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EFFECTS OF METHYLMERCURY ON CENTRAL SYNAPTIC
TRANSMISSION IN RAT BRAIN SLICES

By

Yukun Yuan

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

DOCTOR OF PHILOSOPHY

Department of Pharmacology and Toxicology

1997

ABSTRACT
EFFECTS OF METHYLMERCURY ON CENTRAL SYNAPTIC
TRANSMISSION IN RAT BRAIN SLICES
By

Yukun Yuan

Effects of MeHg on central synaptic transmission were examined in rat
hippocampal and cerebellar slices using electrophysiological methods. Bath
application of MeHg initially stimulated and then suppressed synaptic
transmission in the CA1 region of hippocampal slices. 4 - 100 11M MeHg
blocked action potentials and hyperpolarized and then depolarized CA1
neuronal membranes. The primary sites of action of MeHg appeared to be the
postsynaptic CA1 pyramidal cells, although multiple effects were involved.
Inhibitory synaptic transmission appeared to be more sensitive to MeHg than
was excitatory synaptic transmission. After pretreatment of hippocampal
slices with bicuculline, a GABAA receptor antagonist, MeHg only suppressed
population spikes and excitatory postsynaptic potentials (EPSPs); no early
stimulation of these responses occurred. MeHg also blocked responses evoked
by GABAA receptor agonist muscimol. Thus, a preferential block by MeHg of
GABAA receptor-mediated responses appeared to be primarily responsible for
the initial enhancement of hippocampal synaptic transmission.

Similarly, MeHg caused a biphasic effect on field potentials recorded

from the molecular layer of the cerebellar slices. To identify sites of action,

effects of MeHg on EPSPs evoked by stimulating the parallel or climbing fibers
and repetitive firing of Purkinje cells evoked by injecting depolarizing current
at the soma were compared. MeHg blocked all voltage-dependent responses,
including Na”-dependent, fast somatic spikes and Ca2*-dependent, slow
dendritic spike bursts. MeHg appeared to affect voltage-dependent responses
and glutamate receptor-mediated responses differently. Similarly, MeHg
hyperpolarized and then depolarized Purkinje cell membranes. Moreover,
MeHg changed the patterns of repetitive firing of Purkinje cells from
predominantly Na"-dependent, fast somatic spikes to predominantly Ca2+-
dependent, low amplitude, slow dendritic spike bursts, suggesting that MeHg
may affect Purkinje cell membrane ionic conductances. Apparently, MeHg acts
primarily at the postsynaptic Purkinje cells to block cerebellar synaptic
transmission, multiple effects are involved. Thus, effects of MeHg on

hippocampal and cerebellar synaptic transmission are generally similar.

ACKNOWLEDGMENTS

I would like to thank my mentor, Dr. William D. Atchison, for providing
me with the opportunity to work and study in his lab for the past several
years, which will be a very important period of time in my career. Under his
guidance, I understood a simple but very important philosophy: Learn to walk
before run. I am very grateful for his great efforts and patient in helping me
to improve my oral and writing skills. I would also like to thank the rest
members of my dissertation committee: lDrs. Peter JR. Cobbett, Gerard L.
Gebber, Kenneth E. Moore and Monte M. Piercey, for their great efforts and
helpful advice. Special thanks are extended to Dr. James J. Galligan for his

help and suggestions in the setting up of the electrophysiological experiments.

I would also like to take this opportunity to thank everyone in Dr
Atchison’s lab, John, Tim, Cindy, Michael, Mike, Lynne, Jay, Ravindra, Sue,
Yao, Laura and Youfen, for their assistance and friendship during my stay.
Especially, I’d like to thank Michael, Mike and Jay for teaching me the "kid
language". I also want to thank all the graduate students in this department,
especially my classmates Amy, Courtney, Frederick, Ken and Rosie, for their
support and friendship. Special thanks are extended to the department office

staffs, Diane, Nelda and Mickey for their great help in all the paper work.

I’d also like to thank my family, especially my parents, for their

iv

understanding and constant support. I also want to thank my daughter and

son, Lucy and Daniel, for giving me lots of extra fun.

Finally, I want to express my gratitude to my wife and colleague, Hong,
for her constant support, understanding, suggestion and love. Without her
encouragement and help, I can’t image how difficult it would be for me to

accomplish my work.

TABLE OF CONTENTS

LIST OF TABLES

LIST OF FIGURES

LIST OF ABBREVIATIONS

CHAPTER ONE: INTRODUCTION
A. Methylmercury neurotoxicity
B. Effects of MeHg on peripheral synaptic transmission
C. Effects of MeHg on central synaptic transmission
D. Specific aims

CHAPTER TWO: THE PREPARATION AND USE OF BRAIN SLICES
FOR ELECTROPHYSIOLOGICAL STUDIES OF CENTRAL
SYNAPTIC TRANSMISSION

A. The advantages of in vitro brain slice techniques

B. The organization of neurons and synaptic circuits
the hippocampal slice

C. The organization of neurons and synaptic circuits
of cerebellar slices

D. Preparation of brain slices
1) Preparation of hippocampal slices
2) Preparation of cerebellar slices

E. Methods for electrophysiological studies of synaptic
transmission in brain slices

1) Extracellular recording

2) Intracellular recording

vi

vii

viii

xiii

16

18

19

21

27

34

34

35

39

39

44

3) Sharp single-electrode voltage clamp recording 45

4) Whole-cell patch recording 51
CHAPTER THREE: METHYLMERCURY ACTS AT MULTIPLE SITES

TO BLOCK HIPPOCAMPAL SYNAPTIC TRANSMISSION 55
A. Abstract 56
B. Introduction 58
C. Materials and Methods 60
D. Results 65
E. Discussion 94

CHAPTER FOUR: ACTION OF METHYLMERCURY ON GABAA
RECEPTOR-MEDIATED INHIBITORY SYNAPTIC
TRANSMISSION IS PRIMARILY RESPONSIBLE FOR
ITS EARLY STIMULATORY EFFECTS ON HIPPOCAMPAL

CA1 EXCITATORY SYNAPTIC TRANSMISSION 104
A. Abstract 105
B. Introduction 107
C. Materials and Methods 110
D. Results 113
E. Discussion 142

CHAPTER FIVE: COMPARATIVE EFFECTS OF METHYLMERCURY
ON PARALLEL-FIBER AND CLIMBING-FIBER RESPONSES

IN RAT CEREBELLAR SLICES 150
A. Abstract 151
B. Introduction 154
C. Materials and Methods 157

D. Results 168

vii

E. Discussion

CHAPTER SDI: SUMMARY AND CONCLUSIONS

A. Summary
B. Conclusion

C. Future direction

BIBLIOGRAPHY

viii

209

220

221

237

238

240

Table 2.1.

Table 3.1.

Table 3.2.

Table 6.1.

Table 6.2.

Table 6.3.

Table 6.4.

LIST OF TABLES

Putative neurotransmitter in the major intrinsic
pathways of hippocampus.

Time to MeHg-induced block of action potentials evoked
at the threshold and maximum stimulation of Schaffer
collaterals.

Amplitudes of action potentials and the resting
membrane potentials just before or at the time that
action potentials were blocked by MeHg.

Comparison of time to MeHg-induced block of population
spikes and field excitatory postsynaptic potentials
in the CA1 region of hippocampal slices.

Comparison of times to MeHg-induced block of parallel-
fiber volley, parallel-fiber postsynaptic response and
climbing-fiber response in cerebellar slices.

Comparison of times to MeHg-induced block of action
potentials evoked by stimulating Schaffer collaterals
and by current-injection in CA1 pyramidal cells of
hippocampal slices.

Comparison of times to MeHg-induced block of parallel-
fiber excitatory postsynaptic potentials, climbing-

fiber excitatory postsynaptic potentials and repetitive
firing of Purkinje cells evoked by current injection

in cerebellar slices.

ix

26

68

70

235

235

236

236

LIST OF FIGURES

Figure 1.1. Time course of effects of 4-500 uM MeHg on amplitude of
population spikes, field excitatory postsynaptic
potentials and antidromically-activated population
spikes. 11
Figure 1.2. Time course of reversal of effects of MeHg (100 pM) on
population spikes, field excitatory postsynaptic potentials,

or antidromically-activated population spikes by washing
with MeHg-free ACSF or D-penicillamine.

13
Figure 1.3. Effects of increasing stimulating intensity on population

spike responses in hippocampal slices blocked irreversibly
by 100 11M MeHg.

15
Figure 2.1. Schematic diagram of structure and intrinsic synaptic
circuits of a transverse hippocampal slice. 23
Figure 2.2. Schematic diagram of organization of neurons and synaptic
circuits of the transverse and sagittal cerebellar slices. 29
Figure 2.3. Diagrammatic depiction of arrangement of the agar and
cerebellar tissue blocks on the tissue pedestal of an
oscillatory slicer.

38
Figure 2.4. Representative demonstration of the field potentials

recorded from the CA1 region of a hippocampal slice and

the molecular layer of a cerebellar slice using

extracellular recording techniques. 43

Figure 2.5. Representative example of responses recorded from
individual CA1 pyramidal cells of a hippocampal slice
and Purkinje cells of a cerebellar slice using
intracellular recording techniques. 47

Figure 2.6. Schematic diagram of the principle of sharp single-electrode
voltage clamp technique.

49
Figure 3.1 Diagrammatic depiction of the synaptic circuits of a
traverse hippocampal slice and methods for recording
action potentials in the CA1 pyramidal cells.

64

Figure 3.2. Time courses of effects of 4, 20 and 100 uM MeHg on
action potentials of hippocampal CA1 pyramidal cells.

Figure 3.3. Time courses of 4 - 100 uM MeHg on resting membrane
potentials of hippocampal CA1 pyramidal cells.

67

73

Figure 3.4. The maximum hyperpolarization of CA1 neuronal membranes

caused by 4 - 100 uM MeHg.

Figure 3.5. Effects of increased [Ca2+]e on time to MeHg-induced
block of action potentials evoked at threshold and
maximum stimulation of Schaffer collaterals.

Figure 3.6. Comparison of times to MeHg-induced block of action
potentials evoked by different stimulation methods.

Figure 3.7 Time course of effect of 20 11M MeHg on action potentials
evoked by depolarizing current injection through a
recording electrode at the CA1 pyramidal cell soma.

Figure 3.8. Comparison of times to block by 100 uM MeHg of responses
of CA1 neurons to electrical stimulation of presynaptic
terminals and to iontophoretic application of glutamate
on the apical dendrites of CA1 pyramidal cells.

Figure 3.9. Comparison of time course of effects of 100 11M MeHg on
EPSPs and IPSPs.

Figure 3.10. MeHg-induced changes in IPSP amplitudes at 0, 10 and 35
min and changes in EPSP amplitudes of hippocampal CA1
pyramidal cells at 0, 40 and 60 min.

Figure 4.1. Comparison of times to block of recurrent IPSPs and
EPSPs or IPSCs and EPSCs by 100 uM MeHg or ng”.

Figure 4.2. Time course of effect of 100 uM MeHg on EPSCs recorded
from CA1 pyramidal cells of hippocampal slices using
sharp single-microelectrode voltage-clamp techniques.

Figure 4.3. Effects of 100 11M MeHg on IPSCs recorded at different
holding potentials.

Figure 4.4. Effects of 100 pM Hg2+ on IPSCs recorded at different
holding potentials.

76

80

83

85

88

91

93

115

118

121

123

Figure 4.5. Comparison of effects of 100 pM MeHg and ng+ on the
current-voltage relationship (I/V curve) of IPSCs
recorded in the CA1 pyramidal cells. 125

Figure 4.6. Comparison of effects of MeHg and bicuculline on
population spikes of hippocampal CA1 pyramidal cells. 128

Figure 4.7. Time courses of effects of MeHg on population spikes of
hippocampal CA1 pyramidal cells with or without
pretreatment of slices with bicuculline or strychnine. 131

Figure 4.8. Time course of effects of 100 pM MeHg on EPSPs of
hippocampal CA1 pyramidal cells evoked by stimulating
Schaffer collaterals in the presence of bicuculline. 135

Figure 4.9. Effects of 20 uM bicuculline and 100 uM MeHg on muscimol-
evoked responses in hippocampal CA1 pyramidal cells. 137

Figure 4.10. Comparison of the maximum enhancement of population
spikes of CA1 hippocampal pyramidal cells by strychnine,
bicuculline and MeHg alone, with the combinations of MeHg
with bicuculline or strychnine. 141

Figure 5.1. Diagrammatic depiction of the structure of the cerebellar
cortex in sagittal and transverse slices and methods for
recording field potentials of Purkinje cells following
activation of parallel-fibers or climbing-fibers. 160

Figure 5.2. Effects of DNQX and AP-5 on field potentials evoked
by stimulating the parallel or climbing fibers in
cerebellar slices. 163

Figure 5.3. Effects of stimulus intensity on the parallel and climbing
fiber responses. 166

Figure 5.4. Time course of effects of 100 and 20 uM MeHg on field
potentials recorded from the molecular layer following
stimulation of the parallel fibers in transverse
cerebellar slices. 170

Figure 5.5. Time course of effects of 100 and 20 11M MeHg on field
potentials recorded from the molecular layer following
stimulation of the climbing fibers in sagittal
cerebellar slices. 173

Figure 5.6.

Figure 5.7.

Figure 5.8.

Figure 5.9.

Figure 5.10.

Figure 5.11.

Figure 5.12.

Figure 5.13.

Figure 5.14.

Figure 5.15.

Figure 5.16

Comparison of times to block by 100 and 20 uM MeHg of
field potentials representing the parallel fiber volleys,
postsynaptic responses and climbing fiber responses. 175

Time course of effects of 100 and 20 uM MeHg on field
responses representing the parallel fiber volleys,
postsynaptic responses and climbing fiber responses. 177

Time courses of effects of 100 and 20 11M MeHg on PF-

EPSPs recorded from Purkinje cell soma by stimulation

of the parallel fibers in the transverse cerebellar

slices 181

Time courses of effects of 100 and 20 11M MeHg on CF-
EPSPs recorded from Purkinje cell soma by stimulation
of the climbing fibers in the sagittal cerebellar slices. 183

Time courses of effects of 100 and 20 11M MeHg on resting
membrane potentials of Purkinje cells. 186

Time course of effects of 100 uM MeHg on responses evoked
by injection of depolarizing current through the recording
electrode at Purkinje cell soma. 189

Comparison of time courses of effects of 100 uM MeHg on
PF-EPSPs evoked by stimulation of the parallel fibers and
repetitive responses evoked by injection of depolarizing

current at Purkinje cell soma. 192

Comparison of time courses of effects of 20 uM MeHg on
PF-EPSPs evoked by stimulation of the parallel fibers and
repetitive responses evoked by injection of depolarizing

current at Purkinje cell soma. 195

Comparison of time courses of effects of 100 pM MeHg on
CF-EPSPs evoked by stimulation of the climbing fibers and
repetitive responses evoked by injection of short duration

depolarizing currents at Purkinje cell soma. 197
Comparison of times to block of PF-EPSPs, CF-EPSPs and
repetitive firing responses (Soma) of Purkinje cell by 100

and 20 11M MeHg. 200
Spontaneous repetitive firing of a Purkinje cell just after

xiii

penetration with a glass recording microelectrode. 203

Figure 5.17. Effects of 100 uM MeHg on spontaneous repetitive firing
of Purkinje cells. 205

Figure 5.18. Effects of 100 11M MeHg on spontaneous firing of Purkinje
cells recorded from the Purkinje cell layer of a transverse
cerebellar slice using extracellular recording techniques. 207

ACh
ACSF
AP-5
EF-EPSPs
CFRs
CNS
CNQX
[Ca’*}.,
[032’],
DMSO
DNQX
EPPs
EPSPs
EPSCs
fEPSPs
GABA
GABAA
GABA13
ng+
IP3

LIST OF ABBREVIATIONS

acetylcholine

artificial cerebrospinal fluid
amino-5-phosphonopentanoic acid
climbing fiber excitatory postsynaptic potentials
climbing-fiber responses

central nervous system
6-cyano-7-nitroquinoxaline-2,3-dione
extracellular Ca2+ concentration
intracellular Ca2+ concentration
dimethyl sulfoxide
6,7-dinitroquinoxaline-2,3-dione
end-plate potentials

excitatory postsynaptic potentials
excitatory postsynaptic currents

field excitatory postsynaptic potentials
y—aminobutyric acid

A type of GABA receptor

B type of GABA receptor

inorganic mercury

inositol-l,4,5-tris-phosphate

IPSPs
IPSCs
I/V curve
MeHg
MEPP
min

mM

ms

mV

MQ
NMDA
PF-EPSPs
PFVs
PSRs

SE

sSEVC

11M

v/v

inhibitory postsynaptic potentials

inhibitory postsynaptic currents
voltage-current relationship

methylmercury

miniature end-plate potential

minute

millimole

millisecond

millivolt

megohm

N-methyl-D—aspartate

parallel fiber excitatory postsynaptic potentials
parallel-fiber volleys

postsynaptic responses

standard error of mean

sharp single-electrode voltage-clamp recording
tetrodotoxin

micromole

volume per volume dilution

CHAPTER ONE

INTRODUCTION

A. Methylmercury neurotoxicity

Methylmercury (MeHg) is a well-known environmental contaminant.
Even today, mercury pollution remains an important global environmental
problem (Evans, 1986; Wendro, 1990; Nriagu et al., 1992; N ater and Grigal,
1992; Nriagu, 1993). Sources of mercury contamination are generally from
industrial, agricultural and other anthropogenic activities and natural events
from geological formations. MeHg can be converted from inorganic mercury,
via methylation by microorganisms in the sediments of river and lake bottoms,
and then concentrated in fish tissues within the food chain. This is thought
to be primarily responsible for the chronic events of MeHg poisoning events in
Minamata Bay and the Niigata district of Japan in the 19503. The mean
biological half-life of MeHg is about 70 days in the human body (Nelson et al.,
1971; Birke et al., 1972) and much longer in brain (Komulainen, 1988). MeHg
is soluble in both water and lipid with a high lipid/water partition coefficient,
which confers on it the ability to cross the blood-brain barrier more readily
than other mercurial compounds. Therefore it accumulates in the brain
following chronic exposure. The distribution of MeHg in the brain is generally
uniform, however, the areas which attain the maximum concentrations of
MeHg following subacute or chronic exposure were the cerebral cortex,
hippocampus and the cerebellar cortex (Olserwski et al., 1974; Chang, 1980;
Moller-Madsen, 1990, 1991). Regional variations in distribution of MeHg have

also been demonstrated between experimental animal species, sex and patterns

3
of administration of MeHg (Yoshino et al. , 1966a; Yasutake and Hiyama, 1986;

Omata et al., 1986; Thomas et al., 1986; Moller-Madsen, 1990, 1991). The
critical target organ of MeHg is the nervous system, particularly the central
nervous system (CNS). Acute and chronic exposure to MeHg disrupts sensory
and motor functions and causes a series of peripheral and central nervous
system disorders of human and experimental animals (Takeuchi et al., 1959;
Kurland et al. , 1960; Tokuomi et al. , 1961; Takeuchi et al., 1962; Miyakawa et
al., 1970; Bakir et al., 1973; Rustam and Hamdi, 1974; Chang, 1977, 1980).
The typical symptoms and signs of MeHg poisoning include extremity
weakness, cerebellar ataxia, visual damage (tunnel-vision), loss of hearing,
disturbances of sensory functions and so on (Kurland et al., 1960; Tokuomi et
al., 1961; Bakir et al., 1973; Rustam and Hamdi, 1974; Chang, 1977, 1980).
The underlying mechanisms responsible for these effects remain poorly
understood. Multiple mechanisms may be involved, as MeHg has been
reported to interfere with intracellular homeostasis of Ca2+ (Komulainen and
Bondy, 1987; Kauppinen et al., 1989; Levesque and Atchison, 1991; Hare and
Atchison, 1992b, Hare et al., 1993; Denny et al., 1993; Sarafian, 1993; Hare
and Atchison, 1995a,b; Marty and Atchison, 1997), to affect Ca2+ channels
(Atchison et al., 1986; Shafer and Atchison, 1989; Shafer et al., 1990; Shafer
and Atchison, 1991, 1992; Hewett and Atchison, 1992; Leonhardt et al., 1996;
Sirois and Atchison, 1996), K” and Na" channels (Shrivastav et al., 1976;

Quandt et al., 1982; Shafer and Atchison, 1992; Sirois and Atchison, 1995;

4
Leonhardt et al., 1996), to inhibit protein phosphorylation and synthesis

(Yoshino et al., 1966b; Syversen, 1981; Sarafian and Verity, 1990a,b Sarafian,
1993), to inhibit activity of some enzymes (Taylor, 1963; Tunnicliff and Wood,
1973; Verity et al., 1975; Omata et al., 1982; Dyck and O’Kusky, 1988;
Kishimoto et al., 1995), to affect neurotransmitter release and disrupt
peripheral synaptic transmission (J uang and Yonemura, 1975; J uang, 1976a,b;
Bondy et al., 1979; Minnema et al., 1989; Atchison and Narahashi, 1982;
Atchison, 1986; Traxinger and Atchison, 1987a,b; Levesque et al., 1992), to
induce cell death (Sarafian et al., 1989; Sarafian and Verity, 1990a; Sarafian
et al. , 1994; N agashima et al. , 1996; Kunimoto and Suzuki, 1997), to depolarize
neuronal membranes (J uang, 197 6; Shrivastav et al., 1976; Quandt et al., 1982;
Kauppinen et al., 1989; Hare and Atchison, 1992a) and to have other
neurotoxic effects (Chang, 1980; Atchison, 1987b). It is generally believed that
effects of MeHg on both peripheral and central synaptic transmission may play

an important role in its neurotoxicity.

B. Effects of MeHg on peripheral synaptic transmission.

In experimental animals with subacute MeHg poisoning, pathological
examination indicated that MeHg first or selectively affected the peripheral
nerves, especially the sensory nerve fibers, (Miyakawa et al. , 1970; Chang and
Hartmann, 197 2). In the Iraq MeHg poisoning episode, individuals also

exhibited neuromuscular weakness which was similar to myasthenia gravis,

5
suggesting that MeHg may disrupt peripheral motor synaptic transmission

(Rustam et al., 1975). Thus, early mechanistic studies have extensively
examined the effects of MeHg on synaptic transmission at vertebrate
peripheral synapses such as the neuromuscular junction and autonomic
ganglia. Acute bath application of MeHg irreversibly blocks synaptic
transmission at these synapses (Juang and Yonemura, 1975; Juang, 1976a,b;
Atchison and Narahashi, 1982; Atchison, 1986; Traxinger and Atchison,
1987a,b). In neuromuscular preparations, MeHg caused a biphasic effect on
spontaneous release of acetylcholine (ACh). Release, measured as a change of
miniature end-plate potential (MEPP) frequency, is stimulated and then
depressed to block by MeHg in the concentration range of 4 - 100 uM (Atchison
and Naraharshi, 1982; Atchison, 1986, 1987a; Traxinger and Atchison, 1987
a,b; Levesque and Atchison, 1987, 1988). Effects of MeHg on synaptic function
are not limited to spontaneous release of ACh. MeHg also affects nerve-evoked
release of ACh, measured as changes in amplitude of end-plate potentials
(EPPs) (Atchison and Narahashi, 1982; Atchison et al., 1986; Traxinger and
Atchison, 1987b). In some cases MeHg also transiently increased the
amplitude of EPPs prior to block (Manalis and Cooper, 197 5; Juang, 1976b;
Traxinger and Atchison, 1987b). Using mouse triangularis sterni motor
nerves, Shafer and Atchison (1992) directly examined effects of MeHg on
functions of presynaptic nerve terminal Ca2+ and N a” channels at intact

neuromuscular junctions. At micromolar levels (20, 100 pM), MeHg rapidly

6

and irreversibly blocked both Ca2’- and Na’-mediated potential components
evoked by stimulating presynaptic nerve fibers, suggesting that MeHg may
decrease motor nerve excitability and block neurotransmitter release by
disrupting function of Na+ and Ca2+ channels on nerve terminals. None of
these effects described above were reversed completely by washing
neuromuscular preparations with MeHg-free solution. However, partial
reversal of effects of MeHg on synaptic functions occurred under certain
conditions such as by washing neuromuscular preparations with MeHg-free
solution in conjunction with increasing stimulus intensity or duration or by
increasing extracellular Ca2+ concentrations (Von Burg and Landry, 1976;
Alkhadhi and Taha, 1982; Atchison, 1986; Traxinger and Atchison, 1987b).
The mechanisms responsible for these effects caused by MeHg on peripheral
synaptic transmission are generally considered to be predominantly
presynaptic (Atchison and N arahashi, 1982; Atchison, 1986, 1987; Levesque
and Atchison, 1987, 1988; Shafer and Atchison, 1991, 1992), because at
relatively high concentrations (40 or 100 11M), MeHg has no effects on either
action potentials (twitches) evoked by direct stimulation of muscle fibers or
resting membrane potentials of postsynaptic muscle fibers despite block of
neuromuscular transmission (Juang, 1976a). Moreover, MEPPs of normal
amplitude and duration still occur and responses of end-plates to iontophoretic
application of ACh were unaffected by 100 pM MeHg at times that EPPs were

blocked (Atchison and Narahashi, 1982).

