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• W1969275043 abstract "Polysialic acid (PSA) is a post-translational protein modification that is widely expressed among neural cell types during development. Found predominantly on the neural cell adhesion molecule (NCAM), PSA becomes restricted to regions of neurogenesis and neuroplasticity in the adult. In the mammalian genome, two polysialyltransferases termed ST8Sia-II and ST8Sia-IV have been hypothesized to be responsible for the production of PSA in vivo. Approaches to discover PSA function have involved the application of endoneuraminidase-N to remove PSA and genetic manipulations in the mouse to deplete either NCAM or ST8Sia-IV. Here we report the production and characterization of mice deficient in the ST8Sia-II polysialyltransferase. We observed alterations in brain PSA expression unlike those observed in mice lacking ST8Sia-IV. This included a PSA deficit in regions of neurogenesis but without changes in the frequency of mitotic neural progenitor cells. In further contrast with ST8Sia-IV deficiency, loss of ST8Sia-II did not impair hippocampal synaptic plasticity but instead resulted in the misguidance of infrapyramidal mossy fibers and the formation of ectopic synapses in the hippocampus. Consistent with studies of animal models bearing these morphological changes, ST8Sia-II-deficient mice exhibited higher exploratory drive and reduced behavioral responses to Pavlovian fear conditioning. PSA produced by the ST8Sia-II polysialyltransferase modifies memory and behavior processes that are distinct from the neural roles reported for ST8Sia-IV. This genetic partitioning of PSA formation engenders discrete neurological processes and reveals that this post-translational modification forms the predominant basis for the multiple functions attributed to the NCAM glycoprotein. Polysialic acid (PSA) is a post-translational protein modification that is widely expressed among neural cell types during development. Found predominantly on the neural cell adhesion molecule (NCAM), PSA becomes restricted to regions of neurogenesis and neuroplasticity in the adult. In the mammalian genome, two polysialyltransferases termed ST8Sia-II and ST8Sia-IV have been hypothesized to be responsible for the production of PSA in vivo. Approaches to discover PSA function have involved the application of endoneuraminidase-N to remove PSA and genetic manipulations in the mouse to deplete either NCAM or ST8Sia-IV. Here we report the production and characterization of mice deficient in the ST8Sia-II polysialyltransferase. We observed alterations in brain PSA expression unlike those observed in mice lacking ST8Sia-IV. This included a PSA deficit in regions of neurogenesis but without changes in the frequency of mitotic neural progenitor cells. In further contrast with ST8Sia-IV deficiency, loss of ST8Sia-II did not impair hippocampal synaptic plasticity but instead resulted in the misguidance of infrapyramidal mossy fibers and the formation of ectopic synapses in the hippocampus. Consistent with studies of animal models bearing these morphological changes, ST8Sia-II-deficient mice exhibited higher exploratory drive and reduced behavioral responses to Pavlovian fear conditioning. PSA produced by the ST8Sia-II polysialyltransferase modifies memory and behavior processes that are distinct from the neural roles reported for ST8Sia-IV. This genetic partitioning of PSA formation engenders discrete neurological processes and reveals that this post-translational modification forms the predominant basis for the multiple functions attributed to the NCAM glycoprotein. Polysialic acid (PSA) 1The abbreviations used are: PSA, polysialic acid; NCAM, the neural cell adhesion molecule; endo-N, endoneuraminidase; LTP, long term