Receptor protein tyrosine phosphatase is essential for hippocampal neuronal migration and long-term potentiation
2003; Springer Nature; Volume: 22; Issue: 16 Linguagem: Inglês
10.1093/emboj/cdg399
ISSN1460-2075
AutoresA. Petrone, Fortunato Battaglia, Cheng Wang, Adina Dusa, Jing Su, David Zagzag, Riccardo Bianchi, Patrizia Casaccia‐Bonnefil, Ottavio Arancio, Jan Sap,
Tópico(s)MicroRNA in disease regulation
ResumoArticle15 August 2003free access Receptor protein tyrosine phosphatase α is essential for hippocampal neuronal migration and long-term potentiation Angiola Petrone Angiola Petrone Department of Pharmacology, NYU School of Medicine, 550 First Avenue, New York, NY, 10016 USA Search for more papers by this author Fortunato Battaglia Fortunato Battaglia Nathan Kline Institute for Psychiatric Research, Orangeburg, NY, 10962 USA Department of Psychiatry, NYU School of Medicine, 550 First Avenue, New York, NY, 10016 USA Search for more papers by this author Cheng Wang Cheng Wang Department of Pharmacology, NYU School of Medicine, 550 First Avenue, New York, NY, 10016 USA Search for more papers by this author Adina Dusa Adina Dusa Department of Pharmacology, NYU School of Medicine, 550 First Avenue, New York, NY, 10016 USA Search for more papers by this author Jing Su Jing Su Department of Pharmacology, NYU School of Medicine, 550 First Avenue, New York, NY, 10016 USA Search for more papers by this author David Zagzag David Zagzag Department of Pathology, NYU School of Medicine, 550 First Avenue, New York, NY, 10016 USA Search for more papers by this author Riccardo Bianchi Riccardo Bianchi Department of Physiology and Pharmacology, SUNY Downstate Medical Center, Brooklyn, NY, 11203 USA Search for more papers by this author Patrizia Casaccia-Bonnefil Patrizia Casaccia-Bonnefil Department of Neuroscience and Cell Biology, UMDNJ, Piscataway, NJ, 08854 USA Search for more papers by this author Ottavio Arancio Ottavio Arancio Nathan Kline Institute for Psychiatric Research, Orangeburg, NY, 10962 USA Department of Psychiatry, NYU School of Medicine, 550 First Avenue, New York, NY, 10016 USA Department of Physiology and Neuroscience, NYU School of Medicine, 550 First Avenue, New York, NY, 10016 USA Search for more papers by this author Jan Sap Corresponding Author Jan Sap Search for more papers by this author Angiola Petrone Angiola Petrone Department of Pharmacology, NYU School of Medicine, 550 First Avenue, New York, NY, 10016 USA Search for more papers by this author Fortunato Battaglia Fortunato Battaglia Nathan Kline Institute for Psychiatric Research, Orangeburg, NY, 10962 USA Department of Psychiatry, NYU School of Medicine, 550 First Avenue, New York, NY, 10016 USA Search for more papers by this author Cheng Wang Cheng Wang Department of Pharmacology, NYU School of Medicine, 550 First Avenue, New York, NY, 10016 USA Search for more papers by this author Adina Dusa Adina Dusa Department of Pharmacology, NYU School of Medicine, 550 First Avenue, New York, NY, 10016 USA Search for more papers by this author Jing Su Jing Su Department of Pharmacology, NYU School of Medicine, 550 First Avenue, New York, NY, 10016 USA Search for more papers by this author David Zagzag David Zagzag Department of Pathology, NYU School of Medicine, 550 First Avenue, New York, NY, 10016 USA Search for more papers by this author Riccardo Bianchi Riccardo Bianchi Department of Physiology and Pharmacology, SUNY Downstate Medical Center, Brooklyn, NY, 11203 USA Search for more papers by this author Patrizia Casaccia-Bonnefil Patrizia Casaccia-Bonnefil Department of Neuroscience and Cell Biology, UMDNJ, Piscataway, NJ, 08854 USA Search for more papers by this author Ottavio Arancio Ottavio Arancio Nathan Kline Institute for Psychiatric Research, Orangeburg, NY, 10962 USA Department of Psychiatry, NYU School of Medicine, 550 First Avenue, New York, NY, 10016 USA Department of Physiology and Neuroscience, NYU School of Medicine, 550 First Avenue, New York, NY, 10016 USA Search for more papers by this author Jan Sap Corresponding Author Jan Sap Search for more papers by this author Author Information Angiola Petrone1, Fortunato Battaglia2,3, Cheng Wang1, Adina Dusa1, Jing Su1, David Zagzag4, Riccardo Bianchi5, Patrizia Casaccia-Bonnefil6, Ottavio