All surgical and experimental procedures conformed to the guidelines of the Canadian Council on Animal Care (CCAC) and were approved by the Université de Montréal and Université du Québec à Montréal animal care committees, and the University of Windsor animal care committee, where some of the anatomical experiments were performed.
Experiments were performed on 98 reproductive adult sea lampreys (Petromyzon marinus
) of both sexes provided by the Great Lakes Fishery Commission and the Department of Fisheries and Oceans Canada. In addition, some of the electrophysiology studies were carried out on larval (n
62), newly transformed (n
102), and parasitic (n
7) lampreys. All animals were kept in aerated fresh water maintained at 7°C until used.
For the CNS attached to the intact olfactory epithelium preparation, the animals were anesthetized with tricaine methanesulphonate (MS-222, 100 mg/l, Sigma-Aldrich, Oakville, ON, Canada) and decapitated at the level of the 7th branchiopore. The surgery and all experiments were performed in cold oxygenated Ringer's (8–10°C) of the following composition (in mM): 130 NaCl, 2.1 KCl, 2.6 CaCl2, 1.8 MgCl2, 4.0 HEPES, 4.0 dextrose, and 1.0 NaHCO3, at pH 7.4. The branchial apparatus and the myotomal musculature were removed, with all the soft tissue attached to the ventral side of the cranium. A dorsal incision was made to expose the rostral spinal cord and the brain. The nasal cavity was left attached to the brain through the intact ONs. Care was taken to keep a maximum of the olfactory epithelium intact by simply performing a small window opening on its dorsal aspect, to simply permit faster perfusion and washout of odorants.
The preparation was placed into a recording chamber continuously perfused with cold oxygenated Ringer's at a rate of ~4 ml/min. A minimum of 1 h was allowed for recovery after surgery prior to recording. Using sharp glass microelectrodes filled with 4 M potassium acetate (80–130 MΩ), intracellular recordings were made from RS neurons in the MRRN under visual guidance through a binocular microscope (). The signals were amplified with an Axoclamp 2A (Axon Instruments, Foster City, CA). Only RS neurons displaying a stable membrane potential lower than −70 mV for at least 15 min were considered in this study.
For electric stimulations, we used homemade glass-coated tungsten electrodes (4–5 MΩ with a 10 µm tip exposure) and a Grass S88 stimulator (Astro-Med, Longueuil, QC, Canada). Stimulation was applied every 10 s as single, double, or triple pulses (2–50 µA intensity, 1–2 ms duration, and 20 ms pulse interval). Synaptic responses are presented as a mean of eight consecutive responses to the same stimulation.
To observe “fictive locomotion” (), we recorded from ventral roots in newly transformed lampreys, using suction electrodes filled with Ringer's solution. The signals were amplified using AM systems 1800 dual channel amplifiers (A-M systems Inc., Sequim, WA).
Semi-intact preparations () were dissected as follows: the brain and rostral spinal cord were exposed like previously described 
, whereas the caudal two-third of the body was kept intact to freely swim behind. In this case, because of the presence of cutaneous sensory inputs, the brain was transected at the level of the diencephalon for decerebration purposes before the experiment. The preparation was then transferred into a double compartment recording chamber. Teflon-coated stainless steel microwires (50 µm diameter) were inserted into the segmental muscles for EMG recording.
Calcium Imaging Experiments
RS cells were retrogradely labeled in Ringer's solution for 24 to 36 h by placing Calcium-Green dextran crystals (3000 MW, Invitrogen, Eugene, OR) on the rostral stump of the spinal cord, transected at the first segment. Labeled cells were observed on a Nikon epifluorescent microscope equipped with a 20× (0.75 NA) objective. A fluorescein isothiocyanate (FITC) excitation/emission filter set was used to visualize the neurons. The emitted light was captured with an intensified CCD video camera (Photometrics CoolSNAP HQ, Roper Scientific, Tucson, AZ) and recorded at a rate of two images per second, using Metafluor imaging software (Molecular Devices, Sunnyvale, CA). Calcium responses are expressed as relative changes in fluorescence (ΔF/F%).
Anterograde and retrograde labeling was obtained after unilateral injections of Texas Red-conjugated dextran amines (3000 MW, Molecular Probe). A period of 24 to 36 h was allowed for the transport of the tracer. The preparations were then immersed in a solution of 4% paraformaldehyde/0.4% picric acid in phosphate-buffered saline for 5 to 6 h. They were then transferred overnight into a solution of 20% sucrose in phosphate buffer. Transverse sections of 25 µm thickness were made with a cryostat and mounted on microscope slides with Vectashield (Vector laboratories, Burlington, ON, Canada) for observation under epifluorescence microscopy (Nikon E600 microscope equipped with a DXM1200 digital camera, Nikon, Montreal, QC, Canada; or with a Nikon E800 microscope in Windsor, ON, Canada).
Chemical Stimulations and Drugs
All drugs were purchased from Sigma-Aldrich (Oakville, ON, Canada), except sex pheromones (graciously provided by Dr. W. Li, Michigan State University, MI). They were kept as frozen concentrated stock solutions (at −80°C for pheromones) and dissolved to their final concentration in Ringer's solution prior to their use. For all local ejections, the inactive dye Fast Green was added to the drug solution to monitor the extent of application. Drug application was performed by pressure ejections of concentrated substances through a glass micropipette in the nasal cavity, or the brain tissue, using a Picospritzer (General Valve, Fairfield, NJ). Ejection of Ringer's with Fast Green at the same location was used as control in each experiment. Chemical stimulations of the olfactory epithelium were performed on reproductive adult lampreys during the first days after their capture in the wild.
Data were analyzed using paired t test or a Mann-Whitney test (Sigmastat, SPSS, Chicago, IL, USA). Significance was set at p<0.05. Results are presented as mean ± S.E.M.