potentiation; LTD, long term depression; ES, embryonic stem; RT, reverse transcriptase; BrdUrd, 5-bromo-2′-deoxyuridine; ACSF, artificial cerebrospinal fluid; fEPSPs, field excitatory postsynaptic potentials; TBS, θ-burst stimulation; STP, short term potentiation; HFS, high frequency stimulation; PTP, post-tetanic potentiation; CS, conditioned stimulus; ANOVA, analysis of variance; NMDA, N-methyl-d-aspartic acid; BDA, biotinylated dextran amine; wt, wild type. is a post-translational modification consisting of a homopolymer of α2-8-linked sialic acids that participates in neural development (1Schachner M. Martini R. Trends Neurosci. 1995; 18: 183-191Google Scholar, 2Rutishauser U. Landmesser L. Trends Neurosci. 1996; 19: 422-427Google Scholar, 3Kiss J.Z. Troncoso E. Djebbara Z. Vutskits L. Muller D. Brain Res. Rev. 2001; 36: 175-184Google Scholar). Typically attached to N-glycans, PSA is produced in the Golgi apparatus and is found among a limited number of glycoproteins and predominantly on the neural cell adhesion molecule (NCAM). Enzymatic removal of PSA by using endoneuraminidase (endo-N) induces various neurological abnormalities including deficits in hippocampal long term potentiation (LTP) and long term depression (LTD), spatial learning, cell migration, and axonal targeting as observed in NCAM-deficient mice (4Tomasiewicz H. Ono K. Yee D. Thompson C. Goridis C. Rutishauser U. Magnuson T. Neuron. 1993; 11: 1163-1174Google Scholar, 5Cremer H. Lange R. Christoph A. Plomann M. Vopper G. Roes J. Brown R. Baldwin S. Kraemer P. Scheff S. Barthels D. Rajewsky K. Wille W. Nature. 1994; 367: 455-459Google Scholar, 6Muller D. Wang C. Skibo G. Toni N. Cremer H. Calaora V. Rougon G. Kiss J.Z. Neuron. 1996; 17: 413-422Google Scholar, 7Becker C.G. Artola A. Gerardy-Schahn R. Becker T. Welzl H. Schachner M. J. Neurosci. Res. 1996; 45: 143-152Google Scholar, 8Hu H. Tomasiewicz H. Magnuson T. Rutishauser U. Neuron. 1996; 16: 735-743Google Scholar, 9Cremer H. Chazal G. Goridis C. Represa A. Mol. Cell. Neurosci. 1997; 8: 323-335Google Scholar, 10Seki T. Rutishauser U. J. Neurosci. 1998; 18: 3757-3766Google Scholar, 11Cremer H. Chazal G. Carleton A. Goridis C. Vincent J.D. Lledo P.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13242-13247Google Scholar, 12Cremer H. Chazal G. Lledo P.M. Rougon G. Montaron M.F. Mayo W. Le Moal M. Abrous D.N. Int. J. Dev. Neurosci. 2000; 18: 213-220Google Scholar, 13Bruses J.L. Rutishauser U. Biochimie (Paris). 2001; 83: 635-643Google Scholar). PSA may alter the adhesive property of NCAM in mediating these processes and may also influence cell-cell communication involving integrins, cadherins, and members of the immunoglobulin superfamily (14Rutishauser U. J. Cell. Biochem. 1998; 70: 304-312Google Scholar, 15Fujimoto I. Bruses J.L. Rutishauser U. J. Biol. Chem. 2001; 276: 31745-31751Google Scholar). Expression of PSA is high in embryonic brain and generally reduced in the adult. However, PSA is continuously present in some adult regions such as the olfactory bulb, hippocampus, and hypothalamus, coincident to where neurogenesis and neuronal plasticity persist (16Seki T. Arai Y. Neurosci. Res. 1993; 17: 265-290Google Scholar, 17Kuhn H.G. Dickinson-Anson H. Gage F.H. J. Neurosci. 1996; 16: 2027-2033Google Scholar). Two genes encoding polysialyltransferases ST8Sia-II (STX) and ST8Sia-IV (PST) are independently capable of directing PSA synthesis in vitro (18Eckhardt M. Mühlenhoff M. Bethe A. Koopman J. Frosch M. Gerardy-Schahn R. Nature. 1995; 373: 715-718Google Scholar, 19Kojima N. Yoshida Y. Tsuji S. FEBS Lett. 1995; 373: 119-122Google Scholar, 20Nakayama J. Fukuda M.N. Fredette B. Ranscht B. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7031-7035Google Scholar, 21Scheidegger E.P. Sternberg L.R. Roth J. Lowe J.B. J. Biol. Chem. 1995; 270: 22685-22688Google Scholar, 22Angata K. Fukuda M. Biochimie (Paris). 