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Dynamics of the newly formed neuromuscular synapse

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Dynamics of the newly formed neuromuscular synapse

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DYNAMICS OF THE NEWLY
FORMED NEUROMUSCULAR
SYNAPSE
by
Chuck Zhuo-Kang Li
A Thesis Presented to
THE FACULTY OF THE SCHOOL
OF ENGINEERING
UNIVERSITY OF SOUTHERN
CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
Master o f Science in Biomedical
Engineering
December 1997
Copyright 1997 Chuck Zhuo-Kang Li
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UMI Humber: 1389989
UMI Microform 1389989
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This thesis, written by
Chuck Zhou-Kang Li
under the guidance o f his/her Faculty Com m ittee
and approved by all its members, has been
presented to and accepted by the School of
Engineering in partial fulfillm ent of the re
quirements fo r the degree o f
Master o f Science
Biomedical Engineering
D a te__ c T^b_e. r ___
Faculty Cornmitae
Chairman
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TA BLE OF CONTENTS
List of Figures.......................................................................................................
Acknowledgments................................................................................................
Chapter 1....................................................................................................................
Introduction........................................................................................................
1.1 The Neuromuscular Junction..............................................................
1.2 Background and Significance................................................................
1.3 Specific Aims.........................................................................................
Chapter 2................................................................................................................
Experimental Approaches..................................................................................
2.1 Animals and Preparations.....................................................................
2.2 Drug Preparations and Delivery..........................................................
2.3 Electrophysiology..................................................................................
Chapter 3................................................................................................................
Results..................................................................................................................
3.1 Neurotrophic Factors in Synaptic Transmission and Maintenance...
3.2 Modulation of Svnaptic Transmission.................................................
3.3 Activity and Svnaptic Dynamics...........................................................
Chapter 4.....................................................................................................................
Discussion............................................................................................................
References..............................................................................................................
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LIST OF FIGURES
Number Page
Figure 1. A proposed model for the possible mechanism of L-type VSCC
modulation in newly developed motor nerve terminal............... 6
Figure 2. A schematic of TTX infusion in the rat using implantable osmotic
pump..............................................................................................13
Figure 3. Effect of NT-3 on synaptic transmission in neonatal rat and
regenerating frog muscles............................................................17
Figure 4. Effect of chronic administration ofNT-3 in neonatal rats................18
Figure 5. Effect of GDNF on synaptic transmission in newborn rat...............20
Figure 6. GDNF potentiated synaptic transmission in newborn rats.............. 21
Figure 7. CGRP did not affect EPP amplitude in newborn rats......................23
Figure 8. Substance P decreased EPP amplitude in regenerating but not in
normal frog NMJs........................................................................24
Figure 9. Substance P reduced EPP amplitude in regenerating frog NMJs.... 25
Figure 10. High frequency stimulation prevented the potentiation effect of
nifedipine......................................................................................26
Figure 11. SNX-230 injection was lethal to neonatal rats................................28
Figure 12. Inactivation of muscle did not induce L-type VSCC
functionality................................................................................. 29
iii
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ACKNOWLEDGMENTS
I wish to thank Dr. Chien-Ping Ko for his guidance and generous support, and
my parents for their encouragement throughout the duration of this study.
iv
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C h a p t e r I
INTRODUCTION
1.1 The Neuromuscular Junction
One of the simplest, yet critical elements in neural communication is
the synapse. The classical definition of a synapse is that it composes of a
presynaptic and postsynaptic component, and that transfer of information takes
place between them. The neuromuscular junction (NMJ) is a typical synapse
where nerve communicates with muscle. There are four major components of
the NMJ; they include the presynaptic motor nerve terminal, the postsynaptic
receptor, the synaptic cleft, and extracellular matrix (ECM). There are also
supportive glial cells called perisynaptic Schwann cells (PSCs) that extend
fingerlike processes into the synaptic cleft.
