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Advanced Neurology Dysregulation of cAMP signaling pathway in MT2KO mice

cleared with xylene. Finally, a neutral gum seal was used 2.11. Statistical analysis
for coverslipping. For statistical analysis, differences between the groups
2.8. Golgi staining were tested by analysis of variance followed by the least
significant difference post-hoc test using SPSS 17.0. For a
The mice were anesthetized with 6% chloral hydrate, the single comparison, the significance of differences between
perfusion tube was inserted into the aorta of the mice, and means was determined by t-test. P < 0.05 was considered
the normal saline containing 0.5% sodium nitrite at 37°C statistically significant.
was used for perfusion for about 5 min until the blood of
the mice was removed. The perfusion solution was replaced 3. Results
with 4% polymethyl methacrylate.
3.1. MT2KO and DKO mice, instead of MT1KO mice,
The first 100 mL of the perfusion solution was dripped exhibited a deficit in learning and memory
quickly, and then, the rest was dripped slowly for about 2 h; Due to the weak eyesight of the C3H mice, we chose
next, the perfusion solution was replaced with mordant
solution (1000 mL mordant solution containing 50 g chloral the contextual fear conditioning test to examine
hydrate, 50 g potassium dichromate, and 100 mL 40% hippocampus-dependent learning and memory. A step-
formaldehyde solution). The fixed brain tissues of mice were down inhibitory avoidance task was administered to
immersed in mordant solution at room temperature for all groups, and statistical latency and error times were
measured to determine the deficit in memory. We found
3 days, and then, the specimens were placed in silver nitrate an obvious increase in the errors (Figure 1A) and short
solution in the dark for 3 days, and the solution was changed latency in MT2KO and DKO mice 2 h later (Figure 1B).
every day. Finally, the specimens were washed with ultrapure
water, and the brain slices were cut into 30 μm thick brain Then, we also detected the long-term memory after 24 h
slices with an oscillating microtome. The cut brain slices were and obtained the same results (Figures 1C and 1D).
This demonstrated that MT2KO and DKO mice have a
pasted on a clean slide, dehydrated and cleared, and covered significant deficit in memory.
with a cover slip for observation under an optical microscope.
3.2. High quantity of choline and myo-inositol
2.9. cAMP and Cyclic guanosine monophosphate without change of neuron number in the
(cGMP) ELISA
hippocampus of MT1KO, MT2KO, and DKO mice
cAMP and cGMP ELISA kits were purchased from YaJi NMR results suggested a high quantity of choline (Cho) in
Biological (Shanghai, China). A protein sample was added MT2KO and MT1KO mice (Figure 2A) and a high quantity
to each well of the 96-well plate set at 37°C and was let of myo-inositol (mI) in MT1KO mice (Figure 2B), while no
to stand for 20 min. Antibody with biotin was added to significant change of N-Acetyl-L-aspartic acid (NAA) in
each well and incubated at 37°C for 60 min. Finally, the melatonin receptor knockout mice (Figure 2C). Functional
horseradish peroxidase-labeled avidin working solution magnetic resonance imaging (fMRI) results suggested no
was added and incubated at 37°C for 60 min. The optical statistical difference in hippocampus volume (Figure 2D).
density (OD) value was read at the wavelength of 450 nm.
Furthermore, Nissl staining proved that there was no
2.10. PKA activity assay change in the number of neurons in the hippocampus of
melatonin receptor knockout mice (Figure 2E and 2F).
PKA activity assay kit was purchased from GenMed
Scientifics Inc. (U.S.A) (GMS50059.2.3 v.A). Sixty-five 3.3. Aberrant Aβ accumulation in MT2KO and DKO
microliters of reagent C, 10 μL of reagent D, 10 μL of mice, instead of MT1KO mice
reagent E, and reagent F were added to each well of a The deposition of Aβ was another important
96-well plate. The plate was then shaken gently and mixed pathologic change in the AD brain. We performed
well, and incubated at 30°C for 3 min. Five microliters immunohistochemical staining using 4G8 antibody
of reagent G were added to the blank well, and 50 μg of (Figure 3A) and found a high density of staining in
protein was added to the sample well. The plate was then MT1KO and DKO mice (Figure 3B). Our further results
shaken gently to mix well, and immediately, the OD values showed high levels of Aβ42 in MT2KO mice and high
were read at the wavelength of 340 nm with a microplate levels of Aβ40 and high total levels of Aβ40 and Aβ42 in
reader at 0 min, 5 min, and 10 min. MT1KO mice (Figure 3C). The ratio of Aβ42 and Aβ40
In our system, the activity of PKA can be calculated was very high in MT2KO mice (Figure 3D), indicating
using this formula: A = 10.7 × OD, where A is PKA activity, dysregulation of APP metabolism. Therefore, we used the
and OD is the absorbance value. senile plaque antibody A3981 to explore the deposit of Aβ

Volume 2 Issue 3 (2023) 4 https://doi.org/10.36922/an.0974