Rewiring of hindlimb corticospinal neurons after spinal cord injury

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1 Rewiring of hindlimb corticospinal neurons after spinal cord injury Arko Ghosh, Florent Haiss, Esther Sydekum, Regula Schneider, Miriam Gullo, Matthias T. Wyss, Thomas Mueggler, Christof Baltes, Markus Rudin, Bruno Weber and Martin E. Schwab

2 Dorsal Rostral Supplementary Figure 1. The injury site. A snapshot of a reconstructed lesion site. In grey the lesion, in green the section outline and in red the visible grey matter. Insert: dark field image of a section at the epicenter of injury.

7 Imaging Hindlimb Forelimb VSD imaging BOLD-fMRI Retrograde labelling Hindlimb corticospinal neuron also projecting to forelimb Hindlimb corticospinal neuron with no projections to forelimb Intact a FL M1 FL S1 HL II/III V b One week after thoracic spinal cord injury b II/III V b Three weeks or later after thoracic spinal cord injury c II/III V b Supplementary Figure 6. Legend on next page.

8 Supplementary Figure 6. A schematic summary of the main findings presented in this paper. a: In intact animals the BOLD forelimb (FL) and hindlimb (HL) sensory representations are well separated (shown in dark red and blue). In the forelimb area, sensory and motor functions are spatially separated whereas in the hindlimb area they completely overlap (forelimb M1, under the hashed area). The VSD reveals response (shown in a lighter color) to forepaw stimulation involves forelimb M1 in addition to forelimb S1. Hindlimb corticospinal neurons originate from the hindlimb sensory motor area. The neurons are shown as a layer of droplets in the output lamina Vb. Their axons are shown as red lines on the schematic dorsal view of the rat brain and spinal cord. b: After thoracic injury, the injured hindlimb corticospinal neurons rewire to the forelimb (shown as droplets with blue borders, and their axons on the dorsal view of the spinal cord) in an environment of changing forelimb representations. One week after injury there is increased forelimb sensory motor excitability that is also reflected by faster responses to forelimb stimulation in axotomised hindlimb corticospinal neurons (shown as curved blue arrows, the exact pathways that lead to this activation are uncertain). c: The neurons in proximity to the forelimb area are more likely to remain rewired at longer time points after injury. The exact nature of response of the re wired hindlimb corticospinal neurons to forelimb input and the responses of axotomised hindlimb neurons at longer time points after injury are unknown due to technical limitations. Dotted red boundary depicts original hindlimb sensory motor representation (in b and c). Cortical layers Vb and II/III are indicated on the figure.

9 Supplementary Notes on Electrophysiology Notes on the electrophysiological method to record action potentials from passing through CST Background In 1937 Edgar Adrian detected action potentials in the CST passing through the brainstem prior to entering the spinal cord 1. In his experiments, action potentials were elicited in response to pinching the cat s forepaw. The subsequent years saw several studies devoted to the understanding of sensory input to the motor cortex (leading up to the experiments by Asanuma and Colleagues 2 ). Since Adrian s demonstration and the subsequent experiments by John Swett and colleagues 3, the method of recording corticospinal cells in the cortex itself took precedence over using Adrian s method to record from the CST as it passes through the brainstem or spinal cord 4. However, descending volleys detected by placing epidural electrodes in proximity to the CST is frequently measured in humans to assess corticospinal function 5. As we were interested in both the timing and extent of corticospinal excitation in response to peripheral stimulation we recorded action potentials from the Hindlimb CST. Reasoning Hindlimb CST Our recording site was at T-7 CST in the dorsal funiculus. In this segment majority (> 95 %) of the axons continue to descend and then run through T-8. The site is about 6 segments lower than the lowest spinal segment that is involved in forelimb function 6. Below T-7, the spinal segments are primarily involved in hindlimb and tail functions. The extent of penetration of the CST in rodents, however, is far less in the lower segments involved in tail function. Therefore, we can state the following regarding the CST passing through T-7: 1) except a rare few axons, none target forelimb spinal segments, 2) majority of the axons are involved in hindlimb functions. Moreover, after axotomy at T-8 the progressive retraction of the injured corticospinal axons rarely reaches as far as T-7 at one week after injury 7. Verification of CST recording Supplementary figure 5 shows the three approaches used to verify that our recording was from the CST passing through T-7. Upon cortical ablation that included the hindlimb area, the CST responses to forelimb or hindlimb inputs were eliminated. Next, visualization of the tract left by the DiI coated electrode in the spinal cord showed the presence of the electrode in the CST. Finally, microstimulation of the hindlimb cortex (2 mm lateral, 2 mm posterior to bregma), which is expected to activate the CST both directly and synaptically 4, led to a large increase in the number of action potentials within a short time. The hindlimb cortex was stimulated at a depth of 1.2 mm with a single biphasic pulse lasting 0.5 msec at µa.

