3% Triton X-100 and 10% skimmed milk) or a polyclonal anti-FG raised in rabbit (Bioscience Research Reagents, Temecula, CA; diluted 1:10,000 in PB containing 0.3% Triton X-100 and 10% skimmed milk) for 24–48 h at 4 °C. After several rinses, they were transferred to a biotinylated anti-rabbit secondary antibody raised in goat (Vector, Burlingame, CA; 1:200 dilution) for 2 h at room temperature, rinsed again and exposed to the ABC mixture (Vectastain, Elite ABC Kit, Vector Laboratories; 1:200 dilution) for 2 h at room temperature. The peroxidase reaction product
was visualized by using SCH772984 in vivo the glucose-oxidase procedure (Itoh et al., 1979) and the metal-free 3,3′-diaminobenzidine tetrahydrochloride (DAB) as the chromogen. The sections were mounted on gelatinized slides, air-dried, and dipped in a 0.05% learn more aqueous solution of osmium tetroxide for 20 s to enhance the visibility of the labeling, dehydrated, transferred into xylene and coverslipped with DPX. An adjacent series was stained with thionin. The brain sections were analyzed with a microscope under brightfield and darkfield illumination. The PHA-L and FG injection sites and the distribution of anterograde labeling of representative cases were mapped
by the aid of a computer drawing program (AutoCad, Release 13) combined with a microscope (Leitz, Diaplan, Leica Microsystems, Wetzlar, Germany) and camera lucida aimed at a flat-screen computer monitor. Photomicrographs
were taken with a Spot 2 digital camera. The low power photomicrographs are montages of four fields captured with a ×10 objective. The digitized images were converted to gray scale and contrast and brightness adjusted by using Photoshop software (version 5.5; Adobe Systems, Mountain View, CA, USA). Unless, otherwise specified, the nomenclature and cytoarchitectonic parceling adhere to the rat brain atlas of Paxinos and Watson (2007). We thank Dr. Phospholipase D1 Martin A. Metzger and Dr. Newton S. Canteras for critical reading of a previous version of the manuscript and valuable suggestions, and Ana Maria Peraçoli Campos for expert technical assistance. We are also grateful to Dr. Newton S. Canteras for allowing us full access to his collection of cases with PHA-L injection in the medial amygdaloid nucleus. This work was supported by FAPESP grant 2008/52907-1 (to S.J.S.L) and FAPESP fellowship 2008/50445-0 (to L.S.N). “
“Page 52, column 1, last line and column 2, lines 1-5 should read as follows: [In 1922, Forbes raised the possibility of fiber group rotation during fatiguing contractions but emphasized that it required testing. As described by Bawa and coworkers (2006), motor unit rotation can be characterized as follows: “A motor unit of similar threshold is now recruited, while the fatigued unit cannot continue to discharge. After some minutes, this second motor unit falls silent, and the originally discharging unit resumes tonic discharge”.