Chemical Transmission Between DRG Somata Via Intervening Satellite Cell

Sandwich Synapse: Chemical Transmission Between DRG Somata via Intervening Satellite cell

Hyunhee Kim

A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Physiology and Neuroscience University of Toronto

Sandwich Synapse:

Chemical Transmission between Dorsal Root Ganglion Somata via Intervening Satellite Glial Cell

Hyunhee Kim

Master of Science

Department of Physiology and Neuroscience

University of Toronto

2010 ABSTRACT

The structure of afferent neurons is pseudounipolar. Studies suggest that they relay action potentials (APs) to both directions of the Tjunctions to reach the cell body and the spinal cord. Moreover, the somata are electrically excitable and shown to be able to transmit the signals to associated satellite cells. Our study demonstrates that this transmission can go further and pass onto passive neighbouring somata, if they are in direct contact with same satellite cells. The neurons activate the satellite cells by releasing ATP. This triggers the satellite cells to exocytose acetylcholine to the neighbouring neurons. In addition, the ATP inhibits the nicotinic receptors of the neurons by activating P2Y receptors and initiating the Gproteinmediated pathway, thus reducing the signals that return to the neurons that initiated the signals. This “sandwich synapse” represents a unique pathway by the ectopic release between the somata and the satellite cells.

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ACKNOWLEDGEMENT

First and foremost, I would like to express my most sincere gratitude to Dr. Elise

F. Stanley, my supervisor. All of the work was made possible by her continuous guidance and support. Throughout the course of my study, she has taught me of scientific knowledge, critical thinking skills, the integrity required as a researcher, and most importantly the passion for scientific research. I am also grateful for her hard work needed in order to teach a girl who did not have any experience outside of school. It has been such an honour to be one of her graduate students.

I would like to thank my supervisory committee, Drs. Milton P. Charlton and

Peter Backx. I am in debt of their valuable suggestions and insightful comments that

significantly influenced this work. I am also thankful to my defense committee, Drs.

Melanie Woodin, Diane M. Broussard, and Shuzo Sugita.

A thousand thanks to all my lab members, past and present, Dr. Qi Li, Alex

Webber, Fiona K. Wong and Sabiha Gardezi, Adele Tufford, and Maria Altshuler. It was

a great learning experience for me to work with them. I appreciate all the knowledge and

techniques that they taught me. I also would like to express my gratitude to my friends

who cheered and supported me for the last two years. To my dearest friends, Wenjun,

Jinnie, Melody and Youngjoo: you guys are the best. I am also thankful to Dr.

Schlichter’s lab. I had such a wonderful time with them.

I would like to express my special love and gratitude to Mother, Father and

Namhee who have been always there for me and gave me strength to go further. I love

you, Umma. I would like to dedicate this thesis to you.

Lastly, Thank you, God, for always giving me more than I deserve.

TABLE OF CONTENTS

ABSTRACT ii

ACKNOWLEDGEMENTS iii

TABLE OF CONTENTS iv

LIST OF FIGURES vi

LIST OF ABBREVIATIONS vii

INTRODUCTION 1

PART 1: OVERVIEW OF SOMATOSENSORY SYSTEM 1 Spinal somatosensory pathways 1

PART 2: DORSAL ROOT GANGLION NEURONS 4 Anatomy of DRG 4 The functional celltypes in DRG neurons 4 Morphology of the afferent sensory neurons 5 Ectopic release from DRG neurons 6 Receptor in DRG neurons 7 Nicotinic acetylcholine receptor 7 P2X receptor 8 P2Y receptor 9 Cross excitation between DRG neurons 10

PART 3: SATELLITE GLIAL CELLS 11 Anatomy and physiology 11 The satellite cell as a protective layer of DRG neurons 12 Receptors in satellite glial cells 13 Neuronsatellite cell interactions 13 Role of the satellite cells in neuronal development and regeneration 14 Bidirectional communication between the satellite cells and the neurons 15

GOAL OF THE STUDY 19

HYPOTHESIS 20

METHODS 21 Dorsal root ganglia (DRG) dissection 21 Enzyme Treatment 21 Dissociating and plating of the DRG cells 22

Electrophysiology 22 Single patchclamp recordings 22 Pair patchclamp recording 23 Drug experiment (electrophysiology) 24 Immunocytochemistry 24 Electron Microscopy 26 Statistical Analysis 26

RESULTS 28 Presynaptic markers are localized at the junction between DRG neuron 28 The current fluctuation in a DRG neuron often increases with adjacent neuron excitation 28 Satellite cells reside in between DRG neurons. 34 DRG neuron to satellite cell transmission is purinergic (P2X receptor mediation) 36 Satellite cell to DRG neuron transmission is cholinergic 41 P2Y receptors in DRG neuron inhibit satellite to DRG neuron transmission 45 Relief of Gprotein inhibition in DRG neurons enhances neuronal crossexcitation 55

DISCUSSION 56 The chemical basis of the sandwich synapse 56 The sandwich synapse as the functional unit of DRG neuronal communication 61

REFERENCE LIST 65

LIST OF FIGURES

Figure 1: Simplified diagram of somatosensory pathway 3

Figure 2: Chemical signalling between DRG neurons and satellite cells 17

Figure 3: Hypothesis 20

Figure 4. Synaptic proteins at the junction between the DRG pair 29

Figure 5. DRG neurons display spontaneous inward current transients 31

Figure 6. Inward current transient of DRG pairs become enhanced after stimulation in adjacent neurons 32

Figure 7. Satellite cell separating neighbouring DRG neuronal pair 35

Figure 8. Miniature excitatory postsynaptic currents (mEPCs) in satellite cells. 37

Figure 9. DRG neuron to satellite cell transmission is purinergic. 40

Figure 10. Response of DRG neurons to the purinergic and cholinergic agonists. 42

Figure 11. satellite cell to DRG neuron transmission is cholinergic. 43

Figure 12. mEPClike activities appear in the neurons after the inhibition of G protein. 46

Figure 13. Neuronal mEPCs are cholinergic. 47

Figure 14. ATP applied to the satellite cells triggers mEPC activity in the neuron 49

Figure 15. Gprotein mediated inhibition of the cholinergic transmission is induced by the activation of P2Y receptors. 51

Figure 16. Role of G protein activities in the neuronalpair communication. 54

Figure 17. Schematic diagram of the Sandwich Synapse 62

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LIST OF ABBREVIATIONS

Abbreviation Full name ACh Acetylcholine ATP Adenosine triphosphate Cav2.2 Ntype calcium channel ChAT CholineOacetyltrasferase CNQX 6cyano7nitroquinoxaline2,3dione DRG Dorsal Root Ganglion DTC dtubocurarine EGTA ethylene glycol tetraacetic acid GDPβS guanosine 5'O[gammathio] diphosphate HEPES 4(2hydroxyethyl)1piperazineethanesulfonic acid MEM Minimum essential medium nAChR Nicotinic acetylcholine receptor P2XR Purinergic receptor (ionotropic) P2YR Purinergic receptor (metabotropic) PCB Pancuronium bromide RIM Rab3interacting molecule SGC Satellite glial cell SP Substance P SV2 Synaptic Vesicle Protein 2 TRP Transient receptor potential TTX Tetrodotoxin

INTRODUCTION

PART 1: OVERVIEW OF THE SOMATOSENSORY NERVOUS SYSTEM

The sensory system monitors the state of the organism and changes in its environment. It does so by using external detection via sense organs, such as touch, taste, smell, vision and sounds, and internal detection via mechanical receptors such as proprioception, pressure and temperature. The detected signal is transferred to the brain by different neuronal systems (e.g., visual or olfactory systems). Among these, the somatosensory system governs the recognition of mechanical stimulation, such as touch, temperature, nociception and proprioception (Arezzo et al., 1982).

Spinal somatosensory system pathways

Sensory information, coded as action potentials (APs), passes from the sensory endings to the somatosensory cortex in the brain via a series of neuronal relays through three orders of organization. First order neurons have their cell bodies in the dorsal root ganglion which project axons to the sense organ or ending and to the dorsal horn of the spinal cord where they synapse onto dendrites of the second order neurons. These axons ascend within the spinothalamic tract or dorsal columnmedial lemniscus to the thalamus.

The third order is composed of a neuronal network that connects the thalamus to an area of the cerebral cortex layer IV (Greenstein and Greenstein, 2000; Kandel et al., 2000;

Martini et al., 2004; Randall et al., 2002).

The first order neurons, whose endings express sensory receptors that extend across the periphery, relay the information to the central nervous system as AP trains.

The nerve endings are equipped with mechanoreceptors that transform the physical 2

stimulus to electrical impulses (Greenstein and Greenstein, 2000). Two of the most

common mechanoreceptors are Meissner’s corpuscles and Pacinian corpuscles (Smith,

2008). Deformation of the corpuscle opens Na + channels in the nerve terminal, bringing

up membrane potential to threshold and generating APs (Greenstein and Greenstein,

2000). The APs travel along the afferent axons within the peripheral nerve, through the

dorsal roots to the nerve terminals within the spinal cord. The nerve fibers that carry the

APs to the spinal cord differ in diameter and morphology, with size classes that are

related to sense organ functions. There are two main types of sensory axon: myelinated

“A” type, and the slowly conducting, unmyelinated “C” type. The A type can be further

subdivided into three subtypes: Aα, Aβ, Aγ in order of diameter and conduction velocity,

where the Aα fibers are the largest and fastest (Greenstein and Greenstein, 2000; Lawson

et al., 1993; Lawson, 2002). The nerve terminals of the afferent neurons eventually

transmit the signals to the laminae I to IV of the gray matter. The different fibers

innervate different regions (Smith, 2008).

