Electrophysiological assessment of TrkB signaling in primary sensory neurons following spinal cord injury Público
Jang, Kyeong Ran (Fall 2024)
Abstract
Chronic neuropathic pain after spinal cord injury (SCI) is debilitating, maladaptive pain that remains refractory to current treatments. While the nociceptive processing underlying pain hypersensitivity is well-studied in the spinal cord, the mechanisms at peripheral sites of pain integration, such as the dorsal root ganglia (DRG), are less explored. Previous studies that evaluated the contribution of brain-derived neurotrophic factor (BDNF) and tropomyosin receptor kinase B (TrkB) in nociceptive plasticity after SCI have not identified a specific mechanism, although both central and peripheral changes have been reported. Using TrkBF616A (F616) mice, our lab recently found that, after SCI, systemic inhibition of TrkB signaling with 1NM-PP1 (1NMP) delayed onset of SCI-induced pain hypersensitivity, implicating maladaptive TrkB signaling in pain development.
To identify a potential peripheral mechanism by which TrkB signaling contributes to pain after SCI, I investigate changes in TrkB signaling that may drive sensory neuron hyperexcitability after SCI. My overall hypothesis was that SCI-induced hyperexcitability of small-diameter TrkB-responsive neurons, presumed to be nociceptors or Aδ-low threshold mechanoreceptors (Aδ-LTMRs), underlies neuropathic pain after SCI. To test my hypothesis and accomplish my research goals, I obtained DRG neurons from two transgenic mouse strains (F616 and TrkB::ChR2 mice) to characterize changes in 1) neuronal excitability and 2) TrkB-mediated signaling in small-diameter nociceptors or Aδ-LTMRs following SCI. Whereas F616 mice enable selective pharmacogenetic inhibition of TrkB in sensory neurons, TrkB::ChR2 mice express yellow fluorescent protein (EYFP), that allow selective, visual targeting of TrkB+ neurons.
Patch-clamp recordings from dissociated thoracic DRG neurons revealed that the TrkB agonist 7,8-dihydroxyflavone (7,8-DHF) induced inward currents in both nociceptors and TrkB+ neurons. However, following SCI, TrkB-mediated responses decreased in nociceptors but increased in Aδ-LTMRs. Furthermore, changes in electrophysiological and firing properties suggested that SCI induced nociceptors to be more excitable but not Aδ-LTMRs. In general, the inhibition of TrkB did not reverse nociceptor hyperexcitability. The results revealed that thoracic contusion SCI leads to alterations in nociceptor hyperexcitability, but these changes are partly independent of TrkB activation in DRG neurons. This demonstrates complex interactions involving TrkB signaling, potentially recruiting normally non-nociceptive TrkB+ sensory neurons into post-injury nociceptive pathway.
This study is the first to delineate changes in peripheral TrkB signaling after SCI, offering new insights into the peripheral drivers underlying SCI-induced neuropathic pain. The current findings suggest that peripheral BDNF or TrkB signaling is involved in modulating nociceptive pathways after SCI. Further investigation of potential modulators of TrkB activity, such as voltage-gated sodium channels, that changes neuronal excitability and response amplitude is necessary to gain a deeper mechanistic understanding of pain hypersensitivity and its underlying cellular drivers.
Table of Contents
Distribution Agreement
Approval Sheet
Abstract Cover Page
Abstract
Cover Page
Acknowledgement
Table of Contents
List of Figures & Tables
List of Abbreviations
Chapter 1: Introduction. 1
1.1 The spinal cord: Introduction. 3
1.1.1 The spinal cord: structure and anatomy. 4
1.1.2 Spinal cord injury. 5
1.1.3 The dorsal root ganglia and nociceptors. 6
1.4 Central and peripheral sensitization. 7
1.5 Brain-derived neurotrophic factor and tropomyosin kinase receptor B.. 8
1.6 Summary. 9
1.7 Figures. 11
Figure 1.1: Summary of various components in the periphery implicated for dysregulation of nociceptive circuit after SCI with BDNF-TrkB system as an example. 12
Chapter 2: A review of dorsal root ganglia and primary sensory neuron plasticity mediating inflammatory and chronic neuropathic pain. 14
2.1 Abstract 15
2.2 Introduction. 16
2.3 Overview of pain. 18
2.4 Overview of spinal cord injury. 21
2.4.1 Neuropathic pain after spinal cord injury. 22
2.5. Overview of the dorsal root ganglia (DRG) 24
2.5.1 DRG modulation of sensory information. 25
2.5.2 Overview of sensory neurons. 25
2.5.3 Nociceptors. 27
2.6 Voltage-gated sodium channels in DRG and sensory neuron are critical in pain. 29
