Common goldfish, Carassius auratus, of both sexes ranging from 7 to 11.5 cm in standard length, were purchased from Hunting Creek Fisheries (Thurmont, MD). The animals were housed in a filtered and aerated aquarium at approximately 18˚C in the Animal Resources Center, University of Colorado Denver Health Sciences Center. All procedures reported herein were carried out with the approval of the University of Colorado Denver Health Sciences Center Institutional Animal Care and Use Committee.
Retrograde labeling of reflex interneurons
To retrogradely label the reflex interneurons in the sensory layer of the vagal lobe, biocytin injections were made into the motor layer in goldfish vagal lobe slices. Goldfish were cold anesthetized with ice and the brain was removed quickly from the skull. The brain was blocked in the transverse plane, and mounted on a platform with acrylate tissue glue (Vetbond, 3M, St. Paul, MN). The tissue was embedded in warmed 2% agarose (Type IX, melting point 8–17˚C; Sigma, St. Louis, MO), covered with chilled, oxygenated artificial cerebral spinal fluid (aCSF, 131 mM NaCl, 20 mM NaCO3, 2 mM KCl, 1.25 mM KH2PO4, 2 mM MgSO4, 2.5 mM CaCl2, 10 mM dextrose, pH ~7.4), and sliced at 350 – 800 µm on a vibratome. After the sectioning, vagal lobe slices were put on the plastic petri dish and small crystal of biocytin (Sigma) were applied into the motor layer with a broken glass micropipette under microscopic observation. Brain slices were then incubated in the aCSF for 8–17 hours at room temperature and fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.2) overnight. After rinsing in 20% sucrose in 0.1 M PB at 4˚C for 3–5 hours, the brain slices were embedded in 10% gelatin containing 20% sucrose and postfixed again in the same fixative containing 20% sucrose at 4˚C overnight. After washing in 20% sucrose in 0.1 M PB, the embedded brains were frozen and cut in the transverse plane at 36–40 µm on a cryostat. The sections were first washed in phosphate buffered saline (PBS) containing 0.3% Triton X-100 (PBST) for 30 minutes and then pretreated with 0.3% hydrogen peroxide (H2O2) in methanol for 10 minutes to reduce endogenous peroxidase. After 3 × 10 minutes washes in PBS, the sections were incubated in avidin-biotin HRP complex (1/200, Vector Laboratories, Burlingame, CA) for 1 hour at room temperature and washed 3 times in PBS (20 minutes each). Then the sections were incubated in 0.025% NiCl2, 0.03% H2O2, 0.025% diaminobenzidine (DAB) in 0.1 M PB in the dark for 10 minutes and given 3 washes in PBS (10 minutes each). After washing, the sections were mounted on Superfrost Plus slides (Fisher, Fair Lawn, NJ) and dried overnight at room temperature. Then the mounted sections were counterstained with Giemsa, dehydrated in a graded series of ethyl alcohol, cleared with xylene, and coverslipped with Permount.
Double labeling of reflex interneurons (VRIs) and primary vagal afferent fibers
Double labeling was used to assess the relationship between VRIs and primary vagal afferent fiber terminals. The animals were anesthetized in 0.15 g/l MS-222 (tricaine methanesulfonate, Sigma), and then were placed in a fish surgical restraint to hold them stationary while permitting superfusion of the gills with aerated tap water containing 0.075 g/l MS-222. To expose the vagus nerve, a hole was made in the left side of the cranium just dorsal to the operculum. A small amount of cholera toxin subunit B conjugated Alexa Fluor 555 (Molecular Probes/Invitrogen, Eugene, OR) or 3,000 MW Texas Red or rhodamine-labeled fixable dextran amine (Molecular Probes/Invitrogen) was applied to the vagus nerve and the wound was covered with gelfoam (Upjohn, Kalamazoo, MI) and Vetbond Surgical Adhesive (3M Animal Care Products, St. Paul,. MN).
After 2–4 day survival, living vagal lobe slices were made as described above and observed under the fluorescent microscope to confirm labeling of primary afferent fibers and then biocytin was injected in the motor layer as described above. The slices were fixed and sectioned as above and the resulting tissue sections were incubated in PBST at 30 minutes. The fluorescent signals of cholera toxin, although apparent, were not sufficiently strong to permit visualization of all labeled processes. Therefore, sections were treated with immunohistochemistry with primary antiserum against cholera toxin subunit B (1/5000, polyclonal, produced against recombinant cholera toxin subunit B, Molecular Probes/Invitrogen) to optimize the signals. This antiserum produces no staining in uninjected goldfish. Sections were incubated in blocking solution (2% normal goat serum in PBST) for 2–5 hours at room temperature before incubation in primary antiserum for overnight at 4˚C. The sections then were washed in three changes of PBS (each 20 minutes) and incubated in Alexa 555 goat anti-rabbit IgG (1/1000) and streptavidin, Alexa Fluor 488 conjugate (1/2000, Molecular Probes/Invitrogen). After 3 washes with 0.1 M PB (each 20 minutes), the sections were mounted and coverslipped with Fluoromount-G (Southern Biotechnology Associates, Birmingham, AL).