2003; 85: 195-206Google Scholar). Although ST8Sia-II and -IV share 59% amino acid identity, they are expressed in different spatial and temporal patterns among neural tissues (23Yoshida Y. Kurosawa N. Kanematsu T. Kojima N. Tsuji S. J. Biol. Chem. 1996; 271: 30167-30173Google Scholar, 24Kurosawa N. Yoshida Y. Kojima N. Tsuji S. J. Neurochem. 1997; 69: 494-503Google Scholar, 25Hildebrandt H. Becker C. Murau M. Gerardy-Schahn R. Rahmann H. J. Neurochem. 1998; 71: 2339-2348Google Scholar, 26Ong E. Nakayama J. Angata K. Reyes L. Katsuyama T. Arai Y. Fukuda M. Glycobiology. 1998; 8: 415-424Google Scholar, 27Eckhardt M. Gerardy-Schahn R. Glycobiology. 1998; 8: 1165-1172Google Scholar, 28Takashima S. Yoshida Y. Kanematsu T. Kojima N. Tsuji S. J. Biol. Chem. 1998; 273: 7675-7683Google Scholar). To understand how the mammalian neurological system may be modulated in vivo by altered PSA formation, the genetic bases of PSA formation in vivo must be defined. To date this has been explored by analyzing mice lacking the ST8Sia-IV polysialyltransferase. NCAM expression was unaltered by ST8Sia-IV deficiency, whereas specific brain regions exhibited decreased PSA levels, and adult animals bore a restricted phenotype involving an impairment of LTD and LTP in the hippocampal CA1 region (29Eckhardt M. Bukalo O. Chazal G. Wang L. Goridis C. Schachner M. Gerardy-Schahn R. Cremer H. Dityatev A. J. Neurosci. 2000; 20: 5234-5244Google Scholar). However, unlike NCAM deficiency, no decrease in CA3 LTP was observed, and hippocampal mossy fiber projections were unaltered. Here we report the generation and characterization of ST8Sia-II-deficient mice to investigate the role of this polysialyltransferase in neurological activity and define the biological mechanisms that modulate PSA formation and NCAM function. ST8Sia-II Mutagenesis—The murine ST8Sia-II gene was isolated from a mouse129/SvJ genomic DNA library and used to construct a targeting vector. An EcoRI-XhoI fragment containing exon 4 was flanked by two loxP sites as described (30Marth J.D. J. Clin. Investig. 1996; 97: 1999-2002Google Scholar). Targeted embryonic stem (ES) clones were identified by PCR and characterized by genomic Southern blotting. ES clones bearing the deleted (Δ) ST8Sia-II allele were obtained after transient expression of Cre recombinase. Targeted ES cells were injected into C57BL/6 blastocysts to generate chimeric mice. Mutant ST8Sia-II alleles were bred into the C57BL/6 mouse strain for more than six generations prior to phenotype analysis. RNA Analyses—Total RNA was purified using Trizol solution (Invitrogen) and analyzed by Northern and RT-PCR as described (26Ong E. Nakayama J. Angata K. Reyes L. Katsuyama T. Arai Y. Fukuda M. Glycobiology. 1998; 8: 415-424Google Scholar). The oligonucleotide primers (10 μm) used are as follows: mX-Tg1, 5′-CTGGAGGCAGAGGTACAATCAGATC-3′ (nucleotides 104-128); mX-Tg2, 5′-CCTCAAAGGCCCGCTGGATGACAGA-3′ (nucleotides 646-622); mP-Tg1, 5′-AGGCTGGCTCCACCATCTTCCAACA-3′ (nucleotides 173-197); and mP-Tg2, 5′-CTCTGTCACTCTCATTCCGAAAGCC-3′ (nucleotides 625-601). Western Blot Analyses—Brain tissues were isolated and homogenized with RIPA buffer (150 mm NaCl; 50 mm Tris-HCl, pH 7.4; 1% Nonidet P-40; 0.1% SDS; 5 mm EDTA) containing proteinase inhibitor (Roche Applied Science). Protein was analyzed by SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Millipore). Some tissue extracts were incubated with endo-N (31Hallenbeck P.C. Vimr E.R. Yu F. Bassler B. Troy F.A. J. Biol. Chem. 1987; 262: 3553-3561Google Scholar) for 1 h at 37 °C prior to analysis. Membranes were blocked with 10% dry milk powder in 20 mm Tris-buffered saline, pH 7.6, containing 0.1% Tween 20 (TBST), and incubated