Arancio2,3,7 and Jan Sap 1Department of Pharmacology, NYU School of Medicine, 550 First Avenue, New York, NY, 10016 USA 2Nathan Kline Institute for Psychiatric Research, Orangeburg, NY, 10962 USA 3Department of Psychiatry, NYU School of Medicine, 550 First Avenue, New York, NY, 10016 USA 4Department of Pathology, NYU School of Medicine, 550 First Avenue, New York, NY, 10016 USA 5Department of Physiology and Pharmacology, SUNY Downstate Medical Center, Brooklyn, NY, 11203 USA 6Department of Neuroscience and Cell Biology, UMDNJ, Piscataway, NJ, 08854 USA 7Department of Physiology and Neuroscience, NYU School of Medicine, 550 First Avenue, New York, NY, 10016 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:4121-4131https://doi.org/10.1093/emboj/cdg399 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Despite clear indications of their importance in lower organisms, the contributions of protein tyrosine phosphatases (PTPs) to development or function of the mammalian nervous system have been poorly explored. In vitro studies have indicated that receptor protein tyrosine phosphatase α (RPTPα) regulates SRC family kinases, potassium channels and NMDA receptors. Here, we report that absence of RPTPα compromises correct positioning of pyramidal neurons during development of mouse hippocampus. Thus, RPTPα is a novel member of the functional class of genes that control radial neuronal migration. The migratory abnormality likely results from a radial glial dysfunction rather than from a neuron-autonomous defect. In spite of this aberrant development, basic synaptic transmission from the Schaffer collateral pathway to CA1 pyramidal neurons remains intact in Ptpra−/− mice. However, these synapses are unable to undergo long-term potentiation. Mice lacking RPTPα also underperform in the radial-arm water-maze test. These studies identify RPTPα as a key mediator of neuronal migration and synaptic plasticity. Introduction Protein tyrosine phosphatases (PTPs) constitute a broad gene family, dating back to the earliest stages of animal evolution. They are essential to the reversibility of tyrosine phosphorylation as a regulatory mechanism, and are thus particularly relevant in processes where speed or dynamic turnover are of essence. Phosphorylation/dephosphoryl ation on tyrosine residues in proteins is continuous and highly dynamic, as indicated by the fact that PTP inhibition, even without kinase stimulation, quickly leads to excessive accumulation of phosphotyrosine and activation of signaling pathways. Thus, it is the balance between kinase and PTP activities that determines the phosphorylation level of a protein or site. Tyrosine dephosphorylation is, in turn, regulated in complex and dynamic ways. The size and complexity of the PTP family are of the same order as the tyrosine kinases (Andersen et al., 2001), and PTPs are subject to numerous regulatory mechanisms (Ostman and Bohmer, 2001). Knowledge of the cellular and developmental functions of individual PTPs lags far behind that for tyrosine kinases. Much progress thus far is based on cellular models or genetic approaches in non-mammalian systems. A recurrent finding in cellular studies has been their intimate involvement in cell–substrate or cell–cell interactions; alteration of a cell's PTP profile affects cellular adhesion or migration (Petrone and Sap, 2000). The close link between PTPs and cell–cell or cell–matrix communication is underscored by the existence of multiple receptor PTPs (RPTPs), whose ectodomains can interact with matrix or cell adhesion molecules. Studies of such RPTPs in Drosophila and chicken reveal roles in axonal outgrowth and guidance, but the substrates and molecular mechanisms mostly remain to be elucidated (Stoker, 2001). Current understanding of the role of RPTPs in the mammalian central nervous system (CNS) is limited, and the phenotypes of mammalian RPTP loss-of-function mutants are sometimes subtle. Mice lacking PTPσ display aberrant pituitary development, and a number of architectural abnormalities, many of which are resolved with age, suggesting a developmental delay (Elchebly et al., 1999; Meathrel et al., 2002). Mice deficient for PTPδ lack anatomical abnormalities, but combine strengthened hippocampal long-term potentiation (LTP) with