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DYNAMICS OF FAST SYNAPTIC EXCITATION IN
MYENTERIC NEURONS

presented by
JIANHUA JIM REN

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DYNAMICS OF FAST SYNAPTIC EXCITATION
IN MYENTERIC NEURONS

By

Jianhua Jim Ren

A DISSERTATION
Submitted to
Michigan State University

in partial fulfillment of the requirements
for the degree of

DOCTOR of PHILOSOPHY
Neuroscience Program

2006

ABSTRACT

DYNAMICS OF FAST SYNAPTIC EXCITATION IN MYENTERIC NEURONS
By
J ianhua Jim Ren

The goal of this dissertation is to understand the dynamic properties of fast
excitatory synaptic transmission in myenteric neurons in the small intestine. Three
specific issues have been addressed: 1) identification of purinergic P2X receptor subtypes
expressed in myenteric neurons; 2) characterization of fast excitatory synaptic
transmission during bursts of synaptic activity and 3) the cellular and molecular
mechanisms by which presynaptic 5-HT4 receptors mediate facilitation of fast synaptic
transmission. Two types of electrophysiological recording methods were used: 1)
intracellular microelectrode recordings from single myenteric neurons in acutely isolated
longitudinal muscle-myenteric plexus (LMMP) preparations and 2) patch-clamp

recordings from single myenteric neurons maintained in primary culture.

Identification of P2X receptor subtypes was conducted in acutely isolated LMMP
preparations in vitro from the small intestine of guinea pigs and mice which P2X2 or
P2X; gene was knockout. My data suggest that P2X receptors were P2X; homomers in
myenteric S neurons and P2X2/3 heteromers in AH neurons in mouse ileum. However, in
guinea pig ileum, P2X receptors in myenteric S neurons contained P2X3 subunits. P2X
receptors in AH neurons did not contain P2X; subunits. My data also showed that P2X;
subunit-containing receptors in myenteric S neurons of guinea pig ileum coupled to Ca2+-
activated 1Cr channels to mediate a hyperpolarization that followed agonist-induced

depolarization.

Fast excitatory postsynaptic potentials (fEPSPs) evoked in guinea pig LMMP
preparations in vitro declined (rundown) during trains of stimulation. The rundown rate
was frequency-dependent. Neither desensitization of nAChRs or P2X receptors nor
receptor-mediated presynaptic inhibition contributed to rundown of fEPSP. Depletion of
transmitter vesicles in the readily releasable pool in presynaptic terminals was proposed

as a mechanism to account for synaptic rundown.

5-HT4 receptors are localized to presynaptic terminals in myenteric neurons. In
my study, 5-HT4 agonists increased fast EPSP amplitude during trains of stimulation and
facilitated recovery afier rundown. The effects of 5-HT4 receptor agonists on fEPSPs
were mediated through activation of the adenylate cyclase-cAMP-PKA signaling
pathway. Detector-patch studies in cultured myenteric neurons suggested that 5-HT4
agonists facilitated fast synaptic transmission by increasing neurotransmitter release

probability from single varicosities.

These results provide novel information on pre- and postsynaptic mechanisms
regulating synaptic transmission in the enteric nervous system. Rundown of fEPSPs
during bursts may be a presynaptic mechanism to limit the synapses from over-activity.
After-hyperpolarization mediated by P2X receptors and facilitation mediated by 5-HT4
receptors may provide the cellular mechanisms by which neurotransmitters or drugs that
can mimic neurotransmitters’ action can alter neuronal activity. In the View of practice,
the differential expression of P2X subunits in myenteric neurons will help to select drug
targets for development of novel treatment of GI motility disorders. Studies of the
mechanisms of 5-HT4 receptor-mediated facilitation of synaptic transmission may

provide insights into the neurophysiological basis of GI motility disorders.

ACKNOWLEDGEMENTS

First and foremost I would like to thank my mentor Dr. James J. Galligan for
giving me the opportunity working in his laboratory. 1 have benefited for my scientific
career and daily life so much from his patient teaching, his wise advice and the excellent
environment he provides. These past few years working with him will be an invaluable
experience for rest of my life. I would also like to thank my thesis advisory committee
members for their helpful discussion, constructive suggestions and encouragement. They

were: Drs. Steven Hedeimann, David Kreulen and Mary Rheuben.

I dedicate this dissertation to my lovely wife, as she has always been a loyal

audience for my work.

iv

TABLE OF CONTENTS

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

LIST OF TABLES viii
LIST OF FIGURES ix
INTRODUCTION 1
The enteric nervous system 2
Overview 2
Anatomy of the GI tract and arrangement of the enteric plexuses ---- 4
Morphology and ultrastructure of enteric neurons 7
Electrophysiology of myenteric neurons 8
Functional classification of enteric neurons 15
Intrinsic neural circuits in the myenteric plexus l7
Extrinsic innervation 20
Submucosal neurons 21
Synaptic transmission 23
Overview 23
Slow synaptic transmission in enteric neurons 24 I
Fast synaptic transmission and ligand-gated ion channels in myenteric
neurons 25
Dynamics of neurotransmitter release 31
Recycle of synaptic vesicles 33
5-HT4 receptors in myenteric neurons 36
Research goal 38
Specific aims 39
METHODS 42
Animals 42
Longitudinal muscle myenteric plexus preparations 42
Myenteric neurons maintained in primary culture 43
Intracellular recording from single myenteric neurons 44
Patch-clamp recording from primary cultured myenteric neurons ------------ 45
Immunohistochemistry 46
Drugs and drug application 46
Statistics 49
RESULTS 50
Subtypes of P2X receptor in myenteric neurons in guinea pig and mouse
small intestine 51
Part A. P2X receptor subtypes in myenteric neurons in mouse small
intestine 51
Characteristics of myenteric neurons from P2X2+/+, P2X2'l', P2X3H+ and
P2X;”' mice 51

 

 

 

P2X2 subunits knockout altered P2X-meidated responses in S neurons
59

 

Properties of P2X receptors were not affected by P2X3 subunits
knockout in S neurons 66
or, B-mATP depolarizes AH neurons from P2X?+ and P2X2'l' mice -

 

66

 

ATP- and a, B-mATP- induced depolarization in AH neurons from
szf” and P2X;"' mice 75
Part B. Pharmacological identification of p2X subtypes in guinea pig
myenteric neurons 75
Depolarization induced by ATP and a,B-mATP in AH and S neurons
in guinea pigs 75
Effects of TNP-ATP on fEPSPs and ATP-induced depolarization in S
neurons in guinea pigs 78

 

 

 

 

P2X receptors couple to a calcium-dependent potassium conductance in

 

 

 

guinea pig myenteric neurons 85
An afterhyperpolarization (AHP) is induced by activation of P2X
receptors 85
The ionic basis of the afterhyperpolarization 88
Immunochemical characterization of S neurons with after-
hyperpolarization 93

 

Fast excitatory synaptic transmission during burst of neuronal activity—98

Fast synaptic transmission declined in amplitude during trains of

 

 

 

 

 

electrical stimulation 98
fEPSCs exhibit paired pulse depression 101
Recovery from synaptic rundown 101
The contribution of nAChRs and P2X receptors to rundown of fEPSPs

104
Presynaptic mechanisms 109

Presynaptic 5-HT4 receptor-mediated regulation of fast excitatory

 

 

 

 

 

 

 

 

synaptic transmission 125
5-HT4 receptor agonist increases fEPSPs during trains of stimulation -

125

5-HT4 receptor agonist accelerates recovery time of fEPSP after
rundown 125
Forskolin mimics 5-HT4 receptor agonist effects 130
H-89 abolishes the effects of 5-HT4 receptor agonist I30

5-HT4 receptor agonists increase release probability from single
varicosities 13 5
DISCUSSION 143
P2X receptor subtypes expressed in myenteric neurons 144
Properties of murine myenteric neurons 145

 

vi

P2X receptor-mediated responses in murine AH neurons ------------ 147
Fast synaptic transmission and P2X receptor-mediated responses in

murine S neurons 148
P2X subtypes in myenteric neurons in guinea pig small intestine ---151

 

P2X receptor-mediated hyperpolarization in guinea pig myenteric

 

 

 

 

 

 

 

neurons 153
Ionic basis of afterhypolarization 153
Functional significance of AHP in myenteric S neurons ------------- 157

Rundown of fEPSPs during bursts of neuronal activity 159
Rundown of fEPSPs 159
Receptor desensitization did not contribute to synaptic rundown --- 160
Rundown is not caused by presynaptic inhibition 161
Changes in presynaptic action potentials do not contribute synaptic
rundown 163
Depletion of transmitter accounts for synaptic rundown ------------- 165

Rundown of fEPSPs during bursts of neuronal activity 168
Activation of 5-HT4 receptors attenuates synaptic run-down and
accelerates recovery from run-down I71

 

5-HT4 receptor-mediate presynaptic regulation of synaptic
transmission is through adenylate cyclase-cAMP-protein kinase A

 

 

signaling pathway 173

Activation of 5-HT; receptors increases probability of transmitter

release from single varicosities 174
SUMMARY AND SIGNIFICANCE 178

 

BIBLIOGRAPHY l 83

 

vii

LIST OF TABLES

 

Table 1. Drugs and ligands used in this dissertation 48

Table 2. The electrical properties of myenteric neurons in mouse small intestine -------- 53

 

Table 3. Effects of 5-HT4 receptor agonists on release probability 142

viii

Figure 01.

Figure 02.

Figure 03.

Figure 04.

Figure 05.

Figure 06.

Figure 07.

Figure 08.

Figure 09.

Figure 10.

Figure l I.

Figure 12.

LIST OF FIGURES

 

 

 

The general organization of the enteric nervous system 5
Electrophysiological recordings from single myenteric neurons in LMMP

preparations and neurons maintained in primary culture 10
Myenteric AH and S neurons 12
Intrinsic neural circuitry in the myenteric plexus l8

 

The action potentials evoked in myenteric S neurons from wild type, P2X; and

P2X3 knockout mice 55

 

The action potentials evoked in myenteric AH neurons from wild type, P2X2,

P2X; knockout mice 57

 

Effects of PPADS on fEPSPs from murine myenteric S neurons in wild type

 

and P2X; knockout tissues 60
Effects of mecamylamine on fEPSPs from murine myenteric S neurons in wild

type and P2X; knockout tissues 62

 

Responses of murine myenteric S neurons in wild type and P2X; knockout

64

 

tissues to nicotine, ATP and or, B-mATP
Effects of PPADS on fEPSPs from murine myenteric S neurons in wild type

and P2X3 knockout tissues 67

 

Effects of mecamylamine on fEPSPs from murine myenteric S neurons in wild

type and P2X; knockout tissues 69

 

Responses of murine myenteric S neurons in wild type and P2X3 knockout

71

 

tissues to ATP and or, B-mATP

ix

Figure 13.

Figure 14.

Figure 15.

Figure 16.

Figure 17.

Figure 18.

Figure 19.

Figure 20.

Figure 21.
Figure 22.
Figure 23.
Figure 24.
Figure 25.

Figure 26.

Figure 27.

Responses of murine myenteric AH neurons in wild type and P2X; knockout

 

tissues to or, B-mATP 73

Responses of murine myenteric AH neurons in wild type and P2X3 knockout

 

 

tissues to ATP and or, B-mATP 76
Responses of myenteric S and AH neurons in guinea pig small intestine to
ATP and or, B-mATP 79

Effects of TNP-ATP on ATP-induced depolarization in guinea pig S neurons

 

 

 

81
Effects of TNP-ATP on fEPSP in guinea pig S neurons 83
or, B-mATP induces an after-hyperpolarization following depolarization in
guinea pig S neurons 86
a, B—mATP-induced after-hyperpolarization is Ca2+ sensitive 89

 

or, B-mATP—induced after—hyperpolarization is mediated by an increase in 1C

 

 

 

conductance 9 1
Identification of Kc, subtypes mediating after-hyperpolarization ------------- 94
Identification of neurons that generate afterhyperpolarization 96
Trains of electrical nerve stimulation induce rundown of fEPSPs ------------- 99
Paired stimulation caused synaptic depression 102
Time-dependent recovery from rundown 105

 

Both cholinergic and purinergic component of synaptic transmission rundown

107

 

Postsynaptic desensitization does not contribute to synaptic rundown ------ 1 10

Figure 28

Figure 29.
Figure 30.
Figure 31.
Figure 32.
Figure 33.
Figure 34.
Figure 35.
Figure 36.
Figure 37.

Figure 38.

Figure 39.
Figure 40.

Figure 41.

. Antagonists against presynaptic inhibitory receptors do not alter synaptic

rundown

 

 

Pertursis toxin does not affect synaptic rundown

Action potentials do not fail during the train stimulation at 10 Hz -----------

 

Effects of IBTx on action potentials in S and AH neurons

 

The effect of IBTx on synaptic rundown

Prucarlopride increases cholinergic and purinergic fEPSP

 

 

Renzapride increases fEPSPs elicited by train of stimulation

 

Renzapride reduces recovery time of fEPSP from rundown

F orskolin mimics the effects of renzapride

 

 

H-89 abolishes the effects of renzapride

113

115

119

121

123

126

128

131

133

I36

Activation of presynaptic 5-HT4 receptors increases release probability from

single varicosities

 

139

A proposed model for P2X-mediated afterhyperpolarization in S neurons --154

A proposed model for 5-HT4 receptor-mediated presynaptic regulations ---

169

A summary of neural circuitry in myenteric plexus with emphasis on the new

 

findings in the thesis

180

Note: Images in figures 01, 02, 03, 04, 22, 39, 40 and 41 are presented in color. A black

and white copy may not represent the original information.

xi

INTRODUCTION

THE ENTERIC NERVOUS SYSTEM

Overview

The enteric nervous system (ENS), sympathetic and parasympathetic nervous
systems compose the autonomic nervous system (ANS). The ENS is intrinsic to the
gastrointestinal (GI) tract. The ENS is the collection of neurons and supporting cells
located within the wall of GI tract, including the neurons within the pancreas and gall
bladder (Fumess and Costa, 1987). The ENS is composed of two major interconnected
ganglionic plexuses: the myenteric (Auerbach’s) and submucosal (Meissner’s) plexuses
(Fumess and Costa, 1987). Auerbach and Meissner are the two neuroanatomists who first
provided detailed descriptions of the arrangement of the neurons and nerve fibers of the
plexuses that bear their names. The descriptions of the two groups of neurons as the
myenteric and submucosal plexuses are more meaningful in terms of their anatomical

arrangement and this terminology is currently used widely.

The ENS is highly autonomous as it can function independent of central nervous
system (CNS) control. Although it is innervated by efferent nerve fibers originating in
neurons in sympathetic, parasympathetic ganglia and sensory afferent fibers from
neurons in the nodose and dorsal root ganglia, the ENS can control most GI functions
after these extrinsic nerves have been cut. The ENS is often referred as the “little brain”
or “second brain” to denote its functional autonomy (Gershon, 1999). The ENS can
operate autonomously because it contains all of the components required for most GI

reflexes (Bertrand, 2003; Fumess et al., 2004). The original data supporting this

conclusion can be tracked back to 1899 when Bayliss and Starling observed a pressure-
evoked descending propulsive wave consisting of oral contraction and anal relaxation
(Bayliss and Starling, 1899). They removed sympathetic and parasympathetic
innervations of the intestine and found no interruption of the pressure-evoked reflex.
They proposed the “ Law of the Intestine” which stated that “local stimulation of the gut
produces excitation above and inhibition below the excited spot. These effects are
dependent on the activity of the local nervous mechanisms.” (Bayliss and Starling, 1899).
In 1917 Trendelenburg conducted a further study and observed the same type of reflex in
guinea pig gut in vitro, which Trendelenburg called peristalsis (F umess and Costa, 1987).
These pioneering studies demonstrated that the peristaltic reflex could occur in vitro
when the bowel is obviously isolated from the CNS and sensory ganglia. They provided
indirect evidence that the ENS contains all the elements and complete intrinsic neural
circuits that underlie the coordinated movement of GI smooth muscle. Modern
experimental techniques have facilitated steady progress in identifying these components
and the neural circuits they form (Fumess, 2000; Fumess et al., 2004; Bertrand 2003.
Also see below “classification of myenteric neurons and neural circuits” for details).
Briefly, the individual components include sensory neurons that transduce chemical or
mechanical stimulation to initiate a reflex; intemeurons that relay the inputs and mediate
the reflex and; excitatory and inhibitory motor neurons that cause coordinated contraction
and relaxation of smooth muscle to accomplish peristalsis (Bertrand, 2003; Fumess et al.,

2004).

So, when J. N. Langley formally defined the ANS, he cited anatomical and

functional evidence that led him classify the ENS as separate from the sympathetic and

parasympathetic divisions. In his comprehensive review (Langley, 1921), he pointed out

that:

“...we should expect the cells of A uberbach ’s and Meissner ’s plexuses to be on the
course of the bulbar and sacral nerves, but as there was no clear proof of their central
connection, and as their obvious histological characters diflered from those of any other
peripheral nerve cell, I placed them in a class by themselves as the enteric nervous

D

system... ’

Anatomy of the GI tract and arrangement of the enteric plexuses

The GI tract is a muscular tube extending from the mouth to the anus. In general,
the GI tract includes the esophagus, stomach, duodenum, jejunum, ileum and large
intestine. The duodenum, jejunum and ileum compose the small intestine. The GI tract
has a similar laminar structure in these different segments and consists of two smooth
muscle layers, mucosa and the enteric plexuses (Schofield, 1968; Fumess and Costa,
1987). There is an inner circular and an outer longitudinal smooth muscle layer. The
anatomic arrangement for all these layers is, in order from serosal to lumen side,
longitudinal smooth muscle, myenteric plexus, circular smooth muscle, submucosal
plexus and mucosal plexus (Figure 1). This kind of arrangement provides an anatomical
basis for the function of the two enteric plexuses fimction. The myenteric plexus controls
motility as it exists between two smooth muscle layers. The submucosal plexus regulates

secretory and absorptive function and it lies just below the mucosal layer.

Figure l. The general organization of the enteric nervous system

A schematic diagram adapted from Fumess and Costa (1987) illustrates the
organization of the enteric nervous system as seen in a whole mount of small intestine.
The lumenal mucosa is at the bottom while the serosal surface is at the top. The
submucosal plexus is located between the circular muscle and the mucosal layer. The
myenteric plexus is located between the longitudinal and circular smooth muscle layers.
In guinea pig small intestine, the myenteric plexus is attached tightly to the longitudinal
muscle. Therefore, the longitudinal muscle-myenteric plexus (LMMP) preparation is
used to study myenteric neurons. Synaptic connections in the myenteric plexus that are

present in vivo are preserved in the LMMP preparations.

Figure l

Interganglionic
Myenteric Ganglion Fiber Tract

  
 
   
 
  

Submucosal
Ganglion

Longitudinal
Muscle

Morphology and ultrastructure of enteric neurons

The ENS contains more than 100 million neurons, which are as many as the
number of neurons in the spinal cord. Alex Dogiel (1899) provided a complete and
accurate description of the morphology of enteric neurons. He used methylene blue as
histological stain to reveal neuronal morphology. He demonstrated three groups of enteric
neurons based on soma shape, soma size and the number and distribution of neuronal
processes (Fumess and Costar, 1987). The morphological classification scheme of enteric
neurons is named after him. Dogiel type I cells are flat, with stellate or angular shapes.
The size of cell bodies was 13- 35 pm in length and 9-22 pm in width. They had one
axon (unipolar) and 4-20 or more short dendrites. Because many of these cells sent single
axons to the muscle, Dogiel concluded these neurons were motor neurons. Dogiel type 11
cells were described as angular, star or spindle shaped with multiple long neural
processes. The cells had smooth surfaces with long axes of 22-47 um and short axes of
13-22 pm. These cells projected multiple processes to the mucosa and adjoining ganglia
and were recognized as sensory neurons. Dogiel type III cells were similar to type 1 cells.
They had a uniopolar shape with 2—10 filamentous dendrites. Type III neurons were
suggested to be motor neurons. Later studies have suggested there was an overlap
between type I and III neurons in morphology therefore these two cell types were re-

classified as type I neurons.

Ultrastructural studies of enteric ganglia reveal important differences from other
autonomic ganglia. Enteric ganglia do not have internal capillaries and connective tissue

(Cook and Burnstock, 1976) and the enteric neurons in ganglia are packed tightly without

 

satellite cell sheaths (Llewellyn-Smith et al., 1981). Electron microscopy has identified
eight types of neurons within myenteric ganglia in guinea pig small intestine based on the
size and the intracellular unltrastructure (Cook and Bumstock, 1976). However, the
submucosal ganglionic neurons do not differ from each other in their ultrastructural
appearance as they all have broad and thin dendrites, axo-somatic and axo-dendritic
synapses and similar perikarya of 12-30 pm (Wilson et al., 1981; Komuro et al., 1982).
Ultrastructural studies have also revealed that there are multiple types of synaptic vesicles
that can be differentiated based on size and vesicle content. These observations have led
to the suggestion that enteric neurons use multiple transmitters and some nerve terminals
contain and release more than one type of transmitter (Fumess and Costa, 1987;
Llewellyn-Smith et al., 1989). Myenteric neurons are directly contacted by varicosities.
However the pre-and postsynaptic specializations are not different from synapses in other

autonomic ganglia or in the CNS (Llewellyn-Smith et al., 1989).

Electrophysiology of myenteric neurons

Electrophysiological studies of the ENS began with extracellular recordings from
the myenteric plexus (Wood, 1970). Later, intracellular microelectrode recordings were
made from the myenteric plexus (Hirst et a1 1974; Nishi and North 1973) and submucosal
plexus (Hirst and Mckirdy 1975). Patch-clamp recording techniques have also been used
to record from cultured myenteric neurons (Zhou and Galligan, 1996) and submucosal
neurons (Barajas-lopez et al., 1998) and in acutely isolated LMMP preparations (Rugiero
et al., 2002). Studies in LMMP preparations provide information that represents

Physiological conditions in vivo while studies done in cultured neurons allow

 

investigations of electrophysiological properties in more detail than is possible using the
intact plexus preparation. Figure 2 contains schematic diagrams showing the setups for
these two types of electrophysiological recordings. These studies have characterized the
electrophysiological properties of myenteric neurons and the ion channel basis of these
properties (Galligan et al., 1989; Galligan et al., 1990; Galligan, 20023 and b; Rugiero et

al., 2002; Fumess and Sanger, 2002; Bertrand, 2003).

Most intracellular electrophysiological studies of myenteric neurons have been
done in preparations from the guinea pigs. The reason to choose the guinea pigs as animal
model for GI studies is because guinea pig gut is readily dissected, reasonably long and
mechanically quiescent when studied in vitro. There have also been studies done in
mouse myenteric plexus (F urukawa et al., 1986). Gene manipulation in mice provides an
opportunity to study the electrophysiological properties of specific proteins (Ren et al.,
2003; Bian et al., 2003). Most neuronal properties are similar between guinea pigs and

mice. The following description is mainly drawn from guinea pigs unless noted.

Two distinct types of myenteric neuron have been identified based on their
electrophysiological characteristics. S neurons and AH neurons were described by Hirst
et al (1974); while Nishi and North (1973) found neurons with the same properties but
called these type I and type II neurons (Figure 3). The nomenclature of Nishi and North is
not longer used because of its similarity to the Dogiel classification of cell morphology.
In addition, Hirst’s nomenclature was based on the properties of the neurons and this is
more meaningful. Neurons responding to single focal stimulation of interganglionic nerve
fibers with a fast excitatory postsynaptic potential (fEPSP) were termed S neurons; the

fEPSP is the predominant mechanism of synaptic excitation in the GI tract. Action

Figure 2. Electrophysiological recording from single myenteric neurons

A. Intracellular electrophysiological recordings are made from single myenteric
neurons in LMMP preparations. Myenteric ganglia can be visualized using a microscope
with differential interference contrast optics. A sharp microelectrode filled with 2 M KCl
and with a tip resistance of ~100 M ohm is used to impale single neurons. Synaptic
activity can be evoked by electrical stimulation of interganglionic nerve bundles.
Agonists can be applied locally by pressure ejection from a drug pipette positioned close

to the impaled neuron.

