Stereotaxic surgery was performed 2–3 weeks before behavioral experiments began. Mice were anesthetized with isoflurane (5% induction, 2% maintenance) and placed into a stereotaxic frame (Kopf Instruments). An incision was made in the scalp and the skull was adjusted to place bregma and lambda in the same horizontal plane. Small craniotomies were made above the desired injection site in each hemisphere. AAV was delivered at a rate of 2 nl/s to dCA1 (AP −2.0 mm and ML ±1.5 mm from bregma; DV −1.25 mm from dura) through a glass pipette using a microsyringe pump (UMP3, World Precision Instruments). For stimulation experiments, the AAVs used were AAV9-CaMKIIa-hChR2(H134R)-eYFP (250 nl/hemisphere, titer: 8.96 × 1013, Penn Vector Core) and AAV9-CaMKIIa-eGFP (250 nl/hemisphere, titer: 3.49 × 1013, Penn Vector Core). For inhibition experiments, the constructs were AAV5-CaMKIIa-ArchT-GFP (350 nl/hemisphere, titer: 5.2 × 1012, UNC Vector Core) and AAV5-CaMKIIa-GFP (350 nl/hemisphere, titer: 5.3 × 1012, UNC Vector Core). After AAV infusions, an optical fiber (200 μm diameter, Thorlabs) was implanted above dCA1 in each hemisphere (AP −2.0 mm and ML ±1.5 mm from bregma; DV −1.0 mm from dura). The fiber implants were secured to the skull using dental adhesive (C&B Metabond, Parkell) and dental acrylic (Bosworth Company).
Stereotaxic frame
Manufactured by Kopf Instruments
Sourced in United States, Germany, Italy
The Stereotaxic frame is a laboratory instrument used to immobilize and position the head of a subject, typically an animal, during surgical or experimental procedures. It provides a secure and reproducible method for aligning the subject's head in a three-dimensional coordinate system to enable precise targeting of specific brain regions.
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Lab products found in correlation
Isoflurane, by Baxter (6 mentions)
Vetbond, by 3M (6 mentions)
Nanoject 2, by Drummond (6 mentions)
Heating pad, by Harvard Apparatus (5 mentions)
Cunningham adaptor, by Harvard Apparatus (5 mentions)
6 ohda, by Merck Group (5 mentions)
Metabond, by Parkell (5 mentions)
Microinfusion pump, by Harvard Apparatus (5 mentions)
Hamilton syringe, by Hamilton Company (5 mentions)
Syringe pump, by KD Scientific (5 mentions)
C b metabond, by Parkell (4 mentions)
Surgicel, by Johnson & Johnson (4 mentions)
Microinfusion pump, by World Precision Instruments (4 mentions)
Rose bengal, by Merck Group (4 mentions)
Avertin, by Merck Group (4 mentions)
Rompun, by Bayer (4 mentions)
Ultramicropump controller, by World Precision Instruments (3 mentions)
Infusion pump, by Harvard Apparatus (3 mentions)
Micro4 pump controller, by World Precision Instruments (3 mentions)
Nanoject 3, by Drummond (3 mentions)
536 protocols using stereotaxic frame
1
Stereotaxic Viral Delivery and Optogenetic Manipulations in Mouse dCA1
2
Adenoviral Targeting of GRP78 in Rat VMH and ARC
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Rats were placed in a stereotaxic frame (David Kopf Instruments) under ketamine-xylazine anesthesia. The VMH and ARC were targeted bilaterally using a 25G needle (Hamilton). The injections were directed to the following stereotaxic coordinates: (1) for the VMH, 2.4/3.2 mm posterior to the bregma (two injections were performed in each VMH), ±0.6 mm lateral to midline and 10.1 mm ventral; and (2) for the ARC, 2.8 posterior to bregma, ±0.3 mm lateral to midline and 10.2 mm ventral, as previously reported (López et al., 2008 (link); López et al., 2010 (link); Whittle et al., 2012 (link); Martínez de Morentin et al., 2014 (link); Beiroa et al., 2014 ). Adenoviral vectors (Viraquest) containing GFP (used as control), GRP78 WT (at 1012 particles/ml), GRP78 DN (dominant-negative, at 108 particles/ml) (Hendershot et al., 1995 (link)) were delivered at a rate of 200 nl/min for 5 min (1 μl/injection site) as previously reported (López et al., 2008 (link), 2010 (link); Whittle et al., 2012 (link); Martínez de Morentin et al., 2014 (link); Beiroa et al., 2014 ). Animals were treated for 6–9 days.