impaired learning (Uetani et al., 2000). The paucity of knowledge of the contributions of RPTPs to development and function of the mammalian CNS is surprising given some of their interaction partners and substrates. PTPβ/ζ is a ligand for multiple cell adhesion molecules, and interacts with sodium channels; PTPβ/ζ-null mice display no obvious developmental defects (Harroch et al., 2000), but show impaired recovery from experimental demyelination (Harroch et al., 2002). In the peripheral nervous system, PTPϵ plays a role in myelination by Schwann cells, controlling potassium channel phosphorylation and activity (Peretz et al., 2000). Several considerations led us to address the function of RPTPα (encoded by the Ptpra locus) in the nervous system. First, the protein is abundantly and dynamically expressed during CNS development (den Hertog et al., 1996; Ledig et al., 1999), and remains high in the adult. Secondly, RPTPα associates with potassium channels, controlling their phosphorylation and activity in response to neurotransmittors (Tsai et al., 1999; Imbrici et al., 2000), and with the neural adhesion molecule contactin (Zeng et al., 1999). Thirdly, RPTPα regulates kinases of the Src family (SFKs) (Ponniah et al., 1999; Su et al., 1999; Zheng et al., 2000), by counteracting phosphorylation of their inhibitory C-terminal phosphorylation sites by the tyrosine kinase Csk. As a consequence, these sites are hyperphosphorylated in cells lacking RPTPα, the kinase activity of Src and Fyn in brain lysates and fibroblasts from Ptpra−/− mice is reduced (Ponniah et al., 1999), and integrin signaling in Ptpra−/− fibroblasts is impaired (Su et al., 1999; von Wichert et al., 2003). Csk and its targets Src and Fyn have important roles in multiple aspects of CNS development and function. Inactivation of Csk disrupts early neural tube development (Imamoto and Soriano, 1993). Mice lacking Fyn display anatomical abnormalities in hippocampus and olfactory bulb, and defective LTP (Grant et al., 1992). NMDA-receptor subunits are in vivo substrates for Fyn (Nakazawa et al., 2001), and Src positively modulates NMDA-receptor function (Yu and Salter, 1999). While, individually, the CNS phenotypes of SFK ablation are relatively mild, their functions are highly redundant (Stein et al., 1994). We wished to address the significance of the numerous observations that relate RPTPα to molecules important for CNS function, and assessed the effect of ablating the Ptpra gene on CNS development and hippocampal synaptic plasticity. Because of the functional relationship between RPTPα and SFKs, we expected that this could also contribute new insights into the roles of this family of kinases in the CNS. Results Macroscopic analysis of Ptpra−/− mice Our previously reported approach to generate RPTPα-deficient mice led to complete loss of detectable RPTPα protein (Su et al., 1999). Ptpra−/− animals were born at close to Mendelian ratios, were viable, and had a normal lifespan. Closer scrutiny revealed impaired fertility, and modest reductions in weight and size, but no overt neurological or behavioral phenotypes. Macroscopic observation showed altered morphology of Ptpra−/− forebrain, with the mutant brain displaying a less elongated, more stubby appearance compared with wild-type (WT) animals (Figure 1A). The cerebellum also appeared smaller in size. Figure 1.Abnormal brain structure in adult Ptpra−/− mice. (A) Overall brain morphology. Dorsal view of two WT (top) and two mutant (Ptpra−/−, bottom) brains. Scale is in centimeters. (B) Hematoxylin–eosin/luxol blue staining of parasagittal sections of hippocampus from WT (left) and mutant (right). The lower panels show progressively higher magnifications of SP. The graph shows the relative distribution of cell bodies in SO, SP and SR for WT and mutant (Ptpra−/−) CA1 (error bars indicate standard deviation; n = 7; ***P < 0.001; *P < 0.05; cells in SO + SP + SR = 100%). Cells were classified as belonging to SO when separated from the SP by at least one cell diameter; hence, this graph underestimates the mutant SP's disorganized texture and ragged boundary. (C) Hematoxylin–eosin/luxol blue staining of somatosensory cortex (left, WT; right, Ptpra−/−). Note the recognizable layers IV and V (pyramidal) in both