B. Patch-clamp recordings are made from single myenteric neurons maintained in
primary culture. The picture shows the cultured myenteric neurons visualized using a
microscope with differential interference contrast optics. Cultured myenteric neurons
grow processes that form synapses with other myenteric neurons. Synaptic responses can
be elicited by stimulating presynaptic single nerve fibers. Agonists can be applied locally

by gravity-driven flow from a drug pipette positioned close to the target neuron.

Figure 2

A

STIMULATING
ELECTRODE

Drug pipette var—Tr —— I—, -- RECORDING
’ _, - r, , ELECTRODE

    

   

 

Stimulating Electrode
Recording Electrode

Figure 3. Myenteric AH and S neurons

A. Photomicrographs of an AH neuron morphology and its electrophysiological
properties. AH neurons are bipolar with several dendrites and a smooth cell body. Action
potentials in AH neurons have a long-lasting (1-10 5) hyperpolarization pointed by the
arrow (lower left). Trains of stimulation (as arrow shows) evoke slow EPSPs from AH

neurons (lower right).

B. S neurons have one axon and multiple short dendrites. Single electrical stimuli
(as arrow shows) elicit fEPSPs from S neurons (lower right). Action potentials in S
neurons have a short afterhyperpolarization as the arrow shows (lower left). For
morphological studies, microelectrodes filled with 4% Neurobiotin were used to record

from these neurons. Neurobiotin was localized with a Cy3 conjugated avidin.

12

Figure 3

 

 

A
All neuron
/ Afterhyperpolarization
—2 s f 2 s
St' I t.
Action potential 1mu a Ion sEPSP
B

S neuron

 

#w

 

 

_ "T v "‘ '1 —
2 S / 10 ms
Afierhyperpolarization Stimulation
Action potential fEPSP

potentials recorded from the soma of S neurons were mediated solely by voltage-gated
NaJr channels and they can be blocked by tetrodotoxin (TTX). The action potential in S
neurons is followed by an afterhyperpolarizaion of ~20 ms duration (Hirst et al 1974;
Bomstein et al., 1994). Most S neurons fire action potentials continuously when
depolarized by a current pulse injected through the recording microelectrode (Bomstein
et al., 1994). Inward rectifier potassium current (Ikir) modulates the resting membrane
potential of S neurons (Galligan et al., 1989; Rugiero et al 2002). As myenteric S neurons
receive mainly excitatory synaptic inputs, the membrane potential is unlikely to go to
values more negative than 13.. (~ -80 mV) where Kir channels operate more actively and
potassium ions leave the cell. Under physiological conditions, IKir hyperpolarizes the

neurons to maintain resting membrane potential.

AH neurons are so named because the action potentials generated in soma are
followed by long-lasting (1-10 5) afierhyperpolarization (AHP) (Figure 3) (Nishi and
North, 1973; Hirst et al., 1974; Fumess et a1, 1998). AH neurons cannot fire a train of
action potentials because the AHP limits the firing rate. The AHP is mediated by
activation of an intermediate conductance calcium-dependent potassium channel (Imp)
(Morita et al., 1982; Hirst et al., 1985; Galligan et al., 1989; Vogalis et al., 2002). The
action potential of AH neurons has two components: 1) a TTX-sensitive sodium current
and, 2) a calcium current. Calcium action potentials are mediated by N-type calcium
channels as N-type calcium channel blocker m—conotoxin reduces the duration of action
potentials and the amplitude of the afterhyperpolarization (F umess et al., 1998; Vogalis et

al., 2001; Rugiero et al., 2002). In addition, immunoreactivity for the alB (N-type)

l4

calcium channel subunits is localized to AH neurons, whereas only weak alA (P/Q-type
calcium channel subunit) immunoreactivity was observed (Kirchgessner and Liu, 1999).
R type calcium channels are only expressed in the nerve fibers (Naidoo, personal
communication) therefore it does not seem to contribute to action potential but to
contribute to regulation of synaptic transmission. AH neurons contain calbindin
immunoreactivity, which is a neurochemical marker to differ AH neurons from S
neurons. A cationic current activated by hyperpolarization (1..) appears to be one major
conductance in AH neurons (Galligan et al., 1990; Rugiero et al., 2002). Activation of 1;,
was suggested to increase excitability in AH neurons by keeping the membrane potential
of AH neurons less hyperpolarized in response to activation of [AHP and Ikir. Some AH
neurons receive fast synaptic inputs but slow synaptic transmission is the primary

mechanism of synaptic excitation in AH neurons (Galligan et al., 2000; Fumess et al.,

2004)

Functional classification of myenteric neurons

Classification of myenteric neurons has accomplished using morphological,
electrophysiological or neurochemical properties (Fumess, 2000; Brookes 2001). A
combined classification scheme that includes function would be more significant in a way
that can facilitate understanding the functional significance of the myenteric neurons with
different properties. This revised scheme requires correlating the electrophysiology, the
neurochemistry and the morphology of enteric neurons with their functions. This will

help to uncover the mechanisms underlying neural regulation of GI function.

15

There has been significant progress in defining the types of enteric neurons in the
context of their roles in enteric neural circuitry (Bertrand, 2003; Fumess et al., 2004).
Correlation of electrophysiology, morphology and function has been accomplished by
injecting marker dyes into neurons that had been recorded from and subsequently
examining cell shape and neurochemical content (Hodgkiss and Lees, 1983; Bomstein et
al., 1984; Iyer et al., 1988; Messenger et al., 1994; Clerc et al., 1998; Lomax and Fumess,
2000; Tamura et al., 2001; Nurgali et al., 2003). Based on these kinds of studies it has
been established that the myenteric plexus contains three major types of neurons. They
are motor neurons, intemeurons and intrinsic primary afferent neurons (IPANs). IPANs
are so termed is to differentiate them from the extrinsic sensory neurons whose cell
bodies are in dorsal root and nodose ganglia, which have afferent connections with the
ENS (Fumess et al., 2004). Motor and intemeurons have Dogiel type I morphology and
IPANs have Dogiel type II morphology (Figure 3). Five subtypes of motomeurons, one
subtype of ascending and four subtypes of descending intemeurons have been identified
(Fumess, 2000; Brookes, 2001; Fumess and Sanger, 2002). There are also a small
number of intestinofugal neurons (<1% in number) that send afferent fibers to
prevertebral ganglion and these connections are part of intestinofugal reflex pathways
(Fumess, 2002; 2003). Each type of neuron has neurochemical coding and
electrophysiological characteristics (Fumess, 2000; Fumess and Sanger, 2002; Bertrand,
2003). For example, inhibitory longitudinal motor neurons express nitric oxide synthase
(NOS), vasoactive intestinal peptide (VIP) and GABA while excitatory longitudinal

motor neurons express choline acetyltransferase, calretinin and substance P. IPANs are

16

the only neuron type that expresses calbindin. Electrophysiologically, motor- and

intemeurons are S neurons while IPANs are AH neurons.

Intrinsic neural circuits in the myenteric plexus

Studies of neuron types have provided the information that helps to understand '
how myenteric neurons are organized to form a functional network that underlies the
peristaltic reflex (Figure 4). Because the peristaltic reflex is based on distention-evoked
contraction oral to the point of distention and relaxation anal to the distention point, the
organization of the intrinsic neural circuitry must be polarized accordingly. The
accumulated data suggest that IPANs are excited indirectly by intraluminal chemical or
mechanical stimulation through sensory mediators released from enterochromaffin (EC)
cells in mucosa. The identified mediators released from EC cells are 5-HT and ATP
(Bertrand, 2003). 5-HT acts at 5-HT3 receptors and ATP acts at P2X receptors on nerve
terminals to activate myenteric IPANs antidromically (Fumess et al., 2004). S-HTlp
localized to terminals of submucosal IPANs may also mediate sensory information
(Gershon, 2004). IPANs then activate intemeurons via cholinergic and purinergic
synaptic transmission in the descending pathway and cholinergic synaptic transmission in
the ascending pathway (Galligan, 2002b). Interneurons then activate motomeurons,
which excite smooth muscle orally and inhibit smooth muscle anally to generate the
peristaltic reflex. It is very clear that fast synaptic transmission between these neurons
plays an important role in process sensory information (see “fast synaptic transmission”

for details). However, the mechanisms by which fast synaptic transmission may encode

l7

Figure 4. Intrinsic neural circuitry in myenteric plexus

This diagram a propose model of the enteric neural circuit underlying the
peristaltic reflex. Intrinsic primary afferent neurons (IPANs) are AH neurons while inter-
and motomeurons are S neurons. Intraluminal mechanical or chemical stimulation of the
mucosa releases 5-HT from enterochromaffin cells. S-HT acting at 5-HT3 receptors
localized to nerve terminals of myenteric IPANs excites the IPAN antidromically.
Activation of IPANs releases transmitters to activate intemeurons in orally and anally
directed synaptic pathways. Subsequent activation of intemeurons stimulates inhibitory

or excitatory motor neurons to relax or contract smooth muscle, respectively.

IPAN: intrinsic primary afferent neuron; LM: longitudinal muscle; MP: myenteric

plexus; SM: smooth muscle; SMP: submucosal plexus;

18

Figure 4

 

sensory
motomeuron intemeuron neuron intemeuron motomeuron

 

 

has:
_L
MP éACh ”AC

 
  

 

CM
SMP

Mucosa

   

Enterochromaffin cell

Mechanical or chemical stimuli

and regulate sensory information causing responses to a variety of physiological

stimulation are still unknown.

Extrinsic innervation

Myenteric and submucosal plexuses also receive extrinsic inputs through
parasympathetic and sympathetic nerves. The sympathetic preganglionic cell bodies are
in thoracic (T) and lumbar (L) segments of the spinal cord. Their axons end on the
postganglionic neurons in celiac ganglia, superior and inferior mesenteric ganglia.
Sympathetic postganglionic noradrenergic fibers innervate the myenteric plexus, the
submucous plexus, and the mucosa, but have little input to the muscle layers (F urness and
Costa, 1987; King and Szurszewski, 1989; Llewellyn-smith et al., 1993). The majority of
postganglionic sympathetic neurons innervating the ENS are found in celiac-mesenteric
ganglia. They project via splanchnic nerves to the stomach and the small intestine. Some
sympathetic innervation originates from cervical chain ganglia and projects to the upper

small intestine.

The parasympathetic innervation of the GI tract originates in the cervical and
sacral portions of the spinal cord and terminates in the esophagus, stomach, pancreas,
sigmoid colon, rectum and anus (Kirchgessner and Gershon, 1989). Only few vagal
efferent fibers innervate the small intestine. Sympathetic and parasympathetic innervation
regulates GI function. Sympathetic postganglionic neurons release norepinephrine (NE)
to inhibit enteric excitatory cholinergic neurons through presynaptic mechanism (Hirst

and McKirdy, 1974) by acting at a2-adrenergic receptors (Frigo et al., 1984). Activation

20

of sympathetic nervous system results in decreased motility of GI smooth muscle.
Parasympathetic nervous system provides only excitatory innervation to GI tract.
Stimulation of the parasympathetic nervous systems results in the release of ACh, which

causes an increase in GI activity.

There is also afferent innervation that conveys sensory information from the GI
tract to the CNS where GI reflex is coordinated and integrated with conscious behavioral
responses. The extrinsic sensory innervation originates from enteric plexus and follows
two routes to the CNS. The cell bodies of vagal afferent neurons are located in nodose
ganglia while the cell bodies of spinal afferent neurons are located in dorsal root ganglia.
Pseudo-unipolar sensory neurons in nodose ganglia send their central processes to the
nucleus tractus solitarius (NTS) of the medulla in brain stem and their peripheral
processes to the GI tract via vagal nerve (Berthoud et al., 2004). Vagal afferent is
considered to convey the sensory information from the GI tract that generates the
consciousness, for example satiety (Grundy and Scratcherd, 1997). Spinal afferents enters
spinal cord to make synapses with the second order neurons that convey GI nociceptive
information to the CNS. There is a differential innervation of these afferents throughout
the GI tract. Vagal afferents end largely in the upper GI tract including the esophagus and
stomach (Powley and Phillips, 2002). For spinal afferents, pelvic afferents are limited in

the lower bowel. In contrast, splanchnic afferents innervate the whole GI tract.

Submucosal neurons

The submucosal plexus is a second important component of the ENS. It underlies

the regulation of intestinal secretory reflexes (Cooke, 1999). Submucosal neurons receive

21

their intrinsic innervation mainly from myenteric neurons and partly from other
submucosal neurons (Bomstein et al., 1987). They also receive major extrinsic
innvervation from noradrenergic neurons in the prevertebal sympathetic ganglia (Mihara
et al., 1997). In submucosal plexus, four distinct types of submucosal neurons have been
identified based on neurochemical properties (Mihara et al., 1997; Fumess, 2000;
Brookers, 2001). Some submucosal neurons have Dogiel type II morphology. The other
submucosal neurons have filamentous morphology (Brookes, 2001). Non-cholinergic
secretomotor/vasodilator neurons are immunoreactive for dynophin (DYN), galanin
(GAL) and VIP. These neurons account for 45% of submucosal neurons and they use
VIP as their secretomotor neurotransmitter. Cholinergic secretmotor neurons account for
30% and they are immunoreactive for ChAT, calcitonin gene-related peptide (CGRP) and
neuropeptide Y (NPY). Submucosal IPANs (10%) that have immunoreactivity of ChAT,
DYN and substance P (SP) and they use Ach as transmitter. Cholinergic intemeurons

comprise 15% of submucosal neurons and they are immunoreactive for ChAT and DYN.

Electrophysiologically, submucosal neurons can generate three types of synaptic
potentials in response to focal nerve stimulation (Mihara, 1993). Fast EPSPs can be
recorded from motor and intemeurons afier single electrical stimuli. Fast EPSPs are
mediated by cholinergic inputs from neurons in both myenteric and submucosal plexuses.
Slow EPSPs are evoked by trains of stimulation and slow EPSPs are likely mediated by
SP, VIP and 5-HT (Mihara et al., 1997). Slow IPSPs can be recoded from non-
cholinergic secretmotor neurons: slow IPSPs are mediated by norepinephrine released
from extrinsic sympathetic nerves. Norepinephrine acts at a2-adrinergic receptors

(Surprenant and North, 1987).

22

SYNAPTIC TRANSMISSION

Overview

Neurons communicate with each other through synaptic transmission, which
occurs at a specialized neuronal structure, the synapse, a milestone-like concept coined
by C. Shenington. Enteric neurons use chemical synaptic transmission to mediate neural
signals. Synaptic transmission at chemical synapses is complex in structure, fiinction and
modulation mechanisms (Eccles, 1982; Walmsley et al., 1998). Neurotransmitters
released from presynaptic nerve terminals act at receptors located at postsynaptic neurons
(Ruff, 2003). Transmitter release occurs from nerve terminals after an influx of calcium
produced by an action potential, triggers the excytosis of neurotransmitter vesicles
clustered at the active zone (Smith and Augustine, 1988; Zucker, 1993; Martin, 2002).
Some transmitters are recovered by uptake by specific transporters and some will be
degraded in the synaptic cleft by enzymes (McMahan et al., 1978; Amara and Arriza,
1993). Most chemical synaptic transmission is unidirectional. However, the retrograde
synaptic transmission may occur in some tissues (Jessel and Kandel, 1993; Levenes et al.,
2001; Diana et al., 2002). Synaptic modulation can occur by targeting presynaptic nerve

terminals or/and postsynaptic neurons.

There are two broad mechanisms of chemical synaptic transmission based on
synaptic delay between a presynaptic action potential and the onset of the postsynaptic
change in membrane potential caused by the release neurotransmitter: slow and fast

synaptic transmission.

23

Slow synaptic transmission in myenteric plexus

Slow synaptic transmission occurs through activation of G-protein coupled
receptors (GPCRs) by neurotransmitters. GPCR activation alters the membrane potential
of postsynaptic neurons through activation or inhibition of a number of second
messengers leading to close or open of ion channels. GPCRS are a family of membrane
proteins with seven transmembrane domains (Ji et al., 1998). The third intracellular loop
is the G-protein binding site (Wess, 1997). G-proteins are trimers composed of a, B and 7
subunits (Hamm, 1998). In general, two mechanisms mediate extracellular signals by
activation of GPCRs. The first one is a signaling pathway through activated G protein a
subunit resulting in an increase or decrease in the activity of a target enzyme (typically a
protein kinase) via a second messenger (Gilman, 1987). The protein kinase then
modulates the target effectors such as ion channels and gene expression to alter neuronal
properties (Hille, 1994; Hamm, 1998). In addition, the G protein [3 and 7 subunit complex
can directly act at the ion channels as membrane de-limited factors to modulate ion
channel kinetics (Wickman et al., 1994; Huang et al., 1995; Clapham and Neer, 1997). As
there are multiple-step processes following activation of GPCRs including agonist
binding, the subsequent activation of signaling pathways and the activation of target ion
channels, GPCRs mediate slow synaptic transmission with an onset of 50-100 ms and
effects on neuronal excitability lasting seconds to minutes. Slow synaptic transmission is
an important signaling mechanism in the ENS (Surprenant and North, 1988; Bertrand and
Galligan, 1994; Bertrand and Galligan, 1995; Pan et al, 1997; Alex G et al., 2001;
Galligan, 2002 a, b). In the ENS, examples of GPCRs include NK—3 receptors activated

by substance P (SP) (Holzer and Holzer-Petsche, 2001), 5-HT.p receptors activated by 5-

24

HT (Pan et al., 1997) and P2Y receptors (Bomstein et al., 2002) activated by ATP. Slow
synaptic responses in the ENS are due to a concurrent decrease in potassium conductance
and increase in chloride conductance (Bertrand and Galligan, 1994). Slow synaptic

transmission occurs predominately in AH neurons (Galligan, 20023).

Fast synaptic transmission and ligand-gated ion channels in myentric neurons

Ligand-gated ion channel-mediated fast synaptic transmission is the second broad
class of synaptic transmission in the ENS (Galligan, 2002a). Ligand-gated ion channels
are membrane proteins composed of multiple subunits. The ligand binding sites and the
ion channels are localized within the same protein complex (Lester, 1992). There is no
second messenger needed to convey the primary signals via ligand-gated channels (Uings
and Farrow, 2000; Breitinger, 2001). Therefore, the synaptic delay is less than 1 ms and
the duration of transmitter release and response lasts less than 100 ms (Panas et al.,
2000). Several ligand-gated ion channels mediate fEPSPs in myenteric neurons. The
major receptors include nicotinic cholinergic receptors (nAChRs), purinergic P2X
receptors and 5-HT3 receptors. These receptors have been localized to S neurons and they

receive synaptic inputs from AH neurons and other S neurons to mediate reflex activation
(Galligan, 2002a).
Nicotinic acetylcholine receptors (M C hRs)

Acetylcholine (ACh), acting at nAChRs, is the predominant contributor to fast
synaptic excitatory neurotransmission in the ENS (Nishi and North, 1973; Hirst et al.,

1974; Galligan and Bertrand, 1994; Zhou et al, 2002). In myenteric plexus, application of

25

nAChR antagonists inhibits fEPSPs, at least partly, in 92% of S neurons. In submucosal S
neurons fEPSPs are exclusively mediated by ACh acting at nAChRs (Hirst and Mckirdy,
1975; Suprenant, 1984). In the myenteric plexus, 25% of S neurons generate fEPSPs that
are completely blocked by nAChR antagonists (Galligan et al., 2000); fEPSPs in the
remaining neurons have non-cholinergic components (Galligan and Bertrand, 1994;
Galligan et al., 2000). Cholinergic fEPSPs play a primary role in ascending reflex
pathway in guinea pigs (Johnson et al., 1996; Spencer et al., 2000). Cholinergic fast
synaptic transmission also mediates signals in descending reflex pathways in the rat colon

and guinea pig ileum (Johnson et al., 1996; Bian et al., 2003).

Neuronal nAChRs are heteromeric pentamers that are composed of different
combinations of a and B subunits; each subunit four transmembrane domains. The
electrophysiological and pharmacological properties of nAChRs are determined by the
specific subunit composition (Albuquerque et al., 1996; Jones et al., 1999; Skok, 2002).
Fast EPSPs and ACh-induced responses in myenteric neurons have a reversal potential of
0 mV (Galligan and Bertrand, 1994), which suggests that nAChRs are permeable to
cations. Data from immunohistochemical and pharmacological studies done in myenteric
neurons maintained in primary culture suggest that S neurons do not express ()7 subunit-
containing nAChRs in the cell body but most S neurons express nAChRs that contain
(1301584 subunits (Kirchgessner and Liu, 1998; Zhou et al., 2002). Cholinergic fEPSPs
can also be recorded from AH neurons (Fumess et al., 1998; Galligan et al., 2000). But
the amplitude of fEPSP in AH neurons is smaller than that in S neurons as there may be a
lower density of nAChRs expressed in AH neurons (Barajas-Lopez et al., 2001; Galligan,

2002a). Patch-clamp studies performed in cultured myenteric neurons indicate that

26

nAChR-mediated inward currents have a reversal potential of 0 mV and the current-
voltage relationship is inward rectified. These results confirm that nAChRs are non-
selective cation channels and also suggest that nAChRs operate more actively in
depolarizing the neurons to increase excitability. nAChRs desensitize fast within 0.3
second by 80% in response to steady-state application of ACh. Single channel currents
have linear relationship with the potentials, which suggests that inward rectification of
whole cell currents is due to a decrease in nAChR open probability at positive membrane

potential (Zhou et al., 2002; Brown et al, 2003).

In addition to nAChRs localized to somatodendritic region of S neurons, evidence
indicates that nAChRs may also be localized to nerve terminals. These nAChRs mediate
release of tachykinin peptides in myenteric ganglia and at the motor neuron-
circular/longitudinal muscle junction (Galligan, 1999; Schneider and Galligan, 2000;
Schneider et al., 2000). The mechanisms and the physiological functions of presynaptic

nAChRs remain firrther studied.

Purinergic P2X receptors

The non-cholinergic component of fEPSPs recorded from 67% of myenteric S
neurons is sensitive to the P2X receptor antagonists PPADS or suramin (Galligan and
Bertrand, 1994; Galligan 2002a, b). This indicates that ATP acting at P2X receptors is a
co-mediator with ACh acting at nAChRs in mediating fast synaptic excitation in
myenteric neurons. But the purinergic fEPSPs are not evenly distributed in the whole
gastrointestinal tract. Recordings from different segments of the gut indicate that

purinergic fEPSPs predominate in ileum compared with that in duodenum, jejunum,

27

proximal and distal colon (LePard et al., 1997). There is also a preferred oral-anal
polarity of purinergic projections in myenteric plexus (LePard and Galligan, 1999). In
guinea pig ileum, by stroking the mucosal villi and recording synaptic responses, it has
been concluded that P2X receptors mediate fast synaptic transmission between
intemeurons in descending excitatory reflex pathway (Spencer et al., 2000; Monro et al.,
2002). P2X receptors are also shown to mediate synaptic transmission from intemeurons

to inhibitory motor neurons in guinea pig ileum (Bian et al., 2000).

P2X receptors may also mediate activation in AH neurons and contribute to
initiation of peristalsis (Bertrand and Bomstein, 2002). In this study, ATP applied to the
cell body evoked a large depolarization in most AH neurons. The depolarization was
blocked by PPADS. Application of ATP to the mucosal terminals of AH neurons can
trigger action potentials that propagate back to the soma. The action potentials were also
abolished by PPADS. Although the pharmacology of P2X receptors mediating these
responses need to be further studied, P2X receptors obviously play physiological function
in AH neurons mediating sensory information. However, the localization of ATP sources
remains unclear. Study from vagal neurons has suggested that ATP may be co-transmitter
with histamine in mast cells (Kreis et al., 1998). ATP is also localized in the vesicles in
EC cells (Tamir and Gershon, 1990). In vivo, ATP released from these sources may serve

as stimulator to P2X receptors.

