Download If so, is trkB mRNA in SNB motor neurons

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
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).
Questions
**
(P<0.0056)
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25
0
Sham GDX
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GDX (14 days)
Sham GDX
 If so, is trkB mRNA in SNB motor neurons regulated by androgens?
Methods
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
trkB mRNA in SNB motoneurons
175
*
150
(P<0.041)
125
100
75
50
25
0
Sham GDX
BNDF mRNA in RDLN motoneurons
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trkB mRNA in RDLN motoneurons
(pixels/cm2)
 Do SNB motor neurons contain trkB mRNA?
trkB mRNA in SNB motoneurons
(pixels/cm2)
 If so, is BDNF mRNA in SNB motor neurons regulated by androgens?
GDX + Blank
175
 Do SNB motor neurons contain BNDF mRNA?
200
GDX + T
Sham GDX
trkB mRNA in RDLN motoneurons
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150
125
100
75
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.
50
25
0
GDX (14 Days)
GDX (14 Days)
Sham GDX
GDX (14 Days)
Experiment 1: In situ hybridization histochemistry using 35S-labeled cRNA
probes to detect BDNF mRNA in SNB and RDLN motoneurons
SNB and RDLN motoneurons express BDNF mRNA
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) .
Number of contacts containing both VGLUT1
and trkB immunoreactivity contacting
fluorogold-labeled SNB dendrites
Experiment 2: In situ hybridization histochemistry using 35S-labeled cRNA
probes to detect trkB mRNA in SNB and RDLN motoneurons
 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).
Questions
Do glutamatergic neurons innervate SNB motoneuron dendrites?
Methods
 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 antiguinea pig-Cy5 (Jackson Immuno) , and donkey anti-sheep-Texas Red (Jackson).
Experiment 2: Triple-label Confocal ICC
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.
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 pigCy5 (Jackson Immuno) , and donkey anti-sheep-Texas Red (Jackson).
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Sham
BDNF was expressed extensively through the SNB motoneuron dendrites of Sham GDX (Column 1) and GDX+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.
Castration decreases BDNF in Fluorogold-labeled dendrites of SNB,
but not RDLN, motoneurons
60
50
40
30
20
**
(P<0.0078)
10
0
Sham
GDX + T
GDX + Blank
% fluorogoldlabeled RDLN dendritic
length containing BDNF immunoreactvity
SNB and RDLN motoneurons express trkB mRNA
Is the expression of BDNF or trkB protein in either region dependent on
androgen?
90
0
% fluorogoldlabeled SNB dendritic length
containing BDNF immunoreactvity
If so, do glutamatergic terminals or the glutamatergic post- synaptic region
contain either BDNF or trkB receptor proteins?
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
Triple-label ICC for FG, VGLUT1, and trkB
Number of contacts containing both VGLUT1 and
trkB immunoreactivity contacting
fluorogold-labeled RDLN dendrites
 After bilateral axotomy, BDNF and testosterone are both required to
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200
2
regulation (Al-Shamma and Arnold, 1997).
150
BNDF mRNA in SNB motoneurons
(pixels/cm )
 Following axotomy, animals treated with BDNF show less AR down-
BDNF mRNA in RDLN motoneurons
 Blocking neurotrophic receptors (including trkB) halts masculinization of
SNB motoneurons in testosterone treated females (Xu et al, 2001)
Triple-label ICC for FG, VGLUT1, and BDNF
Castration decreases BDNF and trkB mRNA in
androgen-dependent SNB, but not RDLN, motoneurons
BDNF mRNA contained in SNB motoneurons
(pixels / cm2)
Background: Study 1
60
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GDX + Blank
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Sham
GDX + T
GDX + Blank
Summary and Future Directions
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.
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.
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GDX + T
90
Acknowledgements
Sham
GDX + T GDX + Blank
The project was funded by NIH grant# NS045195 to CLJ and NIH
grant # NS28421 to SMB