7

These model peripheral synapses, which are well characterized
physiologically and anatomically, have been useful systems in which to detail
some of the early effects of MeHg on synaptic function such as neuromuscular
weakness in those Iraqi poisoning patients. However, the clinical symptoms
and signs and pathologic lesions in human and experimental animals with
MeHg poisoning, especially following chronic exposure, suggest that the major
neurotoxic target is the central nervous system, particularly the cerebellum
and the visual cortex of the occipital lobe (Hunter and Russell, 1954; Kurland
et al., 1960; Tokuomi et al., 1961; Takeuchi et al., 1962; Bakir et al., 1973;
Rustam and Hamdi, 1974; Chang, 1977, 1980), although differences in the
sites, degree and sequence of MeHg-induced neuropathologic lesions in the
CNS exist among experimental animal species (Yoshino et al., 1965; Shaw et
al., 197 5). Moreover, central synapses have a number of unique characteristics
such as the presence of significant Ca2*-mediated action potentials in dendrites
of some neurons (Llinas and Hess, 1976; Schwartzkroin and Slawsky, 1977;
Wong and Prince, 1978; Wong et al. , 1979; Llinas and Sugimori, 1980b; Kimura
et al., 1985; Llinas and Walton, 1990; Johnston et al., 1996), which are not
present at the neuromuscular junction. Thus, MeHg may affect central
synaptic function in a manner different from that on peripheral somatic nerve

transmission.

8
C. Effects of MeHg on central synaptic transmission.

The effects of MeHg on central synaptic transmission have not been as
well studied as its effects on peripheral synaptic transmission. Thus, little is
known of the mechanisms by which MeHg acutely and chronically alters
central synaptic functions. In vitro, however, exposure to MeHg alters
neurotransmitter release fiom brain homogenates or synaptosomes (Bondy et
al., 1979; Minnema et al., 1989; Levesque et al., 1992), depolarizes
synaptosomal and intraterminal mitochondrial membranes (Kauppinen et al.,
1989; Hare and Atchison, 1992a), affects Ca2+ channels in synaptosomes and
primary cultures of cerebellar granule cells (Shafer and Atchison, 1989; Shafer
et al., 1990; Hewett and Atchison, 1992; Sirois and Atchison, 1996), and
disturbs intracellular homeostasis of Ca2+ of synaptosomes and intact neurons
(Komulainen and Bondy, 1987; Kaupppinen et al., 1989; Levesque and
Atchison, 1991; Hare and Atchison, 1992b; Denny et al., Marty and Atchison,
1996; 1997). Thus, it is likely that MeHg also blocks synaptic transmission at
intact central synapses.

Using extracellular microelectrode recording techniques I initially
examined the effects of MeHg on the field potentials [including population
spikes, field excitatory postsynaptic potentials (fEPSPs) and antidromically-
activated population spikes] recorded from the CA1 pyramidal cells of
hippocampal slices (Yuan and Atchison, 1993, 1994). Acute bath application

of 4 - 500 1.1M MeHg caused a concentration- and time-dependent biphasic

9

effect on these field potentials (Figure 1.1). Specifically, MeHg initially
increased and then decreased to complete block population spikes, fEPSPs and
antidromically-activated population spikes. However, the characteristics of
block of these field potentials by MeHg differed somewhat in terms of the time
courses and degree of reversibility. Block of orthodromically- and
antidromically-activated population spikes was at best only partially reversible
by washing slices with MeHg-free artificial cerebrospinal fluid (ACSF) or 1 mM
D—penicillamine‘, a MeHg chelator, whereas block of fEPSPs was partially
reversible by washing slices with MeHg-free ACSF and quickly and completely
reversible by D-penicillamine (Figure 1.2). In those slices refractory to reversal
by washing with either MeHg-free ACSF or D-penicillamine, increasing the
stimulus intensity slightly could induce temporary recovery of the population
spikes (Figure 1.3). These results suggest that acute bath application of MeHg
may alter hippocampal CA1 neuronal membrane excitability and act at

multiple sites to disrupt synaptic transmission.

10

Figure 1.1. Time course of effects of MeHg on amplitude of population
spike (PS), field excitatory postsynaptic potential (fEPSP) and
antidromically-activated population spike (Anti-PS). Slices were
perfused continuously with MeHg--containing ACSF at 4, 20 100 and
500 11M. PSs and fEPSPs were recorded at the CA1 pyramidal cell soma
and apical dendritic region, respectively, by stimulation of Schaffer
collaterals at 0.25 Hz. Anti-PSs were recorded at the CA1 pyramidal
soma region by stimulating the alveus. Values obtained before perfusion
with MeHg were considered as pretreatment control. All values are the
mean :t SE of 6-11 individual experiments. Only one slice per rat was

used.

11

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0

12

Figure 1.2. Time course of reversal effects of MeHg (100 uM) on
population spikes (PSs), field excitatory postsynaptic potentials
(fEPSPs), or antidromically-activated population spikes (Anti-PS5) by
washing with MeHg-free ACSF or D-penicillamine. Slices were washed
with MeHg-free ACSF or 1 mM D-penicillamine after the amplitudes of
PSs, fEPSPs or Anti-PSs were reduced by MeHg to 50% of their
pretreatment control level. Values are the mean :t SE of 5-10 individual

experiments.

13

 

 

 

 

 

 

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14

Figure 1.3. Effects of increasing stimulating intensity on population
spike (PS) responses in slices blocked irreversibly by 100 uM MeHg.
After PS amplitude was increased and then decreased to 50% of control,
the slice was washed with MeHg-free ACSF (Top) or 1 mM D-
penicillamine (Bottom). PSs were induced and maintained by repeated
single shock stimulation of 3.2 V (A) or 3.0 (B) at 0.25 Hz. After PSs
were blocked completely, increasing stimulation intensity from 3.2 V to
3.6 V at 5, 60 and 90 min (Top) or fi'om 3.0 V to 3.4 or 3.6 (Bottom)
could still induce PS responses. Upon returning stimulation intensity

to 3.2 V or 3.0 V, PS responses again disappeared.

AMPLITUDE (% of control)

200 '

150 ’

100 "

50

200 '

150 '

100 “

50

 

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16

Due to the limitations of extracellular recording techniques, it is difficult
to specify where and how MeHg acts to block hippocampal synaptic
transmission. In addition, it is unknown if these effects occur similarly in
other brain regions such as the cerebellar cortex. Thus, in order to
characterize further the effects of MeHg on central synaptic transmission,
extracellular and intracellular microelectrode recordings, sharp single-electrode
voltage-clamp recordings and iontophoresis techniques were applied in this
dissertation to examine effects of MeHg on synaptic transmission in both

hippocampal and cerebellar slices of rat brain.

D. Specific aims.

The general objective of this dissertation is to characterize in vitro acute
effects of MeHg on central synaptic transmission in brain slices, to explore the
potential mechanisms underlying these effects and, to collect basic information
for future design of studies of the subchronic and chronic effects of MeHg on
the CNS. Specifically, the questions to be asked in this dissertation are:

(1). Does MeHg affect neuronal membrane excitability or alter the threshold
for neuronal excitation?

(2). Are the mechanisms responsible for effects of MeHg on synaptic
transmission in a given region of brain slices pre- or postsynaptic?

(3). Does MeHg also affect inhibitory synaptic transmission in addition to its

effect on excitatory synaptic transmission?

17

(4). Are effects of MeHg on synaptic transmission in hippocampal and
cerebellar slices similar?
(5). Are effects of MeHg on central synaptic transmission similar to those of
MeHg on peripheral somatic synaptic transmission?

To answer these questions, hippocampal and cerebellar slices prepared
freshly from rat brain were used as the central synaptic circuit model in this

dissertation.

CHAPTER TWO

THE PREPARATION AND USE OF BRAIN SLICES

FOR ELECTROPHYSIOLOGICAL STUDIES OF

CENTRAL SYNAPTIC TRANSMISSION

18

19

Central synaptic transmission can be studied in freshly isolated brain
slice preparations (Lynch, 1980; Schwartzkroin, 1981; Langmoen and
Anderson, 1981; Alger et al., 1984; Johnston and Brown, 1984; Teyler, 1986).
Since its introduction in the 19208, especially after the 19708, the in vitro brain
slice technique has been widely used in electrophysiological, morphological,
biochemical, and pharmacological studies. This is because it offers several
major technical advantages over in vitro invertebrate model, cell culture, whole
brain in situ, and in vivo methods for the investigation of mammalian CNS

neurobiology and neurophysiology.

A. The advantages of in vitro brain slice techniques.

First, the brain slice provides simple and precise control over
experimental conditions such as pH, temperature and concentrations of tested
chemicals, compared to the variables that must be controlled in in vivo studies.
There is no blood pressure to monitor, no expired CO2 concentration to
maintain, no heart rate to stabilize and no anesthetics, paralytics or foreign
agents need be used in slice preparations from small animals and during
experiments.

Second, the brain slice preparation provides direct visual control over
the placement of both recording and stimulating electrodes in the desired sites,
avoiding the hazards and ambiguities of stereotaxic techniques. The neurons

being targeted can be located, identified, and accessed easily.

20

Third, in contrast to most cell culture systems, the normal anatomical
relationships and synaptic circuits remain intact and healthy in a properly-
oriented brain slice. This is of particular advantage in a laminated structure
such as the hippocampus and cerebellar cortex.

Fourth, the brain slice greatly improves the stability of
electrophysiological recording. There are no mechanical disturbances caused
by heart beat and respiration. Therefore, it is possible to make high-quality,
long-lasting intracellular recordings from neurons in isolated brain slices with
relative ease.

Fifth, both side hippocampi from one animal can be sectioned into 5 to
10 slices. Thus, one animal is possibly used to do several experiments with
different objectives such as concentration-dependent effects.

In short, the in vitro brain slice technique combines many of the
technical advantages of whole brain in situ and cell culture simplicity with
normal complex organization of mammalian CNS tissue, and has greatly
facilitate our investigation of the electrical properties and function of neurons
in the CNS. Use of the brain slice has also greatly increased our knowledge
of the effects of many neurotoxic chemicals on the mammalian CNS in the last
decade. The brain slices can be made from almost any region of the brain. In
this dissertation, both rat hippocampal and cerebellar slices were used as
model central synapses to examine in vitro acute effects of MeHg on central

synaptic transmission.

21

B. The organization of neurons and synaptic circuits of the
hippocampal slice.

The hippocampal slice preparation has been the most commonly-used
model for studying central synaptic function, due to (1) its relatively simple
organization of a few major types of cells (pyramidal, granule and basket cells)
with well-characterized synaptic pathways; (2) the entire hippocampus can be
easily removed from the brain with a minimum of manipulation, while its size
is optimal for slice preparation with very simple equipment, and hippocampal
slice is easy to prepare and maintain; (3) the layers of pyramidal and granule
cell bodies and several important fiber tracts (perforant path, mossy fibers, the
commissural-Schaffer collaterals and alveus) can be easily discerned with a
dissecting microscope; (4) most of the major intrinsic and extrinsic hippocampal
fiber systems are organized according to a "lamellar" plane in which they
travel at right angles to the longitudinal axis of the structure (Figure 2.1).
Thus, a transverse hippocampal slice (see "Preparation of hippocampal slices")
will contain a variety of projections whose axons extend for some distance
along that cross section that are amenable to the study of several different
synaptic circuits in a single slice. In addition, interest in hippocampal slice is
also due to the fact that the hippocampus plays an important role in certain
aspects of learning and memory and is a target of some degenerative disorders

such as Alzheimer’s disease and many neurotoxic agents (Walsh and Emerich,

1988).

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The organization of neurons and synaptic connections in the transverse
hippocampal slice are relatively simple (Dingledine, 1984; Franck et al., 1989;
Brown and Zador, 1990; Kennedy and Marder, 1992). It is composed of two
sheets of two principal neurons that are highly interconnected with each other.
The first sheet consists of three contiguous areas of cortex-CA fields (Cornu
Ammonis), the subicular complex, and the entorhinal cortex (Figure 2.1). The
CA fields are composed of a large flap of tightly packed CA1-C4 pyramidal cells
(CA2 and CA4 have rarely been used as designating terms; they are not
substantially different from those pyramidal cells of CA1 and CA3 regions).
The sizes of pyramidal cells become larger as they extend from the CA1 region
to the CA3 region; in the CA3 region the pyramidal cell bodies condense into
monolayer. The second sheet of neurons is the dentate gyrus, in which are
small, round and highly packed granule cells.

The major input to hippocampus is the axons from neurons of the
entorhinal cortex via the perforant pathway to make the first set of synapses
on the dendritic trees of the granule cells in dentate gyrus (perfront pathway
also projects to pyramidal cells in the CA field, not shown in Figure 2.1). The
granule cells send axons, known as mossy fibers, to make the second set of
synapses on the dendrites of CA3 pyramidal cells. One mossy fiber
synaptically contacts a long row of CA3 pyramidal cells; the mossy fiber
contacts are always near the base of the apical dendrites of the CA3 pyramidal

cells. The CA3 pyramidal cells in turn send one axon branch to project out of

25

the hippocampus (principally to the septum), the other axon branches, known
as Schaffer collaterals, extends to make the third set of synapses on the apical
dendrites of CA1 pyramidal cells. These cells send axons, within the alveus,
which is the major output from the hippocampal formation, to the subiculum
and entorhinal cortex to complete the so-called trisynaptic loop. The
principal putative excitatory transmitters in these intrinsic excitatory
pathways are glutamate and/or aspartate (Table 2.1). In addition to these
intrinsic excitatory synaptic pathways, intrinsic inhibitory pathways also exist
between basket cells and granule cells or pyramidal cells. Anytime when
granule cells or pyramidal cells are activated, their axons will in turn activate
basket cells to release y—aminobutyric acid (GABA) to act on GABAA or GABAB
receptors on granule cells or pyramidal cells to cause so-called recurrent
inhibition of granule cells or pyramidal cells via both pre- and postsynaptic

mechanisms.

26

Table 2.1. Putative neurotransmitters in the major intrinsic pathways of hippocampusl

 

 

 

 

 

 

 

 

Synapse Transmitter Receptor Ionic effect
Kainate/AMPA(?) 116M“,
pp-GC2 glutamate
NMDA (?) 1TGC32+vTGNa+,K+
Met, Leu-enkephalin(?) (?) (?)
Kainate/AMPA IIGNw‘K,
mf-CA3 glutamate/aspartate
NMDA 1T(3012+,Tc}Na+.K+
Dynorphin (?) (?) (?)
Kainate/AMPA ITGNMK+
Sch-CA1 glutamate/aspartate
NMDA 1TGCa2+9TCTNa+K+
mGluR T[Ca2*],,lGK,
GABAA “Ge?
BC-GC GABA
GABAB iiGK+
GABAA I’IGC,+
BC-CA3 GABA
GABAB “GK,
GABAA “OJ
BC-CAI GABA

 

 

 

 

 

 

1. Based on references (Dingledine, 1984; Franck et al., 1988; Collingridge and Singer,
1990; Baskys, 1992; Sommer and Seeburg, 1992; Shirasaki et al., 1994; Nadler et al.,
1994; Spuston and Sakmann, 1995).

2 Abbreviations: pp-GC, perforant path-granule cell synapse; mf-CA3, mossy fiber-CA3
pyramidal cell synapse; Sch-CA1, Schaffer collaterals-CA1 pyramidal cell synapse; BC-
GC, basket cell-granule cell synapse; BC-CA3, basket cell-CA3 pyramidal cell synapse;
BC-CAI, basket cell-CA1 synapse; AMPA, a-amino-B-hydroxy-5-methyl-4-
isoxzoleproprionic acid; NMDA, N-methyle-D-aspartate; mGluR, metabotropic
glutamate receptor; GX, ionic conductance; [Ca2+],, intracellular Ca2+
concentration.

27

C. The organization of neurons and synaptic circuits of cerebellar
slices.

The cerebellum is an outgrowth of the pons and consists of a three-
layered cortex overlying deep nuclear groups of cells. Structurally, the
cerebellum consists of (1) a superficial gray mantle, the cerebellar cortex; (2)
an internal white mass, the medullary substance; and (3) four pairs of intrinsic
nuclei (dentate nucleus, globose nucleus, emboliform nucleus and fastigial
nucleus) embedded in the white matter. The cerebellum is divided into a
median portion-the cerebellar vermis, and two lateral lobes-the cerebellar
hemispheres (Anderson, 1989; Shepherd, 1990; Carpenter, 1991; Nicholls et al.,
1992). The cerebellar cortex receives two major sets of afferent, the climbing
and the mossy fibers, and generates a single output system, the axons of
Purkinje cells. The cerebellar nuclei receive collaterals from the climbing and
mossy fibers and are the main targets for the Purkinje cell axons.

The cerebellar cortex is uniformly structured in all parts and is
composed of three layers containing five major different types of neurons.
These layers from the deepest to the surface are (1) the granular layer, ( 2) the
Purkinje cell layer, and (3) the molecular layer (Figure 2.2).

The granular layer is the thickest and deepest, lying adjacent to the
white matter. This layer is tightly packed with granule cells (estimated
number between 1010 and 1011 cells in human brain), which are round or oval

and 5 - 8 pm in diameter. Each granule cell has four or five short dendrites.

28

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Granule cells send their unmyelinated axons outward to the molecular layer
to form Parallel Fibers, which run throughout the molecular layer and are
oriented perpendicular to the sagittal fan-shaped dendrites of the Purkinje
cells. The synaptic contacts between the parallel fibers and the dendrites of
Purkinje cells are called the "cross-over" synapses. The dendrites of a typical
human Purkinje cell may make as many as 200,000 synaptic contacts with
parallel fiber afferents. Also in this layer, mainly in the upper parts of the
granular layer, are larger (9-16 pm in diameter) and less numerous Golgi
cells. Dendrites of these cells extend throughout all layers of the cerebellar
cortex and are contacted by parallel fibers in the molecular layer and by
climbing and mossy fiber collaterals in the granular layer. Axons of Golgi cells
terminate on granule cell dendrites within the cerebellar glomeruli and release
GABA as their major neurotransmitter. The outermost layer, the molecular
layer, is made up primarily of the parallel fibers (bifurcating axons of granule
cells), the dendrites of cells (Purkinje cells and Golgi cells) in deeper layer and
two types of interneuron (outer stellate cells and basket cells). Dendrites of
both cells and axons of the outer stellate cells are confined to the molecular
layer. Axons of outer stellate cells make synaptic contacts with dendrites of
Purkinje cells. Basket cells, located in deep parts of the molecular layer near
the Purkinje cell bodies, give rise to dendrites that ascend into the molecular
layer and unmyelinated axons that form synaptic contacts with the somata of

many Purkinje cells. Both outer stellate cells and basket cells provide

31
inhibitory inputs to Purkinje cells. Separating the granular and the molecular

layers is the Purkinje cell layer. The dendrites of Purkinje cells extend
toward the cortical surface and divide into an elaborate dendritic tree within
the molecular layer. The dendritic tree of Purkinje cells branches profusely,
but it does so primarily in a single plane perpendicular to the long axis of the
folium and thus to the course of the parallel fibers passing though it. The
axons of Purkinje cells are the only output that leaves the cerebellar cortex.
Purkinje cell axons are myelinated, pass though the granular layer and white
matter, and make synaptic contacts with the deep cerebellar nuclei. Most
Purkinje cells contain GABA as their principal neurotransmitter to modulate
activity of the deep cerebellar nuclei.

The major inputs to the cerebellar cortex are the Mossy Fibers and
Climbing Fibers. The mossy fibers originate from many sources, including
the cerebral cortex via the cortico-ponto-cerebellar pathway, the vestibular
sense organs via the vestibular nuclei and vestibulocerebellar tracts, spinal
cord and the reticular formation. Mossy fibers branch when they enter the
cerebellum and send axon collaterals both to the deep cerebellar nuclei and to
the cerebellar cortex. In the cerebellar cortex, mossy fibers lose their myelin
sheath and form excitatory synaptic connections with granule and Golgi cells.
The dendrites of granule cells constitute the postsynaptic element. Golgi cells
function as a negative feedback to the mossy fiber-granule cell relay. The

mossy fibers constitute the principal mode of termination of most cerebellar

32

afferent systems. N 0 candidates have been clearly identified as the
neurotransmitter released from the mossy fibers (Shepherd, 1990). However,
recent evidence indicates that glutamate may be at least one of the
neurotransmitters released from the mossy fibers (Garthwaite and Brodbelt,
1989; Traynelis et al., 1993; D’Angelo et al., 1995; Silver et al., 1996). The
second major input to the cerebellum is the climbing fibers that arise in the
contralateral inferior olive nucleus of the medulla. The main inputs to the
inferior olive are from the spinal cord, the brainstem, the cerebellar nuclei and
the motor cortex. The climbing fibers pass through the granular and Purkinje
cell layers and make excitatory synaptic contacts directly with Purkinje cell
dendrites as they ascend in the molecular layer. A single Purkinje cell receives
terminals from only one climbing fiber axon. However, a given inferior olivary
cell axon may branch to form several climbing fibers and each climbing fiber
can make as many as 200 synapses with each Purkinje cell. Stimulation of a
group of climbing fibers produces a powerful excitation of Purkinje cells. When
a climbing fiber discharges, the Purkinje cell also discharges. However,
stimulation of climbing fibers not only excites Purkinje cells, but also excites
a number of Golgi cells, which then inhibit granule cells and the inputs from
the mossy fibers to granule cells. By this feedforward inhibition, when
climbing fibers fire, their targeted Purkinje cells are dominated by these
climbing fiber inputs. In short, in cerebellar cortex, a single Purkinje neuron

receives two major excitatory inputs: mossy fiber-parallel fiber-Purkinje cell

33

pathway and climbing-Purkinje cell pathway. The mossy fibers presumably
release glutamate and/or other transmitter to excite granule cells, and in turn
the axons (parallel fibers) of granule cells release glutamate as their principal
transmitter to excite Purkinje cells. Simultaneously, parallel fibers also
activate the outer stellate and basket cells, which in turn modulate Purkinje
cell excitability, and Golgi cells to produce feedback inhibition of granule cells.
The climbing fibers presumably release glutamate/aspartate as their
transmitter to excite Purkinje cells. Unlike the majority of neurons, the
Purkinje cells generate two types of responses to these two excitatory synaptic
inputs (Anderson, 1989; Shepherd, 1990). One is a simple, single-peaked
action potential, the simple spike or somatic spike, and the other is a
multipeaked action potential, the complex spike or dendritic spike. It has
been shown that the simple spikes are produced by activation of the mossy
fiber-granule cell-parallel fiber pathway, whereas complex spikes are produced
by activation of climbing fibers. The complex and simple spikes result from
voltage-gated Ca2+ and Na“ channels that are distributed along different parts
of the cell membrane. In cerebellar slice, it has been shown that action
potentials generated in the soma-initial segment region are Na*-dependent
(Llinas and Sugimori, 1980a; Anderson, 1989, Shepherd, 1990; Stuart and
Hausser, 1994) and are blocked by tetrodotoxin (TTX), whereas those
generated in the dendrites are Ca2*-dependent and resistant to TTX (Llinas

and Hess, 1976; Llinas and Sugimori, 1980a,b; Kimura et al., 1985; Ross and

34

Werman, 1987; Llinas and Walton, 1990). Climbing fibers produce a large,
synchronous depolarization of the dendrites, thus they activate the dendritic
Ca2+ channels and initiate action potentials there to generate the complex
spikes. Parallel fibers, however, each produce a small synaptic current that
must sum at the initial segment to produce an action potential. As a
consequence, parallel fiber input leads to N a’-dependent simple spikes whose
frequency is graded as a function of the summed synaptic current from many
parallel fiber synapses. However, a recent study showed that TTX-sensitive
Na” channels are also present in Purkinje cell dendrites at a density high
enough to produce Na+ action potentials and synaptic inputs to the dendrites
via either parallel fibers or climbing fibers can elicit N a+ action potentials in
these dendrites (Regehr et al., 1992). On the other hand, studies also showed
that in Purkinje cells, even single synaptic climbing fiber-mediated responses
produce spike-like increases in [Ca2+]i not only in dendrites but also in a
narrow somatic submembrane shell (Ellers et al., 1995). The sole output of
cerebellar cortex is the axon of Purkinje cell, which projects on the deep nuclei

to modulate ongoing activity of these nuclei.

D. Preparation of brain slices.
1) Preparation of hippocampal slices.
Hippocampi of both sides are isolated quickly from brains of male

Sprague-Dawley rats (180-220 g) after decapitation. One hippocampus is then

35

placed on the platform of the tissue chopper and sectioned transversely to the
longitudinal axis of hippocampus to slices of approximately 400 um thickness
using methods described by Teyler (1980). Slices were transferred immediately
to a recording chamber, where they were superfused continuously with
oxygenated ACSF. The composition of ACSF (in mM): NaCl, 124; KCl, 5;
MgSO4, 2; KHZPO4, 1.25 ; NaHCOa, 26; CaClz, 2 and D-glucose, 20. The pH of
ACSF was adjusted to 7.4 using HCl after solution was bubbled with 95% 02'
5% CO2 for 15-30 min. A humidified gas mixture of 95% O2/5% CO2 was
circulated over the slices by bubbling in water. Slices were incubated in the
recording chamber for at least 60 min before electrophysiological recording

began.

2) Preparation of cerebellar slices.

