Thirteen female albino rats (body weight 190–210 g) with clean ear canals and no sign of middle ear infection were used in this study. These specimens were also used for the study of SPON projections to the IC described in a previous article (Saldaña et al., 2009 (
link)). All animals were cared for and used in compliance with European Union regulations concerning the use of animals in biomedical research, and the experimental procedures were approved and supervised by the Animal Care and Use Committee of the University of Salamanca. For surgical procedures, including the transcardial perfusion of fixatives, all animals were deeply anesthetized with a mixture of ketamine HCl (80 mg/kg body weight) and xylazine (6 mg/kg body weight) administered intramuscularly. Animal suffering was minimized by monitoring the depth of anesthesia often, carefully attending to physiological cues such as rate and depth of respiration and reflex activity. Supplemental doses of anesthetics were given as needed to maintain deep anesthesia throughout all procedures.
Glass micropipettes loaded with the neuroanatomical tracer biotinylated dextran amine (BDA, 10,000 MW, Molecular Probes, Eugene, OR, USA; 10% in 0.1 M sodium phosphate buffer, pH 7.4) were lowered into the SPON of deeply anesthetized rats using stereotaxic coordinates (Paxinos and Watson, 2007 ). To avoid damage to the prominent transverse sinus, the pipettes were lowered into the brain
via a dorsocaudal to ventrorostral approach, so that their trajectory formed a 16° angle with the coronal plane. The tracer was delivered by iontophoresis using a pulsed 5 μA DC positive current (7 s on/7 s off) for 5–15 min. The current was then stopped and the pipette left in place for an additional 15–20 min prior to withdrawal in order to minimize leakage of the tracer along the injection tract.
Following 7–10 days survival, the rats were anesthetized deeply and their brains fixed by transcardial perfusion of buffered 4% formaldehyde (prepared from freshly depolymerized paraformaldehyde) and 0.1% glutaraldehyde. After cryoprotection in 30% sucrose in phosphate buffer, the brains were cut coronally on a freezing microtome at a thickness of 40 μm. To visualize the tracer, the sections were first processed by the avidin–biotin–peroxidase complex procedure following the manufacturer's specifications (ABC, Vectastain, Vector Labs, Burlingame, CA, USA), and then by standard histochemistry for peroxidase, with or without heavy-metal intensification (Vetter et al., 1993 (
link)). For cytoarchitectural reference, every fourth section was counterstained with cresyl violet.
Sections were photographed at high resolution using a Zeiss Axioskop 40 microscope equipped with a Zeiss AxioCam MRc 5 digital camera and 2.5× (NA 0.075), 5× (NA 0.15), 10× (NA 0.30), 20× (NA 0.50), and 40× (NA 0.75) plan semi-apochromatic objective lenses. Image brightness and contrast were adjusted with Adobe Photoshop software (Adobe Systems Incorporated, San Jose, CA, USA), and the illustrations were arranged into plates using Canvas software (ACD Systems of America, Inc., Miami, FL, USA).
To generate the drawings of Figure
2, the sections were first photographed at high resolution with the 5× objective lens. At this magnification, several micrographs were needed to photograph every section. These photographs were then arranged and fitted using Adobe Photoshop software to create a large mosaic image of the section. The resulting digital image was imported into Canvas software. To increase the resolution of the final image, a new layer was created over the digital image and each labeled fiber contained within the original micrograph was redrawn digitally using Canvas’ freehand drawing tool. This digital procedure allowed us to subsequently adjust the thickness of the lines. The new digital layer, without the underlying micrograph, was finally saved as a TIFF file.
A similar procedure was used to produce the plots showing the distribution of presumed labeled synaptic boutons in Figures
6 and
7. To convey a clear impression of synaptic bouton density, each plot of Figure
7 was subsequently transferred to a Photoshop document and blurred using a Gaussian filter with a 20-pixel square matrix.
Viñuela A., Aparicio M.A., Berrebi A.S, & Saldaña E. (2011). Connections of the Superior Paraolivary Nucleus of the Rat: II. Reciprocal Connections with the Tectal Longitudinal Column. Frontiers in Neuroanatomy, 5, 1.