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This is to certify that the

dissertation entitled
MODIFICATION OF IONIC CONDUCTANCE AT THE NEUROMUSCULAR
JUNCTION FOLLOWING EXPOSURE TO THE FARALYTIC AGENT

2,4-DITHIOBIURET.
presented by

JOHN MARTIN SPITSBERGEN

has been accepted towards fulﬁllment
of the requirements for

DOCTORATE degree in PHARMACOLOGY /TOXICOLOGY

 

 

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MODIFICATION OF IONIC CONDUCI‘ANCE AT THE
NEUROMUSCULAR JUNCTION FOLLOWING EXPOSURE TO THE
PARALYTIC AGENT 14.13111110an
By

John Martin Spitsbergen

A DISSERTATION

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

DOCTOR OF PHIIDSOPHY

Department of Pharmacology and Toxicology and Neuroscience Program

1991

ABSTRACT
MODIFICATION OF IONIC CONDUCTANCE AT THE
NEUROMUSCULAR IUNCIION FOLLOWING EXPOSURE TO THE
PARALYTIC AGENT 2,4-DITHIOBIURET
By

John Martin Spitsbergen

Rats treated with small daily doses of 2,4-dithiobiuret (DTB) develop a
delayed onset neuromuscular weakness following 4 to 6 days of treatment. Analysis
of quantal release of acetylcholine using nerve-muscle preparations taken from rats
exhibiting muscle weakness following chronic treatment with DTB, demonstrated a
decrease in quantal content of evoked end-plate potentials, a decrease in the
frequency of spontaneously occurring miniature end-plate potentials and a
prolongation of rise and decay times for end-plate potentials and miniature end-plate
potentials. The studies described herein were designed to answer two questions.
First, at what time following exposure to DTB can the earliest alterations in
transmission be detected? Second, can the alteration of rise and decay times for
synaptic potentials be attributed to effects of DTB on current ﬂowing through
acetylcholine receptor-gated channels at the neuromuscular junction? To answer
these questions synaptic potentials, synaptic currents and membrane noise induced
by iontophoretic currents were studied in hemidiaphragm preparations from rats 1,
4, 8, 24 hours and 7 days following treatment with DTB, or from control rats and
exposed to DTB in the bathing medium. Single-channel currents were recorded

using patch voltage clamp techniques with a clonal cell (G8) line exposed to DTB

in the growth medium. Results of these studies indicate that transmitter release is
altered in hemidiaphragm preparations taken from rats as early as 1 hour after
treatment, or within minutes following exposure of control muscles to DTB in the
bathing medium. Kinetic analysis of the decay of synaptic currents in muscles from
rats treated with DTB and in muscles from control rats exposed to DTB in the
bathing medium revealed a shortening in the decay of these currents. Analysis of
single-channel open- and closed-times using patch voltage clamp and ﬂuctuation
analysis, indicated that exposure to DTB decreased single-channel open-time. In
conclusion, soon after exposure to DTB is initiated alterations in both transmitter
release and the kinetics of current decay can be detected. These alterations, many
of which are observed in muscles taken from chronically treated rats exhibiting
muscle weakness, appear to be early effects of DTB, which with continued exposure

could lead to the observed weakness.

Dedicated to my wife Sandy and my parents for all their help and support.

ACKNOWLEDGMENTS

I am thankful for the assistance of my entire committee for their input and
guidance throughout my studies. Thanks to all the students, student workers and
technicians with whom I worked for making my stay in the department so enjoyable.
Special thanks to Tim Shafer, Sandy Hewett, Mike Hare, Paul Levesque, Mike
Denny and Lynne Ireland for many stimulating discussions (both scientiﬁc and
nonscientific). Most of all I want to acknowledge the assistance of Dr. William
Atchison for putting up with a lot of crap during my stay in his lab, for helping me
throughout my studies, giving me much needed guidance and most of all for being
a very good friend. Thanks Bill.

TABLE OF CONTENTS

Bag:
LIST OF TABLES. iv
LIST OF FIGURES. v
LIST OF ABBREVIATIONS. vii
INTRODUCTION. 1
Chapter 1. The neuromuscular junction. 3
Chapter 2. Introduction to dithiobiuret. 11
Chapter 3. Signiﬁcance of toxicity of DTB. 23
Chapter 4. Materials and methods. 26
Preparations and treatments. 26
Electrophysiological recording procedures. 31
Chapter 5. Effects of acute exposure to DTB on neuromuscular 47
transmission.
Results. 48
Discussion. 62
Chapter 6. Effects of exposure to DTB on end-plate currents. 65
Hypothesis. 68
Methods. 71
Results. 71
Discussion. 84
Chapter 7. Effects of DTB exposure on single-channel currents. 89
Introduction. 89
Methods. 93
Results. 94
Discussion. 107

Chapter 8. Effects of DTB on single-channel current measured 122
- via ﬂuctuation analysis.

Results. 128
Discussion. 137
Chapter 9. Discussion. 139
Conclusion. 148
LIST OF REFERENCES. 150

LIST OF TABLES

Table 1. Summary of effects of DTB.
Table 2. Effect of DTB on single channel conductance.

iv

Figure 1.
Figure 2.
Figure 3.

Figure 4.
Figure 5.
Figure 6.
Figure 7.
Figure 8.
Figure 9.

Figure 10.
Figure 11.
Figure 12.
Figure 13.

Figure 14.

Figure 15.
Figure 16.
Figure 17.
Figure 18.

Figure 19.

Figure 20.
Figure 21.
Figure 22.

Figure 23.

Figure 24.
Figure 25.
Figure 26.
Figure 27.

LIST OF FIGURES

Innervation of muscles.
Neuromuscular transmission.

End-plate potential and miniature end-plate

potential.

The structure of DTB.

Setup for recording under current clamp.

Setup for recording under voltage clamp.
End-plate penetrated with two microelectrodes.
Setup for recording under patch voltage clamp.
Setup for recording ACh-induced membrane noise.
Effect of acute DTB treatment on EPP amplitude.
Effect of DTB treatment on m of evoked EPPs.

Effect of DTB treatment on MEPPs.

DTB treatment increases the incidence of abnormal

MEPPs.

Effect of DTB treatment on rise and decay times

for EPPs and MEPPs.

Effect of bath exposure to DTB on EPP amplitude.

Effect of bath exposure to DTB on MEPP frequency. 58

Bath exposure to DTB increased MEPP frequency.
Bath exposure to DTB alters the distribution of

MEPP amplitudes.

Effect of bath exposure to DTB on rise and decay

times for EPPs and MEPPs.

Effect of DTB treatment on peak EPC amplitude.
Effect of DTB treatment on m of EPCs.
Effect of DT'B treatment on the distribution of

MEPC amplitude.

Effect of DTB treatment on the amplitude of modal

MEPCs.

Effect of DTB treatment on t EPC.
Effect of DTB treatment on t m.
Effect of DTB treatment on 1 WC for modal MEPCs. 82

Effect of DTB treatment on 10-90% rise time for

EPCs and modal MEPCs.

73
76
77

78

81
83

Figure 28.
Figure 29.
Figure 30.

Figure 31.
Figure 32.

Figure 33.

Figure 34.
Figure 35.
Figure 36.
Figure 37.

Figure 38.
Figure 39.

Figure 40.
Figure 41.

Figure 42.
Figure 43.
Figure 44.

Single-channel currents.

Multiple conductance levels for single-channels.
Current vs voltage relationship for single-channel
currents.

Open-time distributions for single-channel currents.
Effect of acute exposure to DTB on single-channel
open-time (SCh2 as agonist).

Effect of chronic exposure to DTB on single-channel
open-time (ACh as agonist).

Closed-time distribution for single-channel currents.
Effect of DTB on the distribution of closed-times.
Model of ACh binding and channel opening.

Effect of DTB on single-channel open-time (single
exponential ﬁt).

Plot of t {I + t {I - t e" vs concentration of DTB.
Exposure to DTB causes an increase in the incidence
of abnormal MEPCs.

Amplitude and duration of iontophoretic current.
Membrane noise observed during iontophoresis

of ACh.

Power spectrum from ACh induced membrane noise.
Values of r from control muscles.

Effect of DTB on 1: .

95
97
98

101
103

105
106
108
114

117
129

131
132

133
135
136

LIST OF ABBREVIATIONS

Acetylcholine

Acetylcholine receptor
Carbamylcholine

Corner frequency

Decay time constant
2,4-Dithiobiuret

5,5’-dithio-bis (2-nitrobenzoate)

' Dithiothreitol

End-plate potential

End-plate current

Ethylene glycol-bis-(B-amino ethyl ether) N,N-tetraacetic acid
Gigaohm

Hertz

Intraperitoneal

Megohm

Micromolar

Millivolt

Miniature end-plate current
Miniature end-plate potential
Nanoampere
N-hydroxyethylpiperazine-N-Z-ethane sulfonic acid
Picoampere

Quanta] content

Rate constant for channel closing
Suberyldicholine

INTRODUCTION

The studies described in the following dissertation were designed to examine
alterations in transmission at the neuromuscular junction of rats treated with the
paralytic agent 2,4-dithiobiuret (DTB). Rats treated with small daily doses of DTB
(1 mg/ kg/ day) become paralyzed following approximately 1 week of treatment,
while rats treated acutely with a single large dose of DTB do not exhibit muscle
weakness. Results of previous intracellular recording studies indicate that transmitter
release is depressed in neuromuscular preparations taken from rats exhibiting muscle
weakness following chronic treatment with DTB (Weiler $131., 1986; Atchison
1989). This depression of release is observed as a decrease in quantal content of
evoked end-plate potentials and a decrease in the frequency of spontaneous and
depolarization evoked miniature end-plate potentials (W eiler £131., 1986; Atchison
1989). Alterations in transmitter release such as these are normally indicative of a
presynaptic site of action. What was not known, at the time when the studies
described herein were initiated, was whether alterations in transmission occur early
on during treatment of rats with DTB and progress to the observed weakness, or

whether effects on transmission are only observed once weakness is observed. It was

2

also not known whether an alteration of postsynaptic processes was related to the
weakness observed in treated rats. The purpose of the studies described herein was
to determine whether early effects on transmitter release could be detected in rats
treated with DTB and to explore the possibility that a postsynaptic effect of DTB was

related to the observed weakness.

Chapter 1. The neuromuscular junction.

The synapse formed between motor nerves and muscle cells is known as the
neuromuscular junction. Axons innervating muscles normally branch prior to
‘synapsing on a muscle ﬁber. Thus each axon innervates more than one ﬁber. At
mammalian neuromuscular junctions each ﬁber is innervated at only one point by a
single nerve. The synapse formed at neuromuscular junctions is composed of a
presynaptic terminal containing transmitter-ﬁlled vesicles and a postsynaptic
' component with a large number of receptor molecules. In vertebrates these vesicles
contain the neurotransmitter acetylcholine (ACh). ACh is synthesized in the nerve
by choline acetyltransferase from the precursors choline and acetyl coenzyme A.
Following synthesis, ACh is stored within synaptic vesicles prior to its release. These
vesicles are found throughout the terminal but normally aggregate around presynaptic
densities, termed active zones. These densities appear as fuzzy dense projections
in most ultrathin sections viewed using a transmission electron microscope. A much
clearer picture of the organization of these particles is obtained by using the freeze-
fracture technique. Utilizing this method the presynaptic particles appear as parallel
lines of intramembranous proteins located across the synaptic cleft from postsynaptic
active regions. Recent evidence indicates that these protein molecules may be
calcium-channels important for calcium (Ca+ *) inﬂux during transmitter release
(Heuser 3,141., 1974). Terminals also contain large numbers of mitochondria and

cisternae of smooth endoplasmic reticulum (Figure 1).

A Nerve

Muscle /
- A: .
\ _4_——/———‘

 

 

 

   
   
 

Synapﬁc
Vesicle

.00 II

Nerve
Terminal

End-plate ,
Acetylcholine

Receptors

Figure 1. Innervation of Muscles.

Each muscle cell is innervated by one or more nerves, each nerve normally
branches to innervate more than one muscle ﬁber and each muscle ﬁber is normally
innervated by a single nerve .(a). The point at which a muscle ﬁber is contacted by
a nerve is known as an end-plate region (b). located within the nerve terminal are
a number of synaptic vesicles containing neurotransmitter (acetylcholine). The end-
plate region is highly folded and contains a large number of receptors which are
located at the tops of the membrane folds.

5

These cistemae are often associated with coated vesicles and a small number of
dense-cored vesicles. Experiments monitoring the uptake of horseradish peroxidase
into terminals following nerve stimulation indicate that the smooth endoplasmic
reticulum cisternae and coated vesicles are involved in the reformation of synaptic
vesicles following transmitter release (Heuser and Reese, 1973).

The region of the muscle at which the nerve makes contact is termed the sole-
plate, or end-plate region. At this site, there are several muscle cell nuclei and
large numbers of mitochondria, all located beneath a highly folded region of the
muscle cell membrane. If one examines sections through a terminal and its
associated postsynaptic structures it can be seen that the folds in the muscle cell
membrane occur beneath the presynaptic active zones. A thickening of the
postsynaptic membrane can be observed at the tops of the folds. This thickened
membrane region at the tops of the folds has been demonstrated to contain ACh-
receptor (AChR) molecules located within the postsynaptic membrane (Gauthier,
1976). Filling the gap between the pre- and postsynaptic components is a ﬁbrous
meshwork known as the basal lamina. Associated with basal lamina are a large
number of acetylcholinesterase (AChE) molecules (AChE is also present on the
postsynaptic membrane). This enzyme, which splits ACh into choline and acetate,
is partially responsible for removing transmitter from the cleft following its release.

Transmission at the neuromuscular junction occurs via release of ACh, a
chemical transmitter, which diffuses across the synaptic cleft, and binds to AChR

on the postsynaptic membrane. Upon excitation of a motor nerve and arrival of an

6

action potential at the nerve terminal there is an electrotonic depolarization of the
nerve terminal. Depolarization of the terminal leads to the activation of voltage
sensitive calcium-channels located within the terminal region. Ca+ ” ﬂows into the
nerve terminal and triggers (in some unknown way) the fusion of transmitter ﬁlled
vesicles with the presynaptic membrane. Fusion of vesicles with the presynaptic
membrane leads to the release of ACh into the synaptic cleft. ACh diffuses across
the synaptic cleft and binds to AChR present in the postsynaptic membrane. Binding
of ACh to its receptors leads to the opening of receptor-associated ion-channels.
These channels open for several milliseconds and allow cations (primarily Na+ into
the muscle cell and K” out) to ﬂow through them. At a normal resting membrane
potential of approximately -90 mV, this predominantly inward cationic current leads
to a depolarization of the end-plate region (Figure 2). The depolarization of the
muscle cell membrane is known as an end-plate potential or EPP. Following nerve
stimulation many ACh ﬁlled vesicles will fuse with the membrane and dump their
contents into the synaptic cleft. The number of vesicles which are released per
stimulation (or nerve impulse) is known as the quantal content (m) of the EPP,
while a single transmitter ﬁlled vesicle is known as a single quantum of transmitter.
It is possible to record an EPP by inserting a very ﬁne microelectrode into the
muscle cell membrane in the region of the end-plate. The amplitude of the EPP
when measured in this way can range from a few millivolts to tens of millivolts.
When recording from an end-plate region, random depolarizations are also observed

which appear to be small EPPs.

 

 

 

 

 

 

Figure 2. Neuromuscular Transmission.

Following stimulation of a nerve an action potential will be propagated down
the axon and lead to a depolarization of the nerve terminal (1). Voltage sensitive
CaM channels open and allow Ca” to ﬂow into the terminal (2). An increase in
intraterminal Ca+ ” triggers the fusion of transmitter-ﬁlled vesicles with the
presynaptic membrane and the subsequent release of transmitter into the synaptic
cleft (3). Transmitter binds to receptor-channel complexes in the postsynaptic
membrane increasing the permeability to Na“ and K+ (4).

stimulus EP P
artifact

/

MEPPs

VI. IL

 

 

Figure 3. End-plate potential and miniature end-plate potential.

Following stimulation of the nerve, many synaptic vesicles will release their
transmitter contents into the synaptic cleft leading to a large depolarization of the
end-plate region. This depolarization of the end-plate region is known as an end-
plate potential or EPP. The spontaneous release of the contents of a single synaptic
vesicle (quantum) leads to a smaller depolarization known as a miniature end-plate
potential or MEPP.

9
These are termed miniature end-plate potentials (MEPPs) and are believed to

represent the spontaneous release of a single quantum of transmitter in the absence
of nerve stimulation.

The nicotinic AChR to which ACh binds following its release is an integral
membrane protein consisting of 5 subunits. These are 2 a subunits and one each of
B, y or e, and 6 . The 7 and e subunits replace one another during different stages
of development. The 1 subunit is expressed in developing or denervated muscle,
while the e subunit is expressed in adult muscle (Mishina $131., 1986). The names
of the subunits were chosen from the distance which they migrated on SDS
polyacrylamide gels, with it having the highest mobility and 6 the lowest (Steinbach
and Ifune, 1989). These 5 subunits combine in such a manner as to form a pore
through the membrane, which when open allows Na” into the cell and K+ out. A
variety of other positively charged molecules have been found to pass through the
channel but do not appear to contribute to the current under physiological
conditions. Not only do the subunits form a channel through the membrane, but
they also contain the binding sites for ACh. These sites are located on the a
subunits, thus each receptor-channel complex contains 2 binding sites. Also present
on the a subunit is a reactive disulﬁde which is located very close to the binding site
for ACh, reduction of which alters binding of ACh to the receptor (Ben-Haim _e_t_al.,
1973).

Due to its high degree of accessibility and stability mm the neuromuscular

junction has been well characterized both biochemically and electrophysiologically.

10

Many aspects of chemical transmission were ﬁrst discovered using this preparation
and have later been found to apply to other chemical synapses. Thus, the
neuromuscular junction is often used as a model in which general aspects of
transmission are studied or in which the effects of different pharmacological and

toxicological treatments on synaptic transmission can be examined.

Chapter 2. Introduction to dithiobiuret.

Historical perspective. 2,4-Dithiobiuret (thioimidodicarbonic diamide, DTB)
is a thiourea derivative with moderate reducing properties (Figure 4) (Preisler and
Bateman, 1947). Compounds related to DTB and derivatives of DTB have been
known since the early 19005, however, the parent compound was not characterized
fully until 1945 (Sperry, 1945). Following its characterization in 1945 a variety of
studies investigated potential uses of DTB in both agriculture and industry. In
agriculture DTB was proposed to be used as a promoter of seed germination and
root growth (Dufrenoy and Pratt, 1948), as an insecticide or insect chemosterilant
(Bousquet and Guy, 1945; Oliver _e_t_al., 1971; Chang M, 1971), as a
rodenticide (Dieke _e_t_a1., 1947), or as a fruit thinner in peach orchards (Keil and
Fogle, 1971). It was proposed to be used in industry as an intermediate in the
production of resins and thermoplastics (Webb, 1952) or as a reducing agent in
organic and analytical work (Preisler and Bateman, 1947). At present DTB is listed

as being manufactured only for "specialty uses” by the chemical industry.

11

.3. .5?
H2N—C—NH—C—NH2
2,4 -Dithiobiuret (DTB)

Figure 4. The structure of DTB.

l3
Paralytic effects. In the early 1940’s, while screening thiourea derivatives for

antithyroid activity, Astwood ( 1943) observed that rats given small daily doses of
DTB in their drinking water became paralyzed and died after approximately 1 week
of treatment. Upon further characterization of the toxicity of DTB, Astwood e131.
(1945) found that rats could be maintained in a paralyzed state for extended periods
of time by decreasing the concentration of DTB in their drinking water at the time
of paralysis, yet upon cessation of treatment paralyzed animals recovered apparently
normal function in approximately 1 to 2 weeks. Results of this group’s studies
indicated that stimulation of motor axons in DTB-paralyzed rats caused contraction
of the associated muscles. Thus they concluded that neither the motor nerve nor
muscle were affected by DTB. Astwood e131, (1945) also reported an absence of
morphological alterations in muscle, spinal cord or brain. Consequently, it was
concluded that DTB most likely acted in the central nervous system on some poorly
characterized aspect of transmission to cause the observed paralysis.

Following these early reports Altschul (1947) examined electroencephalogram
of both dogs and cats treated with DTB in an attempt to characterize central effects
observed in treated animals. In these studies no difference was observed in
electroencephalograms recorded from control animals and those treated with DTB.
An increase in the threshold for electrically-induced seizures was detected in rats
severely paralyzed following treatment with DTB and the type of convulsion observed
following electrical stimulation changed from a tonic-clonic type to a sustained tonic

seizure. In light of these ﬁndings Altschul concluded, as did Astwood 9131, (1945),

14
that DTB appeared to act centrally to cause the observed paresis.

In 1948 Seifter eta}. published the results of a study in which skeletal muscle
electrolyte levels and carbohydrate metabolism were examined in DTB-treated
rabbits and rats. Their results demonstrated no change in the levels of skeletal
muscle water, K“, Na“, Cl', phosphorus or creatine between control and treated
animals. Seifter M. did report increases in blood glucose following both acute and
chronic exposure of rabbits to DTB. These could be antagonized by insulin or
. ergotamine. The conclusions resulting from these studies were that the paralysis
observed in DTB treated animals was not due to an effect at the level of skeletal
muscle and that the hyperglycemic effect was a nonspeciﬁc one mediated by
eatecholamines.