genotypes, and the less distinct nature of the layer I/II boundary in the mutant. Download figure Download PowerPoint Histological characterization of adult Ptpra−/− brain Histological analysis revealed a striking pattern of disorganization in Ptpra−/− hippocampus. Cell bodies in the pyramidal cell layer (stratum pyramidale; SP) in the CA1 region were much less compacted in mutant than WT animals, in extreme cases even showing layer discontinuity (Figure 1B). At the same time, excess cell bodies were found in the white matter of Ptpra−/− stratum oriens (SO), with the mutant SO tending to be thicker than in WT controls. This phenotype was statistically highly significant (Figure 1B). The total number of cell bodies in adult CA1 (i.e. SO + SP + SR) did not differ between WT and mutant (P = 0.20; n = 7). However, whereas in WT 11% of total cell bodies were found in SO, this number was increased to 28% in the mutant (P < 0.001; n = 7/genotype). Furthermore, cell bodies in WT SO were almost always located close to the SP proper, while most cell bodies in mutant SO were located at significant distances from the SP, often closer to the Alveus than to the SP. This phenotype was observed at all adult stages, and was independent of genetic background (it manifested itself equally on a mixed 129SVJ × 129SvEv background, or after five backcrosses to C57/Bl6). No such hippocampal malformation was seen in Ptpra+/− heterozygotes (see Supplementary figure 1 available at The EMBO Journal Online). More subtle abnormalities were observed in other brain regions. Grossly, neocortical lamination appeared normal, with distinguishable layers IV and V in somato-sensory areas; however, transitions between neocortical layers appeared more diffuse. There was a modest but consistent tendency for the boundary between layers I and II to be less regular, and for layer I to be thinner (Figure 1C). The cerebellum displayed a slight reduction in Purkinje cell numbers and disturbed packing of Purkinje cell bodies (data not shown). Ectopic cells in SO have a CA1 pyramidal phenotype The ectopic cell population in SO displayed characteristics of pyramidal hippocampal neurons. Besides expressing the neuronal marker neuN (data not shown), they expressed SCIP (Figure 2A), a specific marker for pyramidal cells in CA1 absent from inhibitory interneurons (Grove and Tole, 1999). They were negative for the interneuronal marker parvalbumin (data not shown). Furthermore, MAP2 staining revealed dendrites emanating from the ectopic cell bodies in SO that extended through the pyramidal cell layer into stratum radiatum (SR; Figure 2C, arrowheads). This cytoarchitecture is similar to that of bona fide pyramidal cells, whose dendrites in SR receive synaptic input from the Schaffer collateral pathway emanating from CA3 pyramidal cells. This staining also revealed that, while the overall direction of dendritic targeting of pyramidal cells in Ptpra−/− animals was normal, their trajectory was more disorganized and meandering than in WT animals (Figure 2C). Lastly, ectopic cells in adult Ptpra−/− SO stained positive for lacZ, indicating that the Ptpra promoter is transcriptionally active in the ectopic cells, as it is in pyramidal cells within SP (Figure 2B). Strong LacZ staining also occurred in pyramidal cells of the phenotypically normal heterozygotes (data not shown). Figure 2.Ectopic cells in hippocampal CA1 express RPTPα and pyramidal cell markers. (A) Staining of Ptpra−/− CA1 for the CA1 pyramidal cell marker SCIP. Neurons in SP, as well as ectopic cells in SO, stain positive. (B) LacZ staining of Ptpra−/− hippocampus. The CA1 region in the top panel (dotted box) is enlarged in the bottom panel. LacZ is expressed from the Ptpra promoter as a consequence of the homologous recombination strategy used (Su et al., 1999). Expression is seen both in bona fide pyramidal cells in SP, and in the ectopic cells in SO. (C) MAP2 immunostaining of WT (top) and mutant (bottom) hippocampus. Arrowheads indicate the presence of radially oriented dendrites in mutant SO. Download figure Download PowerPoint Abnormal radial neuronal migration during development of Ptpra−/− hippocampus A recurrent feature in CNS development is that the eventual positions of neurons are distinct from the areas