cts poster
TRANSCRIPT
Getting a Grip: Walking Surfaces Influence Stepping Phenotype when Long Ascending Propriospinal Neurons are Silenced
Interdisciplinary Program in Translational Neuroscience , Kentucky Spinal Cord Injury Research Center , and Departments of
Anatomical Sciences & Neurobiology , Neurological Surgery , and Bioengineering , University of Louisville
Courtney T. Shepard , Rachel L. Cary , Morgan Van Rijswijck , Amanda M. Pocratsky , Kevin L. Tran , Scott R. Whittemore , David S.K. Magnuson1-4
1 23 4 5
HiRet-TRE-EGFP.eTeNT1. AAV2-CMV-rtTAV163.
retrograde transportEGFP.eTeNT2.
EGFP.eTeNT anterograde transport5.neurotransmission
Blocked7.
Neurotransmission
Cleave VAMP26.
rtTAV164. + DOX
= eTeNT
Adapted from Kinoshita et. al. 2012
0 1 2 3 4 5 6 7 8
C6 Injection
L2 Injection
Pre-DOXDOX On
DOX Off
Weeks
Baseline
Fig 1. TetON two-viral vector system was used to silence ipsilateral and commissural LAPNs through conditional expression of enhanced tetanus neurotoxin (eTeNT). n= 10 adult female SD rats received 4 bilateral injections (A) of HiRet-TRE-EGFP.eTeNT (1.68x10 vp/ml) at C6 and AAV2-CMV-rtTAV16 (2.66x10 vp/ml) at L2. 20mg/ml doxycycline was given ad libitum for 8 days (DOX 1) followed by a 10 day wash out and post-DOX administration testing (B). 3D kinematics and gait assessments were performed in plexiglass and Sylgard tanks at specified time points.
Acknowledgements: We thank Tadashi Isa for vector constructs and Christine Yarberry, Darlene Burke, Johnny Morehouse, Russell Howard, Alice Shum-Siu, Amberly Riegler, and Grace Mahlbacher for outstanding technical assistance. Funding: GM103507, NS089324, The Kentucky Spinal Cord and Head Injury Research Trust, Norton Healthcare, and the Commonwealth of Kentucky Challenge for Excellence.
TetON System to Conditionally Silence LAPNs
Experimental Design
Hindlimb and forelimb alternation during conditional silencing of LAPNs is severely disrupted during stepping in the Sylgard-tank, mildly disrupted in the plexiglass tank, and unaltered during swimming.
DOX On D8 Plexiglass
DOX On D8 Sylgard
No DOX
How does afferent input from different walking surfaces affect the phenotype seen during LAPN silencing?
NFf
Fg
Leather Surface
Plexiglass/Sylgard 184
N
Ff
Fg
µ
= Friction force
= Force gravity
= Normal force
= Degrees from horizontal
= Coefficient of friction
Increased coefficient of friction in the Sylgard-coated tank contributes to the disruption of right-left alternation during conditional LAPN silencing.
Discussion
Plexiglass Tank Sylgard Tank
Changes in alternation were unrelated to differences in speed between tanks or between DOX conditions.
µ = tan θ
θ > 90 o
µ> 1.0
µ = tan θ
θ = 23.7o
µ= 0.43
Fig 2. (A) Diagram of coefficient of friction using a leather surface to represent the rat paw. Coefficient of friction in the plexiglass tank (B) was lower than that of the Sylgard tank (C), indicating a slippery surface on the plexiglass versus a no-slip surface on the Sylgard.
A) B) C)
L2 C6
rtTAV16 EGFP.eTeNT
Hypothesis: Disruptions in left-right alternation seen during LAPN silencing will be independent of the stepping surface.
Previous Work: Silencing of long ascending propriospinal neurons (LAPNs) leads to disruption of left-right alternation in both hindlimb and forelimb during overground stepping.
Sylgard Tank
Muscle
CPG
Sens
ory
Feed
back
Brain
Motor Output
Plexiglass Tank
Muscle
CPG
Sens
ory
Feed
back
Brain
Motor Output
Afferent input from a slippery walking surface such as plexiglass (A) results in more consistent right-left alternation when the LAPNs are silenced, implying a supraspinal influence on spinal locomotor circuitry not present when the animal is stepping on a grippy surface such as Sylgard (B).
DOX-On D8 Plexiglass
*
0.5 Stance
Stance
*
0.5
RRFRFLRLA)
B)
Fig 4. The disruptions in right-left alternation in the hindlimbs (A) or forelimbs (B) were not attributed to changes in walking speed on either the plexiglass or Sylgard tank. The dotted line indicates the average speed at which phase transitions from walking to galloping or bounding.
7
12
1-4
1-3 2, 4 5 52, 3
0
20
40
60
80
100
120
140
160
0.50 0.55 0.60 0.65 0.70 0.75 0.80 0.85 0.90 0.95 1.00
Forelimb Transformed Phase
Spee
d (c
m/s
)
+2 SD0
20
40
60
80
100
120
140
160
0.50 0.55 0.60 0.65 0.70 0.75 0.80 0.85 0.90 0.95 1.00
Hindlimb Transformed Phase
Spee
d (c
m/s
)
+2 SD
A) B)
Stepping
Swimming
RRFRFLRL
Stance
Stance
*
*
DOX-On D8 Sylgard
Stance
Stance
*
*
0,1
0,1
Walk-Trot
0.5
Gallop
0.75
Bound
0,1
0
0.5
1.0
0.5
Phas
e Tr
ansf
orm
atio
n
A)
B)
0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
0.90
0.95
1.00
+2 SD
Baselin
e
Pre-DOX1
DOX1ON -D8
Plexigl
ass
DOX1ON -D8
Sylgard
DOXOFF
Tran
sfor
med
Pha
se V
alue
Hindlimb-Forelimb Coordination
**
Swim Phase
Tran
sfor
med
Pha
se V
alue
Pre-DOX1
Baselin
e0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
0.90
0.95
1.00
+2 SD
DOX1 D
3ON
DOX1 D
5ON
n.s.
0.50
0.60
0.70
0.80
0.90
1.00
0.75
0.85
0.55
0.65
0.95
Tran
sfor
med
Pha
se V
alue
Baselin
e
Pre-DOX1
DOX1ON -D8
Sylgard DOX
OFF
+2 SD
Hindlimb Coordination
DOX1ON -D8
Plexigl
ass
n.s ***
Tran
sfor
med
Pha
se V
alue
0.50
0.60
0.70
0.80
0.90
1.00
0.75
0.85
0.55
0.65
0.95
Baselin
e
Pre-DOX1
DOX1ON -D8
Plexigl
ass
DOX1ON -D8
Sylgard DOX
OFF
Forelimb Coordination
+2 SD
**n.s.C) D) E)
F) G) H)I)
Thus, the balance between the supraspinal and spinal control of alternation during overground stepping appears to be influenced by sensory input from the walking surface. The stepping surface may be important for alternation during recovery of locomotor activity after spinal cord injury.
Fig 3. The four classic locomotor gaits are shown with hindlimb swing-stance graphs and left-right phase relationships (A). Phase data was transformed to range from 0.0-1.0 to 0.5-1.00 (B). Hindlimb (C) and forelimb (D) alternation was not significantly altered in plexiglass tank (E) but was significantly altered in the Sylgard tank (I) during LAPN silencing. Hindlimb-forelimb coordination was also mildly affected but did achieve significance between tanks on DOX (H). LAPN silencing did not affect hindlimb coordination during swimming (F, G). **p<.01, ***p<.005, ****p<.001 with Repeated Measures ANOVA and Bonferroni post-hoc t-tests.