S32357

Zebrafish were anesthetized in 0.03% tricaine methane sulfonate (MS-222, Sigma-Aldrich, E10521). Retrograde labeling of descending spinal cord neurons located in spinal segments 1–3 was achieved through dye injections with biotinylated dextran (3000 MW; ThermoFisher, D7135) into segment 16 or 17. Animals were kept alive for at least 24 h after injection to allow retrograde transport of the tracer, deeply anesthetized with 0.1% MS-222, and the spinal cords were dissected and fixed in 4% paraformaldehyde (PFA) and 5% saturated picric acid (Sigma-Aldrich, P6744) in phosphate-buffered saline (PBS; 0.01 M, pH = 7.4; Santa Cruz Biotechnology, Inc., CAS30525-89-4) at 4 °C for 4–10 h. The tissue was then washed extensively with PBS and incubated in streptavidin conjugated to Alexa Fluor 488 (dilution 1:500, ThermoFisher, S32354), Alexa Fluor 555 (1:500, ThermoFisher, S32355), or Alexa Fluor 647 (dilution 1:500, ThermoFisher, S32357) overnight at 4 °C. Primary and secondary antibodies were applied as described in the “Immunohistochemistry” section. After thorough buffer rinses, the tissue was mounted on gelatin-coated microscope slides and cover-slipped with an anti-fade fluorescent mounting medium (Vectashield Hard Set, VectorLabs; H-1400).

The following antibodies and molecules were used (concentrations used are indicated in parentheses): Chicken polyclonal anti-calreticulin (Thermofisher, PA1-902A, 4,4 µg/mL), Rabbit polyclonal anti-ERp57 (Abcam, ab10287, Dilution 1:100), Mouse anti-CD47 conjugated to Alexa Fluor A647 (Santa Cruz, B6H12, 2 µg/mL), Annexin V conjugated to Biotin (Biolegend, 640904, 2.5 µg/mL), Rabbit Polyclonal anti-Chicken IgY labeled with Alexa Fluor 532 (Cohesion Biosciences, CSA3314, 5 µg/mL), Goat polyclonal anti-Chicken labeled with Alexa Fluor 555 (Thermofisher, A21437, 2 µg/mL), Donkey polyclonal anti-Rabbit IgG labeled with FluoProbes 647H (Interchim, FP-SC5110, 10 µg/mL), Streptavidin conjugated to Alexa Fluor 647 (Thermofisher, S32357 - 0,4 µg/mL).

For identification of cells following recordings, tissue was fixed in 4% paraformaldehyde overnight at 4^oC. Slices were washed with PBS three times, 20 min each, and incubated in Streptavidin-Alexa 488 (S11223) or Alexa 647 (1:500, S32357, Invitrogen) and DAPI (1:1000, in 0.3% PBST) overnight at 4°C. Slices were washed in 0.3% PBST four times and mounted on glass slides with Mowiol 4-88. Mcherry fluorescence in the MEC was examined with confocal imaging and those animals where virus injection missed MEC L5 were excluded.
For staining of the interneuron marker Parvalbumin (PV), slices were prepared as described above, then incubated in primary antibody solution containing mouse anti-PV (1:1000, PV 235, Swant) and 5% NGS in 0.3% PBST for 48 hr at 4°C. Slices were then washed and incubated in secondary antibody solution with Alexa-conjugated Streptavidin and DAPI prepared in 0.3% PBST and mounted on glass slides after overnight incubation.
A Zeiss LSM800 microscope was used for image acquisition of the slices. Pinhole was set to 1 Airy Unit. Objectives used include ×10 (air), ×20 (air), and ×40 (oil) to image the hippocampal formation, morphology of biocytin filled cells, and immunolabelling of interneurons, respectively.

At the end of the in vivo experiments, animals were deeply anesthetized using urethane (2.5 g kg⁻¹ body weight) and transcardially perfused with phosphate-buffered saline (PBS), followed by 4% paraformaldehyde (PFA) in PBS. Following an overnight fixation in PFA, brains were carefully washed in PBS before they were mounted on a vibratome (Leica VT1000S, Leice Biosystems, Wetzlar, Germany) and cut into 100 µm slices. Sections were mounted in either Vectashield (Vector laboratories) or DAPI-containing Fluoroshield (Sigma-Aldrich). For in vitro experiments, slices were transferred to a PFA solution and fixated overnight. Immunoreactions for GAD-67 were carried out using primary mouse antibody (diluted 1:500, MAB5406, Millipore) and secondary Alexa 555 (1:500, A-21424, Invitrogen). Streptavidin was conjugated to Alexa 647 (1:500, S32357, Invitrogen) for visualizing the biocytin. Sections were then mounted in Fluoroshield and imaged using a Leica TCS SP5 confocal microscope (Leica Biosystems, Wetzlar, Germany). Images were analyzed using the free software ImageJ. The location of individual cortical recording sites was assigned to either deep or superficial layers based probe tracks and recording depth