Acetylcholine (ACh) is the neurotransmitter in the NMJ. It is
packaged in synaptic vesicles in units of quantum in the motor nerve terminal.
The spontaneous release of single units of synaptic vesicles was observed by
Del Castillo and Katz (1954). This spontaneous release produces miniature
endplate potential (MEPP) on the muscle fiber, and provides evidence of
quantal release. Upon the arrival of a nerve impulse, an influx of calcium ion
enters the nerve terminal through voltage sensitive calcium channels (VSCCs).
This C a^ influx triggers a cascade of events that leads to evoked synaptic
transmission. Briefly, C a^ dissolves actin filaments that normally hold
synaptic vesicles in place to facilitate the movement of these vesicles toward
the active zone, where they eventually dock and fuse with the plasma
membrane. The final exocytosis of the synaptic vesicles marks the quantal
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release of ACh to the synaptic cleft (Katz and Miledi 1967; Kelly 1987; Kelly
1988). Postsynaptically, ACh molecules bind to acetylcholine receptors
(AChRs) on muscle endplates, thus opening AChR channels permeable to Na^
and K~. The resultant conductance creates a current change on the membrane
of the muscle fiber and generates a graded endplate potential (EPP).
Depending on the amount of depolarization brought by the EPP, nearby
voltage sensitive Na+ channels could be activated and thus allowing the
entrance of Na+ . If there is a sufficient amount of Na^ influx, an action
potential can be elicited, producing muscle fiber contraction.
The NMJ has a very efficient uptake mechanism for transmitters
remaining in the synaptic cleft after they are released. Transmitters can be
quickly recycled by endocytosis or undergo degradation process by
acetylcholinesterase. This removal process shortens the refractory period of
synaptic transmission and receptor desensitization, and therefore it is another
essential factor in synaptic efficacy and robustness.
1.2 Background and Significance
The most well known feature of the NMJ is its simplicity and
accessibility in the study of synaptic transmission. Although there has been a
tremendous amount of work on the NMJ ranging from structure to activity,
only recently has the interest of its ontogeny been emerged. The reason for
such interest lies in the hopes of discovering models of plasticity that are
inspirational to higher functions such as learning and memory. Studies in the
central nervous system (CNS) have been relatively hampered due to
inaccessibility and complexity. Thus, it is advantageous to use simple models
such as those in the NMJ to facilitate the discovery o f fine structures and
functionality of the CNS (Dan et al. 1995).
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One of the most obvious signs of plasticity in the NMJ occurs during
the first couple weeks of neonatal development and nerve regeneration.
Termed synapse elimination, this period is typified by the elimination of
redundant synapses that were formed when initial contacts between the nerves
and muscles were made. After this critical period in NMJ development, every
muscle fiber becomes singly innervated, and these functional units last
throughout adulthood (Redfem 1970; Bennett and Pettigrew 1974; Betz et al.
1979; Dennis and Harris 1979; Dennis et al. 1981).
The phenomenon of synapse elimination is just one of the many
aspects of dynamism in newly formed synapses. One would question its
mechanism, specifically how, and what factors dictate the withdrawal or the
strengthening and maintenance of certain synapses. Several theories have
arisen so far; they include activity, trophic effect, and support cells and
molecules. To explore some of these hypotheses, this investigation will focus
on the roles of neurotrophic factors, calcium channels and neuromodulators,
and activities in the NMJ, and how they affect and modulate synaptic
transmission.
1.3 Specific Aims
(I) Neurotrophic factors, acute and chronic effect.
Neurotrophic factors are believed to be retrograde messengers to
motor neurons, and the retraction of multiple innervation has been speculated
to be related to the availability of trophic agents in the target sites on the
muscles (Colman and Lichtman 1993; English and Schwartz 1995; Jordan
1996). They have also been claimed to promote synapse maturation (Wang et
al. 1995). Some of the likely target derived neurotrophic factors include brain
derived neurotrophic factor (BDNF), glial-cell-line-derived neurotrophic
j
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factor (GDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5). The
expression of these factors have been found in skeletal muscles and limb buds
in the early stages of development (Maisonpierre et al. 1990; Springer et al.