with either mouse anti-PSA 5A5 antibody (Developmental Studies Hybridoma Bank, diluted 1:1000 in TBST) or rat anti-NCAM H28 antibody (Immunotech, diluted 1:200), followed by peroxidase-conjugated anti-mouse IgM (1:4000) or anti-rat IgG (1:3000), and detected by ECL (Amersham Biosciences). BrdUrd Labeling—BrdUrd (20 mg/ml in 0.007 n NaOH and 0.9% NaCl) was delivered by intraperitoneal injection into ST8Sia-II-deficient and wild-type mice (50 mg/kg body weight) five times at 2-h intervals over an 8-h period. One week after the last administration, the mice were deeply anesthetized with Avertin (0.015 ml/g body weight), and brains were removed, fixed with Carnoy (60% ethanol, 30% chloroform, 10% acetic acid), and embedded in paraffin to cut sagittal or coronal sections at 10 μm. Every third section was collected, and 10 sections, which cover 300 μm in the center of the hippocampus, were stained for BrdUrd. BrdUrd-positive cells were detected by BrdUrd-specific monoclonal antibody (Roche Applied Science) and Alexa Fluor® 488 goat anti-mouse IgG1 (Molecular Probes). To measure distribution of embryonic neural stem cells in brains, BrdUrd was administered into pregnant heterozygous mice (100 mg/kg at embryonic day 16, E16) that were mated with heterozygous male mice. Brains from neonatal mice and 10-day postnatal mice were fixed and stained as described above. Histology—For Timm's staining, mice were perfused intracardially with 15-50 ml of 0.37% sodium sulfide solution followed by 4% paraformaldehyde in phosphate-buffered saline. Every third section covering 300 μm in the center of the hippocampus was analyzed as described (9Cremer H. Chazal G. Goridis C. Represa A. Mol. Cell. Neurosci. 1997; 8: 323-335Google Scholar). Immunohistochemical or immunofluorescence staining was performed as described (10Seki T. Rutishauser U. J. Neurosci. 1998; 18: 3757-3766Google Scholar, 26Ong E. Nakayama J. Angata K. Reyes L. Katsuyama T. Arai Y. Fukuda M. Glycobiology. 1998; 8: 415-424Google Scholar, 29Eckhardt M. Bukalo O. Chazal G. Wang L. Goridis C. Schachner M. Gerardy-Schahn R. Cremer H. Dityatev A. J. Neurosci. 2000; 20: 5234-5244Google Scholar). To stain sections with antibodies for β-tubulin (Babco), synapsin I (Chemicon), glial fibrillary acidic protein, or MAP2 (Roche Applied Science), brains were fixed with Carnoy; to stain sections with anti-PSA 12F8 (BD Biosciences), anti-NCAM H28, and anti-calbindin d-28K (Chemicon), brains were fixed with 4% paraformaldehyde in 0.1 m phosphate buffer, pH 7.4. Electrophysiology—Hippocampal slices from 5- to 6-month-old ST8Sia-II-deficient mice and their wild-type littermates were used for recordings. After halothane anesthesia, decapitation, and removal of the brain, the hippocampi were cut with a VT 1000 M vibratome (Leica, Nussloch, Germany) in 300-μm-thick slices in ice-cold artificial cerebrospinal fluid (ACSF) containing (in mm) 250 sucrose, 25 NaHCO3, 25 glucose, 2.5 KCl, 1.25 NaH2PO4, 2 CaCl2, and 1.5 MgCl2, pH 7.3. The slices were then kept at room temperature in a chamber filled with carbogen-bubbled ACSF, containing 125 mm NaCl instead of 250 mm sucrose, for at least 2 h before the start of recordings (modified from Ref. 32Edwards F.A. Konnerth A. Sakmann B. J. Physiol. (Lond.). 1990; 430: 213-249Google Scholar). In the recording chamber, slices were continuously superfused with carbogen-bubbled ACSF (2-3 ml/min) at room temperature. For recordings in the CA3 region, the slices were prepared as for recordings in the CA1 region but with some modifications. Before decapitation, mice were transcardially perfused with ice-cold ACSF, containing (in mm) 250 sucrose, 25 NaHCO3, 25 glucose, 2.5 KCl, 1.25 NaH2PO4, 0.5 CaCl2, and 6 MgCl2, pH 7.3. Slices were cut according to Claiborne et al. (33Claiborne B.J. Xiang Z. Brown T.H. Hippocampus. 