Characterization of Thoracic Sympathetic Postganglionic Neurons Público

Abstract

Thoracic sympathetic postganglionic neurons (tSPNs) comprise the final output of the distributed sympathetic nervous system controlling vascular and thermoregulatory systems. Traditionally considered a non-integrating relay, what little is known of tSPN intrinsic excitability has been determined by sharp microelectrodes with presumed impalement injury. We thus undertook the first electrophysiological characterization of mouse tSPN cellular and synaptic properties using whole-cell recordings and coupled results with a conductance-based model to explore the principles governing tSPN excitability.

Input resistance and time constant values were an order of magnitude greater than values reported elsewhere, leading to much lower rheobase. All cells were capable of repetitive firing, in contrast to prior reports. We used the computational model to explain observed phenomena and to account for the differences in observed properties compared to prior recordings. Frequency of spontaneous events (sEPSCs) was highly variable between cells. Amplitude of sEPSCs averaged 40pA, and evoked synaptic events ranged from approximately the same size as sEPSCs to an order of magnitude greater. Values of time constant were consistent with kinetic properties of α3β4 nicotinic receptors. sEPSCs were not blocked by TTX, indicating that they occur in the absence of action potentials.

We then explored changes that might occur in these neurons after high thoracic spinal cord transection (SCI) that would lead to emergent autonomic dysfunction. Three to six weeks after SCI, tSPN intrinsic membrane properties were comparable to controls, but frequency and amplitude of spontaneous preganglionic synaptic activity was increased, potentially a homeostatic response to SCI-induced decreased presynaptic drive. Overall, this suggests that while the intrinsic excitability of tSPNs is unaltered after SCI, they are likely to be driven more robustly by SCI-induced increases in presynaptic drive.

Taken together, whole-cell recordings reveal tSPNs have more dramatically amplified excitability than previously thought, with greater intrinsic capacity for synaptic integration and with the ability for maintained firing to support sustained actions on vasomotor tone and thermoregulatory function. Rather than acting as a relay, these studies support a more responsive role and possible intrinsic capacity for tSPNs to drive sympathetic autonomic function.

Table of Contents

1 Introduction ................................................................................................................................ 1

1.1 Overview of the sympathetic nervous system .......................................................................... 1

1.2 Anatomical organization ....................................................................................................... 2

1.2.1 Gross anatomy ............................................................................................................... 2

1.2.2 Cellular morphology ....................................................................................................... 6

1.3 Circuitry ............................................................................................................................... 8

1.3.1 Brain and brainstem nuclei ............................................................................................. 8

1.3.2 Preganglionic neurons ................................................................................................... 10

1.3.3 Postganglionic neurons ................................................................................................. 11

1.3.4 Effectors ....................................................................................................................... 12

1.4 Intrinsic properties of SPNs .................................................................................................. 13

1.4.1 Passive and firing properties........................................................................................... 14

1.4.2 Ion channels ................................................................................................................. 15

1.5 Synaptic properties of SPNs .................................................................................................. 16

1.5.1 Types of synaptic input .................................................................................................. 16

1.5.2 Primary and secondary nicotinic synapses ....................................................................... 17

1.5.3 Synaptic integration ...................................................................................................... 18

1.6 Computational modeling ...................................................................................................... 18

1.6.1 Intrinsic membrane properties ....................................................................................... 19

1.6.2 Synaptic and network properties ..................................................................................... 20

1.7 Effect of spinal cord injury on autonomic function .................................................................. 20

1.7.1 Changes in cardiovascular function ................................................................................. 20

1.7.2 Autonomic dysreflexia .................................................................................................... 22

1.8 Summary and goals ............................................................................................................... 23

2 Intrinsic membrane properties of thoracic postganglionic neurons...................................................25

2.1 Abstract ............................................................................................................................... 25

2.2 Significance .......................................................................................................................... 26

2.3 Introduction ......................................................................................................................... 26

2.4 Results ................................................................................................................................. 29

2.4.1 Passive membrane properties ......................................................................................... 29

2.4.2 Rheobase ....................................................................................................................... 31

2.4.3 Repetitive Firing ............................................................................................................ 34

2.4.4 Impalement simulation................................................................................................... 37

2.4.5 Spike rate adaptation ..................................................................................................... 39

2.4.6 Cell firing type classification............................................................................................ 41

2.4.7 Afterhyperpolarization ................................................................................................... 44

2.4.8 Subthreshold conductances............................................................................................. 46

2.4.9 Effect of spinal cord injury .............................................................................................. 51

