Erich N. Ottem, Laurel A. Beck, S. Marc Breedlove, and Cynthia L. Jordan

1. Regulation of brain-derived neurotrophic factor (BDNF) and tyrosine receptor kinase b (trkB) by androgens in the motoneurons of the spinal nucleus of the bulbocavernosus (SNB) Erich N. Ottem, Laurel A. Beck, S. Marc Breedlove, and Cynthia L. Jordan Neuroscience Program, Michigan State University, East Lansing, MI 48824 Triple-label ICC for FG, VGLUT1, and BDNF Triple-label ICC for FG, VGLUT1, and trkB Castration decreases BDNF and trkB mRNA in androgen-dependent SNB, but not RDLN, motoneurons Background: Study 1 GDX + T Sham GDX GDX + Blank • Blocking neurotrophic receptors (including trkB) halts masculinization of SNB motoneurons in testosterone treated females (Xu et al, 2001) • Following axotomy, animals treated with BDNF show less AR down- regulation (Al-Shamma and Arnold, 1997). • After bilateral axotomy, BDNF and testosterone are both required to completely restore dendritic arborization in castrated adult male rats (Yang et al, 2004). • BDNF may act as a classic peptide neurotransmitter, an autocrine factor, or paracrine factor. BDNF may be synthesized in a presynaptic neuron or may originate at a postsynaptic site be retrogradely transported to the presynaptic neuron (Lu,B., 2003). BNDF mRNA in RDLN motoneurons BNDF mRNA in SNB motoneurons Questions • Do SNB motor neurons contain BNDF mRNA? • If so, is BDNF mRNA in SNB motor neurons regulated by androgens? • Do SNB motor neurons contain trkB mRNA? • If so, is trkB mRNA in SNB motor neurons regulated by androgens? trkBmRNA in SNB motoneurons trkB mRNA in RDLN motoneurons Methods Fluorescent ICC using antibodies to detect fluorogold, VGLUT1, and trkB in a Sham GDX rat. Immunorectivity for trkB was observed in SNB motoneurons. Many terminals contacting SNB motoneurons contained VGLUT1 and trkB. Red arrowheads: trkB immunoreactivity in SNB soma. White arrowheads: VGLUT1-positive appositions to SNB dendrites. White arrows: terminal structures positive for both VGLUT1 and trkB. 2 groups of adult (90-100 days) male Sprague-Dawley rats: Sham operated (n=6), sacrificed 14 days after operation Gonadectomized (GDX; n=6), sacked 14 days after GDX Experiment 1: In situ hybridization histochemistry using 35S-labeled cRNA probes to detect BDNF mRNA in SNB and RDLN motoneurons Experiment 2: In situ hybridization histochemistry using 35S-labeled cRNA probes to detect trkB mRNA in SNB and RDLN motoneurons Castration does not change the number of dual labeled VGLUT1 and trkB contacts on SBN or RDLN motoneurons Background: Study 2 • BDNF localized to dendrites is implicated in the targeting of a variety of postsynaptic density proteins to dendritic motors (Schratt et al., 2004). • BDNF enhances glutamatergic signaling via activation of the trkB receptor (Matsumoto et al., 2005). • In some systems BDNF is co-released with glutamate to enhance glutamatergic signaling (Lowenstein and Arsenault, 1996). SNB and RDLN motoneurons express BDNF mRNA Questions • Do glutamatergic neurons innervate SNB motoneuron dendrites? • If so, do glutamatergic terminals or the glutamatergic post- synaptic region contain either BDNF or trkB receptor proteins? • Is the expression of BDNF or trkB protein in either region dependent on androgen? Summary and Future Directions BDNF was expressed extensively through the SNB motoneuron dendrites of Sham GDX (Column 1) andGDX+T (Column 2) male rats. BDNF expression was much reduced in SNB dendrites of the GDX+Blank (Column 3) treatment group (Column 3). Arrowheads: VGLUT1-positive appositions to SNB dendrites. Arrows: BDNF labeling in SNB dendrites. BDNF mRNA (black silver grains) in SNB (A) and RDLN (B) motoneurons from a sham GDX rat detected with 35S-labeled cRNA probes. SNB and RDLN motoneurons were counterstained with cresyl violet (blue stain). All SNB and RDLN detected were positive for BDNF mRNA. Scale bars, 5 mm • Castration decreases BDNF mRNA in SNB motoneurons and BDNF protein in SNB dendrites. • Because BDNF is implicated in the targeting of post-synaptic density proteins (PSDs) in dendrites, castration may also cause a decrease of several PSDs in SNB dendrites. Castration decreases BDNF in Fluorogold-labeled dendrites of SNB, but not RDLN, motoneurons Methods SNB and RDLN motoneurons express trkB mRNA • 3 groups of adult (90-100 days) male Sprague-Dawley rats: • 1. Sham GDX (n=5), BC and footpad muscles inj. 15mg Fluorogold (FG; Fluorochrome • Inc.) , sac’ed d14. • 2. GDX + TP (n=5), BC and footpad muscles inj. 15mg FG, sac’ed d14 • 3. GDX + Blank (n=6), BC and footpad muscles inj. 15mg (FG), sac’ed d14 • Experiment 1: Triple-label Confocal ICC • A. 1o Abs ; Rabbit antI-FG ( Chemicon), guinea pig antI-VGLUT1 (Chemicon), and • sheep anti-BDNF (Chemicon). • B. 2o Abs: donkey anti-rabbit-Alexafluor 488 (Molecular Probes), donkey anti- • guinea pig-Cy5 (Jackson Immuno) , and donkey anti-sheep-Texas Red (Jackson). • Experiment 2: Triple-label Confocal ICC • A. 1o Abs ; RabbIt antI-FG (Chemicon), guinea pig antI-VGLUT1 (Chemicon), and • chicken anti-trkB (Chemicon). • B. 2o Abs: donkey anti-rabbit-Alexafluor 488 (Molecular Probes), donkey anti-guinea pig- • Cy5 (Jackson Immuno) , and donkey anti-sheep-Texas Red (Jackson). References • Xu, J, GingrasK. M. , Bengston, L., Di Marco, A., and Forger, N.G. (2001) J. Neurosci. 21:4366-4372. • Al-Shamma, H.A. and Arnold, A.P. (1997) PNAS.94:1521-1526. • Yang, L.Y., Verhovshek, T., and Sengelaub, D.R. (2004) Endocrinology. 145:161-168. • Lu, B. (2003) Learn. Mem. 10:86-98 • Schratt, G.M. et al., (2004) J Neurosci. 24: 7366-7377. • Matsumoto, T et al., (2005) Mol Cell Neurosci. [Epub ahead of print]. • Lowenstein, D.H. and Arsenault, L. (1996) J. Neurosci. 16: 1759-1759. Acknowledgements trkB mRNA (black silver grains) in SNB (A) and RDLN (B) motoneurons from a sham GDX rat detected with 35S-labeled cRNA probes. SNB and RDLN motoneurons were counterstained with cresyl violet (blue stain). All SNB and RDLN detected were positive for trkB mRNA. Scale bars, 5 mm. The project was funded by NIH grant# NS045195 to CLJ and NIH grant # NS28421 to SMB