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Advanced Neurology MiR-195 regulates MS-dCA1 neural circuit in CBH rat

and incubated with the primary antibodies anti-choline the platform and allowed to relax for at least 20 s. On day
acetyltransferase (ChAT; Catalog. #297013, 1:1,000, 6, the platform was taken out of the water, and each rat
Synaptic Systems) and β-actin (Catalog. #8432, 1:1,000, participated in a single 120-s swim probe session. Next,
Santa Cruz). The membranes were then incubated with the following parameters were tracked: escape latency,
fluorescent secondary antibody. The protein bands were length of swim path, swim speed, frequency of crossing,
captured using the Odyssey Infrared Imaging System and percent of swimming distance in target quadrant of
(LI-COR Biosciences, Lincoln, NE, USA) and quantified overall distance.
using the Odyssey V3.0 software.
2.13. Statistical analysis
2.10. Assessment of ACh concentration The data are presented as mean ± standard error of mean

The guidelines were followed to produce the dorsal (SEM). MatLab 7.0 was used to examine the power spectra
hippocampus samples, which were then utilized for the of LFPs. P < 0.05 was regarded as statistically significant
ELISA assay. To obtain the supernatant, frozen tissues were when comparing two groups using t-test. Our statistical
homogenized in pre-chilled phosphate-buffered saline analysis was performed using SAS 9.1 software (Institute
(PBS) and centrifuged at 3000 rpm/min for about 20 min. Incorporation, serial number: 989155).
Following extraction, the concentrations of ACh were
assessed using the ACh ELISA Kit (Catalog #MBS774123, 3. Results
MyBioSource) in accordance with the manufacturer’s 3.1. Downregulation of basal forebrain miR-195
instructions. Then, the standard curve’s linear regression expression impairs the electrical activity of the
equation was fitted using the concentration of the standard MS-dCA1 neural circuits
sample and the associated optical density (OD) value, and It has been reported that CCH can induce downregulation of
ACh concentration of the test sample was computed using miR-195 expression in the hippocampi and cortices of rats,
this equation (y=aln(x)+b).
which can regulate Aβ deposition, tau hyperphosphorylation,
2.11. Immunofluorescence detection neuronal death, and microglial polarization [7-10] . Here,
we found that miR-195 levels in the MS region were also
Rats were infused with 0.9% sodium chloride (NaCl) and 4% significantly reduced in 2VO rats (Figure 1A). Our recent
buffered paraformaldehyde after the electrophysiological study found that CCH can also impair the function of the
and LFP recordings. The brain tissue was preserved in 4% MS-dCA1 neural circuits in rats . To investigate whether
[12]
paraformaldehyde for 2 days before being sliced into 30 μm miR-195 is involved in CCH-induced impairment of
slices with a Leica oscillating microtome. The brain slices MS-dCA1 neural circuit, we stereotaxically injected lenti-
were sealed with goat serum for 2 h and then incubated AMO-195 into the MS region of rats to establish a rat model
with the primary antibodies (ChAT, #GTX163114, of miR-195 knockdown (Figure 1D). Eight weeks later,
Genetex; GAD67, #MAB5406, Millipore; PV, #MCA3C9, the qRT-PCR result showed that compared with lenti-NC
Encor) at 40C for 12 h. On the next day, the brain slices group, lenti-AMO-195 injection induced a decrease in
were taken out of the freezer and rinsed 3 times in PBS the expression of miR-195 in MS, which was reversed by
for 15 min followed by secondary antibodies conjugated coinjecting lenti-pre-miR-195 (Figure 1B).
to Alexa Fluor 594 (1:200) and DAPI (1:50). Then, brain
samples were examined under bright-field microscopy Subsequently, we assessed the basic electrophysiological
using a Zeiss Axio Scope A1 microscope with ×5, ×20, and properties of the MS-dCA1 neural circuit in vivo
×40 objective. (Figure 1C and E). Compared with lenti-NC-injected
rats, higher stimulation intensity was required to elicit the
2.12. Morris water maze responsiveness of the MS-dCA1 circuit in lenti-AMO-

The Morris water maze consists of a 2.0 m diameter 195-injected rats (Figures 1F). When applying a 1-V-step
pool with a black bottom. As hints, circles, triangles, and stimulation to MS, we found that the amplitude of fEPSPs
squares are draped from a black curtain around the pool. increased with the enhancement of stimulus intensity among
In the middle of the first quadrant, there is a hidden escape lenti-NC, lenti-AMO-195, and lenti-AMO-195 + lenti-pre-
platform with a 20 cm diameter and a top surface that was miR-195-treated rats. However, the fEPSP amplitude in lenti-
2 cm just under the water. The procedure for preparing AMO-195-injected rats was significantly lower than that in
2VO rats is outlined in previous works [7,32] . At 8.00 am, lenti-NC-injected rats, which was rescued by coinjection of
rats were placed in the pool, facing the wall, in the second, lenti-pre-miR-195 in the MS (Figure 1G-I).
third, and quadrants (3-trial per day for 5 days), and they To further assess the presynaptic function of the
had 120 s to reach the platform. If not, they were led to MS-dCA1 neural circuit, we monitored the paired-pulse

Volume 1 Issue 2 (2022) 4 https://doi.org/10.36922/an.v1i2.116