3
Remyelination Potential of Induced and Oligodendrocyte Progenitor Cells
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All animals, including the PBS-treated group, received 5 mg/ml cyclosporine daily starting the day before transplantation. O4+ OPC and iOPC suspensions were prepared at a concentration of approximately 200,000 cells/µl. The cell suspensions (5 µl) were transplanted over a period of 5 min bilaterally to the cerebral ventricles (Coordinates: AP = +0.9 mm, ML = 0.3 mm, DV = −2.8 mm) of the anesthetized animals 15 days after EAE onset. The cells were delivered by a Hamilton syringe (Hamilton, Reno, NV, USA) attached to a stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA). Animals that received the same surgery with an injection of 5 µl of PBS served as controls. To validate their myelination capacity, 500,000 O4+ iOPCs (n = 6) or OPCs (n = 6) were transplanted into the corpus callosum (coordinates: AP = +0.9 mm, ML = 0 mm, DV = −2.8 mm) of 4-week-old homozygous shiverer mice (The Jackson Laboratory, Bar Harbor, ME, USA) using the same procedure. Before transplantation, the mice were randomized into three groups (PBS-treated, iOPCs-engrafted, and OPCs-engrafted mice, n = 6 mice per group)
4
Intracerebral Injection of Mu-Saporin in Mice
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Mice were anesthetized under isoflurane gas and placed into a stereotaxic frame (Kopf instruments, CA, USA). Topical anesthetic (Cryogesic, Acorus Therapeutics Limited Chester, UK) was applied to the scalp before the head was shaved. A midline incision was made and the skull cleaned. A small burr hole was drilled over the left and right lateral ventricles and 0.5 μL of mu-saporin (0.596 μg/μL, Advanced Targeting Systems, San Diego, CA, USA; n = 16) or 0.9% saline (n = 19) was injected into each ventricle (coordinates from Bregma: AP = −0.4 mm, ML = 1.0 mm, DV = −2.3 mm) at a rate of 0.2 μl/min using a 32G Hamilton syringe. The needle was left in situ for 2 min after the injection to allow for diffusion. Analgesia was administered intraperitoneally at the time of surgery (Carprieve, 5% w/v, 0.32 ml/kg, Norbrook, Northamptonshire, UK) and mice were able to self-administer sugar-free jelly (Hartley, Histon Sweet Spreads Limited, Leeds, UK) containing Carprofen (250 μg, Zoetis, London, UK) for 1 week post-surgery.
5
Viral Vector Delivery to Vestibular Nucleus
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Mice (n = 28, 37.4 ± 7 days, 22.1 ± 3.2 g) were anesthetized with isoflurane (5.0 % induction, 1.5–0.7 % maintenance) and placed in a stereotaxic frame (Kopf Instruments). Ketoprofen analgesic solution (5 mg/kg s.c; Sanofi-aventis) and ocular protective gel (Viscotears®) were applied before stereotaxic procedure. Mice received intracranial AAV injections into the vestibular nucleus (VN) at the following coordinates: AP −0.6, ML ± 0.125, DV −0.39 mm from the Bregma, using the correction factor (Bregma - Lambda/4.21). A total of 1.0 μL of AAV-mitoTag vector (0.7 × 1012 viral genomes/mL; 0.5 μL per side) was injected into each hemisphere at a rate of 0.25 μL/min using a 5 μL Hamilton syringe with a 32G blunt needle. Following injection, the needle was kept in place for 4 min post-delivery to allow proper viral vector diffusion and, subsequently, removed at 1 mm/min.