Additional reports on the mechanism of the paralytic effects of DTB were not
published until the early 19805 when Atchison and Peterson (1981) characterized
further and quantitated the time to onset and progressive nature of the DTB-induced
weakness. Their results demonstrated that treatment of rats with DTB (1
mg/kg/ day, ip) led to the loss in ability to walk on a rotating rod (rotarod test) after
4—6 days of treatment; loss of righting reﬂex after 6-9 days of treatment; and death
following 6-10 days of treatment. It was observed that tail-ﬂick, hindpaw withdrawal,
corneal, pinna and vibrissae responses all could be elicited in rats exhibiting marked
muscle weakness, indicating a lack of effect of DTB on sensory nerves mediating
these reﬂexes. Treated rats also showed decreased intake of food and water, weight

loss, a delayed onset diuresis, watery mucoid feces and chromodachryorrhea (a red

15

porphyrin secretion from around the eyes). Pair-fed control rats lost a comparable
amount of weight to the treated rats, thus the decreased food intake and subsequent
weight loss did not appear to be responsible for the weakness.

Atchison and Peterson (1981) also monitored rotarod performance in a group
of rats which received a single 25 mg/ kg dose of DTB, a dose which approaches the
48 hour ID,0 in these animals (29 mg/kg). They found that this single large dose
had no effect on rotarod performance when monitored in surviving rats for up to 3
weeks after treatment. Cholinesterase activity was also examined in rats treated with
DTB to determine whether an alteration in its function was related to the observed
weakness. Results of these studies demonstrated no change in whole blood
Cholinesterase activity.

Atchison and Peterson ( 1981) also reported an age and sex dependance for
the effects of DTB. They found that females were more sensitive to the effects of
DTB than males, while older rats of both sex were more sensitive than younger rats.
Atchison e131. (1981a) discovered that altering the dose of DTB administered to rats
altered the time of onset for rotarod failure. A dose of 0.125 mg/kg/ day failed to
cause muscle weakness after 52 days of treatment, while at doses ranging from 0.25
to 1 mg/ kg/ day weakness was observed following 5-20 days of treatment, depending
on the dose. For this range of doses, it was found that a cumulative dose threshold
of 4 to 5 mg/kg determined the onset of muscle weakness. When rats were given
daily doses from 4—16 mg/ kg/ day the onset of muscle weakness became constant at
4 days.

16

Neuromuscular eﬁects. The ﬁrst evidence that a neuromuscular deﬁcit was
related to the observed weakness was reported by Atchison _e_t_a1. (1981b).
Experiments performed examined the tension of muscle twitches generated in situ,
from muscles of control rats and rats exhibiting muscle weakness following treatment
with DTB. In these experiments, twitches were elicited both by stimulation of the
nerve (indirect) and by direct electrical stimulation of the muscle membrane. Results
of these studies demonstrated that twitches in the gastrocnemius muscle elicited by
indirect stimulation were decreased in treated rats compared to those measured in
pair-fed controls. Tension developed following tetanic stimulation was also less in
treated rats than controls, and muscles from DTB-treated rats displayed a marked
fade in tension during stimulation. When twitch tension elicited by direct electrical
stimulation of the muscle was monitored, no difference was noted between muscles
of control or treated rats. These results indicated, for the ﬁrst time, a possible
impairment of axonal conduction and / or neuromuscular transmission in rats
exhibiting muscle weakness following treatment with DTB. Further evidence for a
prejunctional impairment in rats treated with DTB was observed by Atchison 9131.
(1982). They found in studies utilizing interrupted tetanic stimuli that rats exhibiting
muscle weakness following treatment with DTB were somewhat resistant to the
depression of contractile tension caused by hemicholinium—3, a blocker of high
afﬁnity choline uptake into the nerve terminal (Collier and MacIntosh, 1969;
Guyenet £131., 1973). Concomitant treatment with choline, which normally

antagonizes the hemicholinium-3 induced fatigue (Guyenet £131., 1973), worsened

17
the existing fatigue observed in rats treated with DTB. Atchison M. (1982)

reported that administration of 4-aminopyridine to rats exhibiting muscle weakness
following treatment with DTB caused a return of twitch tension to control levels but
did not overcome DTB-induced rotarod failure. 4-Aminopyridine blocks K“ efﬂux
through the so-called "delayed rectiﬁer" potassium channels within the nerve terminal
leading to an increase in the time that the terminal remains depolarized following
nerve stimulation. This prolongation of terminal depolarization leads to an increase
. in the amount of transmitter that is released following nerve stimulation (Lundh,
1973; Pehlate and Pichon, 1974; Molgo .e_t_al., 1977). Thus, DTB treatment
appears to be associated with a depression of transmitter release from motor nerve
terminals that can be reversed by the actions of 4-aminopyridine. More evidence for
an effect on choline metabolism has come from biochemical determinations of
endogenous ACh and choline levels in rats treated with DTB. Results of these
studies demonstrated an increase in both ACh and choline content in mm
W muscles taken from rats treated with DTB compared to muscles of
control rats. These ﬁndings indicate that DTB does not inhibit choline uptake or
ACh synthesis. Instead it appears to disrupt processes responsible for ACh packaging
or release (Weiler £31., 1986).

Electrophysiological ﬁndings. In light of the above results, several studies
were performed which utilized intracellular recording techniques to characterize
further the alterations in transmitter release detected in the above studies (Weiler

3131., 1986; Atchison 1989). Results of electrophysiological experiments performed

18
on hindlimb muscles taken from animals treated with 1 mg/kg/ day ip of DTB for 5

to 6 days have demonstrated a decrease in m of the evoked EPPs, a decrease in the
frequency of spontaneously occurring and K+ ~evoked MEPPs and an increase in the
incidence of very large amplitude MEPPs with abnormal rise and decay times
(Weiler e131., 1986; Atchison, 1989). An interesting ﬁnding reported by Atchison
(1989) indicated that the decrease in m for EPPs could be partially reversed by
increasing external Ca+ + or by treatment with 4-aminopyridine, however, these
manipulations were unable to restore m completely to control levels. This is in
contrast to what was observed in earlier muscle twitch studies in which 4-
aminopyridine treatment was able to restore twitch tension to control levels in
muscles from DTB-paralyzed rats. One possible conclusion from these results is that
DTB acts at a site after Ca“ + has entered the terminal to cause some of the
observed effects on quantal transmitter release.

Antagonism of DTB-induced muscle weakness. A characteristic of paresis
produced by DTB treatment is that while small daily doses (0.25 to 5 mg/ kg/ day)
reliably cause this weakness, large daily doses (above 12 mg/ kg/ day) do not cause
muscle weakness. Williams 3131, (1991) demonstrated that at a daily dose of 12
mg/kg/ day ﬂaccid neuromuscular weakness is not observed. These animals do
display other signs of DTB intoxication such as watery mucoid feces, weight loss,
chromodacryorrhea and a delayed onset diuresis (Atchison and Peterson, 1981;
Williams 5131,, 1986). Inability to walk on a treadmill is observed in these rats

following 3 to 4 days of treatment, but is not related to muscle weakness. Instead,

19
treated rats appear to be close to death and normally die within 24 hour of treadmill

failure. These results are very interesting in that it appears as though DTB affords
some protection against its own toxic effects when administered at high doses.
Results of an earlier study by Williams 9131. (1986) also demonstrated protection
against DTB-induced muscle weakness by other sulfhydryl containing compounds.
This study demonstrated that the sulﬂrydryl-containing compounds d-penicillamine,
cysteamine, diethyldithiocarbamate and disulﬁram all protected against the DTB-
induced weakness. These compounds could both prevent DTB-induced muscle
weakness if coadministered to animals with DTB and could reverse the DTB-induced
weakness if given once weakness was observed. It was concluded that the protection
against toxicity of DTB by sulfhydryl-containing compounds may indicate that some
of DTBs effects are due to alterations of thiol/disulﬁde status within nerve cells.
Nonmotor alterations in peripheral transmission. Although motor function
becomes severely impaired in DTB-treated rats, little evidence for an effect on
sensory function has been noted. As described earlier, Atchison and Peterson
(1981) observed no effect on tail ﬂick latency, pinna, corneal and vibrissae reﬂexes
in rats exhibiting marked muscle weakness following treatment with 1 mg/kg/ day of
DTB for 6 days. Crofton 3131. (1991) performed an extensive series of tests on
DTB-treated rats to determine whether DTB treatment altered sensory processing
as well as motor function. They found that rats exhibiting muscle weakness following
treatment with DTB demonstrated no change in thermal sensitivity, auditory

function or pattern visual function, indicating that treatment with DTB does not

20

impair sensory systems. They did, however, report alteration in ﬂash evoked
potentials (FEPs) recorded with electrodes placed in the skull over the visual cortex.
The FEPs which they recorded are thought to be generated within superﬁcial layers
of the visual cortex indicating an alteration in processing within the cortex itself, not
a deﬁcit in sensory input to the CNS (Crofton 5131., 1991).

Pathological alterations in DTB induced weakness. A characteristic of many
of the chemically-induced neuropathies including that by acrylamide (Kaplan and
Murphy, 1972), cholinesterase inhibitors (Weeker £131., 1978) and the neurotoxic
hexacarbons such 2,5-hexanedione (Spencer and Schaumburg, 1977) is the presence
of high levels of neurodegeneration. Evidence for pathologic lesions in muscle or
nerve of rats treated with DTB is lacking (Astwood £131., 1945; Atchison 9131.,
1981b). In a study performed at the light microscopic level, pathological alterations
were not detected in liver, kidney, lung, thyroid, skeletal muscle, sciatic nerve,
spinal chord or brain, even in rats maintained in a weakened state for extended
periods of time (Williams .e_t_al., 1987). Kemplay (1984), using the zinc iodide-
osmium staining technique, was able to detect small amounts of degeneration and
nerve terminal remodelling in female rats treated with DTB. She observed both
retraction and clumping of nerve terminals and an increase in terminal sprouting
during the onset of muscle weakness. Axonal degeneration was detected in
intramuscular nerve bundles within the W muscle, while little or no
degeneration was observed in nerves innervating the lnmbrical, 59191.5 and tibialis

anterior muscles. Results of this study are also interesting in that in the past the

21
weakness had been described as ascending in nature (Atchison and Peterson, 1981),

yet these ﬁndings indicate that some of the earliest and most striking changes were
observed in the innervation to the W muscles. This may indicate that the
long axons to the lower extremities are not injured ﬁrst, again differentiating DTB-
induced muscle weakness from many other chemically-induced neuropathies. In a
more recent study by Jones (1989), accumulations of branching smooth endoplasmic
reticulum (SER) were noted within nerve terminals innervating thelumbﬁgal muscles
as early as 2 days following treatment of female rats with DTB. On days 3-5 of
dosing, terminals became swollen and packed with organelles including large
numbers of synaptic vesicles, dense core vesicles and SER. Evidence of nerve
terminal sprouting was also observed at this time, as was terminal retraction and
interposition of Schwann cells in the synaptic cleft. Jones also examined Ca+ + levels
within the terminals and muscle cells in control and treated rats. Results of these
studies indicate a reduction in the intraterminal free Ca+ * pool in nerve terminals
of rats treated with DTB, detected as a decrease in oxalate-pyroantimonate
precipitation of Ca“ * within these terminals.

Summary. Rats treated with small daily doses of DTB begin showing signs
of weakness following 2 days of treatment (Kemplay, 1984; Jones, 1989). By day
4-6 of treatment the weakness has progressed to the point where rats can no longer
walk on a rotating rod or a continuously-moving treadmill (Atchison and Peterson,
1981; Williams M, 1986). Morphological alterations, Observed as an

accumulation of smooth endoplasmic reticulum within nerve terminals are also

22
observed by the second day of treatment with DTB (Jones, 1989), while nerve

terminal retraction and sprouting are observed by day 3 of treatment (Kemplay,
1984, Jones, 1989). Electrophysiological correlates to the observed weakness are
a decrease in m of EPPs, a decrease in the frequency of potassium-evoked and
spontaneous MEPPs and an increase in the frequency of failures of transmission
following nerve stimulation (Atchison, 1989). The frequency of abnormally large
slow MEPPs is also increased in muscles from rats exhibiting weakness (Atchison,
1989). There is some evidence that presynaptic alterations in the handling of Ca+ "
may play a role in the observed alterations in transmitter release. Jones (1989)
observed a decrease in Ca+ * content of terminals by 3-4 days of treatment with DTB.
Atchison M. (1982) observed that administration of 4-aminopyridine, to rats
exhibiting muscle weakness following treatment with DTB, caused a return of twitch
tension to control levels. Exposure to 4-aminopyridine or increasing extracellular
Ca“ + did not completely restore quantal content, which was depressed in muscles
from treated rats, to control levels nor did it cause recovery of normal motor
function. Thus, the observed depression in transmitter release does not appear to

be due solely to alterations in presynaptic Ca” + handling.

Chapter 3. Signiﬁcance of toxicity of DTB.

Neuromuscular junction as a model synapse. As described in Chapter 1 the
neuromuscular junction is often used as a model in which to study synaptic
transmission in general. Many aspects of chemical transmission which are
understood at this time were ﬁrst studied at this synapse. By understanding how a
chemical like DTB disrupts transmission, the maintenance of normal synaptic
contacts and the disruption of transmission during other compromised states may be
better understood.

Novel type of muscle weakness. When examined closely the actions of DTB
and the neuromuscular weakness it causes appear very unique when compared to
those of well characterized agents known to cause weakness or paralysis.
Neuromuscular blocking agents such as d-tubocurarine, pancuronium and
succinylcholine, although they act by different mechanisms, block transmission soon
after administration of a single dose (Miller, 1984). This is in contrast to the actions
of DTB for which the observed weakness is determined both by a minimum time of
exposure and cumulative dose threshold (Atchison _e_t_al., 1981a). Other agents have
been characterized which also require repetitive exposure over prolonged times
before their effects are observed. Compounds such as the neurotoxic hexacarbons
(n-hexane, 2,5-hexanedione) and acrylamide require time after initiation of exposure
before paralysis or weakness is Observed (Kaplan and Murphy, 1972; Spencer and

Schaumburg, 1977). Studies examining pathological alterations during the onset of

23

24

paralysis with these agents in all cases reveal large amounts of neurodegeneration
(Kaplan and Murphy, 1972; Spencer and Schaumburg, 1977). In light of this, the
progression of the changes observed with these agents has been classiﬁed as a ”dying
back” or "distal axonopathy" (Spencer and Schaumburg, 1977 ). By this it is meant
that long ﬁbers innervating muscles of lower extremities are affected ﬁrst, while
effects spread to more proximal nerves with continued exposure. Upon cessation of
treatment or exposure, affected animals do recover normal neuromuscular function.
However, it can take weeks to months for this recovery to occur. This' is not the
case for DTB. Very little neurodegeneration is observed in rats treated with DTB
(Kemplay, 1984; Jones, 1989). The neurotoxic hexacarbons and acrylamide aﬁect
both motor and sensory nerves (Kaplan and Murphy, 1972; Spencer and
Schaumburg, 1977), while in DTB-induced muscle weakness sensory systems appear
to be spared (Atchison and Peterson, 1981; Crofton _e_t_al., 1991). Thus, DTB
appears to have actions which are in many ways different from those of classical
blockers of neuromuscular transmission or other neurotoxic agents which cause
paralysis.

Comparison with human neuromuscular disorders. Similarities exist between
electrophysiological characteristics of weakness observed in rats treated with DTB
and those observed in patients suffering from several neuromuscular diseases.
Electrophysiological analysis of muscle biopsies from patients suffering from one
myasthenic disorder demonstrated decreased MEPP frequency and quantal content

of evoked EPPs and increased duration of MEPP half-decay time (Engle £131.,

25
1977). Results from studies of patients suffering from another myasthenic disorder,

congenital myasthenic syndrome, which is proposed to be due to prolongation of the
open-times for ACh channels at the neuromuscular junction, showed an increase in
the duration and half-decay time for EPPs, MEPPs and miniature end-plate currents
(MEPCs), a decrease in MEPP amplitude and normal quantal content (Engle £131.,
1980; 1982). Following exposure to botulinum toxin (from W)
EPP amplitude is decreased, the incidence of failures in transmission is increased
and frequency of MEPPs decreased. Thus, neuromuscular weakness induced in rats
by DTB may be useful as a model system in which to study neuromuscular deﬁcits

associated with several diverse neuromuscular diseases in humans.

Chapter 4. Materials and methods.

During the course of the experiments described herein three different
electrophysiological recording techniques were utilized. These different techniques
allowed for the acquisition of information regarding the action of DTB on AChR-
channels which could not have been obtained using any one of the techniques alone.
In the following section is a description of the methods used in gathering data using
single electrode current clamp, patch voltage clamp, two microelectrode voltage
clamp and ﬂuctuation analysis using two microelectrode voltage clamp in conjunction
with iontophoresis of ACh. Also included are descriptions of the preparations

studied using the above techniques.
Preparations and Treatments.

Intact neuromuscular preparations. All experiments utilizing single electrode
current-clamp and two microelectrode voltage-clamp were performed using the
isolated phrenic nerve-hemidiaphragm preparation (Bi‘tlbring, 1946) of male rats
(180-250g, Harlan Sprague-Dawley Laboratories, Madison, WI). This muscle was
chosen because it is a ﬂat, relatively thin preparation, and can be maintained for
long periods of time mm. It was important to have a preparation which was only
a few ﬁbers thick so that terminal regions could be readily observed and penetrated

during two-microelectrode voltage clamp and iontophoretic experiments. In many

26

of the studies in which DTB was added to the bathing medium, preparations were
observed for several hours. Thus the nerve and muscle must be stable for several
hours m. Hemidiaphragms, with associated phrenic nerves, were dissected
from control rats and rats treated previously with DTB. Muscles ﬁbers were cut
approximately 4 mm on either side of the intramuscular nerve branch to prevent
contractions due to stimulation of the phrenic nerve (Barstad and Iilleheil, 1968;
Hubbard and Wilson, 1973; Glavinovic, 1979; Traxinger and Atchison, 1987a).
Cutting the muscle in this way causes depolarization of the muscle cell membrane,
yet does not alter cable properties of the muscle or transmitter release (Glavinovic,
1979; Fiekers, 1983; Atchison, 1986). Following transection, muscles were
incubated for 1 hour in ice cold, low K+ buffer and then transferred to a Sylgard-
coated, Plexiglas chamber and pinned out and allowed to equih’brate for
approximately 30 minutes prior to recording to restore normal ionic gradients
disturbed by incubation at low temperatures (Traxinger and Atchison, 1987a,b). The
use of buffer with a lower K+ concentration (2.5 mM vs 5 mM in normal
extracellular buffer) prevents K”, which leaks out of the transected muscle ﬁbers,
from causing conduction block of nerves innervating the muscle. During recording
sessions the diaphragm was superfused continuously with a low K” physiological
saline solution modiﬁed from that described by Liley (1956); it contained (mM):
NaCl, 137.5; KCl, 2.5; MgCl, 1; CaCl, 2; d-glucose, 11; and N-
hydroxyethylpipemzine-N-Z-ethane sulfonic acid (HEPES), 14. Solutions were

continuously oxygenated with 100% 02. All experiments were performed at room

27

temperature and in buffers of pH 7.4.

Rats treated chronically with DTB. Chronically-treated rats were given 1
mg/kg/day ip of DTB dissolved in 0.9% NaCl (1 mg/ml) for 6 to 8 days. Rats
treated in this manner develop muscle weakness after approximately 4-6 days of
treatment and normally fail the rotarod test for 2 consecutive days by 6-8 days of
treatment (Atchison and Peterson, 1981; Atchison et aL, 198la,b). Previous
experiments used 2 days of rotarod failure as an index to insure that all rats had
. attained the same level of weakness prior to their use in electrophysiological
recording experiments (Atchison, 1989).

Rats treated with a single large dose of DTB. To determine the time course
for the onset of effects of DTB on neuromuscular transmission rats were treated with
a single 25 mg/kg (ip) dose of DTB (dissolved in tween-80: ethanol (95%): and NaCl
(0.9%), 1: 1: 8, v/v/v to a ﬁnal concentration of 5 mg/ml). Rats were sacriﬁced
and diaphragms removed 1, 4, 8 and 24 hour after treatment. Although this dose
approaches the 48 hour LD,0 in rats, it has been shown to cause no observable
muscle weakness in rats monitored for up to 30 days following treatment (Atchison
and Peterson, 1981). By using a maximally tolerated dose, which does not cause
muscle weakness, we hoped to observe very early alterations in neuromuscular
transmission which may progress to the weakness observed in chronically treated rats.

Rats treated with a single low dose of DTB. Several of the ﬂuctuation analysis
recordings were performed in muscles taken from rats 24 hour following treatment

with a single 1 mg/kg dose of DTB. From these experiments we hoped to determine

29

whether effects observed in muscles taken from rats exhibiting muscle weakness
following 6-8 days of treatment with 1 mg/ kg/ day could be detected following a
single 1 mg/kg dose.

Bath exposure of control muscles to DTB. The earliest times at which the
effects of DTB could be examined in hemidiaphragm preparations from rats treated
with DTB were approximately 2-2.5 hr following initial exposure to DTB. Most of
this time was required for incubation in cold saline followed by equilibration at room
temperature. To determine whether DTB affected transmission at earlier times than
could be measured in muscles from treated rats, diaphragms were removed from
untreated rats and exposed to DTB in the bathing medium. Diaphragms were
removed from control rats and handled in the same manner as described above.
After equilibration to room temperature, control MEPPs and EPPs (1.0 Hz
stimulation frequency) were recorded for 5 minutes each, after which time the
perfusion solution was changed to one containing DTB. In the initial current clamp
studies two concentrations of DTB were used: the high concentration of 1.85 mM,
was calculated to be the plasma concentration if DTB were to distribute to 10 % of
body weight (plasma alone) and the low value of 200 u M was calculated to be the
plasma concentration if DTB were to distribute to 70 % of body weight (whole body
water), 1 hour following a single dose of 25 mg/kg DTB. Porter 9111, (1983)
reported a plasma DTB concentration of 14.04 p g/ g (approximately 150 u M) 3 hour
following a single 25 mg/ kg dose of DTB, which supports our calculated values for
1 hour after the 25 mg/kg dose. In ﬂuctuation analysis experiments a value of 1 u M

30

DTB was used. This concentration was chosen for two reasons. First, previous
patch voltage clamp experiments had demonstrated an effect on single channel open-
time following exposure to this concentration of DTB. Second, treated rats used in
ﬂuctuation analysis studies received daily doses of 1 mg/ kg/ day, a dose level which
results in a plasma concentration of DTB derived equivalents of approximately 1 u M
24 hour following treatment (Porter 9131., 1983).