1993; 3: 115-121Google Scholar). Exchange of sucrose-containing ACSF with normal ACSF (containing 2.5 mm CaCl2 and 1.5 mm MgCl2) was performed gradually using peristaltic pumps. Schaffer collateral-CA1 extracellular recordings of focal fEPSPs were obtained from the stratum radiatum of the CA1 region in response to stimulation of Schaffer collaterals by an electrode placed ∼400 μm apart from the recording electrode in the stratum radiatum of the CA1 region. Recordings and stimulations were performed with glass pipettes filled with ACSF having a resistance of 2 megohms. Basal synaptic transmission was monitored at 0.033 Hz. The slices were maintained at room temperature. Homosynaptic LTP in the CA1 region was induced by θ-burst stimulation (TBS) applied orthodromically to Schaffer collaterals and recorded extracellularly in the stratum radiatum. A TBS consisted of 10 bursts delivered at 5 Hz. Each burst consisted of four pulses delivered at 100 Hz. Duration of pulses was 0.2 ms, and five TBSs were applied every 20 s to induce LTP (29Eckhardt M. Bukalo O. Chazal G. Wang L. Goridis C. Schachner M. Gerardy-Schahn R. Cremer H. Dityatev A. J. Neurosci. 2000; 20: 5234-5244Google Scholar). The stimulation strength was in the range of 40-70 μA to provide fEPSPs with an amplitude of 50% of the subthreshold maximum. The mean slope of fEPSPs recorded 0-10 min before TBS was taken as 100%. The transient potentiation immediately following TBS (or STP, short term potentiation) was measured as a maximal increase in the fEPSP slope during 1 min after LTP induction. The values of LTP were calculated as increase in the mean slopes of fEPSPs measured 50-60 min after TBS. Mossy fiber-CA3 extracellular recordings and stimulations were both performed with glass pipettes filled with ACSF and having a resistance of 2 megohms with stimulation strength of ∼40 μA. The stimulating electrode was placed close to the internal side of the granule cell layer. The recording pipette was placed in the stratum lucidum of the CA3 region. The mossy fiber responses selected for recording were of 40-60 μV, with a fast rise time and decay of fEPSPs (total duration of fEPSP <10 ms, rise time <3.5 ms), large paired pulse facilitation (>170%), and prominent frequency facilitation (>200%). The selected responses had no hallmarks of polysynaptic activation, such as jagged decay phase with multiple peaks, or variable latencies of fEPSPs. The LTP-inducing high frequency stimulation (HFS) consisted of one train of stimuli applied at 100 Hz for 1 s one time (“weak” stimulation protocol) or repeated four times with an interval of 20 s (“strong” stimulation protocol). To evoke LTP exclusively in mossy fiber synapses, which are known to undergo LTP in an NMDA receptor-independent manner, the NMDA receptor antagonist AP-5 (50 μm; Tocris, Bristol, UK) was applied 15 min before and during HFS. All recorded mossy fiber responses followed presynaptic stimulation of 100 Hz and showed no changes in the shape of responses after induction of LTP. To additionally confirm that the fEPSPs recorded were evoked by the stimulation of mossy fibers and not by the associational/commissural pathway, an agonist of type II metabotropic glutamate receptors (LCCG1, 10 μm; Tocris), which is known to reduce synaptic transmission in CA3 mossy fiber synapses (34Manzoni O.J. Castillo P.E. Nicoll R.A. Neuropharmacology. 