6
Bilateral AAV Injection in mPFC
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For bilateral injection of adeno-associated viruses (AAV) (AAV2/9-CMV-mCherry; AAV2/9-hSyn-hM4Di–mCherry) into the mPFC, male mice (8–10 weeks old) were anesthetized with isoflurane (3% for induction and 1% for maintenance) and placed in a stereotaxic frame (David Kopf Instruments). An incision was made on the top of the skull, and the skin was retracted and connective tissue gently scraped away. After exposing the skull and cleaning the surface with 3% hydrogen peroxide, bilateral craniotomies (∼1 mm diameter each) were made to allow virus delivery (500 nl at 100 nl/min). Stereotaxic coordinates of virus injection were based on Paxinos and Franklin mouse brain atlas (AP: −1.94 mm, L: ±0.4 mm, DV: −2 mm). For EEG/EMG analysis of AAV-injected mice, the EEG/EMG electrode implantation was performed immediately following AAV injection. Clozapine N-oxide (CNO; Cayman Chemical, Item No. 12059) was dissolved in saline. Vehicle (0.9% saline) or CNO (3 mg/kg) was administered by intraperitoneal injection at ZT0 and ZT3.5 before the mouse returned to the home cage.
7
Chronic Intrathecal Catheterization in Rats
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Chronic catheterization of the intrathecal (i.t.) subarachnoid space was performed as described by Yaksh and Rudy (1976) (link). The rats were anesthetized with a ketamine-xylazine (75–10 mg/kg, i.p.) and placed in a stereotaxic frame (Kopf Instruments, United States) with the dorsal part of the head and neck previously shaved. The atlanto-occipital membrane was exposed and pierced, and a polyethylene catheter (PE-10, 9.0 cm length) was inserted intrathecally and advanced caudally to the level of the thoracolumbar junction. The wound was then sutured, and the animals were housed in individual cages to recover from surgery for 5 days before the formalin test.
8
Retrograde Tracing of Neural Circuits
9
Anterograde Tracing of Midbrain Dopaminergic Neurons
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Mice were anesthetized with isoflurane (4 mg/ml) supplemented in the air, while placed in a separate cage, and afterward, they were mounted in a stereotaxic frame (David Kopf Instruments, Tujunga, CA) equipped with a mouse adapter. Mice were kept anesthetized throughout the procedure by inhaling isoflurane at a concentration of 2 mg/ml. Anterograde tracing of the mDA neurons was achieved by injecting a Cre-dependent AAV expressing EGFP (AAV2/2.pCAG.FLEX.EGFP.WPRE.bGH) (26 (link)) in the midbrain of 8-month-old mice. Using bregma as a reference point, 1 μl of the AAV virus was injected unilaterally reaching the SN [anteroposterior (AP): −2.9, mediolateral (ML): −1.25, and dorsoventral (DV): −4.5] and 1 μl reaching the VTA (AP: −3.1, ML: −0.5, and DV: −4.2). All coordinates are in millimeters relative to bregma according to The Mouse Brain in Stereotaxic Coordinates (Academic Press, San Diego, CA, 2012). Virus injection was executed at a dispense rate of 0.2 μl/min. Animals were euthanized 3 weeks after surgery by CO2.
10
Unilateral Cortical Lesion Procedure
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Males 3–4 months old were anaesthetized with 2–3% isoflurane and positioned in a stereotaxic frame (Kopf Instruments, USA), as previously described (Xilouri et al., 2012 (link)). A unilateral lesion was performed to the right hemisphere of the cerebral cortex by inserting a 19-gauge needle at the following coordinates: +0.6 mm anteroposterior, −1.6 mm mediolateral and −3.2 mm dorsoventral from the bregma as previously described (Buffo et al., 2005 (link); Frik et al., 2018 ). The needle was twisted manually and gently retracted. This was repeated thrice in total and the skin was thereafter sutured.
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