Cultured GS muscle cells. A requirement for patch voltage clamp is direct
access to a relatively clean membrane surface (Hamill 5131., 1981). This is seldom,
if ever, attained in intact mammalian neuromuscular preparations. Due to these
limitations all patch voltage clamp studies were performed on a clonal cell line. The
cell line which was used was a mouse myoblast cell line (G8 passage 20-30). These
cells express nicotinic AChR and have been well characterized electrophysiologically
(Morris and Montpetit, 1986; Morris _e1_a1., 1989). A muscle cell line was chosen
over other cell lines which express nicotinic AChR since future studies plan to use
cocultures of nerve and muscle cells to study effects of DTB on synaptic activity
within these cultures. G8 cells were grown in collagen-coated 30 mm culture dishes.
Cells were grown in Dulbecco’s modiﬁed eagles medium containing 10% fetal bovine
serum and 10% horse serum. Differentiation of myoblasts was enhanced by growing
cells in normal growth medium until conﬂuence, then decreasing the serum content
to 2% fetal bovine serum and 2% horse serum. Following a decrease in the serum
content, these cells differentiate from small, mononucleated, spindle shaped cells

to form multinucleated myotubes, which are highly responsive to ACh (Morris 3131.,

31
1989). Electrophysiological recordings were performed on differentiated myotubes

within 3—4 days of differentiation.

Exposure of cells in culture to DTB. Two protocols for exposure of cells to
DTB were used in these experiments. In the ﬁrst, patches were excised from control
cells and single-channel currents were recorded in the presence of agonist (0.1-2 u M
ACh or 100 nM suberyldicholine (SChz) followed by exposure to agonist plus DTB
(1-1000 14M). In the second group of experiments, cells were grown in growth
medium containing DTB (1-10 uM). Cells died when grown in medium containing
DTB at concentrations of 100 u M or more. Following exposure of cells to DTB for

1-3 days single-channel currents were recorded in the presence of 0.1-2 u M ACh.
Electrophysiological Recording Procedures.

Single microelectrode current clamp studies. Synaptic potentials were
recorded using borosilicate glass microelectrodes (15-25 M0 impedance) ﬁlled with
3 M KCl (Figure 5). Synaptic potentials were ampliﬁed and subsequently displayed
on a digital oscilloscope (Model 2040, Nicolet Instruments, Verona, WI) and
recorded on magnetic tape using an FM instrumentation tape recorder (Model B,
Vetter Instruments, Rebersburg, PA). EPPs were elicited via stimulation
(supramaximal constant current, 70 microsecond duration) of the phrenic nerve with
a suction electrode and a 1830 interval generator, 1831 pulse train module, and a

1850A stimulus isolator (WPI Instruments, New Haven, CT).

32

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 5. Setup for recording under current clamp.

Hemidiaphragm preparations were removed from control and treated rats and
transected to prevent contraction following stimulation of the phrenic nerve. Muscles
were pinned down in a Sylgard-coated Plexiglas chamber and perfused with
physiological saline. End-plate regions were impaled with a KCl-ﬁlled
microelectrode and EPPs and MEPPs recorded.

33
EPP amplitude, MEPP amplitude and frequency and rise and decay rates for both

MEPPs and EPPs were analyzed using the analysis program pClamp' (Axon
Instruments, Foster City, CA) on a Zenith microcomputer (Model ZW-248-84).
Rise time was measured as the time elapsed from the start of rise of an event to the
peak of the event, while decay time was measured as the time from peak amplitude
to the resting potential. EPP amplitudes were corrected for nonlinear summation
(MacLachlan and Martin, 1981; Traxinger and Atchison, 1987a) and standardized
to a membrane potential of -50 mV (Katz and Thesleff, 1957) as described by
Atchison (1989). Quantal content was calculated by dividing the mean EPP
amplitude by the mean MEPP amplitude (mean value method, (Del Castillo and
Katz 1954; Hubbard e131,, 1969)). Typically 1-3 end-plate regions were recorded
from in each preparation. Impalement was considered acceptable if rise times for
MEPPs were approximately 1 millisecond, while rise times for EPPs were
approximately 1.5 milliseconds. At each end-plate region, MEPPs were recorded
continuously for 5 min. Following this, EPPs were elicited by electrical stimulation
of the phrenic nerve at frequencies of 0.5, 2, 5, 25 and 50 Hz to determine whether
there was any frequency-dependance to the effects of DTB. Two minutes were
allowed to pass between the ﬁrst three stimulation frequencies and 5 minutes
between the next two for redistribution of Ca+ + following stimulation. For statistical
purposes at least 200 EPPs were recorded at each stimulus frequency.

In muscles which were exposed to DTB in the bathing medium MEPPs were

recorded for 5 minutes and EPPs (1.0 Hz) for 4 minutes prior to DTB exposure.

34
After recording control potentials the perfusing solution was changed to one

containing DTB (200 uM or 1.85 mM) and EPPs (1.0 Hz) were recorded
continuously until block of EPP was observed. In preparations in which block did
not occur within 30 minutes, MEPPs were counted for 5 minutes and at 10 minute
intervals thereafter until block occurred or until the preparation was exposed to DTB
for 60 minutes. After 60 minutes of exposure to DTB, the preparation was washed
with a DTB-free control solution and monitored while stimulating at 1.0 Hz for
approximately 30 minutes. In preparations in which block of the EPP occurred
within 30 minutes, stimulation of the phrenic nerve was halted, MEPPs were
recorded for 5 minutes, and then the preparation was washed with a DTB-free
buffer and nerve stimulation resumed until EPPs returned.

Two microelectrode voltage clamp recordings. Voltage clamp experiments
were conducted using the two microelectrode voltage clamp technique (Takeuchi and
Takeuchi, 1959) and hemidiaphragm preparations prepared as described above
(Figure 6). Voltage recording and current passing electrodes had impedances of 5-10
and 2-4 M0 respectively when ﬁlled with 3 M KCl. Lower resistance microelectrodes
were used for current passage; this allowed the passage of large amounts of current
necessary to maintain space-clamp of the end-plate region of the muscle cells. With
the two microelectrodes in the bath, the series resistance of electrodes can be
neutralized via the bridge balancing circuitry of the ampliﬁer. This also allows
measurement of the electrode resistance directly from the adjustment dial on the

ampliﬁer.

35

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 6. Setup for recording under voltage clamp.

Hemidiaphragm preparations were prepared as described in Figure 5. End-
plate regions were impaled with two microelectrodes. One electrode monitors
membrane potential via a voltage follower circuit. During a change in membrane
potential, which is sensed by the voltage follower, current is passed through the
second electrode to return the membrane potential to its original level. Using this
conﬁguration the membrane potential can be held (clamped) at a constant level and
currents can be recorded which underlie the synaptic potentials described above.

36

End-plate regions were located and impaled with the low resistance current passing
electrode, followed by the high resistance voltage recording electrode. Impalement
in this order was more successful due to the fact that the current recording electrode
has a ﬁner tip and tends to disturb an already impaled cell to a lesser degree than
impaling in the other order. Locating the same cell with both electrodes is done by
passing a small amount of current through the second electrode (the voltage
recording electrode in this case) and penetrating cells in the region until a step
depolarization is observed in the electrode already within the cell (the current
passing electrode in this case). When both electrodes are located within the same
cell a step change in voltage can be observed in one electrode following current
injection from the other electrode and MEPPs with similar rise and decay times
should be observed simultaneously through both electrodes (Figure 7). Following
impalement of a single cell with both electrodes, the capacitive coupling between the
two electrodes can be minimized using the capacitive compensation circuitry on the
ampliﬁer. Capacitive coupling between the electrodes can also be minimized by
maintaining the bath level as close to the surface of the muscle as possible. Voltage
clamp steps were made from a holding potential of -50 mV using the Axoclamp-Z
voltage clamp circuit (Axon Instruments Inc., Foster City, CA). EPCs were elicited
by nerve stimuli (1 Hz, 70 microsecond duration) applied through a suction
electrode with a Grass S88 stimulator and a SIUS stimulus isolation unit (Grass

Instruments, Quincy, MA).

37

microelectrode 7

current
injection
microelectrode 2
electrotonic
MEPPs <\
\ response

Figure 7. End-plate penetrated with two microelectrodes.

Upon successful penetration of an end-plate region with a microelectrode
MEPPs can be observed which have rise times of 1 millisecond or less. By placing
a second electrode in the same cell MEPPs can be observed simultaneously through
both electrodes and current injected through one electrode can be detected with the
second electrode.

38
At each holding potential 20-30 EPCs and 100-200 MEPCs were recorded on FM

tape with a 02.5 kHz bandwidth (Store 4DS, Racal Recorders Inc., Irvine, CA)
and analyzed off-line using a Zenith microcomputer with a maximum sampling rate
of 83 kHz (model ZW-248-84). EPCs and MEPCs were digitized and the decay time
constant (1:) was obtained from a curve ﬁtting routine which ﬁt an exponential curve
to the falling phase of EPCs or MEPCs (pClamp', Axon Instruments, Foster City,
CA). Analysis of modal amplitude MEPCs was accomplished by creating an average
MEPC from all MEPCs recorded at a single end-plate region which had amplitudes
equal to the mode value for that end-plate. These MEPCs were selected ﬁrst by
amplitude and then sorted manually to eliminate MEPCs with prolonged rise and
decay times. The edited MEPCs were then summed to give an average modal
MEPC, and amplitude, 10-90% rise time and s were calculated. 1 MEPC for the
modal MEPCs was obtained from the inverse of the slope of semilog plots of the
decay phase of the summed MEPCs (Pickers, 1981). Separate Gaussian
distributions were ﬁtted to the MEPC amplitude histograms using the pSTAT portion
of pClamp’ (Axon Instruments, Foster City, CA). Quantal content was calculated
by the mean value method (Del Castillo and Katz 1954; Hubbard £131., 1969).
Patch voltage clamp. Single AChR-channel currents were recorded from G8
myotubes using low resistance, ﬁre-polished microelectrodes fabricated from
borosilicate glass (1 mm outside diameter, WPI Instruments, New Haven, CT).
Currents were recorded using standard patch voltage-clamp techniques on cell-free

patches in the outside-out conﬁguration (Hamill £131., 1981) (Figure 8).

39

A Cell .4 trashed

/
I Extracellular Sol
/ intracellular Sol.
0»
Extracellular Sol

ntracellular Sol.

Whole C‘e/l
'3 I
I intracellular Sol.

Extracellular Sol

C Outside - Out Gel/free

e

I intracellular Sol.
Extracellular Sol

Figure 8. Setup for recording under patch voltage clamp.

Formation of a high resistance seal (1-100 gigaohm) between a ﬁre polished
pipet and a cell membrane can be accomplished by touching the pipet to the
membrane and applying negative pressure to the Interior of the pipet. This
procedure isolates a small patch of membrane from the rest of the cell membrane,
and is referred to as a cell-attached patch (A). Additional application .of negative
pressure ruptures the patch of membrane spanning the mouth of the pipet, which
allows both electrical and physical access to the interior of the cell (whole-cell
conﬁguration (B)). Withdrawal of the pipet from the cell leads to the formation of
a cell-free, outside—out patch spanning the tip of the pipet (C).

40
During electrophysiological recording the bathing medium was changed to a

physiological saline; it contained (mM) NaCl, 135; KCl, 5; MgClz, 1; CaClz, 2;
d-glucose, 11 and HEPES, 14 (set to pH 7.3 with NaOH). Patch electrodes (5-10
MD resistance) were ﬁlled with saline containing; (mM); KCl, 135; MgCl, 2; d-
glucose, 10; HEPES, 10; ethylene glycol-bis-(B-amino ethyl ether) N,N-tetraacetic
acid (EGTA), 2 (set to pH 7.3 with KOH). All solutions used were ﬁltered prior
to use to remove small contaminating particles which could impede seal formation
(Whatrnan #1 ﬁlters). Prior to patch formation, positive pressure was maintained
on the inside of the patch electrode (pressure adjustments were made with a 1 cc
syringe attached to the inlet port of the electrode holder) to prevent cell debris in
the perfusing solution from sticking to the end of the microelectrode. During seal
formation the microelectrode was brought into contact with the membrane of a
diﬁerentiated myotube. The point of contact can be determined visually by observing
an indentation of the membrane surface at the tip of the pipet and by a change in
the audio response of the patch clamp circuit during repetitive voltage pulses. In
addition, the amplitude of the current observed during repetitive voltage pulses will
decrease as the microelectrode forms a seal with the membrane (the resistance of
this initial seal is on the order of 50- 100 M0 ). Following initial seal formation
negative pressure was applied to the interior of the microelectrode; this normally
leads to the formation of a very high resistance seal on the order of 1-100 GO (giga-
seal). Upon formation of a "giga-seal" the current step observed during a pulse of

voltage across the membrane will be decreased to a very low level, leaving only

41
capacitive transients at the start and ﬁnish of a step change in voltage change. If

formation of a high resistance seal does not follow the application of negative
pressure to the interior of the microelectrode, imposition of a moderate
hyperpolarization across the patch (10-50 mV) often aided in its formation.
Following formation of a high resistance seal between the microelectrode and cell
membrane, the patch of membrane spanning the interior of the microelectrode can
be ruptured to form what is known as the "whole-cell" conﬁguration. The membrane
patch can be ruptured by increasing the negative pressure within the microelectrode
further, by passing an oscillating voltage across the membrane patch or a
combination of these two methods. Following rupture of the membrane patch within
the microelectrode, the capacitive current changes observed at the start and ﬁnish
of a current pulse were increased in amplitude and duration, indicating an increase
in membrane area from which capacitive current is measured. To obtain a detached
patch from the whole-cell conﬁguration the microelectrode was slowly withdrawn
from the membrane surface. Upon withdrawal of the microelectrode from the
surface of the cell, a small patch of membrane formed across the tip of the
microelectrode. If the microelectrode was pulled away from the cell surface when
in the whole-cell conﬁguration, the patch of membrane at the tip of the
microelectrode had its extracellular face on the outside of the microelectrode
(”outside-out cell-free patch”) (Hamill £1_a1., 1981). The resistance of the cell-free
patch could sometimes be improved by imposing small amounts of positive or

negative pressure to the inside of the microelectrode. Once a high resistance seal

42

was obtained in a cell-free patch, pressure on the inside of the microelectrode
(either positive or negative) should be removed to prevent the opening of pressure
sensitive channels. Single-channel currents were ampliﬁed 100x using an Axopatch-
1D patch clamp ampliﬁer (Axon Instruments, Foster City, CA) and recorded to FM
tape (15 inches per second, Store 4DS, Racal Recorders Inc., Irvine, CA). Single-
channel currents were recorded to two different channels on the tape. One channel
was ﬁltered using a low-pass ﬁlter (Bessel response) with a cutoff frequency of 2
kHz. The second channel was not ﬁltered prior to recording to allow later analysis
in the absence of ﬁltering, or following ﬁltering at different frequencies. During
analysis, current recordings were sampled at 50 microsecond intervals, stored on
hard-disc and analde off-line using the pClamp' analysis software (Axon
Instruments, Foster City, CA) on a Zenith microcomputer (model ZW-248-84).
The opening and closing of channels was detected by a half-amplitude current
threshold (Colquhoun and Sigworth, 1985). Due to the limited bandwidth of the
data analysis system, the minimum duration of event which was detectable was 100
microseconds. This was determined by measuring the minimum duration of a voltage
step (generated by a Grass S88 stimulator) which could be accurately measured using
the acquisition system. Histograms of open and closed-times were constructed from
the idealized open and closed intervals. Mean values for open and closed durations
were determined from exponential curves ﬁt to the distributions. The mean
amplitude of channel openings was determined from gaussian distributions ﬁt to

histograms of channel amplitudes recorded for a given patch. Channel conductance

43

was determined from the slope of the current vs voltage relationship for a given
amplitude class of channels. In most patches multiple conductance levels were
observed, however, channel openings with a slope conductance of 37 pS
predominated. Only this conductance class was used in further analysis.
Fluctuation Analysis. The membrane potential of end-plate regions of
muscles from control rats and rats treated with DTB was clamped using the two
microelectrode voltage clamp techniques described earlier. Following successful
clamp of the membrane potential at an end-plate region, a third microelectrode
(iontophoretic electrode) containing 2 M ACh was brought into the region of the
end-plate (Figure 9). The ACh containing electrode was connected to a WPI
intracellular recording ampliﬁer. Using this ampliﬁer allowed the resistance of the
electrode to be monitored (normally 50-100 M0) and for the passage of current
(depolarizing and hyperpolarizing). When positive current is passed through the
iontophoretic microelectrode, it will force ACh out of the electrode (due to ACh’s
positive charge) into the region of the end-plate. To prevent ACh from leaking from
the electrode when not iontophoresing, a negative retaining current was passed (2-4
pA). By placing the iontophoretic electrode very close to the end-plate region
(actually into the connective tissue), very fast currents could be elicited by
iontophoresis of ACh into the region. However, placement of the iontophoretic
electrode in this manner normally leads to the displacement of one or both of the

voltage clamp microelectrodes from the end-plate region.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 9. Setup for recording ACh-induced membrane noise.

The membrane potential of end-plate regions were held constant using two
microelectrode voltage clamp as descn'bed in Figure 6. A third ACh-containing
microelectrode was brought into the region of the end-plate. By passing a positive
current through the ACh-containing electrode, ACh is ejected into the region of the
end-plate. Steady application of low levels of ACh to the end-plate region causes
membrane noise to increase due to the random opening and closing of a number of
AChR-gated ion channels. Information regarding the conductance and open-time of
these channels can be determined from the amplitude and frequency of the observed
ﬂuctuations.

45

Better results for the recording of membrane noise were obtained by placing the
iontophoretic electrode a short distance from the end-plate region. By passing low
levels of positive current (2-10 pA) from an iontophoretic electrode placed near the
end-plate region, EPCs on the order of 2-20 nA could be elicited which could be
maintained for many seconds (up to 60 seconds). Membrane noise, in the absence
and presence of ACh, was recorded at 14 different end-plate regions in each
hemidiaphragm preparation at holding potentials from -20 to -70 mV. Noise
, containing signals before and after iontophoresis of ACh were recorded to PM tape
(15 ips, Store 4DS, Racal Recorders Inc., Irvine, CA) for storage prior to analysis.
During analysis, noise-containing signals were sampled at 50 microsecond intervals,
stored on hard-disc and analyzed off-line using the program SPAN (Spectral Analysis
Program, J. Dempster, University of Strathclyde, Glasgow, G1) on a Compac
microcomputer. For analysis two channels of noise-containing signal were recorded
to computer. One channel was DC-coupled and ampliﬁed 1 to 10X. This channel
was used to determine the amplitude of the iontophoretic current. The second
channel was AC-coupled and was ampliﬁed 100-1000X. This channel was used to
determine the amplitude and duration of the noise ﬂuctuations in the absence and
presence of iontophoresed ACh. The AC-coupled signal was low-pass (500 Hz) and
high-pass (1 Hz) ﬁltered (modiﬁed Butterworth response) using an electronic ﬁlter
(VBF/ 8 dual channel variable ﬁlter, Kemo Limited, Kent, UK). Power spectra for
the recorded noise was calculated, one or two Lorentzian curves were ﬁt to the

calculated spectra and the decay time constant for channels were determined from

46
the corner frequency (f(c)) of the calculated spectra (all were done using the program

SPAN). Channel conductance could be determined from amplitude of the
macroscopic current and the standard deviation of the ACh induced noise or from

the maximum power level of the calculated spectra (SPAN).

Chemicals. Puriﬁed, recrystallized DTB was obtained from Ash Stevens
(Detroit, MI). HEPES was obtained from United States Biochemical Corporation
(Cleveland, Ohio). ACh and SCh2 were obtained from the Sigma Chemicals
(Milwaukee, WI).

Statistics. Slopes of calculated regression lines were compared using a Host
for the comparison of slopes. Speciﬁc points on these lines were compared using a
t-test for comparing the height of points on separate lines, and the means of
treatment group values were compared to control values using an Analysis of
variance, comparisons between groups were made using Dunnet’s t-test and the
means before and after exposure to DTB were compared using a Student’s t-test

(Steel and Torrie, 1980). Signiﬁcance was set at p < 0.05.

Chapter 5. Effects of acute exposure to DTB on neuromuscular transmission.

Purpose of studies. One possible explanation for the cumulative dose
threshold and latency to onset of paresis is that it takes time for DTB or a
metabolite thereof to accumulate in the rat prior to manifestation of muscle
weakness. This is quite possible when one considers that the plasma half-life of DTB
or DTB-derived equivalents is between 8 and 9 hour in rats (Williams et al., 1982;
Porter et al., 1983). Thus only 88% of the DTB given in a daily dose would be
excreted by the time the next dose was administered. Two sequences of events may
be observed during accumulation of DTB. First, as DTB, or a metabolite thereof,
begins to accumulate subtle changes in transmission may occur. With continued
exposure to DTB these alterations increase in magnitude as the level of the toxic
compound increases within the organism. Conversely, transmission may not be
affected until DTB or a metabolite thereof reaches a toxic level, at which time
transmission is affected and paralysis observed. Accumulation of DTB or a
metabolite may not be critical; instead some process critical to transmission may be
inhibited by DTB. Then continuous exposure to DTB would lead to continuous
interference with the critical process, slowly decreasing transmission over the course
of treatment. That is, DTB treatment may produce a subtle gradual disruption of
synaptic transmission which only becomes evident as frank paralysis after 3—6 days of
continuous dysfunction. Therefore, the purpose of the studies described below was

to determine whether subtle changes in neuromuscular transmission could be

47

48

detected at early times following a single large dose of DTB when no muscle
weakness is observed in the whole animal, or at very early times following exposure
to DTB using a perfusion system and isolated tissue. If so, these effects might
progress to a more generalized weakness with time and continued exposure to DTB.
Conversely, it may take several days of treatment before DTB, or a metabolite
thereof, reaches a level at which transmission is affected. If this is true then no

effects should be observed following acute treatment.