1995; 34: 965-971Google Scholar), was applied at the end of each experiment. Slices in which responses were reduced by at least 70% were selected for analysis. Basal synaptic transmission was monitored at 0.033 Hz. The mean amplitude of fEPSPs recorded 0-10 min before HFS was taken as 100%. Post-tetanic potentiation (PTP) was calculated as the maximal increase in the amplitude of fEPSP after HFS. The values of LTP were calculated as increase in the mean amplitude of fEPSPs measured 50-60 min after HFS. Data acquisition and measurements were performed by using an EPC-9 amplifier and Pulse software (Heka Elektronik, Lambrecht/Pfalz, Germany). Values in electrophysiological experiments are reported as mean ± S.E. Student's t test was used to assess statistical significance using Sigma Plot 5.0 software (Chicago). Nerve Tract Tracing between the Hippocampus and Amygdala—Neuronal tracing was analyzed in eight mice of each genotype by injecting biotinylated dextran amine (BDA, Molecular Probes) into the amygdala. After the mouse was deeply anesthetized with ketamine (1.25 mg/g) and xylazine (0.07 mg/g), BDA (10% in 0.1 m phosphate buffer, pH 7.4; 0.2 μl) was stereotaxically injected into amygdala by using Picospritzer (Parker Instrumentation) and the following coordinates: Anterior, -0.12; Left, -0.27; Dorsal, -0.4 in cm from Bregma (35Franklin K.B.J. Paxinos G. The Mouse Brain in Stereotaxic Coordinates. Academic Press, San Diego1996Google Scholar). The animals were allowed to recover under close observation and were returned to their cage. Six days after injection, brains were fixed as described above, and cryosections were analyzed immunohisto-chemically using fluorescein isothiocyanate-labeled Avidin (Vector Laboratories). Metabolic and Behavioral Parameters—Two separate cohorts of 4-month-old male mice were analyzed. The first consisted of 10 wild-type and 9 ST8Sia-II-deficient littermates. These were assessed in a behavioral test battery modified from that used by McIlwain et al. (36McIlwain K.L. Merriweather M.Y. Yuva-Paylor L.A. Paylor R. Physiol. Behav. 2001; 73: 705-717Google Scholar) and as described previously (37Corbo J.C. Deuel T.A. Long J.M. LaPorte P. Tsai E. Wynshaw-Boris A. Walsh C.A. J. Neurosci. 2002; 22: 7548-7557Google Scholar). This included parameters such as metabolic performance, physical appearance, sensorimotor reflexes, motor activity, nociception, acoustic startle, sensorimotor gating, and assessments of learning and memory. Concern that testing mice in such a large battery could influence behavior in any individual task and that multiple assessments increased the probability of a type I statistical error, a second cohort of mice was also analyzed (wild-type, n = 16; Δ/Δ, n = 15). In the open field test, activity was measured in a 30-min test period in an area of 45 × 45 cm using a Digiscan apparatus (Accuscan Electronics, Columbus, OH). Vertical activity (rearing) and distance (total and center) were recorded. Passive avoidance analysis involved a two-compartment light/dark apparatus (35 × 18 × 30 cm, Coulbourn Instruments, Allentown, PA). Each mouse was placed in the lighted compartment. When the animal entered the dark compartment, a guillotine door closed behind and a foot shock of 0.4 mA was delivered through the grid floor of the dark compartment for 3 s. If the mouse did not enter the dark compartment within 10 min, it was excluded from the retrieval test. In the retrieval trial performed 24 h later, the latency for the mice to enter the dark compartment was recorded. The maximum latency was 600 s. Fear conditioning analyses used chambers (26 × 22 × 18 cm high) made of clear Plexiglas placed in a 2 × 2 array (Med Associates). A video camera was used for recording and analysis (FreezeFrame, Actimetrics, St. Evanston, IL). The conditioned stimulus (CS) was an 85-db, 2,800-Hz, 20-s tone, and the unconditioned stimulus was a scrambled foot shock at 0.75 mA presented during the last 3 s of the CS. Mice were placed in the test chamber for 3 min before recording CS and freezing behavior. Freezing was defined as the absence of movement other than breathing, and thresholds were selected via