AN INVESTIGATION OF SPINAL ECTOPIC EPILEPTIFORM ACTIVITY Open Access

Abstract

Spinal cord injury (SCI) leads to sensory dysfunction including neuropathic pain, which is resistant to treatment with classical analgesics. Previous work has established spinal somatosensory network hyperexcitability as a critical driver of neuropathic pain after SCI. This network hyperexcitability shares common cellular mechanisms with epileptic conditions, including perturbations in ion channels, loss of GABAergic inhibition, and dysregulation of intracellular chloride concentration. Given the mechanistic similarities between the conditions, I sought to determine whether dorsal horn somatosensory circuitry exhibits epilepsy-like (epileptiform) network characteristics after SCI. Here, using a mouse lower thoracic contusion injury model and an isolated spinal cord preparation that enables recordings from sensory axons in multiple segmental dorsal roots (DRs), I demonstrated expression of GABA interneuron-driven ectopic bursting in primary afferents after SCI. This bursting synchronized across dorsal roots, ostensibly driven by dysfunction in primary afferent depolarization (PAD) through conversion from subthreshold inhibition to suprathreshold spiking. Synchronous bursting occurred spontaneously and could be evoked by afferent stimulation, including optogenetic stimulation of non-pain encoding afferents (Aδ-LTMRs and C-LTMRs). Indeed, SCI-induced bursting expressed distinguishing traits of an epileptiform circuit including stereotyped bursting activity that exhibited hypersynchrony, post-burst refractory period, recruitment by afferent stimulation, and functional reorganization of circuitry. Thus, SCI unmasks dorsal horn circuits that drive ectopic epileptiform bursting. All emergent features of bursting after SCI could be reproduced in naïve animals with the convulsant KV blocker 4-aminopyridine (4-AP), the KV blocker tetraethylammonium (TEA), and the KCC2 blocker VU0240551, suggesting that epileptiform burst circuitry expresses degeneracy such that ectopic bursting can arise from multiple alterations. Bursting is ostensibly generated by PAD-evoking GABAergic interneurons and propagates through Aδ and C fibers, including LTMRs, by which it is likely to directly activate pain circuitry. Overall, epileptiform bursting enables profound corruption of sensory signaling, as ectopic bursts propagate bidirectionally to aberrantly activate spinal circuitry and acutely perturb mechanosensitivity. This work contributes to the understanding of post-SCI somatosensory dysfunction by identifying a manifestation of dorsal horn hyperexcitability that is epileptiform, exhibits features indicative of degeneracy, and provides a substrate for crossing of sensory modalities, which could explain features of neuropathic pain after SCI. 

Table of Contents

Chapter 1:    Introduction. 1

1.1     The importance of somatosensation. 1

1.2     Primary afferent populations and somatosensory coding. 2

1.3     Organization of primary afferent projection to the dorsal horn. 4

1.4     Presynaptic inhibition and modulation in sensory coding. 7

1.5     SCI and Neuropathic pain. 10

1.6     Animal models of SCI neuropathic pain. 12

1.7     Hypotheses explaining allodynia after SCI. 13

1.8     Similarities between epilepsy and neuropathic pain. 16

1.9     Pain and the brain. 17

1.10       Summary and goals. 18

Chapter 2:    Epileptiform bursting after SCI. 19

2.1     Abstract 19

2.2     Introduction. 20

2.3     Methods. 22

2.3.1      Animals. 22

2.3.2      Spinal Cord Injury. 23

2.3.3      Measurement of mechanical allodynia. 24

2.3.4      Dissections. 25

2.3.5      Electrophysiology. 26

2.3.6      Models of sensory hyperexcitability. 26

2.3.7      Blockade of synaptic activity. 27

2.3.8      Data and statistical analysis. 28

2.3.9      Code accessibility. 29

2.4     Results. 29

2.4.1      Injured mice preferentially exhibit spontaneous and stereotyped bursting in primary afferents. 29

2.4.2      Burst synchrony arises from unilateral burst generators that can synchronize contralaterally 31

2.4.3      SCI Bursts can be evoked by afferent stimulation after a refractory period. 33

2.4.4      Ectopic bursting can be recapitulated with the convulsant 4-AP. 34

2.4.5      Burst circuitry can be unmasked by 4-AP, TEA, SCI, time, and KCC2 block, suggesting degeneracy in burst-evoking dorsal horn hyperexcitability. 37

2.4.6      Burst frequency is reduced by the anticonvulsant retigabine. 40

2.4.7      Bursting is dependent on GABAA receptors. 42

2.5     Discussion. 45

2.6     Conclusions. 55

2.7     Research Contributions. 56

Chapter 3:    Spinal circuits and burst propagation. 57

3.1     Abstract 57

3.2     Introduction. 58

3.3     Methods. 60

3.3.1      Animals. 60

3.3.2      Spinal Cord Injury. 62

3.3.3      Models of sensory hyperexcitability. 62

3.3.4      Dissections. 62

3.3.5      Intact spinal cord preparation electrophysiology. 64

3.3.6      Skin-nerve preparation electrophysiology. 65

3.3.7      Conduction velocity and collision testing recording configurations. 68

3.3.8      Fos labeling. 68

3.3.9      Data and statistical analysis. 70

3.3.10    Code accessibility. 71

3.4     Results. 71

3.4.1      Bursts are evoked by Aβ, Aδ, and C fiber electrical stimulation. 71

3.4.2      Epileptiform bursting occurs along a continuum of hyperexcitability. 74

3.4.3      C-LTMR afferent stimulation recruits bursting networks. 77

3.4.4      Aδ-LTMR afferent stimulation recruits bursting networks. 82

3.4.5      Bursting recruits spiking in C-LTMR and Aδ-LTMR afferents. 84

3.4.6      Insights into synaptic organization of burst generation. 88

3.4.7      Extrasynaptic GABAergic modulation. 91

3.4.8      Bursting and involvement of dorsal horn circuitry. 92

3.4.9      Spinally driven bursts propagate to the cutaneous periphery. 97

3.5     Discussion. 101

3.6     Conclusion. 113

3.7     Research contribution. 113

Chapter 4:    Discussion and conclusions. 115

4.1     Summary of key findings. 115

4.2     Contributions to the field. 119

4.3     Remaining questions and future directions. 119

4.4     Final words. 122

Appendix A.    Algorithmic burst detection and analysis. ii

Section 1.01     Abstract ii

Section 1.02     Introduction. iii

4.4.1      Data collection. iv

4.4.2      Code development iv

4.4.3      Python libraries. iv

4.4.4      Data. v

Section 1.03     Explanation of program functionality. v

4.4.5      Opening and filtering raw data. v

4.4.6      Stimulus detection and artifact removal vi

4.4.7      Burst identification. ix

4.4.8      Burst quantification. xi

4.4.9      Cross-correlation. xii

4.4.10    Data export and visualization. xiii

4.4.11    Verification and comparison to manual analysis. xiv

Section 1.04     Discussion. xv

Section 1.05     Research contributions. xvii

Appendix B: additional skin-nerve preparation results. xviii

Section 1.01     SCI does not obviously induce chronic changes in cutaneous mechanoreception. xviii

Section 1.02     Trial and error with skin-nerve preparation. xx

Section 1.03     Research contributions. xxi

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Neuroscience
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Neuroscience
Keyword	Spinal cord injury Epileptiform Neuropathic pain Dorsal horn
Committee Chair / Thesis Advisor	Hochman, Shawn , Emory University
Committee Members	Wenner, Peter, Emory University Au Yong, Nicholas, Emory University Garraway, Sandra M. , Emory University

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