Results.

Effects of DTB on animal appearance and behavior. At 1 and 4 hour
following a single injection of 25 mg/kg of DTB, rats appeared normal, while at 8
and 24 hour after treatment they appeared lethargic. Treated animals could move
when disturbed, however their normal exploratory behavior appeared to be
suppressed. Several other signs of DTB intoxication such as diarrhea and lack of
grooming were also observed in these rats. In one rat in which electrophysiological
experiments were not performed the above signs of intoxication were observed up
to 24 hour; by 48 hour this rat appeared normal.

Electrophysiological studies on muscles from rats treated with DTB. Results
from electrophysiological studies demonstrate that increasing stimulus frequency,
from 0.5 to 50 Hz, caused a steady decline in EPP amplitude in muscles taken from

both control and treated rats (Figure 10).

49

+ CON

EPP AMPLITUDE (mV)

 

 

 

STIMULATION FREQUENCY (Hz)

Figure 10. Effect of acute DTB treatment on EPP amplitude.

Effects of acute DTB treatment on EPPs at the rat neuromuscular junction.
Diaphragms were removed 1, 4, 8, and 24 hour after treatment with DTB (25
mg/kg of DTB, ip) or vehicle. EPPs were recorded at stimulation frequencies of
0.5, 2, 5, 25, and 50 Hz from preparations in which the muscle was cut to prevent
contraction due to stimulation of the phrenic nerve. Values are the means of at least
four different preparations. The bar in the lower left corner is the average SEM.
SEM values ranged from 0.3 to 3.3.

50

 

 

 

 

 

—<>- CON
E -+- 'l h
l— —A-- 4 h
E
i- -O- 8 h
2
8 + 24 h
\I~ ~~~~~
21 “‘rﬂ
E _ Lag—$3
<
D I
0 o l l J I j

STIMULATION FREQUENCY (Hz)

Figure 1 1. Effect of DTB treatment on m of evoked EPPs.

Effects of acute DTB treatment on quantal content at the rat neuromuscular
junction. EPPs were recorded at stimulation frequencies of 0.5, 2, 5, 25, and 50
Hz from preparations in which the muscle was cut to prevent contraction due to
stimulation of the phrenic nerve. Quantal content was calculated using the mean
value method. The bar in the lower left represents the average SEM; SEM values
ranged from 0.4 to 3.7.

5 1
EPP amplitude from muscles taken 1 hour after treatment with DTB appeared to be

decreased compared to control, however, this was not signiﬁcant. EPP amplitudes
from muscles taken 4, 8 and 24 hour after treatment were similar to those of control
muscles (Figure 10). Comparisons of quantal content demonstrated no difference
between control and treated groups at any treatment time or any stimulus frequency
(Figure 1 1).

MEPP frequency was signiﬁcantly depressed in muscles taken 1 hour after
treatment with DTB, while for muscles taken at 4, 8 and 24 hour after treatment
these values had returned toward control levels (Figure 12). Even though MEPP
amplitude was not signiﬁcantly altered in muscles taken 1 hour following treatment
with DTB there appeared to be a greater number of MEPPs with amplitudes of 1
mV or more (2.5% 1 hour after treatment compared to 1.4% in control muscles) and
these values tended to occur at multiples of mean MEPP amplitude (Figure 13).

Rise and decay times for EPPs were unaffected by DTB treatment while rise
and decay times for MEPPs were signiﬁcantly prolonged at treatment times longer
than 4 hour (Figure 14).

Effect of bath-applied DTB on EPPS and MEPPs. Due to the long
e ' 'bration time needed following removal of the diaphragm and transection of the
muscle ﬁbers, it was difﬁcult to examine very early effects of DTB on transmission
in diaphragms taken from treated rats. Therefore, diaphragms from untreated
control rats were exposed to DTB in the bathing medium, thus allowing examination

of very early potential effects of DTB on neuromuscular transmission.

52

MEPP

A

l .,
IN/-

 

AMPLITUDE (mV)
in
I-l>

 

 

 

 

 

 

o I
41
B s -
55
>. A A
3
a“ I
o I
Ill
I
IL
0L I

O 8 1 6 24
TIME (hr)

Figure 12. Effect of DTB treatment on MEPPs.

Effects of a single injection of DTB on amplitude (top) and frequency
(bottom) of MEPPs at the rat neuromuscular junction. Rats were treated with 25
mg/kg of DTB, ip, and diaphragms were removed 1, 4, 8, and 24 hour after
treatment with DTB or vehicle. MEPPs were recorded for 5 minutes prior to
stimulation of the motor nerve for EPP recording. Values are the means a SEM of
at least four preparations. The asterisk indicates a signiﬁcant decrease in MEPP
frequency from control values (p s 0.05).

53

1.2

MEPP AMPLITUDE (mV)
2»

 

5 msec

Figure 13. DTB treatment increases the incidence of abnormal MEPPs.

MEPPs recorded from a hemidiaphragm removed 1 hour following a single
dose of 25 mg/ kg of DTB. MEPPs were recorded for 5 minutes, prior to recording
EPPs at various ﬁ'equencies. The ﬁgure contains 25 MEPPs which were recorded
consecutively and superimposed for the purpose of display. MEPP amplitude was
0.35 z 0.02 mV (mean 1 SEM) for this experiment with the majority of MEPPs
being approximately 0.3 mV in amplitude (nonstandardized value); ' however,
MEPPs with amplitudes of approximately 0.6 and 0.9 mV were also observed.

54

N

74

i—r>\—-c
I—D

Rise Time (msec)

Rise Time (msec)

 

 

.a
«5

CD
t

r91

Decay Time (msec)
~I
y.—
Decay Time (msec)
(a)

I l I I

 

 

L I

0 8 16 24 0 8 16 24
Time (hr) Time (M

0

Figure 14. Effect of DTB treatment on rise and decay times for EPPs and MEPPs.

Effects of a single injection of DTB on rise and decay times for EPPs (left
panel) and MEPPs (right panel) recorded at the rat neuromuscular junction. DTB
treatment and uncoupling of muscle contraction from nerve stimulation are as in
Figure 10. Rise and decay times were calculated from EPPs elicited at a frequency
of 0.5 Hz. Values are the means 2 SEM of at least four preparations. The asterisk
indicates a value signiﬁcantly different than control (p s 0.05).

55
The effects observed following exposure to 1.85 mM DTB were similar to

those observed following exposure to 200 u M DTB except that they occurred at
earlier times during exposure. Figure 15 shows that exposure of diaphragms to 1.85
mM DTB had a biphasic effect on EPP amplitude. During the ﬁrst minutes of
exposure there was an increase in EPP amplitude, followed by a decrease with
continued exposure. Block of the EPP was observed in 4 out of 7 preparations after
approximately 10 minutes of exposure to 1.85 mM DTB, while the mean time to
block following exposure to 200 u M DTB was 30 min. The block of the EPP
following exposure to DTB was readily reversed by changing the perfusing solution
to a DTB-free buffer and could be reproduced by washing in a DTB-containing
buffer a second time. In experiments in which block of the EPP was Observed, there
was a tendency for EPPs to return following rest periods during which stimulation
was terminated to record MEPPs. In several experiments in which block of the EPP
was reversed by washing with DTB-free buffer, the amplitude of the EPPs decreased
with continued stimulation. If stimulation was stopped, the amplitude of EPPs
evoked following a short rest period was increased. During block of the EPP there
was a progressive decline in EPP amplitude, yet normal MEPPs were still observed.
Therefore it appeared as though there was a steady decline in quantal content

leading up to the observed block.

56

A CONTROL EPP

wJILL

B DTB, 13 min I

C DTB, 20 min

W

D DTB, 23 min _
E WASH, 55 min

MIA“

5 mV
12.5mSec

Figure 15. Effect of bath exposure to DTB on EPP amplitude.

Effects of bath application of DTB on nerve-evoked EPP amplitude. (A)
Control EPPs were recorded for 4 minutes prior to bath application of DTB (1.85
mM). Following application of DTB there was (B) an initial rise in EPP amplitude
followed by (C) a decrease in amplitude. (D) With continued exposure to DTB,

EPP rise and decay times are increased. (E) Washing with DTB-free solution caused
a partial reversal of DTBs effects.

57
MEPP frequency also exhibited a biphasic response following exposure to

DTB. Soon after exposure to 1.85 mM DTB there was a large increase in MEPP
frequency. With continued exposure, however, MEPP frequency declined toward
control levels (Figure 16). At the time of block of the EPP, MEPP frequency was
still signiﬁcantly elevated (Figure 17).

As was observed in muscles from rats treated with a single large dose of DTB,
large amplitude MEPPs were observed which occurred at multiples of the mean
MEPP amplitude (Figure 18).

Diaphragms exposed to DTB in the perfusion medium also showed a slowing

of the decay time for MEPPs (Figure 19).

58

A CONTROL MEPPs C DTB, 25 min

vmuwﬂﬁdryw' ”WWW M W

WWW
W W
3%W mfg“ WW

8 DTB, 10 min D WASH, 80 min

 

Figure 16. Effect of bath exposure to DTB on MEPP frequency.

Figure 16. Increased MEPP frequency produced by bath application of DTB
(1. 85 mM) at the rat neuromuscular junction. MEPPs were recorded (A) 5 minutes
prior to bath application of DTB (control); (B) after 10 minutes exposure to DTB;
(C) immediately following block of the EPP which occurred between 23 and 24
minutes after washing in DTB; or (D) after 80 minutes of washing with DTB free
solution.

59

 

\\\N~ *

 

 

 

 

 

 

 

MEPP FREQUENCY (hz)

O
k

CON DTB

Figure 17. Bath exposure to DTB increased MEPP frequency.

Effect of bath-applied DTB (1.85 mM) on MEPP frequency at the rat
neuromuscular junction. DTB treatment and uncoupling of contraction from nerve
stimulation are as in Figure 13. MEPPs were recorded for 5 minutes prior to DTB
application (control) and then at 10 minute intervals during bath application of DTB.
Data presented are taken at a mean time of 35 minutes of exposure to DTB. Values
are the mean 1 SEM of seven preparations. The asterisk indicates a value
signiﬁcantly different than control (p s 0.05).

b
O

30

20

10

PERCENT OF TOTAL MEPPs

1

 

 

60

o
‘
0‘.

0.3 0.6 0.9 1.2
MEPP AMPLITUDE (mV)

Figure 18. Bath exposure to DTB alters the distribution of MEPP amplitudes.

MEPP amplitude histograms before and after bath application of 1.85 mM
DTB. Control MEPPs were recorded for 5 minutes at the start of each experiment.
MEPPs recorded in DTB-containing medium were measured at a mean time of 33.5
minutes following exposure to DTB. The data are displayed as percent of total
MEPPs observed at each amplitude. MEPP amplitudes are not standardized to -50

mV.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

lg: TEP%JI:=P CON
E“ Z Z DTB%
% %

612' {VI

 

 

 

 

 

 

 

0

Figure 19. Effect of bath exposure to DTB on rise and decay times for EPPs and
MEPPs.

Effects of bath-applied DTB on (A) rise and (B) decay times of MEPPs and
EPPs at the neuromuscular junction. Diaphragms were removed from untreated
animals, cut, and placed in [low K‘] buffer to prevent contraction due to
stimulation of the phrenic nerve. MEPPs and EPPs were recorded before and after
bath application of DTB (1.85 mM). EPPs were evoked at a frequency of 1 Hz.
Diaphragms were maintained in DTB-containing solution until block of the EPP
occurred or for approximately 60 min, whichever occurred ﬁrst. Data presented are
taken at a mean time of 33.5 minutes of exposure to 1.85 mM DTB. Values are the
means 2 SEM of at least six preparations. The asterisk indicates a value
signiﬁcantly different than control (p s 0.05).

62

Discussion.

Chronic daily treatment with DTB causes ﬂaccid muscle weakness and
depressions in evoked and spontaneous release of ACh. Conversely, acute
administration of DTB does not cause ﬂaccid neuromuscular weakness, even when
given at doses which approach the LDSO. What was unknown was whether acute
administration of DTB could alter neuromuscular transmission in subtle ways that
might not be apparent in the whole animal over that time course. In striated muscle,
a twitch will occur so long as the amplitude of the EPP reaches threshold for
generation of an action potential. Typically, a large safety-factor exists for
neuromuscular transmission (Paton and Waud, 1967). That is, the amplitude of the
evoked EPP is typically much greater than that necessary to elicit an action potential
in the muscle (and hence a twitch). A reduction in safety factor for neuromuscular
transmission during acute DTB-treatment could reduce the EPP amplitude compared
to non-treated end-plates yet not reduce the amplitude below the action potential
threshold, and hence not block muscle contraction. This is particularly true for the
diaphragm, which has a much higher safety factor than do hindlimb muscles (Paton
and Wand, 1967; Waud and Waud, 1972). This no doubt, accounts for the
observation that paralytic effects of DTB in the hindlimb appear to precede those on
respiratory muscles (Atchison and Peterson, 1981). By examining early effects on
neuromuscular transmission, in rats treated with a single 25 mg/kg dose of DTB,
or very early effects on transmission following bath application of DTB, it was hoped

that events responsible for the onset of paresis in chronically treated rats could be

63

determined. In general the effects of bath application of DTB on neuromuscular
transmission resembled those following a single large dose of DTB, the main
difference being that following bath application there was an initial transient increase
in EPP amplitude, MEPP frequency and MEPP amplitude followed by a steady
decline with continued applicatiOn of DTB, whereas following a single large dose of
DTB only a decrease was noted.

With the exception of decrease of quantal content, the alterations in
transmission observed following a single large dose were similar to those observed
following chronic treatment with DTB. These effects included depression of EPP
amplitude (not signiﬁcant), a decrease in MEPP frequency (Weiler £141., 1986;
Atchison, 1989) and a slowing of the rise and decay times for MEPPs (Atchison,
1989). Atchison (1989) found the decrease of quantal content to be due to a
decrease in the readily releasable store of ACh rather than effects on the probability
of release. The fact that neither a reduction in quantal content nor paresis was
observed in acutely treated rats may indicate that the paresis observed in chronically
treated rats is due to a gradual decrease in the releasable pool of transmitter.

In both the bath application and acute dose studies a slowing of rise and
decay times for EPPs and MEPPs was observed which did not recover to control
levels at the same rate as MEPP frequency and EPP amplitude. Atchison (1989) also
reported a similar alteration of the rise and decay times following chronic exposure
to DTB. These alterations of rise and decay times may indicate that DTB has

postsynaptic effects in addition to the presynaptic eﬁects reported by others

64
(Atchison £111., 1982; Weiler £1_al., 1986). A postsynaptic effect would be

expected if DTB were reacting with sulfhydryl groups located on the AChR (Karlin
and Bartels, 1966; Karlin, 1980). Reduction (Ben-Haim £1_a1,, 1973; 1975;
Terrar, 1978), oxidation or sulfonation (Steinacker and Zuazaga, 1981; 1987) of
the postjunctional nicotinic receptor all cause alterations of decay kinetics of the EPP
or EPC.

In conclusion, acute exposure to DTB via a single large dose or bath
application causes deﬁcits in neuromuscular transmission. With the exception of a
decrease in quantal content the effects observed following acute exposure in many
ways resemble those observed in rats paralyzed following chronic treatment with
DTB. From these results it appears that alterations in neuromuscular transmission
occur soon after treatment with DTB is initiated and that with continued exposure
these deﬁcits progress to the more generalized muscle weakness observed in

chronically treated rats.

Chapter 6. Effects of exposure to DTB on end-plate currents.

Summary of results of acute studies. In experiments designed to determine
early effects of DTB on junctional transmission, it was found that as early as 1 hour
following a single dose 25 mg/kg of DTB, at a time when no muscle weakness is
observed, EPP amplitude appears depressed (not signiﬁcant) and MEPP frequency
was decreased (Chapter 5). Rise and decay times for MEPPs were prolonged in a
manner similar to that observed in chronically-treated rats exhibiting skeletal muscle
weakness induced by DTB. The early effects of DTB on EPP amplitude and MEPP
frequency appear to reverse by 24 hour after a single dose of DTB; the slowing of
rise and decay times does not. These early alterations of rise and decay times may
represent effects of DTB on transmission which, with continued exposure, could
lead to the decreased junctional transmission observed in chronically-treated rats.

Prolongation of rise and decay times of synaptic potentials could be due to
presynaptic or postsynaptic eﬁects (Table 1). Prolonged. rise and decay times for
EPPs and MEPPs might be observed if DTB altered binding of ACh to its receptor,
opening and closing of the receptor-gated ionic channel or reduced levels or activity
of AChE (Eccles and MacFarlane, 1949). A DTB-induced depolarization of the
muscle cell membrane would cause a decrease in EPP and MEPP amplitude as well
as alter time constant for decay of current ﬂowing through the ACh-channel

(Magleby and Stevens, 1972).

6S

Table 1. Summary of effects of DTB.

66

 

 

 

 

 

 

 

 

 

 

 

\ 0‘
0° «‘0
“90““ “a"
e \
Fo‘g‘ 0‘9‘0“
Muscle
weakness no yes ? ?
EPP (ml - dec. yes -
Failure of
transmission no yes yes -
MEPP
frequency dee. dee. yes 7
Slow rise
and decay yes yes ? ?
for potentials:
Abnormal
MEPPs yes yes ? 7
(\0 (\o
e? 09
0940 s‘gq“
v‘ 90

 

67
(Weiler £1_a1, (1986) found no evidence of alterations of either postjunctional

membrane potential or muscle input resistance in rats paralyzed after 5 days of DTB
treatment.) A decrease in MEPP amplitude could lead to an apparent decrease in
MEPP frequency by increasing the number of MEPPs lost in background noise.
DTB could inhibit the packaging of ACh into vesicles thus decreasing quantal size
(MEPP amplitude) and in turn EPP amplitude (without affecting m). DTB-induced
terminal retraction or glial cell interposition [pathological observations associated
with chronic treatment with this agent (Kemplay, 1984; Jones, 1989)] could also
prolong rise and decay times by altering the diffusion distance for ACh within the
synaptic cleft to the end-plate receptors. DTB might also cause release of ACh from
sites in the terminal which are not directly apposed to postjunctional invaginations
again increasing the diffusion pathway for ACh to its receptors.

Objectives. The objectives of the studies described in the following chapters
were to determine whether a postsynaptic modiﬁcation was responsible for the
observed effects on EPP and MEPP rise and decay kinetics. More speciﬁcally, the
purpose of these studies was to determine whether DTB altered current ﬂow through
AChR-gated channels in muscles taken from rats exposed acutely to a dose of DTB
which causes no overt signs of neuromuscular weakness at the whole animal level or

chronically to DTB at a regimen that causes observable neuromuscular weakness.

Hypothariv

Treatment of rats with DTB leads to an alteration in current ﬂow through
AChR-gated ion channels present in the muscle cell membrane. The time course of
these effects indicate that they may be partially responsible for the weakness

observed with continued treatment.

69

Introduction to voltage clamp. Following a change in voltage across a
membrane or a change in conductance of channels in a membrane there will be a
current (1,) ﬂowing across the membrane. The measured current has two
components- a capacitive component (L) and an ionic component (L), where:

L=L+L
. In this equation the ionic component (Ii) is equal to the capacitance of the
membrane times the change in voltage across the membrane as a function of time
(dv/dt). By clamping the membrane potential at a constant level the change in
voltage will equal zero, so the capacitive current component drops out of the above
equation. This allows measurement of only the ionic current ﬂowing through
channels in the membrane.

The voltage clamp which was used in these studies utilizes two
microelectrodes placed in the same cell to maintain the membrane potential at a
steady level. One electrode is used to measure membrane potential via a voltage
follower circuit and the second electrode passes current back into the cell. The
voltage clamp ampliﬁer compares the membrane potential measured by the voltage
follower circuit to a reference voltage (holding potential). Current is passed through
the second electrode to bring the membrane potential back towards the reference or
clamped level (Halliwell £1_a1,, 1987 ). Following transmitter release into the

synaptic cleft, during which time an EPP or MEPP would normally be recorded, the

70

voltage follower circuit senses a change in membrane potential which leads to the
passage of current through the current passing electrode. The current required to
clamp the membrane potential at a constant level during the action of transmitter
can then be recorded. This current should be equal and opposite to that ﬂowing
through AChR-channels in the membrane during the action of transmitter.
Utilizing voltage clamp it is possible to determine several characteristics of
ionic currents ﬂowing through the channels within the membrane. First, the peak
current moving through channels at a given membrane potential can be measured.
By measuring the peak current which ﬂows across the membrane at a series of
holding potentials a current vs voltage relationship can be obtained (T akeuchi and
Takeuchi, 1959). Several pieces of information can be gathered from the current
vs voltage relationship. The slope of current vs voltage relationship can give
information concerning the conductance of channels active during the measured
current. Second, the membrane potential at which the end plate current (EPC)
reverses its direction of ﬂow through the membrane (reversal potential) can be
determined. This provides information on the nature of the ions which make up the
active current (T akeuchi and Takeuchi, 1960). In addition, by examining the rise
and decay phases of single EPC’s or MEPC’s one can gain information regarding the

kinetics of channel opening and closing (Magleby and Sevens, 1972a; 1972b; Linder

star. 1984).

