived of food for 24 hr and then introduced into a box ofdimensions 50 cm x 100 cm x 40 cm. The floor was covered with 5 cm of wood shavings and a food pellet was hidden 1 cm below the surface. The time to find the food was measured with a stopwatch. The test was performed twice, with an intertrial interval of 2 hr, during which the mice were returned to their home cage. RESULTS AND DISCUSSION uPA Is Overexpressed in Specific Brain Regions of aMUPA Mice at Both the Transcriptional and the Enzymatic LeAvel. In addition to transgenic expression in the ocular lens as expected from the high tissue specificity of the promoter, aMUPA mice produce the transgenic enzyme in ectopic sites, including retina and brain (29, 30). We have now examined the mouse brain in more detail for expression of transgenic and endogenous uPA. aMUPA and control wildtype (WT) mice were compared through zymogel for PA activity in entorhinal and cingulate cortex and in hippocampal CA1 and dentate gyrus. aMUPA mice exhibited a considerable higher uPA activity in all four regions, while no such difference was observed in the tPA band of the two mouse types (Fig. 1A). We also noted that in WT mice tPA activity was similar in all four brain regions and was always major compared with uPA. To localize transgenic expression in the brain, we compared brain sections of aMUPA and WT mice through in situ hybridization, using an RNA probe that recognized both transgenic and endogenous uPA mRNA. Results obtained with sections through the anterior hippocampus are presented in Fig. 1 B-D. A striking difference between the two mice was found. In the aMUPA section (Fig. 1C) prominent labeling was evident throughout the cortex (short arrow), hippocampus (arrowhead), and amygdala (long arrow). It is also evident that
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FIG. 1. Overexpression of uPA in different brain regions of aMUPA mice. (A) Zymographic analysis for PA activity was performed on samples of homogenates prepared from the entorhinal and cingulate cortex (10 gg of sample protein) and from the hippocampal CA1 and dentate gyrus (3 pg of sample protein), from WT (W) and aMUPA (a) mice. (B-D) RNA localization by in situ hybridization was performed on coronal sections taken from similar positions of WT (B) and aMUPA (C) brain. B and C show darkfield photographs (x3.0) of sections hybridized to the antisense probe. D is a brightfield photograph (x700) of the CA2 region of the aMUPA section. The signal seen in C was absent in adjacent sections hybridized to the sense probe (data not shown).
only a fraction of the cells, 10% at most, were labeled (Fig. 1D). In contrast, in the WT sections no signal for uPA mRNA was detected in the same brain regions (Fig. 1B). aMUPA Mice Are Impaired in Their Performance in Water Maze Learning Tasks. The marked overexpression of uPA in the hippocampus of aMUPA mice made it pertinent to enquire whether the abnormal levels of the enzyme interferes with hippocampus-dependent learning. Water maze tasks are commonly employed to quantify such learning in rodents (36). Several versions of the water maze task can be used. In the basic paradigm, a platform is hidden under water, and the rats or mice, placed in the water, learn to escape onto the platform by identifying its spatial location on the basis of extra-maze topographical cues. This specific task is considered to be hippocampus-dependent. In another version of the water maze paradigm, the island protrudes above water, and the animal can learn the location of the platform by directly observing it. This version of the task is not considered to critically depend on an intact hippocampal formation. In water maze experiments, visual cues are routinely but not solely employed as extra-maze cues (in the hidden-platform version) and as platform markers (in the protruding-platform version). Since aMUPA mice were derived from WT mice carrying a genetic visual impairment, we had to use olfactory and auditory cues, as described under Materials and Methods. Success in the protruding-platform version of the water maze experiments cannot be attributed to spatial mapping, because the platform is shifted from one location to another throughout the experiment. However, because olfactory cues were used by us, and because olfaction is intimately associated with the limbic system in general (40), this specific version of the water maze paradigm cannot be considered to be independent of hippocampal function.