the software of high correlation with human observers. Three CS/unconditioned stimulus pairings were given with 1-min spacing, and freezing during the CS was also recorded. Each mouse was returned to the shock chamber 24 h later, and freezing responses were recorded for 3 min (context test). The chambers were modified to present a different environmental context (shape, odor, color changes), and 2 h later the mice were placed in this novel environment. Freezing behavior was recorded for 3 min before and during three CS presentations (cued conditioning). The time spent freezing was converted to a percent value. The water maze task constituted a pretraining phase during which all mice from both cohorts were tested for 2 days in a straight-swim pretraining protocol. Mice received 16 trials (8 trials over 2 days) in a 31 × 60-cm rectangular tank that was located in a different room than the circular tank used in the hidden platform trials. The platform was located 1 cm below the water opposite from the start location. Latency to climb onto the platform was the dependent measure. Criteria for advancing to the hidden platform trials was completing 6 of 8 trials under 10 s on the 2nd day. This pretraining procedure provided experience with swimming and climbing onto a submerged platform without exposing the mice to the spatial cues used in the hidden platform trials. This procedure both screens for mice with severe motor deficits and reduces behavioral variability often seen on the 1st day of hidden platform testing. All mice successfully passed this pretraining phase. Hidden platform testing followed in which extra-maze visual cues were hung from a curtain located around a 1.26-m diameter circular tank. The water was made opaque with the addition of non-toxic paint. The 10-cm diameter escape platform was located 1 cm below the surface of the water, and a Polytrack video-tracking system (San Diego Instruments) was used to collect mouse movement data (location, distance, and latency) during training and probe trials. Each mouse was given eight trials a day, in two blocks of four trials for 4 consecutive days. After 36 trials, each animal was given a 60-s probe trial. During the probe test, the platform was removed, and quadrant search times were measured. Visual cue testing was performed 1 day after the last hidden-platform training trial, wherein mice were trained to locate a visiblecued platform. The visible cue was a gray plastic cube (9 cm) attached to a pole so that it was 10 cm above the platform. On each trial of the visible platform test, the platform was randomly located in one of the four quadrants. Mice were given eight trials, in blocks of four trials, and the latency to find the platform was recorded for each trial. Metabolic chambers termed CLAMS (Comprehensive Lab Animal Monitoring System; Columbus Instruments, Columbus, OH) automatically recorded metabolic parameters including volume of carbon dioxide produced (VCO2), volume of oxygen consumed (VO2), respiration (respiratory exchange ratio) = VCO2/VO2, and caloric (heat) value ((3.815 + 1.232 × respiratory exchange ratio) × VO2), motion in all three axes in time, and consumption of food and water. Data were collected every 30 min over three 12-h dark cycles and two 12-h light cycles and analyzed as mean values over each 12-h period with the exception of food and water intake which were added to the total during subsequent cycles. Pulmonary function was scored by measurement of the uptake of CO. A carbon monoxide uptake monitor (Columbus Instruments) measured the CO level in a sealed chamber after exposing the mouse to a 60-s interval of air with 0.17% CO. The mean breath per min was also recorded. Each animal was tested once. Blood pressure was determined by a noninvasive blood