where their terminal mitosis has occurred. A major pathway of cell movement in forebrain development involves a radial course from periventricular proliferative zones towards the pial surface (Nadarajah and Parnavelas, 2002). Therefore, the accumulation of pyramidal cells in Ptpra−/− SO, and their relative deficiency in SP, may reflect an underlying migratory abnormality, since SO is the zone through which these cells migrate in order to take up their eventual position in SP. Indeed, the Ptpra−/− phenotype bears similarity to that of other mouse mutants where radial neuronal migration is disturbed, such as heterozygosity for Lis1 (the gene for type I human lissencephaly), inactivation of the genes for VLDR and ApoER2, or hypomorphic alleles of Dab1 (Trommsdorff et al., 1999; Magdaleno and Curran, 2001; Herrick and Cooper, 2002; Nadarajah and Parnavelas, 2002). To test the hypothesis of defective radial migration during morphogenesis of Ptpra−/− hippocampus, we used BrdU birthdating. Pregnant heterozygote females, mated with heterozygote males, were injected with a single pulse of BrdU on day E15.5, the expected peak of radial migration in hippocampus. Because of the short half-life of BrdU, this intervention marks cells undergoing mitosis during a narrow time-window. Since BrdU is diluted out from nuclei of cells that undergo subsequent rounds of mitosis, only cells that underwent their terminal mitosis at the time of injection remain strongly labeled, and their eventual location in the adult can be tracked. This analysis revealed that, as expected, the majority of cells (91%) labeled by BrdU at E15.5 in WT embryos had migrated to the hippocampal SP in the adult, with only 9% taking up residence in the SO (Figure 3A and B). In contrast, in Ptpra−/− littermates, 45% of BrdU-labeled cells did not reach SP but were found in ectopic locations in SO (Figure 3A and B) (P < 0.001; n = 8/genotype). This experiment also allowed us to analyze the fate of BrdU-labeled cells in neocortex. It is well known that radial migration follows an 'inside-out' pattern, with cells born later progressively migrating over longer distances and taking up more apical positions (Nadarajah and Parnavelas, 2002). While this phenomenon holds in both archi- and neocortex, it is most easily observed in the latter. We found that the distribution of labeled cells in Ptpra−/− neocortex largely tracked that of WT littermates (Figure 3C), with early BrdU pulses (E13.5) predominantly labeling deeper layers (data not shown). Thus, the inside-out migratory pattern is conserved in the mutant, and no inversion has occurred. However, in Ptpra−/− animals, there was a consistent tendency for E15.5-birthdated neocortical neurons to assume adult positions that were scattered more broadly over the various neocortical layers (Figure 3C). Figure 3.BrdU birthdating analysis. (A) Anti-BrdU immunostaining on coronally sectioned hippocampi of WT and Ptpra−/− littermates derived from a litter whose mother was injected with a single pulse of BrdU on day 15.5 of gestation. Sections were obtained at the adult stage and stained with anti-BrdU (red) and DAPI (blue) (ALV, Alveus). (B) Quantitation of data obtained in (A). Mean percentage of total number of anti-BrdU-stained cells in SP and SO (cells in SO + cells in SP = 100%) for each genotype that had reached either of these layers. Error bars denote standard deviations (n = 8/genotype; ***P < 0.001). (C) Similar experiment as in (A), but at lower magnification, also showing neocortex. Download figure Download PowerPoint RPTPα expression and radial glial morphology during forebrain development During forebrain development, post-mitotic neurons are born from radial glial precursors, the latter having elaborate radial processes extending from their cell bodies in the periventricular zone to the pial surface, which provide the substrate for neuronal 'gliophilic' migration (Noctor et al., 2002). To determine the reason for the abnormal migration in Ptpra−/− mice, we analyzed the normal pattern of RPTPα expression and radial glial morphology during development of Ptpra−/− embryos. Staining of E15.5 Ptpra+/− heterozygote embryos for the lacZ reporter (introduced downstream of the Ptpra promoter; Su et al., 1999) showed RPTPα