Nitric oxide negatively regulates mammalian adult neurogenesis -- Data Supplement - HTML Page - 3579SuppText.htslp -- Proceedings of the National Academy of Sciences

Supporting Materials and Methods
Surgical Procedures.
Osmotic minipumps (model 2002, Alzet, Palo Alto, CA) (0.5 m l/h for 14 days) were aseptically filled with sterile solutions of 50 mM N^ω-nitro-methyl-L-arginine methyl ester (NAME) (Calbiochem) or N^ω-nitro-methyl-D-arginine methyl ester (D-NAME) (Sigma) in calcium and magnesium-free Hanks’ buffered saline (GIBCO) (pH 7.2, NaOH). The investigator was blinded to the identity of the solution. The pumps were connected via a catheter to right-angle brain-infusion cannulae and positioned at AP –1.2 mm, ML –1.2 mm, and DV –3.6 mm in relation to bregma in six male Sprague–Dawley rats (9 weeks old, ~250 g, Taconic Farms) anesthetized with 90 mg/kg ketamine and 10 mg/kg xylazine. BrdUrd Administration.
BrdUrd (in 0.9% saline containing 7 mM NaOH) was given i.p. to rats (120 mg/kg) twice daily for 7 days starting the day after surgery. The same solution of BrdUrd was given to mice (50 mg/kg) twice daily for 2.5 days. Fourteen days postsurgery, rats were perfused transcardially with Hanks’ buffer containing calcium and magnesium at room temperature and then 4% paraformaldehyde in PBS at 4° C. Mice were perfused in the same way 2 h after the last BrdUrd administration. The brains were postfixed for 4 h and cryoprotected in 6% sucrose in PBS for 2 h and then 30% sucrose in PBS for 4 days, all at 4° C. Sections (10 m m) were cut on a cryostat in a serial manner sagittally from the midline until the olfactory bulb (OB) was completely collected on gelatin-coated slides. Histochemistry.
For diaphorase staining, slides were incubated in fresh 1 mM NADPH/245 m M Nitroblue tetrazolium in 100 mM Tris·HCl (pH 7.4), containing 0.2% (wt/vol) Triton X-100 for 90 min at 37° C, dehydrated, and mounted. For BrdUrd immunohistochemistry slides were washed in PBS before being treated with 2 M HCl 30 min at 37° C. Endogenous peroxidases inactivated with 1% H₂O₂, and the slides, were treated with 2% normal goat serum in PBS containing Triton X-100 (0.1%) (PBS-Triton). The slides then were incubated with anti-BrdUrd antibody [clone BU1/75(ICR1), 1 in 500; Harlan Laboratories (Haslett, MI)] at 4° C for 18 h. After washing, a biotinylated anti-rat secondary antibody (1 in 200, Vector Laboratories) was applied in PBS-Triton for 2 h followed by further washing and incubation in avidin–biotin-complex-horseradish peroxidase complex (Vector ABC Elite) for 1 h before staining with 3,3¢ -diaminobenzidine and H₂O₂. For fluorescent double labeling of sections for BrdUrd with other markers, the H₂O₂ step was omitted, the anti-BrdUrd antibody ascites solution was used at 1 in 200, and the anti-rat secondary antibody used was directly conjugated to Alexa Fluor 488 (Molecular Probes). The sections then were incubated with the next primary antibody for 18 h (mouse anti-βIII-tubulin, clone 5G8, 3.4 m g/ml, Promega), or mouse anti-glial fibrillary acidic protein (clone G-A-5, 4 m g/ml, Roche Molecular Biochemicals). All incubations with primary antibody were at 4° C. The sections were washed, and appropriate secondary antibodies conjugated to Alexa Fluor 594 (Molecular Probes) were applied at 5 m g/ml for 2 h. The Alexa Fluor-conjugated secondary antibodies had been preabsorbed by the manufacturer against sera from the host species of the other primary antibody used for the particular experiment. For neuronal NO synthase (nNOS)/BrdUrd double labeling, the anti-nNOS antibody was incubated with the sections first for 42 h (rabbit anti-nNOS, Z-RNN3, 0.25 m g/ml, Zymed). The sections then were fixed (5 min, 4% paraformaldehyde at room temperature) prior to acid denaturation and BrdUrd immunohistochemistry. For all experiments "no primary" and "no secondary" antibody controls were included, and crossover of spectral emissions under compound and confocal microscopy were checked. To examine apoptotic cells by using the terminal deoxynucleotidyltransferase-mediated dUTP end labeling (TUNEL) assay, the CLONTECH ApoAlert DNA fragmentation assay kit was used per manufacturer directions by using propidium iodide as a counterstain. We quantitated the proportion of TUNEL-positive cells in arbitrary areas in zones 3 and 4 as defined in Fig. 3. Confocal microscopy was performed on a Zeiss LSM 510 machine by using "multitracking," where excitation and emission could not bleed through between channels. Optical slices (0.3–0.4 m m thick) were collected that overlapped through the entire section in the z plane such that projections were continuous. Cell Counting.