After reaching the spinal cord the signal generally ascends to the ventral posterolateral (VPL) nucleus in the thalamus. However, some fibers project into the

ventral root that induce muscle reflexes (Fig. 1; Martini et al., 2004). The APs from the

VPL nucleus eventually pass the posterior limb of the internal capsule and reach the postcentral gyrus. This region of the cerebral cortex is termed the somatosensory cortex

and functions to interpret the stimulus and its source. (Fig 1; Kaas, 2004). Each area in

the gyrus is assigned a particular area of the body, which can be visualized in a map

called the sensory homunculus (Kandel et al., 2000; Martini et al., 2004; Randall et al.,

2002). 3

Figure 1. Simplified diagram of somatosensory pathway . APs generated from the sensory ending travel along the axon of the primary afferent neuron (the first order) and enter the spinal cord. The signal is either transferred onto the cell bodies in the spinal dorsal horn or in the medulla (the second order). Signals eventually pass through the ventral posterolateral nucleus of the thalamus (the third order) and reach the somatosensory cortex where their information is processed (Greenstein and Greenstein,

2000; Kandel et al., 2000; Smith, 2008).

PART 2: DORSAL ROOT GANGLION (DRG) NEURONS

Anatomy of DRG

The dorsal root ganglion (DRG) contains the somata of the afferent neurons. It is located beside each of the spinal segments, forming a nodule at the end of the dorsal root.

The spine is divided into five compartments: 7 cervical vertebrae, 12 thoracic, 5 sacral

(which are fused to form a sacrum) and 4 coccygeal, of which the last 3 vertebrae are fused (Martini et al., 2004). Each of the spinal segments has a pair of ganglia on the lateral sides at the dorsal roots (DR). There are 31 pairs of ganglia in total (Martini et al.,

2004). Each of the segments also has the ventral motor root containing motor neurons that control the muscles. Each DRG innervates a region in the body from which they can gather the sensory information, which generally overlap. The region in the body that is assigned each DRG is called a “dermatome” (Greenstein and Greenstein, 2000).

The neurons in the DRG ganglia originate from the neural crests that gave rise to the peripheral nervous system (Frank and Sanes, 1991). The neuron somata in the DRG have no incoming synapses and it is generally believed that their primary role is to maintain their axonal trees and nerve endings (Martini et al., 2004). Approximately

99.8% of the cytoplasmic and intracellular substances are in the axons and nerve terminal, while only 0.2% are in the somata (Devor, 1999). Each neuron is almost completely enclosed by ‘satellite glial cells’, specialized glia that can be found in ganglia (Lieberman,

1976; Matsumoto and Rosenbluth, 1986).

The functional cell-types in DRG neurons 5

The most common method of classification for somatosensory neurons is by their

‘modality’ or specific sensation. Modality is mainly divided among three types:

nociception, thermoreception and mechanoreception (Arezzo et al., 1982; Lieberman,

1976) which process the information along different pathways. The nociception and

thermoreception are processed through anterolateral system (ALS), while the latter is

handled by the dorsal columnmedial lemniscal system (Greenstein and Greenstein,

2000). The size of the neurons is a simple characteristic that can be used to categorize the

neurons by their functions and varies from 10 to 50 m. Small neurons (10 to 20 m) are

considered to be nociceptive while large neurons (30 to 50 m) are mechanoreceptive

(Lawson et al., 1993; Mense, 1990). The nociceptive neurons look darker when observed

under electron microscopy, due to concentrated Nissl substance in the plasma (Rambourg

et al., 1983). It has been suggested that their specific roles can also be identified through

other characteristics, such as velocity of APs, the diameter of the axons, and the presence

of myelinating Schwann cells wrapping around the axon (Lawson, 2002).

The morphology of the afferent sensory neurons

The morphology of DRG neurons is unique, in which a single axon generated from the soma bifurcates into two processes; one of the processes projects to the peripheral nerve endings while the other enters the spinal cord (Edward, 1992). This form is termed “pseudounipolar” and the location where the axon is bifurcated is called a T junction (Coleman et al., 2003). The APs travel directly from nerve ending to spinal cord and also branch up the T junction into the soma (Devor, 1999). The soma is excitable 6

and produces APs which may influence how the neuron maintains its neuronal structures

and other functions (Fields et al., 1997).

Ectopic transmitter release from DRG neurons

The somatosensory neurons release specific neurotransmitters from their nerve

endings in the spinal cord by typical calciumgated mechanisms. These transmitters

include ATP, substance P (SP), glutamate, somatostatin, vasoactive intestinal polypeptide

(VIP), and galanin, and the specific type is related to their modality (Lawson, 1992).

Moreover, the neurons release several transmitters from the sensitive endings in the periphery (Holzer, 1988; Szolcsanyi, 1988; White, 1997).

Interestingly, they are also capable of releasing transmitters from the soma. This

is termed ectopic release, which is any release occurring outside of the synaptic cleft

(Huang and Neher, 1996; Matsui and Jahr, 2003). They cannot, however makes

synapses with each other within the ganglion due, perhaps, to their individual coatings of

satellite glial cells. Transmitter release is involved in communication between the neuron

and its satellite cells sheath (Zhang et al., 2007b).

The neurons have representatives of all three classes of voltagegated calcium

channels (L, N and T; or Ca V1, Ca V2 and Ca V3) that are involved in a number of calcium mediated intracellular signalling pathways (Forscher et al., 1986; Fox et al., 1987; Hilaire et al., 1996; Kostyuk et al., 1993; Wen et al., 2010). Calcium influx triggers the release of neurotransmitters, such as ATP and SP (Huang and Neher, 1996; Zhang et al., 2007).

Previous studies showed that the release of neurotransmitter can be inhibited by nimodipine (Ltype calcium channel blocker) and botulinum toxins (the neurotoxin from 7

Clostridium botulinum that cleaves SNARE proteins, that mediate transmitter vesicle

fusion), while Bay K8644 (a calcium channel agonist) facilitates the release (Singh, 2006;

Welch et al., 2000).

Receptors in DRG neurons

DRG neurons express many types of receptors, despite the fact that they do not form

synapses with other neurons. These include: 5HT, purinergic (both P2X and P2Y),

cholinergic (both nicotinic and muscarinic), neurokinin, opioid, GABA receptors, and

transient receptor potential (TRP) channels (e.g., vanilloid receptors), (Ault and

Hildebrand, 1994; Borvendeg et al., 2003; Lawson, 1992; Nakamura and Strittmatter,

1996;Obata et al., 2005; Petruska et al., 2000b; Szucs et al., 1999; Zhang et al., 2007a). It

is unclear why these receptors are present in the neurons. However, studies show that

there is a correlation between the receptor expression and the modalities of the neurons,

since it is known that isolectin B4, calcitonin, P2X, vanilloid, and neurokinin receptors

are only expressed in the nociceptive neurons (Aoki et al., 2004; KaiKai, 1989).

Here are the details of some of the receptors that will be discussed in later

sections of this thesis.

Nicotinic acetylcholine receptor (nAChR): Nicotinic receptors are a ligand gated pore activated by ACh and are widely expressed in both the CNS and PNS at neuronal synapses and at neuromuscular junctions. Neuronal nAChR are divided into two classes: homopentameric and heteropentameric. The former is sensitive to α bungarotoxin and highly permeable to calcium, while the latter is not (Fucile, 2004). 8

Each receptor has five transmembrane subunits that are arranged in the shape of a ring to

form a pore in the middle, with a total molecular weight of 290 kDa (Unwin, 2005). The

subunits are categorized into four types, and they are denoted as α, β, γ, and δ (Itier and

Bertrand, 2001) in order of increasing molecular weight. So far, 17 nicotinic subunits

have been identified (Itier and Bertrand, 2001). DRG neurons express a variety of these

nAChR subunits (α2–7 and β2–4) (Genzen et al., 2001; Fucile et al., 2005).

While ACh is the endogenous neurotransmitter that activates AChRs there are a

large number of known agonists, including nicotine or carbachol. In order for the pore to be opened, the receptors have to be bound to two agonist molecules concurrently. In the

heteropentameric receptors found in neuromuscular junctions ((α1) 2β1δγ), the binding sites are located at the junction between α and β subunits (Galzi et al., 1991).

The receptors may be associated with the transmission of pain, because Ca 2+ influx through the receptor negatively modulates vanilloid receptors and that neuropathic rats after axotomy lose functional nAChR channels in their DRG neurons (Dube et al.,

2005; Fucile et al., 2005).

P2X receptor: This receptor is a ligandgated ion channel that can be found in skeletal and smooth muscles, and sensory neurons (Petruska et al., 2000a; Ralevic, 2002).

The channel is known to be composed of three transmembrane subunits (trimer) that are joined to form a pore (Barrera et al., 2005; Nicke et al., 1998). Each of the subunits is composed of two transmembrane domains with an extracellular loop and cytoplasmic C and N terminals (Khakh, 2001). So far, 7 subunits have been discovered, and they are numbered from 1 to 7 (e.g., P2X 1 subunit). The ligand binding site is speculated to be 9

located in the extracellular loop, but the specific location is different between the

receptors (Evans, 2009). Moreover, the degree of difference of the binding site between

the subtypes correlates to their affinity to ATP (Evans, 2009).

P2X receptors are present in primary afferent neurons (Petruska et al., 2000a).

The expression of the P2X receptors is restricted to nociceptors, and the activation of the

receptor can trigger the release of substance P in the nerve endings in the dorsal horn,

suggesting that the receptors play a role in the transmission of pain (Nakatsuka et al.,

2001). Recent studies show that P2X receptors are also present in the enveloping satellite

cells (Zhang et al., 2007b).

P2Y receptor : This receptor is a ligandgated Gproteincoupled receptor that can be found in almost any part of the body, from organ tissues to bone and blood (Moore et

al., 2001). Like all of the Gproteincoupled receptors, it consists of 7 transmembrane

domains (Abbracchio et al., 2006). Ten P2Y receptor types have been identified, with 8

of them found in mammals. All of them can be activated by ATP, ADP, UTP, or UDP,

while their sensitivities to each of the agonists are different (Simon et al., 1995).

Mutagenesis studies suggest that the positively charged motif in transmembrane domains

3, 6 and 7 and the second extracellular loop play a crucial part in nucleotide binding and

activation of the receptor (Erb et al., 1995; Jiang et al., 1997). The Gprotein binding site

is located in the Cterminal of the receptor. The different receptor types activate different

Gprotein types (Gαs, Gαi, and Gαq/11) (Abbracchio et al., 2006).