1994: Choi Lundberg and Bohn 1995; Griesbeck et al. 1995; Suvanto et al.
1996; Wright and Snider 1996; Nosrat et al. 1996). They have also been
implicated in nerve regeneration and survival due to their up-regulation after
axotomy (Funakoshi et al. 1993; Springer et al. 1995; Trupp et al. 1995;
Hammarberg et al. 1996). Rather than expression in target muscles, ciliary
neurotrophic factor (CNTF) is secreted by mylinating Schwann cells. Similar
to other trophic factors, CNTF also up-regulates after nerve damage or
axotomy and enhances nerve regeneration (Curtis et al. 1993; Sahenk et al.
1994; Newman et al. 1996).
There have been numerous studies detailing the effects of these
neurotrophic factors on synaptic activities in vitro (Lohof et al. 1993; Stoop
and Poo 1995; Stoop and Poo 1996). Generally, trophic factors applied to
Xenopus nerve-muscle co-cultures enhanced synaptic transmission. The
effects were salient, with spontaneous synaptic currents increasing more than
two to three folds, and frequency increasing up to eight folds. Evoked
synaptic currents were also affected as well, although not as dramatic as the
spontaneous currents.
While the effects of the in vitro experiments have been convincing, are
they physiological? It is interesting to see if they are reproducible outside of
the culture media. If so, in vitro studies potentially could yield very precise
and efficient models for in vivo studies, and provide us yet another avenue to
answer our questions. Thus, the first aim of the study is to assess the acute and
chronic effects of some o f these neurotrophic factors in vivo and discover their
true physiological roles.
4
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It is proposed that in the acute experiments, intracellular recording to
be performed on regenerating frog and neonatal rat muscles under the
treatment of neurotrophic factors. In chronic experiments, neonatal rats will
be injected with neurotrophic factors during the first two weeks after birth.
The muscles will then be assayed electrophysiologically to determine trophic
effects on synaptic maintenance.
(2) Roles o f voltage sensitive calcium channels (VSCCs) and their
modulators in newly formed synapses.
The many types of VSCC subtypes have been found to be very
dynamic throughout synapse development. For example, transformation of
VSCC subtypes was observed during the development of Xenopas (O’Dowd et
al. 1988) and chick embryos ( McCobb et al. 1989; Gray et al. 1992). Recently
we found that L-type VSCC was involved in the modulation of synaptic
transmission in newly formed synapses (Sugiura and Ko 1997). Although
non-existent or non-functional in mature NMJs (Pancrazio et al. 1989;
Atchison 1989), these L-type VSCCs exist in the presynaptic nerve terminals
of neonatal rat and regenerating mouse and frog synapses. According to our
model (Fig. 1), the transient Ca~^ influx through L-type VSCC triggers the
release of some unknown neuromodulators or neuropeptides which activate a
G-protein on the presynaptic terminal membrane. The activation of G-protein
in turn inhibits transmitter release mediated by P/Q (rats and mice) or N-type
(frogs) VSCCs. This self-limiting feature is believed to exist in immature
NMJs as a means of vesicle preservation because of the paucity of
neurotransmitters and the vulnerability to synaptic depression (Dennis et al.
1981). Thus blocking these L-type VSCCs using dihydropyridines removes
the inhibition mechanism and enhances synaptic transmission. This effect was
attenuated as the NMJs matured.
5
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Neuromodulator
Presynaptic
merrbrane
(-) G protein
V
N or P/Q
VSCCs
/
L-type
VSCCs
Active zone
Figure 1. A proposed model for the possible
mechanism o f L-type VSCC modulation in
newly developed m otor nerve terminal.
