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dissertation entitled

Characterization of an EPSP-Like
Potential in the Ventral-Cord of
'Cricket, Acheta domesticus

presented by

Kamakshi Venkatarao

has been accepted towards fulfillment
of the requirements for

Ph.D. Audiology & Speech Sciences

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CHARACTERIZATION OF AN EPSP-LIKE POTENTIAL IN THE

VENTRAL-CORD OF CRICKET, AQflEIA_DQfl§§IIQQ§

BY

Kamakshi Venkatarao

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements for
the degree of

DOCTOR OF PHILOSOPHY

Department of Audiology and Speech Sciences

1992

ABSTRACT

CHARACTERIZATION OF AN EPSP-LIKE POTENTIAL IN THE
VENTRAL-CORD 0F CRICKET. AQEEIA_DQHE§II§Q§

BY

KAMAKSHI VENKATARAO

In the mammalian auditory system the neuro-transmitter(s),
released from the hair cells in the cochlea by mechano-
electrical stimulation, excite type I afferent auditory
nerve fibers, which can be recorded as post-synaptic
potentials. If these potentials are excitatory they are
called excitatory post-synaptic potentials (EPSP), and if
large enough, will initiate action potentials (AP). A
review of the literature has indicated that few
investigations have addressed questions about the EPSPs
generated by auditory afferent dendrites of the hair
cell/auditory nerve synapse. The EPSP is the generator
potential which leads to the generation of the AP. In fact,
the auditory EPSP is an integral part of the coding
mechanism and, if well characterized, may be used as a

sensitive index of hair cell-auditory nerve functioning.

Among the studies which have investigated the EPSP, there is
a controversy as to whether the recorded EPSP is truly a
post-synaptic potential or a summating potential (DC shift

of the hair cells of the cochlea in response to basilar

membrane motion). To address this question, house crickets
(Acheta ggmesticus, invertebrates which do not possess a
cochlea) were used as experimental subjects. Gross
potential recordings were made to identify EPSP—like
potentials from the terminal ganglion, which is the first
synapse in the cereal hearing system of the cricket, located

in the cricket's ventral-cord.

To identify and characterize the EPSP-like potential several
experiments were conducted in which various stimulus
parameters, tetrodotoxin (a neurotoxin) and kynurenic acid
(an excitatory amino-acid antagonist) were used. Results
indicated that it is possible to identify and characterize
the EPSP-like potential at intensity levels below the
threshold for the AP, at frequencies below 2000 Hz, with a
repetition rate of 3.1/sec and a rise-decay time between 1.5
and 6.0 ms. Tetrodotoxin significantly reduced the AP but
not the EPSP-like potential. Kynurenic acid reduced the
amplitude of the EPSP-like potential within a few minutes of
its application. It is concluded that the EPSP-like
potential is a true neural response and is an integral part
of the coding mechanism for neural transmission within the

crickets cercal system.

Dedication

To my dear husband Gopal, daughter sushmita, and son Tejas,

for their ever present love and support.

iv

ACKNOWLEDGEMENTS

I would like to express my deepest appreciation to my major
professor and chairperson of my dissertation committee Dr.
Ernest. J. Moore for his knowledge, understanding,
inspiration, intellectual stimuluation, invaluable help, and
for the positive role he played as my mentor. I would like
to sincerely thank the members of my dissertation committee
Dr. Jerry. L. Punch; Dr. John. B. Eulenberg; and Dr. John.
I. Johnson for their help, guidance, support and
encouragement. Special appreciation goes to Mr. Randy Robb,
for his unflinching technical support. My special thanks to
Dr. Connie Osborn and Dr. Charles Lowrie who contributed
their time, effort and expertise to this study. I once
again wish to thank Dr. Ernest. J. Moore, past chairperson
and Dr. Michael Casby, present chairperson of the Department
of Audiology and Speech Sciences, Michigan State University
for providing the facilities and financial support which
allowed me to undertake this venture. Finally my very
special thanks go to my husband Gopal, daughter Sushmita,

and son Tejas for their constant encouragement, support and

understanding.

List of
Figures

List of
Tables

List of
Symbols

Chapter

Chapter

Chapter

Chapter
Chapter

Chapter

TABLE OF CONTENTS

andAberViations ............OOOOOOOOOOOOOOOOO

I.

II.

III.

IV.

VI.

INTRODUCTION 0.0.0..........OOOOOOOOOOOO l
REVIEWOF LITEMTURE ......OOOOOOOOOOOOOO 12

Introduction ......................... 12
Synaptic Transmission ................ 13
Excitatory Post-Synaptic Potentials ... 14
Integration of Signals ............... 17
Evolution of Ears .................... 17
Transduction in the Inner Ear ........ 18
Synapses in the Inner Ear ............ 22
Auditory EPSP ........................ 23
Glutamate as the Neurotransmitter .... 31
General Features of House Cricket .... 34
The Cricket Auditory System ........... 36
Cercal Receptors ..................... 38
Anatomy of Cercal Hearing System ..... 39
Electrophysiologic Features .......... 51
Neurotransmitters in SAG ............. 58

INSTRUMENTATION AND PROCEDURE ........... 61
Stimulus Generating Section .......... 61
Analog section 0 O O O O O O O O O O O O O O O O O O O O O O 64
Digital section 0 O O O O O O O O O I O O O O O O O O O O O 64
Display Section ...................... 65
calibration system 0 O O O O O O O O O O O O O O O O O O 66
Materials and Procedures ............. 70

RESULTS ......OOOOOOOOOOOOOO ........... O. 76

DISCUSSION 0 O O O O O O O O O O I O O O O 0 O O O O O O O O O O O O O 130

SUMMARY AND CONCLUSIONS ................. 145

vi

REFERENCES

APPENDICES

Appendix A.

Appendix B.

Appendix C.

Appendix D.

Appendix E.

Appendix F.

Analog waveforms of CAP from four
crickets depicting the effect of
intensity levels ........................

Analog waveforms of CAP from two
crickets depicting the effect of
stimulus frequency ......................

Analog waveforms of CAP from two
crickets depicting the effect of
rise-decay time of the stimulus .........

Analog waveforms of CAP from two
crickets depicting the effect of
repetition rate of the stimulus .........

Analog waveforms of CAP from two
crickets depicting the effect of
phamaCOIOgic agents 0 O O O O O O O O O I O O O O O O O O O

Input-Output functions of No, Po, N1 and

EPSP-like potentials as a function of TTX
and kynurenic acid from two crickets ....

vii

153

171

171

176

179

182

185

188

LIST OF FIGURES

I-lA. Schematic representation of the mammalian
hair-cell auditory afferent synapse ........... 7

I-lB. Schematic representation of the receptor
afferent fiber in the cricket cercal hearing
SYBtem ......OOOOOOOOOOOOOOO......OOOOOOOOOOOO 7

11-1. Electron micrograph of a single cercum from
acr1CRet .....OOOOOOOO0.00.0.0.0...0.0.00.0... 40

III-1. Block diagram of the instrumentation .......... 62
III-2. Block diagram of the calibration system ....... 67

III-3. Diagram representing typical electrode
placement on the cricket ..................... 73

IV-l. Typical electrophysiologic recordings from
two crickets for high intensity, low
frequency acoustic stimuli ..................... 79

IV-2. Electrophysiologic recordings of No, Po and N1
potentials as a function of polarity ........... 82

IV—3. Electrophysiologic recordings of No, Po and N1
potentials as a function of varying the
intensity in 5.0 dB increments ................. 85

IV-4. Electrophysiologic recordings of No, Po and N1
potentials as a function of varying the
intensity 1.0 dB increments ................... 92

IV-5. Electrophysiologic recordings of No, Po and N1
potentials as a function of frequency .......... 94

IV-6. Electrophysiologic recordings of No, Po and N1
potentials as a function of rise-decay time .... 96

IV-7. Electrophysiologic recordings of No, Po and N1
potentials as a function of repetition rate .... 99

IV-8. Electrophysiologic recordings as a function of
Simultaneous maSRing 00............OOOOOOOOOOOOO 102

viii

IV-9A. Input-output mean latency vs intensity
functions for No, Po and N1 potentials from five
criCKets ......OOOOOOOOOOOOO......OOOOOOOOOOOOOI

IV-9B. Input-output mean size (amplitude) vs intensity
functions for No, Po and N1 potentials from
five criCkets O......OOOOOOOOOOOOOOOOOO00......O

IV-lOA.Input-output latency vs intensity (1.0 dB
steps) functions for No, Po and N1 potentials ..

IV-lOB.Input-output size vs intensity (1.0 dB steps)
functions for No, Po and N1 potentials .........

IV-11. Electrophysiologic recordings of No, Po, N1 and
EPSP-like potentials as a function of TTX and
kynurenic aCid 0.00.0.0...OOOOOOOOOOOOOOOOOOOOO

IV-12. Input-ouput functions of No, Po, N1 and EPSP-
like potentials as a function of TTX and
kynurenic aCid 0.0.0..........OOOOOOOOOOOOOOOOOO

IV-13. Electrophysiologic recordings of No, Po, N1 and

EPSP-like potentials as a function of TTX over
a period of 224 minutes ........................

ix

107

109

112

112

120

123

126

TABLE

TABLE

TABLE

TABLE

Table

IV-lA.

IV-lB.

IV-2.

Iv-3 o

IV-4.

LIST OF TABLES

Latency of No, Po and N1 potentials
in crickets 063, 067, 071, 072 and

076 ......OCOOOOOOIOO0.000000000000000... 87

Amplitude of No, Po and N1 potentials
in crickets 063, 067, 071, 072 and

076 00.000.000.000.........OOOOOOOOOOOOOO 89

Mean and SD of latency and size of No,

Po and N1 potentials in five crickets

used for plotting the input-output
latency/intensity functions ............. 105

Latency and size of No, Po and N1
potentials in Cric-067 as a function
of intensity varied in 1.0 dB increments. 114

Slopes and intercepts of No, Po and
N1 potentials for various functions ..... 116

LIST OF SYMBOLS AND ABBREVIATIONS

Action Potential (AP) - The cell membrane of neurons when
excited undergoes permeability changes which produce an
electromechanical all-or-none disturbance that is actively
propagated along the length of the axon. The associated

voltage change is termed a spike or action potential.

Compound action potential (CAP) = CAP is the sum of the
action potentials of many individual neurons which are

firing nearly simultaneously to acoustic stimulation.

Excitatory post synaptic potentials (EPSPs) and inhibitory
post-synaptic potentials (IPSPs) = The electrical effect at
the post-synaptic membrane site is dependent upon the neuro-
transmitter substance released by the pre-synaptic membrane.
Two types of activity exist, excitatory and inhibitory. For
the excitatory post-synaptic activity the transmitter
produces a local depolarization by increasing membrane
permeabiltiy for K+ and Na+. For the inhibitory post-
synaptic potentials the transmitter increases the
permeability to K... and Cl' causing hyperpolarization and a
decrease in neuron excitability. EPSPs are also referred to
as generator potentials, and if large enough will generate

action potentials.

xi

EPSP-like potential = Excitatory post-synaptic potentials.
that are not recorded intracellularly or in a single unit,

but as a gross potential from surface recordings.

Gross potential = A gross potential is interpreted as the
summed effect of massed action potentials travelling in a
volley down the auditory nerve. Electrodes that are too
large to record from a single neural element can record
summed neural activity produced by the simultaneous

activation of many neurons.

No a No is the neural negative wave preceding the EPSP-like
potential.
Po 8 Po is the neural positive wave preceding the EPSP-like

potential and following the No potential.

N1 = N1 is the first dominant negative wave of the gross
neural response recorded to suprathreshold acoustic

stimulation.

N1/P1/N2 complex = The complex consists of the first
dominant negative potential, followed by the first positive
potential and the second negative potential generated by the

nerves during acoustic stimulation at suprathreshold levels.

I' - I' is a positive wave which precedes the wave I
potential in humans. It may correspond to the Po potential

in animals.

'xii

Cochlear microphonic (CM) = The deflection of the basilar
membrane in response to a sound produces a microphonic
potential in outer hair cells, which follows the movement of
the basilar membrane and occurs in the chain of events

before an EPSP or an action potential.

Summating potential (SP) = SP is a baseline shift in the
positive or negative voltage resulting from stimulation of
the cochlear partition and can be recorded as a direct
current electrical response from the cochlea. It is also
considered as a receptor potential generated by the cochlea

hair cells.

xiii

Chapter I

INTRODUCTION

Synaptic potentials are produced by brief alterations in the
electrical properties of the membrane potential. The
membrane potential is maintained primarily by three ionic
species, Na+, K+ and Cl’. The membrane is able to maintain
a separation of charge which is positive outside and is
negative inside, since it acts as a permeability barrier to
the diffusion of ions. The separation of charge is
responsible for the resting negative membrane potential

(Kandel, 1985).

To generate an action potential, the membrane potential must
be depolarized or made less negative by reducing the charge
separation across the membrane. Within a certain range of
membrane potentials, only generator potentials are evoked,
and a small inward-outward current produces a small
depolarization/repolarization. If the inward-outward
current is larger, the depolarization/repolarization is, of
course, larger. As an active process, the all-or-none
action potential (AP) is generated only when the membrane
potential reaches a critical or threshold level (Koester,

1985).

2
Applying the.concept of generalized electrical signaling
properties of nerve cells to auditory afferent nerve fibers,
we know that hair cells, which are receptors of mechano-
electrical stimuli in the organ of Corti, release neuro-
transmitter(s) which activate the peripheral dendrites of

the auditory nerve type I spiral ganglion fibers.

The release of the neuro-transmitter substance is preceded
by a number of stages (Dallas, 1984). Briefly, vibrations
of the basilar membrane of the cochlea, in response to
mechano-electrical stimuli, lead to the bending of hair cell
stereocilia. This bending of the stereocilia opens ionic
channels for passage of ions, the most likely candidate is
K+, which releases intracellular stores of Ca2+ and brings
about depolarization of the hair cells. The depolarization
of the hair cells may be recorded as electrical potentials,
namely, cochlear microphonic (CM), and summating potential
(SP). The depolarization in the hair cells results in the
release of a neuro-transmitter substance. The neuro-
transmitter substance(s) in turn excite the dendrites of the
type I auditory afferent fibers, leading to generation of
post-synaptic potentials. If the post-synaptic potential is
excitatory (EPSP), and if it reaches threshold, this will
lead to the generation of APs (Dallos, 1984; Siegel and

Dallas, 1986).

In reviewing the literature on auditory nerve potentials, it

is of interest to note that identification and

3
characterization of the EPSP generator potential, which
leads to the generation of the AP is not addressed. In
other words, depolarization of hair cells and the subsequent
release of a neuro-transmitter substance(s) does not lead to
an AP, but rather, leads to the generation of an EPSP, which
if adequate, leads to the generation of an AP. Thus, the
EPSP is an important step in the generation of the AP and
should not be ignored. In fact, if the EPSP is adequately
characterized, it may prove to be a more sensitive index of
the activity of the output of the hair cell rather than the

AP of the auditory nerve.

There have been only a few studies which have investigated
the auditory EPSP in animals (Furukawa and Ishii, 1967:
Flock and Russell, 1973, 1976: Furukawa, 1978: Furukawa and
Matsuura, 1978: Furukawa, Hayashida and Matsuura, 1978:
Furukawa, Kuno and Matsuura, 1982: Siegel and Dallos, 1986;
Palmer and Russell, 1986; Sewell, 1990). In humans, Hughes
and his colleagues (Hughes and Fine, 1980: Hughes, Fine and
Gagnon, 1981; Hughes and Fino, 1985) have successfully
recorded an early wave called ”I'”, a potential which
precedes wave I of the auditory brain stem response (ABR)
which is thought to be an EPSP. They have suggested that
the generator for I' may be the most distal part of the
auditory nerve. Benito, Falco and Laura (1984) have
confirmed the presence of a wave before wave I with a
latency of approximately 1.0 msec, which they called the

”IO” wave. Moore and Semela (1985) have also recorded a

4
potential which precedes wave I of the ABR in humans which
they called "BI". It has been determined, however, that 1',
1° and BI are the same response (Moore, Semela, Rakerd, Robb
and Ananthnarayan, 1991). I' is therefore the designation

of choice since it enjoys precedence in the literature.

Moore and his colleagues (Moore, Caird, Klinke and LBwenheim
1988a and 1988b; Klinke, Caird, waenheim and Moore, 1988:
and Moore, Caird, Lowenheim and Klinke, 1989) have recorded
an I'-like potential in the cat and gerbil, called Po.

Using an experimental paradigm in which intracochlear
infusion of tetrodotoxin (TTX) was used, they found a
reduction in the amplitude of the Nl/Pl/Nz complex (first
negative, first positive and second negative potential) of
the AP, but not Po. TTX is known to block Na+ channels
which serve as precursors for generation of the AP. When
kynurenic acid (KYNA) or L-glutamic acid diethyl ester
(GDEE) was infused into the cochlea following TTX, Po
diminished (KYNA and GDEE are antagonists to certain neuro-
transmitter substances). These results would appear to
indicate that Po is a post-synaptic potential whose
appearance is related to the kinetics of a neuro-transmitter
substance. Thus, P0 is perhaps a neural response and is not

a part of the AP response, since it is not affected by TTX.

Another group of investigators (Xi, Dolan and Nuttal, 1989;
Dolan, Xi and Nuttall, 1989) have reported similar results.

