MODULATING SYMPATHETIC OUTPUT: FACTORS INFLUENCING CONDUCTION IN THORACIC PREGANGLIONIC AXONS Restricted; Files Only
Halder, Mallika (Spring 2024)
Published
Abstract
Spinal cord sympathetic preganglionic neurons (SPNs) are crucial for transmitting signals from the CNS to the peripheral postganglionic neurons that control organ function. It has been assumed that SPN peripheral axonal spike propagation along its projections is reliable. To assess this, I developed an ex vivo approach having intact thoracic paravertebral sympathetic chain ganglia and ventral roots maintained in situ. Suprathreshold stimulation of SPN axons in thoracic ventral roots was achieved through optical and electrical methods in ChAT-CHR2 adult mice, allowing capture of evoked population compound action potential responses across multiple ganglia. Variability in axonal recruitment was overt and found to be particularly significant in the slow-conducting unmyelinated axons. That SPN peripheral axon projections have a low safety-factor with demonstrable conduction failures challenges the traditional assumption that SPN spike propagation across its broad distribution occurs without failure.
Focusing on mechanisms affecting spike propagation, slow-conducting SPN axons were found to be particularly susceptible to conduction failures via temperature increases and frequency-dependent conditioning stimuli. Pharmacological block of GABAA receptors and activation of serotonin receptors, acetylcholine receptors, and K2P leak channels depressed conduction in these axons. Conversely, the convulsant 4-aminopyridine greatly facilitated conduction -presumably via block of voltage-gated KA channels.
Overall, this work identified variability of spike conduction in SPN axons as fundamental features of its operation. As autonomic neural drive to organ systems commonly involve continuous activity with slow temporal dynamics, we hypothesize that population-encoded responses utilize small-diameter unmyelinated axons with low safety factor as a metabolically efficient method for population control of peripheral autonomic circuitry. That axonal conduction failures are history dependent and under neuromodulatory controls, suggests that alterations in conduction reliability provide a mechanism of output gain control beyond those encoded by centrally-driven SPN spike recruitment.
Table of Contents
1 Introduction. 17
1.1 Overview of the autonomic nervous system.. 18
1.2 Overview of the sympathetic nervous system.. 20
1.2.1 Circuitry. 20
1.2.2 Anatomical organization of sympathetic preganglionic neurons. 21
1.2.1 Sympathetic preganglionic projections and actions on postganglionic neurons 23
1.3 Impulse Conduction Through Autonomic Ganglia. 26
1.3.1 Principles of Convergence. 26
1.3.2 Principles of divergence. 31
1.3.1 Sympathetic gain control. 32
1.4 Axonal action potential propagation in SPNs. 33
1.4.1 Anatomical features of the preganglionic axon that affect spike propagation. 33
1.4.2 Firing Properties of SPNs. 36
1.4.3 Factors controlling action potential propagation in SPNs. 39
1.5 Summary. 44
2 Isolation and Electrophysiology of Murine Sympathetic Postganglionic Neurons in the Thoracic Paravertebral Ganglia. 46
2.1 Abstract 46
2.2 Background. 47
2.3 Materials and Reagents. 47
2.4 Equipment 49
2.4.1 Surgical tools. 49
2.4.2 Recording equipment for whole-cell recordings. 49
2.4.3 Recording equipment for studies involving multisegmental preganglionic and postganglionic compound action potentials. 52
2.4.4 Image Capture Equipment for Calcium Imaging. 53
2.4.5 Software. 54
2.5 Procedure. 54
2.5.1 Ex vivo mouse dissection for experiments recording multisegmental pre and postganglionic compound action potentials. 57
