In Vivo Regulation of Brain-Derived Neurotrophic Factor in ...
Introduction to Neurotrophin Include: –Nerve Growth Factor (NGF) –Brain-Derived Neurotrophic...
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Transcript of Introduction to Neurotrophin Include: –Nerve Growth Factor (NGF) –Brain-Derived Neurotrophic...
Introduction to Neurotrophin• Include:
– Nerve Growth Factor (NGF)– Brain-Derived Neurotrophic Factor (BDNF)– Neurotrophin T-3 and NT-4
• Receptors:– Low Affinity: p75– High Affinity: TrkA, TrkB, and TrkC
• In development: Regulates neuronal innervation to specific targets
• In adults:– Regulates neuronal plasticity– Regulates the number of neuronal progenitor cells
http://www.devbio.com/article.php?ch=13&id=143
Effects of Exercise Effects of Exercise Following Lateral Fluid Following Lateral Fluid
Percussion Brain Injury in Percussion Brain Injury in RatsRats
Romana R. Hicks, et al.University of Kentuky
Restorative Neurology and Neuroscience 12 (1998) 41-46
Background
• After brain injurymany impairments
• Exercise has shown to:– Maybe maintain memory: increases 5HT and
noradrenaline– With PreTx of Exc: attenuates ischemic damage– Activate different molecular cascades: increase
of EC lactate, increases antioxidant enzymes, and increases BDNFBDNF, NGF, and bFGF
BDNF: •Most prevelant in the brain•Amoung other rolesIs involved in activity-dependent plasticity•Enriched housing increases BDNF mRNA levels in rats
Hypothesis
“…tested whether exercise following a lateral fluid percussion (FP) brain injury could increase BDNF mRNA expression in the hippocampus and attenuate the neuropathology and behavioral deficits that are associated with this model of experimental brain injury in rats.”
Materials and Methods
• Surgery:– Sprague-Dawley rats, n=20– Utilizing the stereotaxic and FP device placed a
unilateral brain injury to the left parieto-occipital cortex
Materials and Methods, cont
• Treadmill Training:– Began the day after FP injury (n=10) with 5min– Time increased 5min per day until 60min reached– Control (n=9): Handled 30-60sec daily
• Behavioral Tasks– MWM: day15-17 post-op after 12 trials, 90sec probe– Inclined plane test– Visual Limb Placing– Vertical Righting Response– Grip Test
Materials and Methods, cont
In Situ Hybridization:
Three sections from dorsal hippocampus were analyzed per animal
Histological Evaluation: Eosin stain•Cortical Volumes (left vs. right)•%lesion volume= (ipsi/contra)-100•CA3 cell loss/injury scored on scale of 0-4
CA3CA3
Results
Morris Water Maze
Histology
**
Battery of Behavioral Tasks
??
Results
Basal Levels of BDNF?
Discussion
• Exercise after FP injury does not:– Attenuate histological, cognitive, or
neuromotor deficits (were there any deficits?)
• Exercise after FP injury does: – Increase hippocampal BDNF mRNA
compared to injured/no exercise group
Even though BDNF has previously been shown to increase solely in response to injury, authors believe that the increase of BDNF mRNA in exercised animals occurred via a separate pathway….exercise induced pathway?
Discussion cont
• Suggestions:– Unanswered questions of benefits vs.
harm of exercise– Stress Response– Motor learning better than repetitive
exercise
Conclusion: “…experimental brain trauma are able to increase neurotrophin levels in the brain in response to exercise.”
questions?