These investigators found that in guinea pigs, TTX abolished

5
the AP, leaving a residual slow negative "EPSP-like"
potential, without altering the CM or the SP. However, when
kainic acid was infused into the cochlea the EPSP-like
potential was eliminated, leaving the CM and SP intact.
They indicate that the origin of the EPSP-like potential may
be the unmyelinated dendrites innervating inner hair cells,
and these potentials may reflect summed EPSPs from

depolarization of afferent dendrites of the auditory nerve.

Since in vertebrates there are concerns as to whether Po (or
the equivalent EPSP-like potential preceding AP) is a true
neural response or a SP, it would be of interest to
determine if an EPSP-like potential exists in an
invertebrate preparation, such as house cricket (ACRES:
ggmestigus). The cricket may serve as a model of mammalian
hearing since it does not possess a cochlea, and thus, the

SP should not be present.

Most work on cricket audition has focused on its role in
intraspecies communication. Acoustic communication mediates
many important social functions including mating courtship,
inter-male aggression, inter-male spacing and detection of
predators. Sound reception in the cricket is accomplished
by two structurally and neurophysiologically different
mechano-receptors (Pumphrey and Rawdon-Smith, 1936: Edwards
and Palka, 1974; Counter, 1976), namely, the tympanal organ
in the tibia of the prothoracic legs and the cerci located

on the abdominal appendages.

6
The abdominal cercus is of interest to us since it appears
that the response in the cereal nerve is similar to that of
the mammalian eighth nerve (Pumphrey and Rawdon-Smith,
1936). The analogy between the mammalian auditory system
and the cricket cercal hearing system is schematically
represented in figure I-l. Figure I-lA represents synaptic
transmission in the mammalian cochlea-afferent auditory
complex, while figure I-lB depicts the synaptic transmission
in the cricket cercal hearing system. Each hair cell
contains freely articulated cilia, while each cercus is
covered with freely articulated trichoid sensilla or hair
mechano-receptors. There are hundreds of mechano-receptors
on each cercus, which respond to light puffs of air and to
sound of adequate intensity over a broad frequency spectrum.
A similar mechanism of response is obtained from mammalian
hair cells. Each of the sensilla are singly innervated by
dendritic processes of cereal sensory nerves while each
inner hair cell is innervated by several afferent fibers.
The axons of the cereal nerve synapse ipsilaterally on
branches of a few giant interneurons in the fifth abdominal
ganglion (SAG) (Gnatzy, 1976). The axons of the auditory
nerve synapse at the spiral ganglion. A group of ten giant
interneurons project from the SAG on the right and left
sides, and run anteriorly in the ventral nerve cord,
synapsing along the way to the prothoracic brain. Auditory
nerve fibers synapse at the level of the cochlear nucleus as
they course through the brain stem on their way to the

auditory cortex

Figure I-lA: Schematic representation of the mammalian
hair-cell auditory afferent synapse. RP = receptor potential,
PSP = post-synaptic potential, AP = action potential,

receptor = hair cell.

Figure I-lB: Schematic representation of the receptor

afferent fiber in the cricket cercal hearing system.

Figure I-lA & 18

A. Mammalian Hair Cell-Auditory Afferent System

Afferent fiber
r-) 050 " AP 1‘ 7’" ‘
Cilia - I

Dendrite Axon

 

 

 

Receptor
Synapse

8. Cricket Cereal Heating System

 

  
  

 

 

Receptor - Sensory’Neuron
(Sengillum) ¥_J
-- ' .Synapse
C t l . .
=i{%5=\\ Cereal Ne ar ~ Giant Interneuron
::— '- M“»—— ‘0. 4-.
*— RI}

Cercum Fifth Abdominal
Ganglion (SAC)

9
Auditory stimulation of the cerci evokes correlated spike
impulses in the cereal nerve and giant post-synaptic
interneurones (Edwards and Palka, 1974). Auditory
stimulation (with appropriate parameters) of the mammalian
ear produces synchronous responses from the auditory
afferent nerve fibers. Electra-physiological recordings
also indicate that the cercal auditory system can decode
stimulus information by narrow tuning in individual cells

and by synchronous discharge patterns (Counter, 1976a).

A thorough review of the results of the literature failed to
reveal any information about the characterization of the
EPSP in the cercal hearing system of crickets. Thus if we
are to understand electrophysiological events within the
cereal hearing system, there is a need to characterize the
EPSP in crickets. Of interest to us also is the nature of
the neuro-transmitter substance(s) at the cereal nerve/

interneuron synapses in the SAG.

The purpose of this investigation was to characterize the
EPSP-like post-synaptic potentials in the cricket. The term
EPSP-like is used instead of EPSP since we are making gross
potential recordings and there is no evidence that the slow
negative potential is a true EPSP. In so doing, the first
step was to identify a Nl/Pl/Nz complex of the cricket
auditory system. Thus first prominent negative deflection
was designated as N1, the positive peak following N1 was

designated P1. If there was a negative peak following Pl it

10
was designated as N2. Careful observations were made to
identify consistent negative-positive waves prior to N1, and
they were designated as No and Po, respectively. In the
presence of N1, it was not possible to identify the EPSP-
like potential. In order to identify the EPSP-like
potential, various stimulus parameters and a neurotoxic
substance were used. Further, a broad spectrum excitatory
amino acid antagonist was used in order to diminish the No-
Po/EPSP-like potentials since it was surmised that they were
due to the action of the neuro-transmitter substance
utilized by the afferent auditory fibers in the fifth

abdominal ganglion.

Questions

The experimental investigation was designed specifically to

answer the following questions:

1. Is it possible to identify an EPSP-like potential in
extracellular recordings of the ventral cord of AQBQLQ
domestigufi, whose different components can be designated

as No-Po/EPSP-like potentials?

2. Following identification of the No-Po/EPSP-like
potentials, what stimulus parameters such as frequency,
intensity and time best characterize the post-synaptic

potentials using latency and amplitude functions?

11
3. Using pharmacologic agents such as tetrodotoxin and
kynurenic acid, how do they assist in the identification of

the No-Po/EPSP-like potentials?

12

Chapter II

REVIEW OF LITERATURE

Introduction

The purpose of this investigation was to characterize the
auditory excitatory post synaptic potential (EPSP or EPSP-
like) which can be recorded from the fifth abdominal
ganglion of the house cricket (Acheta domesticgs). In order
to accomplish this goal, the EPSP-like response and the
compound action potential (CAP) were recorded using gross
potential recordings. The EPSP-like potential was
identified and characterized using various stimulus
parameters. Given, however, that the EPSP is normally
masked by the much larger AP, a neurotoxin tetrodotoxin
(TTX), was used to suppress the action potential (AP). Upon
suppression kynurenic acid (KYNA), a broad spectrum
excitatory amino acid antagonist, was used to investigate

its effect on the EPSP-like potential.

In the following review, I will discuss the various
electrical potentials and their underlying mechanisms, the
auditory EPSP, neuro-transmitters in the mammalian inner
ear, the cricket auditory system and the analogy between the

cricket and mammalian auditory system. The aim of this

13
chapter is to integrate all of the relevant information that
already exists and build a strong basis for the conduct of

this investigation.

Synaptic Transmission

Usually in the nervous system, the formation of a conducting
pathway does not involve direct anatomical continuity
between one neuron and another, or between a neuron and an
effector organ. An axon gives rise to many expanded
terminal branches known as pre-synaptic terminal boutons.
The post-synaptic component of the synapse is usually a
dendrite (axodendritic synapse), body (axosomatic synapse),
or part of another axon (axoaxonic synapse). At the sites
of apposition, both the pre-synaptic and post-synaptic
membranes exhibit a plaque of electron-dense material on
their cytoplasmic surfaces. Beneath this plaque on the pre-
synaptic membrane and within the pre-synaptic bouton, there
are vesicles ranging in diameter from 10 to 50 nM and often
numerous mitochondria. Synapses can be electrically or

chemically mediated (Kandel, 1985).

It is generally accepted that within chemical synapses the
AP does not cross the synaptic cleft, but instead, causes
the release of a neuro-transmitter at pre-synaptic
terminals. The release of a transmitter requires the entry
of extracellular calcium ions into the pre-synaptic terminal

upon arrival of the AP. The transmitter molecule then

14
crosses the gap, binds to receptors on the postsynaptic
membrane, which then gives rise to post-synaptic potentials.
There are at least two kinds of post-synaptic potentials.
One is similar to the spike potential, which tends to
decrease the resting potential. Hence, it is a depolarizing
post-synaptic potential and is referred to as an excitatory
post synaptic potential (EPSP). The other one tends to
increase the resting membrane potential, and thus it is a
hyperpolarizing post-synaptic potential referred to as an
inhibitory post-synaptic potential (IPSP). EPSP and IPSP
depend on the transmitter receptors and are present in

different proportions in different cells.

Excitatory Post Synaptic Potentials (EPSP)

During excitation a potential appears in recordings from the
post-synaptic cell which is a sign of post-synaptic
depolarization. This slow potential change that appears
prior to the AP is called the EPSP. It consists of a
depolarization of the membrane and can best be seen when the
extent of depolarization is insufficient to give rise to an
AP. When the membrane potential falls to a critical value
the post-synaptic neuron fires, and an AP appears
superimposed on the EPSP. Temporal summation of EPSPs is
possible. If two subliminal volleys are transmitted over
the same nerve, each volley produces an effect which is
manifested by an EPSP. The EPSPs will then sum, and if a

critical level of depolarization is reached in time, an AP

15

will be initiated (Eccles, 1959).

Characteristics of the EPSP

The EPSP is monophasic and nonpropagating, and as mentioned
above, represents a depolarization of the post-synaptic
neuron. The EPSP from a neuron usually has no maintained
plateau; the potential returns to rest once the peak is
reached. Unlike the AP, the EPSP is not an all-or-none
response, since it can be augmented by increasing the
intensity of the input stimulus. EPSPs of different inputs
can sum on a post-synaptic cell to produce a greater
depolarization (Eccles, 1959). These characteristics show
that the EPSP is produced by a process which is

fundamentally different from that of the AP.

One difference is that the movement of sodium (Na+) and
potassium (K+) during the AP is somewhat sequential; during
the EPSP it is simultaneous. The differences in ion
movement during the AP and EPSP can be explained by
differences in the molecular properties of the ion channels
responsible for these two signals. During the AP, membrane
depolarization leads to the opening of two independent
channels first, one selective for sodium, and then another
selective for potassium. To produce the EPSP, the
transmitter opens a special channel whose size and shape

allow both Na+ and K+ to pass with nearly equal

permeability. It may also allow larger cations, but anions

16
are excluded. This cation selectivity suggests that the
channel has a negative charge at its mouth that attracts a
variety of cations below a certain size, but repels anions

because of their different charge.

Another difference between an AP and an EPSP is that the
increase in Na+ influx which produces the AP is
regenerative, whereas Na+ influx produced by the EPSP is not
regenerative. The Na+ and K+ channels responsible for the
AP are voltage sensitive, since both are opened by
depolarization and closed by hyperpolarization. The opening
of the channel responsible for the EPSP is not controlled by
voltage, but rather, depends on the concentration of a
specific chemical transmitter. As a result, the
depolarization produced by the transmitter does not lead to
further increases in the number of channels that are open,
which explains to some extent why EPSPs are small and
additive as compared to an AP. Furthermore, the channels
opened by the transmitter for an EPSP differ pharma-
cologically from those opened by the AP. Thus, the

influx of Na+ produced by an EPSP is not blocked by TTX,
while the influx of sodium through the voltage-gated Na+
channel responsible for the AP is blocked (Kandel and

Siegelbaum, 1985).

17

Integration of Signals

In order to generate an AP, the membrane potential has to
reach a threshold critical for spike generation. EPSPs
drive the membrane potential toward threshold: however, the
amount of depolarization that is brought about by a single
EPSP is far less compared to what is required for an AP.
EPSPs have the property of summing spatially and temporally,
enabling the membrane potential to reach threshold for spike
generation (Eccles,1959). The cell bodies of some neurons
cannot generate an AP, thus in those neurons in which bodies
can trigger an AP, the threshold for spike generation in the
cell body is usually high, but in the trigger zone which is
in the initial segment of the axon, it is relatively low.
The trigger zone is called the integrative component of the
neuron since at this region, the excitatory and inhibitory
inputs are summed. The cell will fire only if the
excitation exceeds the inhibition at the trigger zone by a

critical value.

Evolution of Ears

In order to receive sound and vibration, a variety of sense
organs have developed in vertebrates and invertebrates.
When an organ is specialized so that it preferentially
responds to sound, it is then considered an auditory

receptor. Animals that have some type of an auditory organ

18

include the Arthropods among invertebrates, and most
creatures derived from bony fishes among vertebrates
(Dallas, 1984). The fundamental building block of all ears
is the mechanoreceptor sensory cell. In invertebrates this
cell is a primary sensory cell that possess a single
modified cilium. The arthropod ear may consist of only a
single receptor cell or as many as several thousand. These
sensory cells are incorporated within a variety of auditory

accessory structures.

Accessory structures are developed to convert airborne
sounds to mechanical displacements and to deliver these to
the mechanosensitive receptive cells. In vertebrates the
receptor itself is not part of the sensory neuron, but is a
specialized epithelial receptor cell. In this system the
sensory neuron receives its information from the receptor,
generally via a synaptic path. The sensory cell in all
vertebrate auditory organs is a hair cell, its function
being to convert relative displacements between cilia and

the cell body into the outflow of electrical activity.

Transduction in the Inner Bar

The auditory periphery is basically a transduction mechanism
in which acoustic signals are transformed into neural
impulses that are processed by the auditory peripheral
nervous system. The auditory system has a conductive

mechanism, and hydromechanical sensorineural system. The

19
conductive mechanism consists of the external ear and the
middle ear. The external ear is composed of the pinna,
external auditory meatus, and the tympanic membrane which
conducts acoustic signals to the middle ear. The acoustic
resonant properties of the outer ear, in particular the size
and shape of the ear canal, can affect the spectrum of sound
that reaches the tympanic membrane. The middle ear is an
air-filled cavity containing a chain of three ossicles, the
malleus, the incus and the stapes, connecting the tympanic
membrane to the oval window. The middle ear transmits sound
energy from the external auditory meatus to the cochlea

through the vibrations of the three ossicles.

The cochlea is a fluid-filled chamber that is divided into
three compartments, scala vestibuli, scala tympani and scala
media. The division is made superiorly by Reissner's
membrane and inferiorly by the basilar membrane. The
cochlea is coiled in a spiral shape along its length from
the base to the apex. At the base there are two openings
into the middle ear. The stapes fits into the oval window
which opens into the scala vestibuli, the round window opens
into the scala tympani. The scala vestibuli and scala
tympani communicate at the apex and are filled with
perilymph. The scala media contains endolymph, a fluid of
different ionic composition. These fluid-filled
compartments comprise the hydromechanical parts of the
system. The auditory transducer is the spiral organ of

Corti, which is located on top of the basilar membrane, the

20
latter which divides the scala media from scala tympani

(Pickles, 1982).

The sensory hair cells of the spiral organ of Corti convert
mechanical energy into electrical energy and cause an
outflow of synaptic transmitter(s). Thus, hair cells may be
thought of as transducers (Pickles, 1982). Identification
of the chemical properties of cochlear sensory cells and
neurons, and their neuro-transmitters, remain a major goal

for understanding cochlear function.

Briefly, the process of transduction includes the following
stages. As a result of the vibratory motion of the basilar
membrane, internal deformations of the organ of Corti
transmit mechanical energy to the hair cells. The most
commonly accepted concept (Dallas, 1984) is that this
transmission occurs due to a relative motion between the
tectorial membrane and the matrix of the organ of Corti
cells that contains the sensory cells. This relative motion
translates into a bending of the stereocilia and some form
of deformation of the apical pole of the hair cells,
resulting in the generation of receptor potentials, this is
the first stage of transduction. However, the actual site
of the receptive region has not been identified, although
there have been various speculations. Portions of the
infranuclear segment of the cell are specialized as the
presynaptic region, where presynaptic structures such as

vesicles are presumed to store synaptic transmitters.

21
It is considered that the release of synaptic transmitters
is controlled by receptor currents generated at the apical
pole of the cell (Hudspeth and Corey, 1977: Dallas, 1984).
The release of these transmitters by such currents
constitutes the next stage of transduction. The chemical
transmitters that are presumed to diffuse across the
synaptic cleft between the cell body and the afferent nerve
endings depolarize the postsynaptic membrane. The resulting
postsynaptic potential is conducted to the electrically
excitable portion of the nerve fiber, where it initiates a

train of APs.

The nerve fibers within the organ of Corti are classically
divided into two main classes: afferents and efferents.
Afferents refer to the neurons which carry electrical
activity from hair cells to the brain. Efferents refer to
the neurons located in the brain stem which carry electrical
activity from the brain stem to the cochlea. The afferent
system can further be divided into two subtypes, the
dendrites of the large myelinated spiral ganglion cells (90
to 95% of the total population; Spoendlin, 1972) are
radially connected with the inner hair cells (IHC) and are
called type I fibers, while the dendrites of the small type
II ganglion neurons are spirally connected with the outer
hair cells (OHC), one neuron is in contact with a large
number of cells in the three rows of OHC. Similarly, the
efferent system also has two subtypes, the lateral system,

originating essentially in the ipsilateral superior olive,

22
terminating below the IHC and forming synapses with the
radial auditory dendrites. The medial efferent system
arising mainly in the contralateral trapezoid body of the
brain stem makes direct synapses with OHC bases (Pujol and

Lenoir, 1986).