P2Y 1, P2Y 2 and P2Y 4 receptors are found in the DRG neurons (Kobayashi et al.,

2006; Ruan and Burnstock, 2003). The activation of the P2Y 1 receptor leads to inhibition 10 of the P2X receptors as well as the voltagegated calcium channels, which leads to the inhibition of calciummediated exocytosis in the neurons (Gerevich et al., 2007; Gerevich et al., 2004). The receptor further reduces the concentration of intracellular calcium by activating the plasma calcium pump (Usachev et al., 2002). On the other hand, the P2Y 2 receptors potentiate vanilloid receptor activity, and modulate the gene expression by initiating the phosphorylation of CREB (Molliver et al., 2002; Moriyama et al., 2003).

Cross-depolarization between DRG neurons

The findings that DRG neurons exocytose neurotransmitters and also express receptors that are sensitive to these ligands provide at least the elements for inter neuronal, activitymediated signalling. This certainly seems possible, since the ligand gated receptors in the neurons match with the neurotransmitters they release (Holz et al.,

1988; Petruska et al., 2000b; Szucs et al., 1999; Zhang et al., 2007b). Devor and Wall

(1990) examined the possibility of communication between the neurons and showed that retrograde APs generated from a section of DR which enter the neurons depolarize the adjacent passive neurons in the same ganglion. This activity is termed “cross depolarization.” They suggest that while this depolarization itself is not strong enough to reach AP threshold, it may sum with other depolarizing activity to generate AP discharge

(Amir and Devor, 1996). Interestingly, this phenomenon is greatly enhanced after nerve injury. After the injury, the neurons spontaneously fire APs, and generate APs in the neighbouring neurons by crossdepolarization (Blumberg and Janig, 1982; Devor and

Dubner, 1988; Lisney and Pover, 1983). However, ultrastructural studies argue against these ideas; satellite cells wrap each of the neurons individually, preventing them from 11 having direct contact with each other (Lieberman, 1976; Matsumoto and Rosenbluth,

1986b; Shinder et al., 1998). To account for this disparity Devor et al. suggested that the neurotransmitters released by the neurons escape through the space between the satellite cells to activate the neighbouring neurons (Amir and Devor, 1996).

PART 3: SATELLITE GLIAL CELLS

Anatomy and physiology

Satellite glial cells are a type of glial cell found in a peripheral ganglion. The

satellite cells form a thin sheet that ensheathes each of the neurons (Matsumoto and

Rosenbluth, 1986). Each of the neurons has one or more satellite cells that are

functionally coupled by gap junctions, forming a structural unit (Pannese, 2010). They

wrap either as a single layer or in multiple layers and the thickness of the sheath varies between ganglia. Their pooled volumes of the satellite glia correlate with the volumes of

the associated neurons (Pannese, 2010). Satellite cells that envelop more than one neuron

are uncommon (Pannese et al., 1991).

Like neurons, satellite cells are derived from the neural crest (Frank and Sanes,

1991). A study of satellite cell topology suggests the presence of a bipotent precursor that

differentiates into either myelinating Schwann cell or satellite cell, implying that

Schwann cells have the closest linkage to satellite cells in terms of evolutionary

relationships (Baroffio et al., 1991). However, the protein expression of satellite cells is

noted to be similar to that of astrocytes, suggesting that they are similar in function

(Hanani, 2005). Interestingly, the protein expression of the satellite cells in the DRG and 12 sympathetic ganglia in chicks become different at stages as early as embryonic day 6, supporting the idea that they have distinctive properties (Rudel and Rohrer, 1992).

The satellite cells as the protective layer of the neurons

The earliest known functions of satellite cells include forming a protective barrier around the neurons and the uptake of neurotransmitters released from the neurons, consistent with all peripheral glial cells (Hanani, 2005). Because the neurons are not protected by a blood–brain barrier, they are exposed to harmful substances in the bloodstream. However, studies suggest that satellite cells function as a protective barrier.

In extreme situations, like mercury poisoning, satellite cells become more heavily labeled

with the toxic substance than do the neurons, indicating that the satellite cells can serve to protect the neuron (Kumamoto et al., 1986).

In common with almost all glial cells, satellite cells take up neurotransmitters that

have been secreted from the neurons. The most commonly expressed protein in the

satellite cells is glutamine synthetase, whose function is to convert glutamate to

glutamine (Tsacopoulos, 2002). Only satellite cells express this protein in the ganglia,

and its primary role is to eliminate glutamate molecules after satellite cells have them up

from the vicinity of the neurons (Miller et al., 2002). Glutamate transporters are also

found in satellite cells, further suggesting that they reduce glutamate in the intercellular

space that could lead to unwanted signaling in the neuron at a high concentration (Kugler

and Schmitt, 1999).

The receptors in satellite glial cells 13

Like neurons, satellite cells are known to express a variety of ligandgated receptors. So far, the identified receptors are cholinergic (muscarinic), purinergic (P2X and P2Y), endothelin, bradykinin, NGF (trkA and p75), and somatostatin receptors (Bar et al., 2004; Bernardini et al., 1998; England et al., 2001; Holz et al., 1988; Pannese and

Procacci, 2002; Pomonis et al., 2001). Since only a few studies have been done on receptors in satellite cell, the biological significance of these receptors remains unclear.

However, recent studies have shown that some receptors are activated by neurotransmitters released by the neurons. (Thippeswamy et al., 2005; Zhang et al.,

2007b).

Neuron–satellite cell interactions

Glial cells play a crucial part in many neuronal functions, such as development, regeneration and synaptic plasticity. The interactions between interneurons and astrocytes, and neuromuscular junctions and perisynaptic Schwann cells.

Glial cells often make physical contact with a synapse and can enclose the entire structure. They recognize the signals in the synapse and modulate its activities by releasing hormones and gliotransmitters (Jahromi et al., 1992). Glial cells are now viewed as a functional unit of the synapse; pre and postsynaptic neurons and their associated glial cells are collectively denoted as “tripartite synapse” (Feng and Ko, 2008;

Halassa et al., 2009).

Like other glial cells, satellite cells have functions other than protecting the neurons and cleaning up neurotransmitters. The surface of the neuron forms microvilli that increase the surfacetovolume ratio (Lieberman, 1976). This was thought to increase 14 the exchange rate of macromolecules. However, EM images of satellite cells show that the surfaces of the satellite cells fill in the invaginations of the microvilli, dramatically increasing their surface contact with the neurons (Pannese, 2002). This suggests another possible functionality of the villi, which is to increase the surface area for the neuron glial interactions.

The role of satellite glial cells in the development and repair of neurons

Numerous studies show that the glial cells are important modulators of neuronal development and regeneration. In the CNS, astrocytes become activated in response to neuronal damage and secrete neurotrophins and cytokines that could be either beneficial or detrimental to the survival of the neurons (Halassa et al., 2007). Also, in the neuromuscular junction, the sprouting during the regeneration of motor neurons after axotomy is preceded by perisynaptic Schwann cells that ultimately enwrap the entire junction (Koirala et al., 2000). Ultrastructural studies show that Schwann cells react dramatically after axotomy by proliferating and producing NGFs (p75), a polypeptide growth factor that dictates growth and regeneration of neurons (De et al., 1993).

Moreover, the ablation of perisynaptic Schwann cells can induce the retraction of both fully developing and mature motor nerve terminals, indicating that perisynaptic Schwann cell is essential to maintaining neuromuscular junction (Reddy et al., 2003).

Studies suggest that satellite cells also undergo major reorganization when their associated neurons are damaged. In response to damage, satellite cells upregulate the expression of GFAP and proliferate. Also, the electrical signaling between them is 15 augmented by an increased number of gap junctions (Humbertson, Jr. et al., 1969;

Pannese et al., 2003).

The NGF receptors are found in both the neurons and satellite cells. In situ hybridization has suggested that growth factor is made in the satellite cells, not the neurons (Gill and Windebank, 1998). After axonal injury, the production and secretion of

NGF is increased in the ganglion (Lee et al., 1998). The level of NGF production relapses back to the basal level just before full recovery of the neurons, suggesting that the increase in secretion is related to one of the repairing mechanisms for the neurons (Lee et al., 1998). A study further suggests that satellite cells can downregulate neuronal growth.

Isolated ganglion neurons that are treated with NGF can grow dendrites, but these are prevented when cocultured with satellite cells (De et al., 1993). This indicates that the satellite cells not only secrete NGF, but also control the NGFinduced pathway. The mechanism of this regulation remains unclear.

Bidirectional communications between DRG neurons and satellite cells

Several findings support the idea that glial cells and neurons influence each other’s function. In the CNS, glutamate that is released from neurons can activate the astrocytes and initiate calcium signaling (Neary et al., 1988). In return, astrocytes release

ATP and Dserine to modulate neuronal excitation and synchronicity (Hamilton and

Attwell, 2010; Haydon and Carmignoto, 2006; Henneberger et al., 2010). At the neuromuscular junction, stimulation of a presynaptic neuron leads to an increase in intracellular calcium in its perisynaptic Schwann cell (Jahromi et al., 1992; Rochon et al.,

2001; Rousse and Robitaille, 2006). Findings further suggest that ATP released from a 16 presynaptic terminal raises intracellular calcium concentration in the perisynaptic

Schwanns cell by activating P2X and P2Y receptors (Jahromi et al., 1992; Robitaille,

1995; Robitaille et al., 1997). The transmission can also go from Schwann cell to neuron, since Gprotein receptor activation on Schwann cells is effective in reducing low frequency synaptic transmission in neuromuscular junctions (Robitaille, 1998).

The DRG neuron–satellite cell communication pathways that have been described in detail so far are the nitric oxide (NO) and ATP pathways. NO is a transduction molecule that initiates many important second messenger pathways in the body by activating guanylate cyclase and directing the synthesis of cyclic GMP (cGMP) (Ignarro,

1990). NO is a common molecule found in peripheral nerve tissues (Aoki et al., 1993).

