Neurobiotin tracer

Only transfected cells transplanted into injured slices were used for the electrophysiological studies. After 7 days in culture, slices were transferred to a submersion style recording chamber; and perfused with room temperature, aCSF continuously bubbled with 95/5% O₂/CO₂. The aCSF contained (in mM): 126 NaCl, 3 KCl, 1.25 NaH₂PO₄, 2 MgSO₄, 26 NaHCO₃, 2 CaCl₂, and 10 d-glucose. Whole cell recordings used an Axopatch 200B or Multiclamp 700B amplifier, digitized by a Digidata 1322A, and stored on a PC running pClamp version 9 software (Molecular Devices, Sunnyvale, CA, USA). The intracellular solution was composed of (in mM): 130 K-Gluconate, 15 KCl, 5 HEPES, 1 EGTA, 4 Mg–ATP, and 0.3 Na–GTP. In a subset of recordings, 0.2% Neurobiotin™ Tracer (Vector Laboratories, Burlingame, CA, USA) was included in the intracellular solution to allow post hoc visualization of recorded cells. Whole cell recordings took place in the vicinity of the injured region. Cells without any GFP in the injury site and away from it were recorded to serve as a control for electrophysiological characterization.

In five experiments, the patch pipette electrolyte solution was mixed with Neurobiotin Tracer (Vector Laboratories) in order to stain recorded neurons or the tracks made to reach them. The animals were kept under general anesthesia and then transcardially perfused using 100 ml phosphate buffered saline (PBS) followed by 75 ml 4% paraformaldehyde (PFA) solution. The brains were then removed and post-fixed in 4% PFA solution for 48–72 h and stored in PBS. Before sectioning, the brain was submerged in 25% sucrose in PBS for 48 h. Sixty micrometer sections were then cut using a cryostat-microtome and the sections were stained with Streptavidin conjugated to Alexa Flour 488 (Molecular Probes Inc.). A confocal microscope combined with a fluorescence microscope was then used to identify any stained neurons and electrode tracks. This was used together with the atlas by Paxinos and Watson (2006) in order to determine the location of the recorded neuron, which was compared with the location according to stereotaxic coordinates.

Neurobiotin Tracer (Vector Laboratories) was added into the intracellular solution (4 mg/ml) and diffused into the target Glra3-Cre;tdTomato cells during the patch-clamp recording. The diffusion of Neurobiotin was further assisted by injecting depolarizing current pulses (0.2–0.5 nA; duration, 150 ms) into the cell at 2 Hz for 10–15 min. After the filling, the patch pipette was carefully detached from the cell and removed from the recording chamber. The excessive Neurobiotin in the tissue was removed by perfusing the slice for at least 15 more min after the removal of the pipette. The slice was then transported into an Eppendorf tube and fixed in 4% FA (Histolab) overnight at 4°C. Fixed slices were washed with 1× PBS (Fisher BioReagents) 4x 10 min before the staining. Slices were stained for PKCγ using the same procedure described in previous immunohistochemistry section. Additionally, streptavidin Alexa Flour 488 conjugate (Invitrogen) was added to the primary antibody staining solution with 1:1,000 dilution ratio for Neurobiotin staining. The mounted slice was imaged using a ZEISS LSM700 confocal microscope (ZEISS) with 10× and 20× objectives. The morphology of a filled neuron was reconstructed using the Simple Neurite Tracer plug-in in the NIH ImageJ software (National Institutes of Health).

For intracellular recording, two Ag-AgCl wire interfaces were used, one of which served as reference immersed in saline while the other was inserted into a sharp micropipette. Micropipettes were drawn from borosilicate capillaries (0.75 mm ID, 1.5 mm OD, Hilgenberg, Malsfeld, Germany) with a Flaming/Brown filament puller (P-97 Sutter Instrument Company, Novato, CA), and filled with 1 M KCl for electric conduction. Impedances in tissue ranged from 50–200 MΩ. To allow labeling of cells, the tips of the micropipettes were loaded with Neurobiotin tracer (Vector Laboratories, Burlingame, UK, 4% in 1 M KCl) that could be injected iontophoretically (0.5–2 nA, 1–15 min) after recording. Tapped potentials were amplified and band-passed (10×, 20 Hz–20 kHz; SEC 1L/H amplifier, npi electronic, Tamm, Germany) prior to digitization (16 bit / 11.1 kHz; Power1401mkII converter run with Spike2 software, both Cambridge Electronic Devices, Cambridge, UK) and storage. Software for offline analysis was written in MATLAB (MathWorks, Natick, MA, USA).

For experiments requiring high-resolution images of the RGC dendritic arbors (Supplementary Fig. 1a–h) or immunohistochemical labeling of proteins (Fig. 1b, Supplementary Fig. 1a, i, and Supplementary Fig. 12), RGCs were filled with Neurobiotin tracer (Vector Laboratories, SP-1150, ~3% w/v potassium-based internal solution) and fixed in 3% paraformaldehyde solution for 15 min. After performing immunohistochemical labeling and incubation with streptavidin (see “Immunohistochemistry” below), tissues were imaged on a Nikon A1R laser scanning confocal microscope through a ×40 or ×100 oil immersion objective (Nikon Plan Apo VC × 40/ × 60/1.4 NA).