In the two cases in which this hypothesis was tested, by eliminating neuronal cell bodies supplying afferent innervation to BDNF-ir-rich and mRNA-poor areas, the immunoreactive fibers were eliminated

In the two cases in which this hypothesis was tested, by eliminating neuronal cell bodies supplying afferent innervation to BDNF-ir-rich and mRNA-poor areas, the immunoreactive fibers were eliminated. extremely heavy BDNF-immunoreactive fiber/terminal labeling that lacked BDNF mRNA (e.g., medial habenula, central nucleus of the amygdala, bed nucleus of stria terminalis, lateral septum, and spinal cord). The latter observation suggested that in these regions BDNF was derived from anterograde axonal transport by afferent systems. In the RAD26 two cases in which this hypothesis was tested by the elimination of select afferents, BDNF immunostaining was completely eliminated. These data, along with the observation that BDNF-ir was rarely found within dendrites or fibers distribution has not been determined. The recently discovered NT-6 is restricted to nonmammalian species, and its distribution remains largely unknown (Gotz et al., 1994). The cellular localization of neurotrophin proteins in the CNS has proceeded much more slowly, probably reflecting the technical limitations inherent in the immunohistochemical approach. Recently, we have generated antibodies and developed fixation and immunohistochemical protocols permitting the visualization of PI-3065 NGF protein in the normal adult rat brain (Conner et al., 1992). This protocol also has been used PI-3065 successfully to localize NGF in a wide variety of circumstances, including (1) the normal distribution of NGF in human and nonhuman primates (Mufson et al., 1994), (2) changes in NGF accumulation by basal forebrain cholinergic neurons in human Alzheimers tissue (Mufson et al., 1995), (3) PI-3065 the distribution of NGF protein in transgenic mouse lines with NGF gene alterations (Carlson et al., 1995; Ma et al., 1995), and (4) changes in the Affinity-purified rabbit polyclonal antibodies to BDNF used PI-3065 in this investigation were generated and characterized as described previously (Conner et al., 1996; Yan et al., 1997). This antibody preparation was shown to be specific for BDNF by several criteria. (1) In a Western blot, the antibody was capable of recognizing as little as 0.1 ng of BDNF per lane but did not cross-react with NGF, NT-3, or NT-4/5 at concentrations of even 100-fold greater; (2) in a chick dorsal root ganglion bioassay, the antibody specifically interfered with the survival-promoting activity of BDNF but was not capable of inhibiting the actions of either NGF or NT-3; and (3) the specific pattern PI-3065 of BDNF-ir observed in CNS tissues with this antibody was not detected in mice in which the BDNF gene was deleted but was present in mice with a deletion of the NGF gene (J. Conner, unpublished observations). Antisense BDNF mRNA was generated from the rat recombinant plasmid pR1112C8 with T3 TNA polymerase after digestion with Adult male and female Sprague Dawley rats (Simonsen, Gilroy, CA, and Harlan, San Diego, CA) were used (= 5 for normal distribution; = 14 for analysis of lesion effects). All animals were perfused under deep anesthesia with 50 ml PBS followed with 250 ml 2% paraformaldehyde + 0.2% parabenzoquinone in 0.07 mphosphate buffer, pH 7.2. Brains were removed, post-fixed for 2 hr in the same fixative, and cryoprotected overnight in 30% sucrose in 0.1m phosphate buffer (all solutions at 4C). Coronal sections (40 m) were cut on a sliding microtome and stored in Millonigs buffer until they were processed for BDNF-ir as has been described (Conner et al., 1996). In brief, sections (taken every 240 m) were washed in 0.1 m Tris-buffered saline (TBS), pH 7.4, incubated in TBS containing 0.25% Triton X-100, and incubated in TBS + 2% BSA + 5% normal goat serum. Staining was performed by incubating sections with primary antibodies (50 ng/ml anti-BDNF) for 48 hr at 4C, with secondary antibodies (1.5 g/ml biotinylated goat anti-rabbit; Vector Labs, Burlingame, CA) for 3 hr at room temperature, and with an avidinCbiotinCperoxidase reagent (1:250 dilution, ABC Elite; Vector Labs) for 90 min at room temperature. Sections were then reacted with a solution containing 0.04% diaminobenzidine tetrahydrochloride, 0.06% nickel chloride, and 0.06% H2O2 in Tris-HCl buffer. In situhybridization for BDNF mRNA.Sprague Dawley rats (Simonsen) (= 5) were anesthetized by intraperitoneal injection with sodium pentobarbital and killed by perfusion with 50 ml 0.9% saline followed by 350 ml 4% paraformaldehyde in 0.1 m.