Unbiased absolute counts of BrdUrd⁺ cells were made after ABC-peroxidase immunostaining in rats and mice; in rats these were on the hemisphere ipsilateral to the cannula. Counts were made by a blinded observer, and the code was not broken until all the counting was completed. For the OB of mice and dentate gyrus, subventricular zone, and rostral migratory stream of both rats and mice, every eighth section representing steps of 80 m m were counted starting from the section in the series where the lateral ventricle began until no rostral migratory stream was visible (34–38 sections per brain for rats and 12 sections per brain for mice). No nuclei were counted within 0.7 mm of the cannula tract. For the OB of rats, every 24th section was enumerated through the entire structure. Montages of photomicrographs using an ´ 10 objective (Zeiss Axiophot microscope with a charge-coupled device camera) over the entire structure were analyzed by using OBJECT IMAGE, a variant of NIH IMAGE. This program allows the nondestructive scoring of structures in an image by using an overlying layer. In this way cells could be marked and scored and their three-dimensional position recorded. To determine whether a change in size and shape of the BrdUrd nuclei could influence cell counting, we measured these parameters in zones 4 and 7 of experimental and control animals (defined in Fig. 3). The shape, estimated as a ratio between the length and width of nuclei, in zone 4 was 2.124 ± 0.79 (SD) for control animals versus 2.027 ± 0.99 in experimental animals (P = 0.105, Student’s t test), the particles having a mean length of 8.69 m m (n = 65) and 9.15 m m (n = 64), respectively. The shape of nuclei in zone 7 was 2.81 ± 0.74 for control animals versus 2.68 ± 0.86 in experimental animals (P = 0.374), the particles having a mean length of 10.49 m m (n = 65) and 10.14 m m (n = 64), respectively. The mean area of nuclei in zone 4 was 35.81 ± 8.89 m m² (n = 65) in control animals versus 34.36 ± 8.68 m m² (n = 64) in experimental animals and were not significantly different (P = 0.351). The mean nuclei area in zone 7 was 38.42 ± 14.55 m m² (n = 65) in control animals versus 37.99 ± 11.0 m m² (n = 64) in experimental animals (P = 0.847). Thus, no statistically significant differences in the area of the nuclei were found, and no stereological corrections for the sampling error were applied. Scoring of BrdUrd/βIII-tubulin-coimmunostained cells was performed by using confocal microscope z series as described above. Only βIII-tubulin-positive structures that had the morphology of a cell were analyzed. Cells were only scored as double-labeled if the BrdUrd⁺ nucleus was surrounded in all directions by βIII-tubulin and the BrdUrd signal was present in a nuclear-sized hole in the cell with βIII-tubulin staining. This avoided the possibility of scoring BrdUrd⁺ nuclei that were nonneuronal satellite cells of neurons or those that were closely associated with neurons. Gene Targeting.
A single clone containing a 110-kb fragment of the nNOS locus was isolated as a result of PCR-based screening of the 129/Svj BAC mouse embryonic stem (ES) library, release I (Genome Systems, St. Louis) by using a pair of primers from exon 6 of the gene (5¢ -TTTGGCTCCAAGGCCCACATG-3¢ and 5¢ -GCAGCTTGGACCACTGGATCC-3¢ , GenBank accession no. AF534820). Subsequently, sequencing of various subclones containing exon 6 resulted in the isolation of two genomic fragments: a 2.6-kb ApaI–PmlI fragment upstream of exon 6 and a 4.7-kb EcoRI–XbaI fragment downstream of exon 6. These fragments were inserted into the pPNT targeting vector (1). The resulting targeting construct had a 628-bp PmlI–EcoRI fragment of the WT nNOS allele, containing exon 6 and adjacent intronic sequences, replaced by the 1.8-kb phosphoglycerate kinase/neomycin phosphotransferase cassette from the pPNT vector. The NotI linearized targeting construct was transfected by electroporation into 129/Svj ES cells. Fourteen properly targeted ES clones were recovered out of 286 clones resistant to ganciclovir and G418. The homologous recombination event at the 5¢ end was confirmed by Southern blot analysis with NheI digestion and a unique 401-bp fragment as a 5¢ probe (generated by PCR on WT genomic DNA with primers 5¢ -GCTAGCAGCCTTTCAGATGAAG-3¢ and 5¢ -AACCTTCCCGGGGTGACAGG-3¢ , GenBank accession no. AF534819). Homologous recombination at the 3¢ end was confirmed by using ApaI digestion and a unique 383-bp fragment as a 3¢ probe (generated by PCR on WT genomic DNA with primers 5¢ -TGAAGGGTCCTGCCAGCAAG-3¢ and 5¢ -TGCTGGTGCTTCCTAGGTGC-3¢ , GenBank accession no. AF534821). The 5¢ and 3¢ probes were located upstream and downstream, respectively, of the region used in the targeting vector. Two of the 14 correctly targeted ES clones were injected into C57Bl/6 blastocysts, resulting in 10 chimeras. These chimeras were bred with C57Bl/6 mice to establish two independent heterozygous lines, which then were interbred to produce homozygous mice disrupted with nNOS allele (KOex6) . Genotyping, RT-PCR, and Western Blotting.
Adult animals were genotyped by PCR analysis of mouse tail DNA by using two pairs of primers specific to one of each: the WT allele (5¢ -GGCTCATTGACAACTCCTGCT-3¢ and 5¢ -ATGTGGGCCTTGGAGCCAAAC-3¢ , GenBank accession no. AF534820) or to the neomycin phosphotransferase gene of the KOex6 allele (5¢ -TGCCGAGAAAGTATCCATCATGGCTGATGC-3¢ and 5¢ -CAGAAGAACTCGTCAAGAAGGCGATAGAAGG-3¢ ).

