Stereotaxic Infusion of Recombinant Reelin Protein

Offspring were anesthetized with a mixture (i.p.) of medetomidine hydrochloride (0.3 mg/kg; Wako), midazolam (4 mg/kg; Wako), and butorphanol tartrate (5 mg/kg; Wako), and then positioned between the ear bars of a stereotaxic frame (SR-6N; Narishige, Tokyo, Japan). Full-length Reelin (0.2 pmol/0.5 μL) protein or PBS (control) was delivered bilaterally with a Hamilton syringe at a rate of 0.1 μL/min for a total volume of 0.5 μL on each side. The needle was left in place for 5 min. Injection volume and concentration of recombinant Reelin protein was determined using previous studies (Rogers et al., 2011 (link), 2013 (link); Ishii et al., 2015 (link)). The following coordinates were used: -1.75 mm rostrocaudal, -2.0 mm dorsoventral, ±1.0 mm mediolateral from bregma (relative to dura). Immediately after removal of the needle, the skin was closed with tissue adhesive (Vetbond, 3M, St. Paul, MN). The injection point was confirmed by methylene blue microinjection (Supplementary Figure S1).

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Ibi D., Nakasai G., Koide N., Sawahata M., Kohno T., Takaba R., Nagai T., Hattori M., Nabeshima T., Yamada K, & Hiramatsu M. (2020). Reelin Supplementation Into the Hippocampus Rescues Abnormal Behavior in a Mouse Model of Neurodevelopmental Disorders. Frontiers in Cellular Neuroscience, 14, 285.

Publication 2020

medetomidine hydrochloride midazolam butorphanol tartrate SR-6N Vetbond

Bars Butorphanol tartrate Dura Frame Medetomidine hydrochloride Methylene blue Microinjection Midazolam Needle Protein Reelin Reelin protein Skin Syringe Tissue adhesive

Corresponding Organization : Nagoya University Hospital

Other organizations : Nagoya City University, Fujita Health University

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Variable analysis

independent variables

Injection of full-length Reelin protein (0.2 pmol/0.5 μL) or PBS (control)

dependent variables

Not explicitly mentioned

control variables

Anesthesia mixture (medetomidine hydrochloride, midazolam, and butorphanol tartrate)
Stereotaxic frame positioning
Injection coordinates (-1.75 mm rostrocaudal, -2.0 mm dorsoventral, ±1.0 mm mediolateral from bregma)
Injection volume (0.5 μL) and rate (0.1 μL/min)
Leaving the needle in place for 5 minutes after injection
Closing the skin with tissue adhesive after needle removal

controls

Positive control: Full-length Reelin protein (0.2 pmol/0.5 μL)
Negative control: PBS

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