2.5 Discussion ............................................................................................................................ 51

2.5.1 Re-appraisal of physiological consequence of passive membrane properties........................ 51

2.5.2 The physiological relevance of repetitive firing in tSPNs ................................................... 55

2.5.3 Relating observed cellular properties to underlying conductances ..................................... 56

2.5.4 Other factors contributing to modulation of tSPN excitability ........................................... 57

3 Synaptic properties of thoracic postganglionic neurons.................................................................. 58

3.1 Abstract .............................................................................................................................. 58

3.2 Introduction ........................................................................................................................ 59

3.3 Results ................................................................................................................................ 61

3.3.1 Spontaneous EPSCs ....................................................................................................... 61

3.3.2 Comparison of evoked and spontaneous EPSCs ............................................................... 63

3.3.3 Effect of spinal cord injury ............................................................................................. 65

3.3.4 Effect of TTX on sEPSC frequency .................................................................................. 67

3.4 Discussion .......................................................................................................................... 69

3.4.1 Spontaneous events in thoracic neurons ........................................................................ 70

3.4.2 Evoked events may reveal degree of innervation.............................................................. 72

3.4.3 Spontaneous synaptic activity after spinal cord injury ..................................................... 73

4 General conclusions and future directions ................................................................................... 76

4.1 Conclusion ......................................................................................................................... 76

4.1.1 Summary of results ...................................................................................................... 76

4.1.2 Consequence of enhanced excitability of tSPNs .............................................................. 78

4.1.3 Benefits of computational modeling .............................................................................. 81

4.1.4 Consequence of spinal cord injury on postganglionic neuron function.............................. 83

4.2 Future directions ................................................................................................................ 85

4.2.1 Is there a correlation between cell type and firing properties?.......................................... 85

4.2.2 Impact of SCI-induced changes on cell recruitment? ...................................................... 85

4.2.3 Computational model of individual cells? ...................................................................... 86

4.2.4 Impact of SCI on evoked synaptic events? ...................................................................... 86

5 Methods ................................................................................................................................... 87

5.1 Animals ............................................................................................................................. 87

5.2 Spinal cord injury ............................................................................................................... 87

5.3 Immunohistochemistry ...................................................................................................... 88

5.3.1 Neurotransmitter identity ............................................................................................ 88

5.3.2 Cell diameter............................................................................................................... 89

5.4 Electrophysiology .............................................................................................................. 89

5.4.1 Tissue Preparation ...................................................................................................... 89

5.4.2 Whole-cell recordings ................................................................................................. 91

5.4.3 Optogenetic stimulation .............................................................................................. 92

5.5 Data analysis ..................................................................................................................... 92

5.5.1 Analysis of intrinsic membrane properties .................................................................... 92

5.5.2 Analysis of synaptic events .......................................................................................... 95

5.5.3 sEPSC curve fitting algorithm ...................................................................................... 95

5.6 Computational Modeling ................................................................................................... 96

5.6.1 Single Neuron Model .................................................................................................. 96

5.6.2 Impalement simulation............................................................................................... 98

5.6.3 Synapse simulation..................................................................................................... 99

5.6.4 Code accessibility ....................................................................................................... 99

5.7 Experimental design and Statistical analysis ..................................................................... 100

6 Appendix .............................................................................................................................. 103

6.1 Equations for computational model .................................................................................. 103

6.1.1 Fast Na+ current: ...................................................................................................... 103

6.1.2 Delayed rectifier K+ current:...................................................................................... 103

6.1.3 L-type Ca2+ current: ................................................................................................ 104

6.1.4 M-type K+ current: ................................................................................................... 104

6.1.5 Ca2+-dependent K+ current: ..................................................................................... 105

6.1.6 A-type K+ current: ................................................................................................... 105

6.1.7 H-current: .............................................................................................................. 105

6.1.8 Leak current: .......................................................................................................... 106

6.1.9 Injury current: ........................................................................................................ 106

6.1.10 Synaptic current: ................................................................................................... 106

6.1.11 Somatic [Ca2+]: ..................................................................................................... 106

6.1.12 Master equation: ................................................................................................... 107

7 References ........................................................................................................................... 108

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 Biology, Physiology
Palabra Clave	computational model paravertebral ganglia membrane properties whole-cell patch clamp
Committee Chair / Thesis Advisor	Shawn Hochman, Emory University
Committee Members	Astrid Prinz, Emory University Arthur English, Emory University Marie-Claude Perreault, Emory University Sandra Garraway, Emory University

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