Synapses at the IHC Level

The afferent innervation of IHC is provided by fibers of
type I myelinated ganglion cells (Kiang et a1" 1982). These
fibers are called dendrites when they lose their myelin
sheath upon entering the organ of Corti. They course
radially from the habenula perforata to contact the basal
pole of the IHC. Each IHC is connected with at least 20
afferent dendrites. In all species, a constant and typical
synaptic junction has been described. This synapse is
characterized by pre- and post-synaptic membrane densities,
and pre-synaptic specialization which is generally a
synapatic body surrounded by microvesicles. The synaptic
cleft is a narrow gap which is approximately 10 nM in
diameter in the cat (Liberman, 1980) and 20 nM in diameter

in humans (Nadol, 1983).

The question as to the identity of the neuro-transmitter
acting between hair cells and afferent fibers, as well as
efferent terminals, has attracted a great deal of interest.
The incorporation of afferent transmitters into the

postsynaptic membrane causes a depolarization, leading to

23
EPSPs which have been recorded in afferent nerve fibers
(Furukawa and Ishii, 1967: Flock and Russell, 1976:
Furukawa, 1978: Furukawa and Matsuura, 1978: Furukawa,
Hayashida and Matsuura, 1978: Palmer and Russell, 1986:
Siegel and Dallos, 1986: Sewell, 1990). The most likely
transmitters have been identified as L-glutamate and L-
aspartate or a closely amino acid type (Bobbin and Thompson,
1978: Guth and Melamed, 1982: Wenthold, 1985: Klinke, 1986).
However, identity of the receptor subtypes and ion channels

involved in the process need to be further investigated.

Auditory EPSP

Gross potential recordings of the auditory system are of
particular interest since they can be recorded in human
subjects as well as experimental animals. Intracellular
recordings are made only in experimental animals. Signal
averaging techniques are typically used to enhance the
recordings relative to random noise. Sound-evoked voltage
reponses recorded by electrodes placed at a close distance
to the cochlea in animals and humans are a complex summation
of electrical activity emanating from the inner ear. The
response includes the summating potential (SP), cochlear
microphonic (CM), and the whole nerve compound action

potential (CAP).

The CAP is a summation of synchronous discharges of auditory

nerve fibers, presumably each of which is preceded or

24
accompanied by an EPSP (Siegel and Dallas, 1986). The EPSP
is believed to be the result of hair cell transmitter,
opening ligand-activated ion channels in the auditory nerve
dendrites that synapse on inner hair cells (Furukawa and
Matsuura, 1978). In the mammalian auditory system the
peripheral processes of the afferent fibers that proceed
from the habenula perforata to the inner hair cell are
unmyelinated (Spoendlin, 1974). The EPSP generated at the
synapse below the IHC would thus produce a graded potential
that propagates passively in the unmyelinated peripheral
process until reaching the area of the habenula perforata
where myelination begins. At the point of myelination, the

EPSP evokes all-or-none spike or AP discharges.

A thorough review of the literature in auditory physiology
indicates that EPSP generation has not been extensively

investigated. Only a few studies have identified the EPSP
in evoked potential recordings. One possible reason could
be that we do not yet know the optimum stimulus parameters
and recording sites that elicit the best EPSP responses.

Since in the past optimum settings have not been used, the
EPSP may be masked by the more dominant components such as

the SP, CM and the CAP.

It is important to mention, however, that depolarization of
the hair cells (reflected as CM and SP) perhaps leads to
neuro-transmitter release from the hair cells on to the

dendrites of the auditory afferent fibers, resulting in

25
excitation in the nerve fibers and reflected as an EPSP.
The EPSP, if adequate, will lead to AP generation. Thus, it
would appear that the EPSP is an integral part of the coding
mechanism of auditory nerve activity. If we can
successfully characterize the EPSP, it may contribute
significantly to our basic understanding of auditory
neurophysiology. I shall now briefly discuss those studies
that have successfully recorded the EPSP (or EPSP-Like
potentials) by using intracellular or extracellular

recordings.

Furukawa and colleagues (Furukawa and Ishii, 1967: Furukawa
1978: Furukawa and Matsuura, 1978: Furukawa, Hayashida and
Matsuura, 1978: Furukawa, 1981: Furukawa, Kuno and Matsuura,
1982: Furukawa and Matsuura, 1985) recorded subthreshold
potential variations in the goldfish, which they called the
generator potential (GP). The GP was found to be graded in
size, and when its magnitude was adequate, initiated spike
or AP potentials. The GP was found to have a time course
not much different from that of the AP, and its presence was
evident only when it was not obscured by a spike potential.
With continuous stimulation there was a rapid decline of the
GP and the spike potentials, but not of the microphonic
potentials. There was a delay of 0.6-0.8 ms between the
microphonic potentials and the GP. These features suggested
that the GP is the excitatory postsynaptic potential (EPSP).
An interesting observation was that even after the EPSP had

been completely adapted to a continuous sound, a vigorous

26
discharge of new EPSP was observed when the intensity of the
sound was increased. On application of a step decrement in
the sound intensity, the amplitude of the EPSP showed a
decremental response in which the amplitude was reduced, but
soon returned to a new steady level appropriate to the
decreased sound intensity. Statistical analysis revealed
that the decrease in the mean quantal content of the neuro-
transmitter during step decrement and an increase in the
mean quantal content during step increment was directly
associated with the readily available store (which was
proportional to the intensity of the stimulus), rather than

the mean probability of release.

Dallas and Cheatham (1974) described generator potentials as
graded electrical responses that mediate the initiation of
all-or-none discharges in the fibers of the auditory nerve.
The generator potentials presumably arise from the
unmyelinated dendritic portion of the neurons. They suggest
that these potentials have not been identified
experimentally with certainty, however, postulate that a
negative component of the average SP might be a gross,

remote, manifestation of the generator potential.

Flock and Russell (1976) electrically stimulated the
efferent nerve fibers of the lateral line hair cells in the
fresh water cod, the burbot Lotg lgta and recorded the IPSP
intracellularly. The IPSP were accompanied by a decrease in

the resistance of the hair cell membrane and an increase in

27
the intracellular receptor potential. Recordings from
afferent nerve fibers to mechanical stimulation indicated
small spontaneous and evoked EPSPs. The EPSPs were reduced

in amplitude for the duration of the IPSP.

Palmer and Russell (1986), while investigating the high
frequency limit of phase locking in the hair cells and the
cochlear nerve of the guinea pig, found spontaneous and
acoustically evoked voltage fluctuations in afferent nerve
fibers. The time difference between the onsets of hair cell
and post synaptic potentials was found to be 0.83 ms, and
was attributed to the synaptic delay at the hair

cell/afferent nerve fiber junction.

Siegel and Dallas (1986) recorded from the afferent fibers
innervating the IHC in guinea pigs. They could record
spontaneous activity ranging from 1.3 to 136 spikes/sec with
highly irregular interspike intervals. They also found that
in some instances there were EPSPs which failed to elicit
APs. A preliminary assessment of the slopes of the rising
phase of the EPSPs indicated that the EPSPs that did not
trigger all-or-none spikes had a slower rising phase than

those EPSPs that triggered spikes.

Sewell (1990) investigated the EPSP and the AP in the
afferent fibers of the lateral line organ of Xenopus laevis
(African clawed toads). He used micropipette recordings to

study the discharge rates in the afferent nerve fibers

28
innervating the hair cells organized into discrete clusters
called neuromasts. Perfusion of the synapse with a solution
containing cobalt and manganese diminished the discharge
rate in the afferent fibers for the period of time over
which the agents were present. This supports the hypothesis
that cobalt, an agent blocking voltage dependent transmitter
release, affects the spontaneous EPSP, an indication that
the spontaneous discharge in afferent fibers is due to
voltage-dependent release of the transmitter from hair

cells.

Surface Electrode Recordings of the EPSP

Hughes and Fine (1980) and Hughes, Fine and Gagnon (1981)
recorded auditory brain stem responses (ABR) in humans using
piezoelectric earphone. They identified an early scalp
positive deflection at approximately 1.1 ms which they
called I'. They concluded that I' was not a part of CM,
since it did not change in polarity for clicks of opposite
polarity. They stated that I' was more specifically related
to the post-synaptic potential arising in the afferent
terminals of the eighth nerve fibers in response to the
depolarization brought about by the action of chemical

transmitters.

Benito, Falco, and Laura (1984), investigating brain-stem

auditory evoked potentials, demonstrated a wave before wave

29
I with a latency of approximately 1.0 msec. The response
was more readily identifiable at high intensity levels,
which they called "I0".

Hughes and Fine (1985) presented data indicating the
possible generator of I'. They plotted distributions of
amplitude and latency throughout the scalp for each of the
positive and negative waves of the ABR in 20 normal ears.
The isopotential maps for I' were consistent with the
suggestion that the generator may be the distal part of the

eighth nerve.

Moore and Semela (1985) recorded the whole nerve AP using
surface electrodes in humans. They have consistently
identified a positive peak (which they initially designated
as BI) with a latency of 0.6 to 1.2 msec and an amplitude of
30 to 70 nV. When they repeated several stimulus parameters
along the dimensions of frequency, intensity and time, a
consistently graded potential was recorded and was thought

to be generated by the post synaptic region of the cochlea.

Moore, Caird, Klinke and LBwenheim (1988a and 1988b),

Moore, Caird, LBwenheim and Klinke (1989) and Klinke, Caird,
LBwenheim and Moore (1989) recorded the ABR and the I'-like
potential from the round window of cats and gerbils, and
called it "Po”. They found that when the intensity of the
stimulus increased the Po latency decreased and its

amplitude increased. The latency and intensity of P0 varied

in a
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30
in a manner which closely paralleled the behavior of wave I
and II of the ABR. In an interesting paradigm in which they
used intracochlear infusion of TTX, they found a reduction
in the amplitude of the N1/N2 complex of the CAP and ABR
waves, but not Po. However, when a broad-spectrum amino
acid antagonist kynurenic acid (KYNA) or L-glutamic acid
diethyl ester (GDBE) was infused in the cochlea following
TTX, Po diminished along with the N1/N2 complex. Forward
masking experiments showed P0 to have a different masking
time-decay function than both N1 and CM. Simultaneous
masking with high-and low-pass filtered noise showed that P0
is generated at the high frequency end of the cochlea.
These results reveal several concepts: First, these data
would appear to indicate that P0 is a potential that is due
to perhaps transmitter release (post-synaptic), since it is
affected by drugs acting on the synapse. Secondly, it is
perhaps a neural potential but is not the AP, since it is
not affected by TTX, which is known to block fast-acting

sodium channels.

xi, Dolan, and Nuttall (1989) and Dolan, xi, and Nuttall
(1989) have reported results similar to those of Moore et alv
(1988, 1989). They used tone bursts as stimuli to record
the CAP from the round window of guinea pigs. Results
indicated that TTX abolished the N1-Pl-N2 (AP complex),
leaving a residual slow negative potential without altering
the CM or the SP. Kainic acid (RA) eliminated the AP and

‘the slow negative potential, leaving the CM and SP intact.

31
From the results obtained, the investigators postulated that
the origin of the slow negative potential may be the
responses from the unmyelinated dendrites innervating the
inner hair cells, and this may reflect summed EPSPs and
depolarization of the afferent dendrites. The slow negative

potential is referred to as the EPSP-like potential.

Glutamate as the Neuro-Transmitter in the

Inner Ear Afferent System

Excitatory amino acids mediate perhaps as many as four
distinct receptor systems (Cotman and Iversen, 1987:

Watkins and Olverman, 1987). Three of these, the N-methyl-
D-aspartate (NMDA), Kainate (K) and Quisqualate (Q)
receptors, are named after artificial agonists that
preferentially activate a single receptor subtype. Recently
one other type of receptor that has been described is the d:
amino-3-hydroxy-5-methyl-4-isoxazole-proprionic acid (AMPA),
in which the agonist activates an intrinsic channel similar
to the kainate receptor (Barnard and Henley, 1990).
Selective NMDA antagonists are 3-3(2-carboxypiperazine-4-xl-
propyl-l-phosphonic acid) (CPP) and D-2-phosphonopentanoic
acid (D-AP5). Both K and Q receptors are unaffected by

these NMDA antagonists.

Recently the discovery of the Quinoxalinediones (On), a
series of potent and selective antagonists at K and Q amino

acid receptors has opened the possibility for extensive

32
studies of these non-NMDA receptor subtypes (Honore et al.,
1988). Some of the potent non-NMDA receptor antagonists
used so far are, 6,7-dinitro-quinoxaline-2,3-dione (DNQX),
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 2,3-
dihydroxy-6-nitro-7-sulfamoyl-benzo quinoxaline (NBQX),
among several others. Of the described compounds acting at
the Qu site, 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo
quinoxaline (NBQX) has been found to be more potent than
either DNQX or CNQX (Sheardown et a1, 1990). The affinity
of NBQX for the K receptors was about 30 times less than its
affinity for Q receptors, but was found to have a very low
affinity for the NMDA receptors. The fourth class of the
excitatory amino acid receptor is defined by the antagonist
action of L-2-aminophosphonobutyric acid (L-AP4). There are

no potent antagonist for this class of receptor.

Bobbin and Thompson (1978), Bobbin (1979), Comis and Leng
(1979), and Bobbin, Bledsoe and Chihal (1981) investigated
the effects of various putative neuro-transmitter on
afferent cochlear transmission in the guinea pig, and
suggested glutamate and aspartate as the afferent NT. They
also support the contention that the post-synaptic receptor
is not of the NMDA type, but rather it may be a kainate or

quisqualate receptor subtype.

There have been a number of investigators (Jenison and
Bobbin, 1985: Pujol, Lenoir, Robertson, Eybalin and

Johnstone, 1985: Jenison, Winbery and Bobbin, 1986: and

33
Bobbin and Ceasar, 1987) who have injected within the guinea
pig cochlea several non-NMDA antagonists such as kynurenic
acid, y-D-glutamylamino-methylsulfonic acid, etc. The non-
NMDA antagonists were found to reduce the magnitude of the
Compound AP of the cochlear nerve. Puel, Bobbin, and Fallon
(1988) examined the effect of 2-amino-4-phosphonobutyric
acid (2-APB) on the cochlear potentials of guinea pigs, and
found that it had no effect on the auditory nerve
potentials.

Littman et al (1989) investigated the effect of various Qu,
i.e., DNQX, CNQX, 6,7-dichloro-3-hydroxy-2-
quinoxalinecarboxylic acid (DHQX), and 3-hydroxy 2-

. quinoxalinecarboxylic acid (3HOC), on cochlear potentials in
guinea pig. They found dose-related suppression of the CAP,
a prolongation of N1 latency at suprathreshold levels,
elevation of CAP threshold and a decrease in N1 latency at
CAP threshold. None of the drugs had any effect on CM or

SP.

All the above studies have one trait in common, i.e., they
have all investigated the effect of the various antagonists
on the single nerve fiber AP, or CAP. The AP, however, is
not the first post-synaptic potential that can be recorded
from the inner ear. As was stated above, an AP is not
generated if the EPSP does not reach threshold. Thus, it
would be of interest to examine the effect several of these

various antagonists on the EPSP.

34
There have been only a few studies which have investigated
the effects of glutamate antagonists on the EPSP. As cited
previously, Moore et alv(1988, 1989), Xi et alv(1989), and
Dolan et alv(1989) have successfully demonstrated that with
TTX administration it is possible to diminish the amplitude
of the AP. A reduction in amplitude is not seen for the
slow EPSP-like response, which remains until an amino acid
competitive antagonist is administered. However, all the
animals used in these studies were mammals, and thus, they
possess a cochlea. It has been argued that the remaining
slow negative potential could be a part of the receptor
potentials from the hair cells in the cochlea namely SP. A
method which may clarify this ambiguity is to identify the
EPSP in an animal model which does not posess a cochlea.
Hence, I propose using the house cricket (Acheta domesticus)
as a model to characterize the auditory EPSP-like potentials
in the fifth abdominal ganglion, which is the first synapse

in the cercal hearing system of crickets.

General Features of House Cricket (Acheta domesticus)

The body of the cricket is rather slender, and is divided
into three main parts, the head, thorax and abdomen. Mean
body length of medium males is approximately 15.9 mm, and
that of females is about 15.7 mm. The tegmina nearly
reaches the end of the abdomen: hind wings are either short
and covered by tegmina, or extend considerably beyond the

wings. The posterior femur is short and slender and the

me
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35
ovipositor is slightly longer than the posterior femur.
Their general color is brown and head has four dark reddish
brown transverse bars (The New Encyclopedia Britannica,

1984).

House crickets are essentially domiciliary, but may cause
economic damage. Rearing the house crickets for fish bait
and pet food is a large industry in some parts of the United
States. Several of the species are used extensively in

experimental laboratory studies of insect physiology.

Classification

The house cricket is classified as follows (Randell, 1964):
Superphylum, Arthropoda: Phylum, Entoma: Class, Insecta:
Order, Grylloptera: Suborder, Gryllodae, Superfamily,
Grylloidae, Family, Gryllidae: Subfamily, Gryllinae: Genus,
Acheta Fabricius: Species, Acheta domesticus: Common name,

House cricket (cricket).

Crickets are often said to exhibit programmed behavior. The
nervous system controls the behavior which detects, decides
and reacts by triggering appropriate responses. It includes
input elements, such as the sense organs, eyes and ears
‘which sense changes in the surroundings, decision centers
'which evaluate the many inputs in order to choose a response

and output elements which direct that response.

36
The nervous system is an organized network of individual
nerve cells. The neurons are of three functional types,
_ sensory neurons, interneurons and motor neurons. The
individual nerve cells are similar to that of humans, but
are fewer in number. They have neurons clustered together
in a CNS into which sensory nerves extend, and from which
motor signals are sent. The brain is in the head capsule

and the nerve cord is ventral.

