On the mechanisms of presynaptic inhibition of primary afferents Público
Shreckengost, Jacob Michael (2012)
Published
Abstract
Primary afferent neurotransmission is the fundamental first step in the central processing of sensory information and is controlled by pre- and postsynaptic inhibitory mechanisms. Presynaptic inhibition (PSI) is probably the more powerful form of inhibitory control in all primary afferent fibers. A major mechanism producing afferent PSI is via a channel-mediated depolarization of their intraspinal terminals, which can be recorded extracellularly as a dorsal root potential (DRP). Based on measures of DRP latency it has been inferred that this primary afferent depolarization (PAD) of low-threshold afferents is mediated by minimally trisynaptic pathways with pharmacologically identified GABAergic interneurons forming last-order axo-axonic synapses onto afferent terminals. This thesis describes recent and historical work that supports the existence of PAD occurring by more direct pathways and with a complex pharmacology that questions the proprietary role of GABA and GABAA receptors in this process. I show that cholinergic transmission contributes largely to PAD, including the possibility of direct release from primary afferents. I provide evidence for specific physiological actions of cholinergic receptors on distinct afferent class in modulating sensory transmission and discuss the possible role of cholinergic receptor activation in integration of sensory signaling both centrally and in the periphery. Specifically, cholinergic transmission underlies nicotinic receptor subunit specific actions on sensory gating of afferent subpopulations and plays a major role in generation of spiking in afferent axons, with the majority of the actions being on slower conducting low-threshold afferents. Finally, I describe unique expression patterns of nicotinic receptor subunits in defined populations of afferents. Together this work redefines how integration of sensory signaling occurs in primary afferents with implications for development of subunit specific pharmacological targets in the treatment of aberrant afferent signaling following disease or injury.
Table of Contents
CONTENTS
CHAPTER 1: INTRODUCTION...1
1.1 Overview of Presynaptic Inhibition...1
1.1.1 Presynaptic Inhibition in
Invertebrates...1
1.1.2 Presynaptic Inhibition in Vertebrate Spinal Cord...3
1.1.2.1 Presynaptic Inhibition Muscle
Afferents...10
1.1.2.2 Presynaptic Inhibition in Cutaneous Afferents...12
1.1.2.3 Neurotransmitter Systems Implicated in Primary Afferent
Depolarization...12
1.1.2.4 Limitations of Previous Presynaptic Inhibition
Studies...16
1.1.2.5 Other Possible Mechanisms Generating Afferent-Evoked
PAD...18
1.1.2.6 Aims and Objectives...21
1.2 General Methodology...23
1.2.1 Stimulation of Afferents...23
1.2.2 Recordings...24
Chapter 2 Presynaptic inhibition of primary afferents by depolarization: Observations supporting non-traditional mechanisms...25
2.1 Abstract...25
2.2 Introduction...25
2.2.1 Presynaptic inhibition (PSI) of primary
afferents...25
2.2.2 Some limitations with the current model of afferent-evoked
PAD...26
2.3 Further peculiarities of PAD...30
2.3.1 Is GABA really the transmitter producing
afferent-evoked PAD?...30
2.3.2 Is the GABAA receptor really the receptor
activated to produce afferent-evoked PAD?...31
2.3.3 Could another Cys-loop family receptor produce PAD?...32
2.4. Could another transmitter be released to produce PAD?...34
2.4.1 Acetylcholine...34
2.4.2 Taurine and β-alanine...35
2.5 Experimental support for the existence of non-traditional mechanisms serving afferent-evoked PAD...35
2.5.1 The hemisected spinal cord maintained in
vitro...35
2.5.2 Low-threshold DRPs require GABAA-like receptor
activation...38
2.5.3 Bicuculline-sensitive DRPs remain after greatly restricting
synaptic transmission...38
2.5.4 DRPs can remain even after the block of glutamatergic
transmission...38
2.5.5. Typical and atypical pharmacology of the DRP...42
2.5.6. Many primary afferents are probably cholinergic...44
2.5.7. Primary afferents express taurine and
β-alanine...44
2.3. Conclusion...45