P2X receptors are ligand-gated non-selective cation channels. But the molecular
structure is different from nAChRs as P2X receptors may be trimers consisting of
combinations of one or more of seven different subunits (Khakh et al., 2001; North,

2002; Galligan, 2002a, b). Each subunit has two membrane-spanning domains (Khakh et

28

al., 2001). Studies of heterogeneous expression of P2X receptor subunits have generated
a series of data for the properties of different P2X subunit combinations (North, 2002).
P2X receptors can be composed of the same or different subunits to form homomers or
heteromers, respectively. All P2X subunits can form homomers except the P2X6, which
only combines with other subunits, whereas all P2X subunits can form heteromers except
the P2X7, which can only form homomers. The specific composition of the P2X receptor
determines the unique pharmacological and physiological properties. Pharmacological
data have indicated there are or,B-mATP-sensitive P2X subtypes (P2X1 or P2X3) as well
as P2X; subtype in myenteric S neurons of guinea pig intestine (Zhou and Galligan,
1996; LePard et al., 1997). Immunohistochemical studies have shown that P2X2 and
P2X3 subunits are expressed in myenteric neurons (Castelucci et al., 2002; Poole et al.,
2002). However, lack of selective drugs has impaired a full pharmacological

identification of P2X receptor subtypes in myenteric neurons.

Electrophysiological characterization of P2X receptors made in cultured
myenteric neurons has shown similarities and differences to nAChRs. ATP-induced
whole-cell currents show inward rectification, which is due to a decrease in the open
probability of single channels at more positive membrane potentials as single channel
current-voltage relationship is linear (Zhou and Galligan, 1996). The reversal potential of
0 mV indicates that P2X receptors are permeable to cations. A major difference between
myenteric P2X receptors and nAChRs is that ATP induces inward currents that
desensitize by 80% in 7 seconds and ACh currents desensitize by 80% in less than 1 3
(Zhou and Galligan, 1996; Zhou et al., 2002). This difference may contribute to their

distinct fimctions in mediating synaptic signals in enteric neurons.

29

5-HT3 receptors

5-HT3 receptors are ligand-gated cation channels (Derkach et al., 1989; Fletcher
and Barnes, 1998). These receptors belong to a same superfamily with nAChRs in overall
structure. 5-HT3 receptors are pentamers with each subunit having four transmembrane
domains. In the GI tract, 5-HT3 receptors are found in myenteric sensory nerve endings
localized to the mucosal layer where they initiate the motor reflex by responding to 5-HT
released from EC cells when mucosal villi are stimulated (Foxx-Orenstein et al., 1996;
Bertrand et al., 2000; Galligan, 2002 a, b). In addition to triggering the sensory signal, 5-
HT3 receptors also mediate fast synaptic transmission in some S neurons (Zhou and
Galligan, 1999). These 5-HT3 receptors are localized to the cell body of myenteric S and
AH neurons in small intestine where they mediate rapidly developing and desensitizing
depolarization (Zhou and Galligan, 1999; Zhai et al., 1999). Fast EPSPs from about 11%
S neurons have components that are not cholinergic or purinergic (Galligan et al., 2000).
These fEPSPs are inhibited by ondansetron, a 5-HT3 receptor antagonist. S-HT3 receptor-
mediated fEPSPs are involved in both descending and ascending GI reflex pathway

(Monro et al., 2002; Yuan et al., 1994).

The electrophysiological and pharmacological properties of 5-HT3 receptors have
been studied in myenteric neurons maintained in primary culture (Zhou and Galligan,
1999; Zhai et al., 1999). More than 80% neurons generate inward currents in response to
5-HT. These currents desensitize with a double exponential time course of 1.1 and 6.9 s.
5-HT3 receptor-mediated currents do not rectify at positive membrane potentials as the

nAChRs and P2X receptors do. The current-voltage relationship for 5-HT-induced single

30

channel currents is linear, which suggests that change of potentials does not alter single

channel open probability.

Dynamics of neurotransmitter release

A number of chemicals including ATP, ACh, biogenic amines and amino acids
have been identified as neurotransmitters in the ENS. A neurotransmitter has to meet the
following criteria: 1) it is synthesized in the neuron; 2) it is present in the presynaptic
terminal and exerts a defined action on the effectors; 3) it mimics the synaptic response
when applied exogenously and 4) there is a mechanism to stop its action (Kandel et al.,
2000). In the ENS, identified neurotransmitters mediating fEPSP are ACh, ATP, 5-HT,
glutamate (Galligan, 2002a, b). Neurotransmitter release is triggered by calcium influx
produced by an action potential leading to exocytosis of neurotransmitter-filled vesicles
in quanta units. The exocytosis of transmitter vesicles is via the formation of a complex
involving many specific proteins (Hanson et al., 1997). The released vesicles can be
recycled by endocytosis, which is one mechanism for generating new synaptic vesicles

(Schweizer et al., 1995; Murthy and Stevens, 1998; Sudhof, 2004).

Although many vesicles are contained in a synaptic terminal, the vesicles are
organized into firnctionally different pools (Rizzoli and Betz, 2005); 1) the readily
releasable pool, 2) the recycling pool and 3) the reserve pool. A single action potential
triggers release of a small percentage (~5%) of vesicles to release (Tong and Jahr, 1994;
Stevens and Wang, 1995; Zucker and Regehr, 2002). These vesicles are from the readily
releasable pool (RRP) (Stevens and Tsujimoto, 1995; Stevens and Wesseling, 1999;

Zucker and Regehr, 2002). Ultrastructural studies have shown that, the vesicles in the

31

RRP are clocked at active zone sites where calcium channels are localized (Schikorski
and Stevens, 1997). The RP can be depleted rapidly by a short train of electrical
stimulation at high frequency (Elmqvist and Quastel, 1965, Richards et al., 2003). Most
transmitter vesicles (typically 80 - 90%) are contained in the reserve pool, which cannot
be released until they move into the active zone to refill RRP, a process called
replenishment. Release from the reserve pool occurs only in response to intense
stimulation. The recycling pool contains vesicles that are dynamically released and
reused under different conditions of neuronal activity (Murthy and Stevens, 1999; Rizzoli
and Betz, 2005). Vesicles in the reserve pool can also be released and recycled during
periods of prolonged and intense synaptic activity (Murthy and Stevens, 1999). Styryl
dyes such as FM1-43 are used to monitor synaptic vesicle cycling and significant

progress has been made to elucidate these processes (Cochilla et al., 1999; Sudhof, 2000).

Depletion of transmitter vesicles in the RRP is replenished slowly, which is an
explanation for rundown of synaptic transmission (Sudhof, 2000; Zucker and Regehr,
2002). Replenishment of the RRP is also an explanation for synaptic recovery, which is a
calcium-dependent process (Dittrnan and Regehr, 1998; Wang and Kaczmarek, 1998).
Synaptic vesicle trafficking between these pools is dependent on a number of proteins
(Chi et al., 2003). One of most important proteins is synapsin. Synapsin docks vesicles to
the cytoskeleton and is a target protein for phosphorylation by protein kinases (Sudhof et
al., 1989; Chi et al., 2003). Synapsin phosphorylation dissociates vesicles from the
cytoskeleton and mobilizes them for trafficking from the reserve pool to the RRP (Turner
et al., 1999). Activation of 5-HT4 receptors can stimulate protein kinase A (PKA) in the

ENS (see below). Therefore it is a reasonable hypothesis that such receptors can mediate

32

the regulation of neurotransmitter trafficking to affect synaptic activity. In the ENS, the
dynamic processes associated with synaptic transmission during trains of stimulation

have not been studied.

Recycling of synaptic vesicles

Action potentials induce neurotransmitter release by initiating the processes that
lead to fusion of synaptic vesicles with the presynaptic membrane and exocytosis of
neurotransmitter. Afier exocytosis, synaptic vesicles undergo a recycling process
including endocytosis and refilling with neurotransmitters so that they are prepared for
another cycle of transmitter release in response to the next action potential. This process
is an important mechanism that allows synaptic terminals to maintain an active supply of

transmitter-containing vesicles.

The pioneering studies of synaptic vesicle recycling were conducted at the frog
neuromuscular junction (NMJ) using electrophysiology and electron microscopy (Heuser
and Reese, 1973; Ceccarelli et al., 1973). In these studies, NMJ preparations were
stimulated in presence of horseradish peroxidase (HRP) or dextran. Exocytosis Was
assessed by recording the responses of the muscle cells to ACh released from nerve
terminals. Endocytosis was measured as the amount of HRP or dextran containing in
synaptic vesicles after tissue fixation. The membrane-attached objects with the markers
were considered as the endocytosed vesicles. Based on these studies, two models were
proposed for synaptic vesicle recycling. Using stimulation at high fi'equency, Heuser and
Reese (1973) suggested “during stimulation the intracellular compartments of this

synapse change shape and take up extracellular protein in a manner which indicates that

33

synaptic vesicle membrane added to the surface during exocytosis is retrieved by coated
vesicles and recycled into new synaptic vesicles by way of intermediate cisternae This

model became known as the “slow endocytotic pathway”.

Ceccarelli and colleagues, however, proposed a different pathway for synaptic
vesicle recycling. They used stimulation at low frequency and found (a), that synaptic
vesicles fuse with, and re-form from, the membrane of the nerve terminal during and after
stimulation and (b), that the re-formed vesicles can store and release transmitter
(Ceccarelli et al., 1973). This suggestion later leads to a “fast endocytotic pathway”

model.

Later, extensive studies have been conducted to probe the mechanisms of synaptic
vesicle endocytosis. Processes of endocytosis can be directly monitored by measuring
capacitance change (Sun et al., 2002; Sun and Wu, 2001) or by optical recording (Gandhi
and Stevens, 2003). Recycling can be assessed by monitoring the uptake of fluorescent
tracers (Wu and Betz, 1998; Cochilla et al., 1999; Murthy and Stevens, 1999). Based on
these studies, three endocytotic pathways have been proposed (Sudhof, 2004): 1) “kiss
and stay”, 2) “kiss and run”, and 3) clathrin-mediated recycling. The “kiss and stay”
model proposes that synaptic vesicles recycle directly afier closure of the vesicle
membrane-plasma membrane fusion pore formed during exocytosis. In this model,
vesicles are refilled with neurotransmitters while the vesicles remain docked to the active
zone. Vesicle recycling and refilling do not require the formation of a clathrin coat
around the vesicle. The “kiss and run” model also proposes that vesicles endocytose
without a clathrin-coated intermediate. The difference from the “kiss and stay” model is

that the vesicles do not stay in the active zone (Ceccarelli et al., 1973; Richards et al.,

34

2000). After endocytosis, recycled vesicles mix into the reserve pool. "Kiss and stay” and
“kiss and run” pathways are mechanisms for fast recycling of synaptic vesicles (Pyle et
al., 2000; Sun et al., 2002; Rizzoli et al., 2003). The third pathway, clathrin-mediated
endocytosis, is a slow recycling process. Vesicle recycling in this model requires
formation of a clathrin-scaffold that organizes several adaptor and accessory proteins
(Royle and Lagnado, 2003). The electron-dense coats around vesicles are formed by
clathrin (Heuser and Reese, 1973; Cremona and De Camilli, 1997). Clathrin-mediated
endocytosis and recycling may occur directly or via endosomes. Both slow and fast

endocytosises require GTP hydrolysis (Jockusch et al., 2005; Yamashita et al., 2005).

Rapid recycling including “kiss and stay” and “kiss and run” is a primary pathway
for synaptic vesicle retrieval during low frequencies of stimulation (Jockusch et al.,
2005). The slow recycling pathway is triggered during high frequency stimulation (Sun et
al., 2002; Jockusch et al., 2005). In studies done on synapses in the calyx of Held,
capacitance measurements revealed that endocytosis is completed with time constant of
56 ms after a single vesicle exocytotic event. This value increases to 115 ms during
stimulation at frequencies < 2 Hz and the time constant increases to 2.3 s and 8.3 s after
10 stimuli at 20 or 333 Hz, respectively (Sun et al., 2002). In retinal bipolar cells, using
capacitance measurements of membrane retrieval indicated that clathrin-dependent
endocytosis has a slow phase of membrane retrieval with time constant of 10 3 while

clathrin-independent pathway only takes 1 s (Jockusch et al., 2005).

Although the data suggest that the clathrin-dependent slow recycling pathway is
activated selectively upon high—frequency stiumulation and clathrin-independent fast

pathway is stimulated by low-frequency stimulation, the mechanisms responsible for

35

these differences remain unclear. The proteins involved in these two recycling pathways

have not been fully characterized.

S-HT4 receptors in myenteric neurons

5-HT4 receptors belong to the superfamily of GPCRs (Barnes and Sharp, 1999).
5-HT4 receptors couple to the Gs subtype of G-protein activating adenylate cyclase
increasing cAMP leading to activation of PKA. Several 5-HT4 receptor subtypes have
been identified as products of alternative splicing in different species (Medhurst et al.,
2001). All splice variants of the 5-HT4 receptors possess similar N-terrninal sequences up
to residue 359, but differ in the length of the C-terminal segment. This segment is an
important determinant of constitutive activity and receptor regulation (Blondel et al.,
1998). One of the differences is the desensitization rate in which the shorter variants (h5-
HT4a and r5-HT4s) desensitize slowly whereas the longer variants (h5-HT4b and r5-HT4L)
desensitize more rapidly (Claeysen et al., 1998). The small intestine expresses
predominately 5—HT4b receptors (Blondel et al., 1998). Radioligand binding and
functional studies in the intestine suggest that 5-HT4 receptors are present on smooth
muscle cells, excitatory cholinergic motor neurons, and terminals of IPANs. More
specifically, 5-HT4 receptors are located on the terminals of calcitonin gene-related
peptide (CGRP)-containing IPANs. Electrophysiological study indicated 5-HT4 receptor
agonists did not alter resting membrane potential of myenteric neurons or and slow
synaptic transmission when intracellular recordings were made in cell body (Pan and

Galligan, 1995). This result suggested that 5-HT4 receptors were not localized to soma.

36

Electron microscopic evidence also indicates that 5-HT4 receptors are localized to

synaptic terminals (Liu et al., 2005).

5-HT acting at 5-HT4 receptors triggers initiation of the peristaltic reflex (Craig et
al., 1990; Craig and Clarke, 1991; Foxx-Orenstein et al., 1996; Grider et al., 1996; Grider
et al., 1998; Jin et al., 1999). 5-HT4 receptors modulate fast synaptic transmission
(Kibinger and wolf, 1992; Pan and Galligan, 1994), which underlies 5-HT4 receptor
agonist-induced increases in GI motility (Grider et al., 1998). Further studies show that 5-
HT4 receptors couple to facilitation of fast synaptic transmission by enhancing both ACh
and ATP release (Pan and Galligan. 1994; LePard et al., 2004). It has been also shown
that forskolin mimics 5-HT4 receptor-mediated facilitation of fast synaptic transmission
and PKA selective inhibitors blocked 5-HT4-mediated synaptic facilitation (Galligan et
al., 2003). This 5-HT4 receptor-mediated facilitation is used as the neurophysiological
basis for the prokinetic actions of clinical drugs such as 5-HT4 receptor agonists,
cisapride and tegaserod, which are used to treat G1 motility disorders. However, the

underlying molecular and cellular mechanisms remain unknown.

37

RESEARCH GOAL

The goals of the present study were to investigate fast synaptic transmission
during trains of nerve stimulation and to identify mechanisms of synaptic modulation and

plasticity.

Purinergic P2X receptors in postsynaptic neurons mediate fEPSPs and sensory
transduction in the ENS. The subunit compositions that determine P2X receptor
properties affect synaptic transmission and sensory transduction. Identification of the
P2X receptor subtype expressed by enteric neurons will help to understand the functional

significance of fast excitatory synaptic transmission.

Previous studies on fast synaptic transmission have investigated fEPSP induced
by a single stimulation. However, fEPSP triggered by physiological stimulation occurs in
bursts, which represent the situation in vivo. Therefore it is important to investigate
fEPSPs elicited by trains of action potentials to understand the physiological functions of

fEPSPs.

Presynaptic 5-hydroxytryptamine (5-HT4) receptors mediate facilitation of fast
synaptic transmission in myenteric neurons. This dynamic change mediated by 5-HT4
receptors is very important as agonists of 5-HT4 receptors are used as prokinetic drugs to
treat certain GI disorders such as irritable bowel syndrome (138) and gastroesophageal
reflux disease (GERD). Identification of the mechanism underlying 5-HT4 receptor-
mediated facilitation will be useful in understanding the pathology and drug therapy of

these GI motility disorders.

38

Fast synaptic transmission is an important mechanism for enteric neurons to
perform their physiological functions. Alteration of fast synaptic transmission by
abnormal physiological condition can develop into GI disorders. GI disorders are
common problems throughout the world. Although they are rarely life threatening, they
negatively impact quality of life (Fumess and Sanger, 2002). Therefore study of dynamic
change of fast synaptic transmission will, in one way help to understand the neural
circuits underlying the ENS regulation of GI physiological functions; in the other way

provide information for GI pathology and GI disorder drug therapy.

Specific Aims

1. Identification of P2X receptor subtypes in myenteric neurons in small intestine.

This study was done in tissues from mice and guinea pigs. Although
immunohistochemical localization of P2X subunits in guinea pig myenteric neurons has
been done (Castelucci et al., 2002; Poole et al., 2002; VanNassauw et a1. 2002), the
subtypes of P2X receptors mediating fEPSPs in these tissues are not fully identified.
Selective agonists and antagonists were used to evaluate eletrophysiological responses to
identify P2X receptor subtypes in guinea pig myenteric neurons. In addition to using
pharmacological methods, I also took advantage of P2X2 and P2X; gene knockout mice
to investigate the contribution of these two subunits to electrophysiological properties of

myenteric neurons in mice.

2. Fast synaptic transmission elicited by trains of electrical stimulation.

39

This study was conducted in myenteric neurons of guinea pig small intestine in
vitro. Intracellular electrophysiological recording techniques including single electrode
voltage-clamp (SEVC) were used to characterize fast synaptic transmission during trains
of electrical stimulation in LMMP preparations in vitro. Fast synaptic transmission
elicited by single electrical nerve stimulation has been studied extensively. However, fast
excitatory postsynaptic potentials (fEPSPs) occur in bursts in response to physiological
stimulation. These studies have characterized fast synaptic transmission during trains of
stimulation and examined the contributions of ATP and ACh to fEPSPs during a short

train of electrical stimulation.

3. Presynaptic S-HT4 receptor-mediated modulation of fast synaptic transmission.

5-HT4 receptors mediate enhancement of single fEPSPs in myenteric neurons. 5-
HT4 receptors may also regulate the dynamic change of synaptic transmission evoked by
trains of stimulation. These studies have tested the hypothesis that activation of 5-HT4
receptors reduced the decline in amplitude (rundown) of fEPSPs during trains of stimuli
and facilitates the rate of recovery from synaptic run-down. The regulation is mediated by
5-HT agonists activating adenylate cyclase-cyclic 3’, 5’ adenosine monophosphate
(cAMP)-PKA signaling pathway. 5-HT4 receptors mediate facilitation of fEPSPs by
increasing transmitter release but the cellular mechanism remains unclear. These studies
have examined the hypothesis that activation of 5-HT4 receptors enhances the release
probability from single release sites (varicosities). Two experimental methods were used

in these studies. Intracellular electrophysiological recordings were made from LMMP

40

preparations from guinea pig tissue in vitro. Patch-clamp recordings were made in guinea

pig myenteric neurons maintained in primary culture.

41

METHODS

Animals

All the procedures for handling animals were used in this study were approved by

the All University Committee on Animal Use and Care at Michigan State University.

Intact myenteric plexuses were dissected acutely from adult Guinea pigs (Hartley,
male, 250 — 350 g) and Mice. Guinea pigs were available from Bioport Company
(Lansing, MI). Mice were obtained from Roche Bioscience (Palo Alto, CA). P2X; or
P2X3 gene knockout (P2X2'l' or P2X3'l') mice were generated as described (Cockayne et
al. 2000). All mice used in this study have the genetic background 1290la X C57BL/6
(Harlan), and were derived from homozygous F2 crosses. The comparison analysis was
performed between knockout mouse and its wild type from same family. Genotype
confirmation of all animals was carried out by southern blot analysis as previously

described (Cockayne et al. 2000).

Longitudinal muscle myenteric plexus (LMMP) preparation

Guinea pigs were euthanized by halothane inhalation and by severing the major
neck blood vessels. Mice were anesthetized with halothane and then killed by cervical
dislocation. The small intestine was removed from mice or guinea pigs and placed in
oxygenated (95% Oz and 5% C02) Krebs’ solution of the following composition

(millimolar): NaCl, 117; KCI, 4.7; CaClz, 2.5; MgClz, 1.2; NaHC03, 25; NaHzPO4, 1.2;

42

glucose, 11. The Krebs’ solution also contained nifedipine (1 uM) for blocking L-type
calcium channels and scopolamine (1 pM) for blocking muscarinic cholinergic receptors
to inhibit longitudinal muscle contractions. These drugs do not affect normal myenteric
neuronal properties. A 1.5 cm segment of intestine was cut open along the mesenteric
border and pinned out flat with the mucosal surface up in a petri dish lined with silastic
elastomer. A longitudinal muscle-myenteric plexus (LMMP) preparation was made by
peeling away the mucosal, submucosal, and circular muscle layers using fine forceps and
scissors. A 5 mm2 piece of longitudinal muscle myenteric plexus was transferred to a
small (2 ml volume) recording chamber lined with silastic elastomer. The preparation
was stretched lightly and pinned to the chamber bottom using stainless steel pins (50 pm
diameter). The preparation was superfused with 37°C oxygenated Krebs’ solution at a
flow rate of 4 ml/min. Individual myenteric ganglia were visualized at 200x
magnification using an inverted microscope (Olympus CK-2) with differential

interference contrast optics.

Myenteric neurons maintained in primary culture

Tissue for primary myenteric cell culture was obtained from newborn guinea pigs
(~36 h in age; ~70 g in weight). Newborn guinea pigs were sacrificed by severing the
major neck blood vessels and spinal cord after deep halothane anesthesia. The small
intestine was placed in cold (4°C) sterile-filtered Krebs' solution of the following
composition: ll7mM NaCl, 4.7mM KCI, 2.5 mM CaClz, 1.2 mM MgC12, 1.2 mM
NaHzPO4, 25 mM NaHCO3, and 11 mM glucose. The longitudinal muscle myenteric

plexus was removed from the entire length of small intestine and cut into 5-mm-long

43

pieces. The dissected tissues were divided into four aliquots and placed in 1 ml of Krebs'
solution containing 1600 U of trypsin (Sigma Chemical Co., St. Louis, M0) for 25 to
30 min at 37°C. After trypsin incubation, the tissues were triturated 30 times and then
centrifuged at 900g for 5 min with a bench-top centrifuge. The supernatant was discarded,
and the pellet was then be resuspended in sterile Krebs' solution and incubated (25-30
min, 37°C) in Krebs' solution containing 2000 U crab hepatopancreas collagenase
(Calbiochem-Novabiochem, Corp., La Jolla, CA). The suspension was triturated and then
centrifuged for 5 min. The pellet was suspended in Eagle's minimum essential medium
containing 10% fetal calf serum, gentamicin (10 pg/ml), penicillin (100 U/ml), and
streptomycin (50 ug/ml) (all from Sigma). Cells were plated on plastic dishes coated with
poly-L-lysine and maintained in an incubator at 37°C in an atmosphere of 5% C02 for up
to 2weeks. After 2 days in culture, 10 pM cytosine arabinoside was added to the
minimum essential medium to limit smooth muscle and fibroblast proliferation, and the

medium was changed twice weekly thereafter.