expression to be highest in deeper forebrain layers, and widespread among cells in intermediate to ventricular zones. Strikingly, hardly any RPTPα expression was detected among neurons located in the densely packed cortical plate, the destination layer into which neurons are actively migrating and assuming their final positions at this developmental stage; this pattern applied to prospective hippocampus as well as neocortex (Figure 4A and B). Figure 4.Altered morphology of radial glia in developing Ptpra−/− forebrain. (A) LacZ staining of coronally sectioned E15.5 Ptpra+/− forebrain, indicating cells where the Ptpra promoter is active (LV, lateral ventricle; III V, third ventricle). (B) Higher magnification of lacZ-stained E15.5 Ptpra+/− coronal section, showing forebrain cerebral wall only; the transmitted light-image was overlaid with that in differential interference contrast mode, so as to visualize the different layers (MZ, marginal zone; CP, cortical plate; IZ + SVZ, intermediate and subventricular zones; VZ, ventricular zone). (C) Coronal sections of E16.5 WT and mutant embryos stained for BLBP (Feng and Heintz, 1995). In the mutant, note the more wavy appearance of the radial glial fibers in the cortical plate, and their more abundant arborization below the meningeal basement membrane. Download figure Download PowerPoint Using anti-BLBP (brain lipid-binding protein) immunostaining (Feng and Heintz, 1995), we found that, while the overall structure of the radial glial processes was retained during embryonic development, Ptpra−/− radial glia manifested a more wavy and irregular appearance, with a more extensive subpial arborization than in WT. This phenotype was particularly pronounced at E16.5 (Figure 4C). Taken together with the low RPTPα levels in cortical plate neurons, this suggests that the migratory defect in Prpra−/− embryos may occur in the early steps of the migratory path, perhaps with a primary origin in the radial glial cells themselves. Normal basal synaptic transmission but defective LTP in adult Ptpra−/− CA1 We wished to determine whether the aberrant development of hippocampus in Ptpra−/− mice correlated with deficiencies in synaptic function, plasticity and higher-order information processing. This question appeared relevant, since the Ptpra−/− mutation, as opposed to other more catastrophic migratory defects, left the major outlines of hippocampal organization recognizable, and did not lead to an overt neurological phenotype. To determine whether the absence of RPTPα affects synaptic transmission, we measured input–output relationships between the Schaffer collateral pathway and CA1 neurons in WT and Ptpra−/− hippocampal slices. The stimulus–response curves of field excitatory post-synaptic potential (fEPSP) slope versus presynaptic fiber volley amplitude did not differ between WT and mutant slices across a range of stimulus intensities (t-test P = 0.27; Figure 5A). We conclude that, in spite of the anatomical abnormality, basal synaptic transmission remained normal in Ptpra−/− CA1. Figure 5.Lack of RPTPα impairs LTP in hippocampal slices. (A) Summary graph of field input–output relationships for WT (open circles) and Ptpra−/− (closed circles). Both genotypes showed similar relationships (P = 0.27). (B) Summary graph of PPF in WT (open circles) and Ptpra−/− (closed) mice. Facilitation was similar at all interstimulus intervals tested (P = 0.19). (C) Representative values of fEPSP slopes from single experiments taken from WT (open circles) and Ptpra−/− (closed) littermates that underwent potentiation. Insets show traces taken 1 min before (C) and 60 min after potentiation (LTP). (D) Summary graphs of LTP in WT (open circles) and Ptpra−/− (closed) mice. The average fEPSP slope is normalized to the baseline value (ANOVA P = 0.0045). (E) Summary graph of LTP in WT (closed circles) and heterozygote Ptpra+/− (open) mice (ANOVA P = 0.046). (F) Summary graph of PTP (in the presence of 25 μM D-APV) in WT (open circles) and Ptpra−/− (closed). Download figure Download PowerPoint Another synaptic parameter examined was paired-pulse facilitation (PPF), the enhanced response of the second of two closely spaced stimuli. It is thought to reflect build-up of residual Ca2+ due to the action potential from the first