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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 Following
Lateral Fluid Percussion Brain
Injury in Rats
Romana R. Hicks, et al.
University of Kentuky
Restorative Neurology and Neuroscience 12 (1998) 41-46
Background
• After brain injurymany 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 BDNF, NGF, and bFGF
BDNF:
•Most prevelant in the brain
•Amoung other rolesIs 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
CA3
Histological Evaluation: Eosin stain
•Cortical Volumes (left vs. right)
•%lesion volume= (ipsi/contra)-100
•CA3 cell loss/injury scored on scale of
0-4
Results
?
Histology
Morris Water Maze
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.”
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questions?
Voluntary exercise increases
axonal regeneration from
sensory neurons
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 L4-5
Dorsal Root
Ganglion
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%ParaImmuno
– Longest process of >70 neurons for ea. condition was
measured
– DRB included in transcription-independent culture
medium
Materials and Methods, cont
• 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
• 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 exerciseconditioning 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
NOTICE ANYTHING?
7 day exercise
1. Results: Exc. Increases Growth In Vitro
AXONAL OUTGROWTH INCREASED
WITH LONGER PERIODS OF EXERCISE
POSITIVE CORRELATION
2. Results: Transcription-Independent Growth
DRB: mRNA transcription inhibitor
Gene expression
is not required for
rapid neurite outgrowth
not sig.
significant
3. Results: Regeneration In Vivo
7d-Exercise
FlouroGold
Positive
Sedentary Animal
Neurofiliment
50 um
Number of Neurons
%retrogradely-labeled neurons
3. Results: Regeneration In Vivo
Exercised-Injured Nerve
Sedentary-Injured Nerve
20 um
3. Results: Regeneration In Vivo
Uninjured Sciatic Nerve
4. Results: Exercise Influences Gene Expression
BDNF regulates synthesis
of Synapsin I to increase
NT release
GAP43: GrowthAssociated
Protein
Synapsin I: synaptic
vesicle protein
5. Results: Inhibition of Trk Tyrosine Kinase
Synapsin I mRNA levels
Mean Neurite Length
Activation of
neurotrophin
rec. via
exercise
regulates
synapsin I
expression
GAP43 mRNA levels
Decreased growth argues
Neurotrophins are
essential in regulating
growth potential
Grey Bars:
Presence of Trk Inhibitor
Lack of effect
of GAP43
expression with
inhibition of Trk
rec indicates
separate
signaling
pathways
Discussion
Yes. With
1.
Does exercise effect elongation of neurons in vitro?
2.
If exercise-elongation occur, is it also dependent on gene
positive correlation.
No. Rapid neurite outgrowth can
occur without new mRNA.
expression?
3.
If elongation occurs in in-vitro, does exercise conditioning effect
Yes. In response to injury
pretreatment, greater elongation occurs in
exercised condition.
Does exercise-conditioning effect gene expression? Yes.
Increased mRNA; shows direct functional
effect of exercise.
elongation in vivo?
4.
5.
Are exercise conditioning effects truly neurotrophin dependent?
Yes. Activation of neurotrophin pathways
are critical for growth potential.
Discussion cont
• 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
Supports an exercise induced pathway?
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questions?