DRG neurons express NO synthase, while satellite cells have guanylate cyclase that can be activated by NO (Magnusson et al., 1996; Magnusson et al., 2000). After nerve injury, cGMP production increases in the satellite cells (Fig. 2; Magnusson et al., 2000; Shi et al.,

1998). The blocking of NOcGMP pathway in the injured neurons and the satellite cells can induce apoptosis of both cells, suggesting that this pathway is crucial to their survival

(Fig. 2; Thippeswamy et al., 2001). This pathway is also implicated to be linked to nociception.

It has also been reported that DRG neuron–satellite cell can communicate via

ATP and TNFα, respectively. DRG neurons release ATP via exocytosis and activate

P2Y 7 receptors on the satellite cells. This triggers the production and release of TNFα from the satellite cells (Fig. 2; Zhang et al., 2007b). Another study indicates that TNFα can initiate the production of substance P, suggesting that this pathway is functionally important in the transmission of pain (Fig. 2; Ding et al., 1995). 17

Figure 2. Chemical signalling between DRG neurons and satellite cells. At present, two

types of communication have been described. Injured DRG neuron produces enhanced

level of nitric oxide (NO) which activates guanylate cyclase and generates cGMP in the

satellite cells. The cGMP initiates the production of neurotrophins. The release of the

neurotrophins from the satellite cell supports the survival of the neurons. The other pathway begins as ATP is released from DRG neurons and activates the P2X receptors in

the satellite cells. In return, the satellite cells releases TNFα and triggers the production

of substance P in the neurons (Nakatsuka et al., 2001; Thippeswamy et al., 2005; Zhang

et al., 2007b).

Similar to the crossexcitation between DRG neurons, the communication between satellite cells is enhanced by nerve injury (Suadicani et al., 2010). Studies using an inflammation model suggest that the intensity of the calcium signaling between satellite cells is increased twofold (Hanani et al., 2002; Suadicani et al., 2010). Moreover, the satellite cells of injured neurons proliferate and wrap the neurons in several layers like an onion bulb, making it harder for the neurotransmitter from the neurons to diffuse and activate the neighbouring neurons (Shinder et al., 1999). At the same time, cross depolarization between the neurons becomes strong enough to generate APs on the neighbouring cells (Blumberg and Janig, 1982; Devor and Dubner, 1988; Lisney and

Pover, 1983). This suggests that there might be a more efficient route for cross depolarization than simple diffusion, as suggested by Devor and Amir (1996).

GOAL OF THE STUDY

This thesis covers the interactions between DRG somata and glial cells that eventually allow the neurons to transfer signals to other somata. Other studies have indicated that a signal can be transferred in a bidirectional fashion between the neuron and its associated satellite cells, but it is not clear that the signal would also affect the neighbouring neurons that make a physical contact with the satellite cells. The purpose of the study is to find out if somata can transfer their signals to other somata via the satellite cells.

HYPOTHESIS

I hypothesize that the neurons communicate with each other via the intervening satellite cells. DRG neurons activate satellite cells by releasing ATP. In response, the satellite cells activate the neighbouring neurons by releasing ACh.

1. A DRG neuron exocytoses neurotransmitters (ATP?) to activate a

associated satellite cell

2. The satellite cell releases neurotransmitters to activate other associated

DRG neurons

Figure 3. Hypothesis

EXPERIMENTAL PROCEDURE

Dorsal root ganglia (DRG) dissection

The DRG neuron isolation procedure is identical to a previous study described by

Chan and Stanley (2003) and Stanley (1989). A 13, 14, or 15day embryonic chick was extracted from an egg. The chick was decapitated and its DRG were dissected from the lumbar area under a dissection microscope (TYP 355110; Wild; Heerbrugg, Switzerland; magnification range from X10 to X45), using two pairs of fine forceps. Five to six of the ganglia were dissected from the chick and incubated in MEM (GIBCO) mixed with 5 g/L of glucose Connective tissues and axons were removed from each ganglion with a pair of fine scissors. Then, the ganglia were transferred into a culture dish containing 500 µL of

MEM, and nicked with thin needles to allow the dissociating enzymes to enter the ganglia and break down their connective tissues.

Enzyme treatment

30 µL of 10 mg/0.16 µL hyaluronidase (Worthington, 499 U/mg), 30 µL of 2.0 mg/20 µL of trypsin inhibitor type IIO (SigmaAldrich, chicken egg white), 300 µL of

0.6 mg/500 µL of collagenase IV (Worthington, 351 U/mg), and 400 µL of 20.0 mg/500

µL of dispase II (Roche, 0.98 U/mg) were added to the culture dish. The dish then was transferred to a 36°C/8% CO 2 incubator and was incubated for around 1 hour and 15 min

(Stanley, 1989).

Hyaluronidase breaks down hyaluronic acid, which is one of the major components of connective tissues within neural tissues (Ludoweig et al., 1961).

Collagenase IV breaks down collagen, the other major component of connective tissues, 22 while minimizing the possible damage to transmembrane proteins by having a low amount of trypsin (Tryggvason et al., 1992). Trypsin inhibitor suppresses any residual trypsic activities from the collagenase (Baintner, Jr. and Feher, 1974). Dispase dissociates neurons as effectively as trypsin, while having higher cell viability (Frangakis and

Kimelberg, 1984). All of the enzymes are active at 36°C.

Dissociating and plating the DRG cells

The ganglia were washed in fresh MEM warmed to 36°C. The ganglia were then drawn up with 165 µL of MEM by a 200 µL micropipette. They were then relocated to an

Eppendorf tube and triturated using the micropipette.

Six glass coverslips (Fisher Scientific) were cleaned with 99% acetone, and 25 µL of MEM containing the dissociated cells were plated onto each coverslip and transferred back into the incubator. After 1 hour, when most of the cells had settled and bound to the coverslip, the cells were rinsed again with 30 µL of MEM. The plated cells were held in an incubator at 22ºC/8% CO 2 and used within 1 or 2 days.

Electrophysiology

Single patch recordings

The standard patchclamp technique was used to record the wholecell current of the neurons and satellite cells. The external (bath) and internal solutions were similar to the solutions used for wholecell calcium channel currents; the external solution was (in mM): NaCl, 130; CaCl 2, 2; MgCl 2, 0.8; Dglucose, 5; TTX, 0.001; HEPESNa, 10; NiCl2,

1; and the patch electrode internal solution was: Csgluconate, 120; CsCl, 10; EGTACs, 23

10; MgCl 2, 1; HEPESCs, 10; MgATP, 2. The osmolarity of external and patch electrode solutions were adjusted to 310 and 315 mOsmol, respectively and the pH of both solutions was adjusted to 7.4. The coverslips were removed from the incubator and inserted into a Leiden chamber to which 400 µL of external solution at room temperature was added. Glass electrodes were pulled using Sutter Instrument Model P87

Micropipette Puller and filled with the internal solution as above. The tips of the electrodes were firepolished, yielding tip resistances of 3–6 M for neuron recording and 5–10 M for the much smaller satellite cells. The wholecell current signals were obtained from amplifier Axopatch 200A (Axon Instruments). The current signals obtained were digitized and transferred onto computer by Digidata 1330 (Axon

Instruments), and were recorded onto pCLAMP 9.5 software (Axon Instruments). All samples were sampled at 20 µs or 100 µs, and filtered at 2 kHz or 5 kHz, depending on the objective of the each experiment. The obtained traces were filtered again at 960 Hz for display in the figures.

The mEPCs were selected using semiautomated software, AxoGraph X. A peak that fits into the typical morphology of mEPCs (sharp onset and slow decay) was selected from trace and set as the template. The program scanned the whole trace to find all peaks that were similar to the template. Then each peak was analyzed by eye to ensure that they have met the criteria.

Pair patch-clamp recordings

Wholecell currents of either DRG neuron pairs or satellite cell–DRG neuron pairs were recorded simultaneously. The bath solution described as above was used. As 24 for the patch electrode solution, all of the components and their concentrations were identical except the concentration of EGTACs, which had to be decreased to 0.1 mM to attenuate Ca 2+ chelation and allow calciummediated exocytosis to occur in the cells. The

current signals were obtained from amplifier Axopatch 200A and Axopatch 200B (Axon

Instruments). The obtained signals were digitized and transferred onto computer by

Digidata 1330 (Axon Instruments), and were recorded onto pCLAMP 9.5 software (Axon

Instruments). All samples were sampled at 200 µs and filtered at 2 kHz.

Drug experiment (Electrophysiology)

Pancuronium bromide (PCB), dtubocurarine (DTC), suramin, CNQX, NBQX,

ATP, and carbachol were used to identify the neurotransmitters involved in this

communication, while suramin and GDP βS were used to look at the effect of P2Y receptor activity.

The drugs were administered either by adding to the bath solution or by focal application directly onto the target cell with a micropipette. This micropipette was attached to a manipulator (Sutter Instrument, MP285) so that it could be accurately positioned close to the target cell. The drug solution was either added into the bath using

a P100 micropipette or expelled by positive pressure from an attached syringe. The

antagonists (PCB, DTC, suramin, CNQX and NBQX) were administered via the bath

application, while the agonists (ATP and carbachol) were perfused focally onto the target

cell using the micropipette.

Immunocytochemistry

Immunocytochemistry was carried out by Dr. Qi Li. The DRG neurons were extracted from E15 chicks, and dissociated and plated as described above. The staining procedure was as previously described (Li et al., 2004).

Fixation: The fixative base was made with (mM): cyclohexylamine, 300; EGTA,

40; MgCl 2, 40; PIPES, 40. Then the fixative was made by combining 1.0 ml of the fixative base, 0.8 ml of paraformaldehyde and 0.2 ml of doubledistilled water. After removing the MEM from the coverslip plated with the dissociated neurons, 100 L of the fixative was added onto the cells. The cells were left in a fume hood for 40 minutes.