depolarization of the terminal, leading to enhanced transmitter release at the arrival of the second stimulus. As the time between stimuli increases, facilitation approaches zero, since they are seen as independent. Therefore, PPF is considered a measure of presynaptic function. As shown in Figure 5B, PPF was observed for stimuli applied at 10–200 ms intervals in both WT and mutant mice, with no significant difference between both genotypes (P = 0.19). Thus, lack of RPTPα does not compromise the presynaptic mechanism of PPF in CA1. Synapses from the same animal were next tested for their ability to undergo plastic changes. The ability to support LTP, a lasting enhancement of synaptic efficacy, is widely utilized as a model of learning and memory. In CA1 of hippocampal slices, θ-burst-induced LTP (three series of 10 burst trains) was severely affected in Ptpra−/− mice compared with WT littermates (Figure 5C and D). At 60 min after LTP induction, when WT mice showed a potentiated response in the fEPSP slope of 240.9 ± 22.1% of the baseline pre-θ-burst stimulation (n = 10), potentiation was only 144.7 ± 28.2% of baseline value in Ptpra−/− mice (n = 12). This impairment in potentiation in the mutant animals was of high statistical significance (P = 0.0045 by ANOVA). LTP was also impaired in Ptpra−/− mice when it was elicited with a weak tetanus (one series of 10 burst trains); at 60 min after LTP induction using this protocol, Ptpra−/− mice showed 140.4 ± 8.6% potentiation (n = 6) compared with 181.4 ± 9.5% potentiation in WT (n = 6; P = 0.0057; data not shown). Elimination of RPTPα expression thus severely compromises the capability of synaptic plastic change in the CA1 region. Although to a lesser extent, LTP was also impaired in heterozygous Ptpra+/− mice. At 60 min after LTP induction (three series of 10 burst trains), Ptpra+/− mice showed 179.9 ± 19.8% potentiation (n = 6) compared with 229.3 ± 17.9% in WT littermates (n = 6; P = 0.046; Figure 5E). An analysis of the LTP results revealed that post-tetanic potentiation (PTP) of the fEPSP was smaller in Ptpra−/− mice (Figure 5C and D). PTP is believed to be an indication of presynaptic function. It reflects a period of enhanced transmitter release due to loading of the presynaptic terminal with Ca2+ during the tetanus. To further test whether PTP impairment in Ptpra−/− mice was caused by a reduction in transmitter release during the tetanus, we induced PTP in the presence of the NMDA antagonist D-APV to block LTP-inductive mechanisms. We did not see any difference in the degree of PTP between WT and Ptpra−/− mice (Figure 5F), suggesting that impaired transmitter release during the tetanus was not responsible for the LTP reduction. Another possible explanation for the LTP impairment in Ptpra−/− mice is that synaptic fatigue instead of potentiation per se is responsible for the abnormal plasticity. To address this issue, a train of 40 pulses at the same frequency as the θ-burst stimulation (100 Hz) was applied to a group of slices from Ptpra−/− mice and WT controls. No difference in the change of the fEPSP slope during the tetanus was observed between the two genotypes: the percentage of the 40th fEPSP slope over the first during the tetanus was 60 ± 5% in Ptrpa−/− versus 65 ± 4% in WT (n = 7 for both groups; data not shown), suggesting that synaptic fatigue does not account for the LTP impairment. Impaired memory in Ptpra−/− animals We used the radial-arm water-maze task, considered capable of assessing working (short-term) memory, to examine learning in Ptpra−/− mice. In this test, which is more efficient in sample size requirements than other memory tasks typically used for rodents, mice are required to learn and memorize the location of a hidden platform in one of the arms of a maze with respect to spatial clues. The test has been used in the analysis of other types of transgenic mice, and depends upon hippocampal function (Diamond et al., 1999; Arendash et al., 2001). WT mice showed strong learning and memory capacity (Figure 6A, open circles); they averaged about eight errors on the first trial as they sought out the new platform location for that day, but averaged less than one error by acquisition trial f
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