Stereotaxic Injection of the Peptide and In Vivo Investigation

All experimental procedures and animal care were achieved in conformity with the guidelines of the French Government, with the approval of the Local Committee for Ethics on Animal Experiments (Comité d’Ethique en Expérimentation Animale des Pays-de-la-Loire). The peptide localization in the subventricular zone of adult Wistar rats was analyzed following its stereotaxic injection according to a previously described protocol [¹⁹].

Adult Wistar female rats were anesthetized by intraperitoneal injection of a mixture of ketamine 10% (80 mg/kg) and xylazine 2% (10 mg/kg). The rats were then placed on a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA, http://www.kopfinstruments.com), and a sagittal incision was made through the skin to expose the cranium in which a small hole was made using a dental drill at the appropriate coordinates (−0.8 mm anterior and 1.6 mm lateral or 2 mm lateral to the bregma to inject in the right lateral ventricle or in the subventricular zone, respectively). A 20-µl volume of the 5-FAM peptide 1 mmol/l was injected using a 10-µl Hamilton syringe (Hamilton glass syringe 70 RN) with a 32-gauge needle (VWR International, Radnor, PA, http://www.us.vwr.com) at a depth of 4.3 mm from the outer border of the cranium and connected through a cannula to a 100-µl Hamilton 22-gauge syringe (Hamilton glass syringe 810 RN; VWR International) containing the peptide. A slow infusion was performed by convection-enhanced delivery with an osmotic pump (Harvard Apparatus, Holliston, MA, http://www.harvardapparatus.com) at a flow rate of 0.5 µl/minute. After the injection and slow withdrawal of the needle (0.5 mm/minute), the head skin was sutured. The rats were killed 1, 24, or 48 hours after the injection, and the brains were removed and rapidly frozen using isopentane and liquid nitrogen. They were conserved in a −80°C freezer before sectioning them using a Leica cryostat (Leica Biosystems).

For immunohistochemistry, the brain sections (12 µm thick) were fixed with cold methanol for 10 minutes, washed three times in PBS, and blocked with PBS containing 5% bovine serum albumin at room temperature for 1 hour. The sections were then incubated overnight with mouse anti-glial fibrillary acidic protein (GFAP), mouse anti-vimentin (Sigma-Aldrich), mouse anti-nestin (R&D Systems), mouse anti-βIII tubulin (Sigma-Aldrich), rabbit anti-CD133 (Proteintech, Rosemont, IL, http://www.ptglab.com), or rabbit anti-Olig2 (Abcam, Cambridge, U.K., http://www.abcam.com) antibodies diluted 1/200, 1/200, 1/50, 1/200, 1/100, and 1/100, respectively, in PBS with 5% bovine serum albumin. After washing three times in PBS, primary antibodies were revealed using Alexa Fluor 568-labeled anti-mouse or anti-rabbit antibodies (Thermo Fisher Scientific Life Sciences) at a 1/200 dilution in PBS containing 5% bovine serum albumin. They were incubated for 90 minutes at room temperature and then washed in PBS. The brain sections were then counterstained with 3 µmol/l DAPI for 5 minutes and rinsed twice with PBS. Finally, the slides were mounted with the ProLong Gold antifade reagent and observed with an inverted fluorescent microscope (Leica DMI6000; Leica Biosystems) equipped with a CoolSNAP-HQ2 camera and analyzed with the MetaMorph, version 7.1.7.0, software (Molecular Devices) or with a confocal microscope (Leica TCS SP8; Leica Biosystems).