ژ ژ
Voluntary exercise Voluntary exercise increases axonal increases axonal
regeneration from sensory regeneration from sensory neuronsneurons
Raffaella Molteni, Jun-Qi Zheng, et al.University of California, Los Angeles
PNAS June 1, 2004 vol. 101 no. 22 8473-8478
Background• Neuronal Platicity
– Development: amount of activity influences cortical circuitry
– Adulthood: Activity-dependent plasticity is retained– Both require morphological changes of synaptic structures
•Neurotrophin•Role in growth and differentiation of neurons•Regulators of synaptic plasticity
•BDNF and NT-3 important in regulating the function of the muscle- dorsal root ganglion -spinal cord interface•Previously showed that exercise increases BDNF and NT-3 expression in spinal cord and skeletal muscle
Hypothesis
“…asked how voluntary exercise affects the structural plasticity of the DRG [dorsal root ganglion].” via a neurotrophin-dependent mechanism
Isolated L4-5Dorsal RootGanglion
Materials and Methods
• Exercise Conditioning– DRG Culture and Gene Expression Groups: Control(0
day) and Run (3 day, 7day)– Trk Inhibitor Groups: microbeads implantation 12h
before exercise– Nerve Crush Groups: Run(7 day) and Control
• DRG Culture– L4-5 DRG isolated– After 20-22h, fixed with 4%ParaImmuno– Longest process of >70 neurons for ea. condition was
measured– DRB included in transcription-independent culture
medium
• Nerve Crush– Unilateral sciatic crush: 7d-exercised or control– Three days after crush: nerve was transected– Transected end place in cuff containing FlouroGold– Animals sac two days later– Sciatic nerve and L4-5 DRG isolated, fixed, sectioned, and
used for immunoflourescence
Materials and Methods, cont
• mRNA analysis– Quantitative real-time RT-PCR utilized– Analysis completed on total isolated mRNA
Manipulations
1. DRG culture- does exercise effect elongation of neurons in vitro?
2. DRG culture with RNA synthesis inhibitor- if exercise-elongation occur, is it also dependent on gene expression?
3. Nerve Crush- if elongation occurs in in-vitro, does exercise conditioning effect elongation in vivo?
4. Gene Expression- does exercise-conditioning effect gene expression?
5. Trk inhibitor- are exercise conditioning effects truly neurotrophin dependent?
1. Results: Exc. Increases Growth In Vitro
Sedentary Animal 3 day exercise 7 day exercise
NOTICE ANYTHING?
1. Results: Exc. Increases Growth In Vitro
POSITIVE CORRELATION
AXONAL OUTGROWTH INCREASED WITH LONGER PERIODS OF EXERCISE
2. Results: Transcription-Independent Growth
DRB: mRNA transcription inhibitor
Gene expressionis not required forrapid neurite out-growth
significant
not sig.
3. Results: Regeneration In Vivo
50 um
Sedentary Animal 7d-Exercise
Flou
roG
old
Posi
tive
Neu
rofilim
ent
3. Results: Regeneration In VivoN
um
ber o
f Neu
ron
s%
retr
og
rad
ely
-lab
ele
d n
eu
ron
s
20 um
Uninjured Sciatic Nerve
Exercised-Injured Nerve
Sedentary-Injured Nerve
3. R
esu
lts: Regen
era
tion In
V
ivo
4. Results: Exercise Influences Gene Expression
Synapsin I: synapticvesicle protein
BDNF regulates synthesisof Synapsin I to increase
NT release
GA
P43:
Gro
wth
-A
ssocia
ted
Pro
tein
5. Results: Inhibition of Trk Tyrosine KinaseMean Neurite Length
GAP43 mRNA levels
Synapsin I mRNA levels
Grey Bars: Presence of Trk Inhibitor
Activation of neurotrophin rec. via exercise regulates synapsin I expression
Lack of effect of GAP43 expression with inhibition of Trk rec indicates separate signaling pathways
Decreased growth arguesNeurotrophins are essential in regulating growth potential
Discussion1. Does exercise effect elongation of neurons in vitro? Yes.
With positive correlation.2. If exercise-elongation occur, is it also dependent on gene
expression? No. Rapid neurite outgrowth can occur without new mRNA.
3. If elongation occurs in in-vitro, does exercise conditioning effect
elongation in vivo? Yes. In response to injury pretreatment, greater elongation occurs in exercised condition.
4. Does exercise-conditioning effect gene expression? Yes. Increased mRNA; shows direct functional effect of exercise.
5. Are exercise conditioning effects truly neurotrophin dependent? Yes. Activation of neurotrophin pathways are critical for growth potential.
• Elongation vs. Arborization• Exercise activates mRNA expression which
encode proteins important for elongation of axons “Priming Effect”
• Inhibition of mRNA synthesis:– Supports above– Could mean that exercise activates protein
translation pre- or post- synaptically?• Do not distinguish between activation OR
alleviation of inhibition
Discussion cont
questions?
ژ ژ
Supports an exercise induced pathway?