Impaired learning with enhanced hippocampal long-term potentiation in PTPdelta-deficient mice

Abstract

Protein tyrosine phosphatase delta (PTPdelta) is a receptor-type PTP expressed in the specialized regions of the brain including the hippocampal CA2 and CA3, B lymphocytes and thymic medulla. To elucidate the physiological roles of PTPdelta, PTPdelta-deficient mice were produced by gene targeting. It was found that PTPdelta-deficient mice were semi-lethal due to insufficient food intake. They also exhibited learning impairment in the Morris water maze, reinforced T-maze and radial arm maze tasks. Interestingly, although the histology of the hippocampus appeared normal, the magnitudes of long-term potentiation (LTP) induced at hippocampal CA1 and CA3 synapses were significantly enhanced in PTPdelta-deficient mice, with augmented paired-pulse facilitation in the CA1 region. Thus, it was shown that PTPdelta plays important roles in regulating hippocampal LTP and learning processes, and that hippocampal LTP does not necessarily positively correlate with spatial learning ability. To our knowledge, this is the first report of a specific PTP involved in the regulation of synaptic plasticity or in the processes regulating learning and memory.

Fig. 1. Generation of PTPδ knockout mice by gene targeting. (A) The structure of PTPδ protein (upper) and the targeting strategy for the mouse genomic Ptprd locus (lower). PTPδ consists of extracellular immunoglobulin-like (Ig) and fibronectin type III-like (FN III) domains, a transmembrane domain (TM) and two PTP domains, PTPD1 and PTPD2. The exon that encodes the signature motif of PTPD1 was replaced by the neomycin resistant gene (Neo). The DT fragment was ligated at the 5′ end of the vector for negative selection. Arrowheads: PCR primers (P1 and P2). Zigzag line: plasmid DNA. Striped line: the probe for Southern blot analysis. Gray line: the probe for northern blot hybridization analysis. Black boxes: exons. Arrows: DNA fragments obtained by HindIII digestion. H: HindIII, E: EcoRI, P: PstI. (B) Southern blot hybridization analysis of the knockout mice. Genomic DNA from the liver was digested with HindIII and hybridized with the Southern probe. 6-5C and 16-3B are the targeted ES clones from which the knockout mice were derived. The expected fragment lengths for the mutant (Mt) and wild-type (Wt) alleles are indicated by arrowheads. (C) Northern blot hybridization analysis of the knockout mice. Poly(A)⁺ RNA was hybridized with the northern probe that detected the deleted exon. The same filter was also hybridized with a mouse β-actin probe. The position of 28S ribosomal RNA bands is indicated on the left.

Fig. 2. Growth retardation and semi-lethality of Ptprd^–/– mice. (A) Experimental protocol. A food basket was placed on the cage cap under the usual condition (left). The food location was changed from the ceiling to the floor of the cage under the modified condition (right). (B) Semi-lethality of Ptprd^–/– mice under the usual condition. The survival ratios (%) of Ptprd^+/+ (n = 16) and Ptprd^–/– mice (n = 11) under the usual condition, and of Ptprd^–/– mice (n = 6) under the modified condition, are shown. (C) Body weights of control (+/+, +/–) (n = 10) and Ptprd^–/– mice (n = 6) under the modified condition. Open circles (n = 11) show the body weight of Ptprd^–/– mice under the usual condition. Under this condition, most Ptprd^–/– mice died by 35 days of age as indicated by †.

Fig. 3. Histological analysis of Ptprd^–/– mice. Hippocampus sections of 8-week-old Ptprd^+/+ (A) and Ptprd^–/– (B) mice were stained with the KB method. The sections were reacted with anti-synaptophysin antibody (C and D) or non-immune IgG (E). (C and E) Ptprd^+/+; (D) Ptprd^–/–. Hippocampal CA1, CA2, CA3 and dentate gyrus (DG) are indicated. Bar, 1 mm.