The basic anatomical unit of the the cricket nervous system
is the ganglion, and almost every major segment begins with
a mass of nerve cells in an embryonic ganglion. The three
main ganglia in the head are the protocerebrum, the
deutocerebrum and the tritocerebrum, all of which are fused
to form the brain, or supraesophageal ganglion. The
remainder of the ganglionic chain lies below the alimentary
canal against the ventral body surface and consists of the
prothoracic, mesothoracic and metathoracic ganglia. The
abdomen has five ganglia: the fifth abdominal ganglion (SAG)
is of special interest to us, and will be discussed in

greater detail later.

The Cricket Auditory System

Crickets have a highly specialized auditory system capable
of analyzing different temporal and spectral characteristics

of sound in their environment. The auditory system mediates

37
specific phonotactic responses of female crickets to the
calling song of conspecific males, acoustic interaction
between males, and escape reactions of both males and females

to sounds produced by certain predators.

Sound reception in the cricket is accomplished by two
structurally different mechano-receptors (Pumphrey and
Rawdon-Smith, 1936: Edwards and Palka, 1974: Counter, 1976).
One is the tympanal organ in the tibia of the prothoracic
legs, while the other system is located on the abdominal
appendages of cerci, which arise from the embryonic terminal

abdominal segment.

Tympanal Receptors

Tympanal organs involved in the transmission of sound to the
body of the cricket and in its conduction to auditory
structures include the tympanal membranes, spiracles and
trachea (Pumphrey and Rawdon-Smith, 1936: Edwards and Palka,
1974: Oldfield, 1988). In order to excite receptor cells,
sound pressures act on mechanical sound receptors. There
are three potential sound receiving structures in the ear,
the anterior tympanal membrane (ATM), the posterior tympanal
membrane (PTM), and the partition between the anterior and
the posterior tracheal branches. Movement of each of these
air-exposed structures may cause movement of receptor cells

relative to a group of supporting cells.

38
The receptor cells are positioned on the anterior tracheal
branch (ATB) without direct connection to either tympanal
membrane. Each auditory receptor is tuned to a specific
sound frequency. The frequency of sound to which the
receptors are tuned increases progressively from the
proximal to the distal ends of the arrays. The range of
sound frequencies represented in the auditory organ
encompasses those used for acoustic communication, with an
emphasis on specific biological importance. This emphasis
of specific frequencies is achieved by the presence of

several receptors tuned to the same sound frequency.

The auditory receptors have individual axons that project
from the auditory organ to the primary auditory neuropile in
the CNS (Pumphrey and Rawdon-Smith, 1936: Katsuki and Suga,
1960: Oldfield, 1988). These axons are several millimeters
in length and enter the CNS at the level of the prothoracic
ganglion. The ascending interneurons have terminal

arborizations in the subesophageal ganglion.

The Cercal Receptors

The function of the cereal receptors in the class insecta
‘was first investigated by Pumphrey and Rawdon-Smith (1936).
They described the cercus as a primitive hearing organ,
analogous to the hair structures in the mammalian cochlea in
both manner of innervation and function. Pumphrey and

Rawdon-Smith also found similarity in the cereal receptors

39

among the insects.

Anatomy of the Cerci

The cerci of the crickets are paired segmented sensory
organs located at the tip of the abdomen. They are equipped
with numerous sense organs, especially mechanoreceptive

sensilla (see figure II-l).

The sensilla and sensory neurons are formed from epidermal
cells and lie beneath the cuticle. The cerci, the sensilla,
cercal nerve innervation and the terminal abdominal ganglion
are somewhat similar among different types of crickets.

They are not very different from the cockroach as related to
features and functions. Giant interneurones with
considerable morphologic and functional similarities are
present in crickets, cockroaches and locusts (Boyan and
Ball, 1989). Hence, anatomical and physiologic description
of cercal hearing in Acheta domesticus is sometimes also

reinforced by investigations that have dealt with insects
other than Acheta domesticus.

There are three types of exclusively mechanoreceptive
sensilla on the cricket cerci, campaniform sensilla,
filiform hairs and clavate hairs. There is also a

similarity in the fine structure of the dendrite insertion

40

Figure II-1: Micrograph of a cercum from one of the
crickets used in this investigation.
(The electron micrograph was taken by
Willard Hooks Jr, Graduate student, Neuro-
Audiologic Lab, Department of Audiology and

Speech Sciences, Michigan State University).

The long hair structures seen prominently in the picture are
the filiform sensilla: the short round ones represent the

clavate sensilla.

41

F igure II-l

..snvv— '-
..'..r-. t \N“ ' "
. _ ..III'

#1 Filiform sensillum

ttb‘ '128

Clavate sensillum

 

42
in the three sensilla (F wards and Palka, 1974). A
striking feature of the campaniform sensilla is their close
proximity to the sockets of the filiform hairs (Edwards and
Palka, 1974). The campaniform sensilla are evenly
distributed over the dorsal and ventral surfaces, and tend

to be aggregated in transversely aligned groups.

There are approximately 700 filiform hairs on one cercus:
the longest are about 1.5 cm in length and are situated on
the base of the cercus. Approximately one-fourth of the
filiform hairs which have long shafts are flanked by
campaniform sensilla. The sockets of the longer filiform
hairs are surrounded by a membrane which connects the base
of the sockets with the remaining cuticle of the cercus.
This membrane is not equally spread out in each direction,
but has a larger extension in the direction of the tip of
the cercus. All sockets surrounded by such a membrane can
be bent in the direction of its largest extension, which
points to the cercus tip. Upon release, deflected sockets
immediately return to their undeflected position, since the
membrane is highly elastic (Dumpert and Gnatzy, 1977). In
their undeflected position the sockets are to some extent
inclined towards the cercus tip and form an angle.with the
surface of the cuticle of about 70°. The amplitude of the
receptor potential depends on the direction in which the

hair is bent (Gnatzy, 1976).

The filiform hairs on the cercus are arranged in a very

43

characteristic fashion. Hairs on the dorsal and ventral
aspects of the cercus vibrate transversely (T-hairs) to the
long axis of the cercus. Hairs on the medial and lateral
aspects of the cercus vibrate longitudinally (L-hairs).
Bacon and unrphey (1984) extended the description of these
mechanoreceptors by showing that each of the two main hair
population is composed of two sub-populations, L-hairs are

anterior and posterior, T-hairs are lateral and medial.

The club-shaped clavate hair sensilla are gravity receptors.
They are situated ventrally on the base of the cerci which
deflect differentially according to body position. The
distal part of each sensillum consists of a fluid-filled
club, which extends from a thin shaft inserted in a

cuticular socket on the cercus.

The shaft of the sensilla has an opening that lies about
three microns above the vertex of the cuticular wall. This
opening is called the ecdysial canal (Gnatzy, 1976). The
sensillum on the cerci of crickets is innervated by a single
bipolar sensory cell (Gnatzy, 1976). The cell body is
located peripherally and gives rise to a distal dendrite at
its apex and an axon near its base. Within the proximal
portion of the distal sensory process there are numerous
branches of the ciliary root called rootlets. More
distally, these coalesce to form a single root. The
dendrite swells to form the dendrite dilatation, and then

constricts markedly at the level of the ciliary base.

44
within the dendrite the ciliary root crosses the dendrite

dilatation and enters the tubular body.

The tubular body is a highly organized structure consisting
of approximately 700 individual microtubules. The tubular
body is supposed to play an essential role in the
transduction of the mechanical stimuli because of its
location at the site to which the stimulus force is
transmitted. microtubules in the sensory dendrite are
arranged in different patterns, with free microtubules
distributed over the entire length of the dendrite,
microtubules in the ciliary transitional region, and densely
packed microtubules situated in the tip of the dendrite

comprising the tubular body (Erler, 1983).

The oscillating direction of each sensilla is determined by
the structure of the insertion points of the hairs (Gnatzy,
1976). The side of depolarization corresponds to that on
which the ecdysial canal opens to the outside. During
deflection of the sensilla, the cuticular projection at the
hair base presses against the membrane surrounding the
tubular body, leading to bending of the microtubules of the
tubular body.

'The axon of the sensory cell surrounded by accompanying
glial cells joins the cercus nerve. The sensory cell is
surrounded by two glial cells which have their nuclei in the

ibasal region of the sensory cell. The glial cells are

45
completely enclosed by the innermost of the enveloping cells

(Gnatzy, 1976).

The Cercal Nerves

Sensory axons arising from cercal sensilla assemble in
groups which coalesce to form two bundles in the base of the
cercus. These bundles fuse to form the cercal sensory
nerve. More proximally the cercal nerve is variably joined
by sensory nerves from neighboring integumentary structures,
and a group of neurosecretory axons bound for the rectal
wall. The cercal motor nerve which innervates the extrinsic
cercal musculature leaves the ganglion close to the cercal
sensory nerve, but the two are invariably separate (Edwards
and Palka, 1974). Light and electron micrographs indicate
that the distal sections of the cercal nerve contains about
10,500 axons (in human, there are about 30,000 axons of the

auditory nerve).

The cercal nerve is dominated by fibers in the range of 0.1
to 0.5 microns. Longitudinal sections of the cercal nerve
show that individual axons maintain uniform diameter over
distances of at least 2.0 mm. Cercal sensory fibers
degenerate rapidly after severance from their peripheral
cell bodies. Axons appear to travel in spatially associated
patterns in the cercal nerve. As they enter the terminal
abdominal ganglion, however, they interweave in a complex
fashion, raising the possibility that they are sorted

according to systematic pattern before terminating in the

46
region of giant fiber dendrites, the later of which are

located in the SAG (Edwards and Palka, 1974).

Hair Deflection and Stimulus Transmission

with stimulation, the hair shaft moves away from its resting
position, in the direction of greatest mobility within the
plane. The stimulus is conducted to the dendrite as soon as
deflection of the hair shaft begins, the cuticular peg at
the base of the hair moves perpendicular to the long axis of
the dendrite. This leads to compression of the tubular body
(Gnatzy and Tautz, 1980). Since the tubular body is
bilaterally symmetrical, the area affected by the pressure
is several times larger. The depolarizing stimulus for the
receptor cell, which is a local increase in pressure across
the membrane, causes an indentation of the dendrite. Thurm,
Erler, Godde, Kastrup, Keil, Volker, and Vohwinkel (1983)
determined that the movement of the filiform hair base is
towards the dendrite, at least in crickets. In these
sensilla approximately 1.0 A is required to initiate a nerve
impulse. An adequate increase in pressure across the
dendrite induces, after a latency of approximately 50 to 70
us, an increase of the membrane conductance, thus modulating
the receptor current. The sensory signal coded in the
conductance change is transmitted by the receptor current to
the pacemaker site for cercal nerve impulses. In other

words, when the sensilla is deflected in the preferred

direction, impulses are generated by a depolarizing receptor

47
potential, and if threshold is reached, it will lead to
action potentials in the axons. The axons travel in
spatially associated patterns in the cercal nerve and enter
the SAG where they interweave in a complex fashion. These
axons are sorted according to modality before synapsing with

the giant fiber dendrite (Edwards and Palka, 1974).

Fifth Abdominal Ganglion (SAG)

The terminal or the fifth abdominal ganglion is recognizably
larger than the other abdominal ganglia. The 5AG is
embryologically derived from the fusion of five segmental
ganglia (seventh through eleventh), and morphologically is
found to be a simple structure amenable to cell counts
(Gymer and Edwards, 1967). It innervates segments 7 through
10 through dorsal and ventral mixed nerves. The volume of
the 5AG is found to increase about 40-fold from hatchling
(first instar) to adult. The volume of the adult ganglion
was found to be 84.90 x 106 u3. The total number of cells
in the SAG range from 3400 in the first instar to about

20,000 in the young adult.

The neuropile of the terminal ganglion is dominated by the
processes of the giant interneurons, which lie in two
lateral tracts interconnected by anterior and posterior
commissures. Fibers from the cercal sensory nerve terminate
principally in two regions of the terminal ganglion. The

major projection of the cercal nerve, in the normal adult

48
cricket, is to the posterior ipsilateral quadrant where a
loosely organized spherical glomerulus appears to contain
the majority of the cercal axons. Others terminate along
two oblique dorsoventrally oriented lines. All fibers from
the cereal nerve which synapse in the terminal ganglion
appear to do so on the ipsilateral side (Edwards and Palka,

1974: also see figure I-l).

Bacon and Murphey (1984) investigated afferent projections
of the filiform hairs on to the terminal ganglion using
staining techniques. They found that the filiform hair
afferents project only into the cercal glomerulus, a well
defined club shaped region of neuropile. Sections cut
through the glomerulus revealed that it was a hollow, shell-
like structure. Bacon and Murphey also found a strong
correlation between the directional selectivity of a sensory
neuron and the position of its terminals within the
glomerulus. Lateral-T hair afferents were found to project
to the lateral region of the cercal glomerulus. More distal
hairs had afferent neurones which projected more anteriorly
within this region. The medial-T hairs project to the
posterior and dorsomedial regions of the glomerulus.
Anterior-L hairs were found to project to a region of the
glomerulus that extended from a ventrolateral position and
arched up more dorsally towards the midline. Most of the
posterior-L hair afferents projected to a hook-shaped region
of the glomerulus, although some of them projected

contralaterally to the homologous ventromedial region of the

49

neuropile.

Bacon and Hurphey (1984) also examined the structure of the
medial giant interneurones (MCI) and lateral giant
interneurones (LGI) to determine whether the position of
dendrites within the afferent projection is correlated with
their known excitatory receptive fields. They recorded
simultaneously from MGI and a posterior-L afferent hair
afferent to which it appeared to make contact. By
triggering the oscilloscope sweep with the action potentials
of the sensory neurone, they observed EPSP's up to 1.0 mV in
amplitude and at a latency of 3.0 ms. The EPSP's that
exhibited slow rise and fall times, were found to be very
labile. Consistent with the observations of synaptic
connections between single afferents and the interneurones,
the receptive fields paralled predictions based on

structure.

Blagburn, Beadle, and Sattelle (1984) used a cobalt
injection technique in investigating the synapse between an
identified giant interneurone and a filiform hair sensory
neurone in the terminal ganglion of cockroach nymphs, which
have cerci bearing only two functional filiform hair
sensilla. They found synaptic vesicles clustered around
bar-shaped pre-synaptic densities. The average length of
each bar was found to be 150-300 nM, width was 20-30 nM, and
height 30-60 nM, and some thickening of the post synaptic

region was also observed. Horse radish peroxidase (HRP)

50
staining of one giant interneuron showed its dendrites
forming synaptic contacts with the large identifiable axon
of the lateral filiform hair sensory neurone. The giant
interneuron population of the ventral nerve cord comprises a
group of ten axons with diameters between 20 and 40 nM.
These interneurons are the most prominent interneurons in

the cricket nervous systems.

Mendenhall and Murphey (1974) mapped the giant interneurons
in the cricket, using the technique of back-filling with
cobalt chloride from the axons in the connectives. They
found 8-10 giant axons greater than 20 nM in diameter in the
connective anterior to the terminal ganglion with, their
somata and major dendritic processes in the SAG. The giant
interneurons were arranged in three tracts, a dorso-lateral,
a ventro-medial and a ventro-lateral tract. These groups
maintained a constant position in the nervous system from
the terminal ganglion anteriorly through the thoracic
ganglion, with some of the cells projecting as far as the
sub-esophageal ganglion. The giant interneurons were found
to be derived from one of the five segmental ganglia, and
shared several common features. The soma of the giant
interneurons were located laterally near the anterior most
edge of their respective ganglion. The commissural
processes of each interneuron crossed the midline in the
same sequential fashion within the boundaries of each
primitive segment. Major dendritic arborizations were

located in more posterior positions as compared to the soma.

51

Electrophysiology of the Cercal System

Pumphrey and Rawdon-Smith (1936) investigated the anal
cercus of the cricket and the cockroach due to the
similarity between cercal nerve responses of the two animals
and eighth nerve responses in mammals. They described three
important types of cercal hairs. Of the three types, the
long slender hairs distributed on the ventral side of the
cercus were found to be the most important, since
application of petroleum jelly to the ventral surface

abolished the nervous response to sound stimulation.

The cercal receptors were found to be extremely sensitive to
gross movements of the surrounding air. There was a great
deal of similarity between the cercal responses of the
cricket and the cockroach, suggesting that the similar kind
of cercal receptors mediate the response in both. Physiolo-
gically, the response to sound stimuli were found to
resemble in many respects the mammalian cochlear nerve. At
lower frequencies the response were synchronized with the
stimulus frequency. At higher frequencies, synchrony was
partial and persisted only during the initial time of
stimulation. At lower frequencies, i.e., below 400 Hz, the
cerci were found to respond to both phases of the stimulus.
.At frequencies higher than 600 Hz, the opposite responses
ware obtained, i.e., one response per two cycles of the
stimulus, which was attributed to lengthening of the

refractory period.

52
Edwards and Palka (1974) recorded responses from the
abdominal nerve connectives of the cricket to tones,
airpuffs, and substrate vibrations. They found substantial
ongoing spike activity in the range of 1.0-2.0 mV. The
giant fibers found in the connective of the nerve responded
to sound stimuli ranging from about 2000 Hz and below, to
sub-audio frequencies, with greater sensitivity at lower
frequencies, with a peak at approximately 400 to 600 Hz.
The preferred direction of a sound source for the giant
fibers of each side was approximately at right angles to the
shaft of the corresponding cercus. The interneurons were
found to respond rather weakly to substrate vibration, with

a peak sensitivity to about 2000 Hz.