71
Methods.

The methods used for the following experiments are described in detail in the
materials and methods section of this manuscript. Brieﬂy, voltage clamp
experiments were conducted using the two microelectrode voltage clamp technique
(Takeuchi and Takeuchi, 1959) and hemidiaphragm preparations (Billng 1946)
taken from control male rats (180-250 g, Harlan Sprague-Dawley Laboratories,
Madison, WI) and rats exhibiting muscle weakness following chronic treatment with

DTB (1 mg/kg/ day) or rats exhibiting no weakness following a single dose of DTB

(25 Ins/ks)-

Results

Effects of DTB on behavior of rats. Rats treated with 1 mg/kg/day of DTB
for 7 to 8 days exhibited marked muscle weakness and displayed other signs of DTB
intoxication (Atchison and Peterson, 1981; Atchison £131., 1981a; Williams £131.,
1986). Rats given a single 25 mg/ kg dose of DTB failed to develop any observable
weakness for up to 24 hour after treatment and have been shown not to develop
weakness for extended periods of time following treatment (Atchison and Peterson,
1981), however, these rats did develop other signs of DTB intoxication observed in
chronically treated rats.

Effects of DTB on EPC amplitude and quantal content and MEPC amplitude.
Figure 20 shows peak amplitude of EPCs recorded at holding potentials between -100

mV and + 10 mV. In control cut muscles, the EPC reversal potential occurred at

72
a holding potential of approximately -10 mV, a value similar to that reported by

Glavinovic (1979) for cut muscle of the rat, while the current vs voltage relationship
had a slope of 2.26 nA/mV. In several control preparations there was a inward
rectiﬁcation of the current vs voltage relationship at holding potentials more negative
than ~60 mV, a result consistent with previous observations of voltage-dependence
of the EPC at hyperpolarizing potentials for frog neuromuscular junctions (Magleby
and Stevens, 1972b). The current vs voltage relationship for muscles taken from rats
following either acute high dose or chronic low dose treatment with DTB exhibited
a similar pattern to that of controls. The slope of the current vs voltage relationship
was decreased to 1.48 and 1.71 nA/mV in muscles taken 1 and 24 hour respectively
following treatment with a single dose of 25 mg/kg and to 1.1 nA/mV in muscles
from chronically-treated rats. In DTB-treated preparations in which inward
rectiﬁcation of the current vs voltage relationship was observed, the holding
potential at which rectiﬁcation occurred was more negative than in control

preparations, typically occurring in the range of -65 to -75 mV.

73

 

 

 

 

 

 

 

I (DA) .II.

Figure 20. Effect of DTB treatment on peak EPC amplitude.

Effects of DTB on peak end-plate current (EPC) amplitude at membrane
potentials held between -100 mV and +10 mV. EPC amplitude recorded in
hemidiaphragm preparations taken from A) (0) control rats and (O) rats exhibiting
neuromuscular weakness following chronic treatment for 7 days with 1 mg/kg/day,
ip. of DTB or B) (0) control rats and rats treated (t) 1 hour and (c) 24 hour
previously with a 25 mg/kg dose of DTB. Values are the mean of 3-11 preparations.
The error bar represents the average SEM for all values of a given group. The
asterisk (‘) denotes a slope signiﬁcantly different from control (p < .05).

74
Quantal content (m) of EPCs was calculated for EPCs elicited at holding

potentials between -60 and -30 mV. Values of m calculated at holding potentials
more negative than -60 mV were not used due to rectiﬁcation of the EPC observed
in some preparations, while values for m calculated at holding potentials more
positive than -30 mV were not used. This was due to the fact that normal amplitude
MEPCs were lost in background noise in some preparations. Giant MEPCs, which
were more common in muscles from DTB-treated rats, would still be counted thus
biasing mean MEPC amplitude towards the giant MEPC amplitudes. Figure 21
shows that in muscles taken from rats exhibiting paresis following DTB treatment
there was a signiﬁcant decrease (p < 0.05) in m. Although m also appears depressed
in muscles removed 1 and 24 hour following a single 25 mg/kg dose of DTB, this
decrease is not signiﬁcant (p > .05).

In control preparations, mean amplitude of MEPCs recorded at a holding
potential of -50 mV was 1.93 t 0.31 nA, a value similar to that reported for
transected rat diaphragm muscle by Glavinovic (1979). In muscles taken from rats
1 and 24 hour following treatment with a single 25 mg/ kg dose of DTB, mean
MEPC amplitude at -50 mV was 2.75 :t 0.7 and 2.02 a 0.73 nA respectively, while
in muscles taken from rats chronically-treated with DTB, MEPC amplitude was 1.96
:1; 0.3 nA at -50 mV. None of these values differed signiﬁcantly from control. As
early as 1 hour following treatment with a 25 mg/ kg dose of DTB, the incidence of
abnormally large (greater than twice the mode value), slow MEPCs was increased
from 5.1% of all MEPCs in controls to 28.4% in muscles taken 1 hour after

75
treatment (Figure 22). Moreover the incidence of these abnormally large MEPCs

was increased to 32.4% of all MEPCs in muscles taken from rats treated 24 hour
previously with a 25 mg/kg dose of DTB. As described earlier in current clamp
experiments (Atchison, 1989), the frequency of large slow MEPCs was also
increased in muscles taken from rats treated chronically with DTB (12% were greater
than twice the mode amplitude for controls). In addition to the observed increase
in abnormally large, slow MEPCs, there also appears to be an increase in the
number of small MEPCs with amplitudes of 0.25 to 0.75 nA (at -50 mV) both in
muscles from rats treated 24 hour previously with a 25 mg/kg dose of DTB and in
muscles from rats chronically treated with DTB (Figure 22). To determine whether
DTB treatment decreased the amplitude of normal MEPCs, as was reported for
MEPPs earlier (Weiler £131., 1986; Spitsbergen and Atchison, 1990), we examined
the amplitudes of MEPCs with fast rise and decay which appeared to be the mode
of the amplitude distribution, and hence presumably reﬂected single quantum
MEPCs. In both control and treated preparations the amplitude of the mode
MEPCs was slightly smaller than the mean MEPC amplitudes, however, the
amplitude of mode MEPCs in muscles taken from rats 1 hour following a 25 mg/kg
dose of DTB was greater than that for mode MEPCs in controls (p < 0.05; Figure
23).

76

01
O

N
01

 

 

QUANTAL CONTENT (m)

0

)

00/7 7621/, 0’

TREATMENT TIME

Figure 21. Effect of DTB treatment on m of EPCs.

Effects of DTB on quantal content (m) of EPCs recorded in hemidiaphragm
preparations taken from control rats, rats 1 and 24 hour following a single 25 mg/ kg
dose of DTB, or from rats treated for 7 days with 1 mg/kg/day, ip. of DTB.
Quantal content was calculated by dividing mean EPC amplitude by mean MEPC
amplitude for EPCs and MEPCs recorded from the same end-plate region at the
same holding potential. Values are the means a SEM of 3 to 11 preparations. The
asterisk (‘) denotes values signiﬁcantly different from control (p < .05).

77

 

 

 

 

 

 

 

45’ A Control
soI
15»
0)
g 2 . e s
g 45. B 7 hr
30’
q .
E 15 “h
e 2 4 e s
45' C 24 hr
% 30’
k 15-
E 2 4 6 s
g 45, 0 chronic
E 30-»
15’
* 2 4 3 s

MEPC AMPLITUDE {nA/

Figure 22. Effect of DTB treatment on the distribution of MEPC amplitude.

Amplitude distribution histogram of MEPCs recorded at a holding potential
of -50 mV in hemidiaphragms taken from A) control rats, and rats treated 1 B) and
24 C) hour previously with a 25 mg/ kg dose of DTB, respectively and D) rats
treated chronically with 1 mg/ kg/ day of DTB for 7 days. Values are expressed as
percentage of total number of MEPCs counted. Between 100-200 MEPCs were
sampled from at least 3 preparations for each treatment group.

78

 

 

 

 

A IInA) ‘-4
B 5 '° *

Ill

8

I: -2

.J

Q

E -1

O

0..

lg c

Figure 23. Effect of DTB treatment on the amplitude of modal MEPCs.

Effects of DTB on mean MEPC amplitude for modal (single quantum)
MEPCs. MEPCs having the modal value from each end-plate region were summed
and the effects of DTB following acute and chronic exposure were determined. A)
Current/Voltage relationship for MEPCs recorded in hemidiaphragms taken from
(0) control and (O) chronically-treated rats measured at holding potentials from -20
to -90 mV. B) Comparison of MEPC amplitudes recorded at a holding potential of
-50 mV in muscles taken from control rats, rats treated I and 24 hour previously with
a single 25 mg/ kg dose of DTB or rats exhibiting neuromuscular weakness following
7 days of treatment with 1 mg/kg/day of DTB. Values in A are the means of at
least 9 preparations, the single error bar represents the average SEM for each group.
Values in B represent the means 1- SEM of 3 to 11 preparations. Between 50 and
100 MEPCs were sampled at each holding potential in a given preparation. The
asterisk (‘) indicates a value significantly different from control (p < 0.05).

79
Effect of DTB on rise and decay of synaptic potentials. Despite the reported

slowing of decay time for EPPs described earlier, the decay time constant (r) for
EPCs was not greater in muscles from treated rats than controls. In fact, 1: m for
muscles taken 24 hour following a single 25 mg/kg dose of DTB was decreased
compared to controls (Figure 24). Initial examination of 1: MEPC indicated a
signiﬁcant increase (p < 0.05 ) in r for muscles taken from rats exhibiting
neuromuscular weakness following chronic treatment with DTB and a reversal of the
normal voltage-dependence of t (Figure 25). t MEPC for muscles taken following
acute treatment with DTB was unchanged or only slightly increased. If one
calculates r for the mode MEPCs, leaving out the abnormally slow, large amplitude
MEPCs, then r MEPC is actually signiﬁcantly decreased (p < 0.05) in muscles from
chronically-treated rats compared to controls (Figure 26) and the voltage-
dependence of t is normal. In a similar manner, t MEPC for mode MEPCs recorded
at -50 mV in muscles taken 24 hour following a single 25 mg/kg dose of DTB is also
decreased signiﬁcantly (p < 0.05) analogous to the results seen for r EPC for these
muscles.

The 10-90% rise time for EPCs in muscles removed 24 hour following a single
25 mg/kg dose of DTB is shorter than in controls (Figure 27), while 10-90% rise
time for EPCs of muscles taken 1 hour following a single 25 mg/kg dose of DTB,
or those taken from chronically-treated rats have rise times similar to controls. DTB

treatment had no effect on MEPC rise time calculated for the modal MEPCs.

80

N

.5

 

 

TEpc (msec)

- 1 2 0 - 9 O - 6 0 - 3 O
HOLDING POTENTIAL (mV)

Figure 24. Effect of DTB treatment on r EPC'

Effects of DTB on r EPC and its voltage-dependence. Decay time constants (r)
were determined by a computerized curve ﬁtting routine, which ﬁt exponential curves
to the decay phase of the EPCs. 1: was determined for EPCs elicited at holding
potentials from ~30 to ~100 mV in muscles from (0) control rats, in muscles taken (O)
1 and (.) 24 hour following a single 25 mg/kg dose of DTB and muscles from (a)
rats treated for 7 days with 1 mg/kg of DTB. Values are the means of 3 to 11
preparations. Error bars represent the average SEM for each group. In panel A the
asterisk (') denotes a line whose points have signiﬁcantly different heights from those
on the control line (p < 0.05), while in panel B the asterisk (') denotes a value
signiﬁcantly different from control (p < 0.05).

81

 

 

 

5 O '

A I Con 7 day
8 .

m a- 3
g 3 " '[______I—w . 4' O

I 5

8 .. k 0 O U *0
l.l.I
[‘2 1 .

 

 

- 1 0 O - 7 5 - 5 O - 2 5
HOLDING POTENTIAL (msec)

Figure 25. Effect of DTB treatment on t merc-

Effects of DTB on t MEPC and its voltage-dependence. Calculation of t MEPC
was similar to that described for r EPC in Figure 24. MEPCs were recorded and t
calculated at holding potentials of ~30 to ~100 mV in muscles taken from (0) control
rats and from rats (O) chronically treated with DTB. Values represent the mean :r.
the average SEM of at least 6 preparations. Values to the right of the asterisk (‘)
have heights signiﬁcantly different from points on the control line (p < 0.05).

82

01

C0

 

 

TMEPC (msec)

00,,7 be”); a

Figure 26. Effect of DTB treatment on r MEPC for modal MEPCs.

Effect of DTB on rum and voltage-dependence of t for modal (single
quantum) MEPCs recorded at a holding potential of ~50 mV. 1 was calculated from
the inverse of the slope of a senrilog plot of the decay phase vs time for summed
MEPCs having the mode amplitude from a given end-plate region. Values represent
the means 1- SEM of at least 3 preparations. The asterisk (') denotes a value
signiﬁcantly different from control (p < 0.05).

 

 

’5

8

E .5
a?

O

0’:

l

O

'- .75' B
‘2'

I: .5
h

E .25

Figure 27. Effect of DTB treatment on 10-90% rise time for EPCs and modal
MEPCs.

Effects of DTB on the 10-90% rise time of A) EPCs and B) mode amplitude
MEPCs. The 10-90% rise time was calculated for EPCs and MEPCs recorded at a
holding potential of -50 mV in muscles taken from control rats, chronically treated
rats and acutely treated rats. Values represent the mean : SEM of at least 3
preparations.

Discussion.

Although rise and decay times for EPPs and MEPPs were reported to be
prolonged both in muscles from chronically-treated rats exhibiting DTB-induced
muscle weakness (Atchison, 1989) and in muscles taken from rats treated acutely
with a single 25 mg/kg dose of DTB (Spitsbergen and Atchison, 1990), a similar
prolongation of rise and decay kinetics of EPCs and MEPCs was not observed in this
study. Initially it appeared as though t we was increased and the voltage-
dependence of t reversed in muscles from rats exposed to DTB. However, upon
closer examination it became evident that this was an artifact caused by the increased
incidence of abnormal, large amplitude MEPCs with slow rise and decay. This
appears to be the case, since at depolarized holding potentials the slowing of decay
kinetics was most pronounced, an observation which opposes the normal voltage-
dependence of t MEPC observed in frog for the nicotinic ACh channel (Kordas, 1969;
Magleby and Stevens, 1972a). If on the other hand, one calculates t MEPC only for
MEPCs with fast rise and decay, which are still observed in every preparation, then
MEPC decay does not appear to be slowed and the voltage-dependance of t is
normal. Therefore, what appears to be happening is that at depolarized holding
potentials, at which MEPC amplitude is depressed due to a decreased ionic driving
force across the membrane, a higher percentage of normal MEPCs is lost in
background noise thus causing the ratio of abnormal large amplitude MEPCs to
normal MEPCs to increase in the DTB-treated preparations. This apparent increase

in the number of abnormal MEPCs then biases all further calculations of mean

85
MEPC amplitude and rise and decay kinetics toward the abnormal MEPCs.

It is interesting that once the abnormal MEPCs are removed from the
calculation of t MEPC that 1 actually appears to be decreased in muscles from rats
exposed to DTB as does t are for muscles taken 24 hour following a single large dose
of DTB. A DTB-induced decrease in decay kinetics would not be unexpected since
DTB is a mild reducing agent (Preisler and Bateman, 1947) and reduction of the
nicotinic AChR has been reported to cause shortening of decay kinetics for MEPPs
and MEPCs (Ben-Haim £131., 1973; 1975; Terrar, 1978).

In light of the results described above it is apparent that the prolongation of
rise and decay times observed for EPPs and MEPPs following exposure to DTB does
not appear to be due to a DTB-induced prolongation of channel open-time. The
observed prolongation of rise and decay times for MEPPs could easily be explained
by the increase in the incidence of abnormal MEPPs observed following DTB
exposure. In many cases the amplitudes of these MEPPs are no different from the
normal MEPPs, with only the rise and decay times being prolonged, making it
difﬁcult to separate these events during analysis. In all cases, one is able to observe
normal, rapid rise and decay MEPPs or MEPCs interspersed with the abnormal
ones. Therefore, the cause of the observed slowing must not be due to an effect on
all AChR-channel complexes. Instead DTB might alter a portion of ACh release
sites or possibly a portion of the receptor sites, thus affecting only a fraction of the
observed MEPCs. Although Weiler 913.1. (1986) reported no difference in the input

resistance of the muscle membrane between DTB-treated rats and controls, an in-

86
depth study of passive cable properties was not made. It is possible that the reported

slowing of MEPP and EPP rise and decay times could be due to a DTB-induced
alteration in cable properties of the muscle membrane, an effect which would be
lessened in a preparation for which the membrane potential is held constant and only
active current ﬂow measured.

The origin of these abnormally large amplitude, slow decay MEPCs remains
an enigma. They are occasionally observed in control preparations, but both their
incidence of occurrence and their characteristic amplitude and kinetics are much
more pronounced in DTB-treated preparations. The events are observed under
voltage-clamp interspersed with MEPCs having normal amplitude and decay kinetics.
This makes it unlikely that the aberrant MEPCs are due to a generalized
postjunctional phenomenon. It is certainly possible that a fraction of the ACh
channels may be modiﬁed following treatment with DTB, leading to the occurrence
of these abnormal quantal events. On the other hand, an equally likely, and
perhaps more probable explanation is that these events reﬂect alterations by DTB
in the quantal release process. Extremely large amplitude (> 1.5 mV) MEPPs have
been reported at mammalian neuromuscular junctions since the 1950’s (Liley, 1956).
Yet in some pathological states, the incidence of occurrence of the abnormal MEPPs
is increased dramatically and their rise and decay time is prolonged markedly. These
abnormal MEPCs, seen as giant, slow MEPPs in current clamp experiments have
been reported to be present following treatment of neuromuscular junctions with

botulinum toxin (Cull-Candy 3131., 1976), 4-aminoquinoline (Molgo, and Thesleff,

87
1982), following prolonged curarization or paralysis with tetrodotoxin (Katz, 1966),

or following nerve degeneration (Vizi and Vyskocil, 1979). The incidence of small
amplitude MEPPs (sub-MEPPs) is also elevated at neuromuscular junctions poisoned
with botulinum toxin (Cull-Candy £131., 1976) and at developing or regenerating
neuromuscular junctions (Muniak 3,131., 1982). These abnormally large amplitude,
slow MEPPs and small amplitude sub-MEPPs both appear to be associated with
degeneration and regeneration of the terminal region. In DTB-treated rats the
incidence of large, slow MEPCs is elevated as early as 1 hour following treatment,
while the incidence of both large and small amplitude MEPCs is increased by 24
hour after treatment, suggesting early alterations in the transmitter release process
similar to those found in degenerating and regenerating nerve terminals. However,
morphological studies examining muscles from DTB-treated rats have demonstrated
little nerve degeneration even in chronically-treated rats exhibiting marked
neuromuscular weakness, although terminal retraction and sprouting are observed
(Kemplay, 1984; Jones, 1989).

In conclusion, both quantal content of nerve evoked EPCs and t MEPC for the
normal fast rise and decay MEPCs are decreased in muscles taken from rats
exhibiting muscle weakness following chronic treatment with DTB. t MEPC and t are
are both decreased in muscles taken from rats treated 24 hour previously with a
single large dose of DTB, while quantal content of evoked EPCs is not depressed
signiﬁcantly. Thus, following exposure to DTB shortening of the decay of synaptic
currents is observed, possibly indicating a DTB-induced alteration in the duration

88

which ACh remains bound to its receptor or a decrease in the open time for AChR-
gated ionic channels. These postsynaptic alterations occur prior to presynaptic effects
of DTB, which result in a decrease in quantal content of evoked EPCs. Whether
these alterations are ultimately directly responsible for this muscle weakness or lead
to secondary compensatory changes which result in the observed weakness is not yet
clear. These postsynaptic alterations are not likely to be responsible for the early-
occurring large amplitude "slow MEPCs” so characteristic of the neuromuscular

action of DTB.

Chapter 7. Effects of DTB exposure on single-channel currents.

Summary of results on end-plate currents. Results of the previous voltage
clamp experiments indicate that the decay time constant for EPCs and MEPCs is
decreased in muscles taken from rats treated with DTB. These results suggest that
DTB may act at AChR ion-channels present at the end-plate region to decrease the
time which channels remain open. However, the EPCs and MEPCs which were
studied result from transmitter release from the nerve terminal. Alterations in
release processes, synaptic cleft morphology or cholinesterase activity could still alter
these currents and thus obscure, potentiate or confound postsynaptic events.

Objective. The purpose of the following study was to determine directly,
using single-channel recording techniques, whether the observed decrease in 1: m
and 1' MEPC for fast MEPCs is due to an effect of DTB on current ﬂow through

nicotinic AChR—channels.

Introduction.

Patch voltage clamp. Application of a low resistance (1 to 10 M0 ), heat-
polished, microelectrode to the surface of a cell membrane causes the formation of
a seal between the electrode and the cell membrane. Typically the resistance of the
seal which forms under these conditions is on the order of 50 to 100 M0 (N eher and

Sakmann, 1976). By applying negative pressure to the interior of the microelectrode

89

90
following initial contact with the cell can lead to the formation of a very high

resistance seal, on the order of 1 to 100 GO, known as a ”giga-seal” (Hamill e131,,
1981). Due to the restricted area of membrane recorded from and the extremely
high electrical resistance of this seal, background noise is decreased to a level at
which currents can be observed which are due to the passage of ions through single
channels in the membrane (Neher and Sakmann, 1976; Hamill e131,, 1981).
The ability to measure current flow at the single channel level allows one to
. determine accurately whether compounds of interest alter the single-channel
conductance or processes associated with opening and closing of channels (N eher and
Sakmann, 1976; Hamill and Sakmann, 1981; Colquhoun and Sakmann, 1981).
The seal formed following suction is mechanically very stable. This allows several
variations on the traditional cell-attached patch conﬁguration described above. First,
by applying a second pulse of suction following formation of a giga-seal, or by
passing an oscillating voltage across the membrane, the patch of membrane inside
the electrode can be ruptured resulting in what is known as the "whole-cell patch"
conﬁguration. This conﬁguration allows both chemical access to the interior of the
cell and electrical access to the whole cell membrane. Using this technique with
small spherical cells allows one to clamp the membrane potential of the entire cell
and record currents from the whole cell, similar to those recorded using two
microelectrode voltage clamp described earlier. Second, following formation of a
giga-seal (cell-attached patch) the electrode can be pulled away from the cell causing

formation of a small vesicle at the tip of the electrode. Subsequent movement of the

91
electrode tip across the air liquid interface leads to rupture of the outer portion of

the vesicle. This results in the formation of a patch electrode with a portion of
membrane sealed across it, with the intracellular side exposed to the bathing
medium. This conﬁguration is referred to as the ”inside-out, cell-free" patch.
Finally, if one pulls the electrode away from the cell when in the whole-cell
conﬁguration a portion of membrane will pull free and form a patch across the
electrode tip which has its extracellular side out and is thus known as the "outside-
out, cell-free patch” (Hamill M, 1981). An advantage of the cell-free
conﬁguration is the ability to alter constantly or make additions to the solutions to
which the exposed portion of the membrane is in contact with. This allows one to
examine the effects of different compounds on a single patch of membrane and to
examine the reversibility of the observed effects.