Neuromodulator is released upon Ca~ inflax through L-type VSCCs. which binds to
presynaptic G-protein receptor and activates an inhibition mechanism on N or P/Q-type VSCC
mediated neurotransmitter release. (From Sugiura and Ko 1997)
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What remains unclear now is the means of action and the identity of
the neuropeptides or modulators that were released upon calcium influx.
Studies have indicated that dense core vesicles co-localized with synaptic
vesicles in the nerve terminal could be the neuropeptides substance P (SP) and
calcitonin-gene-related-peptides (CGRP) ( Csillik et al. 1993; Andreose et al.
1994; Sala et al. 1995). In addition, it has been demonstrated that exogenous
CGRP potentiates immature (Lu et al. 1993), and inhibits mature NMJs
(Caratsch and Eusebi 1990), suggesting the presence of CGRP receptors
throughout synapse development. While these experiments focused the effect
o f CGRP on the postsynaptic AChRs, they had not addressed their possible
presynaptic roles in developing NMJs in vivo. Thus, part of this aim will focus
on these two neuropeptides and investigate their roles in synaptic transmission
in immature NMJs in vivo. One approach is recording synaptic transmission
while muscles are bathed in solutions containing exogenous neuropeptides.
Another approach is to examine the effect o f L-type VSCC blockers in a
neuropeptide-free medium by recording synaptic transmission after high
frequency stimulation and the depletion of neuropeptides in the NMJs.
The absence of L-type VSCC functionality in mature NMJs intrigued a
question on its role throughout synapse maturation. Are the L-type VSCCs
necessary and critical in developing NMJs? What is their relationship to P/Q-
type VSCCs which directly mediate transmitter release? To answer these
questions, SNX-230, a synthetic version of the naturally occurring co-
conopeptide that blocks P/Q VSCCs (Bowersox et al. 1995; Sugiura et al.
1995) will be injected into neonatal rats to block off neuromuscular
transmission. We hope to observe under such condition, if (a) the modulatory
L-type VSCCs would transform themselves into mediating transmitter release
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as well, and subsequently, if the second messenger system is still intact; (b) the
modulatory role o f L-type VSCCs becomes more dominant and compensatory.
(3) Activity and inactivity, do they have any bearing on synapse
formation and maintenance?
The significance of L-type VSCCs in immature neuromuscular
terminals is unknown, and the mechanism of their disappearance during
maturation remains to be a mystery. Possible explanation could be directly
related to synaptic efficacy and activity.
There are theories that the maintenance of synaptic contact is activity
dependent (Brown et al. 1982; Thompson 1983). Higher activity level of a
synapse results in a more strengthened synapse. Factors that directly affect
synaptic efficacy include calcium channel dynamics and modulation,
efficiency of intracellular calcium storage, docking and packaging of synaptic
vesicles, vesicle exocytosis, and coordination with the postsynaptic receptor.
Is the existence of L-type VSCCs activity dependent? While there is
no doubt for their presence in newly formed synapses, Sugiura and Ko (1997)
also found that after nerve crush, their functionality returned during nerve
regeneration as well. This is the most direct evidence of relating L-type VSCC
functionality to activity. To mimic the event of inactivity after nerve crush, a
tetrodotoxin (TTX)-filled osmotic pump will be implanted in adult rat. Using
silicone tubing and a nerve cuff, TTX is to be infused to a section of the sciatic
nerve continuously for two to four weeks to prevent the conduction of action
potential. Electrophysiological assessment o f the soleus muscle will then
commence after the treatment to examine the presence of L-type VSCC
functionality. One of the major differences between this approach to that of
nerve crush is the TTX treatment allows axonal transport throughout. This
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will enable us to determine the possibility of any kinds o f retrograde and
anterograde messengers that might affect the functionality of L-type VSCCs,
which were not apparent from the nerve crush approach.