2.5.2 Ex vivo mouse dissection for whole-cell patch clamp recordings or calcium imaging. 60
2.5.3 Manufacture of Trumpet-Shaped Tips of Glass Suction Electrodes and Patch Electrodes. 63
2.5.4 Manufacture of Sylgard-coated dissecting dish. 64
2.5.5 Manufacture of suction electrodes (Figure 2.1) 65
2.5.6 Recipes. 67
2.6 Conclusion. 67
3 Characteristics and Variability in SPN Axonal Conduction: Velocity, Myelination, and Branching Patterns. 70
3.1 Abstract 70
3.2 Introduction. 71
3.3 Materials and Equipment 73
3.3.1 Neurobiotin labeling to assess preganglionic divergence. 73
3.3.2 Myelin Basic Protein Stain and Channelrhodopsin/GFP dual immunolabeling. 74
3.3.3 Imaging and analysis for axon counts. Error! Bookmark not defined.
3.3.1 Assessment of axon counts and size. 75
3.3.1 Tissue Preparation. Error! Bookmark not defined.
3.3.2 Quantification and Analysis of SPN volleys for analysis. 76
3.3.1 Quantification of Coefficient of Variation. 76
3.3.2 Electrode preparation and placement 77
3.4 Results. 77
3.4.1 Assessment of complete synaptic block. 77
3.4.2 Recruitment profiles for SPN volleys. 79
3.4.1 Anatomical assessment of CNS preganglionic (SPN) axon composition and projections. 82
3.4.2 Evoked response fidelity is high in the fastest axons whereas the slowest axons have high response variability indicative of high rates of conduction failures. 93
3.5 Discussion. 97
3.5.1 Mouse thoracic SPN axons exhibit broad divergence and are largely unmyelinated. 97
3.5.2 At room temperature, variability of SPN conduction correlates to conduction velocity. 98
3.5.3 Differential recruitment of SPN axons by electrical and optical stimulation. 100
3.6 Conclusions. 101
4 SPN sensitivity to temperature and frequency. 103
4.1 Abstract 103
4.2 Introduction. 104
4.3 Methods. 105
4.3.1 Multisegmental ex vivo paravertebral preparation. 105
4.3.2 Electrophysiology. 106
4.3.3 Pharmacology. 106
4.3.4 Analysis. 106
4.4 Results. 107
4.4.1 Population recruitment of SPN axons is altered at elevated temperatures. 107
4.4.2 Slower conducting SPN axons that branch exhibit depression during a 5Hz train. 110
4.4.3 A sub-population of slow-conducting SPN axons are resilient to 5Hz but exhibit depression at 10Hz. 114
4.4.4 Further comparison of effects of pulse train duration with subsequent assessment of temperature dependence of these changes at 22°C and 36°C. 115
4.5 Discussion. 122
4.5.1 Effects of temperature. 122
4.5.2 Frequency dependent changes in amplitude of evoked responses. 126
4.5.3 Optogenetic vs. electrical stimulation. 128
4.6 Conclusion. 128
5 Modulation of Branch Point Failure. 130
5.1 Abstract 130
5.2 Introduction. 131
5.2.1 Modulation via GABAARs. 131
5.2.2 Modulation via A-current blockers. 132
5.2.3 Modulation via K+ leak channels. 133
5.2.4 Cholinergic and serotonergic modulation of SPN axons. 134
5.3 Methods. 135
5.3.1 Mice. 136
5.3.2 Electrophysiology. 136
5.3.1 Analysis. 136
5.3.2 Pharmacology. 136
5.4 Results. 137
5.4.1 SPN Conduction Block Observed with Broad-Spectrum GABAAR Antagonists. 137
5.4.2 Pharmacological block of voltage-gated K+ channels. 139
5.4.3 Role of K2P leak channels in the temperature-dependent in block of spike conduction. 146
5.4.4 Modulation via 5-HT. 151
5.4.1 Presynaptic nicotinic acetylcholine receptors may be a site for SPN axon modulation. 152
5.5 Discussion. 155
5.5.1 Overview of Key Findings. 155
5.5.2 Modulation via GABAARs. 155
5.5.3 Modulation via 4-aminopyridine and tetraethylammonium (TEA) 156
5.5.4 Modulation via Potassium Leak Channels. 161
5.5.1 Monoaminergic Modulation. 162
5.5.2 Cholinergic Modulation of SPN Axons. 164
5.5.3 Comparative Analysis of Modulatory Effects. 165
5.5.4 Implications for Neurophysiological Understanding. 166
6 Summary and General Discussion. 169
6.1 Conduction failure behavior in sympathetic preganglionic axons. 171
6.2 Thermo-dysfunction. 171
6.3 Modulation via factors in circulation. 172
7 References 174
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