Cerebellar slices were prepared using methods modified slightly from
those of Llinas and Sugimori (1980a); Crepel et al. (1981); Kimura et al. (1985);
Edwards, et al., 1989; Konnerth et al. (1990); Llano et al. ( 1991); Momiyama
and Takahashi (1994) and Mintz et al. (1995). In general, the process of
preparation of cerebellar slices is similar to that of preparation of hippocampal
slices. In brief, the cerebellum was removed quickly from the brain of
Sprague-Dawley rat (15 - 20 days postnatal) and immersed immediately in cold
oxygenated modified ACSF, containing 125 mM NaCl; 2.5 mM KCl; 1 mM

MgC12; 1.25 mM KHZPO4; 26 mM NaHCO3; 2 mM CaCl2 and 20 mM D-glucose

36

(pH 7.4) for 1 - 3 min. A portion of vermis (for sagittal slices), isolated by two
sagittal cuts, or the whole cerebellum (for transverse slices) was glued on the
tissue pedestal of an OTS-3000-05 Automatic Oscillating Slicer (FHC,
Brunswick, ME 04011) with cyanoacrylate glue. The tissue block was then
transected either sagittally or transversely depending on the purpose of an
individual experiments (Figure 2.3). The thickness of cerebellar slices was
about 300-350 um for conventional extracellular and intracellular recording.
Sometimes, 200 um sagittal slices were used for the purpose of identifying
whether or not the parallel fiber-Purkinje cell pathway contaminated
recordings of effects of MeHg on responses of climbing fiber-Purkinje cell
synaptic transmission. One cerebellar slice was transferred to the recording
chamber and the remaining slices were incubated in a holding chamber for
later use. The entire process from decapitating the rat to transferring the
slices into the recording or holding chamber was usually finished in less than
10 min and under 4 °C. The slice in the recording chamber was incubated and
superfused (1 - 1.2 ml/min) continuously with modified ACSF saturated with
95% O2 /5% CO2 for at least 60 min before electrophysiological recordings

begin.

37

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E. Methods for electrophysiological studies of synaptic transmission in
brain slices.

Electrical stimulation of neurons usually generates two main types of
signals that are either gradual changes in the membrane potentials (EPSPs)
or all-or-none spikes (action potentials). These electrical signals can be
recorded by placing a recording electrode close to a cell or a cell population
(extracellular recording) or by penetrating the cell membrane with a sharp
fluid-filled glass electrode (intracellular recording) or by forming gigaohm seal
of a fire-polished fluid-filled glass electrode onto the plasma membrane of an
intact cell and subsequently rupturing the membrane to gain access to the cell
interior (whole-cell patch-clamp recording). Each of these recording techniques
has been successfully applied to the brain slice preparations (Mfiller, 1992;

Henderson, 1993).

1) Extracellular recording.

Extracellular recording refers to recording potential change across cell
membrane without penetrating the cell with a microelectrode. The
extracellular signal is generated due to (1) the conductance or resistance of the
extracellular fluid is not zero; (2) a cell whose membrane potential is changed
non-uniformly so that one part of the membrane is depolarized more than
another will have current flow within it. Corresponding to the intracellular

current, there is certainly a flow of current in the extracellular media to

40

complete the current path. The extracellular current is then can be picked up
with an extracellular recording electrode. In general, extracellular signals are
very small and therefore require extensive amplification. Since the signals
recorded by extracellular recording electrodes are responses of a single neuron
or population of neurons, larger responses can be generated only when the
firing of many neighboring neurons is highly synchronized and the dipole
orientation of these cells is uniform. The responses of extracellular recording
are referred to as field potentials or population spikes. The amplitude of
population spikes is proportional to the number of neurons firing. Usually,
extracellular recording electrodes can pick up several signal components, these
indicate the presynaptic volley corresponding to afferent spike activity,
summed fEPSPs, and population spikes. The shape and polarity of field
potentials in a given slice structure may vary depending on the location of the
recording electrodes. Extracellular recording may sometimes be sufficient to
answer general questions about effects of chemicals on synaptic transmission
in brain slices, depending on the aims of the experiments. For these
experiments, borosilicated glass microelectrodes having impedances of 5 - 10
MD when filled with either ACSF or 3 - 4 M NaCl (K+ is avoid to prevent nerve
depolarization) are generally used as extracellular recording electrodes. A
bipolar or monopolar tungsten electrode is used as the stimulating electrode.
The activity of pyramidal cells in the cortical structure is well suited for

extracellular recording. In hippocampal slices, extracellular recordings can be

41

made in any regions of CA field or dentate gyrus. Figure 2.4A,B
representatively demonstrates the fEPSPs and population spikes recorded from
the apical dendritic and cell soma layers, respectively, of the CA1 pyramidal
cells of hippocampal slice by stimulation of Schaffer collaterals. Extracellular
recordings can be also made in the molecular layer of the cerebellar cortex to
record activities of parallel fiber-Purkinje cell synapses or climbing fiber-
Purkinje cell synapses (Figure 2.4C,D). The major advantage of extracellular
recording is that it is relatively easy to make stable recordings of long
duration. The main disadvantages of extracellular recording are (a) since the
very weak extracellular signals require higher amplification, the noise levels
will be also likely amplified. Therefore it is important to use low-impedance
electrodes, a low-noise amplifier, and good shielding of the recording system;
(b) the technique cannot provide information of changes in some specific
responses such as resting membrane potentials and input resistance; and (c)
it is nearly impossible to compare amplitudes of field potentials from different
experiments, because the overall amplitudes of responses are influenced by the
location of recording electrode. Amplitudes of population spikes may vary
greatly in different slices of the same animals, in different locations of
recording electrodes in the same slice or even in the same region of the same

slice but with different depths of the electrode tips in tissue.

42

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2) Intracellular recording.

Unlike extracellular recording, an intracellular recording electrode
penetrates the cell membrane to measure transmembrane potential response
of a single neuron. Therefore it is possible to measure changes in the electrical
behavior including resting membrane potentials, input resistance, whole cell
capacitance and action potentials of individual neurons. It is also possible to
do quantal analysis of synaptic transmission, estimate the reversal potentials
of a synaptic response and identify if a given chemical affects functions of the
presynaptic nerve terminals. Moreover, equipped with a bridge circuit in the
intracellular recording amplifier, one can inject DC current into cell through
the same recording electrode to change the resting membrane potentials or
directly excite the targeted cell. This allows me to analyze the site and
mechanism of action of a given chemical on individual neurons. The electrodes
used for intracellular recording usually are sharp with impedance of 60 — 120
M52 when filled with 3 M potassium acetate (01' is avoid to prevent diffusion
of large amount of 01' into cell). In my experiments, the impedances of
recording electrodes were 80 - 120 and 60 - 80 M9, respectively, for the
pyramidal cells in the CA1 region of hippocampal slices and for Purkinje cells
in cerebellar slices. The disadvantages of intracellular recording are (a) it is
difficult to obtain a proper cell penetration and maintain it for a long duration
in the small neurons; (b) before penetration of a cell, even during recording,

the bridge balance and electrode capacitance must be properly adjusted and

45

compensated, otherwise all measurements will be inaccurate; (c) resting
membrane potentials are influenced by the electrode tip potentials, and the tip
potentials are influenced by the ionic microenvironment, which can be changed
after penetration of a cell or by test chemicals. Thus, an exact measurement
of the true membrane potential may not be possible, and (d) penetration of cell
may cause some damage to the cell membrane and hence lead to an unstable
recording. Figure 2.5 representatively shows EPSPs and action potentials
recorded from a CA1 pyramidal cell of a hippocampal slice (A, B and C) and
from two Purkinje cells of a transverse (D) and a sagittal cerebellar slices (E
and F).

3) Sharp single-electrode voltage-clamp (sSEVC) recording.

Similar to the conventional intracellular recording or current-clamp
recording, the sSEVC recording also uses a sharp microelectrode to penetrate
the cell membrane. However, the response recorded from individual neurons
by sSEVC recording is current instead of voltage. In sSEVC the single
intracellular microelectrode functions as both the voltage-recording and
current-passing electrode, usually with a duty cycle of 70% voltage recording
and 30% current passing, i.e. the two functions are time-shared and do not
interact (Figure 2.6). The success of a sSEVC recording depends on several
factors. First, the electrode resistance for sSEVC recording should be as low
as possible, but it should not sacrifice the consistency of successful

intracellular penetrations. Second, the capacitance neutralization should be

46

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Figure 2.6. Schematic diagram of the principle of sharp single-electrode voltage
clamp technique. Top circuit: A recording microelectrode (R1) penetrates the cell
(Cell). Voltage (V9) recorded by R1 is buffered by a unitary gain headstage (A1). Ve
is the sum of the membrane potential (Vm) and the voltage drop developed on the R1
resistance and capacitance by the applied current. A sample-and-hold circuit (SHl)
sample Ve at the times indicated by the arrows and hold the values (Vm) for the rest
of the cycle. The sampled potential is compared with the command potential (Vc) in
a differential amplifier (A2). The putput of this amplifer becomes the input of a
controlled current source (CCS) if the switch (S1) is in the current pasing position.
This circuit has a transconductance GT and it injects a current Im into the R1 which
is directly proportional to the voltage at the input of the CCS irrespective of the R1
resistance. Bottom: diagrammatc illustration of the period of applied current
injection. S1 is shown in the current-passing position during which a square pulse of
current is injected into R1. The rate of rise of the electrode voltage is determined by
the R1 resistance, the R1 capacitance, and the A1 input capacitance. S1 then switchs
to the voltage recording position (input to CCS is 0 V). Im become zero and V8 decays
passively. The V6 decays towards zero with a time constant determined by R1
resistance and total parasitic capacitance, while Vm decays towards ist resting level
with a time constant determined by the neuronal membrane. Sufficient time must be
allowed for Ve to reach within a millivolt or less of Vm. This requires the R1 time
constant to be at least an order of magnitude smaller than the cell time constant. At
the end of the voltage recording period a new sample of Vm is taken and a new cycle

begins. (Modified from Redman, 1992).

49

 

 

 

 

 

 

 

 

 

 

 

 

5O

properly adjusted to ensure that the microelectrode voltage decays to
membrane potentials Within the time allotted in each cycle for passive
recording. Third, the sampling frequencies should be as high as possible in
order to maintain stable sSEVC recording. If the sampling frequency is too
low, the sSEVC recording will be unstable due to that long periods of current
passing between sample allows the membrane potential to overshoot the
command potential to result in larger error signals and larger currents with
each cycle. In addition, the gain and the anti-aliasing filter of voltage-clamp
circuit should be also adjusted properly. Compared with the conventional
intracellular recording or current—clamp, the sSEVC does not have the problem
of non-linear summation of the postsynaptic responses to neurotransmitter.
Normally, as the stimulus intensity applied to the presynaptic fibers is
increased, the amounts of transmitter release are increased. In current-clamp,
however, the amount of potential changes evoked in the postsynaptic neurons
do not increase linearly with the amounts of transmitter changes; as the
potential changes in response to the transmitter release, it moves towards the
equilibrium potentials for the response and thus reduces the driving force for
subsequent potential change as transmitter release increases. In voltage-
clamp mode, the current flow through the membrane is measured in response
to neurotransmitter under conditions in which the membrane potential and
thus the driving force are held constant completely. Compared with the two-

electrode voltage-clamp technique, which is somewhat impractical for using in

51

brain slices due to the small size of most CNS neurons and the difficulty in
visualizing individual cell bodies in brain slices, sSEVC recordings can be
applied to any neurons that are suitable for conventional intracellular
recordings. Compared with the whole-cell patch recording, sSEVC has the
major advantage of not suffering an error due to voltage drop across the
electrode resistance. On the other hand, however, the sSEVC is much harder
to set up than whole-cell patch recording and requires frequent fine-adjustment
of the controls as the microelectrode resistance drifts. Also, you generally
cannot clamp very fast responses or very large responses. In addition, the
amount of noise in sSEVC is about two to three times greater than that in
whole-cell patch-clamp recording.

4) Whole-cell patch recording.

The patch-clamp technique is one of the electrophysiological methods
that allows to record macroscopic whole-cell or microscopic single-channel
currents flowing cell membranes through ion channels, including voltage-gated,
receptor-gated and second-messenger-activated channels. Generally, the patch-
clamp technique refers to both voltage-clamp and current-clamp measurements
using microelectrode with lower impedance (usually a few MQ) when filled
with appropriate internal solutions. Voltage-clamp measurement refers to the
technique that allows the investigators to study the voltage-dependence of ion
channels by experimentally manipulating the voltage across the patch or whole

cell membranes, while current—clamp measurement refers to the technique that

52

allows one to monitor membrane potential changes by experimentally
controlling currents flowing across ion channels.

The patch-clamp recording technique has been widely used to study
synaptic transmission in brain slices (Blanton et al., 1989; Edwards et al.,
1989; Honnerth, 1990; Stuart, 1993; Blitzer and Landau, 1994; Sakmann and
Stuart, 1995; Plant et al. , 1995), because it offers many advantages by combing
the brain-slice technique with the power of the patch-clamp techniques.
Synaptic currents can be recorded in both relatively thick (individual cells not
necessarily visualized) or thin (neuron cell soma or dendrites can be visualized
with an upright microscope equipped with Nomaski optics) brain slices. To
date, three main techniques have been developed for making whole-cell patch-
clamp recording in brain slices. The "cleaning" method was first introduced by
Edwards et al., (1989). In this method, the surface tissue and cell debris over
the targeted cells in the thin (150 - 200 11M) brain slices are first teased apart
by gentle application of positive pressure to a broken tip pipette to eject a
stream of bath solution and then removed by careful suction to exposure the
targeted cell membrane. The second method is the so-called "blind" technique
introduced by Blanton et al. , (1989). This procedure is similar to that used for
conventional intracellular recording in thick brain slices (400 - 500 11M) and no
specific optics, physical cleaning or enzymatic treatment of tissue are required.
When the recording patch electrode tip contacts the cell membrane, as

indicated by an increase in apparent resistance, as the pipette advances

53

through the slice, a slight negative pressure is applied to the recording
electrode to form a gigaseal and whole-cell recording. The third technique is
called the " blow and seal" technique (Stuart et al., 1993), and is a hybrid of
the first two methods. In this procedure, the surface neuropile over the
neurons to be recorded from is cleaned by gentle application of positive
pressure to the recording electrode, similar to the "blind" technique, however,
the advancement and placement of recording electrode in brain slices is
performed under visual control as in the "clean" procedure. Whole—cell patch
recording, using the continuous single-electrode voltage-clamp technique, has
several advantages over the sSEVC. First, whole-cell patch-clamp recording
significantly improves the signal-to-noise ratio. Thus, it is better suited for
recording relatively small amplitude events such as spontaneous miniature
EPSCs than are conventional intracellular recording and sSEVC recording
(Henderson, 1993; Blitzer and Landau, 1994). Second, in whole-cell patch
recording, access to the cell interior is much greater. Thus, relatively large
molecules can enter the cytosol by diffusion from the electrode, permitting the
design of experiments involving the intracellular injection of proteins and
peptides. Third, the low-resistance recording electrodes used in whole-cell
recording are capable of passing larger amounts of currents than are sharp
electrodes, especially depolarizing currents. The major problems associated
with whole-cell patch recording are the series resistance and response

rundown. The access resistance is in series with the membrane. Any current

54

passing through the pipette will induce a voltage across this series resistance
and thus an error in the voltage clamp. The contribution of the access
resistance to total series resistance can introduce a substantial error when
whole-cell recording in the slice. Therefore, a proper series resistance
compensation is required to minimize this error. The rundown or gradual
decrease of responses in the whole-cell recording configuration is due to
dialysis of the intracellular solution and loss of energy sources, second
messenger components or cofactors necessary for normal physiological
functions. To minimize rundown of responses, some specific substances such
as ATP and GTP are often added to the electrode internal solution.

Using these electrophysiological recording techniques described above
except the whole-cell patch recording, I examined the effects of MeHg on

central synaptic transmission in hippocampal and cerebellar slices.

CHAPTER THREE

METHYLMERCURY ACTS AT MULTIPLE SITES TO BLOCK

HIPPOCAMPAL SYNAPTIC TRANSMISSION

55

56
ABSTRACT

To explore the mechanisms by which MeHg blocks central synaptic
transmission, intracellular recordings of action potentials and resting
membrane potentials were made in CA1 neurons of rat hippocampal slices. At
4 - 100 11M, MeHg blocked action potentials in a concentration- and time-
dependent manner. MeHg also depolarized CA1 neuronal membranes.
However, this effect occurred more slowly than did block of action potentials
because the resting membrane potentials remained unchanged when threshold
stimulation-evoked action potentials were blocked. Thus, MeHg may initially
alter the threshold level of neuronal membrane excitability and subsequently
depolarize the membrane leading to block of synaptic transmission. To identify
potential sites of action of MeHg, effects of MeHg on the responses of CA1
neurons to orthodromic stimulation of Schaffer collaterals, antidromic
stimulation of the alveus, direct injection of current at cell soma and
iontophoretic application of glutamate were compared. At 20 and 100 uM,
MeHg blocked action potentials evoked by stimulation of Schaffer collaterals
and by current injection at the cell soma at similar times. In contrast, action
potentials evoked by stimulation of the alveus were blocked more rapidly by
100 uM MeHg than were action potentials evoked by current injection at CA1
neuronal soma. MeHg also blocked the responses of CA1 neurons to

iontophoresis of glutamate, but time to block of these responses was slower

57

than block of the corresponding orthodromically—evoked responses by
stimulation of Schaffer collaterals. Compared to EPSPs, inhibitory
postsynaptic potentials (IPSPs) appeared to be more sensitive to MeHg,
because block of IPSPs occurred prior to block of EPSPs. Thus MeHg

apparently acts at multiple sites to block central synaptic transmission.

‘9'”

58
INTRODUCTION

The neurotoxicant MeHg disrupts sensory and motor functions following
both acute and chronic exposure (Chang, 1980). Mechanisms responsible for
these actions have been studied intensively at vertebrate peripheral synapses
and in cells in culture using acute administration of MeHg (Juang and
Yonemura, 1975; Juang, 1976; Atchison and Narahashi, 1982; Shafer and
Atchison, 1991; 1992). However, considerably less is known of the mechanisms
by which MeHg acutely alters central synaptic function. At the neuromuscular
junction, MeHg primarily affects presynaptic mechanisms to disrupt
transmission (Atchison and N arahashi, 1982; Atchison, 1986; 1987; Traxinger
and Atchison, 1987a; 1987b; Shafer and Atchison, 1992). In hippocampal
slices, however, results obtained from extracellular microelectrode recordings
suggest that MeHg may act at multiple sites to block central synaptic
transmission (Yuan and Atchison, 1993; 1994). Acute bath application of
MeHg caused concentration- and time-dependent block of the population
spikes, fEPSPs and antidromically-activated population spikes recorded from
CA1 neurons of hippocampal slices. The characteristics of block of these field
potentials by MeHg differed somewhat in terms of the time courses and degree
of reversibility. It was suggested that MeHg disrupted central neuronal
membrane excitability and synaptic transmission by both presynaptic and

postsynaptic mechanisms. In dorsal root ganglion cells, MeHg suppressed the

GABA-induced chloride current (Arakawa et al.,1991). Thus, in addition to

59

excitatory systems, MeHg may also act on central inhibitory nerve systems to
alter central synaptic transmission.

Because of the limitations of extracellular recording techniques, it is
difficult to specify where and how MeHg acts to block central synaptic
transmission. Thus, to explore the mechanisms underlying these effects of
MeHg, intracellular microelectrode recordings and iontophoresis techniques
were applied at CA1 neurons of hippocampal slices to examine directly the
effects of MeHg on synaptic and action potentials, resting membrane potentials
and responses of CA1 neurons to the excitatory amino acid neurotransmitter-
glutamate. We sought to determine: 1) whether or not MeHg affects neuronal
membrane excitability or alters the threshold for neuronal excitation; 2) which
is (are) the primary site(s) of actions of MeHg to block synaptic transmission;
and 3) whether or not inhibitory synaptic transmission was also affected by

MeHg.

60
MATERIALS AND NIETHODS

Materials. Methylmercuric chloride, purchased from ICN Biomedical,
Inc. (Costa, CA), was dissolved in deionized water to a final concentration of
5 mM to serve as stock solution. The applied solutions (4 - 100 uM) were
diluted with ACSF. MeHg was applied acutely to slices by bath application at
a rate of 1.5 ml/min with a Gilson infiision pump. L-glutamic acid was

purchased from Sigma Chemical Co. (St. Louis, MO).

Preparation of hippocampal slices. Hippocampal slices were
prepared using method described previously in Chapter Two. When recording,
one or two slices were kept in the chamber at a given time. The rest were
maintained in a reservoir chamber for later use. All experiments were

conducted at 33 - 35 0C. One slice per rat was used.

Electrophysiological procedures. Conventional intracellular
recordings were made in the CA1 neurons of hippocampal slices. 3 M0
monopolar tungsten electrodes (FHC, Brunswick, ME) were used as
stimulation electrodes. Borosilicated glass microelectrodes (o.d. 1.0 mm; id 0.5
mm, WPI, Inc., New Haven, CT) having impedance of 80 - 120 MO when filled
with 3 M potassium acetate were used as recording electrodes. Action

potentials were evoked orthodromically by stimulating Schaffer collaterals,

61

antidromically by stimulating the alveus, or directly by injection of positive
current into CA1 pyramidal cell soma through the recording electrode at
threshold levels (Figure 3.1). The latency to onset of action potentials evoked
by threshold stimulation of Schaffer collaterals was measured as the time
interval between the stimulus artifact and the peak of the action potential,
because the rate of rising phase of action potentials is essentially unchanged
at the earlier stage of exposure to MeHg. Intracellular EPSPs were recorded
at CA1 pyramidal cell soma by stimulation of Schaffer collaterals at a level
that did not evoke action potentials. Typically a 0.1 - 0.2 nA negative DC.
current was applied constantly through the recording electrode to maintain the
cell membrane in a somewhat hyperpolarized state and avoid evoking action
potentials. The recurrent inhibitory postsynaptic potentials (IPSPs) (Dingledine
and Gjerstad, 1979; Collingridge et al., 1988) were recorded by subthreshold
stimulation of the alveus. The membrane input resistance was examined by
DC. current injection through the recording electrode. The stimulus pulses
were generated from a Grass S88 stimulator (Grass, Inc., Quincy, MA) at a
frequency of 0.15 Hz and 0.1 msec duration and isolated with a Grass SIU5
stimulus isolation unit (Grass, Inc. Quincy, MA), which was also used to
change the polarity of stimulus pulses. For iontophoretic application of L-
glutamate, a third electrode (50-100 MQ) filled with 500 mM glutamate in 100
mM NaCl (pH 8.0) was positioned at the apical dendrites of CA1 neurons.

Glutamate was ejected by passing a 20 -100 nA negative current for 30 - 40

62

msec with a retaining current of 0 - 5 nA through the electrode. Signals from
recording electrodes were amplified (Axoclamp-Z, Axon Instruments, Inc.,
Burlingame, CA), displayed on a 2090-3 digital oscilloscope (Nicolet
Instruments, Madison, WI) and stored simultaneously on floppy disks and
magnetic tape by using an FM instrumentation recorder (Model B, Vetter
Instruments, Rebersburg, PA) at a speed of 7-1/2 inch per second for later
analysis. Only those cells in which the amplitude of action potentials was
greater than 60 mV, resting membrane potentials were ~55 mV or more
negative, and membrane input resistances were above 20 M9 were used for

analysis.

Data analysis. Data were collected continuously before and during
application of MeHg and analyzed statistically using Student’s paired t-test or
a one-way analysis of variance and Dunnett’ procedure for post-hoe
comparisons (Steel and Torrie, 1980) unless specified. Differences between

values for comparisons were considered statistically significant when p < 0.05.

63

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RESULTS

Effects of MeHg on action potentials and resting membrane
potentials. Acute bath application of 4 - 100 uM MeHg caused a
concentration- and time- dependent block of action potentials recorded at CA1
neurons of hippocampal slices (Figure 3.2 and Table 3.1). Time courses of
effects of 4 -100 uM MeHg on action potentials are illustrated in Figure 3.2.
At 100 11M, MeHg suppressed generation of action potentials evoked by
threshold stimulation of Schaffer collaterals within 20 min. At that time,
increasing stimulation intensity slightly could again initiate action potentials,
however upon returning the stimulation intensity to its original value, action
potentials again disappeared. Five min later, action potentials evoked at the
increased level of stimulation disappeared again. By continuously increasing
stimulation intensity, action potentials could initially still be elicited in the
presence of MeHg. However with continued exposure to MeHg, eventually
action potentials could no longer be evoked regardless of the level of
stimulation. Similar effects were observed in slices exposed to 20 and 4 uM
MeHg, but time to block of action potentials was much longer. Figure 3.2 also
demonstrates that at the time action potentials disappeared, EPSPs were still
observable, suggesting that synaptic transmission may still be functionally
intact at this moment, but the threshold for initiating action potentials was

altered. The mean time to block of action potentials evoked at the threshold

66

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and maximum level of stimulation are summarized in Table 3.1. At 4 11M
MeHg, only 4 of 6 slices exhibited complete block of action potentials within
180 min. However, if exposure of slices to 4 uM MeHg was continued for
longer times, action potentials in the remaining slices would be expected to be
blocked as well. At the time action potentials evoked by threshold stimulation
were blocked, in most cases the amplitudes of action potentials just before
block remained essentially unchanged (Table 3.2). However, with continued
exposure to MeHg and gradual depolarization of membranes (Figure 3.3), the
amplitudes of action potentials declined progressively until eventually they
disappeared completely. For a given stimulus intensity, the latency to onset
of action potentials was gradually prolonged after exposure to MeHg. At 100
11M MeHg, the latency to onset of action potential generation just before block
of action potentials evoked by threshold stimulation of Schaffer collaterals was
2.0 :t 0.8 msec longer than those of the pre-treatment control (5.8 i 0.5 msec).
The shape or rates of rising phase of action potential spike were usually
unchanged at the same time (Figure 3.2). However, at the late stage of
exposure to MeHg, spikes usually arise directly from the base line (resting
membrane potential) level, and often two or more spikes appeared
simultaneously in response to a single stimulus. Spontaneous neuronal
activity seemed less sensitive to the effects of MeHg, since in many slices even
after evoked action potentials were blocked, the spontaneous spikes remained

observable, but often occurred accompanying depolarization of the neuronal

70

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71

membrane. This suggests that the initial effects of MeHg on action potentials
are due to reduced neuronal excitability or altered threshold for initiating
action potentials and the late effects may be due to nonspecific effects of
membrane depolarization. However, after action potentials were blocked
completely, injection of DC. current to return resting membrane potentials to
their original values generally did not restore the action potentials. Thus,
MeHg-induced block could not simply be ascribed to membrane depolarization.

