The effect of spinal cord injury on C-fiber low-threshold mechanoreceptors and sensory afferents that transiently express tyrosine hydroxylase Open Access

Abstract

Chronic pain after spinal cord injury (SCI) greatly decreases the patient’s quality of life, and complementary interventions addressing neural changes in the periphery have the potential to improve the often suboptimal present treatments. Allodynia is a common at-level pain syndrome in which normally innocuous stimuli are perceived as painful, and C-fiber low-threshold mechanoreceptors (C-LTMRs) have been implicated in mediating this neuropathic pain. Tyrosine hydroxylase (TH) has previously been identified as a genetic marker for C-LTMRs. In this study, a transgenic mouse strain expressing Cre recombinase in the tyrosine hydroxylase promoter sequence (TH-Cre) was crossed with a strain containing a Cre-dependent channelrhodopsin fused to a yellow fluorescent protein (ChR2-YFP). The offspring (TH::ChR2-YFP) express the light-activated ChR2 ion channel in a subset of TH-expressing (TH+) neurons, allowing for the recruitment of TH+ C-LTMR action potentials by shining blue light on the skin. I used these animals to pursue two goals:  (a) confirm the recruitment of TH+ C-LTMRs and assess if they are selectively recruited by optogenetic stimuli; and (b) characterize changes in C-LTMR activity and physiology after SCI at and above the level of the injury. I found that TH::ChR2-YFP animals do express the transgene in TH+ C-LTMRs and those sensory neurons can be recruited by cutaneously applied blue light stimuli. Additional myelinated and perhaps unmyelinated neurons also express ChR2 and respond to light stimuli, possibly due to transient developmental expression of TH. I also found evidence that hemisection SCI caused a change in axonal membrane physiology of TH::ChR2-YFP C-fibers one spinal segment above the level of injury, shown by a change in action potential rise slope and peak amplitude. Furthermore, SCI caused a significant decrease in TH::ChR2-YFP sensory neuron activity at the level of injury, including the TH+ C-LTMRs. To our knowledge, this study is the first to identify changes in C-LTMR activity and physiology in response to SCI. These results may help to elucidate the peripheral changes that lead to SCI-induced at-level allodynia.

Table of Contents

Table of Contents

Introduction

Allodynia in patients with spinal cord injury                                                 1

Traits of C-fiber low threshold mechanoreceptors (C-LTMRs)                    2

C-LTMR processing in the dorsal horn and implications for allodynia        3

Evaluating changes in C-LTMR activity after SCI                                       5

 

Method of Approach

Animal subjects                                                                                           9

Experimental methods                                                                               11

Analysis methods                                                                                      15

 

Results

Anatomy of TH::ChR2-YFP                                                                        19

Electrophysiology of TH::ChR2-YFP                                                          22

Action potential properties were different between ChR2-YFP+

units contralateral and ipsilateral to SCI                                          26

Dermatome mapping revealed significant changes in T10 DCN activity    28

 

Discussion

Evaluating the Tg(Th-cre)1Tmd driver for transgene

expression in DRG neurons                                                            33

Effects of SCI on TH::ChR2-YFP cutaneous neurons                               38

 

Conclusion                                                                                               43

 

References                                                                                               46

 

 

 

Figures and Tables

Table 1:  Distribution of animal subjects                                                      9

Figure 1:  Dorsal root ganglia neurons from TH::ChR2-YFP mice             19

Figure 2:  TH and ChR2-YFP expression in dorsal cutaneous nerves       20

Figure 3:  TH::lacZ cutaneous end organ b-gal staining                             20

Figure 4:  Central projections of TH::ChR2-YFP neurons                           21

Figure 5:  Example DCN recordings of optogenetic response                    23

Table 2:  Action potential properties of TH::ChR2-YFP neurons in DCN    24

Figure 6:  Responses to von Frey hair stimuli                                            24

Figure 7:  Responses to mechanical brush                                                25

Table 3:  Action potential measurements after SCI                                    27

Figure 8:  Comparison of action potential properties after SCI                  28

Figure 9:  Dermatome area maps                                                              31

Figure 10:  DCN ipsilateral vs. contralateral relative intensity maps           32

About this Master's Thesis

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Neuroscience
Degree	M.S.
Submission	Master's Thesis
Language	English
Research Field	Biology, Neuroscience Biology, Physiology
Keyword	C-LTMR Spinal cord injury peripheral nervous system optogenetics Cre recombinase chronic pain allodynia Tyrosine hydroxylase C-fiber low-threshold mechanoreceptors PNS
Committee Chair / Thesis Advisor	Hochman, Shawn, Emory University
Committee Members	English, Arthur, Emory University Garraway, Sandra, Emory University

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