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C h a pt e r 2
EXPERIMENTAL APPROACHES
2.1 Animals and Preparations
Isolated diaphragm and soleus muscles of neonatal Sprague Dawley
rats (Charles River, Wilmington, MA) were used in the study of developing
NMJs. The ages o f the newborn rats were classified according to the number
of days after birth with PO denoting the day of birth. Pups were anesthetized
with sodium pentobarbital and sacrificed by decapitation. Muscles removed
were incubated in normal mammalian Ringer’s (NMR) solution consisting of
135 mM NaCl, 5 mM KCI, 15 mM NaHC03 , 1 mM Na2 HP04, 1 mM M gS04,
2.5 mM calcium gluconate, and 11 mM glucose, pH 7.2. In chronic TTX
treatment, male adult Sprague Dawley rats of one month or older were used.
They were anesthetized with sodium pentobarbital during implantation, and
sacrificed with an overdose of sodium pentobarbital. Animals were housed
separately after any surgical operation. In regeneration studies, the cutaneous
pectoris (CP) muscle of frogs (Rana pipiens, 2-3 inches length) was used.
Frogs were anesthetized with 0.1 % tricaine methanesulfonate in chill water
and nerve entering the right side CP muscle was crushed with fine forceps; the
crush was deemed successful by noting the transparent nerve trunk at the crush
site. The left side CP muscle and nerve were untouched. After two weeks of
recovery, the frogs were sacrificed and both CP muscles removed. A muscle
twitch in response to a light pinch above the previous crush site on the nerve
trunk was used as an indicator of successful nerve regeneration. The intact
muscle was saved as contralateral control. The muscles were bathed in normal
frog Ringer’s (NFR) solution consisting of 120 mM NaCl, 2 mM KCI, 1 mM
10
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NaHCOs, 1.8 mM CaCL, and 5 mM HEPES, pH 7.2. All bath solutions were
at room temperature and bubbled with a mixture of 95% Oj/5% CCb.
2.2 Drug Preparations and Delivery
Neurotrophic factors, NT-3 (Regeneron Pharmaceuticals, Tarrytown,
NY) and GDNF (Alomone Labs, Jerusalem, Israel) were dissolved in
phosphate buffered saline (PBS) in stock concentration o f 14.4 mg/ml. They
then were aliquoted to the desired working concentrations for use in the
experiments. Neuropeptides, CGRP and SP (Sigma, St. Louis, MO) were
dissolved in dELO to make a stock of 0.01 M. L-type VSCC blockers of the
dihydropyridines family, nifedipine (Sigma) and isradipine (Research
Biochemicals International, Natick, MA) were dissolved in ethanol to make a
stock concentration of 20 mM. P/Q-type VSCC blocker, SNX-230 (a
synthetic co-conotoxin, Neurex Corporation, Menlo Park, CA), were dissolved
in PBS to the various concentrations used in injection. In acute
electrophysiological experiments, concentration of drugs were cumulatively
applied to the recording bath, and the maximal concentration of ethanol was
kept less than 0.05% to avoid any side effects.
The injection protocol was used in the chronic studies of NT-3 and
SNX-230. Drugs were injected into die soleus muscles of the newborn rat via
a 30-gauge needle. The vehicle (PBS) was injected to the opposite muscle as a
control. The injected fluid was kept to 10 pi. Injection schedule started on PI
for all the experiments, and ended on P14 in the NT-3 experiment. To prevent
experimental bias, a blind approach was taken, i.e., the identity of the injection
solution was revealed only after results were analyzed.
In the chronic study of TTX blockade and L-type VSCC functionality,
an Alzet osmotic pump (Model 2002, Alza Scientific Products, Palo Alto, CA)
11
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was subcutaneously implanted into the back of adult rat, posterior to the
scapulae (Fig. 2). The pump was filled with a stock solution of TTX (500
pg/ml in citrate buffer, Sigma). TTX was delivered via a Silastic tubing (inner
diameter, 0.635 mm; outer diameter, 1.19 mm) that ends with a longitudinally
slit (5 mm) Silastic cuff (inner diameter, 1.575 mm; outer diameter, 2.413 mm)
jointed by a silicone based medical adhesive (Factor H, Inc., Lakeside, AZ).
