As shown in Figure 1 B, the LIPUS was generated by a 1 MHz, focused transducer with 50 ms burst lengths at a 5% duty cycle and a repetition frequency of 1 Hz. Mice were anesthetized (induced with 5% sevoflurane for 2 min and maintained with 2–2.5% sevoflurane in 40% oxygen) and fixed in a brain stereotaxic apparatus (RWD Life Science, Shenzhen, China), then the parietal and occipital skin was shaved, and the surface point of the dorsal hippocampus was marked as bregma (±1.40 mm, caudal−1.94 mm). A calibrating cone was used to adjust the transducer to ensure that the focal point could be on the hippocampus (Figure 1 C). The acoustic intensity distribution of the lateral and axial direction of the sonic field is shown in Figure 1 D. After transcranial attenuation, the spatial-peak temporal-average intensities (ISPTA) 1 mm from the cone tip was 177 mW/cm2. The duration of sonication was alternated bilaterally for 2.5 min and repeated three times with a total stimulation time of 15 min. Ultrasound stimulation was conducted at four time points around the anesthesia/surgery: 8 h before anesthesia/surgery, 4 h before surgery, 4 h after surgery, and 8 h after surgery.
Brain stereotaxic apparatus
Manufactured by RWD Life Science
Sourced in China
The brain stereotaxic apparatus is a precision instrument used in neuroscience research to accurately position and secure the head of an animal during surgical or experimental procedures. The device employs a three-dimensional coordinate system to target specific regions of the brain.
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8 protocols using brain stereotaxic apparatus
1
Transcranial Low-Intensity Ultrasound Stimulation
2
Conditional Modulation of LH Neurons
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Mice were anesthetized with Avertin (200 mg/kg) and then settled in a brain stereotaxic apparatus (RWD Life Science Co., Shenzhen, China). For adeno-associated virus (AAV) injection, 100 nl AAV-DIO-hM3Dq-mCherry, or AAV-DIO-mCherry (OBiO Technology [Shanghai] Corp., Ltd., 1012 titer) was administered bilaterally into the LH of Bdnf-e1−/− mice using a 10-ml Hamilton syringe with a 33-gauge needle. For antibody injection, 200 nl TrkB-agomab (3.4 mg/ml) or mouse normal IgG (3.4 mg/ml) was administered bilaterally into the LH of Bdnf-e1−/− mice using a 10-ml Hamilton syringe with a 33-gauge needle. Infusion of the antibody (200 nl, 20 nl/min) was accomplished by a micro-syringe pump (KD Scientific, Holliston, USA). The following coordinates defined LH location: anteroposterior, −1.58 mm; mediolateral, ±1.0 mm; and dorsoventral, −4.80 mm relative to the bregma. At the end of the infusion, the needle was left in the brain for another 5 min to reduce backflow of the antibody. Shortly after surgery, mice received metacam (1 mg/kg) for analgesia and were translocated to their home cages.
3
Epicranial Electrode Implantation and MCAO Procedure
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Two days before the MCAO operation, all rats were accepted epicranial electrode implantation (Fig. 7 ). The rats were anesthetized with 1% pentobarbital sodium (45 mg/kg, intraperitoneally [i.p.]) and fixed on the brain stereotaxic apparatus (RWD Life Science, China). After removal of the scalp and underlying tissues, a homemade epicranial electrode with a defined contact area of 3.5 mm2 was mounted on the intact skull at the coordinates bregma AP + 2.0 mm and ML + 2.0 mm with glass ionomer dental cement (Ketac Cem, ESPE Dental AG, Seefeld, Germany). The electrode was remained in place during the whole experiment.![]()
Schematic presentation of the treatment regime of the animals
4
PRV Inoculation in Mouse Brown Fat
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Recombinant pseudorabies virus (PRV) inoculation was performed in a biosafety level-2 operating room, according to a previous study [30 (link)]. Mice were anesthetized with Avertin (200 mg/kg) and then were settled in a brain stereotaxic apparatus (RWD Life Science Co., Shenzhen, China). After the interscapular BAT was exposed, three 500-nl injections of a PRV (PRV-614, 109 pfu/ml, BrainVTA, Wuhan, China) were made into the brown fat on one side using a 10-ul Hamilton syringe with a 31-gauge needle. Shortly after surgery, mice received metacam (1 mg/kg) for analgesia and were translocated to their home cages. At the 6th day after microinjection, the animals were deeply anesthetized by Avertin (350 mg/kg) and perfused with phosphate-buffered saline (PBS; 0.1 M) and paraformaldehyde (PFA; 4%).