MeHg also caused concentration- and time-dependent depolarization of
CA1 neuronal membranes (Figure 3.3). At 100 11M, MeHg depolarized CA1
neuronal membranes to 55 % of control at 60 min and to almost 0 % of control
by 90 min. At 120 min, 20 uM MeHg depolarized membranes to 81 % of
control, whereas 4 pM caused a slight hyperpolarization that was not
statistically significant. However, if exposure of slices to 4 1.1M MeHg was
prolonged to 180 min, membranes also gradually depolarized to 72 % of control
(results not shown in Figure 3.3). Most slices exposed to 4 - 100 uM MeHg
showed a slight hyperpolarization prior to depolarization, which is masked in
Figure 3.3 by averaging due to the variation of time courses among individual
experiments. Thus, the maximum hyperpolarization was averaged and
compared from each individual experiment instead of the time-dependent
averages shown in Figure 3.3. In all six slices exposed to 4 pM MeHg, the

average maximum hyperpolarization of membranes was 115 i 6 % of control

72

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74
(p < 0.05). In 12 of 15 slices exposed to 20 11M MeHg and 16 of 22 slices

exposed to 100 uM MeHg, a slight hyperpolarization was also observed prior
to depolarization. The averaged maximum hyperpolarizations were 114 :t 4 %
and 110 :t 4 % of their control for 20 and 100 11M MeHg, respectively. These
also differed significantly from their own pre-treatment control (Figure. 3.4).
During hyperpolarization of the membrane, input resistance was reduced from
33 :1: 4 M9 to 21 :t 1 M!) (p < 0.05). However during depolarization, changes
in input resistance were inconsistent, even though many cells showed an
increase in membrane input resistance at the late stage of exposure to MeHg.
Thus, MeHg typically caused biphasic changes in resting membrane potentials.
In contrast to the actions of MeHg on action potentials, the effects of MeHg on
the resting membrane potentials occurred relatively slowly. At the time that
action potentials evoked by threshold stimulation were blocked by 20 and 100
11M MeHg, the resting membrane potentials remained essentially unchanged
(Table 3.2).

The effects of MeHg on population spikes were at best only partially
reversible (Yuan and Atchison, 1993; 1994). To examine whether or not the
effects of MeHg on action potentials and resting membrane potentials are
reversible, we washed slices with 1 mM D-penicillamine, a MeHg chelator,
after action potentials evoked by threshold stimulation of Schaffer collaterals
were blocked by 100 uM MeHg. Only one of eight slices demonstrated recovery

of the action potential and two of eight slices showed a recovery of resting

75

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membrane potentials. In the remaining slices, washing did not restore action
potentials and also did not prevent membrane depolarization to progress.
Thus, it appears that the effects of MeHg on action potentials and resting
potentials were generally irreversible; this is consistent with the results
obtained from extracellular recordings (Yuan and Atchison, 1993; 1994).

At the superior cervical ganglion of the rabbit, increasing bath Ca2+
concentration from 2.2 to 6.6 mM delayed the onset and slowed the progression
of block of compound action potentials by 20 uM MeHg (Alkadhi and Taha,
1982). However, in the isolated phrenic nerve-hemidiaphragm preparations of
the rat, elevating [Ca2+]e did not significantly change the latency to block by
MeHg of the end-plate potentials (Atchison et al., 1986; Traxinger and
Atchison, 1987a). Because of the importance of Ca2+ currents in dendritic
excitability in the hippocampus (Wong, et al., 1979; Spruston, et al., 1995;
Magee and Johnson, 1995), we sought to determine if increasing [Ca2+]e would
alter the latency to MeHg—induced block of action potentials. To do this, we
compared time to block of action potentials by 20 and 100 uM MeHg in ACSF
containing 2 or 6 mM Ca2+. In ACSF containing 2 mM Ca2+, 20 and 100 uM
MeHg blocked action potentials evoked by threshold stimulation at 107 :t 34
and 22 :1: 7 min and by maximum stimulation at 176 :t 28 and 63 j; 7 min,
respectively. In ACSF containing 6 mM Ca2+, the same concentrations of
MeHg blocked action potentials evoked by threshold stimulation at 92 :1: 29 and

24 :t 7 min and by maximum stimulation at 149 i 21 and 64 :t 4 min,

78

respectively (Figure 3.5). Thus, similar to the results obtained from phrenic
nerve-hemidiaphragm preparations (Atchison et al., 1986; Traxinger and
Atchison, 1987a), increasing [Ca2+]e from 2 to 6 mM did not significantly alter

the latency to MeHg-induced block of action potentials.

Sites of actions for MeHg-induced block of synaptic transmission.
To identify the primary sites of action of MeHg in blocking hippocampal
synaptic transmission, we compared the time courses of MeHg-induced block
of action potentials evoked simultaneously by maximum orthodromic
stimulation of Schaffer collaterals or by maximum antidromic stimulation of
alveus with those by maximum current injection directly through recording
electrodes at CA1 cell soma. At 20 11M MeHg, four of nine recordings
demonstrated that block of action potentials evoked by stimulating Schaffer
collaterals occurred earlier than block of action potentials evoked by current
injection at the cell soma. At 100 uM MeHg, block of synaptically—evoked
action potentials occurred slightly faster than block of current injection-induced
action potentials in only four of twelve recordings. In most slices, block of
action potentials evoked by the two methods occurred at the same time,
usually accompanying rapid depolarization of the membrane. Overall, times
to block of action potentials evoked by stimulation of Schaffer collaterals and
by current-injection at the cell soma were 136 :t 19 and 142 :1: 18 min,

respectively for 20 uM MeHg, and 47 :t 6 and 49 :t 6 min, respectively for 100

79

Figure 3.5. Effects of increased [Ca2+]e on time to MeHg-induced block
of action potentials evoked at threshold (APThr sum) and maximum (APMaul
Stun) stimulation of Schaffer collaterals. Action potentials were recorded
continuously in ACSF containing 2 or 6 Mm CaCl2 before and during
application of 20 and 100 uM MeHg. Values are the mean :I: SE of

recordings from slices of 5 - 6 rats.

 

 

81

pM MeHg. Thus there were no significant differences between the times to
block of action potentials evoked by the two methods. Similarly, no significant
difference was observed between the times to block of antidromically-generated
action potentials and of current injection-evoked action potentials at 20 uM
MeHg, even though three of five experiments did show that MeHg blocked
antidromically-generated action potentials faster than it blocked current
injection-evoked action potentials. Times to block were 164 :t 24 and 172 :t 26
min, respectively. In contrast, six of nine experiments demonstrated that 100
uM MeHg blocked antidromically-activated action potentials faster than it
blocked current injection-evoked action potentials. Times to block were 42 :t
4 and 49 :l: 5 min, respectively. This difference was statistically significant (p
< 0.05) due to the paired nature of the study (Figure 3.6). In addition, similar
to the effect of MeHg on action potentials evoked by stimulating Schaffer
collaterals, initial block of action potentials evoked by threshold current
injection at the CA1 pyramidal cell soma could be restored completely by
increasing current injection. Later, action potentials evoked by the increased
current injection were also blocked (Figure 3.7). These results further
confirmed the conclusion that the initial effects of MeHg on action potentials
are due to reduced CA1 pyramidal cell excitability or altered threshold for
initiation of action potentials.

Next we sought to determine the contribution of presynaptic and

postsynaptic mechanisms to the actions of MeHg on synaptic transmission. To

82

Figure 3.6. Comparison of times to MeHg-induced block of action
potentials evoked by different stimulation methods. Top: times to block
by MeHg of action potentials evoked by stimulating Schafer collaterals
(APSchmer) and by current injection through a recording electrode at the

cell soma (AP ); Bottom: Times to block of action potentials evoked

soma

by antidromic stimulation of the alveus (AP ) and by current injection

anti

through a recording electrode at the cell soma (AP ). Values are the

mean :1: SE of recordings from 5 - 9 rats. The asterisk (*) indicates a
significant difference between time to block of action potentials evoked

by antidromic stimulation of the alveus and current injection at cell

soma (p < 0.05, paired t-test).

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do this, we compared effects of MeHg on responses of CA1 neurons to
presynaptic electrical stimulation of Schaffer collaterals and to direct
iontophoretic application of glutamate onto their apical dendrites
simultaneously in the same cell. At 100 uM, MeHg blocked action potentials
evoked by maximum stimulation of Schaffer collaterals at 55 i 7 min and
blocked iontophoresis-evoked responses at 63 :1: 9 min. This difference was
statistically significant (p < 0.05, Figure 3.8). Responses of CA1 neurons to
iontophoresis of glutamate usually disappeared after membrane potentials
were depolarized below -40 mV. At this point, however, injection of DC.
current to hold membrane potentials at -60 to -70 mV failed to reverse
responses of CA1 neurons to glutamate, suggesting that block of glutamate-
induced responses by MeHg could not be ascribed to membrane depolarization

alone.

Effects of MeHg on EPSPs and IPSPs. In the hippocampus,
inhibitory interneurons release (GABA) onto both pre- and postsynaptic sites
to control or influence excitatory synaptic transmission (Dutar and Nicoll,
1988a; 1988b; Thompson and Gahwiler, 1992; Isaacson et al. , 1993). Therefore,
if MeHg affects these inhibitory interneurons, we may expect that it also plays
a role in alteration of excitatory synaptic transmission. To test this, effects of
MeHg on both EPSPs and antidromically-activated recurrent IPSPs were

assessed. The time course of effects of MeHg on EPSPs

87

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89

or IPSPs recorded at the CA1 neurons of hippocampal slices is shown for two
separate representative cells in Figure 3.9. At 100 11M, MeHg first increased
EPSP amplitudes during the first 10 to 20 min and then gradually decreased
them to complete block at 60 min (Figure 3.9, Left). Often, action potentials
emerged superimposed on the EPSPs during that period when EPSP amplitude
was increased by MeHg (results not shown). This was not observed under
normal (pre-treatment control) conditions, since the EPSPs were activated by
stimulation of Schaffer collaterals at a level that does not evoke action
potentials and under slightly hyperpolarizing conditions. This suggests that
neuronal excitability was initially increased by MeHg. Conversely, 100 uM
MeHg reduced IPSP amplitudes relatively rapidly (Figure 3.9, Right).
Interestingly, at 10 min an increased EPSP phase could be observed prior to
the decreased IPSP in the same recording (Fig. 3.9, Right). However in
general, the time to decreased IPSP amplitudes appeared to correspond to the
time to increased EPSP amplitudes, suggesting that the increased EPSPs may
be due partially to reduced amplitude of IPSPs. The mean time to MeHg-
induced block of EPSPs and IPSPs is shown in Figure 3.10. At 100 uM, MeHg
caused complete block of IPSPs at an earlier time than EPSPs. Thus, IPSPs

may be more sensitive to MeHg than were EPSPs.

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DISCUSSION

The present study was undertaken with the objective of describing in
more detail, the cellular actions of MeHg on central synaptic activity. Results
are consistent with the following conclusions. First, at 4 -100 uM, MeHg
caused a concentration- and time-dependent block of action potentials and
depolarization of CA1 neuronal membranes in hippocampal slices. Second,
effects of MeHg on the resting membrane potentials occurred more slowly than
did effects on action potentials. Third, the time to MeHg-induced block of
action potentials evoked by orthodromic stimulation of Schaffer collaterals or
by current injection at the cell soma was not statistically significant, but block
of action potentials evoked by antidromic stimulation of the alveus occurred
faster than block of action potentials generated by current injection at the cell
soma. Fourth, MeHg also suppressed the responses of CA1 neurons to
iontophoresis of glutamate, but effects of MeHg on these responses were
relatively slow in onset, in contrast to the rapid effects of MeHg on the
responses to presynaptic electrical stimulation. Finally, IPSPs seemed to be
more sensitive to MeHg than were EPSPs.

Using extracellular recording, we demonstrated previously that MeHg
first increased and than gradually decreased to complete block population
spikes recorded from CA1 neurons of hippocampal slices. These effects were

concentration- and time-dependent (Yuan and Atchison, 1993; 1994). In the

95

present study, the time-courses of effects of MeHg on action potentials were
generally similar to those of MeHg on population spikes. However, MeHg
initially blocked action potentials evoked by threshold stimulation without any
significant changes in the action potential amplitudes, i.e. MeHg blocked action
potentials in an all or none manner rather than gradually. In addition, the
resting membrane potentials remained essentially unchanged and EPSPs were
still observable after block of action potentials. Increasing stimulation
intensity at the time of block completely restored the action potentials. These
results suggest that at the time action potentials were blocked, synaptic
transmission may remain functionally intact, but the threshold level for
neuronal excitation may have been altered by MeHg. Using voltage-clamp
techniques in squid axon, Shrivastav et al. (1976) demonstrated that 25 - 100
M MeHg caused a steady increase in the threshold for action potential
generation and eventual block of conduction without changing the resting
membrane potentials. These effects were thought to be due to suppression of
both peak Na+ current and steady-state K“ current by MeHg. Similar effects
of 20 -60 uM MeHg on peak Na+ current and steady-state K+ current were
observed in N1E-115 neuroblastoma cells (Quandt et al., 1982). Shafer and
Atchison (1992) also demonstrated that 50 - 100 uM MeHg disrupted Na“
channel function in mouse triangularis sterni motor nerves. Therefore, the
initial effects of MeHg on action potentials may be mediated by an action on

Na” channels which alters the threshold for generation of action potentials and

96

blocks current conduction. However, effects of MeHg on Ca2+ channels or
homeostasis of Caz”i may also be involved in these effects since MeHg rapidly
and effectively blocks Ca2+ channels in neurons (Shafer and Atchison, 1989;
Shafer et al., 1990; Shafer and Atchison, 1991; 1992; Hewett and Atchison,
1992; Sirois and Atchison, 1996, 1997) and disturbs homeostasis of Ca2+,
(Komulainen and Bondy, 1987; Levesque and Atchison, 1991; Levesque et al.,
1992; Denny et al., 1993; Hare et al., 1993; 1995; Marty and Atchison, 1997).
In this regard, increasing external Ca2+ concentration delayed the onset of
MeHg-induced block of action potentials in rabbit superior cervical ganglia
(Alkadhi and Taha, 1982), supporting the suggestion that effects of MeHg on
Ca2+ channels could be reversed by raising [Ca2+]e. However, our results
suggest that raising [Ca2+]e alone cannot overcome effects caused by MeHg in
hippocampal slices, a situation identical to that at the rat neuromuscular
junction (Atchison et al., 1986; Traxinger and Atchison, 1987a) and in cortical
synaptosomes (Atchison et al., 1986; Hewett and Atchison, 1992). The late
effects of MeHg to decrease amplitude of action potentials to complete block
may be due to the nonspecific effects of depolarization of neuronal membranes
(Juang, 1976a,b; Shrivastav et al. , 197 6; Quandt et al. , 1982; Kauppinen et al. ,
1989; Hare and Atchison, 1992). However, other mechanisms are probably also
involved, because injection of DC. current to hold membrane potentials around
their original values did not prevent or reverse the effects of MeHg on action

potentials.

97

At the neuromuscular junction and peripheral ganglia, 4O - 100 pM
MeHg had no effects on postsynaptic resting membrane potentials ( J uang and
Yonemura, 1975; Juang, 1976a; Atchison and Narahashi, 1982). In the present
study, 4 - 100 uM MeHg typically caused biphasic changes in the resting
potentials of CA1 neurons, i.e. MeHg first hyperpolarized and then depolarized
CA1 neuronal membranes. The hyperpolarizing effect was especially
prominent at lower concentrations of MeHg (4 - 20 uM). A similar
phenomenon was also observed in synaptosomes exposed to 1 uM MeHg
although the difference was not statistically significant (Hare and Atchison,
1992). It may be that the initial plasma membrane hyperpolarization was
caused by activation of Ca2+- sensitive K” channels as a result of MeHg-induced
elevation in [Ca2+],. In NG108-15 cells, the initial effect of MeHg on [Ca2+], is
to increase [Ca2+], due to release of Ca2+ from IP3-sensitive pool (Hare and
Atchison, 1995a). In this cell line, mobilization of this intracellular Ca2+ pool
results in membrane hyperpolarization (Higashida and Brown, 1986). A
similar phenomenon may be applicable to hippocampal slices. In fact,
decreases in membrane input resistance during membrane hyperpolarization
seem to support this possibility. Differences in the effects of MeHg on resting
membrane potentials at neuromuscular junctions and peripheral ganglia and
those reported here may be due to differences in exposure duration and
concentration of MeHg. High concentrations of MeHg (400 or 500 1.1M)

depolarize muscle fibers (Juang, 1976a,b) and squid axons (Shrivastav et al.,

98

1976), whereas prolonged exposure of tissues to low concentration of MeHg (4 -
20 pM) in this study also depolarized CA1 neuronal membranes. However,
these effects of MeHg occurred more slowly than did those of MeHg on action
potentials, suggesting that different mechanisms may be involved in the effects
of MeHg on action potentials and resting membrane potentials.

In general, effects of MeHg on both action potentials and resting
membrane potentials could not be reversed by washing slices with D-
penicillamine. This is consistent to our previous results in hippocampal slices
(Yuan and Atchison, 1993; 1994) and those of others in alternate systems
(Juang, 1976; Shrivastav et al., 1976; Alkadhi and Taha, 1982; Quandt et al.,
1982; Atchison and N arahashi, 1982; Traxinger and Atchison, 1987b).

The effects of MeHg on peripheral synapses have generally been ascribed
to be primarily presynaptic, because at relatively high concentrations (40 or
100 11M), MeHg has no effects on either action potential amplitude or resting
membrane potentials of postsynaptic muscle fibers despite blocking
neuromuscular transmission (Juang, 1976a). Moreover, responses to
iontophoretic application of acetylcholine to end-plates were unaffected by 100
uM MeHg at times that nerve-evoked end-plate potentials were blocked
(Atchison and Narahashi, 1982). However, previous results from extracellular
recordings suggest that MeHg acts at multiple sites, especially postsynaptic
sites, to block hippocampal synaptic transmission, because of differential

sensitivity of block of orthodromically- and antidromically-activated population

99
spikes compared with fEPSPs to reversal with D-penicillamine (Yuan and

Atchison, 1993; 1994). The block of fEPSPs could be completely restored by D-
penicillamine, whereas population spikes were only partially restored. A goal
of the present study was to identify further the primary sites at which MeHg
acts to block hippocampal synaptic transmission. As such we first compared
the time courses of block of action potentials evoked by orthodromic
stimulation of the presynaptic nerve fibers-the Schaffer collaterals, or
antidromic stimulation of the axons of CA1 neurons-the alveus, with the time
course of block of action potentials evoked directly by current injection through
recording electrodes at the cell soma. The rationale was that if MeHg acts
primarily on presynaptic sites, then block of orthodromically-activated action
potentials would be expected to occur earlier than block of action potentials
evoked by current injection at the cell soma, and may also differ from the time
to block of antidromically-activated action potentials. If MeHg acts primarily
postsynaptically, then the time to block as assessed by the three methods
might be similar or could differ depending on the sites and mechanisms of
actions. MeHg blocked action potentials evoked both by orthodromic
stimulation of Schaffer collaterals and by current-injection at a similar time
even though some slices showed a trend that block of action potentials evoked
by orthodromic stimulation was faster than by current-injection, suggesting
that the primary sites of action may be at the cell soma. These results are

consistent with the findings that MeHg heavily accumulated in the cell bodies

100

of CA1 pyramidal neurons, compared with other areas in hippocampus ( M¢11er-
Madsen, 1990; 1991). However, presynaptic action or block of current
conduction from apical dendrites to cell soma could not be excluded completely
based on these data, because membrane depolarization caused by prolonged
exposure to MeHg may mask the real procession of effects of MeHg. In
contrast, 100 uM MeHg blocked antidromically-evoked action potentials
significantly faster than it blocked current injection-evoked action potentials,
suggesting that current conduction from axon to cell soma may be affected by
higher concentrations of MeHg.

To identify whether presynaptic mechanisms were involved in the effect
of MeHg on synaptic transmission, we compared the latencies to block of
responses of CA1 neurons to maximum stimulation of Schaffer collaterals and
the response of CA1 neurons to direct application of glutamate on their apical
dendrites. The rationale for comparing these responses was that iontophoretic
application of glutamate directly onto the dendrites of CA1 cells could mimic
the process of synaptic activation yet bypass the presynaptic release processes.
If MeHg acts primarily at presynaptic sites to influence transmitter release,
then responses of CA1 cells to electrical stimulation of Schaffer collaterals
would be expected to be blocked, whereas responses of CA1 cells to
iontophoresis of glutamate may not be blocked or be blocked more slowly. On
the other hand, if MeHg primarily acts at postsynaptic sites, no matter where

they are, then time to block of responses of CA1 to either electrical stimulation

101

or iontophoresis of glutamate would be similar. MeHg blocked action
potentials evoked by electrical stimulation of Schaffer collaterals significantly
faster than it blocked responses to iontophoretic application of glutamate. This
suggests that MeHg may also affect transmitter release from presynaptic
terminals, albeit less prominently compared to the postsynaptic actions of
MeHg. Block of responses of CA1 neurons to either electrical stimulation of
presynaptic terminals or direct application of neurotransmitter to apical
dendrites of neurons often coincided with progressive depolarization of
membranes. Nevertheless, this effect could not be ascribed simply to
membrane depolarization because injection of current to hold membrane
potentials at approximately their original values did not reverse the responses
to either form of stimulation. Thus, mechanisms other than membrane
depolarization may be involved as well.

Block of IPSPs by MeHg preceded block of EPSPs, suggesting that
inhibitory interneurons may be more sensitive to MeHg than the CA1
pyramidal neurons. MeHg appeared first to increase EPSP amplitude prior to
block. Actually, during the time of increase in EPSPs, action potentials were
often superimposed on the EPSPs by stimulation at what had been a
subthreshold stimulus prior to MeHg exposure, suggesting that excitability was
increased. The time to suppress IPSPs generally appeared to correspond to the
time of increased amplitude of EPSPs and also corresponded to the time of

increased amplitude of population spikes obtained from previous extracellular

102
recordings (Yuan and Atchison, 1993; 1994). High concentrations of MeHg

may act directly on GABAA receptors, since 100 uM MeHg decreased the
GABA-induced Cl' current of dorsal root ganglion neurons (Arakawa et al.,
1991). Thus, MeHg may suppress GABA-induced Cl' current resulting in
decreased IPSPs and lessened inhibitory effects of interneurons on excitatory
synaptic transmission. This disinhibition, in turn, may contribute in part to
the increase in EPSP amplitude, firing of action potentials in response to a pre-
set subthreshold stimulus and occurrence of multiple spikes in response to a
single stimulus. However, both amplitudes of IPSPs and EPSPs are affected
by the resting membrane potentials. Simply hyperpolarizing the cell
membrane could result in decreased IPSP amplitude and increased EPSP
amplitude. Since recordings of EPSPs and IPSPs in the present study were
made under conditions in which the cells were allowed to be hyperpolarized or
depolarized by MeHg, it is possible that the decreased IPSPs and increased
EPSPs caused by MeHg may be due simply to the nonspecific effects of
membrane hyperpolarization. Thus, further study is required to examine the
relationship between the effects of MeHg on inhibitory and excitatory synaptic
transmission.

In conclusion, MeHg initially alters CA1 neuronal membrane excitability
and ultimately blocks hippocampal synaptic transmission. Multiple sites of
action appear to be involved. The primary sites of action of MeHg appear to

be the postsynaptic CA1 neurons, however, presynaptic mechanisms,

103

nonspecific effects of membrane depolarization and suppression of inhibitory
systems and current conduction also appear to contribute to these effects

caused by MeHg.