Permeabilization: The permeabilizing solution was made by adding 1.0 ml of

fixative base, 0.6 ml of doubledistilled water, 0.2 ml of Brij (5%) and 0.2 ml of paraformaldehyde (5%). Then 100 L of the permeabilizing solution was added to each

coverslip. The coverslips were again left in a fume hood for 10 minutes.

Blocking: The fixation was then stopped with Tris buffer (THAM) containing (in

mM): Tris, 150; MgCl 2, 20; and NaN 3, 20. The cells were incubated with the buffer for

40 minutes and then again with Ab buffer containing (in mM): NaCl, 500; MgCl 2, 10;

NaN 3, 10; and Tris, 20; with BSA 0.1% w/v for at least 40 minutes. Donkey serum

diluted to 5% with the Ab buffer was added to block nonspecific bindings.

Primary antibody: The primary antibodies were diluted with the Ab buffer as

follows: SV2 (mono, Hybridoma Bank), 1/1; Ab571 (poly, Stanley lab), 1/200; RIM

(Synaptic Systems), 1/500; and ChAT (poly, Chemicon), 1/100. The cells were incubated

with the primary antibody containing Ab buffer overnight at 4ºC. 26

Secondary antibody: To add the secondary antibodies, the cells were washed with

Ab buffer. Then the cells were again incubated in the diluted donkey serum to prevent nonspecific bindings. The secondary antibodies (labelled with Texas Red and FITC) were diluted at a ratio of 1/100 with Ab buffer. The cells were incubated with the secondary antibodies for 1 hour.

Lastly, the cells were washed with the Ab buffer and mounted on slides with a mounting oil to be imaged under a microscope (Zeiss Axioplan upright microscope).

Electron Microscopy Preparation

Electron Microscopy was carried out by Fiona K Wong. Glutaraldehyde fixative was prepared by combining either 10 mL 8% glutaraldehyde, 13.33 mL 0.2 M PO 4 buffer and 3.33 mL distilled water; or 5 mL 8% glutaraldehyde, 6.66 mL 0.2 M PO 4 buffer and

1.66 mL distilled water. The dissected chick DRGs were incubated overnight in the fixative on a rotator. The DRG were washed with 0.15 M PO 4 buffer and osmicated with

1% OsO 4 in 0.15 M PO 4 buffer for 1 hour. Then, they were washed again in 0.15 M PO 4 buffer.

The DRG were dehydrated with a series of solutions containing ethanol of

ascending concentration (in percentage): 30%, 50%, 70%, 80%; 90% and 100%. They

were immersed twice for 5 minutes up to 90%, and three times for 30 minutes in 100%

The DRG were infiltrated with a series of ethanol and epoxy/resin mixtures

containing different ratios (3:1, 1:1, and 1:3) for 30 minutes each. Then the ganglia were

treated with 100% resin. The ganglia were put in 100% resin in BEEM capsules and

heated in a 65ºC oven to be polymerized. 27

The ganglia were stained with lead acetate and cut into thin sections with a slicer to be observed under the electron microscope.

Statistical analysis

The data are presented in mean±standard error (SE). P values are calculated by oneway ANOVA, paired ttest, and the nonparametric Wilcoxon signedrank test, as specified.

RESULTS

Presynaptic markers are localized at the junction between DRG neurons

DRG neurons were dissociated as previously described (Chan and Stanley, 2003;

Stanley, 1989). After trituration, most of the neurons were completely isolated, but some were clustered in pairs or triples. The neurons were almost always associated with one or more satellite cells.

Previous studies (Huang and Neher, 1996; Zhang et al., 2007) suggested that the

DRG neurons are able to secrete neurotransmitters by vesicle release. The dissociated

DRG cells were immunostained with antibodies against SV2 and RIM, or SV2 and voltagegated Ntype calcium channels (Ca V2.2; Ab571), which are presynaptic proteins, to see where the synaptic proteins were located (Fig. 4; n=18; Li and Stanley). Staining of isolated neurons localized puncta of SV2 and Ca V2.2 within the cytoplasm and also close

to the surface membrane through out the neuron. However, in 15 out of 18 paired neurons

staining was polarized to the junction between the pairs (white arrow), suggesting that

transmitter releasing sites were localized to this area.

The current fluctuation in a DRG neuron often increases with stimulation on an

adjacent neuron

DRG neurons express various ligandgated channels, suggesting that they can be

excited by neurotransmitters (Borvendeg et al., 2003; Fucile et al., 2005; Huang et al.,

2008; Liu et al., 2009; Nicholson et al., 2003; Ohtori et al., 2006; Petruska et al., 2000).

To test whether the neurons receive chemical signals from the neighbouring cells, the

wholecell currents were recorded from dissociated neurons that still remained as part of 29

Figure 4. Synaptic proteins at the junction between the DRG pair.

Representative image of a DRG neuronal pair coimmunostained with antibodies against

RIM (blue) and SV2 (green; Li and Stanley, unpublished). The proteins were concentrated at the junction between the pairs.

30 a cluster. When the neurons were voltage clamped at −80 mV, which is a membrane potential that would not activate the majority of voltage gated channels, we recorded spontaneous inward current fluctuations (Fig. 5A, top and bottom; n=8). The fluctuations were inward and rapidly activating and deactivating. However, an examination of the fluctuations failed to identify activity any that could be attributed to miniature excitatory currents (mEPCs) as would be predicted at a synaptic contact.

We next tested if inward currents could be transmitted from one neuron to its associated neighbour. To test this idea we carried out simultaneous voltage clamp of neuron pairs (Fig. 6A; n=17). Both neurons were held at 80 mV and one was stimulated with a train of 500, 2 ms step depolarizations to 20 mV (Fig. 6A, top). The frequency of the stimulation (50Hz) is a little higher than the frequency of spontaneous discharge in the afferent neuron after spinal injury (36.23±6.63Hz), yet lower than the frequency after ischemia (120~260 Hz) (Kayaalp and Smith, 1982; Su et al., 2009)

Current fluctuations were compared before and after the stimulation in both the stimulated and passive neurons. We termed the stimulated neurons and their partners as cis and transneuron, respectively. We observed an increase in current in 13 out of the 14 neuronal pairs (Fig. 6A, middle and bottom; n=14). These fluctuations did not begin until

1 or 2 seconds after the 10second stimulation was ended but increased thereafter. The changes in amplitude of the current fluctuations were quantified by calculating the average ion influx per second. By integrating the current fluctuation over time, 5second periods of the ion influx of the cis and transneurons were calculated before,

2pA 2sec

2pA 0.05sec

Figure 5. DRG neurons display spontaneous current inward transients.

Representative trace of DRG neurons at resting potential (−80 mV) (top and bottom; n=11). The DRG neurons exhibit spontaneous inward current fluctuations. Current voltage curve of DRG spontaneous fluctuations (right).

Figure 6

Figure 6. Current inward transients of DRG neuronal pairs become enhanced after stimulation in adjacent neurons. (A) Current traces showing the dual patchclamp recordings of DRG neuron pair. One of the pairs was given a train of 500 depolarization steps that had duration of 2 ms (top). Both the stimulated (middle) and its partner neurons

(bottom) displayed the increase in current fluctuation after the stimulation. (B) Current

integral analysis of the cis and transneurons. The lineseries graphs of the cis (left) and

trans (right) neurons, showing the ion influx of the individual neurons before and after

the stimulation. Four of the six pairs (pair 1, 4, 5 and 6) showed greater response in trans

cell immediately after the stimulation. Increased current fluctuations in the transneuron

(*) during stimulation is an artifact created by the pulses given to the cisneuron. (C) The

average ion influx rate of cis (left) and trans (right) neuron. In the cisneurons, the ion

influx increased immediately after the stimulation and continued to increase 10 seconds

after, but the difference was not significantly different (p>0.05, paired ttest; n=7).

However, the transneuron ion influx increased significantly immediately after the

stimulation (p<0.05; paired ttest; n=10).

34 immediately after, 10 seconds after, 20 seconds after, and 30 seconds after the end of the stimulation. Both of the neurons showed the increase in ion influx after the stimulation.

The changes in ion influx for each of the neuronal pairs were observed by plotting them in line series graphs (Fig. 6B; n=6). Four out of the six pairs showed that the ion influx of the transneurons increased much more rapidly than that of their partners. To find out the significance of this difference, the mean values were plotted in bar graphs (Fig 6C). The ion influx of the cisneurons increased in a slow, gradual fashion, from 0.98±0.29 pC to

1.35±0.66 pC per second immediately after the stimulation, then to 4.72±2.51 pC after 10 seconds (Fig. 6C, left; n=7). The fluctuation increased significantly 20 seconds after the stimulation, to 5.92±2.00 pC (p>0.05, paired ttest; n=7). On the other hand, the ion influx of the transneurons increased significantly from 0.99±0.18 pC to 4.72±1.67 pC immediately after stimulation and continued to increase over time (p>0.05, paired ttest;

Fig. 6C, right; n=10). This suggests not only that DRG neurons can transfer their signals onto each other, but also that they have a directionality that renders the communication able to go from the stimulated neuron to the neighbouring neurons without an equivalent retrograde transmission.

Satellite cells in between DRG neurons.

The cellular organization of the DRG was observed under EM. DRG slices of embryonic 12 and 15day chicks were scanned. The neuronal surfaces of the 21 adjecent neurons all were separated by one or more satellite cells (Fig. 7; Wong and Stanley). This finding is consistent with previous studies that satellite cells envelop nearly all of the surfaces (~98%) of the neurons, thus making a physical barrier between the neurons 35

Figure 7. Satellite cell separating neighbouring DRG neuronal pair.

The EM image shows that the neurons are separated by a satellite cell (arrow). N=nucleus

(Wong and Stanley, unpublished). Transmitter vesicles are not visible due to the

resolution.

(Lieberman, 1976; Shinder et al., 1998;Shinder and Devor, 1994). Thus, this result suggests that direct neurontoneuron transmission is unlikely.