Fig. 4. Behavioral abnormalities in Ptprd^–/– mice. (A) Morris water maze task. The time required to reach the hidden platform (mean ± SEM, over the seven testing days) is indicated. Ptprd^+/+, n = 10; Ptprd^–/–, n = 9. One-way ANOVA revealed significantly different performances between the two genotypes [F(1,17) = 27.43, **p <0.0001]. (B) The probe test after the acquisition trials of the hidden platform task. Times spent in each quadrant of the water pool are shown. SE (southeast) is the trained quadrant (closed bar). Ptprd^+/+ mice spent significantly more time in the SE than in the other three quadrants (open bars) [F(3,36) = 19.10, **p <0.0001], while Ptprd^–/– mice did not [F(3,32) = 2.13, p = 0.116]. (C) Number of crosses over the region where the platform was formally located (mean ± SEM) during probe test [U = 20.50, *p <0.05]. (D) Swimming distances (cm) of Ptprd^+/+ (closed bar) and Ptprd^–/– mice (open bar) during the probe test [t = 5.519, **p <0.0001]. (E) Escape latencies (mean ± SEM) of Ptprd^+/+ (squares) and Ptprd^–/– (circles) mice in the visible platform task over three testing days. One-way ANOVA revealed no significant difference between Ptprd^+/+ and Ptprd^–/– mice [genotype: F(1,17) = 4.19, p = 0.057].

Fig. 5. Defects in learning and memory in Ptprd^–/– mice. (A) Diagram of the reinforced alternation task. Sliding doors (broken lines) were placed at the entrance of each arm. Each goal arm had a small well at the distal end to hold a food pellet (circles). (B) Diagram of the radial arm maze task. Food pellets were placed in a well at the tip of each maze arm (circles). (C) Reinforced alternation task. The percentage of correct arm choices is indicated. [*U(10,9) = 15.5, *p <0.05]. A broken line indicates chance level of this task. (D) Running times of sample and choice trials in reinforced alternation task. Running time per trial are represented as mean ± SEM [F(1,17) = 3.776, p = 0.069]. (E) Radial eight-arm maze task. Number of total errors made before obtaining all eight foods (mean ± SEM) is indicated [F(1,17) = 13.387, **p <0.005]. (F) Radial eight-arm maze task. Number of correct choices in the first eight trials (mean ± SEM) is shown [F(1,17) = 6.773, *p <0.05]. (G) Running time in eight-arm maze task. Running times per choice are represented as mean ± SEM [F(1,17) = 2.695, p = 0.119].

Fig. 6. Enhanced synaptic transmission in the hippocampal CA1 and CA3 regions of Ptprd^–/– mice. (A and B) Input–output relationship of fEPSP slope versus stimulus intensity at the Schaffer-CA1 pyramidal cell synapses (A) and at the CA3 pyramidal cell synapses (B) in Ptprd^–/– (open circles, n = 6) and Ptprd^+/+ mice (closed squares, n = 6). Data are presented as mean ± SEM. (C and D) Plots of fEPSP slope versus presynaptic fiber volley amplitude at the Schaffer-CA1 pyramidal cell synapses (C) and at the CA3 pyramidal cell synapses (D) in Ptprd^–/– (open symbols, n = 6) and Ptprd^+/+ mice (closed symbols, n = 6). (E and F) Comparison of PPF at the Shaffer-CA1 pyramidal cell synapses (E) and at the CA3 pyramidal synapses (F) in Ptprd^–/– (open circles, n = 8) and Ptprd^+/+ mice (closed squares, n = 9). Data are presented as mean ± SEM (Student’s t-test, *p <0.05, **p <0.01). (G and H) Comparison of LTP induced by tetanic stimulation in Ptprd^+/+ (closed squares) and Ptprd^–/– mice (open circles). Tetanus was applied at the times shown by arrows. (a and b) Traces show averaged EPSP obtained at the times indicated. (G) LTP at the CA1 pyramidal cell synapses. The amplitude of LTP was calculated as the ratio of average field responses for 40 min after tetanic stimuli to the control field responses before tetanus. Data are presented as mean ± SEM. LTP induction was significantly enhanced in Ptprd^–/– mice (n = 12 in Ptprd^–/–, n = 10 in Ptprd^+/+). (H) LTP at CA3 pyramidal cell synapses. Tetanus was applied three times as shown by arrows. In this region, the LTP was also enhanced in Ptprd^–/– mice (n = 10 in both Ptprd^–/– and Ptprd^+/+ mice).