Slice Preparation and Physiological Experiments

2.2.1 Slice preparation

Tadpoles of Xenopus laevis (stage 51 54; staged afterNieuwkoop and Faber(1967)) were chilled in a mixture of ice and water and decapitated, as approved by the Göt-tingen University Committee for Ethics in Animal Experimentation. A block of tis-sue containing the olfactory mucosa, the olfactory nerves and the anterior two-thirds of the brain were cut out and kept in bath solution containing (in mM): 98 NaCl, 2 KCl, 1 CaCl2, 2 MgCl2, 5 glucose, 5 sodium pyruvate, 10 Hepes; 230 mosmol, pH 7.8. The tissue block was glued onto the stage of a vibroslicer (VT 1000S, Leica, Bensheim, Germany) and cut horizontally into approximately 200 µM-thick slices (Manzini et al., 2002). The slices were transfered into custom built microscopy chambers and stabilized with a grid (Edwards et al., 1989).

2.2.2 Staining protocols Bath incubation

For the staining solution, Fluo-4/AM (Molecular Probes, Karlsruhe, Germany) was rst dissolved in 20% Pluronic F-127 in DMSO, and then diluted in the bath

2.2 Slice Preparation and Physiological Experiments solution to reach the nal concentration. To avoid transporter-mediated destaining of the slices, MK571 (50 µM; Alexis Biochemicals, Lörrach, Germany), a specic inhibitor of multidrug resistance-associated proteins, was added to the staining so-lution (Manzini and Schild,2003b;Manzini et al.,2008). For 2D imaging, the tissue slices were incubated in a solution with dye concentration of 2 5µMfor 30 min, fol-lowed by a post-incubation period of 30 min in ringer solution with MK571 (50µM).

Bolus loading

For 3D imaging, a solution containing 100 - 500 µM Fluo-4/AM was pressure-injected at a depth of approx. 70µmusing patch pipettes (6 8 MΩ, 50 100 hPa for 1 5 min) into two sites per bulb hemisphere (adopted from Garaschuk et al.

(2006); Stosiek et al. (2003)). The progress of the injection was monitored using a 40× water immersion objective. The spontaneous activity of the neurons was investigated after an incubation period of 30 40 min following the last injection.

Staining of ORNs by electroporation

The animal was anesthetized in ice cold water, placed on a preparation dish covered with silicon, and gently xed by restraining its movements with ne needles. The caudal part of the tadpole was covered with wet cellulose tissue. Small crystals (1 10µg) of Fluo-4 dextran (Invitrogen, Karlsruhe, Germany) were placed in both nasal cavities. After the crystals dissolved, two platinum electrodes were inserted in the nasal cavities. The dye was transferred into the cells by electroporation. Six pulses of 20 V and 20 ms duration were applied, with a break of 5 min between the third and fourth pulse. After another 5 min, the animal was placed back into the water. Experiments were carried out 1 2 days later. This staining proce-dure was established by Eugen Kludt, Department of Neurophysiology and Cellular Biophysics, University Göttingen.

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2. Materials and Methods

2.2.3 Application of odorants Application system

For odor application experiments, the recording chamber was constantly perfused by gravity feed from a syringe through a slightly modied version of a funnel applicator described earlier (Manzini,2002;Schild,1985). The tip of the applicator was placed in front of the ipsilateral mucosa using a 10×objective. The odorants were applied into the funnel without stopping the ow of the bath solution using an electronic pipette (HandyStep electronic, Brand, Wertheim, Germany). The pipette was modied to control the outow with the trigger signal (TTL) provided by the control PC. The timing and reproducibility of the stimulus application was measured by adding uorescent dye (1 µM Fluorescein, Sigma, Deisenhofen, Germany) to the stimulus solution, and imaging the outow from the tip of the applicator with a 10×

objective at 400 Hz. The delay was measured to be (363±9) ms, (436±19) ms and (716±108) ms for 10%, 50% and 90% of the maximum concentration, respectively (see Fig. 3.15b). The absolute time of this delay might slightly vary from slice to slice due to the positioning of the applicator relative to the mucosa. Fluid was removed from the recording chamber using a syringe needle connected to a vacuum pump (Hyo Model C vacuum pump, Medcalf Brothers Ltd., Potters Bar, England) via a waste-bottle.

Odor stimuli

The olfactory system was stimulated with solutions containing amino acids or an extract of amphibia food based on Spirulina algae (Mikrozell, Dohse Aquaristik, Bonn, Germany). The amino acids (Sigma, Deisenhofen, Germany) were dissolved in bath solution (10 mM stock) and diluted prior to the experiment to the nal concentrations as indicated in the text. The amino acids were applied individu-ally or as a mixture of 15 amino acids (L-proline, L-valine, L-leucine, L-isoleucine, L-methionine, L-glycine, L-alanine, L-serine, L-threonine, L-cysteine, L-arginine, L-lysine, L-histdine, L-tryptophane, L-phenylalanine), excluding amino acids that could have a direct eect on the neurons in the OB (glutamate, aspartate,

L-2.2 Slice Preparation and Physiological Experiments glutamine and L-asparagine). For the solution containing the food extract, 0.5 g of Spirulina algae were dissolved in 100 ml bath solution and ltered through a single use lter (0.5 µm pore size, Minisart, Sartorius AG, Göttingen, Germany). Ap-plication of bath solution was used as a negative control. Odors were applied in a randomized order, with a minimum interstimulus duration of 1.5 min.

2.2.4 Microscopy

The custom built line-illumination microscope, described in detail in3.1, was used for all image acquisition except for imaging the results of the dye injection ex-periments in 3.2. After dye injection, slices were imaged using an Axiovert 100M equipped with a laser-scanning unit LSM 510 (Zeiss, Jena, Germany). Alexa Bio-cytin 532 was imaged in the red channel (helium/neon laser, 543 nm, NFT 545, LP 560). The green channel (Fluo-4) was imaged for alignment with the physio-logical recordings from the line illumination microscope (argon ion laser, 488 nm, HFT 488/53, BP 505-550). The following objectives were used as indicated in the text: 25× LD LCI Plan-Apochromat 0.8 W; 40× Achroplan 0.8 W; 63× Achro-plan 0.95 W; 40× C-Apochromat 1.2 W; 10× Plan-NeoFluar 0.3 (all Zeiss, Jena, Germany). For each experiment, `dark images' were acquired by closing the laser shutter with otherwise identical acquisition parameters. These images were used for background estimation.

2.2.5 Electrophysiology

Patch clamp was performed using an EPC7 plus amplier (Heka, Germany) and pipettes with a series resistance between 8 and 12 MΩ. Alexa Biocytin 532 (Invitrogen, Germany) was added to the intracellular solution (in mM: 2 NaCl, 11 KCl, 2 MgSO4, 80 K-Gluconat, 10 Hepes, 0.2 EGTA, 2 Na2ATP, 0.1 Na2GTP).

After breaking the seal, the cell was held at -65 mV for one minute to allow diusion of the dye into the cytosol.

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2. Materials and Methods