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

expose herringbone and sagittal suture. The coordinate as the average of two responses under each stimulation
of the injection site located in MS was referenced by intensity.
the rat brain atlas as follows: Anterior-posterior (AP), The paired-pulse ratio (PPR) was recorded using the
0.6 mm; mediolateral (ML), 0.1 mm; and dorsal-ventral stimulation intensity matching to the 50% peak amplitude
(DV), 6.0 mm. After drilling a hole in the skull of MS, of fEPSP. A 5 ms step was used to set the interstimulus
microsyringes containing lenti-pre-miR-195, lenti- interval. The interstimulus interval was set between 20 and
AMO-195, and lenti-NC were fixed to the stereotaxic 70 ms. The amplitude of the second pulse was divided by
instrument and injected into the MS region of rats. The the first pulse to calculate the PPR.
injection rate was set to 1.5 μl/min. The needle was pulled
out slowly after 5 min to prevent the leakage of lentivirus 2.7. Local field potential (LFP) recording
from tissue. The holes in the skull were filled with bone The LFP of hippocampal dCA1 was recorded as the
wax. Then, the scalp incisions of the rats were sutured. previous studies [30,31] . In brief, rats were anesthetized
After surgery, the rats were placed in home cages and by intraperitoneally injecting 20% urethane solution to
irradiated with a warm light source until they were awake. the rats placed on a stereotaxic frame. The animals were

2.5. Neuro-retrotracing technique maintained at a level of anesthesia at which spontaneous
theta rhythm was not presented but could be elicited by a
Rats were anesthetized by intraperitoneal injection of 10% tail pinch. LFPs were recorded by implanting a monopolar
chloral hydrate. Then, holes were drilled in the dCA1 (AP, tungsten electrode into the hippocampal dCA1 pyramidal
−3.84 mm; ML, ±2.1 mm; DV, 2.7 mm; and AP, −3.84 mm; cell layer (AP, 3.8 mm; ML, 2.4 mm; and DV, 2.7 mm). The
ML, ±3 mm; DV, 3.3 mm) and DG (AP, −3.84 mm; ML, reference electrode was placed on the skull 2 mm away from
±2.1 mm; DV, 3.3 mm) areas of the hippocampus bilaterally the recording electrode. The ground electrode was clamped
according to the rat brain atlas. The green retrobeads were to the scalp. The LFP of hippocampal dCA1 was amplified
injected at a rate of 0.25 μl/min in each injection site. The by a ME-1 preamplifier (Chengdu Taimeng Software
needle was pulled out slowly after 5 min to prevent the Company Limited, China), and then, the signals were
green retrobeads from spreading. Next, the holes in the filtered at 0–20 Hz and recorded by BL-420S system. After
skull were filled with bone wax and the scalp incisions the LFP waveform became stable, the basic spontaneous
were closed with 5-0 medical sutures. The subsequent LFP was recorded for 2 min. Subsequently, the vicinity of
experiments were performed 7 days later. tail base was gently pinched with a metal plastic clamp for
1 min to induce theta rhythm. After recording procedure,
2.6. Electrophysiological recording in vivo
the rats were executed for brain tissue or perfused for brain
Following urethane anesthesia (1.2 g/kg) treatments, the extraction.
rats were put on the stereotaxic frame device (DW-2000,
Chengdu Taimeng Software Company Limited, China) 2.8. Real-time polymerase chain reaction (PCR)
for the implantation of electrodes. The rats were put on Utilizing the Trizol reagent to separate the total RNA from
a heating pad to keep their body temperature at 37°C. the basal forebrain regions, TaqMan MicroRNA reverse
Bipolar stimulating electrodes (stainless steel, 0.5 mm) transcription kit was used to perform reverse transcription
were implanted into the MS area (AP, 0.6 mm; ML, 0.1 mm; (Applied Biosystems, Carlsbad, CA, USA). The SYBR
and DV, 6.0 mm). The recording electrodes stuffed by 3 green core reagent kit’s instructions for real-time PCR were
mol/L of NaCl were implanted into the pyramidal cell followed (Applied Biosystems). Settings for the reactions
layer of the dCA1 (AP, 3.8 mm; ML, 2.4 mm; and DV, were 95°C for 10 min, 95°C for 15 s, 60°C for 30 s, and
2.7 mm). To get the best results, specific positions of the 72°C for 30 s, for a total of 40 cycles. In this experiment,
stimulating and recording electrodes were varied [28,29] . all quantitative PCR data were used to calculate the
MS was activated by constant controlled pulses from relative content of target genes by the 2-∆∆Ct method.
the BL-420S stimulus generator after an electrode ∆Ct = each group (Ct target gene-Ct housekeeping gene),
implantation recovery period. A ME-1 preamplifier ∆∆Ct = experimental group ∆Ct - control group ∆Ct.
was used to amplify the field excitatory post-synaptic
potential (fEPSP) obtained in hippocampal dCA1 area. 2.9. Western blot
The signals were recorded by the BL-420S device after Total hippocampal proteins were extracted, and the
being filtered at 1 Hz–1 kHz and digitalized at 20 kHz. To protein content was determined by the BCA Protein
set up the input-output (I/O) curves, electric stimulation Assay Kit. Protein samples fractionated on a 10% sodium
with intensity ranging from 1 to 20 V was applied in the dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-
MS. The amplitude and slope of fEPSP were expressed PAGE) were transferred onto nitrocellulose membranes

Volume 1 Issue 2 (2022) 3 https://doi.org/10.36922/an.v1i2.116