Equus asinus

This study was carried out in strict accordance with the recommendations in the
Guide for the Care and Use of Laboratory Animals of the National Institutes of
Health. All procedures used were approved by the University of Texas
Southwestern Medical Center Institutional Animal Care and Use Committee APN
2007-0065. All efforts were made to minimize suffering.
Ascl1^CreERT2 knock-in mice were generated by
replacing the Ascl1 coding region with
CreER^T2[8] (link) and
Frt-Neo-Frt cassettes. The targeting strategy was the same used to generate
Ascl1^GFP knock-in mice [9] (link). The endogenous ATG was
replaced by a short sequence containing a PacI site and a consensus Kozak site.
The correct targeting event was identified by Southern analysis of EcoRI
digested DNA using 5′ and 3′ probes. After obtaining germ line
transmission in the Ascl1^{CreERT2-Frt-Neo-Frt} mice,
they were crossed with FLPe mice [10] (link) to remove the neomycin
cassette resulting in Ascl1^CreERT2 mice.
For PCR genotyping, the following primers were used: 5′-AAC TTT CCT CCG GGG CTC GTT
TC-3′ (Sense Ascl1 5′UTR) and 5′-CGC CTG GCG ATC CCT GAA CAT
G-3′ (Anti sense Cre) giving a PCR product of 247 bp.
Tamoxifen (TAM) induction of Cre recombinase was accomplished by intraperitoneal
injection of
Ascl1^CreERT2/+;R26R^YFP/YFPpostnatal day 50 (P50) mice with 180 mg/kg/day TAM (Sigma, T55648) in sunflower
oil on five consecutive days. Brains were harvested at the times specified after
TAM and processed as described [7] (link), [11] (link). R26R^YFP and
Nestin::GFP mice have been previously described [12] (link), [13] (link).
For immunofluorescence staining, free floating sections or sections mounted on
slides were incubated in the appropriate dilution of primary antibody in
PBS/3% donkey (or goat) serum/0.2% NP-40 (or 0.2% Triton
X-100), followed by appropriate secondary antibody conjugated with AlexaFluor
488, 568, or 594 (Molecular Probes). Mouse monoclonal antibodies used were:
Ascl1 (1∶750, RDI Fitzgerald, 10R-M106B), NeuN (1∶1000, Chemicon,
MAB377), GFAP (1∶400, Sigma, G3893). Rabbit polyclonal antibodies used
were: GFP (1∶500, Molecular Probes, A6455), GFAP (1∶500, DAKO,
Z0334), Ki67 (1∶500, Neomarker), Sox2 (1∶2000, Millipore). Goat
polyclonal antibodies used were: DCX (1∶200, Santa Cruz) and NeuroD1
(1∶200, Santa Cruz). Chick GFP (1∶500, Aves Lab) was also used.
Confocal imaging was carried out on a Zeiss LSM510 confocal microscope.
Ascl1⁺ fluorescence intensity levels were classified as high
or low using ImageJ and setting a threshold of pixel intensity for
Ascl1^Low (314–599 units) and Ascl1^High (>600
units). For cell number counts, three Nestin::GFP mice were
analyzed to place Ascl1⁺ progenitors in the adult neural stem
cell lineage. For in vivo genetic tracing experiments using the
Ascl1^CreERT2 knock-in line, at least two
Ascl1^CreERT2/+;R26R^YFP/YFPmice per each harvest time point (7, 30, or 180 days post-TAM) were used. For
co-localization data with each stage-specific marker, 150–500
YFP⁺ cells per animal were counted.

Paraffin-embedded sections were dewaxed, hydrated, treated for antigen retrieval, and blocked with 10% donkey serum. Then, the primary antibodies were used for the incubation of the sections at 4 °C overnight. The next day, the slides were stained with relevant secondary antibodies at room temperature for 1 h. Nuclei were stained with DAPI (Servicebio, Wuhan, China). Finally, the sections were observed with a confocal microscope (Olympus, Tokyo, Japan) after sealing with an anti-fluorescence quencher. The antibodies used in this study were as follows: anti-ZO-1 (1:200, Genetex, Texas, America), anti-Occludin (1:200, Genetex), and Alexa Fluor 488-conjugated donkey anti-rabbit IgG (1:200, Antgene, Wuhan, China).

Participants in subcohort I will undergo MRI using a Siemens 3-Tesla Magnetom Prisma scanner. High-resolution structural T1-, T2-, and diffusion-weighted MR images will be acquired as well as ultra-fast functional magnetic resonance encephalography (MREG) asses cardiovascular brain pulsations [67 (link)]. Resting-state and task-based blood oxygen level-dependent (BOLD) fMRI scans will be acquired to measure related brain function. To assess distributed and intrinsic brain functional connectivity patterns, we will acquire a resting-state fMRI scan (10 min), during which participants are asked to close their eyes, let their minds wander and not fall asleep. Participants will complete established tasks to assess processes involved in cognition and mood, e.g., the Cyberball task, a ball-tossing game during which the participant interacts with fictitious characters to simulate experiences of social inclusion, exclusion, rejection and ostracism [68 (link)]. Trained research personnel will instruct participants on how to perform all tasks.

Immunohistochemistry using a rabbit anti-neural cell adhesion molecule (NCAM) antibody (AB5032, Millipore, Burlington, MA) and rabbit anti-Gαo antibody (551, MBL, Tokyo, Japan) was performed using olfactory organ sections from lungfish as described previously [23 (link), 28 (link)]. Sections were incubated with each primary antibody overnight at 4°C, washed, and then incubated with a secondary antibody, Alexa Fluor 488-donkey anti-rabbit IgG (A21208, Thermo Fisher Scientific, Waltham, MA) for 2 h at RT. The sections were mounted in VectaShield mounting medium with DAPI (H-1200, Vector Laboratories, Burlingame, CA).