Rozhkova and Polishchuk (1976) investigated the electrical
activity of the connectives between the fourth and fifth
abdominal ganglia of crickets in response to stimulation of
the cerci using frequencies from 20 Hz to 3.0 KHz. The goal
of the investigations was to determine the orientation of
the cerci relative to the body and its importance to
directional sensitivity. They fixed the cerci in the
necessary position with pins impeding movement, and except
for the cereal nerves, all other strucutures joining the
cerci to the body of the cricket were sectioned. As a
result, the giant neurones completely lost sensitivity to
the direction of the signal and their diagrams of
directional sensitivity became practically uniform. Hence,

they concluded that due to deprivation of information of the

53
position of the cercus, the giant neurones sum the signals

of different receptors equally from all directions.

Counter (1976a) made electrophysiologic intra-and extra-
cellular recordings of the response patterns of the intact
cerci of adult crickets to well-controlled acoustic
stimulation. The focus of the investigation was to obtain
absolute auditory thresholds for the cereal sensory nerve
and the lateral post-ganglionic giant interneuron in
crickets, and compare them with the corresponding post-
ganglionic thresholds in the cercal system of the adult
cockroach. The results indicate a frequency response from
30 to 3000 Hz. It is significant that the pre-synaptic
elements are consistently more sensitive than the post-
synaptic axons, with the cricket response more sensitive
than that of the cockroach. It is also of interest that the
sensory cercal nerve of the cricket is capable of sinusoidal
synchrony from 300 to 600 Hz, is asynchronous above 600 Hz,
but even the most sensitive of the post-ganglionic units
were found to be asynchronous above 60 Hz. Acoustic
stimulation of the cercal receptors evoked characteristic
discharges in several small and a few large fibers anterior
to the terminal ganglion. The conduction velocity of large
and small fiber populations in the postganglionic
connectives was found to be 5.5 and 2.3 H/sec.
Spontaneously active elements in the post-ganglionic units
in a single connective increased their activity

significantly when the cereal nerve contralateral to the

54
connective under study was severed at a point near the
ganglion. Transection of the ipsilateral cercal nerve,
however, resulted in a significant reduction in spontaneous

spike discharges.

To analyze more precisely the acoustic parameters of the
call song of the cricket, Counter (1976b) analyzed
spectrally the call song using digital Fourier
transformation and measured electrophysiologically the
hearing capacity of the cricket's tympanal nerve. The
waveform and spectral analyses showed the call song to be a
stereotyped chirp of three pulses, each of which had a
dominant carrier frequency of 4600 Hz, resulting from the
rate at which the individual teeth of the pars stridens are
struck by the plectrum. The electrophysiologically
determined audiogram of the tympanic nerve responses
indicated that the most sensitive portion of the curve

corresponds well with the carrier frequency of 4600 Hz.

Tobias and Hurphey (1979) investigated the physiology of the
receptor sensory neuron and the directional response of
cricket giant interneurons to an airpuff stimulus. They
made extracellular recordings of the afferent axons of the
cercal nerve, intracellular recordings from the medial giant
interneuron and extracellular recordings of the giant
interneurons. Results were similar to those found by
Edwards and Palka (1974), in which there were two

populations of mechanoreceptive hairs on cricket cerci,

55

those that moved in the longitudinal axis (L), and those in
the transverse (T) axis. Further, Tobias and Murphey found
two subgroups proximal and distal in each of the L and T
fibers, based on their unidirectional response curves. The
giant interneurons were found to be directional only to an
airpuff, but not to acoustic stimuli. They speculate that
the directional response properties of the interneurons is
the sum of excitatory input via the receptors on one cercus

and inhibitory input from the other.

EPSP Recordings

Pichon and Callec (1970) described the oil-gap technique
that they used to record extracellularly, excitatory post-
synaptic potentials in a single post-synaptic giant axon in
the terminal abdominal ganglion of the cockroach. There
were several advantages of using the oil-gap technique
namely, a low-impedance preparation, a low-noise level, good
stability and unequivocal identification of the post
synaptic element at the level of which the recordings were
made. This permitted efficient irrigation of the ganglion
allowing for better pharmacological studies. When
extracelluar EPSP recordings were compared with
intracellular recordings, the former were smaller and
slower: however, the extracellular post-synaptic action
potentials were similar or larger than those recorded with

intracelluar electrodes.

56
Using the same technique, Callec, Guillet, Pichon and
Boistel (1971) investigated input-output post-synaptic
events to stimulation of the cercal mechanoreceptor.
Simultaneous recording of pre-synaptic and post-synaptic
activity revealed that in most cases, each spike at the
receptor initiates an EPSP. There was a delay of about 0.85
ms between the pre-synaptic spike recorded where it enters
the ganglion and the rising phase of the corresponding EPSP.
The 0.85 ms delay corresponded to the conduction time in the

pre-synaptic element and to the synaptic delay.

Using increasing levels of electrical stimulation of the
homolateral cercal nerve, there was a smooth increase in the
EPSP. When the post synaptic depolarization was larger than
3-10 mV, it gave rise to a full-size 100 to 120 mV AP.

Hence in this type of integrative synapse, it was necessary
to summate the action of numerous sensory impulses in order

to trigger a propagating AP.

It was found that even in the presence of an AP, the EPSP
remained, indicating that the site of initiation of the EPSP
and of the AP are likely to be differentially located. The
investigators studied also the effects of repetitive
stimulation at different repetition rates. They obtained
data at sub-threshold levels so as to avoid spikes, and
isolated the ganglion from spontaneous sensory inputs by
severing the cerci. In order to suppress the interference

with depression or potentiation, the rest interval between

57
two volleys was maintained for 3-5 minutes. Repetitive
stimulation of pre-synaptic fibers resulted in a reduction
of the post-synaptic events. Greater repetition rates
resulted in a more rapid decline of amplitude. If the
preparation was unstimulated for a period of about 3 to 5

minutes, the EPSP's could be restored.

Boyan and Ball (1989) examined the responses to cercal
stimulation of the giant interneuron (GINl) in the terminal
abdominal ganglion of the locust. The goal was to
demonstrate that the GINl receives input from primary
afferents and from a spiking local interneuron. Following
dye injection the terminal ganglion was dissected, and the
GINl was found to have a lateral cell body, an axon which
exited the ganglion anteriorly via the contralateral
connective, and an extensively branched posteriorly directed
dendrite. The response of the GINl to wind stimuli directed
at the cerci, or electrical stimulation of the cercal nerve,
consisted of depolarization supporting spikes, evoked by
input from filiform afferents, and a suprathreshold
depolarization evoked by input from an interneuron.
Simultaneous recording of a filiform afferent
intracellularly from its terminals in the ganglion, and
extracellularly from its axon in the cereal nerve, showed
that spikes are conducted to the ganglion in this afferent.
Spikes obtained from the intracellular recordings of a
filiform afferent were used to trigger a signal averager

into which post-synaptic potentials from GINl were led.

58
Triggering from the spikes in the afferent established that
each AP evoked an EPSP 1.4 ms later in GINl. With
mechanical stimulation of the filiform hairs on different
parts of the cercus, intracellular recordings from a
dendritic branch of GINl showed that several filiform hairs
evoked an EPSP in the GINl. The shape of the EPSP evoked
varied with respect to both rise time and duration,
suggesting that the input synapses for the different
afferents were located at different distances from the
recording site. The response of GINl to electrical shocks
applied to the cercal nerve always consisted of an intial
EPSP and a spike of constant short latency followed by a
delayed spike which was most probably evoked by an

interneuron.

Neuro-Transmitters in the Abdominal Ganglion

The chemical nature of neurotransmitter substances in the
cricket ganglion relies on biochemical, ultrastructural and
electrophysiological data. The six criteria for the
identification of transmitter substances have not been
satisfied in this preparation, largely due to the
difficulties of working with a complex tissue. However,
there are several studies which implicate cholinergic

mechanism for excitatory synaptic transmission.

Callec and Sattelle (1973) used the oil gap method to record

59
extracellular potentials from the abdominal ganglion of the
cockroach using pharmacologic agents. Mechanical
stimulation of cercal receptors was brought about by using a
puff of air to initiate depolarization of the post synaptic
cell. Greater intensity of stimulation resulted in further
depolarization, at times reaching threshold for spike
generation. Electrical stimuli also produced EPSP and AP.
Synaptic potentials recorded from both whole ganglion and
isolated post-synaptic fibers provided similar potentials.
The EPSP were smaller in amplitude to AP. Application of
acetylcholine on the de-sheathed abdominal ganglion caused
a reduction in the amplitude of the EPSP. A number of
experiments were conducted in which the acetylcholine was
applied to the abdominal ganglion. They indicate that the

EPSPs are a sensitive monitor for drug action.

Meyer and Reddy (1985) characterized the central cholinergic
binding sites of putative acetylcholine responses (AcHR) in
the terminal ganglion of crickets. They conducted binding
studies using the muscarinic antagonist L-[HJquinuclidinyl-
benzilate (QNB), and the nicotinic antagonist bungarotoxin
(BGT). Results indicate that a large population of putative
AcHRs in the cricket terminal ganglion specifically binds
the QNB, and therefore, is predominantly muscarinic in its
pharmacologic profile. There were also a separate class of
sites in the terminal ganglion that selectively bind to the
nicotinic ligand BGT, and at least a portion of these AcHRs

were found in the cereal giant interneuron pathway.

60
Interestingly, there are no investigations which have
addressed the possibility of excitatory amino acids as
neuro-transmitters in the terminal ganglion of the house

cricket.

In summary, several investigations have addressed the
auditory EPSP in humans and laboratory animals. However,
due to the controversy as to whether the recordings made are
truly EPSPs or receptor potential from the cochlea it is
very essential at this point in time to clarify this
problem. In this study the house cricket is used as a model
to characterize the EPSP, as the cricket does not possess a
cochlea, but exhibits the cereal hearing system. The
receptors for the cereal hearing system are freely
articulated sensilla. There are hundreds of these sensilla
on each of the cercum. The sensilla are innervated by
sensory neurons whose axons combine to form the cercal
nerve, which synapse on to the giant interneurons in the
terminal abdominal ganglion and-the EPSP's are investigated
in this synapse. An analogy is drawn between the cereal
hearing system's first synapse and the mammalian first
synapse, which is in the hair cell-auditory nerve of the
inner ear. In order to be able to use the cricket as a
model, it is essential to first establish a reliable
technique to record, identify and characterize the EPSPs

hence the purpose of the present investigation.

61

Chapter III
INSTRUMENTATION AND PROCEDURE

The instrumentation employed in this investigation can be
basically discussed under the following sections: stimulus

generating, analog, digital and display sections.

W: The stimulus generating section

consists of the following components:
.Computer (IBM PC-AT 80286)
.Power source (Modular Instruments, Inc, or M12)
.Dual function generator (MI2 208)
.Dual attenuator (M12 108)
.Filter (Frequency devices, Low pass filter 901F)
.Amplifier (Technics power amplifier, SE-9060)
.Level discriminator (MI2 104)
.Data controller timer (H12 214)

.Speaker (Realistic Minimus-3.5)

A simplified block diagram of the instrumentation shows the
experimental set-up used in this investigation (Figure III-
1). Basically, tone bursts were generated by a custom-
'written software program, and were fed to the speaker from
the function generator, dual attenuator, low-pass filter,

and amplifier. The data controller timer was used to

62

Figure III-l: Block diagram of the instrumentation

 

 

 

 

 

 

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64
activate the averager simultaneously or with a delay if
needed, with the stimulus onset. The speaker was used to
transduce electric energy to acoustic energy in order to

provide acoustic stimulation.

Anglgg_§ggtigg: This section basically includes the
following equipment for response recording:

.Three silver wire electrodes (silver-silver chlorided

wire, 270 mu in diameter).

.Preamplifier and filter (Data Inc, 2124 Mod 2)
Following elicitation of the bio-electric activity by the
stimulus, the responses were collected and recorded. The
electrodes detected the responses which were routed to the
pre-amplifier for differential amplification. The
amplification factor was set at 5 x 104, which resulted in a
gain of 94 dB. The responses were filtered from 30-3000 Hz
to enhance the quality of the averaged evoked potentials,
and by setting the system to maximally reject any

interfering random signals.

Digital_§g§tign: This section includes the following
equipment for processing responses:

.Analog/Digital converter (MI2 202)

.Input/output bus (MIZ)
Following differential amplification and filtering, the
analog responses obtained from the crickets were converted
to digital form by the A/D converter, which sampled the.

activity and generated integers that approximated the value

65
of the activity at the instant of sampling. This was done
rapidly, at regular intervals. The responses were then
passed through the memory buffer and input/output bus, and
averaged 256 times, for a sweep time of 20 ms, a dwell time
of 10 ns, sample rate of 1 x 105 Hz and 2 x 103 data

points.

Display section: The following equipment were used for

visualizing the stimuli and the recorded responses.
.Monitor (IBM enhanced color monitor, 5154001)
.Oscilloscope (Textronix D15)
.Frequency counter (Hewlett Packard 5314A)
.Plotter (IBM 6180)
The oscilloscope consists of four channels. The first
channel is used to display the tone bursts generated by the
function generator, the second displays the trigger pulse,
the third displays the tone bursts after amplification and
the fourth channel displays the raw electrophysiologic
activity from the crickets. The frequency counter is used
as a monitor to check for the repetition rate of the signal.
The plotter connected to the computer was used to print the

analog wave forms obtained from the crickets.

66

Calibration of Equipment

In order to understand the relation between stimulus
parameters and responses, it is essential to specify
precisely the stimuli used. Hence, the stimulus was
calibrated in the intensity, rate, duration, phase, rise-
decay time and frequency domains. Figure III-2 shows the
instrumentation used for calibration of the equipment. A
description of the procedure adopted for calibration is
described below:
E ' m s :

.Function generator (MI2 208)

.Dual attenuator (MI2 108)

.Amplifier (Technics power amplifier SE-9060)

.Sound level meter (Larson & Davis)

.Speaker (Realistic Minimus-3.5)

.Oscilloscope (Textronix D15)

.Spectrum analyzer (Hewlett Packard 3582A)

.Frequency counter (Hewlett Packard 5314A)

67

Figure II-2: Block diagram of the calibration system

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Calibration Procedure

The sound level meter (SLM) was calibrated using a
pistonphone. A 1 KHz sine wave with a peak-to-peak
amplitude of 100 mV as measured on the oscilloscope was
generated by the function generator and was routed through
the attenuator, amplifier and speaker to the SLM. The
intensity of the sine wave at the function generator was
adjusted so that it read 100 dB SPL at a distance of 5 cm
from the speaker. Tone bursts were generated with an
amplitude reading of 100 mv on the oscilloscope. This level

was referred to as 100 dB P.E. SPL of the tone burst.

The phase, duration and rise/decay time of the signal were
measured and monitored on the oscilloscope which was
connected to the output of the dual attenuator. The output
from the dual attenuator was also led to a frequency counter
which monitored the repetition rate of the signal. The
frequency content of the signal was measured using a
spectrum analyzer. The noise used for simultaneous masking
was measured for its frequency spectrum using the spectrum
analyzer, and was found to have a frequency spectrum of 1-

6000 Hz.

st

Nu

70
Below is an overview of stimulus conditions used in this

study

Number of Crickets Stimulus Parameters Manipulated

1 Control

2 Response similarities between 2 crickets
1 polarity

5 Intensity in dB P.E. SPL

1 Intensity in 1 dB steps

3 Frequency

3 Repetition rate

3 Rise-decay time

2 Masking

3 Tetrodotoxin and kynurenic acid
1 Tetrodotoxin alone

Materials and Procedures

The eXperimental animals were healthy adult male and female
crickets (Acheta domesticus), which were obtained from a
local pet shop. Crickets were used instead of cockroaches
because review of literature has indicated that the cercal
hearing system of crickets are much more sensitive than that
03 the cockroaches (Edwards and Palka, 1974: Counter,
1976a). The higher hearing threshold seen in the cockroach

are attributed to one or more of the following factors: the

71
sensilla on the cockroach cerci are more rigidly fixed and
not freely articulated as in crickets, the sensilla in the
cockroach are shorter and greater in mass, there are less
number of sensilla on the cockroach cerci. For
experimentation purposes, the cricket was immobilized by
placing it on ice for about 10 minutes (control studies
indicated that the optimum time period for the cricket to be
on ice was 10-11 minutes). The cricket was removed from the
ice and its two pairs of wings and three pairs of legs were
removed and the cut ends were covered with petroleum jelly
to prevent the escape of any bodily fluids. The cricket was
then pinned on to bee's wax and opened dorsally, making
certain not to damage any of the cereal parts or the ventral
cord. Experiments were conducted in a sound-treated room on

a vibration-free table.

Three electrodes were used for recording responses (see
figure III-3). The electrodes were silver-silver chloride
Wire with a diameter of 270 uM, bared at the tip and secured
to the nerve via a microdrive and a micromanipulator. The
active electrode was placed on the fifth abdominal ganglion,
the reference in the head, and the ground electrode in the
abdomen. A saline-soaked cotton pellet was placed on the
tip of the electrodes to keep the recording site moist,
Which also assisted in maintaining the preparation for
several hours. The temperature in the room was maintained

at 25°C.