Intracellular electrophysiological recording from myenteric neurons

Individual myenteric ganglia can be visualized at 200x magnification using an
inverted microscope (Olympus CK-2) with differential interference contrast optics.
Intracellular recordings were obtained from single neurons using glass microelectrodes
filled with 2 M KCl and a tip resistance of 80-120 MO. An amplifier with an active
bridge circuit (Axoclamp 2A, Axon Instruments, Foster City, CA) was used to record
membrane potential. In most experiments, the membrane potential was hyperpolarized

using constant DC current to avoid action potentials when evoking fEPSPs. The

44

amplified signals then were filtered at 1 kHz using a four-pole, low-pass Bessel filter
(Warner Instruments, Hamden, CT), and digitized at 2 kHz using a Digitdata 1200
analog/digital converter (Axon Instruments). Data were acquired and stored using

Axotape 2.02 or Axoscope 8.2 software (Axon Instruments).

All fEPSPs were elicited using a glass pipette (tip diameter 40-60 uM) filled with
Krebs’ solution as a focal stimulating electrode. The stimulating electrode was positioned
closely over an interganglionic nerve strand. Nerve fibers were stimulated electrically
using single stimuli of 0.5 msec of duration at a rate of 0.1 Hz. A digital average of eight
individual fEPSPs was used as a measurement of fEPSP amplitude in the absence or

presence of drug treatments.

Patch-clamp recording from primary cultured neurons

Whole-cell and outside-out patch-clamp recordings were obtained via standard
methods. Recordings were carried out at room temperature with patch pipettes with tip
resistances of 3 to 5 M9 for whole-cell and 5 to 10 M9 for single-channel currents in
- outside-out patches; seal resistances were >5 G9. The tips of pipettes used for single-
channel recordings were coated with Sylgard (Dow Corning, Midland, MI). The pipette
solution contained the following: 160 mM CsCl, 2 mM MgC12, 1 mM EGTA, 10 mM
HEPES, lmM ATP, and 0.25 mM GTP, the pH and osmolarity were adjusted to
7.4 (with CsOH) and 315 mosmong (with CsCl), respectively. All recordings were made
with an Axopatch 200A amplifier. Data were acquired with pClamp 8.0 software.
Currents were sampled at 2 kHz and were filtered at 1 kHz (4-pole Bessel filter, Warner

Instruments, Hamden, CT) and stored on a computer hard drive for offline analysis.

45

Immunohistochemistry

After electrophysiological recordings with Neurobiotin-filled microelectrodes, the
myenteric plexus-longitudinal muscle preparation was fixed overnight at 4 °C in
Zamboni’s fixative (2 % (v/v) formaldehyde and 0.2 % (v/v) picric acid in 0.1 M sodium
phosphate buffer, pH 7.0). The fixative was removed using three washes of dimethyl
sulfoxide at 10 min intervals. Tissues were then washed three times with phosphate-
buffered saline (PBS) (0.01 M; pH 7.2) at 10 min intervals. Subsequently the preparation
was incubated overnight with a primary antibody against nitric oxide synthase (NOS) at
room temperature. After primary antibody incubation, tissues were washed three times at
10 min intervals with PBS. Tissues were then incubated (1.5 h at 23 °C) with goat anti-
rabbitt IgG (1:40 dilution in PBS; Jackson Immunoresearch Laboratories, West Grove,
PA, USA) conjugated to fluorescein isothiocyanate (FITC) to recognize NOS
immunoreactivity and Texas Red to recognize the neurobiotin-filled impaled neurons.
Tissues were washed three times with PBS and mounted in buffered glycerol for
fluorescence microscopy. Images were obtained using Nikon, Eclipse TE2000-U (Tokyo,

Japan).

Drugs and drug application

All drugs used in the studies are listed in Table 1. In general, four different

methods were used for drug application.

46

1. Superfusion: Antagonists were applied by superfusion in a known
concentration by addition to the superfusing Krebs’ solution. Antagonists were applied

for 6-20 minutes prior to measuring the amplitude of evoked responses.

2. Pressure ejection: Local application of agonists was accomplished by ejection
from the tip of a micropipette (~20 pm tip diameter) placed within 150 pm of the impaled
neuron. Agonists were applied using short pulses of nitrogen gas (3 to 35 ms, 10 psi)

using a Picospritzer II (General Valve, Fairfield, NJ).

3. Ionophoresis: ACh or ATP was applied by ionophoresis from an electrode (10
MO <tip resistance <20 MO) placed directly above the impaled neuron. The
concentration of ACh or ATP used for ionophoresis was 1M. When ACh ionophoresis
was applied, a retaining current of —6nA was used and a cathodal current pulse of 1-5
msec duration and 100—199 nA intensity were used. When ATP ionophoresis was applied,
a retaining current of +8 nA was used and an anodal current pulses of 1-5 msec duration

and 100-199 nA intensity was used.

4. Gravity-driven flow: Application of agonists or some antagonists to cultured
neurons was accomplished using a linear barrel array of quartz, gravity—fed flow tubes
(320 pm id. and 450 um o.d., Poltrnicron Technologies, Phoenix, AZ). The precise

timing of onset and offset of drug application was controlled by computer-gated solenoid

valves (General Valve, F airfield, NJ).

47

Table 1. Drugs and ligands used in this dissertation

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Ligands Major Effect

Acetylcholine (ACh) Endogenous AChR agonist

a,B-methyleneATP (or,B- Agonist selective to P2X1 or P2X3

mATP) receptors

Apamin Blocker of Kca with small conductance
(SK)

Adenosine triphosphate (ATP) Endogenous P2 receptor agonist

Clotrimazole (CLT) Blocker of Kca with intermediate
conductance (1K)

8-cyclopentyltheophylline Antagonist selective to A. Adenosine

(CPT) receptors

Cytisine nAChR agonist selective for [34 subunit

DMPP nAChR agonist selective for [3; subunit

F orskolin Adenylate cyclase stimulator

H-89 Protein kinase A blocker

Hexamethonium nAChR blocker

Iberori toxin (IBTx) Blocker of Kcal with big conductance (BK)

Idazoxan a2 Adrenergic receptor antagonist

Mecamylamine nAChR antagonist

MLA nAChR antagonist selective for (17 subunits

Nalaxone Opioid receptor antagonist

NAN-190 5-HT. A receptor antagonist

Nicotine nAChR agonist

Nifedipine L-Calcium channel blocker

PPADS P2 receptor antagonist

Pertussis toxin (PTX) Gi/o Protein inhibitor

Prucalopride 5-HT4 receptor agonist

Renzapride 5-HT4 receptor agonist

Scoplamine Muscrinic AChR blocker

Tetraethylammonium (TEA) BK(<1mM), Kir(10mM)

Tegaserod 5-HT4 receptor agonist

Tetrodotoxin (TTX) Voltage—gated Na+ channel blocker

TNP-ATP P2X receptor blocker selective for P2X.
and P2X3

 

48

 

Statistics

All data were expressed as the mean i standard error of the mean (SEM). Data
were analyzed for significance using Student’s t-test for paired data or analysis of
variance. P < 0.05 was used to established significant differences between control and
treatment groups. The specific methods used to analyze data are explained in the related

result parts.

49

RESULTS

50

SUBTYPES OF FUNCTIONAL P2X RECEPTORS IN MYENTERIC N EURON S

Pharmacological identification of functional P2X receptor subtypes in myenteric

neurons were conducted in tissues from mice (Part A.) and guinea pigs (Part B.).

Part A. P2X Receptor Subtypes In Myenteric Neurons In Mouse Small Intestine

This part of studies used electrohysiological and pharmacological methods in
tissues from P2X2 or P2X3 subunit gene deleted or wild type control mice. As these mice
were derived from homozygous F2 crosses, each knockout mouse has its corresponding
wildtype control. So comparisons were made between each knockout and its

corresponding wildtype control.

Characterization of myenteric neurons from PZXZW, P2X2'l', P2X3H+ and P2X3"'

mice

Intracellular electrophysiological recordings were made from single myenteric
neurons in acutely isolated LMMP preparations in small intestine of mice whose genes
encoding P2X2 or P2X3 subunits have been deleted. RT-PCR and immunohistochemical
analysis confirmed that no P2X2, P2X3 subunit mRNA or protein was expressed in small

intestine of P2X2'l’ and P2X3'l' mouse, respectively (Ren et al., 2003; Bian et al., 2003).

There were no data available that describe the electrophysiological properties of
murine small intestinal myenteric neurons. Therefore, I first characterized the

electrophysiological properties of myenteric neurons prior to assessing changes that

51

might be associated with P2X; or P2X3 gene deletion. S neurons were identified by the
presence of fEPSPs and action potentials that lacked long lasting afterhyperpolarizations
(Hirst et al., 1974). AH neurons were identified by the long- lasting action potential
afterhyperpolarization. Recordings were made from 108 S neurons and 12 AH neurons
for P2X3J'Pr /P2X3'/' studies and 93 S neurons and 12 AH neurons for P2X2‘L/+ /P2X2'/'
studies. AH type neurons were infrequently encountered and represented approximately
10 percent of total number of neurons impaled in the study. This observation was similar

to previous report in the mouse colon (F urukawa et al., 1986).

Resting membrane potentials (RMP) and input resistance (Rgnpm) of AH and S
myenteric neurons from P2X3'l', P2X2'l' and their corresponding wild type mice were not
different (Table 2). Action potentials recorded from S neurons in the different mutants
and control mice were similar (Figure 5). The action potentials were blocked by
tetrodotoxin (TTX) and followed by a brief afterhyperpolarization. The action potentials

had durations at half-amplitude of 1.7 i 0.1 ms in tissues from P2X2H+ and P2X;"' mice

and 1.6 i 0.1 ms in tissues from P2X3H+ and P2X3”’ mice.

Action potentials recorded from AH neurons in tissues from these mice were also
similar (Figure 6). The action potential durations at half-amplitude were 2.1:t 0.1 and 2.3
i: 0.1ms (P > 0.05) in tissues from P2X2+l+ and P2X2'/' mice, respectively; 2.0 :l: 0.2 and
2.4 :k 0.1 ms (P > 0.05) in P2X?” and P2X3‘/' mice, respectively. The peak amplitudes of
the action potentials slow afterhyperpolarization were 5.9 i 1.1 and 5.8 :l: 0.7 mV in

P2X2+/+ and P2X;"' mice (P > 0.05), respectively. These numbers were 5.2 i 0.7mV and

5.4 i 0.6mV in (P > 0.05) in P2X)“+ and P2X3'l' mice, respectively. The duration of the

52

Table 2. Electrical properties of myenteric neurons in mouse small intestine

A. Resting membrane potential (RMP) and input resistance (Rim) of AH and S neurons

in P2X3 +/+ and P2X3 "' mice.

 

 

 

 

 

 

 

 

 

 

 

 

AH neurons S neurons
P2X3 *’* P2X3 "' P2X; “i P2X; "'
(n=5) (n=7) (n=22) (n=22)
Mean 632t4 633:2 62i2 59i2
RMP (mV)
Range 50 — 71 57 -— 73 49 — 73 42 ~ 72
Mean 172i35 153i20 121:1:14 127i10
Rinput (M0)
Range 93 — 285 79 — 212 57 — 277 65 — 204

 

53

 

B. Resting membrane potential (RMP) and input resistance (Rum) of AH and S neurons

in P2X2 +/+ and P2X; 4' mice.

 

 

 

 

 

 

 

 

 

 

 

 

AH neurons S neurons
122x2 *’* P2X; "' P2X2 *’* P2X2 "'
(n=3) (n=7) (n=1 7) (n=2 1)
Mean 69i5 62i4 63:1:2 58i2
RMP (mV)
Range 61 -— 80 48 — 81 50 — 74 45 — 80
Mean l36:t35 132i21 122i13 112i9
Rinput (M9)
Range 96 - 207 85 — 221 65 — 241 54 — 167

 

54

 

Figure 5. Action potentials in myenteric S neurons in mouse small intestine

A. Action potentials recorded from myenteric S neurons in tissues from P2X;
knockout mice (P2X2‘l') and wild type mice (P2X2+/+). S neurons have action potentials
followed by short duration afterhyperpolarizations. TTX blocks the action potential in S

neurons.

B. Action potentials recorded from S neurons in tissues from a P2X3’/' and

P2X3‘L/+ mice. These action potentials also have a short afterhyperpolarization (as arrows

point) and they are blocked by TTX.

55

Figure 5

A P2X2 W sz2 -/-

Control L 10 ms

 

 

TTX (0.3 uM)

 

 

 

 

 

 

 

B Control TTX (0.3 pM)
20 mV
0.15 nA
1 5ms
P2X H/«Jk/ j - ALL. 3L. --
T. _. xv w , __
K
5ms I 20 mV
0.5 ms
P2X3"’ {v A __ w_ _ .—e
K

56

Figure 6. Action potentials in myenteric AH neurons in mouse small intestine

A. Action potentials recorded from AH neurons in P2X2'/' knockout and P2X2’L/+

tissues are similar. They have long-lasting afterhyperpolarization (as arrows show).

B. Similarly, there is no difference between action potentials recorded from AH
neurons in P2X3'I’ and P2X3+/+ tissues and they have long-lasting hyperpolarization

(arrows). In addition, action potential recorded from AH neurons is not blocked by TTX.

57

Figure 6

 

 

 

 

 

 

 

A
szz H+ A,
\
20 mV
PZXZ J- I A/
E 5— ms
B Control TTX (0.31M
_I—1 _J—_‘l
20 mV
5—150. 15 nA
5__ ms
P2)(3 +l+ r wr- .--w :fi‘r‘w‘r
v'\
20 mV I
P2X3 --/ A

 

afterhyperpolarization was 6.4 :t 0.9 s in AH neurons from P2X2+l+ mice and 5.7 :i: 0.4 s
(P > 0.05) in AH neurons from P2X2"/' mice. In P2X3H+ and P2X3'/' mice, these numbers

were 6.3 i 1.33 and 6.5 i 0.7 s (P> 0.05), respectively.

P2X; subunit knockout altered P2X receptor-mediated responses in S neurons

Fast EPSPs recorded from S neurons in tissues of P2X2H+ and P2X2'I‘ mice were
not different in the peak amplitude or time course (Table 2, Figure 7). This result
suggests that P2X2 subunits may not contribute to the P2X receptors mediating fEPSPs in
S neurons. However, in tissues from P2X2+Pr mice, nearly all fEPSPs were reduced in
amplitude by the P2 receptor antagonist pyridoxalphosphate- 6-azophenyl-2,, 4,-
disulphonic acid (PPADS, 10 uM) (Figure 7). All fEPSPs were blocked by the combined
application of PPADS and the nicotinic acetylcholine receptor antagonist, mecamylamine
(10 uM). However, PPADS had no effect on fEPSPs recorded from S neurons in tissues
from P2X2'l' mice (Figure 7). Mecamylamine reduced the amplitude of fEPSPs recorded

4'” tissues, but blocked fEPSPs recorded from S neurons in tissues

from S neurons in P2X;
from P2X2'l’ mice (Figure 8). The amplitude of fEPSP in presence of mecamylamine
recorded from S neurons in P2X2+l+ tissues was larger than that in S neurons of P2X;"'
tissues (Figure 8). Puff application of nicotine caused a depolarization of S neurons from
P2X2'l' mice and this response was similar in amplitude to that recorded from S neurons

in P2X2+/+ tissues (Figure 9). Focal application of ATP, but not or,B-mATP depolarized S

neurons in tissues from P2X?” mice (Figure 9). Neither ATP nor or,B-mATP caused a

response in S neurons from P2X2’/' mice (Figure 9).

59

Figure 7. PPADS inhibits fEPSPs recorded from S neurons in tissues from P2X?”

but not in tissues from P2X2'/' mice.

A. Representative fEPSPs recorded from S neurons in P2X2”+ (left) and P2X2'/’
(right) tissues in the absence or presence of PPADS (10 pM). The fEPSPs recorded from
neurons in tissues from P2X2+H mice were inhibited by PPADS (left) while fEPSPs

recorded from neurons in P2X2’l‘ tissues were unaffected by PPADS (right).

B. Pooled data show that the amplitudes of fEPSPs recorded from S neurons in
P2X2+H (n = 5) and P2X2’l' (n = 6) tissues were similar. PPADS did not alter fEPSP
amplitude recorded from P2X2'l' S neurons but it inhibited the fEPSP in S neurons from
P2X2+/+ tissues. Data are mean :i: SEM. “*” Significantly different from P2X;+le control

(p<0.05).

60

Figure 7

P2X2+’+ P2X2'"

/

  
 

 
  
 
 

Control
PPADS

Control
PPADS

'20 mV

20 ms

   

 

B [:3 Control
30 - - PPADS (10 pM)
A 20 -
>
E.
8%
o. 10 r
k!
0

 

 

 

 

 

 

 

P2X *’* P2X "'

61

Figure 8. Mecamylamine reduced fEPSPs in S neurons from tissues of P2X2+H mice

but completely blocked fEPSPs in S neurons from P2X2’/' mice.

A. Representative fEPSPs recorded from S neurons in P2X2”+ (left) and P2X2'/’
(right) tissues. The fEPSP in P2Xz+l+ neuron is only partly inhibited by mecamylamine

(10 uM), but the fEPSP is blocked by mecamylamine in a neuron from a P2X2'l' mouse.

+/+

B. Pooled data show that mecamylamine reduced fEPSP amplitude in P2X;
neurons (n = 5). However, mecamylamine blocked fEPSPs in P2X2‘l’ neurons (n = 9).
“*” Significantly different from P2X?” or P2X2'I' control amplitude (P < 0.05); “#”

+/+

Significantly different from the amplitude of the P2X2 fEPSP in the presence of

mecamylamine (P < 0.05). Data are mean i SEM.

62

Figure 8

 

 

 

 

 

 

 

 

 

 

A
P2x,+/+ P2X2‘"
Control Control
/ , flamine
Mecamylamine
,— '20 mV
‘ 20 ms
B 30 ' 1:: Control
- Mecamylamine (10 pM)
A ,__T__ I
> 20 ..
.5,
0.
if 10
L15 #
*
0 L
P2X,” P2X "'

2

63

Figure 9. Responses of murine myenteric S neurons in PZXZW and P2X2'l‘ tissues to

nicotine, ATP and a, B—mATP

A. Nicotine induced similar amplitude depolarizations of S neurons from PZXZH+

and P2X2'l' mice. ATP depolarized S neurons from P2X2+l+ but not P2X2'I' mice, while
or, B-mATP did not depolarize any S neurons. Drugs were applied (at the arrows) by
pressure ejection from a pipette positioned near the impaled neurons. The concentration

of each drug in the pipette was 1 mM.

B. Mean data from experiments similar to those shown in A. There was no
difference in the amplitude of the nicotine-induced depolarization recorded from S
neurons in tissues from P2X?+ (n = 15) and P2X2'l' (n = 12)mice. “*” The ATP-induced
depolarization was significantly smaller in P2X2”' neurons compared to those in recorded
from P2X2H+ neurons. or, B-mATP did not elicit a response in S neurons from either type

of mouse.

64

Figure 9

Nicotine

P2X2+/+ H TWW

 

 

 

 

 

P2X2"'

 

ATP

 

 

 

 

 

 

 

 

 

N 00
O O

.3
O

Depolarization (mV)

 

 

O

65

 

 

 

 

 

 

 

 

or,B-mATP

 

Properties of P2X receptors were not affected by P2X; subunit knockout in S

neurons

Single stimuli applied to interganglionic nerve strands evoked fEPSPs in S
neurons from tissues of P2X3+I+ and P2X3‘l‘ mice (Figure 10, 11). The mean amplitude of
fEPSPs recorded from neurons in P2X3+l+ and P2X3'/' tissues was not significantly
different (Figure 10, 11). The pharmacology of fEPSPs between P2X3+I+ and P2X3’l’
tissues was similar. All fEPSPs were reduced, but not completely blocked, by the
nicotinic receptor antagonist mecamylamine (10 uM) (Figure 10). Amplitudes of fEPSPs
in presence of mecamylamine were not different in P2X3H+ and P2X3’l' mice. Similarly,
fEPSPs in tissues from P2X3+l+ and P2X3'/' mice were both partly inhibited by PPADS
(Figure 11). Also, fEPSP amplitude in S neurons from P2X3+I+ and P2X3'l' mice were not
different in presence of mecamylamine. ATP caused a depolarization that was similar in
amplitude in S neurons from P2X3+l+ and P2X3'/' mice (Figure 12). However, or, B-mATP

did not cause depolarization in S neurons in tissues from either type of mice (Figure 12).

or, p-mATP depolarizes AH neurons from szf’+ and szz"' mice

Focal application of afi-mATP (lmM), an agonist selective for P2X. and WK;
containing P2X receptors caused a rapidly developing depolarization that was associated
with a decrease in membrane input resistance in AH neurons from P2X2+l+ and P2X2'l'
mice (Figure 13). The or,B-mATP-induced response was mimicked by ATP applied to the

same neurons and was blocked by PPADS. Amplitudes of or, B-mATP-induced

66

Figure 10. Effects of mecamylamine on fEPSPs from murine myenteric S neurons in

P2X3‘i’+ and P2X;"' tissues.

A. Representative recordings show mecamylamine (10 uM) reduced the

amplitudes of fEPSPs in neurons in both P2X3H+ and P2X3'l’ tissues.

B. Pooled data from experiments shown in A. There was no significant difference
in the amplitude of fEPSPs recorded from S neurons from P2X3+l+ and P2X3'/' mice.
Mecamylamine reduced the amplitude of the fEPSP in all S neurons but it did not
completely block the fEPSP. * Significantly different from fEPSP amplitude recorded in

drug free solution.

67

Figure 10

i>2x,+/+

     
     

Control
/

Mecamytamine (10 uM)

 

20 ms

B 30 ' - Control
E Mecamylamine (10 pM)

fEPSP (mV)
9*. {5 <75

LL

 

 

 

 

O
P

 

 

mm” mx+

68

Figure 11. Effects of PPADS on fEPSPs from murine myenteric S neurons in P2X3”+

and P2X3'l' tissues.

A. Representative fEPSPs recorded from S neurons in tissues from P2X3,+/+ and

P2X3"' mice are inhibited but not blocked by PPADS.

B. Pooled data from experiment illustrated in A. PPADS produces a significant
inhibition of fEPSPs but it does not block the synaptic response. There was no difference

in the amplitude of the fEPSPs recorded from S neurons in P2X3+H and P2X3”' tissues.

69

Figure 11

P2X,“+ szy-

__I
10 mV
__I

20 ms

- Control

25_ n=5 [:1 PPADS (10 nM)

204

 

fEPSP (mV)

 

 

 

 

P2X,+/+ P2X3'/'

70

Figure 12. Responses of murine myenteric S neurons in P2X3m and P2X3‘/' tissues to

ATP and a, B—mATP

A. Representative recordings Show that ATP, but not or, B-mATP induced

depolarization in S neurons.

B. Pooled data indicate there were no differences in the amplitude of ATP-
induced depolarizations in S neurons from P2X?” and P2X3’l‘ mice. or, B-mATP did not

induce depolarization in any tested S neurons.

71

Figure 12

 

.

I

l

.

.‘l.‘.,. '1
1‘
III I

‘20 mV
055

on... n... n.

_ATP (2 mM)
1 c: a, B-mATP

 

 

 

 

03
O
A

n=12

Depolarization (mV)
8

 

 

n:

 

 

P2X3+I+ P2X3-l-

72

Figure 13. Responses of murine myenteric AH neurons in P2X?” and P2X2'" tissues

to or, B-mATP

A. Representative recordings of agonist-induced responses from AH neurons in
P2X2+H and P2X2'/' mouse tissues. or, B-mATP was applied by pressure ejection (at the
arrows) fi'om a pipette positioned near the impaled neuron. The concentration of or, B-

mATP in the pipette was 1 mM.

B. Mean data show that the amplitude of the or, B-mATP-induced depolarization

was similar in tissues from P2X2+l+ (n = 3) and P2X2'/' (n = 4) mice.

73

Figure 13

 

 

A
a,B-mATP
P2X2+/+
IW
(x,B-mATP
P2X2'"
"IM—
20 mV
1—' _I10 mV
S 0.5 s
B
25

’S

E,

C

.9

’5

.N

<3

0

O.

(D

D

 

 

szz+l+ sz2 4-

74

depolarizations were similar in tissues from P2X2H+ and P2X2'/" mice (Figure 13).