The cuff was designed to fit around the sciatic nerve in the mid portion of the
thigh. To secure the cuff and the tubing, suture was used to close the slit on
the cuff after the cuff encircled the sciatic nerve trunk and to anchor the tubing
at the opening of the hamstring musculature. The infusion of TTX lasted for
two weeks per pump at a rate of 0.5 pl/hr. For subjects that required more
than two weeks of treatment, the discharged pump was replaced with a new
TTX filled pump on day 15 of infusion. Rats were monitored daily after
surgery, and the effectiveness of TTX blockade was assessed by the absence
of toe extension reflex which occurs when the tail and hind limbs are lifted
above ground. At the end of treatment, the effectiveness of action potential
block along the sciatic nerve was examined again in situ. The cuff and sciatic
nerve were exposed at the site of implantation and a current was applied along
the nerve from a stimulator through bipolar electrode leads. Two criteria were
used as the final judgment on a successful blockade, (1) stimulation at A but
not B (Fig. 2) results in no muscle twitch in the leg, (2) nerve transection at A
but not B results in no muscle twitch in the leg. Similar implantation
procedures were described elsewhere (Spector 1985).
2.3 Electrophysiology
Intracellular recording from skeletal muscle fiber was used as a means
to assess the properties of synaptic transmission in the NMJ. Glass capillary
tubes were pulled into microelectrodes using a micropipette puller (Sutter
12
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osmotic pump
Silastic tubing
sciatic nerve cuff
Figure 2. A schematic o f TTX infusion in the
rat using implantable osmouc pump.
TTX is delivered to the mid-section o f the rat sciatic nerve from an osmotic pump via a Silastic
tubing and a cuff. The effectiveness o f the activity blockade can be assessed by stimulating or
transecting along the nerve trunk in situ.
13
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Instrument, Novato, CA). Electrodes were filled with 3 M KCI solution and
had a resistance range between 30 to 80 MD. Electrodes with resistance
toward the higher range were used specifically for recording from neonatal
muscles to improve signal to noise ratio. Hemidiaphragm and soleus muscles
of neonatal rats were bathed in 10 mM Ca~ mammalian Ringer’s solution,
modified according to Redfem (1970) to enhance recording stability and the
probability of limited transmitter release in newly developed NMJs. To
prevent spontaneous muscle twitch, D-tubocurare (1-10 pM) was applied to
the bath. Intracellular recording from adult rat soleus muscle and frog CP
muscle was performed in a bath of low C a^ and high M g~ Ringer’s solution
(1.2 mM Ca^/6.0 mM M g^ for rats, 0.4 to 0.7 mM Ca""74.0 to 6.0 mM Mg~"
for frogs). Evoked transmitter release was elicited by supramaximal
stimulation of the motor nerve via a suction electrode. EPPs evoked at 0.05 to
0.2 Hz were recorded continuously at 24 episodes per incremental time, while
those evoked at 1 Hz were recorded intermittently at 100 episodes per
increment dependent upon the time required for the collection of MEPPs.
Continuous recording from an identical cell was used in experiments that
required the monitor of pre and post drug effect. Intracellular recording data
was acquired and analyzed using Digidata 1200 and PClamp6 (Axon
Instruments, Foster City, CA).
In the study of high frequency stimulation on L-type VSCC
functionality, recording from an identical cell was attempted before and after a
continuous pulse train (100 Hz. 300 ps pulse width) was applied to the motor
nerve for 10 minutes. Immediately after the cessation of high frequency
stimulation, recording bath was replaced with similar solution as that before
stimulation. It should be noted that the possibility of tetanic contraction of the
muscle due to high frequency stimulation was unavoidable. Often in such a
14
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case, damage or the dislodgment of electrode occurred, thus recording was
resumed on a nearby cell immediately.
