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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. Magnuson 1-4 1 2 3 4 5 HiRet-TRE-EGFP.eTeNT 1. AAV2-CMV-rtTAV16 3. retrograde transport EGFP.eTeNT 2. EGFP.eTeNT anterograde transport 5. neurotransmission Blocked 7. Neurotransmission Cleave VAMP2 6. rtTAV16 4. + 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 F f F g Leather Surface Plexiglass/Sylgard 184 N F f F g 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 = 23.7 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 Sensory Feedback Brain Motor Output Plexiglass Tank Muscle CPG Sensory Feedback 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 RL A) 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 5 2, 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 Phase Transformation 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-DOX1 DOX1 ON -D8 Plexiglass DOX1 ON -D8 Sylgard DOX OFF Transformed Phase Value Hindlimb-Forelimb Coordination ** Swim Phase Transformed Phase Value Pre-DOX1 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 DOX1 D3 ON DOX1 D5 ON n.s. 0.50 0.60 0.70 0.80 0.90 1.00 0.75 0.85 0.55 0.65 0.95 Transformed Phase Value Baseline Pre-DOX1 DOX1 ON -D8 Sylgard DOX OFF Hindlimb Coordination DOX1 ON -D8 Plexiglass n.s *** Transformed Phase Value 0.50 0.60 0.70 0.80 0.90 1.00 0.75 0.85 0.55 0.65 0.95 Baseline Pre-DOX1 DOX1 ON -D8 Plexiglass DOX1 ON -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.