We found that aMUPA mice failed to learn the location of a hidden platform in the water maze under conditions in which WT mice learned the task (Fig. 2A). F1 progeny of a cross between homozygous aMUPA and WT, expressing %50%o of the transgenic uPA of the homozygous aMUPA mice, learned normally (Fig. 2A). Increasing the intensity of training from three to four training trials per day improved the learning performance of the aMUPA mice; even though they still performed significantly worse than WT up to day 3 of training (P < 0.01); the difference became insignificant on day 4 (WT and aMUPA reaching the island within 38 ± 6 sec and 46 ± 7 sec, respectively). The aMUPA mice did not differ from WT in their swimming ability, pattern of their motor movements, and naive coverage of pool area, as determined by water maze open-field analysis (data not shown). The only behavioral difference observed between aMUPA and WT mice was an enhanced tendency of aMUPA mice to jump from the submerged platform back into the water. This was evident at the first training session of each day, when aMUPA jumped z3 times more than WT. Such an enhanced jumping activity was also observed in the a calcium/ calmodulin kinase II-knockout mutant mice reported to fail the water maze task (41). Repeated jumps from the platform may indicate hyperexcitability, and repeated handling and relocation of the mice on the platform by the experimenter may lead to association of the island with negative reinforcement and hence decrease the motivation to climb the platform. However, we found no correlation between the frequency of jumping and the time to reach the platform. Furthermore, the increased tendency to jump from the platform was observed only when the platform was submerged under water; when we placed the unmarked platform 0.5 cm above water, while leaving it in the same location throughout
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Sessons FIG. 2. Performance of aMUPA, WT, and F1 mice in water maze learning tasks. (A) Submerged-island version ofthe task. Mice were trained for three sessions each day. Asterisks here and elsewhere indicate P < 0.05. aMUPA, n = 29; WT, n = 30; F1, n = 10. (B) WT mice learn the spatial location of the hidden platform. The quadrant test described under Materials and Methods was used, in the absence of the platform. START, the point where the mice were introduced into the pool. The square in quadrant 4 indicates the previous position of the submerged platform. n = 10 for each genotype. (C) Performance of aMUPA and WT mice on the odor-cued version of the water maze task. Mice were trained for three sessions each day. The aMUPA mice performed poorly on this version of the maze as well. n = 10 for each genotype.
the experiment, the aMUPA mice did not jump back into the water but still did not learn (data not shown). When the platform is removed from the pool, mice that have learned the spatial location of the platform still spend more time in that quadrant of the pool that earlier had contained it (36). We used this observation to verify that WT mice indeed learn to locate the island by using the spatial information provided to them by the extramaze odors and noises, whereas aMUPA mice fail to form the spatial map. Analysis of the time spent by each strain in the quadrants of the pool after removal of the platform, indeed revealed that in the submerged island version of the water maze task, the WT mice remembered the spatial location of the missing island, whereas aMUPA mice did not (Fig. 2B). We then subjected the mice to the odor-cued platform version of the water maze task. aMUPA mice failed to learn in this task as well (Fig. 2C). The aMUPA mice did not differ from WT in their olfactory and motivational ability as determined by the buried-food retrieval test (129 + 26 vs. 143 + 31 sec to locate the food for WT and aMUPA, respectively). aMUPA Mice Are Impaired in CTA. The water maze experiments indicate that aMUPA mice are impaired in
learning in water maze tasks dependent on spatial and/or olfactory cues. To further determine the generality of the behavioral impairment, we decided to subject the mice to an additional learning task, CTA, which depends on association capabilities and sensory modalities (gustatory, visceral) not taxed by the water maze tasks. CTA tolerates long delays between stimulus (a novel taste) and reinforcement (LiCl injection) and is subserved by several brain areas, including brainstem and gustatory cortex (37, 42). We found that aMUPA mice failed to acquire CTA normally (Fig. 3A). F1 mice behaved normally under the same conditions. Like the behavior observed in the water maze experiments, here, too, enhanced training diminished the behavioral impairment (Fig. 3B). aMUPA mice did not significantly differ from WT in their naive and repeated response to saccharin in the absence of conditioned aversion (Fig. 3C). They also did not differ from normal in their response to LiCl injection, as judged by scoring of the behavioral repertoire following the injection (see Materials and Methods; data not shown). The only behavioral difference found in this context between aMUPA and WT mice related to the normally observed transient suppression of up
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