1. 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 2
3 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-DOX
DOX 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?
N
Ff
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 = tanθ
θ
> 90o
µ > 1.0
µ = tanθ
θ
= 23.7
o
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
SensoryFeedback
Brain
Motor
Output
Plexiglass Tank
Muscle
CPG
SensoryFeedback
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
RR
FR
FL
RLA)
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
Speed(cm/s)
+2 SD
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
Hindlimb Transformed Phase
Speed(cm/s)
+2 SD
A) B)
Stepping
Swimming
RR
FR
FL
RL
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
PhaseTransformation
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
Baseline
Pre-D
O
X
1
D
O
X
1O
N
-D
8
PlexiglassD
O
X
1O
N
-D
8
Sylgard
D
O
X
O
FF
TransformedPhaseValue
Hindlimb-Forelimb Coordination
**
Swim Phase
TransformedPhaseValue
Pre-D
O
X
1
Baseline
0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
0.90
0.95
1.00
+2 SD
D
O
X
1
D
3
O
N
D
O
X
1
D
5
O
N
n.s.
0.50
0.60
0.70
0.80
0.90
1.00
0.75
0.85
0.55
0.65
0.95
TransformedPhaseValue
Baseline
Pre-D
O
X
1
D
O
X
1O
N
-D
8
Sylgard
D
O
X
O
FF
+2 SD
Hindlimb Coordination
D
O
X
1O
N
-D
8
Plexiglass
n.s ***
TransformedPhaseValue
0.50
0.60
0.70
0.80
0.90
1.00
0.75
0.85
0.55
0.65
0.95
Baseline
Pre-D
O
X
1
D
O
X
1O
N
-D
8
Plexiglass
D
O
X
1O
N
-D
8
Sylgard
D
O
X
O
FF
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.