5
Hippocampal EAAT3 Knockdown via rAAV
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Four potential different shRNA sequences (shRNA-mSLC1A1-1 4) targeting mSLC1A1 and the negative control shRNA (shRNA-NC) were designed and synthesized to construct rAAV vectors respectively named rAAV-shRNA-SLC1A1-1 4 and rAAV-shRNA-NC by Gemma Gene Company (Suzhou, China). To identify the most effective shRNA sequence that could knockdown EAAT3 in the hippocampus, we screened 4 different sequences to infect the HT-22 cell line, nding that rAAV-shRNA-SLC1A1-2 displayed the lowest EAAT3 expression level by RT/PCR and western blot (Suppl. Fig. 1).
For stereotactic injection of rAAV vector into the bilateral hippocampus, mice were anesthetized with pentobarbital sodium (70mg/kg) and placed on brain stereotaxic apparatus (RWD Life Science, Shenzhen, China). After exposing the skull via an incision, two small holes were drilled for injection. The stereotactic coordinates were 2.1 posterior, ± 1.7 lateral, and 2.0 ventral from bregma. Injection speed was 50nl/min, and the needle was kept in place for an additional 15 minutes before it was slowly withdrawn.
RNAi group and RNAi+LPS group received bilateral hippocampal microinjection of rAAV-RNAi at 1μL per side (1×10 13 TU/mL), while the NC group and NC+LPS group received an equal volume of negative control rAAV-NC.
For stereotactic injection of rAAV vector into the bilateral hippocampus, mice were anesthetized with pentobarbital sodium (70mg/kg) and placed on brain stereotaxic apparatus (RWD Life Science, Shenzhen, China). After exposing the skull via an incision, two small holes were drilled for injection. The stereotactic coordinates were 2.1 posterior, ± 1.7 lateral, and 2.0 ventral from bregma. Injection speed was 50nl/min, and the needle was kept in place for an additional 15 minutes before it was slowly withdrawn.
RNAi group and RNAi+LPS group received bilateral hippocampal microinjection of rAAV-RNAi at 1μL per side (1×10 13 TU/mL), while the NC group and NC+LPS group received an equal volume of negative control rAAV-NC.
6
Hippocampal EAAT3 Knockdown via rAAV
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Four potential different shRNA sequences (shRNA-mSLC1A1-1 4) targeting mSLC1A1 and the negative control shRNA (shRNA-NC) were designed and synthesized to construct rAAV vectors respectively named rAAV-shRNA-SLC1A1-1 4 and rAAV-shRNA-NC by Gemma Gene Company (Suzhou, China). To identify the most effective shRNA sequence that could knockdown EAAT3 in the hippocampus, we screened 4 different sequences to infect the HT-22 cell line, nding that rAAV-shRNA-SLC1A1-2 displayed the lowest EAAT3 expression level by RT/PCR and western blot (Suppl. Fig. 1).
For stereotactic injection of rAAV vector into the bilateral hippocampus, mice were anesthetized with pentobarbital sodium (70mg/kg) and placed on brain stereotaxic apparatus (RWD Life Science, Shenzhen, China). After exposing the skull via an incision, two small holes were drilled for injection. The stereotactic coordinates were 2.1 posterior, ± 1.7 lateral, and 2.0 ventral from bregma. Injection speed was 50nl/min, and the needle was kept in place for an additional 15 minutes before it was slowly withdrawn. RNAi group and RNAi+LPS group received bilateral hippocampal microinjection of rAAV-RNAi at 1μL per side (1×10 13 TU/mL), while the NC group and NC+LPS group received an equal volume of negative control rAAV-NC.
For stereotactic injection of rAAV vector into the bilateral hippocampus, mice were anesthetized with pentobarbital sodium (70mg/kg) and placed on brain stereotaxic apparatus (RWD Life Science, Shenzhen, China). After exposing the skull via an incision, two small holes were drilled for injection. The stereotactic coordinates were 2.1 posterior, ± 1.7 lateral, and 2.0 ventral from bregma. Injection speed was 50nl/min, and the needle was kept in place for an additional 15 minutes before it was slowly withdrawn. RNAi group and RNAi+LPS group received bilateral hippocampal microinjection of rAAV-RNAi at 1μL per side (1×10 13 TU/mL), while the NC group and NC+LPS group received an equal volume of negative control rAAV-NC.