10 Anesthesia There is extensive discussion in the initial study by Adrian on the impact of anesthesia on CST responses to forelimb sensory input. The anesthesia per say did not determine the presence or absence of responses but the depth did. In our study the blood pressure and heart rate was monitored to ensure the animal was stable under Urethane anesthesia. Urethane is a widely used anesthetic 8. In more recent studies, this anesthetic 9, 10 has been used extensively to record sensory responses in the cortex and more importantly from deep cortical neurons 11. Sensory stimulation threshold In cats, both cutaneous and deep receptors result in activation of corticospinal cells 3. In rodents the threshold of CST activation in response to peripheral input remains unknown. In order to detect the sensory evoked potentials in the hindlimb CST, we stimulated the forepaw with a single current pulse of 6 ma lasting for 1 ms. Stimulation in this range has been used before to elicit action potentials in corticospinal neurons 12. At this amplitude all categories of A and C fibers innervating the paw are expected to be activated. This stimulation intensity therefore allows us to address if sensations from the paw, irrespective of its form, activate corticospinal cells. At currents as low as used in Voltage Sensitive Dye experiments, the signal to noise ratio was much lower resulting in longer acquisition times and the method was now highly sensitive to the position of the electrode in the CST due to the smaller number of axons activated. Using high current we were able to attain more consistent responses whose alteration after injury could be meaningfully interpreted.

11 Associated References: 1. Adrian, E.D. & Moruzzi, G. Impulses in the pyramidal tract. J Physiol 97, (1939). 2. Asanuma, H. Functional role of sensory inputs to the motor cortex. Prog Neurobiol 16, (1981). 3. Swett, J.E. & Bourassa, C.M. Short latency activation of pyramidal tract cells by Group I afferent volleys in the cat. J Physiol 189, (1967). 4. Jankowska, E., Padel, Y. & Tanaka, R. The mode of activation of pyramidal tract cells by intracortical stimuli. J Physiol 249, (1975). 5. Di Lazzaro, V., et al. Corticospinal volleys evoked by transcranial stimulation of the brain in conscious humans. Neurol Res 25, (2003). 6. McKenna, J.E., Prusky, G.T. & Whishaw, I.Q. Cervical motoneuron topography reflects the proximodistal organization of muscles and movements of the rat forelimb: a retrograde carbocyanine dye analysis. J Comp Neurol 419, (2000). 7. Seif, G.I., Nomura, H. & Tator, C.H. Retrograde axonal degeneration "dieback" in the corticospinal tract after transection injury of the rat spinal cord: a confocal microscopy study. J Neurotrauma 24, (2007). 8. Koblin, D.D. Urethane: help or hindrance? Anesth Analg 94, (2002). 9. Simons, D.J., Carvell, G.E., Hershey, A.E. & Bryant, D.P. Responses of barrel cortex neurons in awake rats and effects of urethane anesthesia. Exp Brain Res 91, (1992). 10. Yu, X.J., Xu, X.X., He, S. & He, J. Change detection by thalamic reticular neurons. Nat Neurosci 12, (2009). 11. Helmchen, F., Svoboda, K., Denk, W. & Tank, D.W. In vivo dendritic calcium dynamics in deep-layer cortical pyramidal neurons. Nat Neurosci 2, (1999). 12. McComas, A.J. & Wilson, P. An investigation of pyramidal tract cells in the somatosensory cortex of the rat. J Physiol 194, (1968).

Clarke's Column Neurons as the Focus of a Corticospinal Corollary Circuit. Supplementary Information. Adam W. Hantman and Thomas M.

Clarke's Column Neurons as the Focus of a Corticospinal Corollary Circuit Supplementary Information Adam W. Hantman and Thomas M. Jessell Supplementary Results Characterizing the origin of primary