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Introduction
Brain-derived neurotrophic factor (BDNF)
and neurotrophin-3 (NT3) belong to a family of trophic proteins called neurotrophins,
which promote the differentiation and survival of neurons during development.1 They
act by binding to a family of closely related glycoprotein tyrosine receptor
kinases.1,2 Brain-derived neurotrophic factor preferentially
binds tyrosine receptor kinase B (trkB); NT3 preferentially binds tyrosine
receptor kinase C (trkC), but also binds trkB with a
lower affinity.1 In the peripheral nervous system,
developing neurons send their processes into their target tissue and
compete with each other for neurotrophic
factors provided by the target cells.1 Trophic factors bind
to receptors on the axon terminals and form a complex. This receptor complex is internalized and
transported from the target to the neuron cell body (Figure 1). Neurons that do not
receive adequate trophic support die. Neurotrophic factors were previously thought to
promote neuronal survival in this retrograde manner. However, recent
evidence suggests that anterograde transport of neurotrophins may also
occur in some parts of the
central nervous system.3,4,5,6,7
Figure 1
Schematic diagram mechanisms of neurotrophin transport.
In
the developing rat olfactory system, olfactory axons have an organizing
effect on the developing forebrain. At embryonic
day 12 to 13, olfactory nerve axons arising from the sensory neurons
in the olfactory epithelium contact and encircle the rostral telencephalon.8,9 At
about embryonic day 14, the olfactory bulbs begin to evaginate. By
embryonic day 16, additional
olfactory axons have innervated the forebrain, and the olfactory bulbs
are distinct.9 This sequence of events suggests
that olfactory nerve axons may trigger development and differentiation
of olfactory bulb neurons.
To determine if neurotrophins might play a role in bulb formation, we used in situ hybridization
to localize the mRNAs for BDNF, NT3, trkB, and trkC in the developing
rat olfactory system. Levels of mRNA expression |
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were
examined in embryos ranging from embryonic day 14 to 20. In addition, NT3 and trkB
immunoreactivity were examined in the olfactory epithelium and bulbs of neonatal rats.
The observed patterns of neurotrophin and trk expression suggest
that trophic factors may be anterogradely transported to the developing
olfactory bulbs.
Materials and Methods
Animals:
Timed pregnant, female Sprague Dawley rats were deeply anesthetized using sodium
pentobarbital (100 mg/kg body weight), and embryos at day 14 to 20 (mate
date=embryonic day 0) were surgically removed. Embryos older than embryonic day 16 were
decapitated. The embryos/heads were frozen in 2-methylbutane at -40ºC. Frozen
sections were cut sagittally in a cryostat at 20 mm and collected onto Vectabond-coated
slides (Vector Laboratories). Tissue was postfixed in 4% paraformaldehyde in 0.1 M
phosphate buffer, and slides were air dried and stored at -20ºC until
processed for in
situ hybridization.
For immunohistochemistry analysis, rats at postnatal day 4 (day of birth=postnatal
day 0) were overdosed with sodium pentobarbital (10 mg/100 g) and transcardially
perfused with 2%
paraformal dehyde in 0.1 M phosphate buffer. After decapitation, the heads were
postfixed in 2% paraformaldehyde at 4ºC for 1 h, and then transferred to 20% sucrose in
0.1 M phosphate buffer for 48 h at 4ºC. Coronal sections through
the head region were cut at 30 mm in a cryostat, collected into 0.1 M
phosphate buffer, and immediately
processed for immunostaining.
In Situ Hybridization:
The 35S-labeled cRNA antisense probes for rat BDNF
and rat NT3 were transcribed from cDNA templates in the presence of alpha 35S-UTP. Antisense BDNF mRNA and antisense NT3 mRNA were generated from rat recombinant
plasmids with T3 RNA polymerase following linearization with PvuII. The cRNA probe
for BDNF contains 384 bases complementary to rat BDNF mRNA. The antisense probe for
NT3 contains 550 bases with 392 bases complementary to rat NT3 mRNA. The antisense
probe for trkB was made from a recombinant plasmid cut with EcoRI using T7 RNA polymerase.
The trkB template corresponds to bases 1120 to 1458 of the rat trkB sequence; the
cRNA detects full length trkB mRNA. The trkC template was cut with
HindIII and transcribed using T3 RNA polymerase. Tyrosine kinase C cDNA
encodes a region corresponding
to bases 349 to 490 of the rat trkC sequence.
 
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