Study of Cells in Culture. In the early 1900’s Harrison (1907) made the
discovery that embryonic neural tissue from frog could be maintained for extended
periods of time outside of the organism in a tissue culture environment. The high
degree of control offered by cell culture has led to widespread use of this technique
in the years since Harrison’s original discovery. A wide variety of cells are presently
used for cell culture studies, including acutely dispersed embryonic cells (primary
cell culture) and transformed cell lines (clonal cells). Primary cell culture offers the
advantage of being normal, albeit embryonic cells, taken from some tissue of
interest. Thus, the characteristics of these cells should be similar to those of cells

within the organism. Problems associated with using primary culture are the

92
contamination of the culture with other cells (i.e. ﬁbroblasts or glial cells) making it

difﬁcult to identify the cell of interest. Second, primary cells arise from embryonic
tissue thus, some characteristics of these cells may differ markedly from those of
cells in adult organisms. Clonal cell lines, on the other hand, oﬁer a very
homogeneous population all with similar or identical characteristics. However, since
these cells are transformed cells (neoplastic cells) they can differ in many ways from
normal cells, thus, it is not certain how well ﬁndings in these cells correlate to those
of normal cells.

Rationale for cell culture studies. The use of cells in culture offers several
advantages over the use of intact tissues taken from treated animals. First, in a cell
culture system we can monitor the effects of DTB on only the cells in question
without worrying about toxicity to other systems in the rat. In addition, in cell
cultures we can monitor continuously changes in cell behavior following exposure to
a set level of DTB without worrying about metabolism and excretion. Access to
cells, both pre— and postsynaptic, for both electrophysiological recording and
iontophoresis is far easier in cultures due to the lack of connective tissue and
surrounding cells. Moreover, little metabolism should occur within our culture
systems, thus we can be fairly certain that observed effects are due to the compound
added (either DTB or a metabolite thereof).

Clonal Gs myoblast cells. All single channel recording experiments were
performed on G8 myoblast cells. 68 cells are a clonal cell line from mouse which,

when differentiated to myotubes, express AChR-channels. These cells were obtained

93
from American Type Culture Collection (Rockville, MD) and were grown on

collagen-coated culture dishes in medium consisting of Dulbecco’s modiﬁed eagles
medium containing 10% hOrse serum and 10% fetal bovine serum. Cells were grown
in culture dishes until conﬂuent, at which time the medium was changed to one
containing 2% fetal bovine serum and 2% horse serum. Decreasing the serum
content of the growth medium in this way enhances differentiation of cells to form
multinucleated spindles, which are highly responsive to ACh (Morris 3131,, 1989).
All studies were performed on cells of passage 20-30, however, some differences
were noted in cells later than passage 25. Thus care should be taken to use cells
around passage 20. Several different ACh-induced channel-conductances have been
reported in these cells (Morris and Montpetit, 1986; Morris 3131,, 1989) and were
also observed in the studies reported herein. Channels having a slope conductance

of 37 pS predominated. Only this class was used for further analysis.

Methods.

In the following study standard patch voltage clamp techniques were utilized
on outside-out, cell-free patches (Hamill 5131., 1981) formed from differentiated
G8 myotubes. Channel conductance and open and closed durations for channels
were determined for single-channel currents recorded from membrane patches of
cells grown in DTB for 1-3 days or from cells exposed acutely to DTB in the

recording solution.

94
Results.

Single-channel currents. Figure 28 shows the changes observed in a patch in
which the membrane potential was clamped at -100 mV, following a change in the
perfusing solution from one containing no agonist to one containing 10 u M SChz.
In the absence of agonist there is little or no channel activity. Within 1-2 minutes
of changing the perfusing solution to one containing agonist single-channel currents
can be observed. By 34 minutes following addition of SChz, as the concentration
of agonist in the bath reaches as steady state, a fairly high level of channel activity
can be observed. With continued exposure to this relatively high concentration of
agonist, desensitization of AChR-channels occurs leading to a decrease in the
observed frequency of channel openings (Jackson, 1988). Following desensitization
(5 to 10 min) channel openings occur in bursts of single channel activity followed by
periods of inactivity. This bursting phenomenon termed "nachschlag (second
helping)” is thought to represent a single channel returning from an inactive state to
an active state, opening and closing several times in quick succession and then

returning to an inactive state (Colquhoun and Sakmann, 1981).

95

_ Subay/dicﬁaﬂ'na m M
- 700/» V

7 min

2 min

3 min
5 min

lam/n
ZpA [—

20 In:

Figure 28. Single-channel currents.

Single channel currents recorded from an outside-out membrane patch at a
holding potential of -100 mV. Cell-free patches were formed from GS myotubes
differentiated for 4-6 days in medium containing 2% fetal bovine serum and 2%
horse serum. Following patch formation the perfusing solution was changed to one
containing 10 uM SCh2 and currents were recorded for 20 min. Currents shown are

filtered at a 2 KHz level.

96
In the last trace (10 min) many rapid transitions between the open and closed state

are observed. This is believed to represent open-channel block of the AChR-channel
by agonist. This phenomenon has been observed for all of the different agonist
molecules examined thus far and a variety of other positively charged (and some
neutral) molecules (Neher, 1983; Ogden and Colquhoun, 1985; Marshall 3131.,
1991).

Single-channel conductance. A characteristic of the G8 myotubes used in
these experiments is the presence of several different conductance classes of channels
(Figure 29). These could represent separate channels each with a different
conductance, or a single population of channels which show subconductance states
of their main conductance level. The majority of channels recorded had the same
conductance, which was calculated to be approximately 37 pS. Only these channels
were characterized in further studies. If single-channel currents are measured at a
variety of holding potentials and the amplitude of the currents plotted vs holding
potential, a current vs voltage relationship can be determined for the channels
present within the patch. Figure 30 shows current vs voltage relationships generated
from patches from 5 control cells and 5 cells exposed for 30 minutes to DTB. The
conductance of the channels active in the patches can be determined from the slope
of the line, as well as the reversal potential for the channels from the point at which
the line crosses the x-axis. From this ﬁgure it can be seen that exposure of patches

to DTB for 30 minutes appears to have no effect on single channel conductance.

97

Figure 29. Multiple conductance levels for single—channels.

Multiple conductance levels observed in some patches could represent
subconductance states of channels or multiple channel types. Single channel currents
elicited by 100 nM ACh were recorded in outside-out patches held at -100 mV.
Currents were ﬁltered at a 2 KHz level.

98

 

 

 

4...

2 2-

3' -

E o

LU -

D:

n:_2_ A

8 My
.%
_4tllltltltritlllrltl

-‘100 -80 -6O -4O -2O 0 20 4O 60 80 100

HOLDING POTENTIAL (mV)

Figure 30. Current vs Voltage relationship for single-channel currents.

Effect of DTB (10 u M) on current vs voltage relationship for single channels.
Single channel currents were recorded from outside-out membrane patches at holding
potentials between 4» 50 mV to 1100 mV in the presence of 100 nM ACh. Currents
were recorded in the absence of DTB for 5 minutes then the perfusing solution was
changed to one containing 10 u M DTB and currents were recorded continuously for
30 minutes. The data shown represent currents recorded following 30 minutes of
exposure to DTB. Only the amplitude of the predominant conductance class present
in patches were analyzed. Data shown represent the mean single-channel current of
patches excised from cells from 3 different culture-dishes before and after exposure

to DTB.

99

Table 2. Effect of DTB on single channel conductance.

Treatment Agonist Amplitude

 

 

 

 

 

Control ACh 3.34 i 0.1
30 min 10 ,u.lv1 DTB ACh 3.21 :t0.07
30 min 1 mM DTB ACh 3.26 :l: 0.07
Control SCh 2 3.1 3 :l: 0.06
5 mun 1 MM DTB SCh 2 3.02 :l: 0.07
30 min 1 MM DTB 801 2 3.02 :l: 0.07

==--_ = =__ .......... .
#
Control ACh 2.52 :l: 0.05
24 hr 1 MM DTB ACh 2.94 :l: 0.25
.x.

24 hr 10 MM DTB ACh 3.10:0.15

 

 

# later passage * different from control p<.05

100
Table 2 displays data recorded from cells exposed to DTB in their growth medium

and cells exposed to DTB only during recording. From this it can be seen that
exposure of cells to DTB for 24 hour may have an effect on channel conductance,
however, these cells were of a later passage and the conductance of control cells had
shifted slightly from earlier passages. This makes it difﬁcult to determine whether
this is a true increase in conductance or a shift in the type of channels expressed.
Effects of DTB on Open and closed durations. Upon examination of a current
trace containing single channel events (as in Figure 28) it can be seen that once
open, the duration which channels remain open is quite variable. Many long-lived
channel openings are interrupted by short closures. If one plots the frequency of
channels which remain open for a given duration of time (open-time) vs open-time,
a distribution is formed which describes the duration which channels remain open in
the patch (open-time distribution). Figure 31 (top) shows an open-time distribution
(or histogram) for single-channel currents recorded from a control patch. When this
histogram was ﬁt with an exponential curve it was found that the distribution was
described by the sum of two exponentials. In Figure 31 center and bottom the effects
of exposure to 10 uM and 1 mM DTB for 30 minutes can be seen. Following
exposure to 10 u M DTB the biphasic decay of single-channel currents is more
pronounced (Figure 31 center). At this concentration of DTB it appears as though
the fast time component of open-time (fast t) is decreased while the slow component
(slow 1) is increased or unchanged. Following exposure to 1 mM DTB both the fast

and slow components appear prolonged.

A cute

78

80 '

’
Lhkauuaaee

10

75 '

EVENTS

78'

IO '

25 .

101

Exposure to 0 TB

 

 

 

 

central

Tar dJNﬁ 6577

 

10

10

20 30 40

10 I'M 073

7'4- axno Ill!

20 80 40

0
IMDTB

f l'1kﬁ.lLlJ

L-l LL 1 n-

80 30 40

TIME (ms

-X- Recorded from same patches as control.

Figure 31. Open-time distributions for single-channel currents.

Open-time distributions from patches excised from control cells and exposed
to extracellular medium containing (top) 100 nM ACh, extracellular medium with
(center) 100 nM ACh plus 10 u M DTB or extracellular medium containing (bottom)
100 nM ACh and 1 mM DTB. Orrrents were recorded from outside-out patches at
a holding potential of -100 mV. Each distribution represents data pooled from two
cells in two different culture dishes. The control and 1 mM data come from the
same patches before and 30 minutes after changing the perfusing medium to one

containing 1 mM DTB.

102
Figure 32 shows the effect of exposure to 1 u M DTB on open-time when SCh2 is

used as agonist. SCh2 binds to the AChR with a greater afﬁnity than ACh and
therefore maintains the channel in an open-state for longer periods of time. It was
hoped that by prolonging channel open-time by using SChz as agonist, changes in
open-time following exposure to DTB would become more evident. Following 30
minutes of exposure to DTB, fast t is decreased for channels opening in the
presence of 100 nM SChz, while the slow t appears unchanged (Figure 32). The
duration of the slow events does appear to be decreased following 5 minutes
exposure to DTB (Figure 32). When cells are grown in the presence of DTB, thus
increasing the duration of exposure to 1-3 days, an effect on channel open-time can
be observed when ACh is used as agonist. Figure 33 shows that exposure to DTB
(1 and 10 uM) causes a decrease in fast 1, while appearing to prolong slow 1 .
When closed-time distributions are examined in a similar manner it is
observed that these are also ﬁt by the sum of two exponentials (Figure 34).
Exposure to DTB at concentrations between 1 and 1000 u M prolongs the duration
of both the short and long closed times. Figure 35 shows the effects of exposure of

cells to 1 u M DTB for 24-48 hour on the fast and slow phase of closed durations.

103

A cute Exposure to I u” 0 TB

 

 

 

 

 

 

 

 

 

 

 

 

Subery/dieho/ine
feet 7 slow 7'
3 ' 25 r
E E 20» l
5 2 . E
I- l- 15 1 j
10’ //
O 1 O
5 5 51 VI /
s O L a o ////%
0 5 30 O 5 30
TREATMENT TIME (min) . TREAW M (ruin)

Figure 32. Effect of acute exposure to DTB on single-channel open-time (SCh2 as
agonist).

Effects of exposure to 1 u M DTB on the open-time of single-channel currents
recorded in the presence of 100 nM SChz. Currents were recorded from outside-out
patches held at -100 mV. Open-time histograms were ﬁt with exponential functions
which were the sum of two exponentials. The panel on the left shows the effect on
the fast t , while that on the right displays effects on slow 1. Data shown represent
the mean .+. SEM of currents recorded from patches of control cells from 12 dishes
and currents from patches exposed to DTB from cells in 6 dishes.

104

Chronic Exposure to I 0 [1M 0 TB
Agar/191‘ 700 IM A07

fast 7'

1.“ 15, slow 7'

 

d
o
d
O

 

 

 

MEAN OPEN TIME (ms)
MEAN OPEN TIME (ma)

/

CON DTB CON DTB

 

 

 

 

 

 

 

 

 

 

Chronic Exposure to I p” 0 TB
Aoonist 100 nM ACh

fast 7'

1.“ 10 r slow 7'

I

 

1.0-

 

 

 

0.5 ’ 7

 

 

 

 

s\\\\\\\\\\\\\\\\\\\\

 

 

 

 

 

 

CON DTB CON DTB

Figure 33. Effect of chronic exposure to DTB on single-channel open-time (ACh as
agonist).

Effects of DTB on open-time for AChR-currents recorded from control cells
and cells treated with 1 (top) and 10 u M DTB (bottom) for 24-48 hr. Currents were
recorded at a holding potential of -100 mV from outside-out patches in the presence
of 100 nM ACh. Open-time distributions were ﬁt by a double exponential function.
Values represent the mean 1 SEM of 1 to 3 cells per plate from at least 4 drﬁ‘erent
plates of cells. The asterisk (‘) denotes values signiﬁcantly diﬁ'erent at p < 0.05.

CLOSED-TIME HISTOGFIAM

50
40 r
Z.
[I]
tr 30 -
UJ
CL
(D
1—
% 2O ’ fast phase 3 ms
51 s/ow phase 635 ms

 

 

 

‘I 5 1 1 O ‘l 1 5 ‘I
CLOSED—TIME (ms)

Figure 34. Closed-time distribution for single-channel currents.

Distribution of closed times determined from an outside-out patch from a
control cell held at -100 mV in which currents were elicited due to exposure to 100
nM ACh. The distribution of closed times shown here is ﬁt by the sum of two
exponentials.

106

 

 

 

 

 

 

 

 

 

 

 

 

§3_ // EIBO' 7

Figure 35 . Effect of DTB on the distribution of closed-times.

Effects of DTB on the distribution of closed-times. Channel currents were
recorded in the presence of 100 nM ACh from outside-out patches held at -100 mV.
Currents were recorded from control cells or cells grown for 24-48 hour in medium
containing 1 u M DTB. Closed-time distributions were ﬁt by the sum of two
exponentials. Data shown represent the mean 2 SEM of data gathered from at least
5 different culture-dishes.

107

Discussion.

Multiexponential open and closed distributions. Until recently, the model
for the binding of ACh to nicotinic AChR-channel complexes and subsequent
opening of the associated channel was similar to that shown in Figure 36A (Del
Castillo and Katz, 1957). In this model two molecules of ACh (A in Figure 36) bind
to the receptor (R in Figure 36), the receptor-channel complex undergoes a
conformational change (AZR to AZR‘ in Figure 36) and this leads to opening of the
channel (AzR‘ represents the open AChR-channel complex). After remaining open
for a short period of time proportional to a (a is the rate constant for channel
closing in Figure 36) the channel closes and agonist molecules dissociate from the
receptor-channel complex. It can be demonstrated that the lifetime in any single

state (i.e. ZAR' in Figure 36A) is exponentially distributed where:

the mean lifetime of that state = 1 / (sum of transition rates that lead away from the

state)

(from Colquhoun and Hawkes, 1985). If the distribution of channel open-times for
a channel acting via the model in Figure 36A were constructed, it would be
described by a single exponential (since only 1 transition rate leads away from 2AR‘)
and the mean open-time 1: for the channels would be equal to 1 / a (Colquhoun and

Hawkes, 1985).

108

A

k or
2A+H5—1-AR+R—3A Fin-AA R*
k_1 k_ 2 3 2

B
2A+RvE-1-‘AR+R-k—2—~A Fl
Wt *2 i
1 0‘1 “ B 0,
AR* A2R*

Figure 36. Model of ACh binding and channel opening.

109

In the experiments described above, open-time distributions, for single-channel
events recorded in the presence of 0.1-1 uM ACh or SChz, were described by the
sum of two exponentials. Biphasic distributions for channel open-time have been
reported by several groups examining single-channel currents at low concentrations
of agonist (Jackson 3131., 1983; Sine and Steinbach, 1984; Colquhoun and
Sakmann, 1985). A biphasic distribution for open-times could indicate several
things. There may be two populations of channels present which remain open for
different durations, the same class of channel may switch between two states one-
open for short durations and one open for long durations, or something may be
happening to a portion of the channels to cause them to close prematurely (i.e.
channel block). Several recent ﬁndings support the theory that short duration
openings represent the rapid opening of singly-liganded channels (AR in the model
in 9a). First, the short openings occur more frequently at low concentrations of
agonist (Jackson 3131., 1983; Colquhoun and Sakmann, 1985a; Labarca 3131.,
1985). The ratio of short openings to long is almost always the same for a given
concentration of agonist; thus it is unlikely that a second channel type is responsible
for these observations. If a second channel type is responsible, then this channel
type must always be present in the same ratio in membranes, and patches must
contain a large number of active channels (Colquhoun and Sakmann, 1985a). The
voltage-dependence of open-time is similar for channels with brief open-times and
long open-times indicating that block of the long duration channels (by a charged

molecule) is unlikely. The preponderance of data support the theory that the short

110

duration openings result from activation of a AChR-channel complex by a single
molecule of ACh. The relatively high incidence of these short duration openings at
high agonist concentrations, when the number of AChRs bound to only one
molecule of ACh should be very low, makes it unlikely that all short openings result
from singly-liganded receptors (Sine and Steinbach, 1984; Colquhoun and Sakmann,
1985a).

Multiexponential closed-time distributions have also been reported recently
(Colquhoun and Sakmann, 1981; Sine and Steinbach, 1984; Colquhoun and
Sakmann, 1985a; 1985b), indicating at least 3 different closed states for the AChR-
channel complex. Two of the observed closed states are short lived, with time
constants of 10 to 70 microsecond for the most rapid phase and 0.1 to 1.2
milliseconds for the intermediate phase, depending on agonist. The third time
constant is normally in the range of hundreds of milliseconds (Colquhoun and
Sakmann, 1985a). The fast time constants are thought to represent rapid
ﬂuctuations between the open and closed state which occur within a burst of activity
for a single channel, while the long time constant represents the time which the
channel remains closed between bursts. When the number of closures per burst was
examined it was found that on average each burst contained approximately 2 fast
closures (1.9 2 0.2) when ACh was used as agonist and approximately 4 fast closures
(4.1 t 0.3) when SCh2 was used as agonist. The intermediate closures occurred very
infrequently and therefore, are not well characterized (Colquhoun and Sakmann,
1985a). In the study described herein, the closed durations were ﬁt by a frmction

111

which was the sum of two exponentials. The minimum detectable duration for an
event (open or closed) with our analysis system is approximately 100 microseconds.
The very fast component described by Colquhoun and Sakmann (1985ab) would not
be detected well using our analysis system. Therefore, what is reported as mean
channel open-time actually represents "apparent” channel open-time. These apparent
openings probably represent channel openings within bursts, where the fast
component which we report are closures within a burst and the slow components are
, closures between bursts. In almost all patches which were recorded from more than
one channel was active, which in many case made it difﬁcult or impossible to
determine whether closed periods represented those within or between bursts or
those between bursts of the same channel or two different channels. Only the
apparent open-times could be examined with any reliability.

Figure 368 displays a more recent model which describes binding of agonist
and opening of channels for AChR-channels (Jackson 3131., 1983; Colquhoun and
Sakmann, 1985a; 1985b). Using this model both the short duration openings
observed at low concentrations of agonist and the bursting behavior observed as
agonist concentrations are increased can be described. In this model the normal long
duration channel openings correspond to AIR'. Two agonist molecules (A2) bind to
the receptor (R), and the channel opens for a short duration of time (AzR‘
represents the doubly-liganded open channel) proportional to a . Upon closing one
or both agonist molecules dissociate from the receptor and the cycle can start over

again. When the single channel events are examined using very high resolution data

112

analysis systems, most openings appear as a burst of channel openings separated by
brief closures. It appears as though each burst of openings represents an oscillation
between the closed AZR state and the open AZR‘ state, in which the channel opens,
closes and reopens before the agonist molecule can dissociate. The results of
Colquhoun and Sakmann (1985a) indicate that, on average, each long duration
opening observed contains 2 rapid closures when ACh is agonist and 4 rapid closures
when SCh2 is agonist. This model also can account for openings associated with the
singly-liganded AChR-channel (AR‘). These are normally observed at a low
frequency due to the positive cooperativity of binding of agonist to the AChR. The
AChR has a greater afﬁnity (up to 100X) for binding of the second agonist molecule
once one molecule is bound (Sine 313., 1990).

