Nanoliter injector

40–50 day old C57BL/6 male mice were used. Mice were deeply anesthetized with isoflurane before being placed on a stereotaxic frame fitted with a custom-made isoflurane mask. Ophthalmic gel (Ocry-gel) was placed onto the mice’s eyes and subcutaneous injection of lidocaine (Xylocaine) in the area was made prior to the incision of the skin. Craniotomy was performed with a fine drill bilaterally. Glass capillaries were filled with lentivirus suspensions and lowered into the cerebellar cortex. Injections were performed using a NanoLiter Injector and UMP3000 micro pump and controller (World Precision Instruments), 1 µL per hole was dispensed over a three minutes injection. The capillary was left in place for three minutes and then slowly pulled out over a two minutes period. Tissues were then glued using surgical skin glue (Vetbond). Mice were given buprenorphine (Vetergesic) for analgesia. Animals were kept 10 days before brains were collected for immunohistochemistry assay.

Oocytes isolated from mature female Xenopus laevis were supplied by the European Xenopus Resource Centre, University of Portsmouth, UK. Oocytes were treated with collagenase (0.5 mg/ml, Sigma type 1 A) in Ca²⁺-free solution (96 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 5 mM HEPES, 2.5 mM Na-pyruvate, 100 U/mL penicillin, 0.1 mg/mL streptomycin, pH 7.5) with shaking at 19 °C to defolliculate and remove the connective tissue surrounding the cells. After separation, oocytes were washed 7 times with modified Barth’s solution (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂, 5 mM HEPES, 2.5 mM Na-pyruvate, 0.5 mM theophylline, 50 μg/mL gentamicin, pH 7.5) and kept at 19 °C in the same solution.
Healthy oocytes were injected with cRNA using a Nano-liter Injector (World Precision Instruments Inc, USA). Mixtures of nAChR subunit cRNAs were injected as follows; for heteromeric rat neuronal receptors a 1:1 ratio of α:β at 200 ng/μL; for mouse embryonic muscle a 1:1:1:1 ratio of α:β:γ:δ at 25 ng/μL; human α7 at 100 ng/μL was mixed with RIC-3 at 30 ng/μL. Each oocyte was injected with 50 nL of RNA solution. Injected oocytes were saved in Barth’s solution at 19 °C for two to three days for expression of the target protein. During this time oocytes were regularly checked to remove unhealthy ones.

Reagents were diluted with Danieau's buffer and phenol red to desired concentrations. All reagents were injected at the 1-cell stage unless otherwise stated. Embryos were immobilized on a 1% agar plate, and an injection pipette was forced into the area adjacent to the blastomeres. A solution of the desired volume was injected using a Nanoliter injector (World Precision Instrument, Sarasota, FL, USA).

We used the multi-cell bolus-loading technique^{49 (link)} to label the olfactory bulbs. Fura PE-3 AM (Teflabs, Austin, TX) or OGB-1 BAPTA AM (ThermoFisher, Waltham, MA) were freshly dissolved in 20% Pluronic F-127 in DMSO (ThemoFisher), and diluted to 0.05 mM in 2.5% Fast Green (Sigma, St. Louis, MO) in a solution containing in mM: 150 NaCl, 2.5 KCl and 10 HEPES, pH 7.4 (final Pluronic F-127 concentration ≈ 0.5%). After sonication and filtering (0.45 µm Ultrafree centrifugal filter, Millipore, Billerica, MA), the dye solution was injected into the olfactory bulbs through a broken back glass microelectrode with a tip diameter of ~12 µm using a Nanoliter Injector (World Precision Instruments, Sarasota, FL). Approximately 1 µl dye solution was injected around the edge of the craniotomy. The olfactory bulbs were then covered with 2% agarose and a coverglass. Another window was installed over the olfactory epithelia (Fig. 1C).
Laser stimulation caused significant optical interference with simultaneous two-photon imaging (see below). To minimize this interference, we covered all areas that were exposed during surgery (except windows) with black dental acrylic. A black cardboard screen was also installed between the olfactory epithelium and the olfactory bulbs to block the stimulation laser light from reaching the microscope objective (Fig. 1C).

Green Fluorescent Protein (GFP)-encoding AAVs with a chicken beta-actin (CAG) promoter and tdTomato-encoding AAVs with a CAG promoter (AAV-8-CAG-GFP/tdTomato) were purchased from Vector Biolabs (Malvern, PA). At day 14 after SCI induction, the rats were placed on a stereotaxic frame under anesthesia induced by an IP injection of ketamine (75 mg/kg) and xylazine (10 mg/kg).
Fourteen days post-SCI, a craniotomy was performed to expose the sensorimotor cortex. The GFP-encoding AAV was injected into the right hemisphere. The tdTomato-encoding AAV was injected into the left hemisphere. Five rats per group (five intact, five vehicle-SCI, five MSC-SCI) received both AAV-8-CAG-GFP/tdTomato. Five rats per group (five intact, five vehicle-SCI, five MSC-SCI) received both AAV-8-CAG-tdTomato.
For each hemisphere, six injections for cortex (AAVs; 4.0 × 10¹⁰ genome copy/μL, 0.5 μL per site) were performed at the following coordinates: 1.0 mm lateral; 1.5 mm, 1.0 mm depth; and -1.0 mm, 0 mm, 1.0 mm posterior to the bregma using a nanoliter-injector (World Precision Instrument Inc., Sarasota, FL) attached to a pulled glass pipette.^{27 (link)} The method used in this study allowed us to perform precise microdelivery of viral vectors to localized regions in the brain. The needle was left in place for 3 min before moving to the next site.

Stages V and VI oocytes were surgically removed from Xenopus laevis ovaries (anesthetized with 1.5 g/L ethyl 3-aminobenzoate methanesulfonate salt; Sigma-Aldrich, Israel), prepared and maintained in ND-96 solution (at 18°C) containing 96 NaCl, 2 KCl, 1 MgCl2, 1.8 CaCl2, 2.5 sodium pyruvate, and 5 HEPES (AMRESCO, LLC) mM; 10 mg/ml PEN/STREP, and 50 μg/ml gentamicin, adjusted to pH 7.5. The incisions were closed using absorbable sutures and the animals were returned to the tank. Surgery was performed according to the guidelines provided by the Ben-Gurion University of the Negev ethics committee for the care and use of animals for experimental work (IL-69-12-2011). Within 24 h of surgery, oocytes were injected with one of the two newly synthesized GluN1-1a splice variant cRNAs (5 ng) and the GluN2A subunit cRNA (5 ng), using a nanoliter injector (World Precision Instruments, Sarasota, FL, United States). All cRNAs were produced by Prof. M. Hollmann’s laboratory (Ruhr University, Bochum, Germany). The NMDAR cDNA accession numbers for GluN1-1a and GluN2A are U08261 and AF001423, respectively. Those and other NMDAR subtypes were successfully expressed in the oocytes’ membranes.

Xenopus laevis oocytes were prepared as previously described [30 (link)]. For expression of the different channels oocytes were injected with 50 nl of cRNA one day after defolliculation. Injection was performed with the Nanoliter Injector (World Precision Instruments, Saratosa, FL). Xenopus laevis frogs were housed in 50 L tanks with continuous filtering and water circulation. The room temperature was 19°C. Frogs were anesthetized with 0.1% tricaine solution and killed by decerebration and pithing. 2 frogs were used for the experiments. All experimental procedures involving animals were conducted in accordance with state laws and institutional regulations. All experiments were approved by the Animal Care and Ethics Committee of Semmelweis University (approval ID: XIV-I-001/2154-4/2012).