CHAPTER FOUR

ACTION OF METHYLMERCURY ON GABAA RECEPTOR-
MIEDIATED INHIBITORY SYNAPTIC TRANSMISSION
IS PRIMARILY RESPONSIBLE FOR ITS
EARLY STIMULATORY EFFECTS
ON HIPPOCAMPAL CA1
EXC ITATORY SYNAPTIC

TRANSMISSION

104

105
ABSTRACT

Acute bath application of MeHg (4-100 11M) causes an early stimulation

prior to block of synaptic transmission in the CA1 region of hippocampal slices.
Effects of MeHg and ng+ on IPSPs or inhibitory postsynaptic currents
(IPSCs) and EPSPs or excitatory postsynaptic currents (EPSCs) were compared
to examine whether or not early block by MeHg of GABAA-mediated inhibitory
synaptic transmission and MeHg-induced alterations of the resting membrane
potentials of CA1 neurons contribute to this initial enhancement of excitability.
MeHg affected IPSPs and IPSCs similarly, and more rapidly than EPSPs and
EPSCs. In contrast, while Hg2+ blocked IPSPs more rapidly than EPSPs, times
to block of IPSCs and EPSCs by ng+ were virtually identical when CA1
neurons were voltage-clamped at their resting membrane potential levels.
MeHg increased EPSC amplitudes prior to their subsequent decrease even
when CA1 neuronal membranes were voltage-clamped at their resting
potentials. This suggests that effects of MeHg on CA1 cell membrane
potentials are not a major factor for MeHg—induced early stimulation of
hippocampal synaptic transmission. Effects of MeHg and ng+ on the reversal
potentials for IPSCs also differed. Both metals blocked all outward and inward
currents generated at different holding potentials. However, MeHg shifted the
current-voltage (I/V) relationship to more positive potentials, while Hg” often

caused a transient and slight increase in outward currents prior to suppression

106

and shifted the UV curve to more negative potentials. ng+ was a less potent
blocker of IPSCs and EPSPs or EPSCs than was MeHg. To determine if the
early increase in amplitude of population spikes or EPSPs is due to an action
of MeHg at GABAA receptors, extracellular recordings of population spikes and
intracellular recordings of EPSPs were compared with or without pretreatment
of hippocampal slices with bicuculline. MeHg alone (20 or 100 11M) increased
and then decreased amplitudes of population spikes and EPSPs to complete
block. After pre-incubation of slices with 10 pM bicuculline for 30 - 60 min,
MeHg only decreased the amplitudes of population spikes and EPSPs to block;
no early increase of synaptic transmission occurred. Pretreatment of slices
with strychnine, did not prevent MeHg-induced early increase in population
spikes. MeHg also blocked responses evoked by bath application of muscimol,
a GABAA agonist. Thus, block by MeHg of GABAA receptor-mediated
inhibitory synaptic transmission may result in disinhibition of excitatory
hippocampal synaptic transmission, and appears to be primarily responsible

for the initial excitatory effect of MeHg on hippocampal synaptic transmission.

107
INTRODUCTION

Acute bath application to hippocampal slices of the neurotoxic metal
MeHg causes a concentration- and time-dependent biphasic effect on synaptic
transmission in the CA1 region. Initially MeHg increases the amplitudes of
field potentials recorded extracellularly (Yuan and Atchison, 1993; 1994) and
EPSPs recorded intracellularly prior to suppression of them to block (Yuan and
Atchison, 1995a). MeHg also blocks the recurrent IPSPs (Andersen et
al.,1964a,b) in the CA1 region. IPSPs appeared to be more sensitive to MeHg
than EPSPs, because block of IPSPs occurred earlier than did block of EPSPs.
The time to the early suppression of IPSP amplitudes appeared to correspond
to the onset of the early increase in amplitudes of both population spikes and
EPSPs. This suggests that the reduced IPSPs contribute to the early increase
in amplitudes of population spikes and EPSPs. MeHg suppresses the GABA-
induced chloride current in dorsal root ganglion cells (Arakawa et al., 1991)
and modulates the muscimol-induced increases in the [3Hlflunitrazepam
binding to GABAA receptors in washed cerebellar membranes (Komulainen et
al., 1995). Thus, I hypothesized that block by MeHg of GABAA receptor-
mediated inhibitory synaptic transmission results in disinhibition of
hippocampal excitatory synaptic transmission, and is at least partly
responsible for the initial stimulatory effects of MeHg on CA1 hippocampal

synaptic transmission. However, MeHg also caused biphasic changes in

108

resting membrane potentials, i.e. initial hyperpolarization and then
depolarization of pyramidal CA1 neurons in hippocampal slices (Yuan and
Atchison, 1995a) and rat forebrain synaptosomes (Hare and Atchison, 1992).
This effect alone could influence the observed changes in IPSP and EPSP
amplitudes. Thus, nonspecific effects of MeHg on resting membrane potentials
may also be involved in its early effects on synaptic transmission.

To test this hypothesis, extracellular recordings of population spikes,
intracellular recordings of EPSPs and IPSPs and single-microelectrode voltage-
clamp recordings of excitatory and inhibitory postsynaptic currents (EPSCs,
IPSCs) from CA1 pyramidal neurons were compared with or without
pretreatment of hippocampal slices with bicuculline, a GABAA antagonist.
Effects of inorganic mercury on these responses were also examined for the
purpose of comparison. I sought to determine: 1) whether or not MeHg and
inorganic mercury (Hg2”) affect IPSPs and EPSPs in hippocampal CA1 neurons
differentially, since they differentially affect GABA-mediated chloride currents
in dorsal root ganglion neurons (Arakawa et al., 1991; Huang and Narahashi,
1996) and field potentials recorded in CA1 neurons of hippocampal slices (Yuan
and Atchison, 1994); 2) whether or not the differential block by MeHg of IPSPs
and EPSPs is due to nonspecific effects of MeHg on resting membrane
potentials; and 3) whether or not block of GABAA-mediated IPSPs is primarily
responsible for the early stimulation of hippocampal synaptic transmission.

Since this early stimulation is a characteristic of the effects of MeHg-induced

109

on central synaptic transmission, I sought to understand how this effect
occurs, and how it pertains to the overall process of MeHg-induced

neurotoxicity.

110
MATERIALS AND METHODS

Materials. Methylmercuric chloride, purchased from ICN Biomedical,
Inc. (Costa, CA), was dissolved in deionized water to a final concentration of
5 mM to serve as stock solution. The applied solutions (4 - 500 pM) were
diluted with ACSF. MeHg and other chemicals were applied acutely to slices
by bath application at a rate of 1.2 - 1.5 ml/min with a Gilson (Middleton, WI)
infusion pump. Strychnine hydrochloride and muscimol were purchased from
Sigma Chemical Co (St. Louis, MO). Muscimol (25 - 100 1.1M) was applied to
slices for 15 - 30 sec at an interval of 10 min to avoid desensitization of GABA,
receptors. 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX), 6,7-
dinitroquinoxaline-2,3-dione (DNQX), D(-)-2-amino-5-phosphonopentanoic acid
(AP-5) and (-)-bicuculline methbromide were obtained from Research
Biochemical International (Natick, MA). CNQX or DNQX were dissolved first
in dimethyl sulfoxide (DMSO) and then diluted further with ACSF. The final
concentration of DMSO in the applied solution was less than 0.02% (v/v), which

has no significant effects on synaptic transmission.

Preparation of hippocampal slices. Hippocampal slices were

prepared using methods described previously in Chapter Two and Three.

111

Electrophysiological procedures. Conventional extracellular and
intracellular recordings were made in the CA1 region of the hippocampal slice.
Monopolar tungsten electrodes (3 M9, FHC, Brunswick, ME) were used as
stimulation electrodes. Borosilicated glass microelectrodes (o.d. 1.0 mm; id 0.5
mm, WPI, Inc., New Haven, CT) filled with ACSF (5 - 15 M52) or 3 M
potassium acetate (80 - 120 M0) were used for extracellular or intracellular
recording respectively. Population spikes were evoked by orthodromic-
stimulation of Schaffer collaterals at an intensity level (usually 2 - 4 V) that
gives a population spike amplitude approximately 50% of the maximum
amplitude as evoked by maximum stimulation. Intracellular EPSPs were
recorded at CA1 cell soma by subthreshold stimulation (0.2 Hz) of Schaffer
collaterals; typically a 0.1 - 0.2 nA negative DC. current was applied through
the recording electrode to maintain the cell membrane in a somewhat
hyperpolarized state to avoid evoking action potentials. The recurrent IPSPs
(Andersen et al., 1964a,b) were recorded by subthreshold stimulation of the
alveus. IPSCs and EPSCs were recorded using single-microelectrode voltage
clamp techniques (Johnston et al., 1980; Johnston and Brown, 1981; 1984).
The sample frequency was set at 8 kHz or as high as possible. When
measuring the current-voltage relationship, voltage step commands were
generated from an internal step command generator and manually controlled
by the thumbwheel switch on the front panel of an Axoclamp-2 amplifier. For

each voltage step, the cell was held at that potential for 30 - 40 sec to obtain

112

at least 3 - 5 traces of IPSCs. The membrane input resistance was monitored
by DC. current injection through the recording electrode. All stimulus pulses
were generated from a Grass S88 stimulator (Grass, Inc., Quincy, MA) at 0.2
Hz and 0.1 msec duration and isolated with a Grass SIU5 stimulus isolation
unit (Grass, Inc.). Recorded signals were amplified (Axoclamp-2, Axon
Instruments Inc., Burlingame, CA), displayed on a 2090-3 digital oscilloscope
(Nicolet Instruments, Verona, WI) and recorded simultaneously to both floppy
disks and magnetic tape by using a FM instrumentation recorder (Model B,
Vetter Instruments, Rebersburg, PA) for later analysis. All measurements

reported in this thesis were made based on the peak amplitude of response.

Statistical analysis. Data were collected continuously before and
during application of MeHg and analyzed statistically using Student’s t-test or
paired t-test or a one-way analysis of variance; Dunnetts’ procedure was used
for post-hoe comparisons (Steel and Torrie, 1980). Values were considered

statistically significant at p < 0.05.

1 13
RESULTS

Comparative effects of MeHg and Hg”+ on IPSPs and EPSPs or
IPSCs and EPSCs. As shown in my previous report (Yuan and Atchison,
1995a), 100 11M MeHg blocked IPSPs more rapidly than it did EPSPs; times
to block were 25 :l: 2 and 45 :1: 3 min, respectively (Figure 4.1 Top). In some
slices, both EPSPs and IPSPs were recorded simultaneously in the same
neuron. In these recordings, an early increase in EPSP amplitude or even
ang of action potentials often accompanied the decrease in IPSP amplitude
at the early times of application of MeHg. At the same concentration, ng“
blocked IPSPs with a time course similar to that of MeHg. However, ng“
blocked EPSPS (63 a: 10 min) more slowly than did MeHg.

Both MeHg and Hg“ alter resting membrane potentials of various
excitable cells (Juang and Yonemura, 1975; Juang, 1976a,b; Shrivastav et al.,
1976; Miyamoto, 1983; Kauppinen et al., 1989; Hare and Atchison, 1992). In
hippocampal slices, acute bath application of MeHg or Hg” depolarized the
CA1 neuronal membrane. However, in many slices hyperpolarization occurred
prior to depolarization (Yuan and Atchison, 1995a,b). Since amplitudes of both
IPSPs and EPSPs are affected by the resting membrane potentials, polarizing
the cell membrane could contribute indirectly to effects of MeHg or Hg“ on
synaptic potential amplitude. As such, single-microelectrode voltage-clamp was

used to examine the effects of mercurials on the IPSCs and EPSCs, and thus

114

Figure 4.1. Comparison of times to block of recurrent IPSPs and
EPSPs (Top) or IPSCs and EPSCs (Bottom) by 100 uM MeHg or Hg”.
IPSPs and EPSPs were recorded in CA1 pyramidal cell soma by
stimulating the alveus or Schaffer collaterals. IPSCs were recorded at
their resting membrane potential levels in the presence of 20 11M AP-5
and 10 uM DNQX. EPSCs were evoked by presynaptic stimulation of
Schaffer collaterals and recorded at CA1 pyramidal cell soma at their
resting potentials levels. All values are the mean :t SE of 5 - 12
individual experiments. The asterisk (*) indicates a significant
difference between times to block of IPSPs and EPSPs or IPSCs and
EPSCs (p < 0.05). The black dot (0) indicates a significant difference
between times to block of IPSCs or EPSCs by MeHg and Hg” (p < 0.05,

student’s t-test)).

         

    

MeHg Wm 4,, Hg2+

.
*

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116
determine whether nonspecific effects of MeHg or ng+ on CA1 pyramidal cell

resting potential contributed to the observed changes in EPSP and IPSP
amplitude. IPSCs were recorded after pretreatment of slices for 30 min with
and in the continuous presence of 20 pM AP-5 and 10 1.1M CNQX or DNQX in
ACSF to block NMDA receptor- and non-NMDA receptor-mediated excitatory
synaptic transmission. When CA1 neuronal membrane potentials were held
at their resting levels (- 67 :t 2 mV), times to block of IPSCs and EPSCs for
MeHg (100 uM) were virtually identical to those for block of IPSPs and EPSPs
(Figure 4.1 Bottom). ng” (100 uM) blocked EPSCs with a time course similar
to that on EPSPs, however, it blocked IPSCs more slowly than it did IPSPs.
Times to block of IPSCs and EPSCs by Hg2+ were 69 :1: 12 and 65 .+. 12 min,
respectively. Moreover, MeHg still caused an early increase in EPSC
amplitude prior to suppressing it even under voltage-clamp conditions (Figure
4.2). Thus changes in resting membrane potentials are not a primary factor
for effects of MeHg on IPSPs and EPSPs or effects of Hg2+ on EPSPs. Effects
of Hg“ on IPSPs however may be due in part to alterations of resting

membrane potential.

117

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Comparative effects of MeHg and Hg” on current-voltage

relationship of IPSCs Figures 4.3 and 4.4 compares the effects of 100 uM
MeHg and Hg” on a family of IPSCs evoked at potentials of ~40 to -90 mV.
Figure 4.5 depicts the current-voltage relationship (I/V curve) for these IPSCs.
The IPSC reversal potential is approximately -75 mV in the absence of MeHg
which is close to the equilibrium potential for CI' as predicted by the Nernst
equation, and similar to these values obtained by Benardo (1994) and Pitler
and Alger (1994), indicating that these IPSCs are primarily GABAA receptor-
mediated chloride currents. MeHg suppressed both outward and inward
currents; this effect usually started after 5 min of application of 100 11M MeHg.
As shown in Figures 4.3 and 4.5 (left) exposure of slices to 100 uM MeHg for
15 min, resulted in depression of all IPSCs evoked at holding potentials of -40
to -90 mV. The UV curve and the reversal potential were shifted to more
positive potentials. In contrast, whereas 100 uM Hg” suppressed both
outward and inward currents, Hg” initially caused an increase in the outward
current prior to suppressing it. Moreover, Hg” shifted the UV curve and the
reversal potential to a more negative potential direction (Figure 4.4 and 4.5
right). At 20 uM the respective effects of MeHg or Hg” were similar but the

latency to onset of action was much longer than at 100 11M (results not shown).

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Comparative effects of MeHg and bicuculline on population
spikes. Since MeHg suppresses the GABAA-mediated chloride currents in
hippocampal CA1 neurons, I sought to determine if its effects are similar to
those of bicuculline, a selective GABAA receptor antagonist. To test this, I
compared the effects of 20 - 500 uM MeHg and 10 uM bicuculline on
population spikes. I used these higher concentrations of MeHg because I
previously showed that the higher concentrations of MeHg induced a more
rapid and noticeable increase in population spikes which was often
accompanied by repetitive firing (Yuan and Atchison, 1993). At 20 - 500 uM,
MeHg caused a concentration- and time-dependent early increase in
amplitudes of population spikes prior to blocking them (Figure 4.6). Higher
concentrations (100 and 500 uM) of MeHg induced repetitive firing in response
to single shock stimuli, suggesting that membrane excitability was increased.
The early stimulatory effects of MeHg on population spikes were similar to

those of bicuculline on population spikes.

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stimulation of synaptic transmission. The early increase in excitatory
synaptic transmission may be due primarily to MeHg-induced suppression of
GABAA receptor-mediated chloride currents. This in turn may lessen the
inhibitory effects of interneurons on excitatory synaptic transmission. If so,

then pretreatment of slices with bicuculline to block GABAA receptor-mediated

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chloride currents should eliminate or suppress the MeHg—induced early
increases in population spike amplitudes. To test this, I compared effects of
20 and 100 11M MeHg on population spike amplitudes in the presence or
absence of bicuculline. Incubation of slices with 10 uM bicuculline for 5 - 10
min increased the amplitudes of population spikes significantly; moreover the
single spike response gradually changed to multiple spike responses. After 30-
60 min of bicuculline, population spike amplitudes typically increased to and
stabilized at 150 - 200% of control (Figure 4.6D). At this point, two sets of
experiments were designed to examine the effects of bicuculline on the early
stimulation by MeHg of excitatory synaptic transmission. In the first set of
experiments 20 or 100 11M MeHg plus 10 1.1M bicuculline was added to the
ACSF with no change in stimulus intensity. Under these conditions, MeHg
still suppressed population spike amplitude as did MeHg alone, but in 4 of 6
slices caused no further significant early increase in population spike
amplitudes (Figure 4.7 Left). The second set of experiments was performed
under reduced stimulus intensity. The reason for doing this was that we were
concerned that pretreatment of slices with bicuculline might increase
population spike amplitudes to a ceiling amplitude, above which MeHg was
unable to cause further increase, thus masking the actual effect of MeHg on
population spikes. Thus, the stimulus intensity was reduced to a level that
gave population spike amplitudes approximately equal to the control level prior

to bicuculline treatment, after the bicuculline-induced increase had stabilized.

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MeHg (20 or 100 uM) plus 10 uM bicuculline were then applied to the slices.

As seen with the results of the first set of experiments, MeHg did not cause
significant early increase in population spike amplitudes but reduced or
blocked completely population spikes in 3 of 4 and 5 of 7 slices at 20 and 100
uM MeHg respectively (Figure 4.7 Right). In the remaining slices there was a
10 - 15% early increase in population spike amplitudes prior to block by MeHg.
This effect was not significant, and is masked in Figure 4.7 due to averaging
of the time courses from the individual experiments. Without pretreatment of
slices with bicuculline, 20 and 100 uM MeHg caused the typical biphasic
changes in amplitudes of population spikes, although the early increase in
amplitude induced by 20 uM MeHg was not as prominent as that caused by
100 11M MeHg (Figure 4.7). Due to variations in time course of effects of MeHg
among the individual experiments, Figure 4.7 does not show any decrease in
population spike amplitudes after exposure to 20 uM MeHg alone for 100 min.
However, prolonging exposure of slices to 20 11M MeHg to 150 - 180 min,
caused suppression or block of all population spikes (Figure 1.1 and 4.6). It
appears that MeHg blocked responses more rapidly in slices treated with
bicuculline than in slices not pretreated with bicuculline. To test if bicuculline
would prevent early increase in EPSP amplitude induced MeHg, effects of
MeHg on EPSPs were examined in the presence of 10 11M bicuculline.
Normally, EPSPs were evoked by subthreshold stimulation of Schaffer

collaterals to avoid generation of action potentials. After application of 10 uM

133

bicuculline for 30 - 60 min, EPSP amplitude increased dramatically and
induced multiple spikes (Figure 4.8). Once the increase in EPSP amplitude
reached a stable level, the stimulus intensity was then reduced to a level that
gave a measurable EPSP but did not initiate action potentials. It was
generally quite difficult to do this after pretreatment of slices with bicuculline,
because either action potentials were generated or the EPSP at a given
stimulus was not measurable. Thus I am only able to obtain a few successful
recordings for this experiment. However, in those experiments MeHg failed
to cause a significant early increase in EPSP amplitude in the presence of 10
1.1M bicuculline as was seen in Figure 4.7 for field potential recordings. Thus
MeHg—induced early increases in amplitudes of population spikes and EPSPs
appear to be related to its actions on GABAA receptors.

The results of the previous experiment do not rule out the possibility
that MeHg directly affects GABA release from interneurons via a presynaptic
mechanism. Thus, to test if the effects of MeHg are due to a direct action on
GABAA receptors I examined the effects of MeHg on responses evoked by
muscimol, a GABAA agonist. Bath application of 25 — 100 uM muscimol to
slices for 15 - 30 sec caused a concentration-dependent depolarization of CA1
pyramidal neurons. It usually took about 6 to 10 min of wash to restore the
depolarized membrane back to the pre-muscimol application baseline. The
muscimol-evoked responses were blocked rapidly by 20 11M bicuculline (Figure

4.9 Top), suggesting that they are GABAA receptor-mediated responses. MeHg

134

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also blocked these muscimol-evoked responses (Figure 4.9 Bottom), which was
consistent with the report of Komulainen et al., (1995). Times to block by 100
uM MeHg of muscimol-evoked depolarization varied from 20 to 50 min in 11
experiments, depending on the concentration and duration of muscimol
application. MeHg blocked the depolarization evoked by 25 uM muscimol more
rapidly than that evoked by 50 or 100 uM muscimol. In these experiments, it
was difficult to determine the exact time to block by MeHg of muscimol-evoked
responses due to the long time interval required for washing out muscimol
from slices before the next application. However, the times to block of
muscimol-evoked responses, especially those evoked by lower concentrations
of muscimol, were generally similar to those to block of IPSPs or IPSCs by
MeHg. This suggests a direct action of MeHg at the GABAA receptor sites

although additional presynaptic mechanisms could still occur.

Effects of strychnine on MeHg-induced early stimulation of CA1
synaptic transmission. GABA is generally believed to be the major
inhibitory transmitter in the mammalian CNS. However, glycine also serves
as an inhibitory transmitter in the CNS, especially in the spinal cord and brain
stem (Aprison and Daly, 1978; Pycock and Kerwin; 1981; McCormick, 1990).
Additionally, glycine can potentiate the action of glutamate at NMDA receptors
(Johnson and Ascher, 1987), although this response is generally assumed to be

strychnine-insensitive (Kishimoto et al., 1981). To test whether or not a

139

putative glycine receptor also plays a role in MeHg-induced early stimulatory
effects on hippocampal synaptic transmission, slices were perfused with 50 uM
strychnine, a glycine receptor antagonist, prior to and during exposure to 20
or 100 uM MeHg. In a similar manner to that of bicuculline, strychnine also
caused a significant increase in population spike amplitudes and induced
repetitive firing, although not as prominently as did bicuculline. However,
unlike the effects of MeHg on population spikes in the presence of bicuculline,
MeHg caused a further significant increase of population spike amplitude
above that already elevated by strychnine. This effect occurred irrespective of
whether or not stimulus intensity was reduced (Figure 4.7). Thus glycine
receptors do not play a major role in the MeHg-induced early stimulation of
hippocampal synaptic transmission. Figure 4.10 summarizes the effects of
MeHg, bicuculline and strychnine alone and in combination with MeHg on
population spike amplitude. Clearly, MeHg, bicuculline and strychnine all
increase population spike amplitudes significantly. However, pretreatment of
slices with bicuculline prevented the MeHg-induced early increase in
population spike amplitudes, whereas pretreatment of slices with strychnine

failed to do so.

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142
DISCUSSION

Previously I showed that acute bath application of MeHg caused an
initial stimulation of hippocampal synaptic transmission prior to suppression
to block (Yuan and Atchison, 1993, 1995a). Under similar conditions, Hg”
blocked synaptic transmission in the CA1 region of hippocampal slices but did
not induce the early stimulatory effects (Yuan and Atchison, 1994). The
primary objective of the present study was to identify the potential factor(s)
responsible for the early stimulatory effects of MeHg on hippocampal synaptic
transmission. Previous results of microelectrode current-clamp recordings
suggested that effects of MeHg on inhibitory synaptic transmission and on
resting membrane potentials may be involved in the MeHg-induced early
stimulation of hippocampal synaptic transmission, because MeHg blocked
IPSPs more rapidly than it did EPSPs, and caused biphasic changes in resting
membrane potentials of CA1 pyramidal neurons (Yuan and Atchison, 1995a).
In the present study, I reconfirmed that IPSPs are more sensitive to MeHg
than are EPSPs, and demonstrated that this effect is not related to MeHg-
induced changes in resting membrane potentials of CA1 neurons, because
times to block by MeHg of IPSCs and EPSCs recorded under voltage-clamp
conditions were similar to those for block of IPSPs and EPSPs recorded under
current-clamp conditions. Moreover, voltage-clamp of neuronal membranes at

their resting potential levels failed to prevent the MeHg-induced early increase

143
in EPSC amplitude. In contrast, Hg” also blocked IPSPs more rapidly than

it did EPSPs. However, it blocked IPSCs and EPSCs similarly when CA1
neuronal membranes were voltage-clamped at their resting potentials,
suggesting that the early block by Hg2+ of IPSPs compared with that for EPSPs
may be due simply to changes in resting membrane potential. Thus, MeHg
blocked inhibitory synaptic transmission more preferentially, although it also
blocked excitatory synaptic transmission, whereas Hg’+ blocked both inhibitory
and excitatory transmission to the same extent and relatively slowly. This is
consistent with our previous observations that MeHg caused early stimulatory
effects on hippocampal synaptic transmission, while ng“ did not (Yuan and
Atchison, 1994).

In dorsal root ganglion neurons, MeHg suppressed GABA-mediated
chloride currents, while Hg2* greatly enhanced these currents in a
concentration-dependent manner (Arakawa et al. , 1991; Huang and Narahashi,
1996). In the present study, the IPSCs recorded at CA1 neurons appear to be
primarily GABAA-mediated chloride currents, since their reversal potentials
are close to the equilibrium potential of Cl’ and these currents can be blocked
by bicuculline. At 20 and 100 uM, MeHg suppressed all inward and outward
currents generated at different holding potentials and shifted the UV curve to
more positive potentials, suggesting that MeHg may block the GABAA-
mediated chloride channels. MeHg has also been shown to inhibit muscimol-

stimulated agonist binding in cerebellar P2 membrane fractions (Komulainen

144

et al., 1995). In contrast, whereas ng‘“ also suppressed to block all inward and
outward Cl' currents, it took longer to do so than did MeHg. Unlike the effects
of MeHg on GABAA-activated Cl' currents, Hg2+ initially caused an increase in
GABAA-mediated outward Cl' currents prior to suppressing them, indicating
that Hg2+ may, as it did to the GABAA-mediated chloride channels in dorsal
root ganglion neurons (Arakawa et al., 1991; Huang and Narahashi, 1995),
initially increase the open probability of GABAA-activated chloride channels.
Moreover, similar to its effects on the tetrodotoxin-, bicuculline- and picrotoxin-
insensitive slow inward currents induced in dorsal root ganglion neurons
(Arakawa et al. , 1991), ng+ shifted the IN curve and the reversal potential to
more negative potentials, indicating that ions other than 01' may be also
involved. These differential effects of MeHg and ng+ on GABAA receptors may
explain why MeHg causes the early stimulatory effects on hippocampal
synaptic transmission, While ng+ does not.