Figure III-3:

72

Diagram representing typical electrode
placements on the crickets (modified from
Counter, 1976a). Act = Active electrode,
Ref = Reference electrode, Gnd = Ground
electrode, To = Tympanal organ, Tm =
Tympanal membrane, PG = Prothoracic ganglion,
SAG = Fifth abdominal ganglion, and

CN = Cercal nerve

73

Figure III-3 ‘

 

 

 

 

 

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1e,

 

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74
The acoustic stimuli generated by the function generator
were delivered via a condensor speaker placed 5 cm behind
the cerci, at an angle of 90° to the longitudinal axis of
the cerci. The frequency and repetition rates of the tone
bursts were changed when they were made the independent
variables. Stimulus intensity ranged from 115 to 85 dB P.E.
SPL in 10.0, 5.0 or 1.0 dB steps. The stimulus polarity was
either rarefaction, condensation, or alternating. The rise-
decay time, frequency, repetition rate and the duration of
the tone burst were changed as necessary to achieve the
desired experimental conditions. A trigger delay of -2.0 ms
was used in all of the recordings to enhance the clarity of
the earlier waves. The 2.0 ms recording prior to stimulus
presentation provided an estimate of the noise background of

the experimental instrumentation.

Initially, compound action potentials were obtained for
various stimulus dependent parameters with no pharmacologic
treatment. This was followed by application of TTX
(Concentration of 0.1 mM) on the ventral cord anterior to
the fifth abdominal ganglion. Recordings were made until
the N1 potential diminished below visual detection level
(VOL). TTX was applied into a plastic barrel (inner
diameter - 2.0 mm) which was placed just anterior to the
fifth abdominal ganglion. Petroleum jelly was applied on
the outer surface of the barrel to ensure that the TTX
aPPlied into the barrel did not spread beyond the point of

aPplication. Further, kynurenic acid was applied on the

75
fifth abdominal ganglion, and recordings were made until the

No-Po/EPSP-like potential diminished below VDL.

Data Analysis

The threshold for the most prominent peak, N1, was defined
as the lowest intensity required for visual detection and
was measured as a 0.1 uV difference between any two
consecutive peaks (as used by Moller and Blegvad, 1976:
Jerger and Mauldin, 1978: Schweitzer, 1987: Amedofu, 1989).
Peak-to-peak amplitude was measured from the peak to the
following trough for a positive wave and from the trough to
the following peak for a negative wave. Latency was
measured from the onset of the stimulus to the peak of

interest.

The mean and standard deviation for latency and amplitude of
peaks No, Po and N1 were calculated. Composite data was
computed for amplitude and latency of waves No, Po and N1 as
a function of stimulus intensity. Composite data was also
computed for size (relative amplitude) of waves No, Po and
N1 as a function of the time following administration of
various pharmacologic agents. Data were displayed, graphed

and plotted using a microcomputer workstation (Dell-325,
80386).

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76

Chapter IV

RESULTS
The research questions posed in this investigation are:
1. Is it possible to identify an EPSP-like potential in
gross potential recordings of the ventral cord of Acheta
domesticug, whose different components can be designated as
No-Po/EPSP-like potentials? (EPSP-like potential is the
gross generator potential which, if large enough, leads to
the generation of action potentials. No and Po potentials
are the negative and positive potentials preceding the EPSP-
like potential. All of these potentials No, Po, EPSP-like
and N1 are known as post-synaptic potentials).
2. Following identification of the No-Po/EPSP-like
potentials, what stimulus parameters such as frequency,
intensity and time best characterize the post-synaptic
potentials using latency and amplitude functions?
3. Using pharmacologic agents such as tetrodotoxin and
kynurenic acid, how do they assist in the identification of

the No-Po/EPSP-like potentials?

In order to answer these questions, several experiments were
conducted on 110 crickets. Among the 110 crickets, some
were used for control studies, some did not yield good
results due to either bad preparation or faulty equipment.
Results from 25 crickets were chosen to constitute a final
Sample. Where applicable, descriptive and inferential

Statistics were used.

77

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Subjects: Adult male and female crickets were used as the
experimental preparation. All appeared to be active and
moved around normally prior to being placed on ice for about
10 minutes. Cercal sensitivity was screened by presenting a
400 Hz tone at 110 dB P.E. SPL. The responses obtained were
found not to be very different from each other in terms of
dynamic range, amplitude, onset latency and morphological

characteristics.

Procedure: The stimulus consisted of 1500 Hz tone bursts
with a nominal duration of 3.3 ms, a repetition rate of
3.1/sec and an intensity of 110 dB P.E. SPL. Control
recordings without acoustic stimulation were obtained (see
trace 1, figure IV-1). The electrophysiologic activity was
averaged 256 times using an analysis time of 20 ms. A
trigger delay of -2.0 ms was used so as to obtain pre-
stimulus background activity. A rest period of 1.5 minutes
was provided between experimental runs so as not to cause
neural fatigue. Three silver-silver chlorided wire
electrodes with a diameter of 270 uM were used, with the
active electrode placed on the fifth abdominal ganglion, the
reference electrode placed in the head between the two eyes

and the ground electrode in the abdomen.

78
Responses: Figure IV-l shows the responses obtained from two
animals. The first recording is a control recording with no
acoustic stimulus. Following this are recordings obtained
when the crickets were acoustically stimulated. The stimuli
used for the second and third recordings consisted of a 1500
Hz tone burst, 110 dB P.E. SPL, 3.1/sec repetition rate, 3.3

ms duration, 20 ms analysis time and 256 averages.

The responses consist of a series of negative and positive
waves. The waves from both crickets exihibit somewhat
similar morphologic characteristics. The first prominent
and stable negative deflection was designated N1. The
positive peak following N1 was designated P1. At times
there was a negative peak following P1 and it was designated
as N2. Having identified the N1/P1/N2 complex, careful
observations were made to identify any consistent positive
and/or negative waves prior to N1. Interestingly, it was
possible to identify a positive peak, Po, which occurred
prior to N1. Although not as obvious as Po, there was, at
times, a negative peak preceding Po and it was termed No.

It is difficult, however, to identify an EPSP-like
potential, i.e., a slow negative wave following Po. The
probable reason is that at such a high intensity level the
EPSP-like potential is masked by N1. Hence, in the study to
follow, the stimulus intensity was decreased systematically
until it was insufficient to trigger N1. Thus an EPSP-like
potential was identified and labeled. Since there is no

direct evidence that the slow negative potential which

Figure IV-l:

79

Electrophysiologic recordings of one control
condition and two stimulus conditions. The
first recording depicts the background activity
of a cricket to no acoustic stimuluation.
Recordings two and three are responses from two
crickets to alternate tone bursts of 400 Hz,
110 dB P.E. SPL, a repetition rate of

3.1/sec, a duration of 3.3 ms, and averaged

256 times. STIM = stimulus.

Figure IV— 1

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81
remains is a true EPSP, the term "EPSP-like" potential is

being used.

Summary: So far we have been successful in recording the
action potential complex, namely, the Nl/Pl/NZ and also its
two preceding potentials, the No and Po potentials.
However, the EPSP-like potential was still obscure, masked

by the large action potential.

Stimplus parameters characterizing the No-Po/EPSP-like
essential

In order to characterize the No-Po/EPSP-like potentials, the
intensity, frequency and temporal parameters of the stimulus
were varied. This experimental paradigm was used in order
to determine stimulus dependent characteristics for these

responses .

Polarity: Figure IV-2 (Cric-075) shows the effects of
stimulus phase on No, Po and N1 to rarefaction, condensation
and alternating tone bursts. All other stimulus parameters
were held constant, i.e., intensity was 110 dB P.E. SPL,
repetition rate was 3.1/sec and frequency was 400 Hz. These
recordings indicate that for rarefaction and condensation
phases, there were earlier responses which changed in
polarity with phase, but were absent for the alternating
phase. The earlier potentials were perhaps stimulus

artifacts or some other condition in which the responses are

 

Figure IV-2:

 

82

Electrophysiologic recordings of No, Po and
N1 potentials as a function of polarity of
the stimulus. Frequency = 400 Hz,
repetition rate = 3.1/sec and intensity =

110 dB P.E. SPL. STIM = stimulus

 

 

 

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stimulus dependent, but cancel during the alternating phase.
However, the potentials of interest, i.e., No, Po and N1
show no reversals, strongly suggesting that these are neural
responses. It is well known that neural responses have no
opposing polarity and do not average out for alternating

stimuli.

Intensipy: Figure IV-3 (Cric-063) shows the effects of
intensity on the potentials. Results from four other
crickets (Cric-071, Cric-072, Cric-076 and Cric-092) are
presented in appendix A. It can be seen that the responses
are quite systematic with variations in intensity. The
amplitude of No, Po and N1 decreased, and latency increased
as the stimulus level was decreased. At high intensity
levels the waves were readily identifiable, but they became
less distinguishable as intensity of stimulation decreased.
This trend was consistent in all the crickets tested (Table
IV-lA depicts the numerical values of latencies in five
crickets, Cric-063, Cric-071, Cric-072, Cric-076 and Cric-
092, and table IV-lB depicts the numerical values for
amplitudes in these crickets). At 100 dB P.E. SPL level, N1
could no longer be identified but in its place the EPSP-like
potential could be seen. This perhaps indicates that the
EPSP-like potential can be identified only at low intensity
levels when N1 is not present. That is, when the intensity
is increased the EPSP-like potential is perhaps masked by
N1, and is no longer observable. At low intensity levels in

which the threshold of N1 is not reached, it is possible to

Figure IV-3:

 

85

Electrophysiologic recordings of No, Po and
N1 potentials as a function of varying the
intensity in 5.0 dB steps. Frequency -
1500 Hz, repetition rate 8 3.1/sec, and

phase = alternating. STIM = stimulus

 

86

 

 

 

 

 

 

 

 

 

 

 

87

Table IV.1A: Latency values of No, Po and N1 potentials
in Cric-063, Cric-067, Cric-071, Cric-072
and Cric-076 as a function of intensity

level (dB P.E. SPL) of the stimulus.

 

 

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Amplitude values of No, Po and N1 potentials
in Cric-063, Cric-067, Cris-071, Cric-072
and Cris-076 as a function of intensity

level (dB P.E. SPL) of the stimulus.

 

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identify the EPSP-like potential.

In order to examine more closely the transition from N1 to
the EPSP-like potential, 1.0 dB steps were incorporated in
our next study. The effect of varying the intensity in 1.0
dB steps is depicted in figure IV-4 (Cric-067). It is seen
that 1.0 dB steps were more effective in depicting the N1 to

EPSP-like transition.

Epggpgnpy: Tone bursts at frequencies ranging from 400 Hz
to 2500 Hz were used. The results indicated that the
responses for lower frequencies appeard to elicit more
distinct waveforms. Tone bursts of frequencies of 2000 Hz
and above elicited responses of which the potentials were
prolonged in latency and decreased in amplitude. Figure IV-
S (Cric-067) depicts the responses to 400, 600 and 1500 Hz.
Responses from two other crickets (Cric-054 and Cric-075)
are presented in appendix B. The results obtained in this
study agree favorably with the results of literature which
show that the lower frequencies tend to elicit more
distinct responses than higher frequencies (Edwards and

Palka, 1974: Counter, 1976a).

31§§;dgpay_pimg: Figure IV-6 (Cric-094) indicates that with
an increase in the rise-decay time of the stimuli, the
potentials increased in latency and decreased in amplitude.
It is well known from previous studies that evoked responses
are influenced by various rise-decay times of the input

signal (Moore, 1983). A point is usually reached wherein

Figure IV-4:

92

Electrophysiologic recordings of No, Po and
N1 potentials as a function of 1.0 dB
increments in intensity. Frequency 8 400 Hz,
repetition rate - 3.1/sec, and duration -

3.3 ms. STIM - stimulus.’

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Figure IV-S: Electrophysiologic recordings of No, Po and
N1 potentials as a function of frequency.
Intensity = 110 dB, repetition rate -
3.1/sec, phase = alternating.

STIM = stimulus.

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Figure IV-6: Electrophysiologic recordings of No, Po and

N1 potentials as a function of rise/decay

time. Intensity 110 dB, repetition rate
3.1/sec, phase = alternating.

STIM = stimulus.

 

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with any further increase in the rise-decay time, response

identification became extremely difficult. This effect is

typical of neural responses, since neural impulses are best

excited by fast-rising stimuli. Results from two other

crickets (Cric-065 and Cric-076) are presented in appendix
C. From the results obtained it would appear that response

identification improves when the rise-decay time is

increased from 1.0 ms to 1.5 ms. Rise-decay times greater

than 6.0 ms causes the waves to diminish in amplitude,
prolong in latency and become less distinct in their

morphological characteristics. This supports the view that

the responses depend on the synchrony of the nerve

discharges, and faster rise-decay stimuli elicit better

sYncl‘nc'ony in the nerve fibers.

W: Figure IV-7 (Cric-075) depicts the effects

of repetition rate on the potentials (see appendix D for
Cric-o76 and Cric-107) . As is typical of neural responses,
the potentials decreased in amplitude and increased in
latency as repetition rate was increased, which, in essence,

is also a reflection of decreasing the inter-stimulus

interval .

The It|<>1'.‘phology of the responses changed also as repetition
rate was increased. In fact, as the repetition rate was
increased from 1.1/sec to 3.1/sec (as seen in responses from
criC‘076 in appendix E) the potentials increased in
amplitude. However, when the repetition rate was increased

to
a rate greater than 3.1/sec, the potentials decreased in

Figure IV-7:

99

Electrophysiologic recordings of No, Po and
N1 potentials as a function of repetition
rate. Frequency = 400 Hz, intensity 8

110 dB, phase a alternating. It is noted
that on these data, there is high frequency
energy superimposed on the responses.

STIM = stimulus.

100

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101
amplitude and the morphology became less distinct in most of
the recordings. It can be seen that the data obtained in
this study are not significantly different from previous
studies of auditory brain stem recordings in humans (Don,
.Allen and Starr, 1977: Moore, 1971) which indicated that as
:repetition rates were increased beyond 10/sec the responses

became less distinct, but the effect did not go unnoticed at

rates below lO/sec .

W3: One purpose of masking experiments is

tn: differentiate between mechanical and neural responses
(Abbas, 1984) . Figure IV-8 (Cric-096) depicts the effect of
simultaneous masking on the potentials. The first recording
was obtained to 1.0 KHz tone bursts at 100 dB P.E. SPL with
In: masking, while the second recording was obtained to the
same stimulus presented simultaneously with a wide-band (1-
6000 Hz, -3 dB), 100 dB P.E. SPL random masking noise. The
tliird recording depicts the response when the masking noise
Was increased to 104 dB P.E. SPL, while keeping the
jJ'I‘tensity of the tone burst constant. In the first
recording, N1 and the earlier part of the EPSP-like response
can be readily seen. In the second recording, however, in
the presence of noise, the potentials were masked

considerably, resulting in a decrease in the amplitude of N1

and a shift in latency.

 

Figure IV-8:

102

Electrophysiologic responses as a function of
simultaneous masking. Stimulus frequency -
1000 Hz, repetition rate - 3.1/sec, phase -
alternating. Masking noise was 0 dB P.E. SPL
for the first recording, 100 dB P.E. SPL for
the second recording and 104 dB P.E. SPL for

the third recording. STIM - stimulus.

103

 

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This effect became even more obvious when the masking noise

was increased to 104 dB P.E. SPL, again, an effect typical

of neural responses. The EPSP-like potential which is co-

incidental with N1 is not seen in the recordings at high

stimulus intensity levels. With masking noise, however, the

N1 potential reduced in amplitude and prolonged in latency,
enhancing the appearance of the EPSP-like potential. It is
to be noted here that the tone burst was initiated at the

beginning of the trace, rather than 2.0 ms after the

computer was initiated.

m ' ude un t'o s

The mean latency and amplitude (size) data from five

crickets for tone bursts at 100, 105, 110 and 115 dB P.E.

SPL are shown in table IV.2. Figures IV-9A and IV-9B depict

the input-output functions of the waveforms. From the
input-output functions it can readily be seen that as the
jJitensity of the stimulus is increased, all waves show a
sYstematic decrease in latency (Fig IV-9A) and an increase
in size (Fig IV-9B) . Input-output latency functions for
each potential of interest, as seen in figure IV-9A,
indicate that as intensity is increased, the standard
deviation of the waves decreased indicating less variability
in the responses. A gradual sloping configuration of the
input-output plot is seen for all waves. Variability is not

represented in the size/intensity function since, a relative

measure was used to represent the size on the y-axis.

 

105

Table IV.2: Mean and standard deviation values of latency
and size of No, Po and N1 potentials in
Cric-067, Cris-071, Cric-072, Cric-76 and

Cris-92.

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Figure IV—9A: Input-output mean latency vs intensity
functions for No, Po and N1 potentials from
five crickets are shown (for better clarity

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Figures IV-lOA and IV-lOB depict the input-output
latency/intensity and size/intensity functions when the
intensity was varied in 1.0 dB steps. Numerical data of
latency and size values for No, Po and N1 potentials are
represented in table IV.3. Here again the potentials No, Po
and N1 decreased in latency and increased in amplitude when

the intensity was increased.

Table IV-4 shows the slope and intercept of the three
potentials, No, Po and N1, represented in figures IV—9A, IV-
98, IV-lOA and IV-lOB. It is seen that the slopes for No,
IPO and N1 are not very different from each other. The
intercepts however, are different for each of the
jpctentials, indicating that their origins are different
(these waves are temporal characteristics of spatial
representation of the potentials). The correlation co-
efficients for the No, Po and N1 potentials for the
latency/intensity function for the 5.0 dB steps were -0.989,
-0.997 and -0.974 respectively. For the 1.0 dB steps data,
the correlation co-efficients were -0.910, -0.920 and
-0.938. For the size/intensity function, the co-efficients
Of correlation for No, Po and N1 for the 5.0 dB steps were
0.964, 0.993 and 0.962, and for the 1.0 dB steps they were
0.905, 0.982 and 0.967 respectively. The negative
correlation values for the latency/intensity functions were
indicative of decreasing latency as a function of intensity.
The positive correlation values for the amplitude (size)/
intensity functions were indicative of increasing amplitude

as a function of intensity. Similar functions investigated

 

112

Figure IV-lOA: Input-output latency vs intensity (in 1.0
dB steps) curves for No, Po and N1
potentials.

