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?