2.6.1 Types of voltage-gated sodium channels. 29
2.6.2 VGSCs play a role in mediating injury-induced pain. 31
2.7 Overview of glutamate and glutamate receptors. 32
2.7.1 Glutamate and glutamate receptors in pain hypersensitivity. 35
2.7.3 Presynaptic NMDA receptors contribute to pain. 36
2.8 Overview of neurotrophins. 37
2.8.1 Brain-derived neurotrophic factor modulates nociceptive and neuropathic pain. 40
2.8.2 TrkB receptor 40
2.8.3 BDNF’s expression and function in sensory neurons and spinal cord. 44
2.8.4 BDNF’s contribution to injury-induced pain. 46
2.8.5 Peripheral BDNF-TrkB signaling in mechanotransduction and pain. 46
2.9 Other events that contribute to inflammatory or injury-induced plasticity of DRG.. 48
2.9.1 Effect of peripheral inflammation on DRG plasticity. 48
2.9.2 Effect of SCI on pain and DRG plasticity. 50
2.9.3 Peripheral changes after SCI that contribute to pain. 52
2.9.4 Other SCI-induced changes: non-nociceptors such as C-LTMRs contribute to pain. 53
2.9.5 Sympathetic efferents and sensory neurons interact to produce pain. 54
2.9.6 Central sensitization. 56
2.10 Conclusion. 57
2.11 Figures. 59
Figure 2.1: Examples of some review literature on pain, SCI, neurotrophins, and nociceptors through the past 30 years. 59
Figure 2.2: Comparison of nociceptive and neuropathic pain. 62
Table 2.1. 65
Chapter 3: TrkB agonist (7,8-DHF)-induced responses in dorsal root ganglia neurons are decreased after spinal cord injury: implication for peripheral pain mechanisms. 66
3.1 Abstract 67
3.2 Introduction. 68
3.3 Materials and Methods. 70
3.3.1 Subjects. 70
3.3.2 Drug administration and pharmacological verification. 71
3.3.3 Surgical procedure. 71
3.3.4 Dissociation of DRG neurons. 72
3.3.5 Whole cell recording from dissociated DRG neurons. 73
3.3.6 Assessment of electrophysiological properties. 74
3.3.7 Preparation and application of drugs. 75
3.3.8 Western blot for TrkB expression. 76
3.3.9 Immunocytochemistry. 77
3.3.10 Statistical analysis. 77
3.4 Results. 78
3.4.1 Morphologies and properties of DRG neurons from uninjured and SCI F616 mice. 79
3.4.2 TrkB agonist induces inward currents in small-diameter DRG neurons. 80
3.4.3 Decreased TrkB-mediated inward currents in the DRGs following SCI 82
3.4.4 SCI increases latency to peak of TrkB induced current 83
3.4.5 Small-diameter DRG neurons exhibit differential changes in excitability after SCI. 83
3.4.7 Decrease in TrkB expression in the DRGs around the lesion following SCI 86
3.5 Discussion. 87
3.6 Figures. 97
Figure 3.1: Illustration of the experimental design and electrophysiology protocol for assessing DRG neuronal response to 7, 8-DHF following SCI and acute and systemic 1NMP treatment 98
Figure 3.2: Characterization of acutely dissociated DRG neurons. 101
Figure 3.3: Electrophysiological recordings of small diameter dissociated DRG. 104
Figure 3.4: Electrophysiological recordings of small diameter dissociated DRG following SCI. 106
Figure 3.5: Changes in DRG neuronal response to 7,8-DHF and electrophysiological properties after SCI 108
Figure 3.6: Capsaicin-induced inward currents in small DRG neurons also decrease after SCI. 109
Figure 3.7: DRG expression of TrkB decreases following SCI. 110
Table 3.1: Summary of Animals used. 112
Table 3.2: Summary of electrophysiological properties. 114
Chapter 4: Increased excitability in TrkB-expressing primary sensory neurons following spinal cord injury. 115
4.1 Abstract 116
4.2 Introduction. 117
4.3 Materials and Methods. 118
4.3.1 Subjects - generation of transgenic mice. 118
4.3.2 Surgical procedures. 119
4.3.3 Behavior assessments. 119
Assessment of mechanical hypersensitivity. 119
Assessment of thermal hypersensitivity. 120
4.3.4 Dissociation and whole cell patch-clamp recording of DRG neurons. 120
4.3.5 Preparation and application of drugs. 121
4.3.8 Western blot for TrkB expression. 121
4.3.9 Statistical Analysis. 122
4.4 Results. 122
4.4.1 Electrophysiological properties of EYFP-positive DRG neurons from Uninjured and SCI TrkB::ChR2 mice. 123
4.4.2 Increased TrkB-mediated inward currents in the EYFP-positive DRGs following SCI 124
4.4.3 No change in TrkB expression in the DRGs around the lesion following SCI 125
4.4.4 EYFP-positive neurons are categorized by their response to capsaicin. 125
4.5 Discussion. 126
4.6 Figures. 130
Figure 4.1: Electrophysiological properties of EYFP-positive DRG neurons from uninjured and SCI mice. 130
Figure 4.2: Capsaicin-induced inward currents in EYFP-positive DRG neurons decrease after SCI. 132
Figure 4.3: Changes in TrkB-expressing DRG neuronal response to 7,8-DHF and electrophysiological properties after SCI. 133
