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Advanced Neurology Dysregulation of cAMP signaling pathway in MT2KO mice

drawing a region of interest (ROI) representing the bilateral Na VO •12H O 0.5 mM, NaF 50 mM, benzyl benzene
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hippocampus on the photograph. The total volume of the 1 mM, and phenylmethylsulfonyl fluoride 1 mM. The
hippocampus can be determined by adding up the volume tissue was added to one-third sample buffer containing
measured from each anatomical image. 8% sodium dodecyl sulfate (SDS), tris 200 mM, and 40%
All proton NMR ( H NMR) experiments were performed glycerol and boiled for 10 min. Lysate was centrifuged at
1
on a Bruker-500 spectrometer at room temperature. 12,000 ×g for 10 min at 4°C, and then, the supernatant was
A pulse-acquisition sequence was used with a 90° flip stored at -80°C. The supernatant’s protein concentration
angle, a 13 s relaxation delay to ensure full relaxation, a was detected using the BCA method. The same amount
spectral width of 10,000 Hz, 32 k data points, and 64 scans. of protein was separated by SDS-polyacrylamide gel
The spectra were zero-filled to 64 k, corrected manually for electrophoresis (10%) and then transferred to the
phase and baseline, and referenced to the chemical shift of nitrocellulose filter membrane. The membranes containing
the trimethylsilyl-propionic-2,2,3,3-d4 acid (TSP) methyl the protein were blocked by the 3% bovine serum albumin
peaks at 0 ppm. Peak area integration was performed (BSA) and incubated with the antibody at 4°C overnight.
using standard routines provided by the Topspin software Using goat anti-mouse secondary antibody, conjugate
package (version 2.0, Bruker). Using TSP as the external to IRDyeTM (1:10000, LI-COR Biosciences), combined
reference, the absolute concentrations of metabolites were with mouse-derived primary antibody, or goat anti-rabbit
determined from the spectra and calculated in the unit of secondary antibody, conjugate to IRDyeTM (1:10000,
mmol/kg wet tissue weight. LI-COR Biosciences), combined with rabbit-derived
primary antibody, the nitrocellulose membrane was
2.4. Antibodies and kits incubated with the corresponding secondary antibody
NR2B antibody against N-methyl-D-aspartic acid at room temperature for 1 h and then scanned using
receptor (NMDA) receptor subunit 2B was obtained Odyssey Imager (LI-COR Biosciences).
®
from Abcam (Cambridge, UK). NR2A subunit of NMDA
receptors (NR2A) antibody against NMDA receptor 2.6. Immunohistochemistry
subunit 2A glutamate receptor type 1 (Glur1) antibody Mice were anesthetized with 6% chloral hydrate and
against was obtained from Millipore. Glur1s845 antibody perfused with 300 mL saline rapidly and then perfused
against phosphor-AMPA receptor 1 at serine 845, PKAα with 500 mL 4% paraformaldehyde for 2 h. The brain
antibody against PAK catalytic subunit α isoform, and was removed from the skull and sliced into 30 μm with
PKAβ antibody against PKA type Iβ regulatory subunit Vibratome (Leica, VT1000S, Germany) after being post-
were obtained from Santa Cruz. Postsynaptic protein-95 fixed in the same fixative in a 50 mL centrifuge tube. The
(PSD95) antibody against postsynaptic density protein brain slice was soaked in the PBS-0.5%Triton-0.3% H O
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95 and postsynaptic protein-93 (PSD93) antibody against to remove the endogenous hydrogen peroxidase and then
postsynaptic density protein 93 were obtained from block with 3% BSA for 30 min. After that, the slice was
Abcam. pS133-CREB antibody against CREB at serine incubated with an antibody (1:200) for 48 h at 4°C. Then,
133, CREB antibody against CREB, and P-APP antibody the slice was incubated with a biotin-labeled secondary
against APP at threonine 668 were obtained from cell antibody for 1 h in a 37°C oven and dyed with the
signaling. Vesicle-associated membrane protein 2 antibody diaminobenzidine tetrachloride system (Bei Jing, ZSGB,
against vesicle-associated membrane protein 2 and 9032). The images were taken with a light microscope
MunC18 antibody against MunC18 were obtained from (Olympus BX60, Tokyo, Japan).
Abcam. Synaptophysin antibody against synaptophysin
was acquired from Sigma. 4G8 antibody against APP was 2.7. Nissl staining
obtained from Covance. 22C11 antibody against APP The slides were dewaxed in xylene I and xylene II for
N-terminus was obtained from Millipore. A3981 antibody 15 min in each solution. Then, the slides were hydrated
against senile plaque was acquired from Sigma. Anti- in a series of gradient alcohol (100% alcohol I, 100%
rabbit IRDye and anti-mouse IRDye were purchased from alcohol II, 95% alcohol, 90% alcohol, 80% alcohol, 70%
Li-Cor Biosciences (Lincoln, NE, USA). The BCA kit was alcohol, and 50% alcohol) for 5 min in each alcohol. The
purchased from Pierce (Rockford, IL, USA). slides were then soaked in distilled water 3 times, for 5 min
each time. Then, the slides were put in a 60°C incubator
2.5. Western blot and dyed with 1% toluidine blue for 40 min (or dyed
Hippocampus was removed from the brain and with tar violet for 30 s). After the excess dye was washed
homogenized in tissue homogenate buffer containing away with distilled water, the slides were dehydrated in
NaCl 50 mM, Tris 10 mM, EDTA-Na •2H O 1 mM, 70%, 80%, 95%, and 100% ethanol, respectively, and then
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Volume 2 Issue 3 (2023) 3 https://doi.org/10.36922/an.0974