ATP— and or, B-mATP— induced depolarization in AH neurons from P2X3J"+ and

P2X3'l' mice

Local application of ATP (2 mM) from a pipette positioned near the neurons
caused a depolarization that was similar in amplitude in neurons from P2X3H+ and P2X3’l'
mice (Figure 14). The ATP-induced response was blocked by PPADS and was associated
with a decrease in membrane input resistance (Figure 14). PPADS blocks some P2X
receptors and P2Y. receptors for ATP so responses by ATP in murine neurons could be
mediated by either P2X or P2Y. receptors. To address this possibility, I tested the effect
of or, B-mATP, an agonist of P2X. and P2X3 subunit-containing P2X receptors. It was
+/+

found that or, B-mATP caused depolarization in AH neurons in preparations from P2X3

but not P2X3'l' mice (Figure 14).

B. Pharmacological Identification Of P2X Receptor Subtypes In Guinea Pig

Myenteric Neurons

Depolarization induced by ATP and or,B-mATP in AH and S neurons in guinea pigs

Intracellular electrophysiological recordings were obtained from 112 myenteric S
type neurons from guinea pig ileum. 85% of the neurons (95 cells) were depolarized by

local application of a, B-mATP, an ATP structure analog identified as an agonist selective

75

Figure 14. Responses of murine myenteric AH neurons in P2X?” and P2X3’" tissues

to ATP and or, B—mATP

A. Representative responses induced by ATP and or, B-mATP. ATP depolarized

AH neurons from both types of mice. or, B-mATP evoked depolarization in AH neurons

in tissues from P2X3‘L/+ but not P2X3'/' mice.

B. Pooled data from experiments illustrated in A. The amplitude of ATP-induced
depolarizations was similar in AH neurons from P2X3H+ and P2X3'l' mice whilst or, [3-

mATP induced depolarization in AH neurons from P2X3+H but not P2X3'/’ mice.

76

Figure 14

 

A
ATP mp-mATP
V V >
E
P2X3+/+ 8
g I . , 1 s
”XS/W
B -ATP (2 mM)
25_ E: (X, B'mATP (2 mM)
1 _
§ 204 "-5 ”=3 "=6
e . l
C
.9 15‘
‘c'u' .
-E'
g 10.
0 d
8 5
c: .
o.

 

   

 

 

P2X3+’+ P2X,-/-

77

for P2X. and P2X3 subunits containing P2X receptors. The rest 15% of the S neurons (17
cells) were insensitive to ATP or a, B-mATP. They were cholinergic neurons and fast
synaptic excitation was mediated solely by ACh acting at nAChRs as mecamylamine (10
uM) completely blocked fEPSPs recorded from these neurons. The amplitudes of ATP
and a, B-mATP-induced depolarization were 21.5 mV and 22.5 mV (P>0.05) (Figure 15),
respectively, which were blocked by PPADS (10 pM).

A total of 28 AH neurons was studied; 17 neurons were tested for ATP
sensitivity. 65% of these neurons were depolarized by ATP. All 28 AH type neurons
were tested for a, B-mATP sensitivity and only 17% of them were depolarized by local
application of a, B-mATP. The amplitudes of depolarization caused by ATP and a, B-

mATP were 14.7 mV and 11 mV, respectively (Figure 15).

Effects of TNP-ATP on fEPSPs and ATP-induced depolarization in S neurons

TNP-ATP (10 pM), an ATP analog, is an antagonist selective for P2X], P2X; or
P2X2/3 receptors. It was found that TNP-ATP reversibly reduced depolarizations induced
by ATP (Figure 16). Superfusion of TNP-ATP also reversibly reduced amplitude of
fEPSPs recorded from S type neurons (Figure 17). These data also suggest that P2X

receptors of S neurons contain P2X., P2X3 or P2X2/3 subunits.

78

Figure 15. Depolarization induced by ATP (lmM) and or, B—mATP (1 mM) in S and

AH myenteric neurons in guinea pig small intestine

A. Representative recordings of depolarizations induced by or,B-mATP and ATP
in AH neurons. The arrowheads refer to pressure injection of or,B-mATP or ATP. Only

17% of the tested AH neurons have similar responses to these two agonists.

B. Representative recording traces show depolarization induced by ATP and (1,13-

mATP in S myenteric neurons. The depolarizations were blocked by PPADS (10 uM).

85% S neurons have similar responses to these two agonists.

C. Pooled data indicated the amplitudes of or, B—mATP and ATP-induced
depolarization in S neurons are similar, which are bigger than these in AH neurons. Data
are the mean :t SEM obtained from 5 AH neurons and 12 S neurons. “*” indicates a

significant difference from responses in AH neurons.

79

Figure 15

A o, B-mATP ATP
|20mV
,~ ~ 1 s

 

 

 

PPADS (10 M)

 

Ezra, B-mATP (1 mM)
- ATP (1mM)

 

 

Depolarization (mV)

 

 

   

 

 

 

AH Neurons S Neurons

80

Figure 16. Effect of TN P-ATP on depolarizations induced by ATP in S neurons

A. Representative traces show that TNP-ATP reduced amplitude of ATP-induced

depolarizations. The effect of TNP-ATP was reversible. Arrowheads indicate pressure
ejection of ATP. B. Data are mean i SEM from experiments (n=7) similar to that shown

in A. “*” indicates a significant difference from control and wash groups.

81

Figure 16

A

ATP

Control

Depolarization (mV)

00
O

N
O

_x
O

J 10 mV
ATP ATP 1 S
V
TNP-ATP (10 pM) Wash

 

 

Control TNP—ATP Wash

82

Figure 17. Effect of TNP-ATP on fEPSPs in S neurons

A. Representative recording traces indicate TNP-ATP reduced the amplitude of
fEPSPs. The effect of TNP-ATP on fEPSP amplitude was reversible. B. Data are mean i

SEM from experiments (n=4) similar to that shown in A. “*” indicates a significant

difference from control and wash groups.

83

Figure 17

A
Control TNP-ATP (10 |JM) Wash
_J 10mV
10ms
B 25 {

fEPSP (mV)

 

 

Control TNP-ATP Wash

84

P2X RECEPTORS COUPLE TO CALCIUM-DEPENDENT POTASSIUM

CONDUCTANCE IN GUINEA PIG MYENTERIC NEURONS

An afterhyperpolarization is induced by activation of P2X receptors

Activation of P2X receptors by a, B-mATP induced a depolarization in myenteric
S neurons of guinea pig small intestine. This resulted from cations flowing through P2X
receptors as input resistance (Rinpm) was reduced during depolarization. However, it was
observed that, in 52 of 95 (55%) S type neurons, (1, B-mATP-induced depolarizations
were followed by a hyperpolarization (Figure 18). PPADS abolished both the
depolarization and hyperpolarization. The amplitudes of depolarization and
hyperpolarization were positively correlated (Figure 18). It was also observed that
membrane conductance was increased during hyperpolarization, which suggested that
opening of ion channels mediates the after-hyperpolarization. To eliminate the possibility
that a, B-mATP-elicited hyperpolarization was due to change of membrane potential,
single electrode voltage clamp (SEVC) recordings from similar preparations were made
to confirm a, li-mATP-induced depolarization and hyperpolarization. In SEVC recording,
inward and outward currents were recorded in S neurons in response to a, B-mATP,
which underlie the depolarization and hyperpolarization, respectively (Figure 18).
Similarly, the amplitudes of inward and outward currents were also positively correlated

and they were blocked by PPADS.

85

Figure 18. or, B—mATP—induced a biphasic response in S neurons.

A. Representative recording showing that or, B-mATP induced a depolarization
followed by a hyperpolarization (resting membrane potential = —50 mV). Downward
deflections in the trace are voltage responses caused by hyperpolarizing current pulses. A
decrease in the amplitude of these responses during the depolarization and
hyperpolarization indicates an increase in membrane conductance. Arrowheads indicate

pressure ejection of a,B-mATP.

B. Current recording in SEVC mode shows a similar result as in A. or, B-mATP
induced an inward current followed by an outward current at a holding potential of —50
mV. The downward deflections are currents caused by voltage steps. An increase in the
amplitude means an increase in membrane conductance. Arrowheads indicate pressure

ejection of or, B-mATP.

C. There is a positive correlation between the amplitude of the hyperpolarization

and the depolarization.

86

Figure 18

 

 

A B
|20mV
23 N
I 0.2nA
25
C
30-
E 25- e
v _ e e.
8 20+ 0 U ,,/’
"a » r w"
.2. ‘15- /./0.0 .
.9 . . e)” . 0.0
O 10- // e
D. x
L. I!!!“ O. . O
8. 5- ° 0 e e
f .
0 x l L

o 510.15‘20‘25‘30
Depolarization (mV)

87

The ionic basis of the afterhyperpolarization

One mechanism for increased conductance in mediating hyperpolarization could
be an efflux of K+ ions. It has been reported that intermediate Cazl-activited K+ channels
mediate the action potential afterhyperpolarization in AH neurons (Vogalis et al., 2002;
Fumess et al., 2004; Neylon et al., 2004). In myenteric S neurons, a depolarization
induced by nicotine is followed by a hyperpolarization, which is also due to activation of
calcium-activated potassium channels (Tokimasa et al., 1983). a, B-mATP-induced

hyperpolarizations in S neurons might also be mediated by Cay-activated K+ channels.

Calcium dependency of the a, B-mATP after-hyperpolarization was examined by
changing extracellular calcium concentration from normal level of 2.5 mM to 0.25 mM.
Lowering extracellular [Ca2+] at 0.25 mM almost abolished fEPSP and reduced (1, B-
mATP-induced depolarization from 20.6 mV to 17.8 mV (Figure 19). As a consequence,
it reversibly and dramatically reduced the hyperpolarization from 14.6 mV to 1.8 mV

(Figure 19).

The subsequent experiment was conducted to examine whether the u,B-mATP
afterhyperpolarization or outward current was carried by KL SEVC recordings were
made from LMMP preparations. The inward and outward currents were induced by a, B-
mATP. The relationships between holding potential and inward and outward currents
were linear (Figure 20). The reversal potentials of inward and outward currents were 3.3
mV and —79 mV, respectively. These data indicate that the inward current underlying

depolarization was carried by non-selective cations through P2X receptors as the reversal

88

Figure 19. a, B—mATP-induced afterhyperpolarization is Ca2+ sensitive

A. Low extracellular Ca2 I (0.25 mM) abolished fEPSPs. Restoring extracellular

Ca2+ to 2.5 mM restored fEPSP amplitude. Ca2+ at 0.25 mM reduced the depolarization
induced by or, B-mATP. The after-hyperpolarization was also reduced. Restoring Ca2+ to

2.5 mM restored the depolarization and hyperpolarization. B. Pooled data (mean i

“*9,

SEM) obtained from similar experiments (n=7) to that shown in A. indicates a

significant difference from 2.5 mM Ca2+ conditions.

89

Figure 19

 

   
 

A
2.5 mM Ca2+ 0.25 mM Ca2+ 2.5 mM Ca2+
10 mV
r 20 ms
J20 mV
" N 2 s
B - Normal Krebs (2.5 mM Ca2+)
25 - Low Ca Krebs (0.25 mM Ca2+)
9
E
o
'0
3
'6.
E
<1:
*

 

 

 

Depolarization Hyperpolarization

90

 

Firl

 

lull

\'Ol

10

Figure 20. 0., B—mATP—induced afterhyperpolarization is K+ dependent.

A. Representative recordings show inward and outward currents at different
holding potentials. B. Current voltage relationship of the inward current. C. Current
voltage relationship for outward current induced by or, B-mATP in S-neurons. Current
voltage relationships were linear. The reversal potential for the inward current was 3 mV.
The reversal potential for the outward current was -79 mV. Data are mean 3: SEM

obtained from 7 neurons.

91

Figure 20

 

 

 

 

 

 

92

A B
“'B’mATP Membrane potential (mV)
-100 -80 -60 -40 -20 0
-30 mV 1,. "
'f-0.1
, O
S
--o.2 a
. 5’.
1.03 3
--0.4
J
-70 mV
-90 mV
. 0.08
9
- 0.04
3
E.
’5
. n . . l . 1 x n . 1 000 3
mimbrane potential (mV) ‘
-0.04

potential is close to 0 mV. The outward current mediating the afterhyperpolarization was
carried by KL because the reversal potential is approximately same as K“ equilibrium

potential of -80 mV.

1 then began to identify the Cay-activated K+ channel (KCa) subtypes that
mediated the afterhyperpolarization or outward currents in myenteric S neurons. Three
toxins for major KCa subtypes were used (Vergara et al., 1998). Iberiotoxin (IBTx) at
concentration of 0.1 uM selectively blocks Kc, with large conductance (BK). Apamin at
0.1 uM selectively blocks Kca with small conductance (SK). Clotrimazole at 10 uM
selectively blocks Kca with intermediate conductance (1K). Application of these

antagonists had no effects on AHP induced by a, fi-mATP in S neurons (Figure 21).

Immunochemical characterization of S neurons with after-hyperpolarization

P2X3 receptors are expressed primarily in the inhibitory motor neurons ((LePard
et al., 1997; Poole et al. 2002; Van Nassauw et al. 2002). As shown above, not in all S
type neurons but in an approximately half of them, P2X3 receptors coupled to a Kc, that
mediates the after-hyperpolarization. One hypothesis could be this subset S neurons may
utilize the P2X; receptors to accomplish their unique function of those neurons. Here I
tested the idea that these S neurons were descending inhibitory motor neurons. The
descending inhibitory motor neurons can be labeled by an antibody against NOS. The
results indicated that the S neurons with a, B-mATP-induced hyperpolarization are either
NOS-ir positive or NOS-ir negative. There was no significant association between this

subset of S type neurons and descending inhibitory motor neurons (Figure 22).

93

Figure 21. Lack of effect of KC, channel blockers on the a,B—mATP-induced

afterhyperpolarization.

A. IBTx, apamin and Clotrimazole, selective blockers of big, small and
intermediate conductance calcium-activated potassium channels, respectively, had no
effect on the afterhyperpolarization induced by or, B-mATP. Data are mean i SEM

obtained from 6, 4 and 7 neurons in IBTx, apamin and Clotrimazole, respectively.

B. Clotrimazole blocked action potential afterhyperpolarization in AH neurons in
the same preparations from which S neurons were studied. These data confirmed the

effectiveness of application of Clotrimazole.

94

Figure 21

 

 

 

 

 

 

 

 

 

A
20 —
A I:IContro|
E ' _Treatment
v 16 -
r:
.9 '
s 12-
5 .
o
e 8 —
a)
Q .
E‘
o 4-
a:
<
lberoritoxin Clotrimazole Apamin
0.1pM 1O uM 0.1pM
B

|20mV
25

control

 

“‘1 - ._... Clotrimazole

 

 

fl wash

95

Figure 22. Identification of the neurochemical phenotype of S neurons that generate

the a, B-mATP induced afterhyperpolarization

There was no association between S neurons generating afterhyperpolarization
and NOS-ir neurons. Green fluorescence indicates NOS-ir while red fluorescence reveals
neurobiotin used to mark neurons from which electrophysiological recordings were
obtained. A neuron generating the afterhyperpolarization contained NOS-ir (A) while C
shows an example of a neuron with an afterhyperpolarization but that did not contain
NOS-ir. A neuron that did not show an afterhyperpolarization contained NOS-ir neuron

(B) while D shows an example of a neuron without NOS-ir neuron (D).

96

Figure 22

S neuron with AHP S neuron without AHP

 

97

FAST EXCITATORY SYNAPTIC TRANSMISSION DURING BURSTS OF

NEURONAL ACTIVITY

Fast synaptic transmission declined in amplitude during trains of electrical

stimulation

First fEPSPs were recorded when a train of electrical stimulation was applied on
presynaptic nerve fibers at different frequencies. Fast EPSPs showed a decline in their
amplitudes during test stimulus train at 5, 10 and 20 Hz. However, trains of stimulation
could also elicit slow excitatory post-synaptic potentials (sEPSPs) in S and AH neurons
in the ENS (Grafe et al., 1980; Morita and North, 1985; Wood and Kirchgessner, 2004).
The depolarization occurring during sEPSPs would reduce the electrochemical driving
force for Na+ and Ca2+ influx through nAChRs and P2X receptors thereby reducing
fEPSP amplitude during a stimulus train. Therefore, I used single electrode voltage clamp
(SEVC) method to hold the membrane potential at -60 mV in order to record fEPSC
amplitude at a constant membrane potential. Holding the membrane potential constant
would eliminate changes in driving force as a cause for alterations in the amplitude of
fast synaptic responses during trains of stimulation. The SEVC experiments showed the
similar results. At 0.5 Hz, the fEPSC declined in amplitude by 50% after the first
stimulus but was maintained throughout the remainder of the stimulus train (Figure 23).
However, at stimulus frequencies of 5, 10 and 20 Hz, the amplitude of fEPSCs declined
to zero and the rate of rundown was frequency-dependent and was fitted by a single

exponential decay (Figure 23). The time constants (r) for rundown of the fEPSCs at 5, 10

98

Figure 23. Trains of electrical nerve stimulation cause synaptic rundown.

A. Representative recordings of fEPSCs during trains of stimulation at 5, 10 and
20 Hz. B. Pooled data show that the rate of rundown in fEPSC amplitude was frequency-
dependent and was fitted by a monoexponential decay (solid lines). Data are the mean i:

SEM. obtained from 21 neurons at 5 Hz, 32 neurons at 10 Hz and 19 neurons at 20 Hz.

 

99

Figure 23

 

A
5 Hz
200 ms
10 Hz
100 ms
20 Hz
50 ms
lnA
B
1.2 -
c
”O
:3
'a 0.9 -
E
(U
(I)
0
‘3 06
e ' '
U
m b
._u_
2 0.3L
5 I 5H2
Z .
20 H2 _ 10 Hz
0.0 l l 1 l

 

 

 

0.0 0.2 0.4 0.6 0.8 1.0
Time (s)

100

and 20 Hz were 0.35 i 0.05, 0.2 i 0.02 and 0.1 i 0.02 s, respectively. These data indicate
that fast excitatory synaptic responses rundown in amplitude during trains of stimulation

and that synaptic rundown is not caused by changes in driving force that might occur

during sEPSP.

fEPSCs exhibit paired pulse depression

Paired pulse facilitation (PPF) is a property of many synapses in the peripheral
and central nervous systems. I tested for the presence of PPF at synapses in the myenteric
plexus by measuring the ratio of the amplitude of fEPSCs elicited by pairs of stimuli
applied at intervals of 200, 100 and 50 ms (5, 10 and 20 Hz frequencies, respectively)
(Figure 24). The second fEPSC in the pair was always smaller than the first indicating
that synapses in myenteric plexus exhibit paired pulse depression (PPD) rather than PPF
(Figure 24). The second fEPSC evoked at 10 and 20 Hz exhibited less depression than

that evoked at 5 Hz (Figure 24).

Recovery from synaptic rundown

Synaptic recovery is another important indicator of synaptic properties. It reflects
synaptic vesicle trafficking alter depletion. 1 next investigated the time course of recovery
from synaptic rundown following a 10 Hz train of stimulation. In these experiments, a
train of 20 stimuli at 10 Hz caused fEPSPs to decline in amplitude to 0 mV. Recovery
from rundown was tested by waiting for periods of 1 to 12 s before a single stimulation

was used to elicit a fEPSP (Vtest); this amplitude was expressed as a

101

Figure 24. Paired stimuli cause synaptic depression

A. The second fEPSC in a pair evoked by stimuli at 5 (200 ms interval), 10 (100
ms interval) and 20 (50 ms interval) Hz was smaller in amplitude than the first fEPSC.
B. Mean data from experiments similar to that shown in A. Data are the ratio of the
second fEPSC vs. the first fEPSC (Isecond/Ifirst) in a stimulus pair. * Indicates that
synaptic depression at 10 (n=32) and 20 (n=l9) Hz is less than that occurring at 5 Hz

(n=21). Data are mean i SEM.

102

Figure 24

 

A I 2001115 1
100 ms
50 ms
lnA
B
1.0-
0.8-

second first

/

 

 

10 Hz 20 Hz

5H2

103

fraction of the amplitude of the first EPSP in the preceding stimulus train (Vfirst) (Figure
25). Fully recovery from synaptic rundown occurred in less than 12 s and the recovery

time course was fitted by a monoexponential function; the recovery time constant was 7 i

2 s (Figure 25).

The contribution of nAChRs and P2X receptors to rundown of fEPSPs

As ACh, acting at nAChRs, and ATP, acting at P2X receptors, contribute to most
fEPSPs recorded from myenteric neurons in the guinea-pig ileum, 1 next examined if
there was selective rundown of one of these fEPSP components during a lO-Hz train of
stimulation. The P2X-mediated component of the fEPSP was isolated by using
mecamylamine (10 uM), to block nAChRs. Under these conditions, it was found that the
remaining fEPSP declined in amplitude with a time course (1’ :03 i 0.07 5, n=6) that was
identical to that occurring in the absence of mecamylamine (r =0.3 i 0.07 s, n=6) (Figure
26). The cholinergic component of the fEPSP was isolated by using PPADS (10 uM) to
block P2X receptors. The cholinergic fEPSP ran down with a time course (1 =0.4 i 0.08
5, n=6) similar to that measured under control conditions and in the presence of
mecamylamine (Figure 26). More than 90% of the fEPSP was abolished by co-

application of mecamylamine and PPADS (Figure 26) indicating that the rundown was a

mixed cholinergic—purinergic fEPSP.

ACh and ATP ionophoresis were utilized to investigate if fEPSP rundown was

due to postsynaptic nAChR and/or P2X receptor desensitization. ACh and ATP

104

Figure 25. Time-dependent recovery of fEPSPs from synaptic rundown

A. Diagram of the protocol used to measure the time course of recovery from
synaptic rundown. A conditioning train of 20 stimuli at 10 Hz was used to cause fEPSP
rundown. A single test stimulus applied at various intervals after the conditioning train
was used to elicit a fEPSP to determine the extent of recovery. At is the time interval
between the last stimulus in the conditioning train and the test stimulus. Recovery is
expressed as the ratio of the amplitude of the fEPSP caused by the test stimulus (V103,) vs.
the amplitude of the fEPSP caused by the first stimulus (Vma) in the conditioning train.
B. The time course of recovery was fit by a single exponential function with a I =7 2t 2 3.

Data points are the mean i SEM. of observations made from 14 neurons.

105

Figure 25

A

 

Sfl rst 8test

I -- - l
fig” ................

é-m— -— ----—-— $56 >
10 Hz train of 20 stimuli At=1, 2, 3 s...

 

 

 

 

Vtest/V 1 st
.0 .o .-*
«h (I) N

.0
o

 

 

0'2'4 é'é'1'0f1'2
Time(S)

106

Figure 26. Mecamylamine or PPADS does not alter fEPSP rundown

A. The amplitude of fEPSPs was reduced by mecamylamine or PPADS (each at
10 uM) but the time course of rundown was not changed by the antagonists. The rate of
synaptic rundown was fitted by a monoexponential function with s values of 0.3 2’: 0.07,
0.3 i 0.07 and 0.4 d: 0.08 3 under control conditions and in the presence of
mecamylamine and PPADS, respectively. Each point is the mean i SEM. (n=6). B. Co-
application of mecamylamine or PPADS inhibited the fEPSP by more than 90%. Data are

mean :1: SEM (n=6).

107

Figure 26

fEPSPs (mV)

      
 
   
 

 

 

 

 

20-
I control
A15" . PPADS
E L A mecamylamine
:10-
35 t
0..
la! 5”
0 l J r . 4 . 1 1.. l .
0.0 0.2 0.4 0.6 0.8 1.0
Time (s)
20 -
15 -
' II Control
10 _ o PPADS+MecamyIamine
5 _
0

 

108

Ant-‘1! -

ionophoresis caused depolarizations that were similar in amplitude and time course to
fEPSPs recorded from the same neurons, which suggests the same group of receptors
were activated by ACh/ATP and by synaptic stimulation (Figure 27). Then ACh and ATP
were applied as trains (10 Hz) of ionophoretic pulses and fEPSPs were evoked in the
same neurons using a 10 Hz stimulus train. As same as described above, fEPSPs declined
in amplitude during the stimulus train. However, in the same neurons, neither ACh nor
ATP responses declined in amplitude during a train of agonist application (Figure 27).
These data indicate that synaptic rundown is not due to postsynaptic receptor

desensitization.