Table 4.1: Summary of animals used in the experiment. 135
Table 4.2: Summary of electrophysiological properties from uninjured or SCI TrkB::ChR2 mice. 136
Chapter 5: Comparative outcomes in TrkBF616 and TrkB::ChR2 mouse models: Advancing insights into peripheral TrkB signaling in SCI-Induced pain hypersensitivity. 137
5.1 Abstract 138
5.2 Introduction. 139
5.3 Comparison of results obtained from F616 and TrkB::ChR2 mice. 141
5.3.1 Electrophysiological characteristics of TrkBF616 and TrkB::ChR2 mice. 141
5.3.2 TrkBF616A neurons are more excitable than EYFP-positive TrkB::ChR2 neurons. 142
5.3.3 TrkBF616A neurons produce greater inward current responses to 7,8-DHF compared to EYFP-positive TrkB::ChR2 neurons even though the channel kinetics are the same. 143
5.3.4 Capsaicin. 143
5.3.5 Differences in the TrkB expression in the DRGs. 144
5.3.6 Pain behaviors. 145
5.4 Discussion of strain differences. 145
5.5 Figures. 151
Figure 5.1: Comparison of firing and electrophysiological properties between F616 and TrkB::ChR2 DRG neurons. 151
Figure 5.2: Comparison of 7,8-DHF and capsaicin-induced responses between F616 and TrkB::ChR2 DRG neurons. 154
Figure 5.3: Comparison of behavioral pain sensitivity between F616 and TrkB::ChR2 mice. 156
Table 5.1: Summary of animals used in the experiment. 157
Table 5.2: Summary of electrophysiological properties from TrkBF616, TrkB::ChR2, and WT mice. 158
Table 5.3: Summary of 7, 8-DHF- and capsaicin-induced inward current amplitudes and latencies to response onset and peak in F616, TrkB::ChR2, and WT DRG neurons. 160
Chapter 6: Discussion, Future Directions, and Broader Implications. 161
6.1 Summary. 162
6.2 Discussion. 164
6.2.1 Significant findings. 165
6.2.2 Experimental approach and potential pitfalls. 167
Challenges of using multiple transgenic mouse models. 167
Sex-differences. 170
Dissociated DRG neurons. 171
Meloxicam.. 172
6.3 Future Directions. 173
6.3.1 Changes in voltage-gated sodium channels (VGSCs) 173
6.3.2 Changes in TrkB isoforms after SCI 176
6.3.2 Pharmacological inhibition of native TrkB receptors. 176
6.4 Broader implications for the peripheral changes and role of peripheral TrkB in neuropathic pain after SCI 177
Conclusions. 178
Appendix. 180
Works Cited. 181
List of Figures & Tables
Chapter 1
Figure 1.1: Summary of various components in the periphery implicated for dysregulation of nociceptive circuit after SCI with BDNf-TrkB system as an example.
Chapter 2
Figure 2.1: Examples of some review literature on pain, SCI, neurotrophins, and nociceptors through the past 30 years.
Figure 2.2: Comparison of nociceptive and neuropathic pain.
Chapter 3
Figure 3.1: Illustration of the experimental design and electrophysiology protocol for assessing DRG neuronal response to 7, 8-DHF following SCI and acute and systemic 1NMP treatment.
Figure 3.2: Characterization of acutely dissociated DRG neurons.
Figure 3.3: Electrophysiological recording of small diameter dissociated DRG.
Figure 3.4: Electrophysiological recordings of small diameter dissociated DRG following SCI.
Figure 3.5: Changes in DRG neuronal response to 7,8-DHF and electrophysiological properties after SCI
Figure 3.6: Capsaicin-induced inward currents in small DRG neurons also decrease after SCI.
Figure 3.7: DRG expression of TrkB decreases following SCI.
Table 3.1: Summary of animals used in the experiment.
Table 3.2: Summary of electrophysiological properties.
Chapter 4
Figure 4.1: Electrophysiological properties of EYFP-positive DRG neurons from uninjured and SCI mice.
Figure 4.2: Capsaicin-induced inward currents in EYFP-positive DRG neurons decrease after SCI
Figure 4.3: Changes in TrkB-expressing DRG neuronal response to 7,8-DHF and electrophysiological properties after SCI.
Table 4.1: Summary of animals used in the experiment.
Table 4.2: Summary of electrophysiological properties from uninjured or SCI TrkB::ChR2 mice.
Chapter 5
Figure 5.1: Comparison of firing and electrophysiological properties of between F616 and TrkB::ChR2 DRG neurons.
Figure 5.2: Comparison of 7,8-DHF and capsaicin-induced inward currents between F616 and TrkB::ChR2 DRG neurons.
Figure 5.3: Comparison of behavioral pain sensitivity between F616 and TrkB::ChR2 DRG neurons.
Table 5.1: Summary of animals used in the experiment.
Table 5.2: Summary of electrophysiological properties from TrkBF616, TrkB::ChR2, and WT mice.
Table 5.3: Summary of 7,8-DHF- and capsaicin-induced inward current amplitudes and latencies to response onset and peak in F616, TrkB::ChR2, and WT DRG neurons.
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