The effects of adrenergic, serotonergic, and cholinergic modulation on hairy skin low threshold mechanoreceptors Open Access

Abstract

Low threshold mechanoreceptors (LTMRs) play a vital role in interactions with our physical environment. They convey information regarding gentle touch, movement across the skin, and hair follicle deflection, among other things. Perception of these types of stimuli can become disrupted in neuropathic pain states after spinal cord injury (SCI), particularly in allodynia, which is characterized by painful sensations in response to innocuous tactile stimuli. While central sensitization is known to play a role, alterations in peripheral sensory processing may also contribute. In particular, recent evidence has implicated C-LTMRs, which normally encode for pleasant touch, in the development and maintenance of allodynia. Several factors may contribute to altered peripheral processing after injury, including inflammation, sympathetic activity, and neuromodulation. The effects of neuromodulation on hairy skin LTMRs have not been thoroughly examined. This project set out to determine the effect of adrenergic, serotonergic, and cholinergic modulation on LTMR activity, with particular emphasis on C-LTMRs. Additionally, we examined the effect of SCI on C-LTMR response properties. We developed a novel electrophysiological recording setup and used an in-vitro skin-nerve preparation to record from dorsal cutaneous nerves while stimulating the outside of hairy skin in naïve, sham, and SCI mice. We used an optogenetic approach to selectively recruit C-LTMRs, and used puffs of air at calibrated forces to broadly recruit all hairy skin LTMRs. To assess neuromodulation, we bath-applied a reuptake or cholinesterase inhibitor, followed by norepinephrine, serotonin, and acetylcholine, respectively. Adrenergic modulation, including reuptake inhibition, decreased the response magnitude of optogenetically recruited C-LTMRs and mechanically recruited LTMRs. Serotonergic modulation had a similar effect in both stimulation paradigms. Cholinergic modulation did not significantly reduce the activity of C-LTMRs. Application of acetylcholine, but not the cholinesterase inhibitor, reduced the total response magnitude of LTMRs recruited at certain forces. These neuromodulators can be released in the skin via sympathetic efferents and immune cells, and it is well-known that SCI alters sympathetic drive and results in inflammation. Neuromodulation via inflammatory mediators or altered sympathetic output might constitute a mechanism by which altered signaling in the periphery contributes to maladaptive pain processing following spinal cord injury. 

Table of Contents

Introduction. 1

Mechanoreceptors. 1

Aβ- Low Threshold Mechanoreceptors. 3

Aδ- Low Threshold Mechanoreceptors. 3

C- Low Threshold Mechanoreceptors. 4

C-LTMRs and the affective touch hypothesis. 5

Spinal cord injury and neuropathic pain. 6

Altered peripheral sensory signaling. 7

Sympathetic activity. 8

Inflammation. 9

Neuromodulation. 9

Acetylcholine. 10

Norepinephrine. 11

Serotonin. 11

Altered C-LTMR activity and tactile allodynia. 12

Methods. 13

Transgenic Mouse Models. 13

Tamoxifen treatment 13

Immunohistological Processing. 14

Contusion SCI. 15

Skin-nerve preparation. 16

Recording dish. 17

Recording setup. 18

Optogenetic stimulation. 19

Mechanical stimulation. 19

Experimental design. 20

Optogenetic Recruitment of C-LTMRs. 20

Mechanical Recruitment of Mechanoceptors. 22

Statistical Analysis. 23

Optogenetic Recruitment of C-LTMRs. 23

Recruitment & Fatigability of C-LTMRs in SCI versus Sham animals. 24

Neuromodulation of C-LTMRs. 24

Drug Order. 24

SCI versus Control 25

Adrenergic, Serotonergic, & Cholinergic Modulation. 25

Mechanical Recruitment of Mechanoceptors. 25

Results. 26

Assessment of changes in optogenetically recruited C-LTMRs. 26

Evidence that the optogenetic approach selectively recruited C-LTMRs. 26

SCI leads to a reduction in recruitment of C-LTMRs. 27

SCI does not appear to alter fatigability in C-LTMRs. 28

Neuromodulation-induced changes in C-LTMRs. 28

Adrenergic modulation reduces C-LTMR recruitment 28

Adrenergic modulation does not alter C-LTMR fatigability. 29

Serotonergic modulation reduces C-LTMR recruitment 30

Serotonergic modulation does not alter C-LTMR fatigability. 31

Cholinergic modulation does not significantly affect C-LTMR recruitment 31

Cholinergic modulation does not alter C-LTMR fatigability. 33

Mechanical recruitment of Low Threshold Mechanoreceptors. 33

Adrenergic Modulation of LTMRs. 35

Serotonergic Modulation. 36

Cholinergic Modulation. 37

Discussion. 38

Novel Recording setup and genetic lines. 39

Effect of Spinal cord injury on C-LTMRs. 40

Neuromodulation of C-LTMRs. 42

Norepinephrine. 42

Serotonin. 43

Acetylcholine. 44

Neuromodulation of LTMRs. 45

Norepinephrine. 45

Serotonin. 46

Acetylcholine. 47

Potential limitations. 48

Future Directions. 49

References. 52

Tables and Figures

Table 1: Cutaneous mechanoreceptor types………………………………………………………………………….2

Table 2: Animal subjects……………………………………………………………………………………………………13

Figure 1: Location and organization of mechanoreceptors………………………………………………….….2

Figure 2: Cre-mediated expression in TH+ C-LTMRs…………………………………………………………..15

Figure 3: Recording setup…………………………………………………………………………………………………17

Figure 4: Ratio of airflow to force……………………………………………………………………………………….19

Figure 5: Example of optogenetic stimulation recording………………………………………………………20

Figure 6: Experimental design…………………………………………………………………………………………..21

Figure 7: Example of an air stimulation recording………………………………………………………………22

Figure 8: Evidence of prep viability and suction quality……………………………………………………….25

Figure 9: C-LTMR baseline fatigability……………………………………………………………………………...27

Figure 10: C-LTMR recruitment – SCI vs Sham………………………………………………………………....28

Figure 11: C-LTMR fatigability – SCI vs Sham…………………………………………………………………….28

Figure 12: Adrenergic modulation of C-LTMRs………………………………………………………………..…29

Figure 13: Serotonergic modulation of C-LTMRs………………………………………………………………..30

Figure 14: Cholinergic modulation of C-LTMRs………………………………………………………………….31

Figure 15: Effect of cholinesterase inhibitor on C-LTMRs……………………………………………………32

Figure 16: Ratio of force to recruitment at baseline..............………………………………………………...34

Figure 17: Ratio of force to recruitment is conserved across washouts…………………………………..34

Figure 18: Adrenergic modulation of LTMRs…………………………………………………………………..….35

Figure 19: Serotonergic modulation of LTMRs………………………………………………………………..….36

Figure 20: Cholinergic modulation of LTMRs………………………………………………………..…………..38

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
Keyword	Spinal Cord Injury Low Threshold Mechanoreceptors Neuromodulation
Committee Chair / Thesis Advisor	Shawn Hochman, Emory University
Committee Members	Peter Wenner, Emory University Sandra Garraway, Emory University

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