Presynaptic mechanisms

Presynaptic inhibitory receptors. Nerve terminals in the myenteric plexus express
a number of receptors that mediate presynaptic inhibition of neurotransmitter release.
These receptors include muscarinic M2 muscarinic receptors, 5-HT1A receptors, opioid
receptors, (12 adrenergic receptors and adenosine A1 receptors. It is possible that during
trains of stimulation, myenteric nerves release the endogenous agonists for one or more
of these receptors and that presynaptic inhibition of ACh and/or ATP is responsible for
fEPSP rundown. To test this hypothesis, fEPSP rundown was measured before and after
application of antagonists to each of the receptors listed above. The exception being an
antagonist for the M2 muscarinic receptor as scopolamine (1 uM), which blocks all
muscarinic receptor subtypes, was present in the Krebs’ solution in all studies (see

Methods and Materials). I used naloxone (10 pM) to block opioid receptors, NAN-190

109

Figure 27. Postsynaptic desensitization does not contribute to synaptic rundown

A. ACh applied by ion0phoresis caused a depolarization that mimicked the
amplitude and time course of the fEPSP. During a 20 Hz stimulation train, the fEPSP ran
down while responses caused by a 20 Hz train of ionophoretic pulses of ACh did not.
Data are mean i SEM (n=4). B. ATP applied by ionophoresis caused a depolarization
that mimicked the fEPSP. Responses caused by a 20 Hz train of ionophoretic pulses of
ATP did not run down while fEPSPs caused by a 20 Hz stimulus train did. Data are Mean

4: SEM (n=4).

110

Figure 27

A

 

 

 

fEPSP ACh ionophoresis
k '10 mV
'7 20 ms
12 _ ACh ionophoresis
§ ' ’ ' 9 ' i i i i i
§0.8- i ’ "=5
93 i fEPSP
g 0.4- i i i E i
s i i
‘r 0. 0
0.0 0.102 03 0.4 0.5
Time(s)
fEPSP ATP ionophoresis
Bk k 5mV
20 ms
1.2r . _
3 ATP IonophoreSIs
c I
8.0.8- 3”” ii
(D
“.3
g 0.4. i fEPSP "=5
E l I I g i
ci’

 

 

0.0 W
0.0 0.1 0.2 0.3 0.4 0.5

Time (s)

111

(0.5 mM) to block S-HTIA receptors, idazoxan (10 pM) to block (12 adrenergic receptors,
and 8-cyclopentyl-theophylline (CPT, 10 uM) to block A1 adenosine receptors. None of
the antagonists tested altered fEPSP rundown during a IO-Hz stimulus train (Figure 28).
It is possible that antagonists tested above have not blocked the receptor(s) responsible
for presynaptic inhibition leading to synaptic rundown. In order to comprehensively
eliminate presynaptic inhibition as a mechanism of synaptic rundown, I attempted to
block the intracellular signaling pathway coupled to presynaptic inhibitory receptors.
Most inhibitory presynaptic receptors are G-protein coupled receptors, which link to the
pertussis toxin (PTX) sensitive Gi/Go subsets of G-proteins (Zamponi, 2001). So PTX
was used to block the signaling pathway to confirm the effects of these receptors on
synaptic rundown. Blockade of the inhibitory postsynaptic potential (IPSP) from
submucosal S neurons can be an indicator of the effectiveness of the PTX-treatment
protocol. Previous work has shown that the IPSP is mediated by norepinephrine acting
a2-adrenergic receptors which couple via a PTX-sensitive G-protein to activation of a
potassium channel (Surprenant and North, 1988). I showed that IPSPs were blocked in
PTX pretreated submucosal preparation while fEPSPs recorded from the same cells were
unchanged (Figure 29). The same protocol was used to treat myenteric plexus
preparations with PTX. In these tissues, it was found that fEPSPs still exhibited rundown

that was similar to that observed under control conditions (Figure 29).

Action potentials. Another possible reason that results in synaptic rundown is that

action potentials are not able to follow stimulus train. In order to test the hypothesis that

112

Figure 28. Antagonists of presynaptic inhibitory receptors do not alter synaptic

rundown

Each figure shows the mean i SEM. of successive fEPSPs normalized to the first
fEPSP in the train before and after drug treatment. There were no differences between the

control and drug treatment values. Data are mean 2‘: SEM (n = 4—5).

113

Figure 28

 

 

 

 

l Adenosine A1 receptors
1.0- I
. I Control
0 CPT (10 pM)
0.5- i
I
O. ‘ I
a. i 3
°- 00 ’
Ell . . . . . v
0.0 0.2 0.4 0.6 0.8 1.0
8 T'
.5 Ime(s)
E
E .
2 l (12 adrenergic receptors
1.0- I
I Control
i . ldazoxan(10 pM)
0.5- g g
l i i g
0.0 - . - . . . . :J—l—
0.0 0.2 0.4 0.6 0.8 1.0
Time (s)

114

 

 

 

 

l Opioid receptors
1.0- I
i I Control
I Naloxone (10 pM)
0.5- i
i a i
. 2 . i
0.0 . . - r- . . . L4—
0.0 0.2 0.4 0.6 0.8 1.0
Time (s)
l 5-HT1 A receptors
1.0« I
g I Control
3 E . NAN-190(0.5 pM)
0.5- i E E
i i i
0.0 0.2 0.4 0.6 0.8 1.0
Time(s)

Figure 29. Pertussis toxin (PTX) does not affect fEPSP rundown

A. PTX pretreatment abolished the IPSP but not the fEPSP in submucosal
neurons. Downward deflections in the IPSP traces are voltage responses caused by
hyperpolarizing current pulses. A decrease in the amplitude of these responses during the
IPSP indicates a decrease in membrane resistance. B. Successive fEPSPs recorded from
myenteric neurons with and without PTX pretreatment. There were no differences

between values obtained in treated and untreated tissues at any time point. Data are mean

:l: SEM (n=8).

115

Figure 29

A Control

-60 mV

 

 

 

 

 

 

 

PTX Treatment

l/\

 

 

 

 

 

 

l
I

III] Control (n=6)

 

 

 

 

 

 

20 . - PTXITreatment (n=3)
3‘ 15 -
E l
8 10
= l
E
O.
E 5-
< h
0 '-— _—
fEPSP IPSP
B
>
e
8
1.0 _ 100 ms
0.
a)
a- p
1:!
g 0.5 -
.‘E \ +
(a!) i Ma
0.0 - . - - - . .‘9

 

0.4 0.6 0.8 1.0
Time (S)

116

axonal action potential failure is not a mechanism of fEPSP rundown, we made
recordings from neurons in which the focal stimulating electrode was positioned on the
axon of the neuron from which the fEPSP was recorded. This allowed us to make
simultaneous recordings of axonally propagated action potentials and fEPSPs during a 10
Hz stimulus train. These experiments were done in 10 neurons and it was found that
antidromic action potentials were maintained throughout the stimulus train while fEPSPs

declined in amplitude during the same stimulus train (Figure 30).

BK channels. The activity of large conductance calcium-activated potassium
(BK) channels in nerve terminals can modulate neurotransmitter release (Robitaille and
Charlton, 1992). Calcium entering the nerve terminal during the action potential opens
BK channels. The Opening of BK channels shortens action potential duration and
hyperpolarizes the nerve terminal; both effects can reduce transmitter release. I used
iberiotoxin (IBTx 100 nM), a selective antagonist of BK channels (Wanner et al., 1999)
to determine if activation of these channels contributed to fEPSP rundown. The effect of
IBTx on somal action potential duration was studied in 12 S neurons and 5 AH neurons.
The duration of somal action potentials triggered by an intracellular current pulse was
broadened by IBTx in 8 of 12 S neurons; in these cells, the action potential duration at
half amplitude increased from 1.3 i: 0.1 to 2.0 5: 0.2 ms (P<0.05) (Figure 31). IBTx did
not change action potential duration in AH neurons; the duration at half amplitude was
2.0 i 0.08 and 2.1 i- 0.07 ms (P>0.05) before and after IBTx treatment, respectively
(Figure 31). lBTx did not change the resting membrane potential or input resistance in

any neuron tested. IBTx did not significantly change the amplitude of the first fEPSP in a

117

I

IO-Hz train of stimulation but in 8 of 12 neurons IBTx attenuated PPD. In the absence of
IBTx, the amplitude of the second fEPSP in a train was 66 i 7% of the first fEPSP while
in the presence of IBTx this value increased to 92 i 6% (P<0.05) (Figure 32). Although
IBTx reduced PPD, fEPSP rundown was not altered as the ratio of amplitudes of the first
and 10th fEPSP was similar in the absence and presence of IBTx (Figure 32). Similarly,
IBTx did not change the time constant of fEPSP rundown. The I value for rundown at 10
Hz before IBTx was 0.4 i 0.1 s and in the presence of IBTx, this value was 0.5 i 0.1 s

(P>0.05) (Figure 32).

118

Figure 30. Action potentials do not fail during the train stimulation at 10 Hz

A. Recordings of antidromic action potentials and fEPSPs elicited by a 10 Hz
train of stimulation. The fEPSP declined in amplitude while the antidromic action
potential followed each stimulus in the train. B. Examples of the antidromic action

potential and fEPSPs were shown on an expanded time scale.

119

Figure 30

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

fEPSP

   

lst 2nd 10th 20th 5 ms

120

Figure 31. Effects of IBTx on action potentials in S and AH neurons

A. Action potentials triggered by an intracellular depolarizing current pulse were
broadened by IBTx in S but not AH neurons. B. Pooled data from experiments similar to
that shown in (A) indicate that the action potential duration at half maximum amplitude is
lengthened by lBTx in S (n=8) but not AH neurons (n=5). * Indicates a significant

difference from control (P<0.05). Data are mean :t SEM.

121

. i

Figure 3 1

 

 

 

 

 

A
-50 mV
_J2o mV
5ms
-50 mV
B
I: Control
2.5 — IBTx(100nM)
2.0
75
E, 1 5
a) 1.0
I: 0.5
0.0
S neurons AH neurons

122

Figure 32. Effects of IBTx on fEPSPs during a 10 Hz stimulus train

A. Representative fEPSPs before and after IBTx treatment. B. The second fEPSP
in the train of fEPSPs is not depressed after IBTx treatment but the 10th fEPSP decreased
to the same level occurring in the absence of lBTx. Data are the ratio of the second
fEPSP vs. the first fEPSP (Vsccond/Vfirst) in a stimulus pair and the ratio of the 10th fEPSP
vs. the first fEPSP in the stimulus train. * Indicates significantly different from control
(P< 0.05). (C) Frequency-dependent rundown of fEPSPs before and after lBTx treatment.

lBTx did not alter the time course of fEPSP rundown. Data are mean :1: SEM (n=8).

123

 

Figure 32

 

 

 

Mu. mMNMLJmmv

0.1s

 

 

 

 

 

 

 

.. NM Hwa

       

 

 

 

 

 

B

12 30.
n- 25:
E 08 :1 Control A20_
9! ' -IBTx(1OO nM) E .
'8 :15~
g 310’
m0.4- LU .
E .. .
é 5-

0‘ ‘ i i ' i 2 16

2nd fEPSP 10tthPSP 0-0 0.4 0.8 1. .

Time (s)

124

PRESYNAPTIC 5-HT4 RECEPTOR-MEDIATED REGULATION OF FAST

SYNAPTIC TRANSMISSION

5—HT4 receptor agonist increases fEPSPs during trains of stimulation

First, I tested the effect of S-HT4 receptor agonist on cholinergic and purinergic
synaptic transmission induced by single electrical stimulation. Renzapride at 0.1 uM
increased both cholinergic and purinergic fEPSPs when presence of purinergic blocker,
PPADS and cholinergic blocker, mecamylamine, respectively (Figure 33). Then the
effect of renzapride on the fEPSPs elicited by trains of stimulation was examined. Under
control condition, fEPSPs declined to zero at about the tenth stimulation during a 20
pulses conditioning stimulation at 10 Hz. The first fEPSP amplitude was 17 i 2 mV and
fEPSP amplitude at the 20th stimulus was 0.7 :1: 0.3 mV (n=9). In the presence of
renzapride, the amplitude of the first fEPSP was 28 i 3 mV and this declined to a steady
state level at the 20 stimulus of 6 i 2 mV (n=9). However, the time constants of decline
were similar. They were 0.6 i 0.1 s and 0.6 i 0.1 s before and afier renzapride (Figure

34).

S—HT4 receptor agonist accelerates recovery after rundown

The effect of renzapride on time of recovery from synaptic rundown following a
10 Hz train of stimulation was also investigated. As described above, in these
experiments, a train of 20 stimuli at 10 Hz caused fEPSPs to decline in amplitude to 0

mV at control condition. Recovery from rundown was tested by waiting for periods of 1

125

Figure 33. Prucalopride increases non—cholinergic and cholinergic fEPSPs recorded

from myenteric neurons

A. Representative fEPSP recorded in the absence of drug (control) and in the
presence of mecamylamine (10 uM) to isolate the non-cholinergic component of the
fEPSP. Subsequent addition of prucalopride (0.1 uM) caused an increase in the amplitude
of the non-cholinergic fEPSP. B. The P2 receptor antagonist, PPADS (10 11M), was used

to isolate the cholinergic component of the fEPSP in the same neuron. Subsequent

addition of prucalopride increased the amplitude of the cholinergic component of the

fEPSP.

126

Figure 33

 

 

 

A
i 10 mV
AN“ 20 ms
ll .
Control Mecamylamine Mecamylamine
+ Prucalopride
25-
’>‘
E,
n.
a)
at
H!
Control Mecamylamine Mecamylamine
+ Prucalopride
B

i10mV

20 ms

   
  

Control PPADS PPADS + Prucalopride

 

 

Control PPADS PPADS +
Prucalopride

127

Figure 34. Renzapride increases fEPSPs elicited by trains of stimulation

A. Representative recordings of fEPSPs during trains of stimulation. Fast EPSPs
declined to zero at the tenth stimulation (arrow) under control conditions. Renzapride
increased the amplitude of all fEPSPs during the stimulus train. The fEPSPs did not
decline to zero at the last stimulation (arrow). B. Pooled data show that renzapride
increased fEPSPs (lower left) but did not alter the time course of synaptic rundown

(lower right). Data were fit by a mono-exponential funtion.

128

 

Figure 34

fEPSP (mV)

N 03
O O

.3
O

O

 

 

Control
4 LL11 I
1 F l
Renzapride ‘23:! 20 mv

 

1.0»

I value (8)

 

 

 

 

0.0 0.5 ‘ 1.0 7 1.5 2.0 ' Com. Renzapride

129

[E A....' ‘52. 3.

to 12 s before a single stimulus was used to elicit a fEPSP (Vmso; this amplitude was
expressed as a fraction of the amplitude of the first EPSP in the preceding stimulus train
(Vfim) (Figure 35). Renzapride accelerated fEPSP recovery rate from rundown. The
recovery time constants as calculated by exponential fittings were 7 i 2 3 under control

condition whereas in the presence of renzapride the recovery time constant was reduced

to 1.6 i 0.2 s (P < 0.05).

F orskolin mimics 5-HT4 receptor agonist effects

5-HT4 receptors couple to Gs-protein. Activation of S-HT4 receptors stimulate a
cascade including activiting adenylate cyclase, cAMP and PKA. To investigate if S-HT4
receptor agonist increases fEPSP through stimulating adenylate cyclase, forskolin, an
adenylate cyclase stimulator, was superfused into the Krebs’ solution. It was found that
forskolin mimicked the effects of S-HT4 receptor agonists. Forskolin increased fEPSPs
during 10 Hz train of electrical stimulation (Figure 36). Fast EPSPs still declined with
forskolin treatment however the decline curves were parallel before and after forskolin
treatment (Figure 36). As 5-HT4 receptor agonists did to fEPSP recovery, forskolin also
reduced fEPSP recovery time from rundown (Figure 36). Since S-HT4 receptors couple to
the Gs-protein and forskolin mimics 5-HT4 receptor agonists, effects of 5-HT4 receptors

on rundown and recovery must be through activation of adenylate cyclase.

H-89 abolishes the effects of 5-HT4 receptor agonist

The function of adenylate cyclase is to catalyze coversion of ATP to cyclic AMP

(CAMP) which activates PKA. PKA will then phosphorylate a number of targets

130

Figure 35. Renzapride accelerates recovery from fEPSP rundown

A. Renzapride accelerated recovery rate after fEPSP rundown. V was the first

first

fEPSP in the train and Vtest was the fEPSP evoked by a test stimulus at various time

intervals after the stimulus train. The recovery time course was fitted by

monoexponential function. B. The recovery time constant was reduced by renzapride.

131

 

Figure 35

A Renzapride

.0
o:

.O
on

Vtest/V1 st

 

 

P
o

 

o z 4 e £3 11072
Time (8)

n=14

1: of recovery (Sec)

 

 

Control Renzapride

132

Figure 36. Forskolin (FSK) mimics the effects of renzapride

A. F SK increased the fEPSP amplitude during a stimulus train. The fEPSPs did
not decline to zero at the last stimulation in presence of FSK. B. FSK accelerated

recovery rate after fEPSP rundown.

133

>

 

 

     
 

30 —
25 —
g 20 i . Control
g 15 _ o FSK
% ’ i % ‘6
_ ésiiiiiéiiii
o~. . . . . °i‘?*ao~.
0.0 0.5 1.0 1.5 2.0
Time (8)
B
1.2 -
0.9 -
$0.6 -
277)
>~ 0_3 . Control
. FSK
0.0 ~

 

 

Time (S)

134

such as proteins and ion channels. H-89, a PKA blocker, was used to examined if
activation of PKA is needed for S-HT4 receptor agonist effects. As shown in Figure 37,
renzapride increased fEPSPs and accelerated the recovery rate. Co—application of H-89
and rezapride abolished the renzapride effects (Figure 37). This result suggested that 5-
HT4 receptor-mediated facilitation of synaptic transmission depended on its downstream

signaling molecule PKA.

S-HT4 receptor agonists increase release probability from single varicosities

The above studies have shown that activation of S-HT4 receptors facilitated
synaptic transmission through activating adenylate cyclase and PKA. But how PKA
increases fEPSPs was not known. Did it increase release probability from single release
site? Those experiments were conducted in acutely isolated LMMP preparations in vitro.
In these studies, synaptic transmission recorded was triggered by electrical stimulation on
a bundle of nerve fibers. However, in the ENS, transmitters are released from varicosities
along single nerve fibers. The mechanism that S-HT4 receptor agonists increase
transmitter release in single varicosities cannot be obtained from such studies. To address
this question, the “sniffer” patch (detector patch) technique was employed in cultured

myenteric neurons.

Outside-out patch clamp recordings were made from myenteric neurons
maintained in primary culture. ACh and ATP-induced inward currents were recoded to

make sure there are nAChRs and/or P2X receptors in the outside—out patches. Then this

135

Figure 37. H-89 abolishes the effects of renzapride on rundown and recovery rate

after fEPSP rundown.

A. Renzapride increased the amplitude of all fEPSPs during stimulus train (10
Hz). Co-application of renzapride and H-89 did not alter fEPSP amplitude. B. Renzapride
accelerated recovery rate afier fEPSP rundown. The acceleration was abolished by

addition of H-89.

136

 

 

 

Figure 37

>
N
.b

fEPSP (mV)
KS 5

05

1.2

0.9

rst

test

0.3

0.0

 

fl T I

1

 

Control
Renzapride
Renzapride + H89

0.

n
v
D

 

/ 1
. Control

 

_ o Renzapride
. Renzapride + H89
0 2 4 6 8 1 0
Time (s)

137

 

patch was used as a detector to measure transmitter released from single varicosities. The
transmitter release was triggered by stimulating a single nerve fiber. The currents flow
through the detector patch represented the activity of a single release site. Twenty
electrical stimuli were given in an interval of eight seconds. Because transmitter release
from a given varicosity is intermittent, only some of twenty stimuli can trigger transmitter
release to induce currents that can be recorded in the detector patch. Under control
conditions, 27% stimulation elicited transmitter release (Figure 38; Table 3). Renzapride
increased the ratio to 52% (Figure 38; Table 3). Similarly, another 5—HT4 receptor agonist
teagasrod increased release probability from 27% to 49% (Figure 38; Table 3). This result
suggests that activation of 5-HT.; receptors increases neurotransmitter release probability

and this effect leads to facilitation of synaptic transmission.

I38

 

Figure 38. Activation of presynaptic 5-HT.. receptors increases release probability

from single varicosities

Representative recordings of single channel events evoked by electrical
stimulation of a single nerve fiber (as arrows show) before and after S-HT4 receptor
agonists renzapride (Figure 38a) or tegaserod (Figure 38b). Renzapride or tegaserod

increased single channel open probability.

139

Figure 38b

Tegaserod (0.15 nM)

\ Control \W

W
\Wmm
W
W \W
W \W

20 ms

141

Table 3. Effects of S-HT4 receptor agonists on release probability.

Treatment of renzapride (A) or tegaserod (B) increased transmitter release

probability evoked by electrical stimulation (see text for details).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

A.
Failure events Successful events
Control 73 27
Renzapride 48 52*
n=5, P < 0.001
B.
Failure events Successful events
Control 58 22
Tegaserod 41 39*
n=4; P < 0.005

142

 

 

 

DISCUSSION

143

P2X RECEPTOR SUBTYPES EXPRESSED IN MYENTERIC NEURONS

In this section, identification of P2X receptor subtypes of myenteric neurons was
accomplished in the small intestine of guinea pigs and mice. Guinea pigs have been used
as the animal model for studying G1 physiology and pharmacology broadly because the
gut is readily dissected, reasonably long and mechanically quiescent when studied in
vitro. Immunohistochemical localization of P2X receptor subtypes has been done in
whole mount preparations of guinea pig small intestine (Pools et al., 2002; Castelucci et
al., 2002). Pharmacological properties of P2X receptors in myenteric neurons have also
been characterized in cultured cells (Zhou and Galligan, 1996; Khakh et al., 2000).
However, no data are available about the function of P2X receptors in acutely isolated
LMMP preparations. It is important to identify P2X receptor subtypes so that a complete
characterization of synaptic mechanisms in neural circuits can be accomplished. I
attempted to characterize pharmacological properties of P2X receptors in myenteric
neurons in guinea pig tissues using limited selective agents. As a supplement to these
pharmacological studies, I also investigated P2X receptor subtypes in tissues from mice
in which P2X; and P2X; genes have been deleted. The advantage of gene deletion would

produce precise information.

My data indicate that, in guinea pig small intestine myenteric S neurons express
P2X; receptors whereas AH neurons express P2X; receptors. In mice small intestine,
myenteric S neurons express P2X; homomers whereas AH neurons express P2X2/3

heteromers. The differences may be due to selective expression of P2X subunits by

144

different species. But I cannot exclude the possibility that lack of specific P2X receptor

ligands limited the conclusion made in guinea pigs.