Effects of DTB on single AChR-channels. The results described above
demonstrate that exposure of AChRochannels to low concentrations of DTB (1-10
uM) alters the time which the channels remain open. When ACh is used as agonist
(0.1 to 1 u M) it appears as though channels which remain open for short periods of
time (fast t) close faster in the presence of DTB and channels which normally
remain open for longer periods of time (slow t ), appear to have prolonged open-
times. When the effects of DTB are examined on single-channels observed when
SCh2 (100 nM) is used as agonist, DTB induced changes appear somewhat different.
Following exposure of patches to DTB (1-10 nM) for 30 minutes there is a decrease
in fast t but no effect on slow t . Five minutes after exposure to DTB there appears

to be a decrease in slow t but no change in fast 1:. The effects of DTB on channel

113

open-time become less complicated when examined in a slightly different manner.
When determining fast and slow r from the observed distributions several criteria are
used to determine whether the distribution is ﬁt by a single exponential or the sum
of multiple exponentials. The 1:2 statistic can be used as one criterion. This statistic
determines how well the observed data points ﬁt the generated curve. A goodness
of ﬁt ratio is also determined by the curve ﬁtting routine when calculating
exponential functions to ﬁt the data. This ratio gives information on how many
iterations are necessary to obtain a curve which describes the data. Perhaps the best
criterion for determining how well an exponential function describes a distribution
is to examine by eye how well the generated function appears to ﬁt the actual data
(Colquhoun and Sigworth, 1985). In many patches it was difﬁcult to determine
whether the distribution for channel open-time was ﬁt better by a single exponential
or the sum of two exponentials and data from several patches were deﬁnitely ﬁt best
by a single exponential. When the effects of DTB on open-time are examined in
distributions which are ﬁt by a single exponential the changes observed become much
more consistent. Figure 37 displays the effects of DTB (1 u M) on channel open-time
when all distributions were ﬁt with single exponentials. When examined in this
manner a decrease in apparent channel open-time is observed following exposure of

cells or patches to DTB.

ll4

CHANNEL OPEN-TIME
A (700 nM ACh}

 

 

 

MEAN OPEN TIME (ms)
*

 

 

 

 

CON DTB

Figure 37. Effect of DTB on single-channel open-time (single exponential ﬁt).

Effect of DTB on channel open-time when open-time distributions are ﬁt with
a single exponential function. Single channel currents were recorded as described
in Figure 33, however, the distributions of open-times were ﬁt with a single
exponential function instead of the sum of two exponentials. Values shown represent
the mean 1 SEM of patches from at least 5 culture-dishes.

1 15
Results of these studies indicate that DTB has a direct effect on single

channels to decrease the time which they remain open. This effect of DTB could
explain the observations in previous voltage clamp experiments in which t are and
t MEPC were found to be decreased in muscles from rats treated with DTB.

DTB could interact with the AChR-channel complex in several ways to cause
the observed decrease in open-time.

Block of open channels by DTB. Many compounds have been found to block
the AChR-channel following its opening. All of the agonist molecules tested thus far
have been found to block the channel (Ogden and Colquhoun, 1983; Sine and
Steinbach, 1984), moreover, agents such as d-tubocurarine, thought of as classical
receptor blockers, also block the ion channel (Colquhoun 11a], 1979). In addition,
a variety of other charged and uncharged molecules (N eher and Steinbach, 1978;
Ogden 3131., 1981; Neher, 1983) also appear to block the channel physimlly.
During open-channel block, effects on both macroscopic (i.e. EPCs and MEPCs) and
single-channel currents can be observed which are very characteristic for this
mechanism. In the presence of compounds which can block open channels the
single-channel currents exhibit very characteristic bursts of activity. These bursts
(Figure 28 last panel) represent a single channel opening and becoming blocked
and unblocked in rapid succession, followed by channel closure. The eﬂ‘ects on
EPCs and MEPCs of open-channel block are observed as a biphasic decay of the
current. In the presence of open-channel blockers the normal number of ion-

channels open following release of transmitter into the synaptic cleft, thus the initial

116

amplitude of the current is normal. Soon after opening a portion of the channels
become blocked. This leads to a very rapid initial decay of the current. The
prolonged phase results from channels becoming unblocked over time and allowing
current to ﬂow through them prior to their ﬁnal closure (Adams and Sakmann,
1978; Lambert 3131., 1980). When the effects of DTB on channel opening are
examined neither the characteristic bursting of single channel currents, nor biphasic
decay of EPCs or MEPCs is observed. This probably indicates that DTB does not
block the Open channels. In addition, a plot of t,’1 + t," - tc'l vs concentration of
DTB will be linear if DTB is blocking the open channels (where; t, = fast 1: and
t' = slow t, in the presence of DTB and 1:c is the single control 1) (Ogden 3131.,
1981). Figure 38 shows a plot of t {1 + 1:;1 - tc'l vs concentration of DTB for single-
channel currents measured in the presence of 100 nM ACh. This plot is not linear,
thus, DTB does not appear to act via open-channel block to decrease the open-time

of single-channel currents.

117

 

 

4..
IE0 3-
T.I
lu—
I~ 21
‘_+
I“)
1L
\A
O I ﬁH-LAJ—l

 

.OO 1 .O 1 1
CONCENTRATION OF DTB (mM)

Figure 38. Plot of tf'l + ts'l - t c'1 vs concentration of DTB.

Plot of 1:;1 + t," - tc’l vs concentration of DTB where; 1: f = fast 1 and t,
= slow t, in the presence of DTB and re is the single control 1:. The data graphed
represent open-times for single channels recorded in outside-out patches held at -100
mV with 100 nM ACh as agonist. Channel currents were recorded in the absence
and presence of 1-1000 u M DTB. Linearity in this plot would indicate that DTB is
aﬁecting channels via open-channel block.

1 18
Effects on the afﬁnity of receptors for agonist. Previous studies have

demonstrated the presence of disulﬁde bonds within the receptor region of the
AChR-channel complex which are critical to normal function (Karlin and Winnik,
1968; Rang and Ritter, 1971). Reduction of disulﬁdes present on the receptor,
with the reducing agent dithiothreitol (DTT), decreases the responsiveness to ACh
while reoxidation, with the oxidizing agent 5,5’-dithio-bis (2-nitrobenzoate) (DTNB),
reverses these effects when examined in electroplax preparations (Karlin and Winnik,
. 1968) and chick muscle (Rang and Ritter, 1971). Walker 3131. (1981) examined
both agonist binding and ion ﬂux in membrane bound vesicles containing AChRs
from 112119119 electroplax. They observed that reduction of the receptors with DTT
decreased the binding afﬁnity of AChRs for carbamylcholine (CCh) and shifted the
dose-response curve for CCh-induced increases in 22Na+ permeability to higher CCh
concentrations. Effects of thiol-group modiﬁcation on ion ﬂux activation and
inactivation kinetics were examined further by Walker 3131. (1984). In these studies,
CCh binding and “Rb inﬂux into vesicles with reconstituted AChR-channels puriﬁed
from W were measured before and after reduction with DTT.
Results of these studies demonstrated that the main effect of DTT reduction was to
shift EC,o values for activation and slow inactivation to higher agonist concentrations.
These ﬁndings are consistent with a decrease in binding afﬁnity for CCh previously
described by this group. More recently Bernstein 3131. (1988) examined the effects
of DTT and DTNB on muscarinic AChRs puriﬁed from pig brain. They observed

that in the presence of DTI‘ the afﬁnity for muscarinic ligands was decreased without

1 19
altering the total number of binding sites. Electrophysiological studies performed on

muscles exposed to DTT have demonstrated that following exposure to DTT, the
amplitude and decay times for EPPs and MEPPs were decreased (Ben-Haim 3131.,
1973; Terrar, 1978). Studies utilizing ﬂuctuation analysis demonstrated that
following reduction of AChRs with DTT, the time which single-channels remain
open and the conductance for these channels are decreased. Results of the above
studies indicate that reduction of critical disulﬁde groups located on the AChR leads
to a decrease in afﬁnity of the receptor for agonist, and associated with reduction
is a decrease in single-channel conductance and open-time. It is possible that effects
of DTB, which is a moderate disulﬁde reducing agent (Preisler and Bateman, 1947),
on channel-open time are due to reduction of disulﬁde groups in a manner similar
to those observed following reduction with DTI‘.

Mechanism for a decrease in affinity decreasing open-time. In the model
(Figure 36b) there are 2 steps for agonist binding with forward and reverse rate
constants of k1, k4, k2 and kg. The dissociation constants for these two binding
steps would be K“1 = ,I/k, and Kd2 = k,z/k2. In normal channels the majority of
openings are going to result from AR binding a second agonist molecule to become
A2R; this combination will open giving AzR‘. During the average opening there will
be two brief closures (Colquhoun and Sakmann, 1985a), in which the AChR-
channel oscillates between A2R‘ and A2R. During the third brief closure (on
average) the agonist molecule dissociates from the receptor and it remains shut.

When a higher afﬁnity agonist is used, it binds tightly enough to the receptor that

120

the channel is able to oscillate through 4 brief closures prior to dissociation of the
agonist (Colquhoun and Sakmann, 1985a). Following reduction of disulﬁdes located
on the receptor the afﬁnity for agonist decreases (Kd increases) (Walker 3131.,
1981). If the rate of dissociation increases, then the agonist molecule may dissociate
during the ﬁrst or second brief closure, thus decreasing the time that the average
channel remains open.

The effects on closed time following DTB exposure complicate this
mechanism to a certain extent. The prolongation of the long closed durations ﬁts
this scheme very well. If the long closed durations represent closures between bursts
when both agonist molecules dissociate from the receptor, then these could be
expected to be prolonged if the afﬁnity of the receptor for agonist is decreased (i.e.
fewer bindings and openings per unit time). If the short bursts represent rapid
transitions between the AZR‘ state and the AZR state, then a change in afﬁnity
should have no effect on this duration. An increase in the duration of short closures
is observed in this study. One possibility which could explain this is that brief
closures represent transitions between the AIR‘ state and the AR state. In this case
changes in afﬁnity would alter both the duration of the short and long closures as
well as open-times.

Conclusion. The decrease in channel open-time observed in this study is
consistent with a direct effect of DTB on the AChR-channel complex. The decrease
in t MEPC for fast MEPCs observed in earlier studies could be explained by such an

effect. It is possible that by reducing critical disulﬁde groups on the AChR-channel

121
complex DTB decreases the afﬁnity for binding of agonist to the receptor, which

then leads to the observed decrease in open-time.

Chapter 8. Effects of DTB on single-channel current measured via ﬂuctuation

analysis.

Summary of DTBs effects on singlerchannel currents. Results of the previous
patch voltage clamp studies indicate that exposure of cultured muscle cells to DTB,
at concentrations approximating those measured in the plasma of rats treated
chronically (1 mg/kg/day) with DTB (Porter 3131., 1983), leads to a decrease in the
apparent open-time for nicotinic AChR-channels. Due to the fact that these studies
were performed on muscle cells grown in culture in the absence of any presynaptic
input, the observed results represent an action of DTB on the AChR-channel itself.
The limited area of membrane recorded from and the high resistance of the seals
formed make it unlikely that the effects observed represent changes in cable
properties of the membrane or ineffective space-clamp of the membrane under study.
The purpose of the following study was to determine whether channel open-time is
similarly affected in intact preparations taken from rats exhibiting no signs of muscle
weakness 24 hour following a single 1 mg/ kg dose of DTB or from rats exhibiting
muscle weakness following 6-7 days of treatment with DTB (1 mg/kg/ day). The
technique used measures channel activity in the presence of exogenously applied
agonist. Therefore, alterations in presynaptic structure or function should not

inﬂuence the observed results.

122

123
Introduction to ﬂuctuation analysis. The patch voltage clamp technique has

proven to be extremely valuable in describing the ldnetics of channel opening and
closing in a variety of cell types (Hamill 3131., 1981; Neher, 1983; Sine and
Steinbach, 1984). Patch voltage clamp techniques are limited by the fact that the
membrane surface to be patched must be readily accessible and relatively free of
contaminating material, such as connective tissue or glial cell processes. Due to
these constraints, channel-kinetics in a variety of preparations, such as intact
neuromuscular junctions, are difﬁcult to study using this technique. One method
used quite frequently to study the behavior of channels at the neuromuscular junction
is to cut the nerve to the muscle of interest and to patch clamp the muscle once the
nerve has degenerated (Neher and Sakmann, 1976; Reiser and Miledi, 1989).
Following denervation there is a change in the number and type of receptor channel-
complexes present on the surface of the muscle which makes it difﬁth to correlate
any observed behavior with the channels originally present (Axelsson and Thesleff,
1959; Katz and Miledi, 1973; Neher and Sakmann, 1976). An alternate technique
which has been very useful in analyzing channel kinetics in intact preparations is
ﬂuctuation analysis. Fluctuation analysis or "noise analysis” as it is sometimes called,
is a technique by which both single-channel conductance and mean open time for
channels are determined from spontaneous, random variations in membrane noise
observed during the opening and closing of channels (Katz and Miledi, 1972;
Anderson and Stevens, 1973; Stevens, 1975). This technique was ﬁrst used to
characterize the AChR-gated ion-channel in the early 19705 by Katz and Miledi

124
(1970; 1972; 1973). They observed that in the presence of a constant application

of ACh to frog end-plate regions there was an increase in the noisiness of the
recording. By analyzing spectra generated from the random ﬂuctuations in noise they
could gain information about the opening and closing of channels underlying the
observed noise.

Determination of mean channel open-time with ﬂuctuation analysis. An
electrical signal which varies with time (time-varying signal) can be characterized by
its frequency spectrum. Analysis of the frequency components of a waveform is
known as fourier analysis. According to Fourier’s theorem, time-varying signals of
the type most commonly encountered can be decomposed into a sum of sine and
cosine waves of various frequencies. By performing fourier analysis, a time-varying
signal is transformed into its spectrum, which is the amplitude of each frequency
component in the signal plotted against frequency. Spectra generated using this
analysis technique are normally displayed as the power density spectrum, which is
the power of each frequency component (watts /Hz) plotted against the frequency
(Hz). When displaying spectra it is common to plot the log power vs log frequency
to compress the axis and allow one to display information over many orders of
magnitude on a single ﬁgure. The different spectral components present in the signal
can be determined in several ways. The original method used was to record a
segment of signal containing data of interest, to ﬁlter this signal sequentially with
narrow band-pass ﬁlters, and to measure the power of the component which is

allowed through the ﬁlter. By changing the frequency which is allowed to pass

125
through the ﬁlter, all components of the signal can be measured. Presently, the

preferred procedure is to use digital computers and an algorithm known as the Fast
Fourier Transform (FFI'). In order to analyze data on digital computers the signal
of interest must be converted from a continuous signal to a digitally sampled one via
an analog to digital converter. To be certain that all frequencies of interest are
retained, and contaminating frequencies not of interest removed, several things
must be accounted for during the process of digitization. First, care must be taken
. when sampling to record long enough samples of signal to insure that low frequency
components of the signal are included. For example, the lowest frequency
component present in a 10 sec long recording will be 0.1 Hz. Second, the signal
must be sampled at a high enough rate to deﬁne high frequency components
contained within the signal. The Nyquist sampling theorem provides a quantitative
basis for the rate at which a signal must be sampled to insure that all components
of interest are retained in the digitized signal. This theorem states that if a band-
limited signal is sampled at twice the highest frequency component (Nyquist
frequency), the sample values will exactly describe the original signal (Malmstadt 31
31., 1981). Thus, a signal with frequency components up to 100 Hz should be
sampled at twice this rate or 200 Hz, a rate which corresponds to 5 milliseconds per
data point. In addition, it is important to ﬁlter the signal of interest prior to
digitization to remove unwanted components in the high frequency regions. If high
frequency components are not removed from the signal an error known as aliasing

can occur. When aliasing occurs, high frequency components above the Nyquist

126

frequency are undersampled and are recorded as spurious low frequency components
in the digitized signal. A good rule to follow is to use a low-pass ﬁlter (removes high
frequency components) with a cutoff frequency equal to one half that of the sampling
rate. If a signal of interest is to be sampled at 1 KHz, then this signal should be
ﬁltered using a ﬁlter which has a cutoff frequency of 500 Hz. This removes
frequency components from the signal, prior to sampling which would lie outside
of the spectra to be generated.

Determination of channel amplitude from ﬂuctuations in noise. The single
channel conductance can be calculated by measuring the amplitude of the
ﬂuctuations in mean current level and comparing these to the amplitude of the mean
macroscopic current. The maximum value (power) of the generated spectra should
be related to the amplitude of the unitary changes. Thus, both the standard
deviation of the noise and the maximum power of the spectra can be used to
calculate the conductance of the single channels.

Iontophoresis. Using iontophoresis, charged molecules can be ejected from
a high resistance electrode (100-200 MD resistance) by passing a constant ejection
current through the electrode. A low level braking current, of opposite polarity
from the ejection current, must be applied to the electrode when not ejecting drug
to prevent leakage from the tip of the electrode and hence receptor desensitization.

The amount of compound ejected over time can be estimated from the formula:

N=F'I‘k‘t

127
where: N equals the number of molecules released, F equals Faraday’ s constant, I

is the current passed through the pipet, k is the transport constant of the pipet
(approx. 0.25 for ACh ejected from a pipet of 100 megohm resistance) and t is the
time of release (Dreyer 3131,, 1978; Dionne 3131,, 1978; Warner and Bate, 1987).
To record fast voltage or current changes with this procedure the iontophoretic pipet
should be brought to within 15-20 pm of the end-plate. For noise analysis better
results are obtained if the iontophoretic pipet is placed approximately 100 u m from
the end-plate. Increasing the distance between the iontophoretic electrode and the
end-plate region dampen out rapid ﬂuctuations in ACh concentration preventing
rapid ﬂuctuations in macroscopic currents.

Experimental methods. The experimental design and methods used are
described in detail in the materials and methods of this manuscript. Brieﬂy,
hemidiaphragm preparations were removed from control rats, rats treated for 1 day
with a single 1 mg/ kg dose of DTB and from rats exhibiting muscle weakness
following chronic treatment with 1 mg/ kg/ day of DTB. In addition, several muscles
taken from control rats were exposed to 1 u M DTB for up to 4 h in the bathing
medium. End-plate regions were clamped, using the two microelectrode voltage
clamp described earlier. A third microelectrode, containing 2 M ACh, was brought
into the region of the end-plate and ACh forced from the microelectrode by passing
positive current out of the ACh containing microelectrode (iontophoresis). For these
experiments end-plate current was recorded at two different ampliﬁcation levels.

The ﬁrst, a DC coupled channel, was ampliﬁed from 1-10X. This signal allows the

128

magnitude of the iontophoretic current to be determined. The second, a high
ampliﬁcation (100—1000X), AC coupled, recording allows the random current
ﬂuctuations due to the opening and closing of ACh gated ion channels to be
characterized. The 100-1000X signal should also be high- and low-pass ﬁltered prior
to analysis to remove unwanted components from the spectra.

Results.

Early effects of DTB on MEPCs. Several different populations of MEPCs,
some with extremely altered amplitudes and rise and decay kinetics, have been
observed at end-plate regions of muscles taken from rats exhibiting muscle weakness
following chronic treatment with DTB (see results in Chapter 6). No
electrophysiological studies have been performed on muscles from rats treated for
1 day with 1 mg/ kg/ day of DTB (the daily dose administered during chronic
treatment). It would be of interest to determine whether MEPCs are altered at end-
plate regions of these muscles. Recordings from these muscles indicate that as early
as 1 day following a single 1 mg/kg dose, the frequency of abnormal MEPCs is
increased. When control muscles are exposed to In M DTB in the bathing medium

for 4 hour the incidence of abnormal MEPCs is also increased (Figure 39).

129

Normal
A MEPCs
\

 

Figure 39. Exposure to DTB causes an increase in the incidence of abnormal
MEPCs.

Hemidiaphragm preparations were prepared as in Figure 20 and MEPCs
recorded at a holding potential of -70 mV. MEPCs were recorded from
neuromuscular preparations taken from A) control rats, B) rats treated 24 hours
previously with a single 1 mg/kg dose of DTB and C) from control rats and exposed
to 1 14M DTB in the bathing medium.

130

Fluctuations in iontophoretic current. Following a change in the current
ﬂowing through the iontophoretic electrode from -4 pA tO + 10 pA a current can be
measured at the end-plate region which has an amplitude on the order Of 5-50 nA
(Figure 40). If there are 10‘5 channels present at the end-plate region and 103 Of
these open during iontophoresis Of ACh, then probability of a single channel being
Open will be P = 103/10‘S = 0.001. Thus, the standard deviation Of the number Of
Open channels is given by [NP(1-P)]1/2 = 31.6. The number Of channels which will
be Open at equilibrium, during iontophoresis, will not be constant but will = 1000
.+_ 31.6 (from Colquhoun and Hawkes, 1985). A ﬂuctuation in the steady current
will be Observed which represents the Opening and closing Of approximately 30
channels in the case above. This ﬂuctuation in channel Opening can be Observed as
an increase in the variance in the current level (Figure 41A) and as an increase in
the level of noise Observed (Figure 413) within the actual signal.

Determination of the decay time constant 1 . Following transformation Of the
signal, using a fast fourier transform, a power spectrum can be generated (Figure
42). From this it can be seen that the frequencies Of noise which contain the most
power are in the range Of 1 tO 100 Hz, while higher frequency components have
lower and lower power levels. This power spectrum can then be ﬁt with a Lorentzian
function, similar to the ﬁt Of single-channel events with exponential functions. This
allows several characteristics Of the events underlying the current ﬂuctuations to be
determined. The decay time constant t for channels generating the noise can be

determined from the corner frequency (f) Of the spectra (or Lorentzian function).