DRG neuron to satellite cell transmission is purinergic (P2X receptor mediation)

Due to the associated satellite cells, direct contact between the neurons is extremely rare; thus, the direct transmission between the neuron is unlikely. Since usually only satellite cells make physical contact with the neurons, it is possible that the satellite cells play an active role in this communication by transmitting and receiving chemical signals. To find out whether the satellite cells receive any chemical signal from the vicinity, the satellite cells that were still associated with the neurons were patchclamped at 80mV to record any spontaneous current activity that occurs (Fig. 8A). Two types of spontaneous inward current fluctuations were revealed (Fig. 8A, far left): singlechannel activity and mEPCs (Fig. 8A, center left and far left; n=8). The presence of the mEPCs suggested that the satellite cells were receiving the signals either from themselves or from the enveloped neurons.

Administration of nicotinic (pancuronium, 200 M; data not shown; n=3) and glutamate antagonists (CNQX, 10 M; Fig. 7A, center left; n=3) did not affect these fluctuations, whereas P2 antagonist seemed to reduce their amplitudes (suramin, 500 M;

Fig 8B; n=5). Yet it is not clear whether suramin was reducing mEPCs, because the singlechannel activity was much more frequent and similar in size to the mEPCs, rendering difficult to pick the mEPCs by threshold detection. To isolate the mEPCs, the fluctuation peaks that fit the characteristics (sharp onset and slow decay) were selected 37

Figure 8

Figure 8. Miniature excitatory postsynaptic currents (mEPCs) in satellite cells . (A) A

satellite cell associated with a DRG neuron also exhibits spontaneous inward current

transients (far left). Two types were identified, mEPCs (far right) and singlechannel

activity (center right). The application of a glutamate blocker (CNQX, 10 µM) did not reduce the amplitudes of the fluctuations (center, left). (B) Current traces before (left) and after the P2 antagonist (suramin, 500 µM; right). (C) mEPCs were detected from the current trace amongst the single channels using computer software, AxoGraph X.

Overlays of the detected mEPCs before (left) and after the suramin application (right).

The amplitudes of the mEPCs were reduced after the treatment. (D) Histogram of the satellite cell mEPCs (bin width = 0.3 pA). The graph was fitted with a Gaussian distribution (left). The mean amplitude of the Gaussian peak was 3.06 pA. The peak was shifted to the left after suramin application (mean of 1.98 pA). Reduction of amplitude, not frequency, indicates that the effect was postsynaptic. (E) Focal ATP (10 µM) perfusion onto a satellite cell from the tip of a micropipette positioned close to the cell.

Current fluctuations increased immediately upon perfusion. The inward current creep prior to the puff was attributed to some leakage of the ATP solution as the pipette approached the cell.

39 using specialized software (AxoGraph X ver. 1.3.5). The superimposed trace of the detected mEPCs showed that suramin indeed reduced their amplitude (Fig. 8C). To find out their quantal nature, the mEPC amplitudes were further plotted onto amplitude versus frequency histograms (Fig. 8D, right; n=5). Although the distributions were slightly skewed to the left (towards smaller events), they were mostly unimodal. The histograms were then fitted into a Gaussian distribution. The mean was 2.91±0.39 pA, suggesting that the mean quantum size of this chemical signal is close to this value (Fig. 8D; left; n=5). The amplitude versus frequency histograms of the mEPCs before and after the application were plotted together to demonstrate the decrease of the peaks after the application. The mean became 1.60±0.32 pA after the application, which was significantly smaller (p>0.05, paired ttest; Fig. 8D, right; n=5), indicating that the mEPC was elicited by the activation of P2X receptors in response to ATP release. Suramin could not eliminate the mEPCs completely at the concentration tested. However, the application of suramin led to a shift in mean to the left without disturbing the overall shape of the histogram, suggesting that the mEPCs were induced by the activation of P2X receptors in the satellite cells. To show the presence of P2X receptors in the satellite cells,

ATP was focally perfused onto them by a closely positioned micropipette. Upon application, the current fluctuation of the satellite cells increased dramatically, confirming the presence of functional ATPsensitive receptors (Fig. 8E; n=3).

In order to examine whether the quantal secretion of ATP originated from the neurons, the wholecell currents of the neurons and their associated satellite cells were recorded simultaneously. The 10second stimulation, which was used on the neuronal pairs previously, was also applied to these neurons (Fig. 9A, top; n=4), and this evoked 40

Figure 9. DRG neuron to satellite cell transmission is purinergic.

(A) Representative recording of a DRG neuron–satellite cell dualrecording while the

DRG neurons were stimulated with a train of depolarizing pulses (top). The mEPC activity of the satellite cell increased immediately after stimulation (bottom). (B) Suramin inhibits of the neuron–satellite cell transmission. The stimulation of DRG neuron was run twice, before and after bath application of suramin (100 M). The DRG neuron–satellite cell transmission was visibly reduced after the application Increased current fluctuations in the satellite cell (*) during stimulation is an artifact created by the pulses given to the neuron. 41 an increase in the mEPC frequency of the enveloping satellite cells (Fig. 9A, bottom).

The increase usually began even before the 10second stimulation ended, indicating that the transmission from the neurons to the satellite cells is faster than that of the neuronal pairs. This increase did not occur in the presence of P2 antagonist (100 M, Fig. 9B;

n=2),confirming that the mEPCs induce the quantal release of ATP from the DRG

neurons. This further demonstrated that the release can be increased when the neuron is

excited

Satellite cell to DRG neuron transmission is cholinergic

If the DRG paircommunication is really mediated by the satellite cells, the

satellite cells must be able to transmit signals to the neurons as well. An isolated DRG

neuron was again patchclamped at the holding potential of −80 mV. Agonists that

activate ionotropic receptors were perfused focally onto the neurons by a micropipette positioned close. A nicotinic agonist (carbachol, 10 M) induced a dramatic increase in

the fluctuation (Fig. 10A), whereas ATP (100 M) induced inhibition (Fig. 10B).

Since ATP can activate the P2X receptors of the satellite cells, experiments were

done to test whether this activation leads to any change in the inward current fluctuation

of the associated neurons. While the wholecell currents of the neurons were recorded,

ATP (100 M) was perfused focally onto the associated satellite cells (Fig. 11A, left).

The inward current fluctuation was increased 10.4±3.0 seconds after the perfusion began

(Fig. 11A, right; n=15). This increase was reversed when the perfusion stopped (Fig. 11A,

right; n=3). A selection of antagonists that block the activities of ionotropic receptors was

added into the bath. The increase was not affected by glutamate antagonists (CNQX and 42

Carbachol Carbachol (10uM) Pipette Inside EXT sol n 10pA 10sec

Pipette Recovery removed

B ATP (100 μM)

20pA 20sec

Figure 10. Response of DRG neurons to the purinergic and cholinergic agonists.

(A) Representative current trace of a DRG neuron during focal perfusion of carbachol(10

M), a nicotinic agonist, in presence of P2 receptor blocker (suramin 100M). The

neuron displays the increase in the current fluctuation. (B) Focal perfusion of ATP (100

M) onto a neuron inhibited spontanteous inward current fluctuations.

Figure 11

Figure 11. Satellite cell-DRG neuron transmission is cholinergic. (A) Diagram of ATP focal perfusion apparatus (left). Wholecell currents of the neurons were recorded at the resting potential while ATP (100 M) was focally perfused onto the associated satellite cell from a micropipette positioned close the cell (right). Inward current activity increase in neuron ~9 seconds after the onset of perfusion. (B) As in A, right panel with the addition of the cholinergic antagonist (dtubocurarine; 200 M; blue line) after the onset of the ATPinduced inward current. The gap indicates an intervening period of ~60 seconds. The increase in the current activity of the neuron was returned to the basal level after DTC application. A transient large increase of current (*) is an artefact caused by the bath application of DTC (C) Bar graphs showing the ion influx of DRG neurons

(measured by integrating currents per second, right) after ATP and DTC application.

Focal perfusion of ATP onto the associated satellite cells increased the fluctuations of the neurons while the application of DTC reduced the fluctuation back the basal level. (D)

Immunostaining of a DRG neuronal pair (NA and NB) with an antibody against ChAT

(green). Note that the staining is concentrated in the satellite cell in between the neuronal pair (Li and Stanley, unpublished).

NBQX, data not shown; n=3), but markedly reduced by a nicotinic antagonist (d tubocurarine (DTC), Fig. 11B; n=3). To quantify the effects of ATP and DTC, the ion influxes of the neurons before and after the ATP perfusion, and after DTC application were calculated by integrating the current fluctuation over time. The ion influx per second increased from −0.85±0.68 pC to −6.09±2.68 pC during the ATP perfusion, and reduced to −0.94±0.49 pC after the DTC application. However, this change was not statistically significant (p>0.05, oneway ANOVA and Wilcoxon signedrank test; Fig.

10C; n=3), due, it seems, to a high variability (Fig. 11B).

As an independent test for cholinergic secretion from satellite cells we used immunocytochemistry to test if these cells stain for cholineOacetyltransferase (ChAT)

(Fig 11D; Li and Stanley). Dissociated DRG was immunostained with an antibody against ChAT (goat, polyclonal). The satellite cells that are in between the neuronal pairs were stained with ChAT (green), suggesting that the protein was expressed in the cells.

P2Y receptors in DRG neuron inhibit satellite to DRG neuron transmission

The double recordings of the neuronal pairs suggest that this neuronal communication has directionality in which the excitation in the transneurons is significantly greater. One possibility for this phenomenon is that the communication is regulated by a second messenger–mediated pathway. To test this, all of the Gprotein activities in the neurons were inhibited by utilizing a nonhydrolysable GDP analogue,

GDP βS . GDP βS (200 µM) was infused into the cells through a recording electrode. In the presence of GDP βS , the neurons revealed two types of inward current fluctuation that could not be seen without GDP βS , mEPC and singlechannellike activities (Fig. 12A; 46

Figure 12. mEPC-like activities appear in the neurons after the inhibition of

G protein. (A) A representative current trace of DRG neurons in the presence of intracellular GDP βS (left). Two types of inward current transients were detected, mEPC

(right) and singlechannellike activity (middle). (B) Traces of the neuron before and after the bath application of Gd 3+ (200 µM). The singlechannel activities were visibly

reduced after the application.