The assessment of synaptic maintenance after the injection of NT-3 to
neonatal rats was performed based on the concept that multiple synapses have
different activation thresholds (Dennis et al. 1981). Specifically, soleus
muscles were removed after the predefined treatment time and prepared in
recording bath as previously described. By slowly adjusting the stimulation
voltage, graded EPPs of various sizes as a result of transmitter release from
different axonal terminals were revealed. Assuming the stimulation voltage
range covered the spectrum of activation thresholds of multiple axons
innervating the same muscle fiber, distinct size levels of EPPs were used to
estimate the multiplicity of innervation on a muscle fiber.
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C h a p t e r 3
RESULTS
3.1 Neurotrophic Factors in Synaptic Transmission and
Maintenance
In an attempt to duplicate the in vitro experiment of neurotrophic
factors in synaptic transmission in vivo, NT-3 was applied to neonatal rat and
reinnervated frog muscles in a series of acute electrophysiological
experiments. Although NT-3 potentiated synaptic transmission in Xenopus
nerve-muscle co-culture (Lohof et al. 1993), no obvious synaptic enhancement
was observed in all the muscles tested. EPP size was not affected in NMJs
under the acute treatment of up to 300 ng/ml NT-3. Figure 3A shows a
sample record in which a rat pup (P8) soleus muscle was treated with 100 and
200 ng/ml NT-3; similarly, a record for a reinnervated (two weeks) frog CP
muscle is shown in Figure 3B. Because of the use of curare in neonatal
muscle recording, MEPPs were collectable only from the frog muscle. They
too, did not show any change in size and frequency.
In chronic in vivo experiment, NT-3 (100 ng/g) was injected
intramuscularly into the soleus muscle of rat pups from age PI to P8 daily to
evaluate its roles in synaptic maintenance. By assaying the muscles
electrophysiologically, it was found that the treatment with NT-3 did not affect
synapse elimination during the first two weeks of neonatal development (Fig.
4). There was no significant difference in the amount of multiply innervated
junctions between untreated (normal) and NT-3 treated (P>0.05, one way
analysis of variance and Dunnett’s test), untreated and PBS (vehicle) treated
16
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200 ng/ ml
100 ng/ml
O
T J
3
£ 3
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o • - .................................
-10 0 10 20 30 40 50 60
Time (min)
0 • - ' • - ....
-5 0 5 10 15 20 2 5 30 35 40 45 50 55 60 65 70 75 80
Time (min)
Figure 3. Effect o f NT-3 on synaptic
transmission in neonatal rat and
regenerating frog muscles.
100 and 200 ng/ml o f NT-3 were applied to the recording bath cumulatively as indicated by
arrows. Each data point represents an average o f 24 consecutive EPPs recorded from the same
junction. Sample continuous recording is from P8 rat soleus muscle (A), and 14 day
reinnervated frog CP muscle (B).
17
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■Normal
■NT-3
■PBS
P4 P5 P6 P7 P8 P9 P10P11 P12P13P14
Age
Figure 4. Effect o f chronic administration o f
NT-3 in neonatal rats.
Daily intramuscular injection o f 100 ng/g NT-3 (from PI) did not affect synapse elimination in
newborn rats. There is no significant difference between untreated (normal) and NT-3 treated
pups, and there is no difference between PBS (vehicle) treated and NT-3 treated pups.
Percentage of multiply innervated NMJs is the percentage o f number o f muscle fibers that
exhibited multiple axonal inputs over the total number of fibers observed.
18
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(P>0.05, one way analysis of variance and Dunnett’s test) soleus muscles. All
experimental groups seemed to follow the same natural timeline o f synapse
elimination.
Although NT-3 did not exhibit significant effects in vivo, another
neurotrophic factor, GDNF, was claimed to be much more potent (Yan et al.