Properties of murine myenteric neurons

Murine myenteric neurons have similar electrophysiological properties to guinea
pigs. In the mouse ileum, AH neurons had a broad action potential that was only partly
reduced by TTX. The action potential has a prominent calcium component that accounts
for the long duration and action potential shoulder in AH neurons from guinea-pig ileum
and colon (Fumess et al. 1998; Nuragli et al. 2004), rat colon (Browning and Lees, 1996),
pig ileum (Cornelissen et al. 2001), mouse colon (Furakawa et al. 1986) and human colon
(Brookes et al. 1987). Therefore, the calcium shoulder is a general property of AH
neurons in the gastrointestinal tract of a number of species. Although not tested in this
study, the long duration and TTX-resistance of mouse ileal myenteric AH neurons
suggest that there is also a calcium component to the action potential. The action
potential in mouse ileal myenteric AH neurons was followed by an afterhyperpolarization
that lasted more than 2 s. This property is also similar to that in AH neurons in the
myenteric plexus of the guinea-pig ileum and colon (Hirst et al. 1974; Fumess et a1.
1998;) rat colon (Browning & Lees, 1996), pig ileum (Cornelissen et al. 2001), mouse
colon (Furakawa et al. 1986) and human colon (Brookes et al. 1987). In guinea pig and
pig intestine, fEPSPs can be evoked in some AH neurons (Liu et al. 1997; Comelissen et
al. 2001) while sEPSPs can be evoked in nearly all AH neurons in the guinea-pig
gastrointestinal tract (Fumess et al. 1998). I found that single electrical stimuli did not

elicit synaptic responses in mouse ileal AH neurons. As I recorded from only a small

145

number of AH neurons, it is possible that I did not sample those neurons receiving fast
synaptic input or that I did not stimulate the interganglionic connectives providing the
fast synaptic inputs to the neurons that I did sample. However, I was able to elicit sEPSPs
in all AH neurons studied. Slow synaptic excitation is the predominant synaptic
mechanism in guinea pig AH neurons (Kunze et al. 1993). Taken together, these data
indicate that the properties of AH neurons in the mouse ileal myenteric plexus are similar

to those identified in the gastrointestinal tract of other species.

S type enteric neurons have an action potential that is completely blocked by TTX
and all S neurons exhibit fEPSPs following single stimuli applied to the interganglionic
connectives in the myenteric plexus (Hirst et al. 1974; Bomstein et al. 1994; Galligan et
al. 2000). In the mouse myenteric plexus, I identified neurons that received fast excitatory
synaptic inputs and the action potential in these neurons was completely blocked by TTX.
Therefore, I concluded that these were S neurons. I also found that the fEPSP in S
neurons was only partly blocked by mecamylamine, a nicotinic cholinergic receptor
antagonist, and that fEPSPs were also inhibited by PPADS. These data indicate that both
acetylcholine and ATP are fast synaptic transmitters in the mouse small intestine as they
are in the guinea-pig intestine (LePard et al. 1997; Johnson et al. 1999) but there may be

additional fast neurotransmitters including 5-HT (Galligan et al. 2000).

Murine myenteric AH neurons were encountered infrequently using a random
impalement strategy with intracellular microelectrodes. This observation is similar to that
reported previously in a study of myenteric neurons in the mouse colon (Furukawa et al.
1986). If AH neurons are sensory neurons in the mouse intestine, as they are in the guinea

pig intestine (Fumess et al. 1998), then normal intestinal reflex behaviors may require

146

only a small number of sensory neurons in the mouse. Alternatively, non-AH neurons
may serve as sensory neurons in the mouse intestine. A previous study, for example, has
shown S neurons in guinea-pig distal colon serve as mechanosensory neurons that are

sensitive to stretch (Spencer and Smith, 2004).

P2X receptor-mediated responses in AH neurons

ATP caused a rapidly developing depolarization in all murine myenteric AH
neurons. This depolarization was associated with a decrease in input resistance and was
blocked by PPADS. These data are consistent with this response being mediated by either
a P2X or P2Y. receptor. a, B-mATP is an agonist at homomeric P2X], homomeric P2X3
or heteromeric P2X2/3 receptors, but it does not activate P2Y receptors (North and
Surprenant, 2000). I found that ATP caused a depolarization of AH neurons in P2X3+H
and in P2X3’/' tissues, while or, B-mATP depolarized AH neurons in P2X3+l+ but not in
P2X3’l’ tissues. These data indicate that P2X3 subunits contribute to the P2X receptor
expressed by murine AH neurons. However, sensory neurons and some sympathetic
neurons also express P2X2/3 heteromeric receptors (Lewis et a1. 1995; Zhong et al. 2000),
and the loss of response to on, B-mATP in P2X3’l' mice could result from the loss of
P2X2/3 heteromeric receptors in murine AH neurons. Other subunits must also be present
in order for an ATP-induced depolarization to persist in AH neurons from P2X3'I' mice.
P2X; subunits remaining in P2X3’/' mice could account for this ATP sensitivity. a, B-
mATP caused similar amplitude depolarizations in AH neurons from PZXZH+ and P2X2’/'

mice. As a, B-mATP activates P2X receptors containing P2X. or P2X3 subunits (North

and Surprenant, 2000), it is possible that AH neurons express P2X receptors composed of

147

P2Xl or/and P2X3 subunits. However, my work has also shown that a, B-mATP does not
depolarize AH neurons in the myenteric plexus of P2X;"' mice while ATP depolarized
the same neurons. Therefore, the P2X receptors in murine AH neurons are likely to be
P2X2/3 heteromers. My suggestion that the P2X; subunits are expressed in AH neurons is
also supported by recent immunohistochemical studies in guinea-pig gastrointestinal
tissues. These studies showed that P2X; subunits are localized to neurons that also
contain the calcium binding protein, calbindin (Castelucci et al. 2002). Calbindin is a
protein marker for most AH neurons in the guinea—pig GI tract (Fumess et al. 1998).
However, it has also been shown that P2X; subunits are not found in calbindin-containing
neurons in the guinea-pig GI tract but that these subunits are localized to calretinin- and
NOS-containing cells (Nassauw et al. 2002; Poole et al. 2002). Calretinin and NOS are
contained in S type neurons and are markers for intemeurons and motoneurons (Brookes,
2001). It is possible that receptor expression and subunit composition of P2X receptors
in subsets of neurons are different in the mouse and guinea pig small intestine. A detailed
study of the neurochemical content and receptor expression of subsets of murine enteric

neurons is needed to address this issue.

P2X receptors in AH neurons do not mediate fEPSPs as fEPSPs are not recorded
from AH neurons. It may be due to no purinergic inputs to AH neurons. The functions of

AH neuron somal P2X receptors remain to be addressed.

Fast synaptic transmission and P2X receptor-mediated responses in S neurons

148

I hypothesized that a P2X receptor contributes to the fEPSP recorded from S
neurons in the mouse small intestine. This hypothesis was based on the finding that
fEPSPs were only partly inhibited by mecamylamine, indicating that a neurotransmitter
in addition to acetylcholine mediates the fEPSP. As fEPSPs were also reduced by
PPADS, the additional neurotransmitter in the mouse myenteric plexus is likely to be
ATP. It is unlikely that P2X3 receptor subunits contribute to the P2X receptor expressed
by S neurons as the properties of fEPSPs in tissues from P2X3H+ and P2X3'l' mice were

+/+ -/- -
and P2X3 mice were

identical. Furthermore, S neurons in tissues from P2X3
depolarized by ATP but not by a, B-mATP. a, B-mATP insensitivity suggests that the
P2X receptor in S neurons is a P2X; homomeric receptor (Zhou and Galligan, 1996).
The fEPSPs recorded from S neurons in tissues from P2X?+ mice were partly inhibited
by the nicotinic cholinergic receptor antagonist mecamylamine and by the P2 receptor
antagonist PPADS. These results indicate that ACh and ATP mediate fast synaptic
responses in the murine myenteric plexus as also occurs in the guinea pig intestine
(Galligan and Bertand, 1994; LePard et al. 1997; Johnson et al. 1999) and colon (Nurgali
et al. 2003). However, the fEPSP in P2X2'/' mice was blocked by mecamylamine while
PPADS had no effect on fEPSPs in P2X2'/‘ tissues. These data indicate that P2X2 subunits
are a critical component of the P2X receptor expressed by S neurons. Although P2X2
subunits mediate the purinergic component of fEPSPs, the average fEPSP amplitudes
recorded from S neurons in P2X2'/' and P2X2‘i/+ tissues were not different. This result may
be related to the findings from previous studies that showed a functional interaction

between P2X and nicotinic acetylcholine receptors (Nakazawa, 1994; Zhou and Galligan,

1998; Searl et al. 1998; Barajas-Lopez et al. 1998; Khakh et al. 2000). In these studies it

149

was found that simultaneous activation of nicotinic and P2X receptors produces a
response that is smaller in amplitude than the predicted sum of responses caused by
individual activation of each receptor. It was concluded that there is a functional link
between P2X and nicotinic receptors that results in cross-inhibition of responses
mediated by these receptors (Nakazawa, 1994; Zhou and Galligan, 1998; Searl et a1.
1998; Barajas-Lopez et al. 1998; Khakh et a1. 2001). Deletion of the P2X; subunit gene
would remove cross-inhibition and synaptic responses mediated by nicotinic receptors in
P2X2'l' mice would be larger in amplitude than those occurring in neurons co-expressing
the P2X; subunit. Lack of P2X2- nicotinic receptor cross-inhibition could account for the

maintained fEPSP amplitude in P2X2'/' tissues.

S neurons in tissues from PZXZH+ mice were depolarized by ATP but not by or, B-
mATP. This result suggests that S neurons express P2X; homomeric receptors as 01,13-
mATP does not activate P2X; homomers, but does activate P2X3 homomeric and P2X2/3
heteromeric receptors (Lewis et al. 1995; North and Surprenant, 2000). In addition, ATP
failed to elicit a depolarization in S neurons from P2X2'l' mice. Based on these results, I
conclude that the P2X receptor mediating fEPSPs in murine S neurons is a P2X;
homomeric receptor. In the guinea pig intestine, P2X; subunit immunoreactivity has been
localized to NOS-containing neurons (Castelucci et al. 2002). NOS—containing neurons
are inhibitory motomeurons and descending intemeurons, and both of these classes of
neuron would have S-type electrophysiological properties (Brookes, 2001). If the
chemical coding of murine myenteric neurons is similar to that in the guinea pig intestine,

these data would indicate that inhibitory motomeurons (Johnson et al. 1999) and some

150

descending intemeurons (LePard & Galligan, 1999; Bian et al. 2000; Monro et al. 2002)

receive fast excitatory synaptic input mediated in part by P2X2 homomeric receptors.

P2X subtypes in myenteric neurons in guinea pig small intestine

Extensive immunohistochemical studies have attempted to identify P2X subunit
compositions in guinea pig myenteric neurons (Poole et al., 2002; Castelucci et al., 2002).
In these studies, it has been shown that P2X; subunits are localized to inhibitory motor
neurons and AH neurons in myenteric plexus. P2X; subunits are localized to inhibitory
motor neurons and S neurons. They are not found in AH neurons. In inhibitory motor
neurons, which are NOS immunoreactive, P2X receptors are P2X2/3 heteromers. The
pharmacological studies in other paper have found there are or, B-mATP-sensitive P2X
receptor subtypes (P2X. or P2X3) contributing fEPSP in S neurons of guinea pig intestine
(LePard et al. 1997). However patch-clamp recordings from myenteric neurons
maintained in primary culture indicate that only in a small subset (10 %) of myenteric
neurons, or, B-mATP caused a rapidly developing and desensitizing inward current,
suggesting that P2X receptors contain P2X] or P2X3 subunits (Zhou & Galligan, 1996).
In present study, all S neurons that were sensitive to ATP were depolarized by a, [3-
mATP but only 17% AH neurons were sensitive to a, B-mATP. A selective antagonist for
P2X] or P2X3 receptors, TNP-ATP reduced ATP-induced depolarization as well as
reducing fEPSP amplitude in S neurons. These results support immunohistochemical
identification and studies in acute isolated LMMPs in guinea pigs. However, these results
are not consistent with guinea pig myenteric neurons maintained in primary culture and

murine myenteric neurons. Most of neurons in primary culture do not desensitize to a, B-

151

mATP may be due to the alteration of P2X3 receptors in cultural condition. The
difference between myenteric neurons in guinea pigs and mice may be due to the
differentiation expression of P2X receptor between these two species; or myenteric S
type neurons in mice and in guinea pigs may play different functions. These data in the
present study suggest that, in guinea pig ileum, P2X receptors in myenteric S neurons
contained P2X3 subunits while P2X receptors in AH neurons were composed of few

P2X3 subunits.

152

P2X RECEPTOR-MEDIATED AFTERHYPERPOLARIZATION IN GUINEA

PIG MYENTERIC S N EURON S

Ionic basis of afterhypolarization

An important finding in the present study was that the depolarization induced by
a, B-mATP was followed by a hyperpolarization in 55 percent of tested S neurons. The
hyperpolarization was mediated by activation of potassium conductance because the
reversal potential of its underlying outward current was equal to K+ equilibrium potential.
The K+ conductance was gated by calcium because reduction of extracellular Ca2+
concentration greatly reduced the hyperpolarization. The influx of Ca2+ that activates K+
conductance was through P2X receptors as PPADS blocked depolarization therefore
abolished hyperpolarization consequentially. The proposed model for P2X receptor-
mediated hyperpolarization is shown as Figure 39. It has been known that P2X receptors
have a predominant Ca2+ permeability that equals to or greater than those of the 100
times more Na+ (Bumashev, 1998). We also noticed that the amplitudes of
hyperpolarization and depolarization were positively correlated although the other cations
are involved. It may still be arguable that Ca2+ could move into the neurons through
voltage-gated channels. It is unlikely the case in the present study since we recorded the
KCa-mediated outward currents in single electrode voltage-clamp recordings. The influx
of Ca2+ can have broad physiological effects by acting at various downstream targets,

which are Caz+-dependent K+ channels (K5,) in the present study.

153

Figure 39. A proposed model for P2X-mediated afterhyperpolarization in S

neurons

P2X3 subunit-containing receptors and calcium-dependent potassium channels are
expressed in myenteric S neurons in guinea pig small intestine. Specifically, they are
located closely within a micro-domain (shown in dashed box). Activation of P2X3
receptors by 0L,B-mATP induces calcium influx. The calcium ions entry activates KCa that

allows potassium flowing out of the cell, which results in afterhyperpolarization.

154

Figure 39

 

 

P2X3 Receptor

 

 

 

155

I attempted to identify the subtypes of K0, channels that mediate
hyperpolarization in S neurons. Large conductance KCa (BK) and small conductance KCa
(SK) have been described in the nervous system (Vergara et al., 1998; Faber and Sah,
2003; Stocker, 2004). Intermediate conductance Keg (1K) has been identified earlier in
non—neural peripheral tissues, particularly smooth muscle tissues (V ergara et al., 1998).
Recently [K is also documented in the enteric nervous system (Fumess et al., 2004).
Further studies have shown that IK is expressed in myenteric AH neurons and mediates
action potential aflerhyperpolarization in AH neurons (Vogalis et al., 2002; Neylon et al.,
2004). 1K is activated when Ca2+ flows into the neurons during action potential firing,
which mediates the long lasting action potential aflerhyperpolarization (Hirst et al., 1974;
Fumess et al., 1998; Vogalis et al., 2001). The selective toxins iberiori toxin (IBTx),
apamin and Clotrimazole against BK, SK and 1K were utilized to identify Kca subtype in
S neurons (Vergara et al., 1998). lBTx broadened the duration of action potential by
blocking BK in S neurons (Figure 31). Clotrimazole blocked action potential
afterhyperpolarization in AH neurons (Figure 21B), which indicated the effectiveness of
Clotrimazole on blocking lK. However none of these toxins affected P2X receptor-
mediated afterhyperpolarization. This result suggests that KCa in S neurons does not

belong to any of these three major types.

Present study provides evidence that KCa channels are expressed in S neurons. KCa
should mediate an afterhyperpolarization in S neurons as it does in AH neurons. However
action potentials are not followed by long lasting hyperpolarization in S neurons. There is
no action potential afterhyperpolarization in S type neurons because there is no Ca2+

influx during the action potential (Hirst et al., 1974), and not because there are no KCa

156

channels in S neurons. Ca2+ moves into the neurons during fEPSPs and a
hyperpolarization should be observed after flSPSP. However a single purinergic fEPSP
evoked by electrical stimulation on presynaptic nerve fibers was not followed by an after-
hyperpolarization. One explanation would be that the amount of Ca2+ influx through P2X
receptors during fEPSP is not sufficient to activate Kca channels. In addition, fast EPSPs
are mediated by P2X3 receptors in most of myenteric S neurons (see discussion above).
Ca2+ permeability of P2X receptor is dependent on different subtypes (Egan and Khakh,
2004). P2X; receptors have relative low permeability comparing with the other P2X
subtypes. The other possibility would be that the P2X3 receptors expressed at the synaptic
area do not couple to KCa channels. For example, immunohistochemistry staining
indicates that only 20% Dogiel type 1 neurons express IK channel immunoreactivity and

it is only seen in cell soma (Neylon et al., 2004).

Functional significance of AHP in myenteric S neurons

Another interesting observation was that only approximate half of tested S
neurons displayed the agonist-induced afler-hyperpolarization. I proposed that ligand-
gated hyperpolarization might be associated with a specific subset of S neurons that have
the same function. S type myenteric neurons are motor or intemeurons (Fumess, 2000).
This is based on the studies using combined electrophysiological and
immunohistochemical methods (Fumess, 2000). Purinergic synaptic transmission
contributes to the descending and ascending excitatory nervous reflex pathway as well as
inhibitory descending pathway (Spencer et al., 2000; Fumess and Sanger, 2002). P2X

receptors coupled to KCa may be a mechanism by which the neuronal firing rate is

157

controlled. 1 tested if this group of neurons belongs to a specific functional category of
purinergic S neurons. I examined the co-localization of these neurons with NOS neurons,
which are inhibitory motor neurons. However, my results did not show any association of
NOS-ir neurons with the neurons that displayed a,B-mATP-induced ligand-gated

hyperpolarization.

A previous study has documented that a depolarization induced by activation of
nicotinic AChRs is followed by a hyperpolarization in myenteric S neurons (Tokimasa et
al., 1982). The underlying mechanism is similar, which is due to calcium entry and
subsequent opening of Kc... Nicotinic AChRs and P2X receptors are both ligand-gated
cation channels, which are permeable to non-selective cations including Ca”. They are
the two primary ligand-gated ion channels in myenteric S neurons and have similar
functions in mediating fast synaptic transmission. The similar hyperpolarization they

induced may have similar physiological function.

158

RUNDOWN OF fEPSPS DURING BURSTS OF NEURONAL ACTIVITY

In this section, fast synaptic transmission during trains of electrical stimulation
was investigated. I found that fEPSPs declined during stimulus train and there was no
contribution of desensitization of nAChRs and P2X receptors. Action potential failure,
presynaptic inhibitory receptors or BK channels did not account for synaptic rundown. I

proposed that synaptic rundown is due to depletion of vesicles in RP.

Rundown of fEPSPs

In isolated segments of guinea-pig ileum, it has been shown that mechanical
stimulation of mucosal villi generates short bursts of fEPSPs that occur in the frequency
range of 15—40 Hz (Bomstein et al., 1991). In similar experiments, it was shown that if
the mechanical stimulus is applied at intervals of less than 10 s, the amplitude of fEPSPs
declined during repetitive stimulation (Smith et al., 1992). Chemical stimulation (low pH
or S-HT application) of the mucosa also elicits short bursts of fEPSPs (average of 14
fEPSPs per burst) occurring at frequencies between 5 and 20 Hz (Bertrand et al., 1997). I
studied the dynamics of fast synaptic excitation during short trains of electrical
stimulation of presynaptic nerve fibers. The frequencies of the stimulus trains that I used
mimicked the frequencies of fEPSPs evoked by the physiological stimuli described
above. In most neurons, these trains of electrical stimulation elicited fEPSPs that declined
in amplitude in a frequency dependent manner. The conclusions are based on data
obtained in S neurons, as these cells received fast excitatory synaptic input, and the action

potentials were not followed a long-lasting (>1 5) action potential afterhyperpolarization.

159

My data differ from those obtained in studies of fast synaptic input to AH type neurons in
the pig intestine where it was found that fEPSPs did not rundown when evoked by trains
of stimulation at frequencies up to 20 Hz (Cornelissen et al., 2001). Similarly, in the rat
colon, fast synaptic input to most myenteric S neurons is maintained during lO-Hz trains
of stimulation (Browning and Lees, 1996). These observations suggest that there are
species or tissue differences in the mechanisms regulating the availability and release of

stores of fast synaptic transmitters in the myenteric plexus.

Receptor desensitization did not contribute to synaptic rundown

ACh acting at nAChRs and ATP acting at P2X receptors mediate most fEPSPs in
myenteric S neurons in the guinea pig ileum (Galligan et al., 2000). Previous work
showed that nAChRs expressed by guinea-pig myenteric neurons desensitize faster than
P2X receptors expressed by the same cells (Zhou and Galligan, 1998; Brown and
Galligan, 2003). Based on this difference in desensitization rate, I proposed that nAChRs
and P2X receptors would make a differential contribution to the fEPSPs during trains of
stimulation. My hypothesis was that the nicotinic component would desensitize early in a
train of stimulation while P2X receptors would maintain synaptic transmission as the
stimulus trained continued. However, the data indicate that this does not happen, as
selective blockade of nAChRs or P2X receptors did not alter the rate of fEPSP rundown
during a 10 Hz train of stimulation. Furthermore, when ACh and ATP were applied by
trains of ionophoretic pulses to mimic fEPSPs during same stimulus train, the
ionophoretic depolarizations did not decline in amplitude. These data indicate that little

desensitization occurs during the brief occupation of nAChRs or P2X receptors by either

160

synaptically released or ionophoretically applied agonist. It was also found that
mecamylamine or PPADS applied individually each reduced fEPSPs by about 35% while
coapplication of these two blockers reduced the fEPSP by 90%. This result is consistent
with the previous finding that P2X receptors and nAChRs are linked in a mutually
inhibitory manner in guinea-pig ileum myenteric neurons (Zhou and Galligan, 1998;
Khakh et al., 2000). That is simultaneous activation of the two receptors produces a
response that is smaller than the predicted sum of responses caused by individual
activation of the two receptors. Blocking either the nAChR or the P2X receptor removes

cross-inhibition allowing a larger amplitude response at the unblocked receptor.

Rundown is not caused by presynaptic inhibition

Presynaptic inhibition is a predominant mechanism for modulating synaptic
transmission in the ENS. a2—Adrenoceptors are localized to cholinergic neurons and
these receptors mediate inhibition of acetylcholine release onto myenteric S neurons
(Stebbing et al., 2001; Scheibner et al., 2002). Opioid receptors are expressed on cell
bodies and processes of myenteric neurons and activation of opioid receptors inhibits the
electrically evoked release of acetylcholine (Stemini, 2001). Muscarinic M2 receptors
also mediate presynaptic inhibition of fast synaptic transmission in the myenteric plexus
(North et al., 1985). Adenosine A1 (Christofi and Wood, 1993) and S-HTIA (Pan and
Galligan, 1994) receptors also couple to presynaptic inhibition of fast synaptic excitation
in the ENS. Acetylcholine, norepinephrine, enkephalins and 5-HT-containing nerve fibers
are present throughout the myenteric plexus (Costa and Fumess, 1984; Costa et al., 1996;

Brookes, 2001) and trains of nerve stimulation can release all of the substances.