Triple labeling of reflex interneuron terminals, vagal motoneurons, and calretinin
To label the vagal motoneurons, dextran tetramethylrhodamine and biotin (3,000 MW, micro-ruby, Molecular Probes/Invitrogen) was applied to the vagus nerve as mentioned above. Then a paste of dextran fluorescein and biotin (3,000 MW, micro-emerald, Molecular Probes/Invitrogen) was applied into the superficial portions of the vagal lobe to anterogradely label the axon terminals of the reflex interneurons. After 3 days survival, the animals were reanesthetized in 0.15 g/l MS-222 and perfused transcardially with teleost Ringer’s solution followed by 4% paraformaldehyde in 0.1 M PB. The brains were removed from the skulls and postfixed in the same fresh fixative at 4˚C for 2–3 hours, then rinsed in 20% sucrose in 0.1 M PB at 4˚C for 2–5 hours. The brains were embedded in 10% gelatin containing 20% sucrose and postfixed as described above, and sectioned in the transverse plane at 32 µm on a cryostat. After preincubation in blocking solution, the sections were incubated in primary antisera against calretinin (1/4000, polyclonal, produced against recombinant human calretinin, Swant, Bellinzona, Switzerland) for 36 hours at 4˚C. The sections were then washed in three changes of PBS (each 20 minutes) and incubated in Cy-5 donkey-rabbit IgG (1/1000, Jackson Laboratories, West Grove, PA). After 3 washes in 0.1 M PB (20 minutes each), sections were coverslipped with Fluoromount-G.
Calcium imaging of vagal motoneurons
To label the vagal motoneurons with calcium sensitive dye, calcium green-1 dextran (CaGD, 10,000 MW, Molecular Probes/Invitrogen) was applied in vivo to the vagus nerve with the same surgical procedure described above. After 2–5 days survival, 300 µm brain slices of the vagal lobe were made by vibratome and set in the recording chamber. CaGD fluorescence was observed by a fluorescence microscope (BX50WI, Olympus) equipped with 4x dry (NA 0.28) or 40x water (NA 0.80) objective lenses. Excitation illumination was by means of a xenon lamp with a band-pass excitation filter (460–500 nm), a dichroic mirror (505 nm), and an emission filter (510–560 nm). Fluorescence images were collected by CCD camera (MiCAM 02, Brain Vision, Japan) with a Videoscope VS4–1845 image intensifier. To evoke motoneuron responses from the reflex interneuron inputs, teflon-coated nichrome bipolar electrodes were placed on the sensory layer and single pulse (0.4 ms in duration) stimulation was applied. Optical images were obtained every 10 ms and total recording time was 2 s (200 frames). In order to obtain adequate baseline data, electrical stimulation was begun 800 ms after starting recording. One trial consisted of 3 rounds of electrical stimulation and recording with 30 seconds intervals. Fluorescence responses of each trial are shown as an average of the 3 recordings.
During the recordings, the chamber was superfused continuously with fresh-oxygenated aCSF at a flow rate of 1.5 ml/min. For pharmacological experiments for non-NMDA receptors, brain slices were incubated with aCSF containing 10 µM 6,7-dinitroquinoxaline-2,3-dione (DNQX, Sigma). To test the role of NMDA receptors, Mg2+-free aCSF (MgSO4 was omitted from normal aCSF) was perfused and 10 µM D(−)-2-amino-5-phosphoovaleric acid (AP-5, Sigma) was added to the Mg2+-free aCSF.
The relative activation of motoneurons was expressed as ratio of the fractional changes in fluorescence intensity (ΔF/F). To permit quantitative inter-trial analysis, we measured peak of ΔF/F within 30 ms after electrical stimulation. In the pharmacological experiments, responses were normalized to control response prior to drug application. Paired, two-tailed Student’s t tests were performed on responses from all pharmacological experiments to test the significance of the pharmacological treatments. p < 0.01 was considered significant.
Brightfield photomicrographs were captured digitally on a SPOT RT camera (Diagnostic Instruments, Sterling Heights, MI) attached to on Olympus upright or dissection microscope. Fluorescent micrographs were captured by using an Olympus Fluoview confocal laser-scanning microscope. Digital images were then processed in Photoshop (Adove Systems, Mountain View, CA), adjusting only brightness, contrast, and color balance.