Chapter 3 Bicuculline-sensitive primary afferent depolarization remains after greatly restricting synaptic transmission in the mammalian spinal cord...48
3.1 Abstract...48
3.2 Introduction...49
3.3 Methods...50
3.3.1 Whole-cell patch clamp
recordings...51
3.3.2 Data Analysis...52
3.4 Results...52
3.4.1 PAD requires synaptic transmission,
TTX-sensitive afferents, and is blocked by GABAA receptor
antagonists...52
3.4.2 Mephenesin and high divalent cation solution largely restrict
transmission to monosynaptic actions...53
3.4.3 Bicuculline-sensitive DRPs remain after restricting synaptic
transmission...55
3.4.4 Further evidence of minimal synaptic transmission
requirements in afferent-evoked PAD...60
3.5 Discussion...60
3.5.1. Is PAD generated monosynaptically or via unconventional non-spiking microcircuits?...61
CHAPTER 4 EFFECTS OF CHOLINERGIC TRANSMISSION AND RECEPTOR ACTIVATION ON PRIMARY AFFERENT DEPOLARIZATION...66
4.1 Abstract...66
4.2 Introduction...67
4.2.1 Is the acetylcholine transmitter system
involved in afferent-evoked PAD?...67
4.2.2 Nicotinic receptors are expressed in primary
afferents...69
4.2.3 ACh acts on ionotropic and metabotropic ACh
receptors...69
4.3 Methods...72
4.3.1 Overview of sensory afferent
classes...72
4.3.2 Isolation and recording of dorsal roots and peripheral
nerves...72
4.3.3 Stimulation of peripheral nerves and dorsal roots...74
4.3.4 Dorsal Root Reflexes...75
4.3.5 Extracellular Field Potentials...76
4.3.6 Drugs...77
4.3.7 Antibodies...81
4.3.8 Determination of soma diameter...82
4.3.9 Data Analysis...82
4.4 Results...89
4.4.1 Cholinergic transmission is involved in afferent evoked DRPs, EFPs, and DRRs...92
4.4.1.1 Block of muscarinic receptors does not
affect cutaneous afferent evoked DRPs or EFPs...92
4.4.1.2 The α9 nAChR contributes to DRPs and EFPs evoked by
afferent stimulation...94
4.4.1.3 ACh and nicotinic receptor agonists depress DRPs and EFPs
likely through depolarization block of afferent signaling...105
4.4.2 Application of ACh and nicotinic agonists leads to a depolarization of primary afferents...117
4.4.2.1 Cholinergic depolarization of primary afferents is mediated by activation of α3, α6, and α9 containing nAChRs...125
4.4.3 Nicotinic depolarization of primary afferents is independent of spinal synaptic actions...125
4.4.3.1 d-tubocurarine and mecamylamine, but not DHβE depress the direct cholinergic depolarization of primary afferents...127
4.4.4 ACh induced DC depolarization is NOT due to overlapping pharmacology with GABAA receptors...130
4.4.4.1 GABAAR mediated PAD is not
affected by d-tubocurarine...131
4.4.4.2 Cholinergic PAD is not affected by picrotoxin...134
4.4.4.3 ACh + neostigmine, but not muscimol leads to a
depolarization of primary afferents after collapse of the chloride
gradient...134
4.4.5 Immunohistochemical detection of nicotinic receptors in primary afferents...137
4.4.5.1 α3-containing nAChRs...140
4.4.5.2 α6-containing nAChRs...143
4.4.5.3 α7 nAChRs...147
4.4.5.4 α9-containing nAChRs...152
4.4.5.5 Summary of salient points regarding nAChR labeling
patterns...154
4.5 DISCUSSION...155
4.5.1 Cholinergic effects on afferent evoked DRPs, EFPs and DRRs...155
4.5.1.1 Atropine block of muscarinic AChRs does
not depress the DRP, but it does show facilitation on
washout...156
4.5.1.2 Nicotinic Antaonist effects on afferent evoked DRPs, EFPs
and DRRs...157
4.5.1.3 Nicotinic agonist depress afferent evoked DRPs and EFPs
through depolarization block of afferent signaling...163
4.5.2 ACh-induced DC depolarization is distinct
from GABAergic PAD...164
4.5.3 Significance of depolarization of primary afferents via nAChR
activation...166
4.5.3.1 Reversal potential implies nAChRs are
involved in spike generation in afferents...167
4.5.3.2 Antidromic spiking and inflammation...167
4.5.3.3 Analysis, Significance and Statistical Power...168
Chapter 5 General Discussion and Summary...171
5.1 Summary and Discussion of Key
Findings...171
5.3 Nicotinic Involvement in Primary Afferent
Depolarization...174
5.4 Roles of Nicotinic Presynaptic Inhibition versus
Facilitation...177
5.5 Implications for Therapeutic Pharmacological Control of Sensory
Integration...179
5.7 Future Directions...180
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