pressure tail-cuff system (Columbus Instruments) that measures systolic blood pressure in addition to heart rate and relative changes in diastolic and mean blood pressure. Individual mice were placed in a small cylinder chamber; occlusion and sensor cuffs were placed on the tail, and the tail was warmed to 37 °C. Mice were first acclimated to the restraining chamber, tail cuffs, and the heat fan for 30 min for 2 days prior to testing. The mean of four measurements on the 3rd day was reported and analyzed by the Student's t test. ST8Sia-II Inactivation in the Mouse Germ Line—The ST8Sia-II gene is highly conserved among mammals and includes multiple exons that compose the catalytic domain (21Scheidegger E.P. Sternberg L.R. Roth J. Lowe J.B. J. Biol. Chem. 1995; 270: 22685-22688Google Scholar). Exon 4 encodes a significant portion of the sialyl motif L, a peptide sequence that is essential for sialyltransferase activity (38Datta A.K. Paulson J.C. J. Biol. Chem. 1995; 270: 1497-1500Google Scholar). DNA encoding exon 4 of the ST8Sia-II gene was targeted for elimination from the mouse genome (Fig. 1A). Targeted ES cells that bore the exon 4 deletion were injected into C57BL/6 blastocysts to obtain chimeric mice. The mutant allele was transmitted into the germ line and bred into the C57BL/6 strain (Fig. 1B). Mice homozygous for the deleted ST8Sia-II allele (Δ) were produced by heterozygous parental genotypes at close to Mendelian ratios (wt/wt:wt/Δ:Δ/Δ = 29:50:21%; n = 255). No differences in body weight or brain size were noted despite the fact that highest expression of ST8Sia-II occurs in wild-type embryos at early developmental stages. Inactivation of ST8Sia-II because of deletion of exon 4 was investigated by RNA and enzymatic analyses. RT-PCR revealed that the truncated form of ST8Sia-II mRNA lacking exon 4 was produced in both heterozygous and the ST8Sia-II null mice (Fig. 1C). The mutant ST8Sia-II cDNA was isolated and found unable to produce polysialyltransferase activity (data not shown). RNA analyses by RT-PCR at various post-natal ages further demonstrated that the disruption of ST8Sia-II does not affect the level of ST8Sia-IV mRNA (Fig. 1D). PSA Expression and Neurogenesis—PSA expression was reduced in the olfactory bulb and cerebral cortex of adult ST8Sia-II-deficient mice (Fig. 2). There was no quantitative alteration of PSA among the hypothalamus, hippocampus, and cerebellum, in contrast to the pattern of PSA deficiency found among mice lacking ST8Sia-IV (29Eckhardt M. Bukalo O. Chazal G. Wang L. Goridis C. Schachner M. Gerardy-Schahn R. Cremer H. Dityatev A. J. Neurosci. 2000; 20: 5234-5244Google Scholar). However, closer examination of the hippocampus of ST8Sia-II-deficient mice revealed a PSA deficit in the dentate gyrus. Neurogenesis takes place throughout adulthood in the dentate gyrus, and progenitor cells that migrate into the granule cell layer express high levels of PSA. There is also an association of neuronal precursor cells and PSA expression with other regions supporting neurogenesis in the adult brain, including the subventricular zone. Progenitor cells expressing PSA in the granule layer were greatly reduced and often absent in ST8Sia-II-deficient mice (Fig. 3, A and B). In the subventricular zone, there was no difference in PSA expression in the anterior region; however, PSA expression was reduced among newly" @default.
• W1969275043 created "2016-06-24" @default.
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• W1969275043 date "2004-07-01" @default.
• W1969275043 modified "2023-10-02" @default.
• W1969275043 title "Sialyltransferase ST8Sia-II Assembles a Subset of Polysialic Acid That Directs Hippocampal Axonal Targeting and Promotes Fear Behavior" @default.
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