Total RNA samples from the whole brains of adult mice of the various genotypes were isolated by using TRIzol reagent (Invitrogen). One-microgram RNA aliquots were analyzed by RT-PCR. Reverse transcription was performed by using random hexamers and murine leukemia virus reverse transcriptase (Applied Biosystems). PCR amplification of the 661-bp fragment of nNOS cDNA was performed by using a pair of primers from exons 4 (5¢ -CTCGGGCAAACAGTCTCCTAC-3¢ , GenBank accession no. AF534819) and 8 (5¢ -TGTGAACTCCACATTAGCTGG-3¢ , GenBank accession no. AF534821). Control RT-PCR experiments were performed without the addition of reverse transcriptase (data not shown).

Protein extracts from adult mouse brains were prepared as described (2). Protein was measured by using the BCA reagent system (Pierce) to normalize the assays. Fifty-microgram protein aliquots were analyzed by Western blotting with either carboxyl-terminal anti-nNOS rabbit polyclonal antibody (R20 sc-648, 1 in 1,000, Santa Cruz Biotechnology) or amino-terminal rabbit polyclonal anti-nNOS (Z-RNN3, 1 in 3,000, Zymed) followed by an incubation with horseradish peroxidase-conjugated anti-rabbit secondary antibody (170-6515, 1 in 5,000, Bio-Rad). Immunoblots were developed by using the SuperSignal detection kit (Pierce). Control experiments were performed without the addition of primary antibodies.

NOS Activity Determination.
An assay for the activity of NOS by using the conversion of [³H]arginine to [³H]citrulline was performed according to standard methods (2). Briefly, 10 m l of the brain extract (15 m g of total protein) was added to 150 m l of the reaction mixture (performed in triplicate for each sample). The samples were transferred to 37° C for 30 min, and the reaction was terminated with 1 ml of ice-cold 2 mM EDTA in 20 mM Hepes (HCl, pH 5.5). An ion-exchange resin (AG 50W, 0.5 mg/ml, Bio-Rad) was added to absorb unreacted arginine, and the free [³H]citrulline was measured by using scintillation. Control experiments showed that the assay was linear with respect to time (data not shown). To test the stability of L-NAME, a 50 mM solution of the inhibitor was aliquoted in Eppendorf tubes and incubated at 37° C. At various times over 16 days an aliquot was taken and serially diluted to a final concentration of 5 m M to 100 nM. At these concentrations, L-NAME blocked 20–90% of the NOS activity of a mouse brain extract taken as a standard (21.5 pmol/min per mg). For this experiment, 10 m l of standard extract was added to the 150-m l reaction mixture, which had been premixed with 10 m l of the inhibitor dilution, and each point was assayed in triplicate.

1. Tybulewicz, V. L., Crawford, C. E., Jackson, P. K., Bronson, R. T. & Mulligan, R. C. (1991) Cell 65, 1153–1163.

2. Knowles, R. G. & Salter, M. (1998) in Nitric Oxide Protocols, ed. Titherage, M. A. (Humana, Totowa, NJ), pp. 67–73.