Figure IV-1OB: Input-output size vs intensity (in 1.0 dB

steps) curves for No, Po and N1 potentials.

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Table IV.3: Latency and size values for potentials No, Po
and N1 in Cric-067 as a function of intensity

(1.0 dB step).

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Table IV.4: Slopes and intercepts for No, Po and N1
potentials for latency/intensity and

size/intensity functions.

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118
in humans and cats have indicated that latency is inversely
proportional to intensity, and amplitude is directly

proportional to intensity (Moore and Semela, 1985; Moore et

al 1988a and 1988b, 1989).

Summary: By varying the stimulus parameters, it was observed
that the No-Po/EPSP-like potentials can best be seen at
lower intensities just below the threshold for N1.
Alternating tone bursts of frequencies below 2000 Hz, with a
rise-decay time between 1.5 ms and 6.0 ms, and a repetition

rate of 3.1/sec enhance the post-synaptic potentials, No, Po

and N1.

EXperiments designed to investigate the latency/intensity
and amplitude/intensity functions on crickets have indicated
that No, Po and N1 exhibit similar trends i.e., as intensity
Was increased the latencies of all the potentials decreased
Eind the amplitude (size) increased. The slopes of their
functions were not very different from each other, but their
intercepts were different, indicating different sites of
Origin of these waves. The correlation co-efficients were
also similar for No, Po and N1 potentials. This trend seen
in the No, Po and N1 potentials in crickets is similar to
the trend seen in the EPSP-like and N1 potentials in humans

Eind cats.

119

Characterization of the EPSP-likepotential using

Pharmacologic agents

In order to identify the EPSP potential, the classic
approach is to minimize the effects of the action potential
which is usually superimposed on the EPSP. In neuro-
pharmacology, tetrodotoxin (TTX) is an agent which acts
Selectively on fast sodium channels, thus inhibiting the
propagation of action potentials. TTX does not interfere
with the post-synaptic potentials generated by the dendrites

of the post-synaptic fibers.

IF‘igure IV-ll (Cric-O67) depicts the effect of pharmacologic
Eigents on the potentials. Recordings from Cric-058 and
(:rdcw063 are presented in appendix E. In figure IV-ll the
ifirst recording is a control record which was obtained prior
‘tlo any treatment. A prominent N1 can be seen, along with
'tlhe No and Po potentials. Following this, 1.0 ul of TTX at
‘Ei.concentration of 0.1 mM was applied anterior to the
ilfifth abdominal ganglion, and recordings were made at
‘\rarious intervals of time. Thirty minutes after application
<:>f TTX (depicted as TTX + 30), the N1 amplitude decreased,
ialnd at 70 minutes after application of TTX (TTX + 70) N1
<:lisappeared, but in its place a broad, low frequency wave

Could be identified which was termed "EPSP-like" potential.

I<ynurenic acid (KYNA) , which is a broad spectrum excitatory
amino acid antagonist was applied on the SAG. Following the

application of 1.0 111 of KYNA at a concentration of 1.0 mM,

120

 

Figure IV-11: Electrophysiologic recordings of No, Po, N1
and the EPSP-like potential as a function of
tetrodotoxin and kynurenic acid.

STIM = stimulus.

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recordings were made at various intervals of time.
Recordings made ten minutes after application of KYNA (Kyna
+ 10) indicated that the EPSP—like potential and the No/Po
potentials diminished in amplitude, and at twenty minutes

post application (Kyna + 20), no major peaks were

ident i fied .

The same data are represented as input-output functions in
Figure IV-12 (Cric-067) . The input-output functions for
Grid-058 and Cric-063 are presented in appendix F. The
graph in figure IV-12 depicts the size (amplitude in 11V) of
Potentials as a function of time. The first vertical line
“8X1: to the y-axis represents the time at which TTX was
applied, and the second vertical line represents the time at
which KYNA was applied. Following the application of TTX
the Potentials decreased in amplitude; however, they did not
reach a zero percent level until KYNA was applied.
F0110wing application of KYNA all the potentials decreased
in Size. The diamonds representing the EPSP-like potential
which was present (size = 100%) prior to application of
“NA: decreased in size immediately after KYNA was applied.
The dashed lines connecting the function of the EPSP-like
POterltials (diamonds) indicate that these data were
BXtraPolated, since it is not possible to identify the EPSP-
like Potential in the presence of N1. In the next
experiment the behavior of the potentials subjected to TTX

alone was investigated .

123

Figure IV.12: Input-output functions of No, Po, N1 and
EPSP-like potentials as a function of

tetrodotoxin and kynurenic acid.

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125
Figure IV-13 depicts the effect of TTX on the potentials
over a period of 224 minutes (3 hours, 44 minutes). N1
decreased in amplitude; however, even at 224 minutes the
EPSP-like potential remained, indicating perhaps that it is
resistant to TTX. This study suggests that the decrease of
potentials to almost a zero percent amplitude (Figure IV-12)
is most probably due to the KYNA, and is not due to the

long-term effects of TTX.

Summary: The experiments designed to investigate the effect
of TTX and Kyna indicate that TTX has a strong effect on the
N1 POtential but not on the EPSP-like potential. However,

within a few minutes after application of kyna the EPSP-like

potent; ial reduced drastically .

From previous studies we know that the cercal hearing
SYSteIn, which is one of the two main hearing systems in
crickets, is comprised of hundreds of freely articulated
sensilla that are innervated by sensory neurons whose axons
combine to form the cercal nerve. The cercal nerves synapse
on t0 the dendrites of the giant interneurons in the
terminal abdominal ganglion. It is the EPSP-like potentials
gefierated by the dendrites in the terminal abdominal
ganglion that we set out to record. This is of

in"—'31=’est since the cricket does not possess a cochlea. If we
are 8macessful in recording the EPSP-like potential in such
a m"“191. it may strengthen the earlier studies which have
indicated that the potential Po appearing prior to the N1

potential in animals (Moore et a1 1988, 1989) and wave 1' in

126

Figure IV-13: Electrophysiologic recordings of No, Po, N1
and EPSP-like potentials as a function of
tetrodotoxin over a period of 224 minutes.

STIM = stimulus.

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128
humans is perhaps an EPSP potential (Hughes and Fino, 1980;
1985 and Hughes et a1 1981), and is not a cochlear receptor

potential, i.e., a summating potential.

Our results indicate that with appropriate techniques it is
possible to record the EPSP—like potential in the cricket's
cercal hearing system. Experiments conducted in which
frequency of the tone bursts was the independent variable,
indicated that frequencies below 2000 Hz evoked greater
responses. Intensity was found to have an important role in
the identification of the EPSP-likepotential. Higher
intensities elicited larger APs but masked the EPSP. Hence,
lower intensities that were unable to evoke APs were
necessary to prevent enough depolarization of the axons,
t1“18, allowing the EPSP-like potential to emerge. As
t"Ypical of neural responses the potentials recorded showed
variations with changes in repetition rate and rise-decay
time of the stimuli. With an increase in repetition rate
and rise-decay time No, Po and N1 amplitude decreased,
l"itency increased and morphology changed. Simultaneous
h1='<>ad--band masking noise effectively masked the N1 response
and the masking effect increased with an increase in the

1“tensity of the masking noise.

1mOther successful method resulting in identification of the
I‘31’Sl’-1:ike potential was to use pharmacologic agents. With
the application of TTX (a neurotoxin which blocks fast Na"
channels) anterior to the terminal ganglion, we were able to

decrease the magnitude of the N1 potential and in its place

129
could be seen the low frequency negative EPSP-like
;potential. Further, with application of a broad spectrum
excitatory amino acid antagonist KYNA on the terminal
(ganglion, No, Po and the EPSP-like potential diminished

below visual detection level.

In this study, we have seen that with appropriate stimulus
parameters, instrumentation and pharmacologic agents it is
possible to identify and characterize the EPSP-like
potential even in the cricket, which does not possess a
«cochlea. This supports the view that EPSPs can be recorded
successfully in a variety of animals from different
phylogenetic stages, e.g., crickets, gerbils, cats and

humans (Moore et al., 1991) .

130

CHAPTER V

DISCUSSION

In the mammalian inner ear, it is well known that the hair
cell is an active transducer utilizing the incoming
vibratory energy to modulate a local energy pool that is
maintained by metabolic processes. It is also well accepted
that due to the shearing motion of the basilar and tectorial
membranes the hair cell stereocilia bend, resulting in the
generation of receptor potentials (Davis, 1961; Dallos,
1984). The electrical changes in the hair cell result in
'the release of neuro-transmitter substance(s) glutamate
.and/or aspartate (Bobbin and Thompson, 1978: Guth and

Melamed, 1982; Klinke, 1986) , on to afferent nerve dendrites

{of the eighth cranial nerve.

'The neuro-transmitter(s) depolarize the post-synaptic
Imembrane, resulting in the production of post-synaptic
jpotentials. These post-synaptic potentials are associated
‘with the occurrence of transmissional activity at junctions
loetween hair cells and the dendrites of the afferent nerve
fibers. The post-synaptic potentials are neurally evoked
lootentials produced at the excitable electrogenic membrane
(of the post-synaptic afferent nerve dendrites. If the post-

ssynaptic potentials are excitatory they are referred to as

131
excitatory post-synaptic potentials (EPSP). If the EPSPs
are adequate in magnitude, they will lead to the generation
of action potentials (AP). The production of the EPSP is
thus essential for the generation of APs and its importance
cannot be overlooked. In fact, if the EPSP is well
characterized, it may be a more sensitive indicator of
synaptic activity, rather than the all-or-none AP. The high
sensitivity of the post-synaptic potential to the synaptic
activity, which, in turn, is sensitive to many varieties of
drugs, makes the post-synaptic potential an useful tool for

investigating synaptic activity (Callec et al., 1971).

Potentials conjectured to be EPSPs of the auditory system
have been recorded in humans (Hughes and Fino, 1980, 1985;
Hughes, Fino and Gagnon, 1981; Moore and Semela, 1985) and
in a variety of laboratory animals, e.g., gold fish, burbot,
turtle, bullfrog, gerbil, cat and guinea pig (Furukawa and
Ishii, 1967; Flock and Russell, 1976; Crawford and
Fettiplace, 1980; Furukawa and Matsuura, 1985; Palmer and
Russell, 1986; Siegel and Dallos, 1986; Moore, Caird,
LBwenheim and Klinke, 1988a, 1988b, 1989; Klinke, Caird,
flowenheim and Moore, 1988; Sewell, 1990). It has been
suggested that the EPSP may be the generator potential of
the auditory nerve, having as its origin auditory nerve
dendrites (Dallos and Cheatham, 1984; Siegel and Dallos,
1986). From the recordings of auditory evoked potentials in

humans and laboratory animals, wave I in humans and wave N1

132
in animals have been shown to originate from the axonal
portion of the auditory nerve (Antoneli-Candela and Kiang,
1978: M¢ller and Janetta, 1985). A few investigators have
shown that the Po/EPSP-like potential precedes wave N1 in
cat (Moore et al., 1988a, 1988b, 1989; Klinke et al., 1988)

and guinea pig (Dolan et al., 1989: Xi et al., 1989).

In order to suppress the afferent activity of the axonal
part of the auditory nerve, Moore et al., (1988a and 1988b)
applied tetrodotoxin (TTX), which blocks the influx of Na+
of nerve membranes. This led to the elimination of N1 and
N2 responses leaving intact the EPSP-like potential.
Further, with application of Kynurenic acid (KYNA) or L-
«Glutamic acid diethyl ester (GDEE, broad-spectrum excitatory
amino acid receptor antagonists), which have been previously
shown to suppress auditory nerve activity (Bledsoe, Bobbin
and Chihal, 1981; Bobbin, Bledsoe and Chihal, 1981; Bobbin
and Ceasar, 1987), the EPSP-like potential diminished. Xi,
Dolan and Nuttall (1989) and Dolan, Xi and Nuttall (1989)
lhave demonstrated a Po/EPSP-like potential in the guinea
jpig, which was resistant to TTX, but was eliminated with the

application of kainic acid.

There exists a controversy as to whether the recorded EPSP-
like potentials are truly generator potentials leading to
.APs, or receptor potentials from the cochlea. In order to

clarify this argument, an animal which does not possess a

133
cochlea was used, i.e., the house cricket (Acheta
domesticus), an invertebrate model. The aim of this study
was to identify and characterize the EPSP-like potential in
the cercal hearing system of the cricket at its first
synapse, which is at the level of the fifth abdominal

ganglion.

In order to identify and characterize the EPSP-like
potentials in the cricket cercal hearing system, two sets of
experiments were conducted. The objective of the first set
of experiments was to vary a number of stimulus parameters
and to specify a set of optimum stimulus parameters as
related to intensity, frequency and time, which will best
characterize the EPSP-like potential. So far little is
known about the effects of acoustic stimulus paramaters on
the cercal hearing system of crickets. Some of the acoustic
parameters relevant to fundamental neuro-physiological
processes are: Sound pressure level (SPL) and rise-decay
time, which apply to the spatio-temporal configuration of
activity in neural aggregrates; repetition rate, as it
refers to the pulses within a signal envelope, applies to
the integration of acoustic energy; interstimulus interval
as it bears upon successive characteristics at varying
levels of the auditory system, applies to the relative
distribution of excitatory and inhibitory mechanisms; and
frequency which applies to the locus of stimulation of the

afferent pathways (Eisenberg, 1965). The above parameters

134
were manipulated in the first set of experiments. In the
second set of experiments pharmacologic agents such as TTX

and KYNA were used to characterize the EPSP-like potential.

We observed that the responses consisted of a series of
negative and positive waves. The waveform morphology,
thresholds, latencies and amplitudes were not similar in all
the crickets tested. However, there were some general
features that were commonly seen in all the waveforms and
were designated as follows: The first prominent and stable
negative deflection was designated N1, the positive peak
following N1 was designated P1. The positive peak preceding
N1 was designated Po, and the negative wave preceding Po was
termed No. This method of designating the positive and
negative waves was earlier adopted by a number of
investigators during the process of identifying cochlear
action potentials and auditory brain-stem responses (Sohmer
and Feinmesser, 1967: Jewett and Williston, 1971; Moore,

1971).

In order to ascertain that these responses were purely
neural responses, the polarity of the tone bursts was
varied. The No, Po and N1 potentials appeared regardless of
the polarity. In addition to the No, Po and N1 potentials,
there appeared several early responses for condensation and
rarefaction stimuli which reversed in polarity, but were

cancelled with the alternating stimuli. The absence of the

135
earlier responses for the alternating stimuli suggests that
these responses are phase dependent or stimulus artifacts,
which then cancel during the alternating phase. The
insensitivity of No, Po and N1 potentials to stimulus
polarity suggests that these potentials are neural rather

than electro-mechanical in nature.

At high intensity levels peaks No, Po and N1 could be easily
identified, however, the EPSP-like potential could be
identified only at lower intensity levels, i.e., at levels
in which the N1 response was not triggered. When the
intensity was increased the EPSP-like potential was masked
be the larger N1 response. The transition phase from N1 to
'the EPSP-like potential was better seen when 1.0 dB
intensity increments were incorporated. This agrees with
‘the results from the study by Callec and Sattelle (1973)
‘where they stimulated the cercal receptors in the cockroach
‘with mechanical and electrical stimulation, and made
recordings from the terminal ganglion. Stimulation of the
receptors resulted in the depolarization of the neurons and
jproduction of EPSPs, as the stimulation was made stronger it
resulted in greater depolarization, reaching the threshold

for spike generation.

'The auditory system in mammals is capable of performing
excellent temporal resolutions and frequency analysis

(Keidel, Kallert, Korth and Humes, 1983). Experiments

136
conducted with the frequency of the tone burst as the
independent variable suggest that the responses for lower
frequencies (most often lower than 1500 Hz) elicited more
distinct waveforms. The responses for frequencies above
2000 Hz were found to be less distinct in all our
recordings. This agrees well with earlier studies, which
have indicated that the cereal hearing system in the cricket
is more sensitive to lower frequencies than high frequencies
(Edwards and Palka, 1974; Counter, 1976a). Although there
is disagreement in the specific adaptive functions assigned
to the auditory sensitivity of the cerci, it is not
unreasonable to say that the cerci perhaps play an important
role in the detection of low frequency rustling noises of
predators as well as other similar biologically significant
sounds (Counter, 1976a). Earlier, katsuki and Suga (1960)
had suggested that the cercal sensilla of many orthoptera
were used in conjunction with the tympanal organs to provide
a mechanism of sound discrimination. The cerci were found
to respond to low and middle frequencies and the tympanal
organs to the middle and high frequencies. other than the
conspecific chirps of the crickets, most biologically
significant sounds in the cricket's environment are complex

low frequency signals detected by the cerci.