Figure 13

Figure 13. Neuronal mEPCs are cholinergic. (A) Current trace of the neurons before and

after the bath application of a general glutamate receptor blocker, CNQX (100 M; top)

or the nAChR blocker PCB (200 M). CNQX did not affect the amplitude of the mEPC,

whereas PCB reduced them. (B) Overlays of mEPCs before (left) and after PCB

application (right). The mEPCs were identified from the current trace, with the semi

automated software AxoGraph X (see Methods). (C) Amplitude histogram of mEPCs in

a DRG neuron (bin width = 0.3 pA). The histogram was fitted with two Gaussian

distributions with means of 3.29 and 5.01pA. The peaks were shifted to the left after PCB

application (200 M) with means of 2.04 and 3.15 pA.

Figure 14. ATP applied to the satellite cells triggers mEPC activity in the neuron .

(A) A current trace of DRG neuron after ATP perfusion to the enveloping satellite cells.

The mEPC activity was increased after the ATP perfusion but was reduced by the nAChR blocker PCB. A transient large increase of current (*) is an artefact caused by the bath

application of DTC (B) A bar graph showing the current changes of GDP βSinfused DRG

neurons after ATP and PCB applications (n=3). Charge influx in DRG neurons

(measured by integrating currents per second) during the enveloping satellite cell

excitation was significantly higher than the control and PCB treatment (p< 0.05). 50 n=10). Gadolinium (Gd 3+ , 200 µM), a potent blocker of various cation channels, was administered to isolate the mEPCs (Babinski et al., 2000; Cho et al., 2002; Hase et al.,

1995; Lansman, 1990). This resulted in the attenuation of the singlechannel activity (Fig.

12B; n=7). In order to identify the neurotransmitter responsible, a selection of receptor antagonists (suramin, CNQX, pancuronium) that block ionotropic channels involved were added to the bath. A purinergic (suramin, 100 M; n=3) and a glutamate (CNQX;

Fig. 12A, top; 100 M; n=3) antagonist did not reduce the overall mEPC amplitude (data not shown), whereas the application of cholinergic antagonist (pancuronium, PCB) did

(Fig. 13A, bottom; 200 M; n=7).

If they are really mEPCs, their amplitudes must be centered of a single value whose frequency distribution can be fitted into Gaussian curves. To test this, the inward peaks that fit into the general characteristics of mEPCs (sharp onset and slow decay) were

selected by AxoGraph. The superimposed mEPC traces showed that the amplitudes of the

mEPCs were reduced by PCB (Fig. 13B). Their amplitudes were plotted in amplitude

versus frequency histograms. The peaks of the histograms were able to be fitted into one

or two Gaussian distributions (Fig. 13C, left; n=7). The mean of the first Gaussian curve

was 3.22±1.27 pA, suggesting that the mean quantum size of this transmission is close to

this value. The mEPCs before and after the PCB application were compared by plotting

them together in the amplitude versus frequency histograms (Fig. 13C, right; n=7). The

Gaussian curves were shifted left and the mean was changed to 1.64±0.35 pA, which was

significantly smaller (p<0.05, paired ttest), suggesting that the transmitter responsible for

inducing mEPCs was ACh. To find out whether the mEPCs were released by the satellite

cells, the ATP (100 M) was perfused focally onto the associated satellite cells. The ATP 51

Figure 15. G-protein mediated inhibition of the cholinergic transmission is induced by the activation of P2Y receptors. (A) Current trace recording from a DRG neuron that was associated with a satellite cells in the presence of suramin, a general P2 receptor antagonist (100 M, left; n=4). The mEPCs were revealed, even in absence of

GDP βS, by the application of suramin (right). (B) Current traces of a DRG neuron during the application of the nAChR agonist carbachol (10 M; green line) which induced tonic inward current activity. Focal perfusion of ATP (100 M; red line, left) reduced the activity (as discussed). Application of suramin (100 µM; blue line), which blocks P2Y receptors, eliminated the effect of ATP (100 M; red line, right; n=3).

52 led to an increase in current fluctuation in the neurons. The increased fluctuation also had the characteristic appearance of mEPC, indicating the increase was made by the mEPCs overlapping onto one another. The fluctuation was again reduced after the PCB application (200 M; Fig. 14A). The ion influx of each of the conditions was calculated to quantify the change in the current fluctuation. The ion influx increased from

−0.17±0.21 pC to −3.87±0.88 pC after the ATP perfusion and was reduced back to

0.12±0.61 pC after the PCB application (p<0.05, oneway ANOVA; Fig. 14B, right; n=3).

Suramin is also a P2Y receptor antagonist. When DRG cells were incubated with suramin (100 M), their current recordings also revealed mEPCs (Fig. 15A; n=4), suggesting that the inhibition of the nicotinic receptors may be initiated by P2Y receptor activation. To test whether the P2Y receptors can effectively inhibit the nicotinic receptor activity, ATP was perfused focally onto the neurons after the nicotinic receptors were activated. The current fluctuation which was increased by the application of carbachol

(10 M) was markedly reduced when the neurons were focally perfused with ATP (100

M; Fig. 15B; n=3). However, in the presence of suramin (100 M), ATP was ineffective in reducing the fluctuations, supporting the idea that P2Y receptors inhibit the activity of the nicotinic receptor. However, when the ATP perfusion stopped, the fluctuation often became greater than before the ATP perfusion, indicating that the perfusion may also induce some unspecific responses as well.

Relief of G-protein inhibition in DRG pairs enhances the response of cis-neurons

Lastly, the final part of this study was to find whether Gprotein activity plays a vital part in this communication. To do so, the wholecell currents of the neuronal pairs 53

were recorded simultaneously, while the pair were infused with GDP βS (200 µM; n=20).

Both cis and transneurons experienced an increase in current fluctuation after the

stimulation (Fig. 16A; n=4). To quantify the current increase, the ion influx was

calculated by integrating their current fluctuation over time. The ion influx in the cis

neurons increased significantly from −3.00±1.51 pC to −7.98±2.88 pC immediately after

stimulation (p<0.05, Wilcoxon signedrank test; Fig. 16C; n=4), and continued to

increase to −19.52±4.96 pC after 20 seconds. The current fluctuation of the transneuron

increased significantly from −1.94±1.47 pC to −11.51±2.35 pC 20 seconds after the

stimulation (p<0.05, Wilcoxon signedrank test; Fig. 16C; n=4). The increases between

the cis and transneurons were not significantly different. Moreover, the magnitude of

the increase in the cisneurons was greater than those of their partners. The overall results

indicate that DRG paircommunication loses directionality of transmission (cistotrans

transmission) in the absence of the inhibitory action of the P2Y receptor, suggesting this

inhibition is a key factor in determining the polarity of the DRG paircommunication.

Figure 16

Vstimulated 20mV 80mV

Istimulated (GDPβS)

20pA 10sec Iadjacent (GDP βS)

Before stimulation After stimulation

10pA

10msec

Figure 16. Role of G protein activities in the neuronal-pair communication. (A)

Superimposed diagram showing the dual patchclamp recordings of the neuronal pair.

Both neurons were infused with GDP βS (200 M) Current fluctuation increased in both of

the neurons after the stimulation. Increased current fluctuations in the transneuron (*)

during stimulation is an artifact created by the pulses given to the cisneuron. (B) The increase was formed by the mEPCs, which were much increased in frequency. (C) Bar

graphs showing the current changes of cis and transneuron in the presence of GDP βS before, immediately after, and 20 seconds after the stimulation. Ion influx of the cis

neuron increased immediately after stimulation (n=4), and continued to increase after 20

seconds. The current fluctuation of the transneurons also increased 20 seconds after the

stimulation. The results indicate that cis neurons become equally responsive to the

stimulation in the absence of Gprotein activity.

DISCUSSION

This study reports that DRG neurons can transmit signals to a neighbouring neuron other through the intervening satellite cell. The DRG paircommunication is composed of two parts. The neuron exocytoses ATP and activates the P2X receptors in the satellite cells. This subsequently triggers the satellite cells to exocytose acetylcholine and activates the neighbouring neuron. We term this newly discovered transmission mechanism a “sandwich synapse.” In this report, we were able to demonstrate that DRG paircommunication can occur between the somata by utilizing the dual patchclamp technique. The trituration procedure by which the DRG neurons were dissociated was originally designed to preserve the synapse in ciliary ganglions (Chan and Stanley, 2003;

Stanley, 1987; Stanley and Cox, 1991). This procedure allowed some of the DRG neurons to still be intact with the neighbouring neurons and their associated satellite cells after the dissociation. The simultaneous patchclamping of pairs provided us an advantage to decipher the chemical basis of the pathway between the pairs, since the pairs are completely isolated from other cells that could release chemicals. Thus it was certain that any signals were caused by either the pairs or the satellite cells in between.

The communication between DRG pair is not certainly via simple diffusion of transmitters between the neurons, because the current increase of the neuron that received the signals is significantly greater than its partner in all of the pairs. If it were a simple diffusion, the response of the neuron that initiated the communication would have been larger due to autocrine signaling. 57

All recordings were done using voltage clamp. In order to study the physiological characteristics of this communication, further study must be conducted by current clamp, which allows observing the natural activities of the neuronal pairs.

The chemical basis of the sandwich synapse

DRG paircommunication begins as the neuron releases ATP to its associated satellite cell. ATP is involved in the neuronglial interaction in different parts of the PNS.

(Jahromi et al., 1992; Robitaille, 1995). ATPcontaining vesicles are present in the neurons and their axons, and the neurotransmitters are released in the spinal cord where their synaptic terminals meet the interneuron of the dorsal horn (Bardoni et al., 1997; Jahr and Jessell, 1983; Soeda et al., 1997). Our results on DRG neuronsatellite cell transmission support the previous study by Zhang and his colleagues (2007b). We further characterized this transmission by presenting physiological evidence that the signals received by the satellite cells can recorded as mEPCs.