161

Adenosine is a breakdown product of neurally released ATP (Westfall et al., 2002) and it
will accumulate in the myenteric plexus during trains of nerve stimulation. Therefore, fast
synaptic rundown could be due to presynaptic inhibition caused by the accumulation of
endogenous agonists for presynaptic inhibitory receptors. However, the data show that
fast synaptic rundown persists in the presence of muscarinic, a2 adrenergic, opioid, Al
adenosine and 5-HT1A receptor antagonists. Based on these data, I conclude that
presynaptic inhibition mediated by one or more of these receptors are not responsible for
fEPSP rundown. However, although I tested the contribution of activation of several
presynaptic receptors to fEPSP rundown, there could be many other inhibitory
presynaptic receptors localized to myenteric nerve terminals. Inhibitory presynaptic
receptors in the ENS belong to the family of G-protein coupled receptors which link to
PTX-sensitive mechanisms and inhibition of neurotransmitter release (Zamponi, 2001).
In order to eliminate presynaptic inhibition mediated by G-protein coupled receptors, I
studied fast synaptic rundown in myenteric plexus preparations that had been treated with
PTX to inactivate the Gi/Go class of G-proteins. Fast synaptic rundown was identical in
control and in PTX-treated tissues indicating that presynaptic inhibition does not account
for fast synaptic rundown. Although PTX-treatment did not alter synaptic rundown, the
treatment protocol was effective in blocking the target G-proteins. This conclusion is
based on the data showing that the a2-adrenoceptormediated IPSP recorded from
submucosal S neurons was blocked in preparations that had been treated using our PTX
incubation protocol. IPSPs in submucosal neurons are mediated by a2-adrenoceptors that

couple directly to a K+ channel via a PTX-sensitive G-protein (Surprenant and North,

1988)

I62

Changes in presynaptic action potentials do not contribute synaptic rundown

Frequency-dependent decreases in nerve terminal Ca2+ influx could result in
synaptic rundown. Decreased nerve terminal Ca2+ entry could be due to axonal action
potential failure during high frequency nerve stimulation (Cunnane and Stjame, 1984). I
made recordings from neurons in which fEPSPs and antidromically propagated action
potentials were recorded simultaneously. These experiments showed that action
potentials followed each stimulus in a 10 Hz train while fEPSPs declined in amplitude.
These data indicate that action potential propagation into the nerve terminal is maintained
throughout a train of stimuli and action potential failure does not contribute to fEPSP
rundown. Frequency-dependent shortening of action potential duration could also
contribute to synaptic rundown. This could occur either through Ca2+ channel inactivation
or though a Cay-dependent activation of K+ channels which would accelerate action
potential repolarization (Franciolini et al., 2001). Inactivation of Ca2+ channels in
myenteric neurons occurs in the time range of 0.1— l s (Bian et al., 2004). As an
individual action potential duration is 2 ms or less, it is unlikely that Ca2+ channel
inactivation makes a significant contribution to fEPSP rundown in myenteric neurons.
However, in the absence of direct data about calcium channel dynamics in the nerve
terminal, I cannot categorically rule out a contribution of Ca2+ channel inactivation to
fEPSP rundown. BK channels are a class of Ca2+-activated K+ channels expressed in
nerve terminals and these channels function to limit nerve terminal action potential
duration and Ca2+ entry (Robitaille and Charlton, 1992). IBTx, a BK channel blocker
(Wanner et al., 1999), did not alter the time course of fEPSP rundown indicating that BK

channel-dependent shortening of action potential duration do not contribute to fEPSP

163

rundown during trains of stimulation. However, I did find the IBTx inhibited PPD in a
subset of neurons as the decline in amplitude of the second fEPSP in the presence of
lBTx was less than that occurring in the absence of the toxin. This result suggests that
BK channel activation contributes to PPD during the first two fEPSPs in a train of
stimulation but that other processes mediate fEPSP rundown as the train duration
lengthens. IBTx increased action potential duration in a subset of S neurons. These data
indicate that BK channels regulate action potential duration in some S neurons and that
Ca2+ must enter these neurons during a single action potential. Direct measurements have
shown that short trains of action potentials can cause measurable increases in intracellular
Ca2+ in some myenteric S neurons; N-type Ca2+ channels mediate this response
(Shuttleworth and Smith, 1999). Although single action potentials did not evoke a
measurable Ca2+ increase, changes caused by a single spike may be under the detection
limits of the assay or there may be local increases in Ca2+ that were not detected. These
local increases in Ca2+ could be restricted to sights near the BK channel as previous
studies in hippocampal neurons have shown that there is a close spatial and functional
relationship between N-type Ca2+ channels and BK channels (Marrion and Tavalin,
1998). A similar relationship may exist in some myenteric S neurons. This relationship is
specific for some S neurons as IBTx did not alter the action potential in AH neurons. This
result is consistent with previously published work showing that IBTx does not alter

action potential duration in AH neurons (Kunze et al., 1994).

164

Depletion of transmitter accounts for synaptic rundown

When nerve fibers were stimulated at a lower frequency (0.5 Hz), fEPSP
amplitude declined by 50% after the first stimulus but was maintained for the remainder
of the stimulus train. Higher frequencies of stimulation resulted in complete rundown of
the fEPSP. There are two models that could explain these data. Firstly, there may be two
separate neurotransmitter pools in myenteric nerve terminals. A readily releasable pool
(RRP) is available for exocytosis during the first action potential in a train and at low
stimulation frequencies; a reserve pool will partly replenish the RRP prior to subsequent
stimuli (Rosenmund and Stevens, 1996; Pyle et al., 2000; Sudhof, 2000; von Gersdorff
and Borst, 2002; Zucker and Regehr, 2002). The release probability of the RRP is very
high as a single action potential can deplete this pool. In addition, we never observed PPF
whose occurrence is most prominent at low probability synapses (Thomson, 2000;
Zucker and Regehr, 2002). At stimulation frequencies >5 Hz, fEPSPs decline in
amplitude because transmitter release rate exceeds the RRP refilling rate (Stevens and
Tsujimoto, 1995; Rosenmund and Stevens, 1996; Dobrunz and Stevens, 1997; Lin et al.,
2001). In myenteric nerve terminals, the refilling rate of the RRP is relatively slow (7 s)
and the slow filling rate will limit the duration and frequency at which bursts of fEPSPs
can occur. The mechanism by which vesicles are replenished after short-term depletion
may vary among synapses with different recovery time constants. For example, the t for
recovery is 5 s at synapses between hippocampal CA3 and CA1 pyramidal neurons
(Debanne et al., 1996), 3 s at synapses between cerebellar granule and Purkinje cells, 4 s
at the calyx of Held in the brain stem (von Gersdorff et al., 1997), and 2.8 s at synapses in

the rat superior cervical ganglion (Lin et al., 2001). The recovery rate depends on

165

 

trafficking between vesicle pools and the mechanisms of vesicle movement at different
synapses may be specialized to meet the particular demands of those synapses. Vesicle
mobilization from a reserve pool can be a principal mechanism for synaptic recovery
after rundown in the myenteric plexus. Mobilization from the reserve pool is relatively
slow (Pyle et al., 2000) and this would account for synaptic rundown during trains of
stimulation >5 Hz. A second model that would account for our data involves vesicle
recycling (Sudhof, 2004). In this scheme, the RRP is released by the first action potential
in a train. At low frequencies of stimulation (0.5 Hz), some of these vesicles recycle and
are then available for release prior to the next action potential. At stimulation frequencies
>5 Hz, the release rate exceeds the recycling rate and the RRP becomes depleted. Rapid
vesicle recycling (1:<l s) contributes to maintained transmission at synapses in the
hippocampus where the recycling rate is rapid enough to maintain transmission at a 10
Hz stimulation frequency (Pyle et al., 2000; Sara et al., 2002; Fernandez-Alfonso and
Ryan, 2004). If vesicle recycling occurs in the myenteric plexus, it must occur at a much

slower rate than in the hippocampus.

To summarize these studies, trains of electrical stimulation evoke fEPSPs that
decline in amplitude in a frequency-dependent manner in the guinea-pig ileum myenteric
plexus. Rundown of fEPSPs is not due to postsynaptic receptor desensitization,
presynaptic inhibition mediated by G-protein coupled receptors or to changes in
presynaptic action potentials. The data presented here are consistent with a model in
which there are two pools of neurotransmitter in myenteric nerve endings. There is
readily releasable pool of transmitter that has a high release probability; this pool is

depleted following a single action potential. At a low stimulation frequency (0.5 Hz), fast

166

 

 

 

#4.

synaptic transmission is maintained either by recycling of released synaptic vesicles or by
replenishment of the readily releasable pool by a reserve pool of transmitter. At high
frequencies of stimulation (20 Hz), fEPSPs decline in amplitude because the stimulation
rate exceeds the rate at which the readily releasable pool can be restored either by

recycling or by refilling from a reserve pool.

 

 

I67

S-HT4 RECEPTOR-MEDIATED PRESYNAPTIC REGULATION OF FAST

SYNAPTIC TRANSMISSION

This section of my dissertation investigated the regulation of fast synaptic
transmission by 5-HT4 receptors. My data show that S-HT4 receptor agonists enhance
both fast cholinergic and purinergic synaptic transmission. During a short train of nerve
stimulation, activation of 5-HT.; receptors increased amplitudes of each fEPSPs and
prevented fEPSP from declining to zero. But the rundown rate was not altered by 5-HT4
receptor agonists. Stimulation of 5-HT4 receptors also accelerated the recovery rate of
fast synaptic transmission after rundown. Further study in cultured myenteric neurons
indicated that 5-HT4 receptor agonists promote fast synaptic transmission by increasing
neurotransmitter release probability in single varicosities. The mechanism of S-HT4

receptor-mediated facilitation of fEPSPs can be summarized in Figure 40.

5-HT.; receptors are localized to nerve terminals of CGRP IPANs (Grider, 2003;
Liu et al., 2005). 5-HT4 receptor stimulation facilitates peristaltic reflexes by increasing
release of CGRP and ACh (Grider et al, 1998; Grider, 2003; Gershon, 2004). 5-HT4
receptors are a target for drug treatment of GI motility disorders. For example, the S—HT4
receptor agonist, teagaserod (Zalnorm), is used as prokinetic agent to treat constipation-
predominant IBS (IBS-C) and chronic constipation. However, the underlying mechanism

is not entirely clear.

As discussed above, fast synaptic transmission occurs in bursts and it declines in

response to physiological stimulation. Therefore investigation of the effects of 5-HT4

168

Figure 40. A proposed model for S-HT4 receptor-mediated presynaptic regulations

Agonists of presynaptic 5-HT.; receptors activate PKA through stimulating AC
and formation of cAMP. PKA phorsphorylates the proteins in the processes of vesicle
trafficking and releasing to facilitate neurotransmitter release. The target proteins remain

unknown.

169

hut-__I-

 

Figure 40

  

5-HT4 Receptor

   

S neuron

 

Actin cytoskeleton

I70

receptor agonist on fast synaptic transmission during a train of stimulation could provide
more precise information in physiological condition. In addition, I proposed that the
rundown of fEPSPs was attributed to depletion of vesicles in readily releasable pool
(RRP) and there was no direct proof for it. As S-HT4 receptor couples Gs-protein to
stimulate adenylate cyclase-cAMP-PKA cascade and synaptic vesicle trafficking and
releasing in nerve terminal can be affected by products of this cascade, identification of
5-HT4 receptor-mediated regulation of fEPSPs rundown and recovery could help to

discover more evidence for understanding mechanisms of fEPSP rundown.

Activation of S-HT4 receptors attenuates synaptic run-down and accelerates

recovery from rundown

Under control conditions, fEPSPs decline to zero amplitude at stimulus trains at 5,
10 and 20 Hz. Renzapride, a selective 5-HT4 receptor agonist, increases ACh and ATP
release elicited by a single shock. Renzapride also increased all fEPSPs elicited by 20
stimuli in a 10 Hz stimulus train. However, the fEPSPs still decline in amplitude. The
decline of fEPSPs was also fit by monoexponential equation. This suggests that
renzapride acting at S-HT4 receptors altered the same process resulting in synaptic
rundown. Interestingly, the rundown rates before and after renzapride were similar. As
we have discussed above, fEPSP declines during stimulus train and the synaptic rundown
is very likely because of the depletion of vesicle pool. Although renzapride increased all
fEPSP amplitudes, it did not affect the rate of rundown but maintain fEPSP to a higher
non-zero level. In the experiment of examining synaptic recovery, it was observed

renzapride extremely reduced the time that takes fEPSP to recover after rundown.

l7l

Based on depletion model, synaptic rundown and recovery represent the processes
of vesicle trafficking and release (Thakur et al., 2004; Sudhof, 2004; Rizzoli and Betz,
2005). At synaptic terminals of myenteric neurons, the size of readily releasable pool
(RRP) is small and/or releasing probability is high since the PPD was observed (Zucker
and Regehr, 2002). Activation of S-HT4 receptors mobilizes synaptic vesicles in
recycling pool and facilitates vesicle trafficking to RRP, which is evidenced by reduced
recovery time. The accelerated replenishment of RRP maintains fEPSPs at certain
amplitude. However, the time constant of synaptic rundown did not change before and
after renzapride treatment. It suggests that activation of 5-HT4 receptors also stimulates
transmitter release or increases releasing probability from a given single releasing site

(Schneggenburger et al., 2002).

Postsynaptic current (PSC) is determined by “Npq”, where q is postsynaptic
quantal size and p is probability of release (Schneggenberger et al., 2002; Zucker and
Regehr, 2002). “N” can be the number of vesicles or the number of morphologically
defined release sites. In sympathetic neuromuscular junction, transmitters are released
from single varicosities and each single varicosity is a synapse with a single active zone
(Bennett, 2000). Therefore “ N ” can be regarded as the number of release sites in the
ENS. The increase of transmitter release may be due to increased release probability by
enlarged pool size of RRP and/or facilitation of vesicle releasing protein complexes.
Because synaptic potentials decline at the similar rate after renzapride treatment, the
processes of vesicle trafficking and release should be regulated in an equivalent manner.
This result provides an indirect evidence that synaptic rundown is a result of vesicle

depletion.

I72

S-HT4 receptor-mediate presynaptic regulation of synaptic transmission is through

adenylate cyclase-cAMP-PKA signaling pathway

Significant progress has been made in mechanisms of metabotropic receptor-
mediated regulation of transmitter release in the CNS (Varma et al., 2001; Sudhof, 2004;
Rizzoli and Betz, 2005) but not many data have been accumulated in the ENS. There are
quite a few presynaptic mechanisms to increase synaptic vesicle mobilization and
neurotransmitter release (Engelman and MacDermott, 2004). Briefly, two broad models
have been proposed: 1) Cay-dependent, increase of Ca2+ entry presynaptically can
promote transmitter release (Brown et al., 2004); and 2) Ca2+ independent, the releasing
complex including proteins or other molecules can also be regulated to facilitate
transmitter release (Blackmer et al., 2001; Takahashi et al., 2001). In both cases, the

targets are common: vesicle recycling and vesicle releasing.

My data indicate activation of 5-HT4 receptors stimulates vesicle mobilization and
facilitates transmitter release. These regulations were mediated by stimulation of
adenylate cyclase and PKA as forskolin mimics renzapride and H-89 abolished effects of
renzapride. Because CAMP is a second messenger that activates PKA, cAMP must also
be a mediator. PKA can phosphorylate many protein targets, some of which are
suggested to regulate vesicle trafficking and release in the CNS (Chavez-Noriega and
Stevens, 1994; Weisskopf et al., 1994; Sudhof, 2004). For example, synapsin is a vesicle
phosphorprotein in nerve terminals (Chi et al., 2001; Sudhof, 2004). It has been
suggested that synapsin functions to bind vesicles to actin cytoskeleton (Sudhof et al.,
1989; Chi et al., 2003). Three synapsin subtypes have been identified and all three

subtypes have a common N-terminal domain that is a phosphorylation site for PKA.

173

Phosphorylation of synapsin controls dynamic mobilization of synaptic vesicles to
regulate vesicles pool size (Hosaka et al., 1999; Turner et al., 1999). In hippocampal
neurons, PKA facilitates transmitter release by acting directly at the proteins of the
neurotransmitter releasing machinery (Capogna et al., 1995; Trudeau et al., 1996).
SNAP-25 is a target for PKA phosphorylation to regulate transmitter release (Nagy et al.,
2004). Another example is Snapin, a SNAP-25 binding protein. Snapin has been shown a
target that can be phosphorylated by PKA to increase transmitter release in hippocampal
neurons (Thakur et al., 2004). Here my data show that activation of PKA is necessary for
S-HT4 receptors to facilitate transmitter release and synaptic recover. Although the
targets in myenteric nerve terminals for PKA need further identification, 5-HT4 receptor-

activated PKA must affect the processes of both transmitter mobilization and release.

Activation of 5-HT4 receptors increases probability of transmitter release from

single varicosities

The studies of 5-HT4 receptor-mediated synaptic regulation were carried out in
acutely isolated LMMP preparations. Electrical nerve stimulation used to evoke synaptic
responses activates multiple nerve fibers instead of a single one. Under such a recording
situation, fast synaptic potential or current represents the neurotransmitter released from
multi-nerve endings/releasing sites. Different from the CNS, neurotransmitter in the ANS
is released from single varicosities. For example, sympathetic nerves end in strings of
varicosities, which form individual synapses with smooth muscle cells. In the CNS, each
nerve terminal has multi-releasing sites (active zones). The Gaussian distribution of

spontaneous release recorded from a nerve terminal in the CNS is actually an indication

174

 

of transmitter release from multiple active zones. However, in the autonomic nervous
system, each varicosity has one active zone (Bennett, 2000). Studies of synaptic
potentials from varicosities in the vas deferens indicated that the neurotransmitter release
from a single varicosity is positively skewed and transmitter release at different
varicosities has a nonuniform probability (Bennett, 1998; Bennett et al., 1995; Brain et
al., 2002) and therefore transmitter release from in varicosities is highly intermittent
(Brain et al., 2002; Bennett, 1996). This intermittent is not due to intermittent failure of
action potential propagation in the nerve terminal (Jackson et al., 2001; Bennett, 1996).
To identify how S-HT4 receptor stimulation increases transmitter release in the ENS, it is

necessary to investigate the transmitter release from one single varicosity.

Varicosities can be visually identified docking along the processes of myenteric
neurons maintained in primary culture. Cultured myenteric neurons express P2X and
nACh receptors. The synaptic responses are also maintained. “Detector patch” technique
can be utilized in the cultured neurons to monitor single varicosity activity. In my
experiments, a patch containing P2X or/and nACh receptors was used as a detector. For
one given single varicosity, there was no spontaneous current observed in detector patch.
This does not necessarily mean there is no spontaneous release. The sensitivity in the
experimental setup could limit observation of current induced by spontaneous release.
However, electrical stimulation evoked transmitter release and the evoked release
probability is 27%. Application of 5-HT4 receptor agonists, renzapride and tegaserod
increased evoked release probability to 50%. The increase of release probability in the

given varicosity may be due to an enlarged RRP size or to a direct regulation of proteins

175

in release machinery or both. As discussed above, the increase of release probability is

through PKA phosphorylation of proteins in processes of trafficking and releasing.

Studies in neuromuscular junction led to a classic model of quanta release and this
model has been widely accepted to be true also in the CNS. The Katz’s model defined the

number of release site (N), probability of transmitter release (p) and quantal size (q) (del

Castillo and Katz, 1954; Dittman et al., 2000).

Considering the equation (Schneggenburger et al., 2002):

PSP=npq; where

PSP: postsynaptic potential;

n: the number of releasing sites (varicosities or actiVe zones);

p: probability of release from one releasing site (varicosity or active zone);

q: postsynaptic quantal size.

Postsynaptic responsive quantal size is independent of release probability and p is
the only factor responsible for short—term synaptic plasticity (Biro et al., 2005). In the
ANS, a varicosity can be considered as equivalent to an active zone in one nerve terminal
since it is one releasing site. Activation of 5-HT.; receptors increases release probability
(p) from the single release sites. All varicosities sitting in the same nerve fiber and in the
range that postsynaptic receptors can be activated can be regarded as the different

releasing sites in one “terminal”. The transmitter release from multi-release sites is

heterogeneous upon arrival of an action potential. This release is at low probability.

176

6‘ 9,

Renzapride or tegaserod acts at 5-HT4 receptors to enhance p value by activating PKA

and therefore increases PSP.

177

SUMMARY AND RESEARCH SIGNIFICANCE

In this dissertation, I have identified P2X receptor subtypes in myenteric neurons
in the small intestine of guinea pigs and mice. 1 have found a novel calcium-dependent
potassium channels that underlie afierhyperpolarization induced by P2X receptor
agonists. I have characterized fast synaptic transmission elicited by stimulus train. I have
also investigated regulation of fast synaptic transmission mediated by presynaptic S-HT4
receptors and identified the underlying mechanism. All these issues were addressed

around a central topic: the dynamic properties of fast synaptic transmission.

In guinea pig small intestine, myenteric S neurons express P2X3 containing
receptors while AH neurons express P2X; homomers. However, in mouse small intestine,
myenteric S neurons express P2X2 homomers; AH neurons express P2X3 subunit-
containing receptors. This difference may be due to the difference in species, which
implies the possible different firnction of these myenteric neurons. P2X3 receptors in half
number of guinea pig myenteric neurons mediate an after-hyperpolarization, which is
mediated by a novel calcium-dependent potassium conductance. But its physiological

function is not clear.

Fast synaptic transmission is depressed during a short stimulus train at 5, 10 and
20 Hz. Synaptic depression is frequency dependent. There is no postsynaptic receptor
desensitization, presynaptic inhibition or action potential failure for the depression.
Synaptic vesicle depletion is proposed as a mechanism. The synaptic depression can be

attenuated by activation of 5-HT4 receptors. Activation of 5-HT4 receptors also

178

accelerates synaptic recovery after depression. PKA has been indicated as a mediator for
5-HT4 receptor-mediated regulations. The targets for PKA remain unknown. Further
study in cultured myenteric neurons indicates that 5-HT4 receptor agonists facilitate

transmitter release by increasing release probability from single varicosities.

All these findings are summarized in Figure 41.

The significance of my findings can be discussed from two aspects: neurobiology

and gastroenterology.

Neurobiology focuses more on the neural basis of behavior. How the neurons
process information to generate a corresponding behavior is a fundamental and also an
ultimate question for neurobiologists. The ENS underlies GI behaviors. So one of my
goals is to understand the way enteric neurons control GI behaviors. Synaptic encoding is
one of mechanisms by which neurons process input signals. For example, synaptic
depression has been shown to be a mechanism that regulates synaptic activity in the CNS.
My data show there is also synaptic depression in the ENS, which probably has a similar
function in limiting synapses from over activity. Vesicle depletion is the mechanism for
this regulation. This is also a target for presynaptic modulation such as by presynaptic 5-

HT4 receptors.

In the view of gastroenterology, the pathological change of GI functions
draws more concerns. Gastroenterologists would like more to look into the
neuropathology of GI and pursue a best therapy for GI disorders. Understanding the

fundamental properties of the ENS will provide the basis to identify neurological

179

Figure 41. A summary for receptors studied in this dissertation in myenteric

neurons

In myenteric plexus, synaptic transmission is polarized as shown in the figure.
Synaptic activation is coordinated with oral contraction and anal relaxation, which
initiates peristalsis. Receptors are expressed accordingly to accomplish the functions.
P2X receptors primarily mediate descending pathway (the anal direction). In guinea pig
small intestine, myenteric AH neurons express P2X2/2 homomers and S neurons express
P2X2/3 heteromers. However, in mouse small intestine, myenteric AH neurons express
P2X2/3 heteromers and S neurons express P2X2/2 homomers as pointed by dashed lines. In
guinea pig small intestine, fifiy percent myenteric S neurons also express calcium-
activated potassium channels, which mediate an afterhyperpolarization. 5-HT4 receptors
are expressed in nerve terminals. 5-HT4 receptor agonists, renzapride or tegaserod

increases fEPSP by increasing synaptic transmitter release probability.

. S-HT4 receptors; . P2X2/2 homomers. P2X2/3 heteromers. Calcium-activated
potassium channels; The dashed arrows indicate receptors in boxes activated by the

agonists in mouse tissue.

LM, longitudinal muscle; MP, myenteric plexus; CM, circular muscle; SMP, submucosal

plexus; IPANs, intrinsic primary afferent neurons.

180

 

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181

disorders and facilitate the treatment. As shown in my data, P2X receptor subtypes are
expressed differentially in the different types of myenteric neurons. So P2X receptor
subtypes can be selected as potential drug target. 5-HT4 receptor-mediated dynamic
changes of transmitter trafficking and release provide a basis for pathology of some GI
motility disorders as 5-HT4 receptor agonists are used as prokinetic drugs. For example,
the patients who suffer from GI motility disorders probably have an abnormal alteration
of vesicle trafficking and releasing processes in the myenteric nerve terminals.

Meanwhile this also offers more opportunities to find ways to treat these diseases.

Finally, the ENS represents a unique case in the mammalian nervous system. It is
a highly isolated system and controls a well-defined range of functions. Therefore the
ENS can be considered as a very appropriate model to challenge many questions in the
area of fundamental neuroscience and therefore help to understand neuropathology of GI

disorders.

I82

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