If effects of MeHg on GABAA receptors are indeed responsible for the
MeHg-induced early increase in population spike or EPSP amplitude, then
pretreatment of slices with the GABAA antagonist bicuculline should eliminate
the early increased phase in either population spikes or EPSPs. After
pretreatment of slices with bicuculline, MeHg no longer caused an initial
increase in population spike and EPSP amplitudes but still decreased them to
block. The failure to induce the early increase in amplitude of population

spikes was not due to a ceiling effect caused by bicuculline, although

145
bicuculline significantly increased population spike amplitude to 180 - 200%

of control. At the time bicuculline-stimulated amplitudes of population spikes
reached maximal levels, increasing stimulation intensity still caused a further
increase in population spike amplitude. Moreover, MeHg failed to cause the
early stimulatory effects even under conditions in which the stimulation
intensity was reduced to pre-bicuculline control level after bicuculline had
increased population spike amplitude to a stable level. The most likely
explanation for these results is that MeHg may directly act at GABAA
receptors to cause disinhibition in a similar manner to the effects of bicuculline
on GABAA receptors. This explanation was further supported by the
observation that MeHg blocks responses evoked by bath application of
muscimol with a similar time course to that of block of IPSPs or IPSCs.

In the hippocampal CA1 region, at least two subtypes of GABAA
receptors coexist in pyramidal neurons (Pearce, 1993; Gordey et al., 1995).
One type is located at the soma or initial segment of the axon. When
activated, it hyperpolarizes the CA1 pyramidal cell membrane. The other type
is located in the dendrites. When activated, it depolarizes the CA1 pyramidal
cell membrane (Gordey et al. , 1995). The responses evoked by bath application
of muscimol in the present study are likely to represent a net response of both
types of GABAA receptor to muscimol. Thus, block of responses evoked by bath
application of muscimol indicated that MeHg affects both types of GABA,-

mediated responses. We cannot exclude the possibility that presynaptic effects

146

of MeHg on the interneurons or some factors other than GABAA receptors
contribute to the increase effect in hippocampal excitability, since in some
slices pretreated with bicuculline, MeHg still caused a delayed increase of
about 10 - 15 % in population spike amplitude, although this was not
statistically significant. In hippocampus, in addition to GABAA receptors,
GABAB receptors are located both pre- and postsynaptically in the CA1 region
and regulate synaptic transmission (Dutar and Nicoll, 1988a,b; Thompson et
al., 1992; Otis et al., 1993; Isaacson et al., 1993; Pitler and Alger, 1994; Wu
and Saggau, 1995). At postsynaptic CA1 neurons, GABAB receptors are
coupled to K+ channels via a G-protein to cause hyperpolarization of cells. This
is expressed as the slow EPSP (Dutar and Nicoll, 1988b; Thompson and
Gahwiler, 1992; Otis et al. , 1993; Pitler and Alger, 1994). Perhaps the delayed
increase in population spike amplitude by MeHg-induced in the presence of
bicuculline was due to an effect on GABAB receptors. Alternatively, effects of
MeHg on intracellular Ca2+ homeostasis may also be involved in the early
stimulatory effects of MeHg on hippocampal synaptic transmission, since
MeHg increases intracellular Ca2+ concentrations in several types of neurons
(Denny et al., 1993; Hare et al., 1993; 1995; Marty and Atchison, 1997). In
fact, in hippocampal slices after block of voltage-dependent N a+ channels using
the local anesthetic QX—314, MeHg also caused an initial increase in Ca2+ spike
amplitudes prior to decreasing them to block (Yuan and Atchison, unpublished

observation).

147

Earlier findings from ligand binding studies (Young and Snyder, 1973)
and autoradiography (Zarbin et al., 1981; Frosholm and Rotter, 1985; Probst
et al., 1986) using [3Hlstrychnine indicated that glycine receptors are
predominately confined to the spinal cord, brain stem and other areas of the
lower neuraxis. However, recent studies using immunocytochemistry with
monoclonal antibodies (Van den P01 and Gorcs, 1988; Becker et al., 1988),
autoradiography with [3H]glycine (Bristow et al., 1988), Northern blot
hybridization (Grenningloh et al., 1990; Kuhse et al., 1990a,; Malosio et al.,
1991) and polymerase chain reaction (Kuhse et al., 1990a,b; 1991)
demonstrated a wide distribution of glycine receptors in the higher regions of
the CNS including cerebral cortex and hippocampus. These glycine receptors,
differ from those in the spinal cord and brain stem which primarily express the
0.1 subunit, a component of the "classical" strychnine-sensitive glycine receptor
(Bristow et al. , 1986; Becker et al. , 1988; Belz, 1990a). Instead, these receptors
express a different ligand binding subunit (a2), which displays only low affinity
for binding of strychnine (Bristow et al., 1986; Becker et al., 1988) or low
sensitivity to strychnine upon heterologous expression in Xenopus oocytes
(Kuhse et al., 1990a). To date, however, we are unaware of any direct report
of the existence and the physiological role of functional glycine receptors in
hippocampal CA1 neurons, although the above evidence suggests their
presence in the hippocampus. In the present study, pretreatment of slices with

strychnine caused a dramatic increase in population spike amplitude and

148

induced multiple spike responses, although it was not as effective in this
regard as was bicuculline. This suggests either that there may be a small
population of strychnine-sensitive subtype of glycine receptors located in the
CA1 hippocampal region, or that strychnine cross-reacts with GABAA receptors,
since they both belong to a superfamily of ligand-gated ion channels and share
significant sequence similarity in primary structure and transmembrane
topology (Grenningloh et al., 1987; Schofield et al. , 1987; Langosch et al. , 1988;
Schmieden et al., 1993). The latter possibility seems less likely, inasmuch as
strychnine did not prevent or suppress the MeHg-induced early stimulation,
as bicuculline pretreatment did. Another possible explanation for the failure
of strychnine pretreatment to block MeHg-induced early stimulation of
synaptic transmission is that these heterologous glycine receptors in
hippocampal neurons may be not blocked completely by strychnine due to their
low sensitivity to strychnine as suggested by previous studies (Young and
Snyder, 1973; Frostholm and Rotter, 1985; Probst et al., 1986; Bristow et al.,
1986; Kuhse et al., 1990). This may be one of the reasons why strychnine was
less potent in increasing population spike amplitude than was bicuculline.
This possibility also seems less likely, because pretreatment of slices with
bicuculline alone completely suppressed MeHg-induced early increase in field
potentials. Thus, if there are glycine receptors located on postsynaptic
membranes, they do not play a primary role in MeHg-induced early

stimulation of hippocampal synaptic CA1 cell transmission.

149
In conclusion, the preferential block by MeHg of inhibitory synaptic

transmission, mediated primarily by GABAA receptors, appears to be primarily
responsible for the MeHg-induced early stimulatory effects on hippocampal
synaptic transmission. The importance of this disinhibition caused by MeHg

to its overall neurotoxicity also remains unknown.

CHAPTER FIVE

COMPARATIVE EFFECTS OF METHYLMERCURY ON PARALLEL-

FIBER AND CLINIBING-FIBER RESPONSES

IN RAT CEREBELLAR SLICES

150

151
ABSTRACT

Previous studies showed that MeHg blocked both inhibitory and
excitatory synaptic transmission in the CA1 region of hippocampal slices.
However, following exposure to MeHg in vivo, the primary target in the CNS
for neurotoxicity is the cerebellum. Thus, in the present study, effects of MeHg
on synaptic transmission between parallel fibers and Purkinje cells and
between climbing fibers and Purkinje cells were compared in 300 - 350 um
cerebellar slices using extracellular and intracellular microelectrode recording
techniques. Field potentials of parallel-fiber volleys (PFVs) and the associated
postsynaptic responses (PSRs), presumably evoked by glutamate released from
parallel-fiber terminals, were recorded in the molecular layer by stimulating
parallel fibers in the same layer in transverse slices. The climbing-fiber
responses (CFRs) were also recorded in the molecular layer by stimulating
white matter in sagittal slices. At 20, 100 and 500 uM, MeHg blocked both
PFVs and the associated PSRs, however, it blocked PSRs more rapidly than it
did PFVs. Times to block of PSRs by 500, 100 and 20 M MeHg were 6 :2: 0.5,
32 :t 4, and 101 :t 24 min, respectively, while times to block of PFVs by 500,
100 and 20 11M MeHg were 10 :l: 0.5, 51 :t: 5 and 184 :t 27 min, respectively.
In addition, MeHg caused an initial slight increase in PFV and PSR
amplitudes prior to suppressing them to block. Similarly, MeHg first increased

and then decreased amplitudes of CFRs to complete block. Times to block of

152
CFRs by 100 and 20 uM MeHg were 45 :t 3 and 115 :t 18 min, respectively.

Thus, MeHg blocks both parallel-fiber and climbing-fiber responses. However,
it blocks the glutamate-evoked PSRs and CFRs more rapidly than it does
PFVs. This suggests that MeHg may either affect the process of
neurotransmitter release from the presynaptic fibers or act at the postsynaptic
Purkinje cells. As a means of identifying the primary sites of action of MeHg
in blocking these field potentials, intracellular recordings of excitatory
postsynaptic potentials evoked by activation of parallel fiber (PF-EPSPS),
climbing fibers (CF-EPSPs) and repetitive firing of Purkinje cells evoked by
direct injection of depolarizing current at the somata were compared. At 100
and 20 uM, MeHg blocked all voltage-dependent responses with a similar time
course. This included the Na*-dependent fast somatic spikes evoked by
parallel fiber stimulation or by direct depolarization of Purkinje cells as well
as the Ca2*-dependent slow dendritic spike bursts evoked by climbing fiber
stimulation or by direct depolarization of Purkinje cells. MeHg appears to
block voltage-dependent responses more rapidly than it does glutamate-
mediated responses. Thus, it may affect voltage-gated ion channels and
glutamate-activated channels differently. MeHg also hyperpolarized and then
depolarized Purkinje cell membranes, suppressed current conduction from
parallel fiber or climbing fibers to dendrites of Purkinje cells and blocked
synaptically-activated local responses. MeHg might affect Purkinje cell

membrane ion conductances because it switched the patterns of repetitive

153

firing of Purkinje cells generated spontaneously or by depolarizing current
injection at Purkinje cell somata from one of predominantly N a"—dependent,
fast somatic spikes to one of predominantly Ca2*-dependent, low amplitude,
slow dendritic spike bursts. Thus, in the cerebellum, as in the hippocampus,
acute exposure to MeHg causes a complex pattern of disruption of synaptic
function. The time course of block of synaptic function in the two different
regions is generally similar. Multiple sites of action appear to be involved,
however, MeHg appears to act primarily at the postsynaptic Purkinje cells to
block synaptic transmission between parallel fibers and Purkinje cells and

between climbing fibers and Purkinje cells.

154
INTRODUCTION

Previously, I demonstrated that acute bath application of MeHg
disrupted neuronal membrane excitability and synaptic transmission in the
CA1 region of hippocampal slices in a concentration- and time-dependent
manner (Yuan and Atchison, 1993, 1994, 1996 and 1997). MeHg appears to
act at multiple sites to cause these effects; it blocked excitatory synaptic
transmission, inhibitory synaptic transmission and antidromically-activated
responses, it hyperpolarized and then depolarized the CA1 pyramidal cell
membranes; and possibly also affected the process of presynaptic release of
neurotransmitter. The primary sites of actions of MeHg on hippocampal
synaptic transmission in the CA1 region appear to be the postsynaptic CA1
pyramidal neurons, at least at the early stage of exposure to MeHg. However,
presynaptic mechanisms, nonspecific effects of membrane depolarization, and
suppression of current conductance may be also involved in the actions of
MeHg in blocking hippocampal synaptic transmission.

Among the specific brain regions, the cerebellum, especially the
cerebellar cortex, appears to be a primary target of MeHg in the CNS.
Chronically, MeHg accumulates most in the cerebellum, particularly in the
Purkinje cells and the Golgi cells in the granular layer, and to a lesser extent
in the granule cells, stellate cells and basket cells (Glomski, 1971; Olszewski

et al., 1974; Chang, 1977, 1980; Moller—Madsen, 1990, 1991; Leyshon-Sdprland,

155

1994). However, pathological examination of patients and experimental
animals with acute and chronic MeHg poisoning indicated that the cerebellar
cortex, especially the granule cells, was particularly sensitive to MeHg (Hunter
and Russell, 1954; Takeuchi et al., 1962; Chang, 1977, 1980; Syversen et al.,
1981). In MeHg poisoning, especially in chronic cases, there was a
characteristic atrophy of the cerebellar cortex, particularly the granular layer
in the lateral lobes and in the vermice, due to extensive loss of granule cells
(Hunter and Russell, 1954; Takeuchi et al., 1962; Chang, 1977, 1980). In
addition, the basket cells, climbing and parallel fibers were also severely
involved. The Purkinje cells were more resistant, but were also typically
affected in chronic cases (Hunter and Russell, 1954; Takeuchi, 1962; Chang,
197 7, 1980). Possibly, the interactions of MeHg with cerebellar neurons are
responsible for the motor deficits caused by acute and chronic exposure to
MeHg. Thus, a specific examination of effects of MeHg on cerebellar synaptic
transmission may aid our understanding of potential mechanisms of MeHg-
induced neurotoxicity.

To date, to my knowledge, no direct study of the effect of MeHg on
cerebellar synaptic transmission has been reported, although many studies
done in this and other labs have shown that in vitro acute exposure to MeHg
affects function of cells or cell fractions derived from the cerebellum. For
example, MeHg reduced influx of 45Ca2+ induced by depolarization of rat

cerebellar synaptosomes (Y an and Atchison, 1996), reduced currents carried

156
through K+ and Ca2+ channels (Sirois and Atchison, 1995, 1996, 1997) and

increased [Ca2+]i in primary cultures of rat cerebellar granule cells (Marty and
Atchison, 1997). MeHg also affected protein phosphorylation and synthesis in
cerebellar granule cells (Sarafian and Verity, 1986, 1990a,b, 1992; Sarafian,
1993), inhibited migration of granule cells in cerebellar organotypic cultures
(Kunimoto and Suzuki, 1997), and induced rapid death of cerebellar granule
cells (Sarafian et al., 1989; Nagashima et al., 1996). The unique architecture
of the cerebellar cortex suggests that Purkinje cells may be a key element in
the cerebellar synaptic circuitry, since they receive and integrate synaptic
inputs from both parallel-fibers and climbing-fibers. Furthermore, their axons
are the only output from the cerebellar cortex to the deep cerebellar nuclei to
modulate activities of these nuclei. Thus, I feel it would be important to
examine if Purkinje cells are functionally sensitive to MeHg, even though
pathologically they are more resistant to MeHg compared to granule cells
(Hunter and Russell, 1954; Takeuchi et al., 1962; Syversen et al., 1981). As
such, the present study was designed to determine if MeHg affects synaptic
transmission between the parallel- or climbing-fibers and Purkinje cells and
whether or not MeHg affects these two synaptic pathways differently since
their electrophysiological characteristics differ. Additionally, I sought to
compare whether or not MeHg affects cerebellar synaptic transmission

differently from its effects on hippocampal synaptic transmission.

157
MATERIALS AND METHODS

Materials. Methylmercuric chloride, purchased from ICN Biomedical,
Inc. (Costa, CA), was dissolved in deionized water to a concentration of 5 mM
to serve as stock solution, which was used for one week. The applied solutions
(20-100 1.1M) were diluted with modified ACSF consisting of (in mM): 125 NaCl,
2.5 KCl, 2 CaClz, 1 MgClz, 1.25 KHZP04, 26 N aHCO3 and 20 mM d-glucose (pH
7.4) just before superfusion. MeHg and other chemicals were applied acutely
to slices by bath application at a rate of 1.2 - 1.5 ml/min with a Gilson infusion
pump (Middleton, WI). DNQX and AP-5 were purchased fi'om Sigma Chemical
Co. (St. Louis, MO). DNQX was dissolved first in DMSO and then diluted
further with modified ACSF. The final concentration of DMSO in the applied
solution was less than 0.02% (v/v), which has no significant effects on synaptic

transmission.

Preparation of cerebellar slices. Cerebellar slices were prepared
using the methods described previously in Chapter 'IVvo. One cerebellar slice
was transferred to the recording chamber and the remaining slices were
incubated in a holding chamber for later use if needed. The entire process
from decapitating the rat to transferring the slices to the recording or holding
chamber was finished in less than 10 min and under a temperature of 4 0C.

The slice in the recording chamber was incubated and superfused (1.2 - 1.5

158
ml/min) continuously with the modified ACSF saturated with 95% O2 /5% CO2

for at least 60 min before the electrophysiological recordings began. All
experiments were conducted at room temperature. Only one slice per rat was

used.

Electrophysiological procedures. A concentric bipolar metal electrode
or monopolar tungsten electrode (3 MO, FHC, Brunswick, ME) was used as the
stimulation electrode. Borosilicated glass microelectrodes (o.d. 1.0 mm, id 0.5
mm, WPI, Inc., orlando, FL) filled with ACSF (5 - 15 M0 impedance) or 3 M
potassium acetate (60 - 80 M0 impedance) were used as extracellular or
intracellular recording electrodes, respectively. Conventional extracellular
recordings were made in the molecular layer of the cerebellar cortex. To record
extracellular responses of Purkinje cells to local extracellular activation of the
parallel fibers, the stimulating electrode was positioned on the surface of the
molecular layer of a transverse slice (S1 in Figure 5.1), just below the pia, and
a recording electrode (R1 in Figure 5.1) was positioned on the same track
which parallel fibers travel along, so-called "on beam" (Crepel and Delhaye-
Bouchaud, 1978; Crepel et al., 1981), in the molecular layer (R1 in Figure 5.1).
The typical extracellular response evoked by activation of parallel fibers
consists of an initial triphasic potential with positive-negative-positive
components and followed by another prolonged negative potential. The initial

triphasic component corresponds to the current generated by action potentials

159

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propagating along parallel fibers, defined as parallel-fiber volley (PFV in
Figure 5.1). The prolonged negative potential corresponds primarily to the
postsynaptic excitatory potentials evoked by glutamate released from parallel
fibers onto the molecular layer dendrites of Purkinje cells, and defined as the
postsynaptic response (PSR in Figure 5.1). It can be blocked reversibly by the
kainate/AMPA type glutamate receptor antagonist DNQX (Figure 5.2) (Salin
et al., 1996). To record field responses of Purkinje cells to activation of
climbing fibers, the stimulating electrode was positioned on the white matter
immediately at the base of the folium in a parasagittal slice (S2 in Figure 5.1);
a recording electrode was positioned in the molecular layer (R2 in Figure 5.1).
The field potentials evoked by stimulation of the climbing fibers are also
glutamate-evoked responses because they are blocked by DNQX (Figure 5.2).
Normally, amplitudes of these field potentials require stabilization for at least
20 - 30 min before beginning the experiments, because most of recordings
showed increases in amplitudes of the field responses within the first 30 min
after establishment of recordings in the absence of any treatment. For
intracellular recordings of parallel- or climbing-fiber excitatory postsynaptic
potentials (PF-EPSPs or CF-EPSPs), the positions of stimulating electrodes
were similar to those for extracellular recordings, however, the recording
electrodes were positioned in the somata of an identified Purkinje cell with the
aid of an Olympus BHWI upright microscope (Olympus Optical 00., Tokyo,

Japan) equipped with Normaski optics and 10 X and 40 X water-immersion

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objective lenses. The stimulus pulses were generated from and isolated using
a Grass S88 stimulator and a Grass SIU5 stimulus isolation unit (Grass, Inc.,
Quincy, MA) at 0.15 Hz, 0.1 ms duration at an initial intensity that produced
approximately 50 - 60 % of the maximum response for a given slice. Recorded
signals were amplified (Axoclamp-Z, Axon instruments Inc., Foster City, CA),
displayed on a 2090-3 digital oscilloscope (Nicolet Instruments, Verona, WI)
and recorded simultaneously to a 5X86 computer at a digital sampling interval
of 0.2 or 1.0 ms. The 1 ms digital sampling interval was used to record those
responses generated by injection of long duration of depolarizing current pulses
at Purkinje cell soma. The responses of Purkinje cells to activation of parallel-
fibers and climbing-fibers were identified further based on their
electrophysiological properties (Llinas and Sugimori, 1980a; Crepel et al. , 1981;
Stuart and Hausser, 1994). The responses, recorded extracellularly or
intracellularly, generated by activation of parallel-fibers are graded events
(Figure 5.3A,B), whereas responses evoked by stimulating climbing fibers are
so-called all or none complex spikes regardless of the stimulus intensity
(Figure 5.3C). Directly depolarizing Purkinje cells by injection of 500 - 1000
ms positive current pulses at the threshold intensity using the recording

electrode in the somata generate Purkinje cell-specific repetitive spikes (Figure

5.3B,C).

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Statistical analysis. Data were collected continuously before and during
application of MeHg and analyzed statistically using student’s paired t test and
one-way analysis of variance. Dunnetts’ procedure was used for post hoc

comparisons. Values were considered statistically significant at p < 0.05.

168
RESULTS

Comparative effects of MeHg on field potentials evoked by
stimulating parallel fibers or climbing fibers. The pattern of effects of
MeHg on field potentials recorded from the molecular layer of cerebellar slices
was similar to that on potentials recorded from the CA1 pyramidal neurons in
hippocampal slices (Yuan and Atchison, 1993, 1994). Acute bath application
of 20, 100 and 500 uM MeHg caused a concentration- and time-dependent
biphasic effect on the PFV and the associated postsynaptic responses-PSRS
generated by stimulation of parallel fibers in transverse cerebellar slices.
Initially, 20, 100 and 500 uM MeHg increased amplitudes of PFVs and PSRs
(Figure 5.4 and 5.7, data for 500 uM are not shown to simplify the figures).
The time-dependent averages of amplitudes of these field potentials are shown
in Figure 5.7. The percentages of peak increases in amplitudes of PFVs and
PSRs, averaged from each individual experiment, were 157 :t 17% and 128 i
8% of control for 100 uM MeHg and 211 :1: 25% and 178 :1: 19% of control for
20 11M MeHg, respectively. The mean times to peak stimulation of PFVs and
PSRs averaged from each individual experiment were 21 :t 4 and 9 :1: 2 min for
100 uM MeHg and 91 i 22 and 49 :t 12 min for 20 11M MeHg, respectively. As
exposure of slices to MeHg was increased, both PFV and PSR amplitudes were
reduced progressively until complete block occurred. In general, MeHg blocked

PSRs more rapidly than it did PFVs. As shown in Figure 5.4, at 30 or 150 min

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after exposure to 100 (Top) or 20 uM MeHg (Bottom), the PSR components

were completely blocked while the PFV components remained essentially
unchanged. The mean times to block PSRs by 500, 100 and 20 uM MeHg were
6 i 0.5, 32 :L- 4 and 101 :1: 24 min, respectively; the mean times to block PFVs
by 500, 100 and 20 uM MeHg were 10 :t 0.5, 51 i 5 and 184 :t 27 min,
respectively (Figure 5.6). Differences between times to MeHg-induced block of
PSRs and PFVs were statistically significant (p<0.05). Thus, the glutamate-
mediated PSRs appear to be more sensitive to MeHg than were the presynaptic
PFVs.

Similarly, 20 and 100 11M MeHg initially stimulated the amplitude of
field potentials evoked by stimulation of climbing fibers in sagittal slices prior
to blocking them (Figure 5.5). The percentages of peak increases in CFR
amplitudes stimulated by 100 and 20 uM MeHg were 128 i 6% and 131 1 7%
of control (p<0.05), respectively. Times to block of CFRs by 100 and 20 uM
MeHg were 45 :t 3 and 115 :1: 18 min (Figure 5.6), respectively. These values
are similar to those for MeHg-induced block of the PSRs, and appear to be
more rapid than those for MeHg-induced block of the PFVs although the
differences were not statistically significant. Figure 5.7 summarizes the time-
courses of effects of MeHg on PFVs, PSRs and CFRs. MeHg first increased
and then decreased amplitudes of all three responses to complete block.

Whereas the effect of MeHg on PSRs appeared to be slightly more rapid than

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Figure 5.7. Time course of effects of 100 (Top) and 20 uM (Bottom)
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obtained prior to exposure to MeHg are considered as control.

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its effect on CFRs, the difference was not statistically significant. Block of
both PSRs and CFRs occurred earlier than did block of PFVs. Thus, MeHg
blocked glutamate-mediated postsynaptic responses activated either by
stimulating parallel fibers or climbing fibers similarly, suggesting that MeHg
may affect either the postsynaptic glutamate receptors or the process of
transmitter release from parallel- or climbing-fiber terminals to block synaptic

transmission by these two pathways.