7
Hippocampal EAAT3 Knockdown via rAAV
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Four potential different shRNA sequences (shRNA-mSLC1A1-1 4) targeting mSLC1A1 and the negative control shRNA (shRNA-NC) were designed and synthesized to construct rAAV vectors respectively named rAAV-shRNA-SLC1A1-1 4 and rAAV-shRNA-NC by Gemma Gene Company (Suzhou, China). To identify the most effective shRNA sequence that could knockdown EAAT3 in the hippocampus, we screened 4 different sequences to infect the HT-22 cell line, nding that rAAV-shRNA-SLC1A1-2 displayed the lowest EAAT3 expression level by RT/PCR and western blot (Suppl. Fig. 1).
For stereotactic injection of rAAV vector into the bilateral hippocampus, mice were anesthetized with pentobarbital sodium (70mg/kg) and placed on brain stereotaxic apparatus (RWD Life Science, Shenzhen, China). After exposing the skull via an incision, two small holes were drilled for injection. The stereotactic coordinates were 2.1 posterior, ± 1.7 lateral, and 2.0 ventral from bregma. Injection speed was 50nl/min, and the needle was kept in place for an additional 15 minutes before it was slowly withdrawn.
RNAi group and RNAi+LPS group received bilateral hippocampal microinjection of rAAV-RNAi at 1μL per side (1×10 13 TU/mL), while the NC group and NC+LPS group received an equal volume of negative control rAAV-NC.
For stereotactic injection of rAAV vector into the bilateral hippocampus, mice were anesthetized with pentobarbital sodium (70mg/kg) and placed on brain stereotaxic apparatus (RWD Life Science, Shenzhen, China). After exposing the skull via an incision, two small holes were drilled for injection. The stereotactic coordinates were 2.1 posterior, ± 1.7 lateral, and 2.0 ventral from bregma. Injection speed was 50nl/min, and the needle was kept in place for an additional 15 minutes before it was slowly withdrawn.
RNAi group and RNAi+LPS group received bilateral hippocampal microinjection of rAAV-RNAi at 1μL per side (1×10 13 TU/mL), while the NC group and NC+LPS group received an equal volume of negative control rAAV-NC.
8
Knockdown of EAAT3 in Hippocampus
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Four potential different shRNA sequences (shRNA-mSLC1A1-1 4) targeting mSLC1A1 and the negative control shRNA (shRNA-NC) were designed and synthesized to construct rAAV vectors respectively named rAAV-shRNA-SLC1A1-1 4 and rAAV-shRNA-NC by Gemma Gene Company (Suzhou, China). To identify the most effective shRNA sequence that could knockdown EAAT3 in the hippocampus, we screened 4 different sequences to infect the HT-22 cell line, finding that rAAV-shRNA-SLC1A1-2 displayed the lowest EAAT3 expression level by RT/PCR and western blot (Suppl. Fig. 1).
For stereotactic injection of rAAV vector into the bilateral hippocampus, mice were anesthetized with pentobarbital sodium (70mg/kg) and placed on brain stereotaxic apparatus (RWD Life Science, Shenzhen, China). After exposing the skull via an incision, two small holes were drilled for injection. The stereotactic coordinates were 2.1 posterior, ± 1.7 lateral, and 2.0 ventral from bregma. Injection speed was 50nl/min, and the needle was kept in place for an additional 15 minutes before it was slowly withdrawn. RNAi group and RNAi+LPS group received bilateral hippocampal microinjection of rAAV-RNAi at 1μL per side (1×10 13 TU/mL), while the NC group and NC+LPS group received an equal volume of negative control rAAV-NC.
For stereotactic injection of rAAV vector into the bilateral hippocampus, mice were anesthetized with pentobarbital sodium (70mg/kg) and placed on brain stereotaxic apparatus (RWD Life Science, Shenzhen, China). After exposing the skull via an incision, two small holes were drilled for injection. The stereotactic coordinates were 2.1 posterior, ± 1.7 lateral, and 2.0 ventral from bregma. Injection speed was 50nl/min, and the needle was kept in place for an additional 15 minutes before it was slowly withdrawn. RNAi group and RNAi+LPS group received bilateral hippocampal microinjection of rAAV-RNAi at 1μL per side (1×10 13 TU/mL), while the NC group and NC+LPS group received an equal volume of negative control rAAV-NC.
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