The DRG neuron releases ATP into the narrow space abutting the satellite cell.

ATP has two actions: it activates P2X receptors in the satellite cells and also P2Y autoreceptors. P2X activation leads to an inward cation current in the satellite cell while the P2Y receptors of the DRG neuron inhibit AChR, presumably via the release of activated G protein subunits. The reduction of mEPC amplitudes in the satellite cells by suramin supports the involvement of P2X receptors. The incomplete block of mEPCs with 500M suramin may be due to heterogeneity in P2X receptor expression. Although suramin is a general P2 blocker, some receptor variants are relatively insensitive, such as

P2X 4 (K d greater than 100 µM) and P2X 7 (K d greater than 300 M and 70µM in rats and 58 human, respectively) (Anderson and Nedergaard, 2006; Buell et al., 1996; Jones et al.,

2000; North, 2002;). Previous studies have also localized P2X7 receptors to this synaptic contact (Zhang et al., 2007b). However, without further studies we cannot be entirely sure that the residual mEPCs reflect a second, suramininsensitive, neurotransmitter type.

Our results suggest that satellite cells activate nicotinic receptors of nearby DRG neurons by exocytosing acetylcholine. This has not been reported previously. There are several possible explanations for this omission. First, transmission may have been affected by the relatively harsh enzyme conditions, including trypsin (either added directly or as a component of many collagenases) used in other studies to isolate their cells. Second, it is possible that under the conditions in previous studies the P2Y receptors were activated and inhibited the AChR. Lastly, and we believe most likely, the cholinergic transmission is most evident at sandwich synapsetype contacts which, to our knowledge, have not been explored by electrophysiology previously. We also show that

ACh release onto the neurons is quantal, and hence most likely occurs by controlled exocytosis as at other synapses. This quantal activity was only revealed after inhibiting

G proteins in the neurons, consistent with the autoinhibition of these receptors via P2Y receptors.

Our study is the first to provide evidence for satellite cell–neuronal transmission involving classical transmitters. Cholinergic transmission from glia to neuron was, however, first reported at the frog neuromuscular junctions where it was observed that even after the motor nerve had been cut and the axon had degenerated miniature end plate potentials (mEPPs) were still observed. Further analysis concluded that these were secreted from perisynaptic Schwann cells that had colonized the postsynaptic apparatus 59

(Miledi and Stefani, 1970;Reiser and Miledi, 1988). At the satelliteDRG neuron contact mEPCs that appeared after G protein inhibition were sensitive to PCB, providing additional evidence that the neurotransmitter was ACh (Maestrone et al., 1994;Ocana et al., 1992). The mEPC activity was not fully blocked after the PCB treatment probably again because of the involvement of different species of cholinergic receptors (Genzen et al., 2001) with different sensitivities to this drug. We also observed multiple peaks

(generally, two) in the mEPC amplitude versus frequency histograms. Since these were noted for spontaneous activity and their amplitudes were not simply multiples, they probably do not reflect release of one and two quanta. Instead we suspect that either secretion is occurring to sites with different density or subtype of receptor (Leech and

Sattelle, 1992). We cannot, however, rule out the idea that the secretory vesicles exist in two distinct forms with different ACh content.

Focal application of ATP onto the satellite cells, activating their P2X receptors, increased mEPC activity in the attached neuron, providing additional evidence that ACh is released from the satellite cells and via P2X receptor activation. This finding is particularly important as it supports the hypothesis that the exocytosis of acetylcholine in the satellite cells is triggered by the ATP released from the neuron. The P2X 7 subunit,

which is commonly found in the satellite cells, is known to increase cytosolic [Ca 2+ ] and regulate the release of neurotransmitters and other molecules in glial cells in the CNS and immune cells (Qu and Dubyak, 2009; Suadicani et al., 2006; Zhang et al., 2007a). The current influx in the neurons was reduced by the application of ATP, which is the opposite of what happened when the enveloping satellite cells were perfused. This is reasonably explained by considering the spontaneous fluctuations to be caused by 60 nicotinic receptor activation, since the perfused ATP activates the P2Y receptor in the neurons and subsequently inactivates the nicotinic receptors.

Previous studies in frog and rat have reported that ATP induces an inward current in a subpopulation of DRG neurons via P2X 3 and P2X 4 receptors (Li, 2000; Petruska et

al., 2000; Ueno et al., 1999). Thus, we would have predicted that ATP application or

secretion from the neurons should have resulted in a similar inward current in our studies but we only observed an inhibition of resting current noise. There are two likely explanations for different result. Chick neuronal P2X receptors have been reported to be inhibited via P2Y receptors (Gerevich et al., 2007). Thus, we suppose that the P2Y inhibition pathway is much stronger in chick than rat, in effect blocking any ATP dependent excitatory input.

The immunocytochemistry of the dissociated DRG showed that ChAT is present in the satellite cells, suggesting that they synthesize ACh. This strengthens the hypothesis that the satellite cells activate the neurons. Some studies have reported that ChAT is also present in DRG neurons (Sann et al., 1995), although a review of these papers suggests that the extent is far less than claimed since many of the stained profiles appear to be satellite cells. It is further indicated that the neurons possess pChAT, the peripheral splice variant (Bellier and Kimura, 2007). Our results also showed that the neurons were occasionally positive for ChAT. However, this does not mean that the mEPCs were induced by autocrine cholinergic activity, since a subset of the primary afferent neurons is known to release Ach in the spinal cord, and the AChs made in those neurons are transported to the CNS to be released (Matsumoto et al., 2007). 61

The simultaneous recordings of the neuronal pairs showed that chemical transmission between DRG neurons occurs and has directionality, in which the excitation after the stimulation is much greater in the neuron that received the transmission than in the neuron that initiated the transmission. The cisneurons had equivalent increase of the current fluctuation after the stimulation when the Gproteinmediated pathway was inhibited in both neurons. This autocrine activity is biologically important, since this activity renders the neuron that initiates the communication insensitive to ACh, subsequently allowing the paircommunication to be polarized to transneurons.

The results further suggest that the directionality is induced by the activation of

P2Y receptors in the neurons. This study is the first to show that nicotinic receptor activation can be inhibited by the activity of P2Y receptors. Previous studies demonstrated that P2Y receptors can inhibit Ntype calcium channels, inwardly rectifying

K+ channels, NMDA receptors and P2X receptors by a Gproteinmediated pathway

(Borvendeg et al., 2003; Filippov et al., 2004; Gerevich et al., 2007a; Luthardt et al.,

2003). The carbacholinduced current of the neurons was reduced by the application of

ATP. The current fluctuation often noticeably increased after the perfusion of ATP stopped. The fluctuation is probably due to an unspecific response of other receptors or the activation of the P2X receptors that were usually suppressed. The most prevalently found subunit in the neuron is P2Y 1 receptors, and the concentration used in this

experiment is extremely high for activating the receptors, whose effective K d value is

around 140 nM (Gerevich and Illes, 2004; Nakamura and Strittmatter, 1996).

Figure 17. Schematic diagram of the Sandwich Synapse . DRG neuron (cis) releases

ATP and activates P2X receptors in the associated satellite cell. In response, the satellite

cell releases ACh and activates the nicotinic receptors in the adjacent neurons (cis to

trans). At the same time, the P2Y receptors in the cisneuron become activated by the

ATP. They subsequently inhibit the nicotinic receptors, thus preventing the retrograde

signalling (the transmission back to the cisneuron).

The sandwich synapse as the functional unit of DRG neuronal communication

Bidirectional communication between neurons and glia can also be demonstrated in the CNS and PNS. Studies further suggest that this communication is important in modulating neuronal excitation and synchronicity (Hamilton and Attwell, 2010;Haydon and Carmignoto, 2006). However, the sandwich synapse is essentially different from this system, by which the satellite cell is the sole route for the DRG neuronal communication.

Thus, ours study is the first to show that the satellite cell actively relays neuronal information by receiving transmissions from a nearby neuron and releasing neurotransmitters to another neuron.

As discussed above, two types of the neuronenveloping satellite cells exist in

DRG, ones that envelop a single neuron and others that envelop multiple neurons. The former are much more common, indicating that the majority of the cells are not connected by a single satellite cell (Pannese et al., 1991). Yet, some of satellite cells are coupled by gap junctions. A recent study showed that Ca 2+ signaling occurs between 36%

(31/85) of the satellite cells and is reduced to 4% (1/24) after the application of carbenoxolone, indicating that the communication is by gap junction couplings

(Suadicani et al., 2010).

This study is not the first to demonstrate excitation between the neuron in the

DRG. Devor and Wall (1990) showed that the spike activity in one neuron can depolarize a neighbouring making the partner neuron more likely to fire APs in response to sub threshold depolarization. Their findings suggest that this form of neurontoneuron communication is chemically mediated, raising the possibility that sandwich synapse type transmission is involved. Yet, this communication is different in a few ways. The 64 onset of the crossdepolarization described by Devor and Wall occurs at the beginning of the stimulation train, whereas the communication between the neurons via the sandwich synapse starts after the end of the train and continue to increase over time. Moreover, the former has been reported in 95% of neurons sampled whereas sandwich synapses are only present only in a small portion of the neurons. Considering these differences, it seems unlikely that crossdepolarization described by Devor and Wall can be attributed to sandwich synapse activity.

The functionality of the communication pathway among DRG somata still remains unclear. Previous studies suggested that the membrane excitability of DRG neurons directs many important cellular pathways in the neuron, from the neurotransmitter vesicle cycling and gene expression to the modulation of signals in the case of nerve injuries (Fields et al., 1997; Wall and Devor, 1983). The sandwich synapse introduces the possibility that these pathways can be more intricate than previously thought, since the membrane excitability is influenced by activities of the neighbouring neurons. Deciphering the role of the sandwich synapse in influencing the overall functions of the neurons may open doors to new knowledge, from understanding the regulatory pathways in somatosensory signals to therapeutic strategies for neuropathy.

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