By the room (PVN)
Regarded as nuclear (SON)
Although OT is known for its role in milk
Recent studies have shown that OT is involved in the expression of social behavior, memory processing, regulation of fear and stress response.
Proof that OT affects accessory behavior (such as parental care and reproduction) and reduces anxiety leads to speculation that it may play a role in mood disorders.
Evidence from pharmacology studies indicates that the OT system is regulated by chloramine and believes that OT is an intermediary of the chloramine re-absorption inhibitor (SSRIs)
In this study, we studied OT-
Labeled cells and five serotonin transporters (5-HTT)
Immune Response (IR)
Using biochemistry and double-
Immune fluorescence technology
Consistent with previous reports, distribution of OT
Labeled cells in the lower part of the visual Hill are limited to PVN and SON.
In these nuclei, we prove that 5-HTT-
Label fibers follow the distribution of OT-labeled cells. Overlap of OT-
Labeled neurons and 5-HTT-
IR fibers appear next to cells, between cells, in the back, and in the subarea of the rear of PVN. In the SON, 5-HTT-
Label fiber and OT
The labeled cells overlap in the medial segment of the abdomen and the "SAC" section of the dorsolum SON.
These findings provide neuroanatomical support for ssris's therapeutic effects on social and anxiety, which may be partially mediated by the components of the OT system. One female (MFJ7)
Five men (
MNJ1, MNJ2, MNJ3, MNJ5, MNJ6)
Used in these studies.
All the experiments were conducted in accordance with the guidelines of the National Institutes of Health.
The experimental design and techniques were designed to minimize the use and pain of animals and were reviewed and approved by the University of Rochester Animal Research Committee.
The initial anesthesia was to inject ketamine ketone through the muscles (10u2009mg/kg)
, And intravenous injection of e-obarbital (
Initial dose 20 mg/kgv.
And maintain as needed).
The animals were subjected to deep anesthesia and were put to death after heart perfusion with normal saline containing 0.
Heparin sulfate of 5 ml, followed by 0 in more than 4% formaldehyde solutions.
1 µm phosphate buffer (pH 7. 4).
Then take out the brain and freeze protection with an increase in sucrose gradient (10, 20 and 30%).
Cut a continuous slice of 50 μm on the frozen slicer and transfer to 0.
1 m phosphate buffer or solution of low temperature protective agent.
Sections of adjacent compartments were selected for immune staining with OT (
Hudson star, Hudson, WI, 1: 10 pmand 5-HTT (
Monoclonal antibody technology
, Stone Mountain, GA, mouse monoclonal 1: 150 2016000)
Staining of antibodies and phenol Violet.
Also selected a compartment for 5-
Comparison of HT immune staining with 5-HTT IR (
Immune star, Hudson, WI, 1:100, 000, rabbit, rabbit).
Dilution curves were performed before the actual experiment to determine the optimal concentration of each primary antibody.
The control sections that omitted primary antibodies did not result in IR staining of cells or fibers.
Rinse the slices thoroughly first in 0.
1 µm phosphate buffer (pH 7. 2)with 0. 3% Triton X-100 (PB-T).
After treatment with endogenous peroxide inhibitors (EPI), and more PB-
T rinse, tissue pre-incubated in 10% normal goat serum (NGS)
Dilute with PBT (NGS-PB-T)for 30u2009min. In the 5-HTT and 5-
In HT experiments, 1% of bovine serum was added to the NGS to reduce background staining.
Then put the tissue into the primary resistancesera to OT, 5-HTT, and 5-
HT was incubated for 4 nights at 4 °c.
After washing with PB-
T, incubated the slices with the appropriate biomarker secondary antibody and used avinitin-
Biotin complex reaction (
Vector lab, Vector lab, Vectastatin ABC kit, Burlingame, CA).
The staining was enhanced by incubation of 1-3 min in 3,3′ diamino B 4 salt (DAB)and 0.
03% hydrogen peroxide.
The slices are mounted on sub-bed slides, dehydrated in an increasing alcohol gradient, cleared in PX, and the lid slides.
Several immune-stained compartments were re-stained with phenol purple.
The adjacent part of the mark, an extra compartment for a woman (MFJ7)and two male (MFJ2 and JFJ5)
The monkey was selected as a double label for OT and 5-HTT. For double-
Using the DAB mark, the slices of OT and nickel are processed first
Strengthen in OT to produce blue-black reaction productlabeled cells.
5-2 processing parts
HTT without nickel reinforcement gives a light brown label of 5-HTT-Positive fibers.
Dilution curves were first performed in a single-label fluorescence assay to optimize the fluorescence signal and minimize background staining.
After treatment with EPI, incubated in 10% NGS and incubated in mixed OT for four nights (
Rabbit, 1:10and 5-HTT (Mouse, 1: 75 000)
Antibody at 4 °c
Then, thoroughly clean the tissue in PB-
TX, incubated in 10% NGSPB-
TX 30 min, incubated dark for four hours in filtered Alexafluor 546 (anti-
Rabbit immunoglobulin antibody
And Alexafluor 488 (anti-
Mouse IgG antibody(
Molecular probe company, Eugene, OR)
, With OT and 5-
HTT resistance, respectively.
Control experiment of fluorescent secondary antibody being "reversed (
Such as Alexafluor 546 defense
5-mouse IgG antibody
HTT 1 resistance;
Alexafluor 488 defense
Rabbit IgG antibody against OT 1)
This is also done to ensure a similar label mode.
The tissue is then rinsed for 1 Thanh, mounted on the slide and covered in the water medium (
Vector lab). For DAB-
Immune response compartment, distribution of OT
The labeled cells in the lower Hill are hands-
Draw in bright
Use a stretch tube attached to the microscope for on-site lighting (× 10 objective). Hand-
Then enter the drawn chart into the computer using the application Canvas 5. 0 (
Victoria ACD Systems, BC, Canada)
Used with a drawing tablet (
Wacom TechnologyVancouver, Washington). 5-HTT-
The fiber of the label is handmade.
Drawing under bright and dark lighting, also using camera lucida technology (× 10 objective).
Then, use the program Adobe Photoshop 6 to scan a paper chart of the marked fibers and surrounding landmarks, including fiber bundles and blood vessels, into the computer. 0 (300u2009dpi)(Adobe Inc. , San Jose, CA)
And import into the drawing program Canvas 5. 0.
Finally, the boundary between PVN and SON in the adjacent Nissl-
The dyed part is also drawn, input the computer with the help of the drawing tablet and in OT and 5-HTT-
Part of the mark.
Landmarks such as blood vessels and fiber bundles are used for alignment. 5-HTT and OT-
In order to confirm these relationships, the labeled slices that were re-dyed with the phenol Violet were examined.
Slices with 5-immune response
Evaluation of 5-with HTT and phenol Violet re-dyeing-
HTT staining of trans-subnuclear.
Double the additional part
Marked as the immune response of OT and five serotonin transporter (5-HTT-IR)
The use of DAB technology was studied at high power using oil immersion.
The analyzed DAB-
Label section, additional section double-labeled for 5-
HTT/OT with immune fluorescent antibodies was evaluated using a fluorescence microscope to further confirm the relationship between these markers.
Use the Optronic Microfire color CCD to take the image and format it in Adobe Photoshop.
In addition, Z-is used using a co-focused laser microscope-Stack analysis (
2 μm distance between pieces)(
Leica Microsystems, Exton, PA).