Ferrets aged P18-23 were anesthetized with isoflurane (delivered in O2). Atropine was administered and a 1:1 mixture of lidocaine and bupivacaine was administered SQ. Animals were maintained at an internal temperature of 37 degrees Celsius. Under sterile surgical conditions, a small craniotomy (0.8 mm diameter) was made over the visual cortex (7-8mm lateral and 2-3mm anterior to lambda). For imaging of GABAergic axons, we injected 5-30 nl of AAV1.mDlx.GCaMP6s at 400 and 200 mm below the pia. In some experiments, we co-injected AAV1/2-hSyn-jRGECO1a (~1uL). For STOMPM (see below), we injected ~1μl of AAV1-mDlx-ChR2-Flag-Kv2.1-p2a-H2b-CyRFP. Finally, the craniotomy was filled with sterile 1 % w/v agarose (Type IIIa, Sigma-Aldrich) and the incision site was sutured.
Type iiia
Manufactured by Merck Group
Type IIIa is a specialized laboratory equipment designed for precise and accurate measurements. It is intended for use in controlled environments, such as research laboratories or quality control settings. The core function of this product is to provide reliable and consistent data collection capabilities for a variety of scientific applications.
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14 protocols using type iiia
1
Anesthetic Ferret Preparation for Visual Cortex Imaging
2
In Vivo Imaging of Mouse Cortex and Cerebellum
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The C57BL6/J male mice (postnatal days 28–61) were anesthetised by intraperitoneal injection of 1.9 mg/g urethane (for neocortical imaging), or 100 mg/kg ketamine and 10 mg/kg xylazine (for cerebellar imaging). The depth of anesthesia was monitored regularly by observing whisker movements and the pinch withdrawal reflex of the hindlimb; additional doses of anesthetic were given as needed. Body temperature was kept at 37 °C with a heating pad (FHC, Bowdoin, ME, USA). The mouse was fixed on a custom stereotaxic apparatus under a surgical microscope (Leica). A skin incision was made along the midline and the skull was partly exposed. A stainless steel frame was glued to the skull with superglue and dental cement. A small craniotomy (˜2 mm in diameter) was made to expose the left barrel cortex (3 mm lateral to the midline and 1.5 mm caudal to the bregma) or the left Crus IIa of the cerebellum (4 mm lateral and 2 mm posterior to the occipital bone line) using a high-speed drill (ULTIMATE500; NSK, Kanuma, Tochigi, Japan). The dura was carefully removed, and the craniotomy was filled with 1.5% agarose (type IIIa, Sigma-Aldrich) in a solution of the following composition (in mm ): 150 NaCl, 2.5 KCl, 10 HEPES, 2 CaCl2, 1 MgCl2, pH 7.3. A coverslip was placed over the agarose to minimise brain movements. The mouse was then transferred to the animal stage under the two-photon microscope.
3
Ferret Visual Cortex Manipulation
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Briefly, female ferrets aged P18–23 (Marshall Farms) were anesthetized with isoflurane (delivered in O2). Atropine was administered and a 1:1 mixture of lidocaine and bupivacaine was administered SQ. Animals were maintained at an internal temperature of 37°C. Under sterile surgical conditions, a small craniotomy (0.8 mm diameter) was made over the visual cortex (7–8 mm lateral and 2–3 mm anterior to lambda). A mixture of diluted AAV1.hSyn.Cre (1:25,000–1:50,000) and AAV1.Syn.FLEX.GCaMP6s (UPenn) was injected (125–202.5 nl) through beveled glass micropipettes (10–15 micron outer diameter) at 600, 400, and 200 microns below the pia. Finally, the craniotomy was filled with sterile agarose (Type IIIa, Sigma-Aldrich) and the incision site was sutured.
4
Ferret Visual Cortex Viral Infection
5
Sparse GCaMP6s Expression in Ferret V1
6
Craniotomy for Barrel Cortex Imaging
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Imaging windows were installed above the barrel cortex in mice about 2 months old. Mice were deeply anesthetized with an isoflurane–oxygen mixture. A craniotomy (1.5-mm diameter) was opened above the right barrel cortex (1 mm posterior from bregma; 3.5 mm lateral from midline), leaving the dura intact. The dura was covered with 1.8% agarose (Type-IIIA, Sigma) dissolved in cortex buffer (Holtmaat and Svoboda 2009 (link)), and covered with a 5-mm custom-made coverslip (Matsunami Glass) that was sealed into place with dental cement. A custom-designed head-mounting apparatus was attached to the animal's skull to reduce motion-induced artifacts during imaging. For a subset of animals, the position of the imaging window was mapped relative to the layer 4 barrels using standard histological methods.
7
Viral Labeling of Visual Cortex Neurons
8
Viral Labeling of Visual Cortex Neurons
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Female ferrets (n = 3) aged P18–23 (Marshall Farms) were anesthetized with isoflurane (delivered in O2). Atropine was administered and a 1:1 mixture of lidocaine and bupivacaine was administered SQ. Animals were maintained at an internal temperature of 37° Celsius. Under sterile surgical conditions, a small craniotomy (0.8 mm diameter) was made over the visual cortex (7–8 mm lateral and 2–3 mm anterior to lambda). A mixture of diluted AAV1.hSyn.Cre (1:25000 to 1:50000) and AAV1.Syn.FLEX.GCaMP6s (UPenn) was injected (125 – 202.5 nL) through beveled glass micropipettes (10–15 μm outer diameter) at 600, 400, and 200 μm below the pia. Finally, the craniotomy was filled with sterile agarose (Type IIIa, Sigma-Aldrich) and the incision site was sutured.
9
Viral Injection for In Vivo Imaging in Ferrets
10
Sparse GCaMP6s Expression in Ferret V1
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Eight juvenile female ferrets (Mustela putorius furo, Marshall Farms) aged P21–P23 were anesthetized with ketamine (50 mg/kg, IM) and isoflurane (1–3%) delivered in O2, then intubated and artificially respirated. Atropine (0.2 mg/kg, SC) was administered to reduce secretions and 1:1 mixture of lidocaine and bupivacaine administered subcutaneously in the scalp. Animals were placed on a feedback-controlled heating pad to maintain internal temperature at 37°C. Under sterile surgical conditions, a small craniotomy (0.8 mm) was made over the visual cortex 7–8mm lateral and 2mm anterior to lambda. AAV2/1.hSyn.Cre (1.32*1013 GC ml−1, Penn Vector Core) was diluted to 1:50000 in phosphate-buffered saline (Sigma) and mixed with AAV2/1.CAG.Flex.GCaMP6s (7.31*1012 GC ml−1, Penn Vector Core) to sparsely express GCaMP6s in layer 2/3 cortical neurons. Beveled glass micropipettes (~15 μm outer diameter, Drummond Scientific Company) were lowered into the brain, and 55 nl of virus were injected over 5 minutes (Nanoject II, Drummond Scientific Company) at 400 and 250μm below the pia. To prevent dural regrowth and attachment to the arachnoid membrane, the craniotomy was filled with 1% w/v agarose (Type IIIa, Sigma-Aldrich).
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