131

 

Mean nA
é

 

0 100 200 300 400

Record NO.

Figure 40. Amplitude and duration Of iontophoretic current.

Amplitude and duration Of current induced following iontophoretic application
Of ACh. The membrane potential Of end-plate regions was clamped at a constant
holding potential Of ~70 mV using two microelectrode voltage clamp. A third
electrode containing 2 molar ACh was placed in the region of the end-plate and an
iontophoretic current was elicited by passing positive current through the ACh
containing electrode. Each record number on the x-axis represents 250 microseconds.

132

 

 

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'5
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0 100 200 300 400

Record No.

MICROSCOPIC CURRENT FLUCTUATION
Before & After Iontophoresis of ACh

B -ACh

WWW

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WWW

Figure 41. Membrane noise Observed during iontophoresis Of ACh.

Microscopic ﬂuctuations in current Observed during iontophoresis Of ACh to
end-plate regions. Panel A displays the variance in the microscopic current
ﬂuctuations Observed during the ACh evoked current shown in Figure 40, each
record represents 250 microseconds. Panel B displays membrane noise Observed
before and during ACh evoked current. Currents were ampliﬁed 100x and band-
pass ﬁltered from 1-500 Hz.

133

 

 

10 air
5 o
— ‘5 .
—I O ‘
—I * i " + co“.
44— N
10 .*+
.=_ 1+.
N : o»
E —1 .9”
-— tr
5" T no
< ~55“ ..
c 10 *0.
= f
a “a
"I .‘b
nun-4 .
10 1 In"
I 1 l 7 I I l I l I I I I I l I I I\r:
10 to2

Figure 42. Power spectrum from ACh induced membrane noise.

Power spectrum generated from ﬂuctuations in current recorded during
application Of ACh to end-plate regions. The membrane potential Of end-plate
regions was clamped at -70 mV and current was recorded before and after
application Of ACh. Current signals were ampliﬁed 100X and band-pass ﬁltered
between 1-500 Hz. Power spectra were generated by fast fourier transform Of the
recorded signal.

134
Fe is the frequency at which the power in the components Of the signal is one half

Of the maximum power. From fc the time constant t can be calculated using the
equation t = 1/21: fc (Anderson and Stevens, 1973). Figure 43 shows values Of 1-
determined for end-plate regions, clamped at potentials between -20 and ~70 mV,
Of muscles taken from control rats. When muscles are taken from rats exhibiting
muscle weakness following chronic treatment with DTB, values Of 1: calculated from
spectra are decreased compared to control values (Figure 44A). Values Of t are
also decreased in muscles taken following a single dose Of DTB indicating that
channel Open-time is affected before muscle weakness is Observed (Figure 44A). 1
is also decreased in muscles from control rats exposed to 1 u M DTB in the bathing

medium for 4 hours when measured at a holding potential Of -70 mV (Figure 44B).

 

135

APPARENT OPEN-TIME

fluctuation ens/yells

 

24
g is
UJ
g +Oon
l
E
8 8 l.
T
l
O

 

 

--80 -60 —4O -20 O
HOLDING POTENTIAL (mV)

Figure 43. Values Of t from control muscles.

Values Of 1 calculated from hemidiaphragms Of control rats. ACh induced
ﬂuctuations in end-plate current were recorded at holding potentials from ~20 mV
tO ~70 mV and spectra were generated as described in Figure 42. The corner
frequency (f) was determined from a single lorentzian curve ﬁt to each spectra and
1 calculated from: = 1/21r fc.

136

APPARENT OPEN-TIME APPARENT OPEN-TIME

ﬂuent/en analysis f/uctwtian arre/ysis

 

 

 

   

 

 

 

 

2“ A 2“ B
E 16 16 I I
—9— Con

Li] —0— O.“
I + 4h
é —ts— 1 a
o 8 8

o o

—80 —60 —4o —20 o -80 —60 ~40 —20 o

HOLDING POTENTIAL (mV) HOLDING POTENTIAL (mv)

Figure 44. Effect Of DTB on t.

Effects Of DTB on channel Open-time (t ). ACh induced current ﬂuctuations
were recorded at membrane potentials between ~20 and ~70 mV in hemidiaphragm
preparations taken (A) from rats treated 24 hours previously with a single 1 mg/kg
dose Of DTB and from rats treated chronically with DTB or (B) control rats and
exposed to 114M DTB in the bathing medium. Values Of t were determined as in

Figure 43.

137

Discussion.

By combining two microelectrode voltage clamp recording techniques with
prolonged applications Of ACh, via iontophoresis, we were able tO record changes
in the level Of membrane noise during the application Of ACh tO end-plate regions.
Spectra generated from the ﬂuctuations in membrane noise, Of control preparations,
were similar to those Observed by other groups examining ACh induced noise
(Anderson and Stevens, 1973; Adams 3131., 1981; Cull-Candy 3131., 1988).
Values Of 1 reported by Cull-Candy (1988) Of 11 z 0.9 at ~80 mV are similar to
values Obtained in these studies. When compared to values Of t determined from
single channel studies described earlier, these values appear high. It is questionable
whether closures which occur within a burst Of channel activity would be detected.
Values Of 1: Obtained from ﬂuctuation analysis are believed tO represent the duration
Of bursts for channels (Cull-Candy 3131., 1988). r values which were determined
in the previous single channel studies (which described apparent Open-time for
individual Openings within bursts) would be expected to be shorter than those
determined in this study.

When the effects Of DTB on channel behavior were examined it was found
that the decay time constant t calculated from muscles taken from rats exhibiting
muscle weakness following chronic treatment with DTB (1 mg/ kg/ day) were
decreased compared to r values from control muscles. Similar effects on t were
Observed in muscles taken from rats treated 24 hour previously with a single dose Of

DTB (1 mg/kg). When 1 u M DTB is applied in the bathing medium to muscles

138
from control rats s also appears to be affected (at hyperpolarized holding potentials).

Conclusion. These results indicate that bursts Of AChR-channel activity
observed following iontophoresis Of ACh onto muscles of rats treated with DTB are
shorter than those Observed in muscles Of control rats. This is very similar to what
was Observed in the previous patch voltage clamp studies and could easily be
explained by a similar mechanism. These effects are Observed within 1 day following
treatment (1 mg/ kg Of DTB) or as early as 4 hour following bath exposure to DTB
(1 n M) as well as in muscles from rats treated chronically with DTB. When effects
are monitored at the level Of the single channel, changes in Open-time are Observed
as early as 30 minutes after exposure tO DTB (1 u M) and possibly as early as 5
minutes following exposure. The early times at which these effects are Observed
indicate that early changes begin tO occur soon after exposure to DTB is initiated,
and that these alterations may in part be responsible for the weakness Observed with

continued exposure.

Chapter 9. Discussion.

The initial Observation that the rise and decay times for MEPPs were
prolonged following exposure to DTB indicated that DTB may alter postsynaptic
function. This effect was ﬁrst Observed in muscles taken from rats within 24 hour Of
a single 25 mg/kg dose. It was subsequently demonstrated that a similar
prolongation Of rise and decay times could also be detected in muscles taken from
rats exhibiting muscle weakness following chronic treatment with DTB. Atchison
(1989) reported that rise and decay times for EPPs were prolonged as well as for
MEPPs. DTB could act in a variety of ways to cause the Observed prolongation Of
synaptic potentials. Several potential modes Of action for DTB will be examined in
more detail in the following sections.

Cholinesterase inhibition. Following its initial release and binding to
receptors further action Of ACh is prevented by the presence Of the enzyme AChE.
This enzyme, which has an extremely high turnover rate for ACh, is also present
in very high concentrations within the synaptic cleft (Cooper 3131., 1982). This
combination leads to the removal Of presynaptically released ACh from the synaptic
cleft within a few hundred microseconds. In most cases transmitter is released from
the nerve terminal, diffuses across the synaptic cleft, binds a single time to receptors
on the postsynaptic membrane leading to channel Opening and then upon unbinding
from the receptor is broken down very rapidly by AChE. It has been Observed in

several preparations that inhibition of AChE, by a variety Of inhibitors, leads tO an

139

140

increase in the amplitude and a prolongation Of rise and decay times Of synaptic
potentials (Eccles and MacFarlane, 1948; Katz and Miledi, 1975; Laskowski and
Dettbarn, 1979). Several lines of evidence go against DTB acting to prolong
synaptic potentials via an inhibition Of AChE. Nonspeciﬁc cholinesterase levels,
both in plasma (Atchison and Peterson, 1981) and brain (Astwood 3131., 1945),
were found to be unchanged in rats exhibiting muscle weakness following chronic
treatment with DTB. Following AChE inhibition there is an increase in MEPP and
EPP amplitude, however, in muscles taken from rats following treatment with DTB
EPP amplitude is decreased, while MEPP amplitude is either unchanged or
deceased (Weiler 3131., 1986; Atchison, 1989; Spitsbergen and Atchison, 1990).
Cholinesterase inhibition also leads to pathological alterations in skeletal muscles
which can be Observed as early as 30 minutes following AChE exposure and which
progress to encompass up to 7% Of muscles in the body with chronic drug
administration. Following 30 minutes Of exposure to paraoxon (an irreversible
cholinesterase inhibitor) mitochondria located within end-plate regions Of muscle
become dilated, sarcoplasmic reticulum is increased, subsynaptic folds become
widened and fused and there is an increase in coated cleft vesicles. 24 hr following
exposure to paraoxon, muscle ﬁber architecture displays a generalized disruption and
there is an accompanying inﬁltration Of phagocytes (W ecker 3131., 1978). In
contrast no evidence for pathological alterations are Observed in muscles Of rats
exhibiting muscle weakness following chronic treatment with DTB (Astwood 3131.,
1945; Atchison 3131., 1981b; Williams 3131., 1987). It is interesting that at early

141

times following exposure Of control muscles to a relatively high concentration Of DTB
(1.85 mM) in the bathing medium EPP amplitude, MEPP amplitude and MEPP
frequency are increased and rise and decay times for MEPPs are prolonged. With
continued exposure, EPP amplitude becomes decreased and block Of evoked release
is observed after approximately 30 minutes Of exposure to DTB. At the time Of
block Of transmitter release, MEPP frequency is still elevated from control levels
(Spitsbergen and Atchison, 1990). An almost identical progression Of events is
Observed following exposure Of neuromuscular preparations tO the cholinesterase
inhibitor paraoxon. Within 30 minutes following paraoxon exposure, evoked release
Of transmitter is blocked, while MEPP frequency is increased and rise and decay
times for MEPPs are prolonged (Laskowski and Dettbarn, 1979). AChE from
lqmdg electroplax has been shown to contain a single free sulﬂiydryl group on its
catalytic subunit which can react with a number of sulﬂrydryl reagents. Modiﬁcation
Of this single sulﬂiydryl group by thiOl reagents has been found to inactivate the
enzyme (Steinberg 3131., 1990). It is possible that a component Of the acute effects
Observed following bath application Of high concentrations Of DTB is due to
alterations in the activity Of AChE. It appears unlikely that these effects are related
tO the effects Observed in chronically-treated rats.

Alterations in synaptic morphology. At normal end-plates the structure of the
synapses is very well deﬁned. Presynaptic nerve terminals contain large numbers Of
transmitter-ﬁlled synaptic vesicles which are concentrated at specialized release sites

known as active zones. These presynaptic active zones are located directly across

142
from postsynaptic specializations which contain large numbers Of AChR molecules.

In regions Of synaptic contact pre- and postsynaptic specialized regions are separated
by a very short distance, which allows transmitter released from the terminal to
diffuse rapidly to postsynaptic receptors and initiate a depolarization Of the end-plate
region. Several studies which have examined the ﬁne structure Of neuromuscular
junctions during the onset Of muscle weakness in rats treated with DTB have
reported alterations in terminal and synapse morphology following exposure to DTB.
Kemplay (1984) demonstrated that retraction Of the terminal arborization which led
tO a clumped appearance for nerve terminals was Observed within 3 days Of treatment
Of rats with DTB. Also Observed at this time was an increase in terminal sprouting.
Following 4 days of treatment with DTB terminal retraction and interposition Of
Schwann cell ﬁlopodia were Observed (Jones, 1989). During terminal retraction or
sprouting ACh could be released from regions which were no longer directly apposed
to postsynaptic receptor sites. During retraction or sprouting the distance which ACh
would have to diffuse prior to binding to receptors would be increased. It is possible
that alterations in synapse morphology could be responsible for the Observed
prolongation Of synaptic potentials Observed following exposure tO DTB. Effects on
rise and decay times for MEPPs have been Observed within 24 hour following a
single dose Of DTB (Spitsbergen and Atchison, 1990), a time at which no alterations
in the structure Of terminals or synapses are Observed. Recent voltage clamp studies
indicate that EPCs and the majority Of MEPCs decay more rapidly in muscles from

treated rats, an effect which would be unexpected if terminal retraction had

143
occurred. It is unlikely that the effects Of DTB on postsynaptic function, which are

responsible for the more rapid decay Of synaptic currents, can account for the
abnormally large, slow MEPCs which are Observed in treated preparations. Instead
it is likely that these aberrant MEPCs result from abnormal release Of transmitter
from retracting or sprouting terminals or from sites on normal terminals which do
not normally release transmitter.

Eﬂ'ects on AChR-channels. Alterations in MEPP amplitude (W eiler 3131.,
1986; Spitsbergen and Atchison, 1990) and rise and decay times for synaptic
potentials (Atchison, 1989; Spitsbergen and Atchison, 1990) were both indicative
Of possible alterations in postsynaptic function following exposure to DTB. It is
known that the AChR contains disulﬁde groups which are critical to its ftmction
(Karlin and Bartels, 1968; Rang and Ritter, 1971). Finally, previous studies
indicated that alterations in thiOl/disulﬁde status within cells may be related tO toxic
effects Of DTB (Williams 3131., 1986). With these facts in mind, the studies
described in previous chapters Of this manuscript were undertaken. Results Of these
studies indicate that DTB does affect postsynaptic function.

Signiﬁcance of effects on channel open-time. The results described in the
previous sections Of this manuscript indicate that exposure of AChR-channels tO DTB
alters these channels to decrease the time which they remain Open. These alterations
Of decay kinetics appear to have little direct effect on transmission since the Observed
changes are similar following 1 day Of treatment, when transmission does not appear

to be affected and 7 days Of treatment, when transmission is markedly affected. One

144
conclusion which could be drawn ﬁom these ﬁndings is that the decrease in single

channel Open-time has no physiological signiﬁcance. This effect is Observed in rats
exhibiting weakness as well as those displaying no signs Of weakness so it need not
be related tO the weakened state. Conversely, it could be concluded that these
effects are some Of the earliest changes Observed following exposure tO DTB and that
by continuously altering these processes changes occur which lead to the Observed
weakness. The remainder Of this discussion will be concerned with examining
possible mechanisms by which subtle changes such as those Observed could
potentially lead to the Observed weakness.

Trophic relationship between nerve and muscle. Traditionally the
communication between muscle and nerve is thought to consist Of transmitter release
from the nerve terminal leading tO depolarization Of muscle end-plate region and
muscle contraction. It is becoming more and more Obvious that there is a fair
amount Of "cross-talk” between muscle and nerve. For instance, following block Of
transmission between nerve and muscle changes occur both at the level of the nerve
and muscle, in an attempt to reestablish transmission. At the level Of the muscle
there is an increase in the number and distribution Of AChR within the muscle
membrane (Axelsson and Thesleff, 1959; Neher and Sakmann, 1976). At the level
Of the nerve terminal, sprouts are formed which can spread out from the original
terminal and form new synapses on the same muscle ﬁber or surrounding ﬁbers
(Ironton 3131., 1979; Kemplay, 1984). Following reinnervation, muscle ﬁbers Often

become polyinnervated during the early stages Of this process. Once transmission is

145
reestablished all multiply-innervated muscle ﬁbers will become singly-innervated

(GoriO 3131., 1983). Recent evidence concerning the peptide transmitter calcitonin
gene-related peptide (CGRP) indicates that this transmitter / modulator may act as
a trophic message at the neuromuscular junction. CGRP is found in motor nerve
terminals stored in dense-core vesicles (these dense core vesicles are increased in
number in DTB treated rats (Jones, 1989)) which appear to be released under
different stimuli from the small ACh ﬁlled vesicles (Matteoli 3131., 1988). Effects
of CGRP on neuromuscular transmission include a decrease in EPP and MEPP
amplitude, quantal content Of EPPs and sensitivity Of the postsynaptic membrane to
iontophoresed ACh (Caratsch and Eusebi, 1990), and an increase in the
spontaneous release Of MEPPs (J innai 3131., 1989). CGRP has been reported to
prevent disuse-induced terminal sprouting (T sujimoto and MO, 1988). Thus,
CGRP appears to play a role in the modulation Of both pre- and postsynaptic
function. Several studies have demonstrated that neuromuscular junctions which are
regenerating, degenerating or physiologically-compromised exhibit an increase in
very large slow MEPPs or MEPCs compared tO normal junctions (see discussion Of
chapter on acute effects Of DTB). It has been proposed that these abnormal MEPPs
serve as a trophic signal between nerve and muscle. Evidence for this comes from
studies examining neuromuscular junctions following botulinum toxin poisoning. In
botulinum toxin-treated preparations the abnormal large MEPPs are the predominant
form Of transmitter released. It has been Observed that even though neuromuscular

transmission is completely blocked with this poisoning, the nerve and muscle

146

maintain trophic communication. If these preparations are treated with curare or
pancuronium the trophic interactions are blocked indicating that ACh binding at
nicotinic receptors is responsible for this communication (Mathers and Thesleff,
1978).

It is possible that the early subtle changes in channel behavior following
exposure to DTB, which do not appear to alter transmission tO a very large extent,
could interact in some way with trophic communication between nerve and muscle.
. By continuously altering some signal between nerve and muscle, DTB could cause
compensatory changes which then lead tO the alterations in transmitter release
Observed in rats exhibiting muscle weakness. Another interesting mode Of
modulation has been proposed by Labarca 3131. (1985), in which the nonquantal
release Of transmitter at neuromuscular junctions (Katz and Miledi, 1977) may
regulate the responsiveness Of the AChRs to the evoked release Of transmitter. Here
again the subtle changes which occur following exposure to DTB could over time
cause compensatory changes which lead tO the Observed weakness.

Presynaptic nicotinic acetylcholine receptors. In early studies in which the
effects Of curare on transmission were examined, changes in transmission were
Observed which appeared tO represent presynaptic effects Of curare on transmitter
release. These early studies indicated that ACh appeared to act presynaptically tO
mobilize transmitter stores (feed-forward mechanism) and that curare blocked this
effect causing a decline in transmitter release during repetitive stimulation (Hubbard

and Wilson, 1973; Glavinovic, 1979). Recently more evidence has been provided

 

147

that both nicotinic and muscarinic receptors are present presynaptically at the
neuromuscular junction and that these receptors play a rOle in the modulation Of
transmitter release (Wessler, 1989; Bowman, 3131., 1990). There is still debate
whether these receptors, if present, exert positive or negative feedback control over
transmitter release (Storella and Bierkamper, 1990).

If AChRs are present on the nerve terminal and if binding Of ACh to these
receptors modiﬁes transmitter mobilization or release, then this could represent a
target for DTB at which the subtle changes at postsynaptic receptors could have a

far greater impact on transmitter release.

DTB-induced muscle weakness as a model of human neuromuscular
disorders. The neuromuscular weakness Observed in rats treated chronically with
DTB is accompanied by changes in transmission which are similar in many ways tO
those Observed in human patients suffering from a variety Of neuromuscular
disorders. Similarities which exist include a decrease in m for nerve evoked EPPs
and EPCs, which is also Observed in Lambert Eaton Myasthenic Syndrome,
Congenital AChE deﬁciency, Botulism and several other disorders related to
myasthenia gravis (Engle 3131., 1977; 1990). A decrease in MEPP amplitude which
is Observed in muscles from treated rats and is also Observed in Congenital AChE
deﬁciency, slow channel Myasthenic Syndrome and Congenital AChR deﬁciency.
A slowing Of the MEPP decay is Observed in Slow-channel Myasthenic Syndrome,

while more rapid decay Of synaptic currents is Observed in High—conductance fast-

 

148
channel Syndrome and AChR deﬁciency and short channel Open-time (Engel 3131.,

1982; 1990). Although the alterations in transmission Observed in rats exhibiting
weakness following treatment with DTB dO not exactly mimic all Of the deﬁcits
Observed in any one Of the aforementioned syndromes, they do provide many
similarities tO a number Of these states. A common aspect Of most Of these
syndromes is the reduction in the safety factor for transmission in patients afﬂicted
with these diseases. Patients suffering from these syndromes appear normal when
at rest, however, during even moderate activity they exhibit increasing degrees Of
skeletal muscle weakness. A similar reduction in safety factor has been Observed in
muscles from rats treated with DTB. Atchison (1990) demonstrated that a reduction
in safety factor and abnormal quantal secretion could be detected prior tO muscle
weakness in neuromuscular preparations from rats treated with DTB. By
understanding how DTB interacts with different components Of the nerve and muscle
and how these interactions lead to the weakness Observed in treated rats a better

understanding Of the causes Of the weakness in the human disorders may be gained.

The results Of the experiments described above indicate that exposure to DTB
alters the time which nicotinic AChR-channels remain open. Changes in channel
Open-time are some Of the earliest signs Observed during treatment Of rats with DTB.

It is possible that the changes Observed if maintained by continued exposure to DTB

 

149
could lead to the alterations responsible for the weakness Observed in chronically

treated rats. Perhaps by altering trophic communication between nerve and muscle
or by altering sensitivity Of presynaptic nicotinic receptors which modulate release Of

ACh.

 

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