Effects of MeHg on parallel-fiber EPSPs (PF-EPSPs) and
climbing-fiber EPSPs (CF-EPSPs). To explore the mechanisms underlying
the effects of MeHg on field potentials recorded in the molecular layer by
activation of parallel fibers or climbing fibers, intracellular recording
techniques were applied to the Purkinje cells to examine effects of MeHg on
PF-EPSPs, CF-EPSPs and resting membrane potentials. Stimulation of the
parallel fibers in the molecular layer of the cerebellar cortex in transverse
slices initiates a negative parallel-fiber volley followed by a PF-EPSP. The PF-
EPSPs were graded amplitude responses with a range of 1.5 - 2.3 ms latencies
from the stimulus artefact to the onset of EPSPs, depending on the stimulus
intensity and the distance between the stimulating and recording electrodes.
After exposure to 100 and 20 uM MeHg, these latencies were prolonged,
suggesting that current conduction from the parallel fibers to Purkinje cells

was affected. Unlike the biphasic effects of MeHg on those field potentials, 100

179
and 20 uM MeHg blocked the PF-EPSPs without causing an early increase in

EPSP amplitude, although a transient slight increase in amplitude of EPSPs
occurred in some slices prior to suppression of the EPSPs. Times to block of
EPSPs by 100 and 20 1.1M MeHg were 33 :t 7 and 63 :t 3 min, respectively.
However, as shown in Figure 5.8 (Left), the parallel-fiber volley responses
appeared less sensitive to MeHg than did PF-EPSPs since PFV amplitudes
remained essentially unchanged when EPSPs were reduced significantly (10
min) or blocked completely by MeHg (100 pM). This was consistent with
results obtained from extracellular recordings in that PFVs were less sensitive
to MeHg than were the associated PSRs. In addition, the early block of EPSPs
by MeHg could be partially restored by increasing the stimulus intensity
(Figure 5.8 Left), suggesting that MeHg may initially suppress neuronal
membrane excitability and/or transmitter release.

In contrast, stimulation of climbing fibers in sagittal cerebellar slices
usually generated a full antidromically-activated action potential followed by
a typical all or none complex spike response or CF-EPSPs (Figures 5.3, 5.9).
The complex spikes consist of several small spikes superimposed on a
pronounced plateau of depolarization. In some recordings, the typical complex
spikes did not occur because the resting membrane potentials were more
depolarized than -60 mV. The latency from the stimulus artefact to onset of
CF-EPSPs was 1.9 :t 0.3 ms. After exposure of slices to 100 uM MeHg, the

latency was prolonged to 2.7 :t 0.4 ms (p < 0.05), indicating that impulse

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conduction from climbing fibers to Purkinje cells was affected by MeHg. Also
shown in Figure 5.9, at 100 and 20 11M MeHg, the amplitudes of the steady-
state depolarization were almost always reduced prior to block of the complete
climbing fiber response. Moreover, the complex spikes appeared to be more
sensitive to MeHg than were the antidromically-activated action potentials
because the antidromically-activated action potentials remained after complex
spikes were blocked completely at 30 min and 50 min by 100 and 20 uM MeHg,
respectively. Initially, block of CF-EPSPs could be restored by increasing
stimulation intensity, which was similar to effects of MeHg on PF-EPSPs.
Times to complete block of CF-EPSPs by 100 and 20 uM MeHg were 36 :t 4
and 67 :1: 16 min, respectively, which were similar to those for block of PF-
EPSPs. Thus, MeHg appears to affect the PF-EPSPs and CF-EPSPs similarly.

The resting membrane potentials of Purkinje cells recorded from 24
slices were —60 :t 4 mV under our experimental conditions. Similar to effects
of MeHg on hippocampal CA1 pyramidal neuronal membrane potentials, 100
and 20 11M MeHg first hyperpolarized and then depolarized Purkinje cell
membranes (Figure 5.10). At 100 and 20 11M MeHg, 11 of 15 and 8 of 9
recordings showed a hyperpolarization prior to depolarization of Purkinje cell
membranes. In most cases, decreases in amplitudes of PF-EPSPs or CF-EPSPS
to complete block were usually accompanied with a gradual progressive
depolarization of Purkinje cell membranes. However, injection of current to

restore the membrane potentials to their original levels after PF-EPSPs and

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CF-EPSPs were blocked completely failed to cause recovery of either PF-EPSPS

or CF-EPSPs, indicating that while membrane depolarization caused by MeHg
may contribute to the effects of MeHg on PF-EPSPs and CF-EPSPs, it was not

the primary cause of MeHg-induced block of PF-EPSPs and CF-EPSPS.

Effects of MeHg on somatic action potentials evoked by direct
depolarization of Purkinje cells. A characteristic electrical property of
Purkinje cells is that direct depolarization of Purkinje cell somata by current
injection just above the threshold level through the recording electrode usually
generates regular, repetitive firing. The patterns of repetitive firing can be
changed by alterations of the amplitude and duration of the injection current
pulses (Llinas and Sugimori, 1980a). To determine if MeHg acts directly on
Purkinje cells to affect their electrophysiological activity, its effects on evoked
repetitive firing activity of Purkinje cells were examined. Bath application of
MeHg also altered the patterns of repetitive firing of Purkinje cells. Under
conditions similar to those described by Llinas and Sugimori (1980), injecting
500 - 1000 ms positive current pulses at a level slightly higher than the
threshold generated a form of regular repetitive firing with a frequency of 30 -
4O spikes/second (Figure 5.11). After exposure to 100 uM MeHg, the firing
pattern was changed in several respects as shown in Figure 5.11. First, the

frequency of firing or the number of so-called fast somatic spikes, described by

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Llinas and Sugimori (1980a), was gradually reduced. Second, the late half
portion of the steady-state depolarization became more pronounced and the
fast somatic spikes were virtually abolished (at 5, 10 and 15 min in Figure
5.11); replacing them were small amplitude, burst-like spikes (as indicated by
arrows). Third, at the late stage of exposure to MeHg, oscillatory bursting
activity or so-called depolarizing spike bursts (Llinés and Sugimori, 1980a)
were superimposed on the late half of the steady-state depolarization (20 min
in Figure 5.11). Finally, all responses were blocked completely by MeHg.
Increasing current injection did not restore these responses. Clearly, these
results demonstrated that MeHg acted directly on Purkinje cells to block their
electrophysiological activity.

The question is then what is(are) the primary site(s) of action of MeHg
in blocking PF-EPSPs or CF-EPSPs. To test this, I compared the time-courses
of MeHg-induced block of action potentials and PF-EPSPs evoked by
stimulation of parallel fibers with responses evoked simultaneously by direct
depolarization of Purkinje cell soma through the recording electrode. At 100
uM, MeHg blocked action potentials evoked by stimulating parallel fibers at
a level slightly higher than threshold within 15 min (Figure 5.12 Left). At this
time, slightly increasing the stimulus intensity could restore an action
potential. Ten min later, action potentials evoked at the increased level of
stimulation were blocked again; further increasing stimulus intensity only

evoked a low-amplitude and narrow-duration EPSP or local responses without

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an action potential superimposed on them. The residual synaptic responses
were blocked eventually at 35 min. At the same time, repetitive firing of
Purkinje cells evoked by depolarizing current pulses were first reduced
significantly in number, then changed to burst-like firing and finally blocked
completely at 25 min (Figure 5.12 Right). In almost all recordings, block of
action potentials evoked by stimulating parallel fibers and of repetitive firing
evoked by current injection occurred at the same time, suggesting that they
resulted from the same effect. However, complete block of the residual PF-
EPSPs or local responses required a slightly longer time. The same was true
for effects of 20 uM MeHg on these responses. As shown in Figure 5.13, at 35
min the repetitive firings evoked by depolarizing current pulses were blocked
completely by 20 uM MeHg (Figure 5.13 Right), whereas the residual PF-
EPSPs could still be initiated at 40 min and were blocked completely only after
60 min exposure to MeHg (Figure 13 Left). We also compared time-courses of
block of CF-EPSPs evoked by stimulating climbing fibers with responses
evoked simultaneously by direct depolarization of Purkinje cell soma through
the recording electrode. In Figure 5.14, the somatic repetitive firings were
evoked by a short current pulse (50 ms). At 100 uM, MeHg blocked action
potentials or complex spikes evoked by stimulating climbing fibers and the

somatic repetitive firings evoked by direct depolarization of Purkinje cells

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through current injection similarly. However, block of the residual synaptic
responses evoked by stimulating climbing fibers took slightly longer (Figure
5.14). In this case, complete block of the residual synaptic responses occurred
at 55 min. Thus, these results suggest that the voltage-dependent responses
or action potentials evoked by stimulating parallel fibers, climbing fibers or by
direct depolarization of Purkinje cells were equally sensitive to MeHg.
However, the residual synaptic responses or local responses evoked by
stimulating parallel or climbing fibers were slightly less sensitive to MeHg
than were those voltage-dependent responses. Thus, the primary sites of
action of MeHg in blocking PF—EPSPs and CF-EPSPs appear to be the
postsynaptic Purkinje cells. Figure 5.15 summarizes the times to block of PF-
EPSPs, CF-EPSPs and responses evoked by current injection. MeHg blocked
PF-EPSPs and CF-EPSPs similarly but slightly more slowly than it blocked

somatic responses if the residual synaptic responses are taken into account.

Effects of MeHg on spontaneous activity of Purkinje cells. In
addition to those unique evoked responses, Purkinje cells also displayed
another well-described electrical property-spontaneous firing or autorhythmic
oscillatory activity, which was observed in both extracellular and intracellular
recordings (Llinas and Sugimori, 1980a,b; Aubry et al., 1991; Chang et al.,
1993). In hippocampal slices, spontaneous activity appeared to be less

sensitive to MeHg than were evoked responses in CA1 pyramidal neurons

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(Yuan and Atchison, 1993). To test if this was also the case for Purkinje cells,

effects of MeHg on spontaneous firing activity were examined simultaneously
with those on evoked responses. Under my experimental conditions, in
general, within the first few minutes after penetration of their membranes,
Purkinje cells displayed a mixed form of spontaneous firing including the so-
called Na*-dependent, fast somatic repetitive action potentials, Ca2*-dependent
slow dendritic spikes or bursting, as described by Llinas and Sugimori
(1980a,b) (Figure 5.16). After recordings were stable for 5 - 10 min, the
pattern of spontaneous firing became predominantly the repetitive, fast
somatic spike form. However, after exposure to 100 uM MeHg, initially, the
number of fast somatic spikes was reduced and then the patterns of
spontaneous firing became predominantly autorhythmic bursting activity
separated by interburst hyperpolarizations at 15 - 20 min (Figure 5.17).
Subsequently, all spikes were blocked and the remaining responses were the
slow rate of rise, low-amplitude oscillatory local responses. At the same time,
all action potentials or repetitive spikes evoked by stimulating parallel fibers
or climbing fibers or by direct depolarization of Purkinje cell soma and even by
antidromic stimulation of Purkinje cell axons were blocked as well, except for
residual PF-EPSPs and CF-EPSPS. Later, the remaining responses including
the low-amplitude oscillatory local responses, the residual PF-EPSPs and CF-
EPSPs were also blocked (Figure 5.17 ). These results suggest that all these

somatic action potentials or repetitive spikes either occurring spontaneously

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208

or evoked were equally sensitive to MeHg. Interestingly, in many cases after
responses recorded from intracellular recordings were blocked completely,
withdrawing recording electrodes out of cells could still pick up some
extracellular spontaneous firing activity. Thus, I examined effects of MeHg on
the spontaneous activity obtained from extracellular recordings. Figure 5.18
shows representative extracellular spontaneous firing activity. After exposure
of a transverse slice to 100 uM MeHg for 5 to 30 min, stimulating parallel
fibers could initiate population spikes, as indicated by the arrows. After 40
min, no fithher evoked responses could be observed, however, spontaneous
firing remained until complete block occurred at 60 min. Thus, spontaneous

activity appears to be less sensitive to MeHg than are evoked responses.

209
DISCUSSION

Previously, we demonstrated that MeHg affects both excitatory and
inhibitory synaptic transmission in hippocampal slices. However, a more
sensitive and primary target of MeHg in the CNS is the cerebellum, especially
the cerebellar cortex. Thus, a direct examination of effects of MeHg on
cerebellar synaptic transmission should be especially relevant to its in vivo
neurotoxicity. As a first step, the objective of the present study was to
determine if MeHg differentially affects synaptic transmission between
parallel-fibers or climbing-fibers and Purkinje cells. Acute bath application of
20 and 100 uM MeHg caused a biphasic effect-namely an initial increase of
amplitude followed by a decrease to block of the field potentials recorded from
the molecular layer of cerebellar slices. This pattern was observed for PFVs,
PSRs and CFRs evoked by stimulation of parallel fibers and climbing fibers.
Moreover, this pattern is a characteristic effect of MeHg also seen in
hippocampal slices. MeHg appears to block the glutamate-mediated
postsynaptic responses PSRs and CFRs with a similar time course and more
rapidly than it did PFVs. Intracellular recordings supported this conclusion
as MeHg blocked both PF-EPSPs and CF-EPSPs with similar time courses.
The primary site of action of MeHg in blocking these responses appears to be
the postsynaptic Purkinje cells because MeHg blocked responses evoked by

direct depolarization of Purkinje cell soma with a similar time course to its

210
effects on PF-EPSPs and CF-EPSPs. Moreover, MeHg also hyperpolarized and

then depolarized Purkinje cell membranes and suppressed spontaneous
activity.

Purkinje cells differ from most neurons in the CNS in that a single
Purkinje cell receives two major excitatory synaptic inputs: the parallel fibers
and climbing fibers. One Purkinje cell may make synaptic contacts with as
many as 200,000 parallel fibers. On the other hand, a given Purkinje cell only
makes synaptic contact with one climbing fiber, however, as many as 200
contacts may be formed between each Purkinje cell and each climbing fiber
(Llinas and Walton, 1990). When activated, both synaptic responses can be
recorded easily in the molecular layer of the cerebellar cortex using
extracellular recording techniques. In addition, when parallel fibers are
activated, action potential propagation along the parallel fibers can be picked
up and recorded as the PFV by an extracellular recording electrode in the
molecular layer. As expected, exposure of cerebellar slices to MeHg caused
stimulation and then suppression to complete block of these field potentials.
In hippocampal slices, MeHg caused similar effects on population spikes
recorded from CA1 pyramidal neurons. The early stimulatory effects of MeHg
on hippocampal CA1 excitatory synaptic transmission are apparently due
primarily to a preferential action of MeHg on GABAA receptor-mediated
inhibitory synaptic transmission leading to disinhibition of excitatory synaptic

function (Yuan and Atchison, 1997). The same mechanism may apply to the

2 1 1
stimulatory effects of MeHg on the glutamate-mediated postsynaptic responses

in Purkinje cells such as PSRs evoked by stimulating parallel fibers and CFRs
evoked by stimulating climbing fibers. Purkinje cells also receive inhibitory
inputs directly from two types of GABAergic interneurons- the stellate and
basket cells and indirectly from Golgi cells (Llinas and Walton, 1990). Golgi
cells, which are excited by mossy, climbing and parallel fibers, exert an
inhibitory action on granule cells and indirectly modulate activity of Purkinje
cells. It is possible that this disinhibition in response to MeHg is also
responsible for the early increase in PFV amplitude. It may be also related to
changes in the long-term depression induced by interaction among the parallel-
fiber, climbing-fiber excitatory pathways and Purkinje cells (Sakurai, 1990;
Crepel and Jaillard, 1990; Konnerth et al., 1992; Kano et al., 1992; Linden et
al., 1993; Aiba et al., 1994; Schotter, 1995). The unexpected result is that
MeHg blocked the presynaptic PFVs apparently more slowly than it blocked
the glutamate-mediated PSRs and CFRs. Pathologically, the granule cells are
well-known to be highly sensitive to MeHg (Hunter and Russell, 1954;
Takeuchi et al., 1962; Chang, 1977, 1980; Syversen et al., 1981) and their
axons, which form the parallel fibers, are unmyelinated and injured by MeHg
during chronic exposure. Thus, theoretically, initiation and propagation of
action potentials along parallel fibers should be affected by MeHg at least as
readily as were the postsynaptic responses. It is unclear why PFVs are

relatively less sensitive to MeHg than are PSRs and CFRs. Perhaps, it is in

212

part due to the relatively high N a+ channel density in the presynaptic parallel
fibers making them more resistant to MeHg, or perhaps it simply reflects a
preferential effect of MeHg on either the glutamate release processes from
parallel fibers and climbing fibers or postsynaptic glutamate receptor functions.

To determine further how MeHg caused the effects on the field
potentials, PF-EPSPs and CF-EPSPs were examined using intracellular
recording techniques. Consistent with the results obtained from extracellular
recordings, MeHg blocked both PF—EPSPs and CF-EPSPs with similar time
courses, although the two responses were generated from two different
synaptic pathways with distinct electrophysiological characteristics. The PF-
EPSPs generated by stimulation of parallel fibers are single-peaked and graded
amplitude responses, the simple spike. Each parallel fiber, when activated,
produces a small synaptic current that must sum at the initial segment of
Purkinje cells to produce an action potential. Thus, activation of parallel fibers
leads to generation of voltage- and Na*-dependent simple spikes graded as a
function of the summation of synaptic currents from many parallel fiber
synapses. Conversely, the CF-EPSPs generated by stimulation of climbing
fibers are all or none Ca2*-dependent responses (Crepel and Delhaye-
Bouchaud, 1978; Llinas and Sugimori, 1980a,b; Crepel et al., 1981, 1982;
Humura et al., 1985; Anderson, 1989; Llinas and Walton, 1990; Llano et al.,
1991; Stuart and Hausser, 1994). Stimulation of climbing fibers produces a

large synchronized depolarization of Purkinje cell dendrites, which then

213

activates the dendritic Ca2+ channels to initiate the slow dendritic Ca2+ spikes.
It is not surprising that MeHg blocked both responses since it reduces currents
carried through voltage-dependent Na+ channels (Shrivastav et al., 1976;
Quandt et al., 1982; Shafer and Atchison, 1992; Leonhardt et al., 1996) and
Ca2+ channels (Shafer and Atchison, 1989; Shafer et al., 1990; Shafer and
Atchison, 1991, 1992; Hewett and Atchison, 1992; sirois and Atchison, 1996;
Leonhardt et al., 1996). The similarity in blocking both responses suggests
that MeHg acts via a similar mechanism to block both PF-EPSPs and CF-
EPSPs. In CF-EPSPs recordings, on the other hand, the early suppression of
the depolarization plateau and block of complex spikes almost always occurred
before block of the antidromically-activated spikes, suggesting that the
orthodromically-activated synaptic responses may be more sensitive to MeHg
than were antidromically-activated responses and implying that effects of
MeHg on the process of synaptic transmission were involved. Unlike the
effects of MeHg on EPSPs recorded from the CA1 region of hippocampal slices,
overall, MeHg did not cause a significant early stimulatory effect on PF-EPSPs
and CF-EPSPs although it caused a slight and transient early increase in some
slices. These results were also inconsistent with those obtained from
extracellular recordings, in which MeHg caused a significant early increase
prior to suppression of the field potentials. The difference between results
obtained from extracellular and intracellular recordings may indicate that

MeHg does not affect the response of an individual Purkinje cell to a given

214

stimulus, but rather affects the recruitment of additional Purkinje cells which
fire synchronously at the early stage of exposure to MeHg.

To determine if MeHg preferentially acts on Purkinje cells to block PF-
EPSPs or CF-EPSPs, effects of MeHg on responses evoked by direct
depolarization of Purkinje cells with current injection at the somata were
examined and compared with those of MeHg on PF-EPSPs and CF-EPSPs.
Normally, injection of a short (50 - 100 ms) or long current pulse (500 - 1300
ms) at the threshold levels in Purkinje cell somata generates regular, repetitive
firing, fast somatic spikes. As the intensity of current injection pulse is
increased, particularly for long current pulses, the regular repetitive firing
form was replaced, near the end of the pulse, by the complex, low-amplitude
spike burst-depolarizing spike burst (Llinas and Sugimori, 1980a). The fast
somatic spike is a low-threshold, voltage- and Na*-dependent response, which
is blocked by removal of extracellular N a* or by application of TTX. The
depolarizing spike burst, on the other hand, is a slow rate of rise, high-
threshold, voltage- and Ca2*-dependent response, which is TTX-insensitive and
blocked by removal of extracellular Ca2+ or by application of Ca2+ channel
blockers such as Coz“, Cd2+ or an+ (Llinas and Sugimori, 1980a,b; Aubry et al. ,
1991; Chang et al., 1993). However, in the presence of MeHg and without
changing the amplitude of threshold current injection, the patterns of the
repetitive firing induced were altered such that they resembled responses

caused by increasing current pulses under normal conditions. Thus, MeHg

215

appears to alter Purkinje cell membrane ion conductances in the same way
that they are altered by increasing stimulus intensity. Normally, generation
of this Ca2*-dependent depolarizing spike burst requires higher stimulus
intensity because of its high-threshold nature. However, exposure of slices to
MeHg induced an identical response even at normal threshold stimulus level.
That suggests that MeHg may initially change the threshold level for
activation of Ca2+ channels. Again, as it did with CA1 pyramidal cells in
hippocampal slices, MeHg blocked all voltage-dependent responses including
both Na*- and Ca2*-dependent spikes evoked by stimulation of parallel or
climbing fibers or by direct current injection at Purkinje cell somata with a
similar time course. This suggests that MeHg primarily acts at the Purkinje
cells via a similar mechanism to block these voltage-dependent responses.
However, a slightly longer time was required for complete block by MeHg of
the synaptically-activated local responses or the residual PF-EPSPs or CF-
EPSPs compared with those required for blocking voltage-dependent responses.
This implies that glutamate receptor functions may be also affected by MeHg,
although they appear to be relatively less sensitive to MeHg than are those
voltage-dependent channels. This is consistent with the results that MeHg
blocked orthodromically-activated synaptic responses more rapidly than it did
antidromically-activated responses. In addition, the prolonged latencies from
stimulus artefact to onset of PF-EPSPs and CF-EPSPs suggest that the current

conduction from parallel fibers or climbing fibers to the dendrites of Purkinje

216

cells were affected by MeHg. Thus, once again MeHg apparently acts at
multiple sites to block synaptic transmission between parallel fibers or
climbing fibers and Purkinje cells. The primary sites appear to be the
postsynaptic Purkinje cells, although the presynaptic actions may be also
involved.

In addition to effects of MeHg on the evoked repetitive firing of Purkinje
cells, MeHg also changed the patterns of spontaneous repetitive firing of
Purkinje cells. Normally, the spontaneous repetitive firing consists of
predominantly the Na*-dependent fast somatic spikes and some low-amplitude
Ca2*-dependent slow dendritic spike bursts (Llinas and Sogimori, 1980a). After
exposure to MeHg, the patterns of spontaneous firing changed to
predominantly Ca2*-dependent autorhythmic burst. This effect of MeHg is very
similar to effects of TTX on Purkinje cell spontaneous firing (Aubry et al. , 1991;
Chang et al., 1993). Application of TTX, a specific N a"-channe1 blocker, to
cerebellar slices suppressed Na‘” spikes of Purkinje cells and induced Ca2*-
dependent oscillatory firing activity. This oscillatory firing activity was
thought to be maintained by an intrinsic property of Purkinje cells inasmuch
as it remained after block of both excitatory and inhibitory synaptic inputs to
Purkinje cells (Chang et al. , 1993). The mechanism proposed to be responsible
for TTX-induced oscillatory activity of Purkinje cells was block of Na“ channels
leading to activation of the Na‘lCa2+ exchanger with a net gain of intracellular

Ca2+ (Aubry et al., 1991; Chang et al., 1993). Compared with TTX, MeHg

217

blocks both N a+ and Ca2+ channels, and increases [Ca2+], in a variety of cells
(Komulainen and Bondy, 1987; Kauppinen et al., 1989; Hare and Atchison,
1992b; Denny et al., 1993; Hare et al., 1993, 1995) including primary cultures
of cerebellar granule cells (Marty and Atchison, 1997). Thus, the MeHg-
induced oscillatory burst activity may be related to an effect on regulation of
[Ca2+]i of Purkinje cells. Perhaps, initially, MeHg blocks Na+ channels in the
same way as TTX does to activate the Na*/Ca2+ exchanger (Aubry et al., 1991;
Chang et al., 1993), which unmasks intrinsic oscillatory activity of Purkinje
cells by an unknown mechanism, increases [Ca2+]i and depolarize Purkinje cell
membranes to generate the slow Ca2+ spikes. Subsequently, the increase in
[Ca2+], activates Ca21-dependent K“ channels leading to hyperpolarization of
Purkinje cells to return their membrane potentials toward the resting level.
As the K” channels close, another cycle begins. However, at the late stage of
exposure of slices to MeHg, all action potentials or voltage-dependent responses
were blocked and what was left were only those low-amplitude oscillatory local
responses. These local oscillatory responses were later blocked completely
along with the synaptically-activated local responses (residual PF-EPSPS and
CF-EPSPs) at the same time, suggesting that the Ca2*-dependent oscillatory
burst activity of Purkinje cells is of dendritic origin. This is consistent with
the conclusion of Llinas and Sugimori (1980a,b). Spontaneous firing activity
is also observed in extracellular recordings. Interestingly, MeHg blocked the

spontaneous responses more slowly than it did the evoked responses. In

218

addition, in many cases, after evoked responses recorded by intracellular
recording were blocked, the extracellular spontaneous responses often
remained, suggesting that the spontaneous responses were less sensitive to
MeHg than were evoked responses. This is consistent with the effects of MeHg
on neuromuscular transmission (Atchison and Narahashi, 1982), in which
spontaneous release of ACh remained observable at the time of evoked release
of ACh were blocked completely by MeHg. Thus, mechanisms responsible for
block by MeHg of spontaneous and evoked responses were differently.
Moreover, it appears that MeHg takes longer time to block extracellular
responses than it does intracellular responses. This may be due simply to (1)
in intracellular recording, penetration of cell with recording electrode causes
injury of membrane, which accelerates the action of MeHg on cell; (2)
accessibility of MeHg to cells located on the surface and in the deep tissue of
slices differs; extracellular recording electrode picks up firing from a population
of cells which may include those located in both surface and deep tissues. For
those cells in the deep tissue, it will take a longer time for MeHg to access
them, and hence a longer time to block their responses.

