The regulation of the potassium chloride co-transporter KCC2 in spinal motoneurons following peripheral nerve injury Público
Akhter, Erica (Summer 2019)
Abstract
Following many types of injury, the potassium chloride co-transporter KCC2 is dysregulated on injured neurons or those associated with neurons that have been injured. This changes driving forces for GABAergic and glycinergic inhibitory synapses and in general increases excitability in the affected neurons and networks. This phenomenon has been described in dorsal horn sensory-associated interneurons following peripheral nerve injury (PNI), as well as on motoneurons after spinal cord injury. In these cases, decreased expression of KCC2 is associated with neuropathic pain and spasticity, respectively. However, increasing activity with exercise or electrical stimulation has previously been shown to aid in motor axon regeneration,and KCC2 has not yet been investigated on spinal motoneurons following PNI. Previous studies in the brainstem have indicated that KCC2 is also downregulated in motoneurons axotomized in peripheral nerve injuries, but the significance of this downregulation and the mechanisms regulating it have received less attention. In this dissertation, we investigate the extent and regulation of KCC2 depletion on spinal motoneurons following PNI using a variety of mutant mice, pharmacological interventions, and injury paradigms. Following sciatic nerve injury, kcc2 mRNA was downregulated in motoneurons within three days of their axotomy. In all cases, two weeks following injury, KCC2 protein was also consistently and drastically depleted on axotomized motoneuron somata and proximal dendrites. This depletion occurs independently from the mechanisms previously established in other regions of the spinal cord after PNI; it is not dependent on microgliosis, motoneuron BDNF release, or TrkB signaling. However, KCC2 is restored if the sciatic nerve is repaired and regeneration is allowed. This restoration is dependent on the presence of an intact neuromuscular junction, but not the functional connection with the muscle. We illustrated that blocking neuromuscular signaling or exercising mice to drive more neural activity had no impact on KCC2 expression. Our results illustrate that KCC2 regulation on axotomized motoneurons occurs via a different mechanism than previously established in other cell types or injury paradigms. This work lays the groundwork for pursuing further questions about the role of downregulation in KCC2, likely accompanied by increasesin activity, in aiding motor axon regeneration.
Table of Contents
Chapter 1: General Introduction .
1.1 General Introduction. . . . . .2
1.2 Peripheral nerve injury as a model and clinical conundrum. . . . . .3
1.3 Neuronal hyperexcitability: Is it only an unhappy accident? . . . . . .5
1.4 The mysterious shift in motoneuron excitability . . . . . .8
1.5 Synaptic changes on regenerating motoneurons . . . . . .9
1.6 KCC2 depletion is a mechanism for altered inhibitory signaling in motoneurons. . . . . .10
1.7 Thesis Overview: does KCC2 decrease on axotomized motoneurons have implications for regeneration? Completing the peripheral picture. . . . . .13
1.8 References. . . . . . . .14
Chapter 2: General Methods
2.1 General Methods . . . . . . . .22
2.2 Animals . . . . . . . .22
2.3 Tamoxifen treatment. . . . . . . .22
2.4 Retrograde tracer injections. . . . . . . .23
2.5 Surgeries. . . . . . . .23
2.6 Tissue collection, processing and immunocytochemistry. . . . . . . .24
2.7 Quantification of KCC2 immunoreactivity on the cell surface . . . . . . . .25
2.8 Statistical analyses. . . . . . . . 26
2.9 Tables and Figures. . . . . . . .27
2.10 References. . . . . . . .29
Chapter 3: Characteristics of KCC2 loss on motoneuron somata and proximal dendrites following axotomy.
3.1 Abstract. . . . . . . . 31
3.2 Introduction. . . . . . . .31
3.3 Methods
3.3.1 Time course of KCC2 loss on motoneuron somata following axotomy. . . . . . . .33
3.3.2Qualitative analyses of KCC2 in motoneuron dendritic arbors . . . . . . . .33
3.3.3In situanalysis of KCC2 mRNA using using RNA-Scope® . . . . . . . .35
3.4 Results
3.4.1 KCC2 depletion is significant and sustained after 14 days. . . . . . . .36
3.4.2 Reliability of KCC2 quantification. . . . . . . .37
3.4.3 KCC2 is preferentially preserved on distal dendrites. . . . . . . .38
3.4.4 KCC2 regulation on axotomized motoneurons occurs at the transcriptional level. . . . . . . .39
3.5 Discussion. . . . . . . .41
3.6 Tables and Figures. . . . . . . .46
3.7 References. . . . . . . .55
Chapter 4: Investigation of the role of established regulators of KCC2 following nervous system injury, namely microglia, BDNF and TrkB.
4.1 Abstract. . . . . . . .59
4.2 Introduction. . . . . . . .59
4.3 Methods
4.3.1 Genetic approaches to interfere with microglia and BDNF release. . . . . . . .61
4.3.2 Pharmacological block of TrkB activation: ANA-12 Treatment. . . . . . . .63
4.3.3 Pharmacogenetic TrkB inactivation . . . . . . . .65
4.4 Results
4.4.1 KCC2 depletion is independent of microglia. . . . . . . .65
4.4.2 KCC2 depletion is independent of motoneuron-BDNF. . . . . . . .66
4.4.3 KCC2 depletion is independent of TrkB activation. . . . . . . .68
4.5 Discussion. . . . . . . .70
4.6 Tables and Figures. . . . . . . .74
4.7 References. . . . . . . .80
Chapter 5: KCC2 regulation by a muscle-derived signal
5.1 Abstract. . . . . . . .85
5.2 Introduction. . . . . . . .85
5.3 Methods
5.3.1 Animals. . . . . . . .88
5.3.2 Exercise paradigm. . . . . . . .88
5.3.3 Alpha-bungarotoxin administration. . . . . . . .89
5.3.4 Motor endplate quantification. . . . . . . .89
5.3.5 Electromyography . . . . . . . .90
5.4 Results
5.4.1 KCC2 restoration is dependent on muscle reinnervation. . . . . . . .91
5.4.2 KCC2 depletion occurs independent 0f exercise. . . . . . . .92
5.4.3 KCC2 depletion & alpha-bungarotoxin. . . . . . . .95
5.5 Discussion. . . . . . . .97
5.6 Tables and Figures. . . . . . . .101
5.7 References. . . . . . . .109
Chapter 6: General Discussion & Future Directions
6.1 Mechanisms of KCC2 depletion on spinal motoneurons following PNI. . . . . . . .114
6.2 The impact of KCC2 loss on spinal motoneurons following spinal cord injury and brainstem motoneurons after PNI. . . . . . . .116
6.3 Does increased inhibitory synaptic activity promote motor axon regeneration after PNI?. . . . . . . .119
6.4 Future strategies for investigating the importance of altered KCC2 and inhibitory signaling in axotomized motoneurons. . . . . . . .121
6.5 High chloride promotes regeneration in axotomized sensory neurons. . . . . . . .122
6.6 Conclusions. . . . . . . .123
6.7 References. . . . . . . .125
Appendix 1: Regulation of KCC2 by miRNA’s
A.1 Abstract. . . . . . . .130
A.2 Introduction. . . . . . . .130
A.3 Methods
A.3.1 Animals. . . . . . . .131
A.3.2 Animal procedures. . . . . . . .131
A.3.3Histology, immunohistochemistry and DICER KO quantification. . . . . . . .132
A.4 Results
A.4.1KCC2 regulation occurs independently from miRNA upregulation after PNI. . . . . . . .132
A.5 Discussion. . . . . . . .133
A.6 Tables and Figures. . . . . . . .135
A.7 References. . . . . . . .138
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