Typical neural responses are influenced by the rise-decay
times of the input signal. Distinct waveforms depend upon a

synchronous discharge of the nerve fibers. Longer rise-

137
decay times cause a delayed neural discharge, hence
affecting the morphology, latency and amplitude of the
waveforms. Responses to tone bursts at various rise-decay
times indicate that rise-decay times less than 1.5 ms, but
greater than 3.0 ms caused an increase in latency and a
decrease in the amplitude of the waveforms. This indicates
that there is an optimum range for rise-decay time in which
response identification is best, beyond a 6.0 ms rise-decay
time the response identification becomes difficult. The
underlying reason for this observation may be that beyond
the optimal levels, there could be less synchrony of neural
responses, as well as delayed neural discharges. In
studying the effect of various rise-decay times on the ABR
in humans, Moore (1983) noted that as rise-decay time
increased, the response identification became more difficult

to a point where peak identification was not possible.

The stimulus repetition rate or better, the inter-stimulus
interval is of high significance for the amplitude of the
evoked potentials (Keidel et al., 1983). With an increase
in repetition rate, as is typical of neural responses, the
potentials decreased in amplitude and increased in latency.
We know from previous studies on humans and laboratory
animals that increasing the repetition rate of a signal
increases latency but decreases the amplitude of the
waveforms (Moore, 1971; Don, Allen and Starr, 1977;

Campbell, Picton and Wolfe, 1981; Amedofu, 1989). However,

138
in humans and laboratory animals such an effect is more
pronounced for repetition rates greater than 11.1/sec. In
crickets the effect is seen much earlier, i.e., around
9.1/sec, although we are not certain as to why this occurs,
we would surmise that the unmyelinated nerve fibers in the
crickets are less capable of transducing these more rapid
stimuli. What is really happening with increase in
repetition rate is that the inter-stimulus interval is being
shortened which in effect leaves less time for the nerve
fibers to recover, thus the system gets overloaded. Earlier
studies mentioned above have also indicated that the effect
of repetition rate is restricted only to neural responses,
and other mechanical responses including cochlear
'microphonics do not decrease with increase in repetition

rate.

Simultaneous masking experiments indicated that the N1
potential can be successfully masked with adequate broad-
band masking noise. The N1 potential diminished in
magnitude with an increase in the masking noise. The EPSP-
like potential which is co-incident with N1 is not seen in
the recordings at high intensity levels, however, with
masking noise N1 reduced in amplitude and prolonged in
latency, enhancing the appearance of the EPSP-like

potential.

Latency/intensity functions for No, Po and N1 exhibited a

139
strong negative correlation between latency and intensity
for 5 dB increments (r= -0.989, -0.997 and -0.974
respectively), and also for 1 dB increments (r= -0.910, -
0.920 and -0.938 respectively). Size (amplitude)/intensity
functions for No, Po and N1 exhibited a strong positive
correlation between size and intensity for 5 dB increments
(r= 0.964, 0.993 and 0.962 respectively), and also for 1 dB
increments (0.905, 0.982 and 0.967 respectively). The
slopes were similar, but the intercepts were different for
No, Po and N1 potentials (slopes for latency vs intensity
functions were -0.06, -0.05 and -0.08 for 5 dB increments,
and -0.17, -0.07 and -0.03 for 1 dB increments. Slopes for
size vs intensity functions were 3.91, 5.43 and 5.07 for 5
dB increments and 13.33, 18.11 and 13.87 for 1 dB
increments. Intercepts for latency vs intensity functions
were 7.72, 7.15 and 11.53 for 5 dB increments, and 15.76,
8.25 and 5.73 for 1 dB increments. Intercepts for size vs
intensity functions were -355.52, -527.57 and -491.0 for 5
dB increments, and -982, -1348 and -1009.2 for 1 dB
increments), indicating possibly different anatomical sites
of origin of the three waves. Similar functions
investigated in humans and cats have indicated that latency
is inversely proportional to intensity, and amplitude is
directly proportional to intensity. In humans Moore and
Semela (1985) have noted that the 1' potential (an EPSP-like
potential in humans) closely paralleled the latency

intensity function of waves I and II (equivalent to N1 and

140
N2 potentials in animals). Experiments conducted by Moore
et al (1988a, 1988b, 1989) have also yielded similar trends.
When intensity was increased Po (an EPSP-like potential in
animals), N1 and N2 potentials increased in amplitude and
decreased in latency. Po closely paralleled the input/
output function of N1 and N2. The results of these
investigations indicate that the No, Po and N1 potentials in
crickets have similar latency/intensity and amplitude/
intensity functions and that they are comparable to the Po

and N1 potentials in mammals.

TTX is among the deadliest poisons known to man. It is
found in the largest quantities in the gonads of some fish.
TTX interferes with the early transient conductance increase
that is associated with downhill movement of Na+ in most
common excitable cells (Kao, 1972). Application of TTX
anterior to the terminal ganglion brought about a
significant change in N1 amplitude and latency over time.
Similar effects have been seen in mammalian studies (Moore
et al., 1988a, 1988b, 1989; Dolan et al., 1989; and Xi et
al., 1989). In the mammalian studies, as well as in the
present study, in order to identify the EPSP-like potential,
the AP was reduced in size with the application of TTX.
Recordings made at regular intervals after application of
TTX indicated that the N1 potential started to diminish in
amplitude, and eventually what remained was the broad low

frequency EPSP-like potential. In order to determine if the

141
EPSP-like potential disappeared over time, in one of the
control experiments post-TTX recordings were made for 3
hours and 44 minutes. Responses indicated that even after
224 minutes after application of TTX (where the experiment

ended) the EPSP-like potential remained.

These experiments suggest that the EPSP-like potential has
characteristics different from the N1 potential and is not

generated by the fast conductivity of axonal sodium

...-sunning... q

. ‘1‘:
f

channels. In the previous studies (Moore et al., 1988a,
1988b, 1989; Dolan et al., 1989; Xi et al., 1989, 1989) a
similar paradigm was used. Intracochlear infusion of TTX in
the mammalian inner ear brought about little change in the
CM or electromechanical component. However, the compound
APs N1 and N2, declined to Zero over a period of about 30 -
60 minutes, resulting in the appearance of a slow negative
wave. This wave which was resistent to TTX was identified
as an "EPSP-like" potential (Dolan et al., 1989; xi et al.,

1989).

It is significant that the slow wave remnant normally masked
by the much larger AP emerges after application of TTX, and
is therefore a candidate for the remainder of the EPSP
(Moore et al., 1988a 1988b, 1989; klinke et al., 1988; Xi et
al., 1989; Dolan et al., 1989). Further, it is of interest
to know that the No-Po/EPSP—like potential can be abolished

by transmitter blockers, which suggests that these

142
potentials are post-synaptic potentials, yet are different

from APs.

In the experiments conducted in this study, after
application of TTX the N1 potential diminished and in its
place the EPSP-like potential could be identified.
Following this, KYNA, a quinoline derivative and a broad
spectrum excitatory amino acid antagonist was applied on the
fifth abdominal ganglion. Antagonist is a substance that
tends to nullify the action of another drug that binds to a
cell receptor, and does not elicit a biological response
(Kao, 1972). The reason of our choice of KYNA over other
antagonists was because previous studies have demonstrated
that KYNA did not significantly interact with other neuro-
transmitters, did not have nonspecific actions on cellular
excitability, and did not alter the input resistance
significantly (Gribkoff and Dudek, 1988, 1990). The
mechanism of action of KYNA as synaptic blocker is probably
via a post-synaptic block of transmitter receptors because
of its antagonist properties (Ganong, Lanthorn and Cotman,

1983).

The EPSP-like potential that was resistant to TTX began to
diminish in size with time after kYNA was applied, and
finally disappeared. The similarities in the results
obtained in mammalian studies and this study with crickets,

indicate that the EPSP-like responses can be recorded by

143
reducing the magnitude of the AP, and the EPSP-like
potentials are perhaps post-synaptic potentials, and the

presence of a cochlea is not necessary for their production.

It is also interesting to note that KYNA applied on the
terminal ganglion reduced the amplitude of No, Po and N1
potentials to almost zero in a short time (20 - 50 minutes).
This strongly suggests that excitatory amino-acids are
likely transmitters at the first synapse in the cricket's
cercal hearing system. Earlier studies (Callec and
Sattelle, 1973; Meyer and Reddy, 1985) have shown the
possibility of acetylcholine receptors in the giant neurons,
but no mention has been made of amino-acid receptors. KYNA
is a broad spectrum excitatory amino—acid antagonist, but it
does not clearly distinguish between different excitatory
amino acid receptors. So, in order to characterize amino-
acid sub-receptor types, studies with more specific agonists

and antagonists will need to be conducted.

There is also a need to attempt to block the EPSP-like
potential with a potassium blocker, such as tetraethyl-
ammonium, in order to rule out the possibility that the
EPSP-like potential is the potassium generated negative
after potential. Is the EPSP-like potential a membrane
potential? This is an important question, and should be
tested by using a membrane blocker such as Joro spiker

toxin.

A

144
Although the role of EPSPs in auditory nerve functioning is
not well established, it may not be erroneous to conclude
that the No-Po/EPSP-like potentials are the generator
potentials that initiate the action potential. The
generator potential has its origin auditory nerve dendrites,
while action potentials have as their origin the axons of

the auditory nerve. Hence, the No-Po/EPSP-like potential is

an integral part of the afferent auditory mechanism, and

this study suggests that crickets can be used as a suitable

... ...
All“
3.4 1

model for the study of synaptic and post-synaptic
activities.

145

CHAPTER VI

SUMMARY AND CONCLUSIONS

In the mammalian auditory system the hair cells, which are
the receptors of sound stimuli in the organ of Corti,
release neuro-transmitters to activate the peripheral
dendrites of the spiral ganglion fibers (Dallas, 1984). The
neuro-transmitters in turn excite the dendrites of the
spiral ganglion fibers, leading to generation of post-
synaptic potentials which, if excitatory in nature, are
called excitatory post-synaptic potentials (EPSP). If and
only when the EPSP reaches threshold will it lead to the
'generation of action potentials (AP). The EPSP, which is
‘the first potential among the post-synaptic potentials, is a
'good indicator of synaptic activity, and, if well
<characterized, could be used as a sensitive index of

synaptic activity.

.Although the importance of the EPSP is generally accepted,
‘very little is known about the auditory EPSP. There are
only a few studies that have investigated the auditory EPSP.
In human beings the EPSP is recorded as the "I'" potential,

the potential preceding wave I of the auditory brain stem

- 1 i

146

response (Hughes and Fino, 1980; Hughes, Fino and Gagnon,
1981; Benito et al 1984, Hughes and Fino, 1985; Moore and
Semela, 1985). In laboratory animals the EPSP was recorded
and designated as the "Po" potential (Moore et al., 1988a
and 1988b; Klinke et al., 1988; Moore et al., 1989). Xi et
al., (1989) and Dolan et al., (1989) recorded the EPSP
potential and named it "EPSP-like" potential. However,
among the investigators a controversy arose as to whether $"i

the recorded potential was truly an EPSP or the summating

‘ '4

potential (a receptor potential from the cochlea). This
study was undertaken in which the house cricket (Acheta
domesticus) was used as a model to study the EPSP potentials
in their cercal hearing system. The cricket basically has
two important hearing systems, the tympanal hearing system
and the cereal hearing system. The receptors for the cereal
hearing system are freely articulated hairs called sensilla,
which are innervated by sensory neurons. The axons of the
sensory neurons combine to form the cercal nerve and synapse
on the giant interneurons in the fifth abdominal ganglion

(also called the terminal ganglion).

The EPSP are studied at the first synaptic level, i.e., in
the terminal ganglion where the cercal nerve fibers synapse
on to the dendrites of the giant interneurons. The
potentials recorded from the terminal ganglion are gross
potentials and not single unit recordings, hence the term

EPSP—like potential is used rather than the term EPSP.

147
Twenty five crickets were used in this study to answer the

following questions:

1. Is it possible to identify an EPSP-like potential in
gross potential recordings of the ventral cord of Agneta
dgmggtigug, whose different components can be designated as
No-Po/EPSP-like potentials?

2. Following identification of the No-Po/EPSP-like
potentials, what stimulus parameters such as frequency,
intensity and time best characterize the post-synaptic
potentials using latency and amplitude functions?

3. Using pharmacologic agents such as tetrodotoxin and
kynurenic acid, how do they assist in the identification of

the No-Po/EPSP-like potentials?

Adult male and female crickets serving as subjects were
immobilized in ice. Three pairs of legs and two pairs of
wings were removed and petroleum jelly was applied on the
cut ends so as prevent escape of bodily fluids. The cricket
was pinned on to bee's wax glued to a vibration-free table

in a sound-treated room.

Three 270-micron silver-silver chlorided wires were used as
electrodes. The active electrode was placed on the fifth
abdominal ganglion, the reference electrode in the head and
the ground electrode in the abdomen. Acoustic stimulation

was provided by tone bursts from a speaker which was located

. ...

r‘ .
'I

148
at a distance of 5 cm, at an angle of 900 to the

longitudinal axis of the cerci.

In order to answer the questions posed, two sets of
experiments were conducted. In the first set, various
stimulus parameters such as stimulus intensity, frequency,
repetition rate, polarity, masking noise and rise-decay time
were varied. Compound action potentials were recorded using

various experimental conditions.

The results of the experiments indicated that an EPSP-like
potential can be identified and characterized using
appropriate stimulus parameters. The No, Po and N1
potentials were easily identified at high intensity levels;
however, the intensity had to be lowered in order to
identify the EPSP—like potential since, EPSP-like potential
was obscured in the presence of N1. At low intensity levels
the stimuli were insufficient to trigger N1, and in its
place was seen the EPSP-like potential. The input/output
intensity/latency curves and intensity/amplitude curves
indicate that No, Po and N1 potentials decreased in latency

and increased in amplitude with an increase in intensity.

The No, Po and N1 responses were better seen at lower
frequencies, i.e. below 2000 Hz. As the repetition rate was
increased from 1.1/sec to 3.1/sec, the potentials increased

in amplitude and decreased in latency. With further

149
:increase in repetition rate the latencies increased and
amplitude decreased. Similarly, with an increase in rise-
ciecay time, as is typical of neural responses, the
[potentials decreased in amplitude and increased in latency.
‘Vith an increase in simulataneous masking of the tone burst

vwith broad band noise, the N1 potential gradually decreased
:in amplitude.

{The latency/intensity and size (amplitude)/intensity
functions indicated similar trends in No, Po and N1
potentials. The trend seen in this study was similar to the
'trend seen in other human and cat studies (Moore and Semela,
1985; Moore et al 1988a and 1988b; Klinke et al, 1988;
IMbore et a1 1989). There exists a negative correlation
between latency and intensity and a positive correlation
between size and intensity for all the potentials tested,

namely, No, Po and N1.

The second set of experiments was conducted so as to
characterize the EPSP-like potentials using pharmacologic
agents. In these experiments the stimulus parameters were
held constant. In order to identify the EPSP-like potential
the N1 potential was decreased in amplitude by applying
tetrodotoxin (TTX, which is a neurotoxin known to affect Na+
channels) into a 2.0 mm plastic barrel placed anterior to
the fifth abdominal ganglion. Recordings made at different

intervals of time after application of TTX indicated that

_ -1

150
saith time, the N1 potential gradually decreased in
amplitude. In its place we could see a low frequency EPSP-
Ilike potential which was highly resistant to TTX. We were
able to show that even 224 minutes after application of TTX,
the EPSP-like potential remained. Application of kynurenic
acid (a broad-spectrum excitatory amino-acid antagonist) on
‘the fifth abdominal ganglion brought about a gradual

decrease in the EPSP-like potential, and within 25-30 J

‘7

Ininutes there were no potentials that were recognizable.

fl.—

frhe potentials recorded from various crickets indicated that
‘there is variability in the morphology, latency, amplitude
and threshold measurements. Although we are not certain of
'the underlying reasons for the variability, we can speculate
that it could be due to the differences in age, size and the

genetic make up of the crickets.

Suggestions for Further Research

Based on the results obtained in this study, the cricket can
be used as a model for investigating synaptic activity and
post-synaptic potentials. The following recommendations are
suggested for additional research:

1. An investigation similar to the present investigation
should be conducted using crickets whose age, size and
genetic make up are known. Such crickets can be obtained

from known suppliers such as Fluckers Cricket Farm, P.O. Box

151
378, Baton Rouge, LA 70821.
2. A similar investigation should be conducted at the level
of the prothoracic ganglion, which is the first synapse in
the tympanal hearing system in crickets.
3. A similar investigation with electrical stimulation
should be conducted in order to determine whether
electrical stimulations evoke similar responses to those of
acoustic stimuli.
4. Single unit recordings of dendrites and giant
interneurons in the ventral cord of the cricket nervous
system should be made so as to obtain more discrete
recordings of synaptic activity.
5. Patch-clamp studies need to be conducted on the giant
interneuron dendrites so as to investigate individual
currents responsible for depolarization and generation of
the EPSP.
6. A mechanical system should be developed which will be
capable of stimulating an individual sensillum, while making
single-unit recordings, so as to investigate contributions
from more discrete portions of the cercus.
7. There is a need to use more specific NMDA and non-NMDA
antagonists in order to identify possible receptor sub-types
of the post-synaptic membranes.
8. Studies using excitatory amino acid agonists are needed
in order to determine excitatory effects of these compounds.
9. There is a need to use nicotinic and muscarinic

antagonists in an attempt to identify possible sub-

152
populations of receptors of this class.
10. Investigations need to be conducted which utilize
tetraethylammonium (TEA) to block K+ efflux, and Joro spider

toxin to block the membrane response.

F ‘1». mm #71
‘

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153

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APPENDICES

Appendix A:

171

Analog waveforms of CAP recordings from four
crickets (Cric-071, Cric-072, Cric-076 and

Cric-092) depicting the effect of decreasing
intensity levels.

 

 

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Appendix B: Analog waveforms of CAP recordings from two
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