In conclusion, MeHg caused biphasic effects on synaptic transmission
between parallel-fibers or climbing-fibers and Purkinje cells in cerebellar slices.
MeHg appears to act primarily at the postsynaptic Purkinje cells to cause
these effects because it blocked responses evoked by directly depolarizing

Purkinje cells. However, multiple actions including hyperpolarizing and

219

depolarizing the Purkinje cell membranes, blocking current conduction and
affecting glutamate receptor functions may be also involved in these effects.
MeHg blocked spontaneous firing of Purkinje cells and acted in a manner
similar to TTX to induce a Ca2*-dependent, spontaneous oscillatory burst
activity in Purkinje cells. This is superficially consistent with the findings that
MeHg increases [Ca2+]i in a variety of cell types. In general, the effects of
MeHg on electroresponsesiveness of Purkinje cells in cerebellar slices are

similar to those on CA1 pyramidal cells in hippocampal slices.

CHAPTER SIX

SUMMARY AND CONCLUSION

220

221
A. SUMLIARY

Previous studies have extensively examined effects of MeHg on
peripheral synaptic transmission at the neuromuscular junction and autonomic
ganglia. However, little is known of the effects and underlying mechanisms
of MeHg on central synaptic transmission. Using extracellular recording
techniques I previously demonstrated that acute bath application of MeHg to
hippocampal slice preparations disrupted CA1 neuronal membrane excitability
and synaptic transmission (Yuan and Atchison, 1993, 1994). However, due to
the limitations of extracellular recording techniques, it is difficult to identify
where and how MeHg caused these effects. In addition, to date, there are no
reports of effects of MeHg on cerebellar synaptic transmission even though it
is well-known that the cerebellum, and especially the cerebellar cortex, is one
of the major neurotoxic targets of MeHg in the CNS. Also, it is unclear
whether or not the data obtained following acute exposure of the hippocampus
to MeHg can be used to predict effects of MeHg on other CNS targets. Thus,
the present study was designed primarily to compare and characterize the in
vitro effects of acute exposure to MeHg on synaptic transmission in both
hippocampal slices and cerebellar slices and to explore the potential
mechanisms underlying these effects. To do this, conventional
electrophysiological recording methods including extracellular and intracellular
microelectrode recording, SEVC recording and iontophoresis techniques were

used.

222

Concentrations of MeHg used in this dissertation varied from 4 to 500
uM, which are similar to those used in other studies in isolated cells, tissues,
etc. The concentrations of 4 and 20 uM MeHg was used because they are
within the range of those reported to be found in blood of patients poisoned
with MeHg during acute exposure episode in Iraq. My previous results have
demonstrated that the effects of MeHg on those field potentials recorded from
CA1 region of hippocampal slices were concentration- and time-dependent.
Moreover, the characteristics of effects of MeHg at lower (4 and 20 11M) or
higher concentrations (100 and 500 11M) on hippocampal synaptic transmission
are generally similar (Yuan and Atchison, 1993, 1994), except that latencies
to onset of effects of MeHg occurred at lower concentrations of MeHg were
much longer than those that occurred at higher concentrations of MeHg. Thus,
relatively higher concentrations of MeHg (100 and 500 uM) were also used in
the present study to shorten the latency to onset of effects of MeHg on
electrophysiological responses and to examine the concentration-dependence
of any responses observed. This is especially helpful when intracellular
recordings were made in relatively small neurones, because it is usually
difficult to maintain a stable, long duration of intracellular recording in small
neurons.

Several new findings presented in this dissertation are consistent with
the following conclusions: (1) acute bath application of MeHg blocked central

synaptic transmission in brain slice in a concentration- and time-dependent

223
manner; (2) MeHg initially stimulates and then suppresses excitatory synaptic

transmission in both hippocampal and cerebellar slices; (3) MeHg
hyperpolarizes and then depolarizes both hippocampal CA1 pyramidal and
cerebellar Purkinje cell membranes; (4) it appears that inhibitory synaptic
transmission is more sensitive to MeHg than is excitatory synaptic
transmission in hippocampal slices, which may be primarily responsible for the
early apparent stimulatory effects of MeHg on hippocampal synaptic
transmission. (5) MeHg appears to act at multiple sites to block central
synaptic transmission, however, the primary sites of action of MeHg on
synaptic transmission in the tested synaptic pathways (Schaffer collateral-CA1
pyramidal pathway in hippocampal slice, parallel fiber- and climbing fiber-
Purkinje cell pathways in cerebellar slices) appear to be the postsynaptic
neurons (CA1 pyramidal and Purkinje cells); (6) MeHg appears to affect
voltage-dependent responses more rapidly than does synaptically-activated
responses; (7) MeHg appears to block evoked responses more rapidly than
does the spontaneous responses.

One of the characteristic effects of MeHg on central synaptic
transmission is that MeHg caused a concentration- and time-dependent
biphasic effect on synaptic transmission in both hippocampal and cerebellar
slices. In hippocampal slices, 4 - 500 uM MeHg initially increased and then

suppressed amplitudes of population spikes and EPSPs to complete block.

Similarly, MeHg caused a transient stimulation prior to suppression of field

 

224

potentials recorded from the molecular layer of cerebellar slices by activation
of either the parallel or climbing fibers. Thus, the biphasic effects of MeHg on
synaptic transmission in both hippocampal and cerebellar slices appear to be
a general feature of effects of MeHg in the CNS. However, such biphasic
effects of MeHg on synaptic transmission were not limited in the CNS; they
were also observed at peripheral synapses. At neuromuscular junctions, MeHg
first stimulated and then suppressed to block of spontaneous release of ACh
from the presynaptic nerve terminals (Juang and Yonemura, 197 5; Juang,
1976b; Atchison and Narahashi, 1982; Atchison, 1986, 1987; Traxinger and
Atchison, 1987a, b; Levesque and Atchison, 1987, 1988). In addition, in some
cases, MeHg also transiently increased EPP amplitude prior to block (Manalis
and Cooper, 1975; Juang, 1976b; Traxinger and Atchison, 1987b). Thus, the
biphasic changes caused by MeHg in synaptic transmission may be a general
characteristic of its effects in both central and peripheral nervous systems.
The question is what factor(s) is(are) responsible for these early stimulatory
effects of MeHg on central synaptic transmission. Data obtained from
intracellular microelectrode and sSEVC recordings in CA1 pyramidal neurons
of hippocampal slices indicated that an effect of MeHg on the resting
membrane potential was not a major factor in causing the early stimulatory
effects on hippocampal synaptic transmission, because MeHg affected synaptic

responses evoked under both current clamp (EPSPs) and voltage clamp

(EPSCs) similarly, and still caused a biphasic effect on EPSCs even though the

 

225

CA1 pyramidal cell membrane was voltage-clamped at its resting membrane
potential. However, by comparison of effects of MeHg on IPSP or IPSCs and
EPSPs or EPSCs, I found that a preferential block by MeHg of inhibitory
synaptic transmission was primarily responsible for the early apparent
stimulatory effect of MeHg on hippocampal synaptic transmission.
Pretreatment of hippocampal slices with bicuculline, the GABAA receptor
antagonist, completely eliminated the initial increase in amplitudes of
population spikes and EPSPs induced by MeHg. Normally, neuronal
membrane excitability of central neurons is regulated by the integrated activity
from both excitatory and inhibitory inputs. If inhibition on a given cell is lost
or reduced for any reason, this cell will become overexcited. Perhaps, MeHg
preferentially suppresses GABAA (maybe also GABAB) receptor-mediated
inhibitory synaptic transmission, which results in disinhibition of excitatory
synaptic transmission via both pre- and postsynaptic mechanisms and leads
to an initial hyperexcitation of CA1 pyramidal cells. Three possible events
might occur following the disinhibition: ( 1) increased release of transmitter
from presynaptic terminals due to loss of presynaptic inhibition; (2) individual
neurons become more excited; and (3) more neurons are recruited to fire
synchronously in response to a given stimulus. One or all of the these events
may be involved in the early stimulatory effect of MeHg on hippocampal CA1
synaptic transmission. To date, no such experiments have been done in the

cerebellar slices. It is reasonable to predict that effects of MeHg on inhibitory

226

synaptic transmission in cerebellar slices will be also primarily responsible for
the early stimulation of cerebellar synaptic transmission, because similar
inhibitory synaptic circuits exist in the cerebellum. However, the lack of an
early increase in amplitude of both PF-EPSPs and CF-EPSPs recorded from
individual Purkinje cells suggests that the early stimulatory effects on field
potentials recorded from the molecular layer of cerebellar slices may be due
primarily to increased recruitment of more Purkinje cells to fire synchronously.
In neuromuscular junction and autonomic ganglion preparations, the early
increase in spontaneous release of ACh or frequency of MEPPs was postulated
to be the result of depolarization of the presynaptic nerve terminal membranes,
which caused the opening of N a" and Ca2+ channels leading to increasing Ca2+
influx, and subsequent increases in spontaneous release of ACh (Atchison and
Narahashi, 1982).

The second feature of effects of MeHg on central synaptic transmission
presented in this dissertation is that MeHg initially reduced neuronal
membrane excitability or altered the threshold level for initiation of action
potentials and subsequently depolarized neuronal membranes leading to
complete block of synaptic transmission. In hippocampal slices, at the time
action potentials evoked by threshold stimulation of Schaffer collaterals or by
directly depolarizing CA1 pyramidal cells were initially blocked, simply
increasing the stimulus intensity slightly could temporarily again initiate

action potentials. In addition, the amplitude of action potentials recorded just

227

prior to conduction block remained essentially unchanged (all or none manner).
The same was true for effects of MeHg on action potentials evoked by
stimulating the parallel fibers or CF-EPSPs evoked by stimulating the climbing
fibers in cerebellar slices. Normally, an action potential is generated at the
initial segment of the axon when the neuronal membrane is depolarized to
reach a threshold level that leads to an explosive opening of Na” channels.
Theoretically, the threshold for a given cell can be changed by a variety of
factors. MeHg may alter the distance between resting membrane potentials
and the threshold by hyperpolarizing the cell membrane such that the resting
membrane potential is farther from its threshold level or by moving the
threshold level farther from a given resting membrane potential level. In
either situation, a higher stimulus intensity will be required to initiate action
potentials. MeHg did cause an initial hyperpolarization prior to depolarization
of hippocampal CA1 pyramidal and cerebellar Purkinje cell membranes,
however, the fact is that the resting membrane potentials were very close to
the pre-MeHg treatment control level at the time action potentials evoked by
threshold stimulation were blocked. Therefore, hyperpolarization alone cannot
explain how MeHg changes the threshold level. Another possibility is that
MeHg moves the threshold level farther from the resting membrane potentials.
Using voltage-clamp techniques in squid axons, Shrivastav et al., (1976)
demonstrated that 25 - 200 uM MeHg caused a steady increase in the

threshold for initiation of action potentials and eventual block of conduction

228

without significant changes in the resting membrane potentials. The authors
proposed that the increased threshold levels were due to MeHg-induced
suppression of both peak Na+ currents and steady-state K+ currents. If this is
also true in hippocampal and cerebellar slice preparations, the amplitude of
action potentials would be expected to decline progressively. This is true for
the effects of MeHg on action potentials at the late stage of exposure to MeHg.
However, at the early stage of exposure to MeHg in my experiments,
amplitudes of action potentials just before block remained essentially
unchanged and moreover, amplitudes of action potentials regenerated by
increased stimulus were the same as those before block. One possible
explanation is that MeHg may initially change the open probability of N a+
channels or increase the threshold for opening Na+ channel via certain
mechanisms to result in the increased threshold for initiation of action
potentials. Another possible explanation is that MeHg may suppress
neurotransmitter release from the presynaptic terminals so that a higher
stimulus intensity is required to release enough transmitter to act at the
postsynaptic receptors and cause membrane depolarization toward the
threshold level. Additionally, effects of MeHg on Ca2+ channels or Ca2+
homeostasis may be also involved, since Ca2+ plays a crucial role in both
neurotransmitter release and maintenance of neuronal membrane excitability.

Much is known of effects of MeHg on resting membrane potentials in a

variety of types of cells. Exposure to MeHg caused depolarization of muscle

229
fibers (Juang, 1976), squid axon membranes (Shrivastav et al. 1976),

neuroblastoma cell membranes (Quandt et al., 1982) and synaptosomes
(Kauppinen et al. 1989; Hare and Atchison, 1992). Results presented in this
dissertation consistently demonstrated that MeHg initially hyperpolarizes and
then depolarizes both hippocampal CA1 pyramidal cell and cerebellar Purkinje
cell membranes in a concentration- and time-dependent manner. Actually, a
similar effect also occurred in synaptosomes exposed to 1 11M MeHg (Hare and
Atchison, 1992). Leonhardt et al. (1996) also demonstrated that MeHg caused
a biphasic change, a transient inward current followed by a larger, sustained
outward current, in the holding membrane current or the "resting membrane
current" at the potential of -80 mV in 25 % of the experiments in rat dorsal
root ganglion neurons. The ionic conductances responsible for the inward and
outward currents are unknown. However, the authors hypothesized that these
effects may be related to functional changes in some ion channels such as K+
and Cl' channels that are regulated directly by increased [Ca2+li. The same
may be true for effects of MeHg on hippocampal CA1 pyramidal cells. The
initial membrane hyperpolarization induced by MeHg may be due to activation
of Ca2*-sensitive K” channels as result of MeHg-induced elevation in [Ca2+],.
In rat synaptosomes, NG108-15 cells and primary culture of cerebellar granule
cells, MeHg caused two phases of elevation in [Ca2+], (Denny et al.1993; Hare
and Atchison, 1995; Marty and Atchison, 1997). The first phase of increase in

[Ca2+]i was believed to be due to release of Ca2+ from intracellular Ca2+ pools

230
(eg. IP3-sensitive pool) (Hare and Atchison, 1995; Marty and Atchison, 1997).

It was believed that in NG108-15 cells, mobilization of an intracellular Ca2+
pool activates K+ channels and subsequently results in membrane
hyperpolarization (Higashida and Brown, 1986). In addition, there is a
difference in the effects of MeHg on resting membrane potentials at somatic
motor end-plates or autonomic ganglia and those presented in this dissertation
in that lower concentrations of MeHg (40 - 100 11M) had no effects on
postsynaptic resting membrane potentials (J uang and Yonemura, 1975; J uang,
1976; Atchison and N arahashi, 1982). Depolarization of cell membranes only
occurred at very high concentrations (500 11M) of MeHg (Shrivastav et al.,
1976). However, in both hippocampal and cerebellar slices, lower
concentrations of MeHg (4 - 20 11M) caused typical hyperpolarization and
subsequent depolarization of CA1 pyramidal and Purkinje cell membranes.
Differences between the effects of MeHg on resting membrane potentials in
peripheral and central postsynaptic membranes may be due to differences in
site of cells, underlying conductances (for example skeletal muscle cells have
a very large endogeous Cl' conductance) or duration and manner of exposure
to MeHg. Alternatively, it may be that excitable cells in the CNS are more
sensitive to MeHg than those at peripheral synapses, or at least with regards
to the resting membrane potentials of postsynaptic muscle fibers.

MeHg appears to block voltage-dependent responses more rapidly than

synaptically-activated local responses in both hippocampal and cerebellar

231

slices. At the time that action potentials evoked by threshold stimulation of
Schaffer collaterals in hippocampal slices were blocked by MeHg, EPSPs
remained observable. Similarly, when action potentials (both N a”-dependent,
fast somatic spikes and Ca2*-dependent, slow dendritic spike bursts) evoked by
stimulating parallel or climbing fibers were blocked completely, synaptically-
activated local responses or EPSPs remained observable. These results suggest
that the voltage-dependent responses may be more sensitive to MeHg than are
the synaptically-activated or glutamate-mediated responses. Thus, MeHg may
affect both voltage-gated ion channels and glutamate receptors to block
synaptic transmission. Further experiments to compare effects of MeHg on
responses evoked by iontophoretic application of glutamate and by injection of
depolarizing current at Purkinje cell soma should be designed to test this
conclusion.

MeHg blocks both evoked and spontaneous responses in hippocampal
and cerebellar slices. However, MeHg appears to block the evoked responses
more rapidly than it does the spontaneous responses. In many cases, after
evoked responses were blocked by MeHg, spontaneous responses remained
observable in both intracellular and extracellular recordings. Similar
phenomena were also observed in the neuromuscular preparations (Atchison
and Narahashi, 1982). At neuromuscular junction, normal amplitude and
duration of MEPPs remained to occur at the time EPPs were blocked. These

results suggest that the mechanisms by which MeHg blocks the evoked and

232

spontaneous responses may differ. Perhaps, the differential effects of MeHg
on evoked and spontaneous responses are resulted from different requirements
and process for spontaneous and evoked release of neurotransmitters.

Apparently, multiple sites and effects were involved in MeHg-induced
disruption of central synaptic transmission in both hippocampal and cerebellar
slices, however, the mechanisms responsible for these effects appear to be
predominantly postsynaptic. Thus, the mechanisms by which MeHg blocks
neurotransmission in brain slices and at neuromuscular junction appear to
differ; the effects of MeHg on transmission at the neuromuscular junction are
generally considered to be primarily presynaptic. The difference between
effects of MeHg on neurotransmission in brain slices and neuromuscular
junction may be due to their different synaptic components. In central
synapses, both pre— and postsynaptic parts consist of the neuronal components,
while at neuromuscular junction, the postsynaptic components are muscle
fibers, which may be more resistant to MeHg than are neurons.

In CF-EPSP recordings, MeHg almost always blocked the steady-state
depolarization evoked by stimulating the climbing fibers first prior to
suppression to block of N a"-dependent action potentials evoked by antidromic
stimulation of Purkinje cell axons, i.e. the orthodromically-activated synaptic
or climbing fiber responses are more sensitive to MeHg than are the
antidromically-activated responses. These results further support the

conclusion that effects of MeHg on synaptic process are involved. These results

233

may also indirectly imply that the Ca2+-dependent responses (climbing fiber
responses) are more sensitive to MeHg than are Na*-dependent responses
(antidromically-evoked action potentials), because voltage-activated Ca2+ and
K+ channel currents were five times more sensitive to MeHg than were voltage-
activated Na+ channel currents in dorsal root ganglion neurons (Leonhardt et
al., 1996).

Thus, the in vitro effects of MeHg on synaptic transmission in the tested
pathways in hippocampal and cerebellar slices are generally similar. However,
differences between effects of MeHg on hippocampal synaptic transmission and
cerebellar synaptic transmission also existed at least in the following aspects:
(1) MeHg appears to block evoked responses in Purkinje cells more rapidly
than it does those evoked in hippocampal CA1 pyramidal cells, especially at
lower concentrations of MeHg (Table 6.1, 6.2). However, the results obtained
from hippocampal slices and cerebellar slices in this dissertation were
conducted in separated experiments. Thus, direct comparison under similar
conditions may be required before making a final conclusion about their
sensitivity to MeHg; (2) due to Purkinje cells receiving inputs from both
parallel and climbing fibers, characteristic effects of MeHg on repetitive firing
of Purkinje cells which occurred spontaneously or was evoked by injection of
depolarizing current were not observed in the CA1 pyramidal cells; and (3) in
most hippocampal slices, MeHg caused an initial increase in amplitudes of

EPSPs evoked by activation of Schaffer collaterals, whereas in most cerebellar

234

slices, MeHg did not induce early increase in amplitudes of PF-EPSPs or CF-
EPSPs.

Effects of MeHg on the central synaptic transmission in brain slices also
share many similarities with those of MeHg on peripheral neuromuscular
transmission such as similar time courses, concentration-dependence and

reversibility.

235

Table 6.1. Comparison of times to MeHg-induced block of
population spikes (PSs) and field excitatory postsynaptic
potentials (fEPSPs) in the CA1 region of hippocampal
slices.

 

 

 

 

 

 

[ PSs 139 a; 16(5/8)“ 41 :1: 4(8) 9 :t 0.8 (11)
l fEPSPs 177 :l: 3(3/8) 42 4 4(7) 9 :l: 0.5 (8)

 

 

L

 

 

 

" Mean :1: SE(n), fraction (5/8 or 3/8) means that in 5 or 3 of 8
slices, PSs or fEPSPs were blocked completely during 180
min application of 20 uM MeHg. The mean values are
averaged based on the 5 or 3 experimental results. Actual
times to block of PSs or fEPSPs by 20 pM should be longer
than these means.

Table 6.2. Comparison of times to MeHg-induced block
of parallel-fiber responses (PFRs), parallel-fiber post-
synaptic responses (PSRs) and climbing-fiber responses
(CFRs) in cerebellar slices.

 

 

 

 

 

 

 

 

 

 

 

MeHg (11M) 20 100 500
PSRS 110 :1: 27(7)a 40 :1: 4(8) 6 :t 0.5(3)
PFRs 200 :1: 26(7) 61 3: 3(9) 10 :t 0.5(5)
CFRs 113 :t 15(6) 47 :1: 3(8) NDb

 

 

“ Mean 1 SE(n) min.
b Not determined.

236

Table 6.3. Comparison of times to MeHg-induced block of
action potentials evoked by stimulating Schaffer collaterals
(APSch) and by current-injection (APSoma) in CA1 pyramidal
cells of hippocampal slices.

 

 

 

 

 

 

 

 

 

 

MeHg (11M) 20 100
APSch 136 :t 19(9)a 47 :1: 6(12)
APSoma 142 :1: 18(9) 49 :1: 6(12)

 

a Mean :1: SE(n).

Table 6.4. Comparison of times to MeHg-induced block of
parallel fiber excitatory postsynaptic potentials (EPSPPF),
climbing fiber excitatory postsynaptic potentials (EPSPCF)
and repetitive firing of Purkinjec ells evoked by current
injection (AP ) in cerebellar slices.

Soma

 

 

 

 

 

 

 

 

MeHg (pM) 20 100
EPSPPF 57 :1: 6(3)“ 33 :1: 7(7)
EPSPCF 60 :1: 8(7) 36 :t 4(8)
APSoma 36 :1: 4(3) 23 :9: 5(5)

 

 

 

 

 

a Mean :1: SE(n) min.

237

B. Conclusion.

Acute bath application of MeHg caused a concentration- and time-
dependent biphasic effect on central synaptic transmission in both hippocampal
and cerebellar slices. Multiple effects or sites of action of MeHg, including
presynaptic mechanisms, nonspecific membrane depolarization, suppression of
current conductances and inhibitory synaptic transmission, are apparently
involved, however, the primary sites of action of MeHg on central synaptic
transmission appear to be the postsynaptic, which is different from those of
effects of MeHg on peripheral neurotransmission at neuromuscular junction
and autonomatic ganglia. The effects of MeHg on synaptic transmission in
both hippocampal and cerebellar slices are similar in terms of the time course
and concentration-dependence. This result suggests that under in vitro
conditions and acute exposure, hippocampal pyramidal and cerebellar Purkinje
cells, and perhaps other central neurons, may have similar sensitivity to
MeHg. Also, these effects are also generally similar to those of acute exposure
to MeHg of neuromuscular junctions in terms of the time course, concentration-
dependence and reversibility. Thus, a similar and nonspecific mechanism may
be responsible for acute effects of MeHg on neurotransmission in both central

and peripheral nervous systems.

238

C. Future direction.

In short term, the following questions should be asked. (1) are the
inhibitory synaptic circuits also more sensitive to MeHg than are the excitatory
synaptic circuits in cerebellar slices? If so, do efiects of MeHg on the inhibitory
synaptic transmission contribute to the early stimulatory effects of MeHg on
cerebellar excitatory synaptic transmission? (2) Are Cab-dependent responses
more sensitive to MeHg than are Na*-dependent responses? Earlier block of
depolarization plateau evoked by stimulation of the climbing fibers than action
potentials evoked antidromically indirectly implied that Ca2*-dependent
responses may be more sensitive to MeHg than Na*-dependent responses; (3) Are
cerebellar granule cells functionally more sensitive to MeHg than are Purkinje
cells? and (4) are there any relationships between effects of MeHg on Ca?+
homeostasis and synaptic transmission?

In long term, since previous studies of effects of MeHg on Ca2+
homeostasis, synaptic transmission and ion channels (Ca2+ channels) were
performed in different experimental systems, a combination of Ca2+ imaging
techniques, electrophysiological techniques and molecular biological techniques
should be used to study effects of MeHg on central synaptic transmission in
brain slices. In addition, previous and present studies primarily focus on the
in vitro acute effects of MeHg on synaptic transmission, future research should

be designed to examine the chronic effects of low concentrations of MeHg on

239

central synaptic transmission using brain slice cultures and eventually in vivo

systems.

240
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