1995). However, so far there are no studies of GDNF on synaptic
transmission in vitro and in vivo. Similar to that of NT-3, various amounts of
GDNF (20,40, 60, and 80 ng/ml) were applied to newborn rat diaphragm
muscle and intracellular recording was performed. GDNF concentration at as
low as 20 ng/ml was enough to potentiate EPP amplitude while higher
concentration did not show additive effect (Fig. 5). In a total of five
experiments on rat pups (P0 to P3), the average EPP amplitude increased from
0.78 ± 0.31 mV before GDNF treatment to 1.33 ± 0.52 mV after reaching the
plateau of GDNF effect (Fig. 6). Paired t-test (two-tailed) revealed that the
probability of the null hypothesis being true, that there was no difference from
GDNF treatment, was less than 0.109 (P < 0.109).
3.2 Modulation of Synaptic Transmission
From the model we devised earlier (Fig. 1), a neuropeptide or
neuromodulator was believed to initiate the signal transduction pathway to
provide the self-limiting feature in synaptic transmission of the the immature
NMJ. One of the likely neuropeptides was CGRP. TotestCGRP’s
involvement. 2 pM of human CGRP fragment was applied to the recording
bath where a neonatal rat hemidiaphragm muscle was submersed. If CGRP
were indeed the neuropeptide responsible for the activation of the G-protein,
one would expect the application of exogenous CGRP to cause a reduction of
19
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60 ng/ ml
80 ng/ml
2.5
>
£ 2
a >
■ o
3
i l 15
g 20 ng/ml
i ’ ^
0.5
0 • - . . . . .
-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160
Time (min)
Figure 5. Effect o f G D N F on svnapac
transmission in newborn rat.
Cumulative application o f 20, 40,60, and 80 ng/ml GDNF (indicated by arrows) increases EPP
amplitude in this sample recording from the hemidiaphragm muscle of a P0 rat. Each data
point is the average o f 24 consecutive EPPs recorded from the same junction. Note that GDNF
at 60 and 80 ng/ml did not produce any additive enhancement.
20
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<
Before GDNF After GDNF
Figure 6. G D N F potentiated synaptic
transmission m newborn rats.
Acute application o f GDNF to neonatal rat (P0-P4) muscles resulted in the enhancement of
synaptic transmission. Data denotes the average o f five experiments and SEM. The average
values were obtained by taking the mean of several data points during a stable state before and
after drug application.
21
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transmitter release. Contrary to our expectation, CGRP did not inhibit
transmitter release at all. Figure 7 shows a sample of a contunuous recording.
Substance P was the other neuropeptide that was believed to be co
released with ACh. Similar to the idea behind CGRP, if SP were involved as
the neuropeptide that activates the G-protein, this exogenous SP should reduce
transmitter release by intensifying the self-limiting mechanism of synaptic
transmission unique to imature NMJs. Our results indicate, contrary to CGRP,
exogenous SP was able to reduce transmitter release in reinnervated frog
NMJs (Fig. 8A), and it did not affect transmitter release in the normal
junctions (Fig. 8B). The reduction onset was generally rapid and observable
within the first few minutes after drug application. In five experiments
performed on 14 days reinnervated frog CP muscles, the average EPP
amplitude was significantly reduced, from 2.70 ± 0.77 mV before to 1.26 ±
0.41 mV after 100 pM SP application (P < 0.04) (Fig. 9).
To further test the involvement of neuropeptides and their roles in the
self-limiting property in synaptic transmission of immature NMJ, the effects of
L-type VSCC blockers were evaluated again after high frequency stimulation
of the nerve terminal. It has been shown that high frequency stimulation can
rapidly remove neuropeptides such as CGRP from nerve terminals (Sala et al.
1995), thus replacing the recording bath after high frequency stimulation with
fresh solution could potentially eliminate the actions of these neuropeptides.
According to our original model, the removal of neuropeptides effectively
abolishes the inhibition mechanism of the G-protein, thus blocking L-type
VSCCs at this point should not produce any synaptic enhancement. As shown
in a sample recording, the application of 10 pM nifedipine after 10 minutes of
high frequency stimulatin and replacement of recording bath indeed did not
result in any potentiation of EPP amplitude (Fig. 10A). In contrast, as
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.
1
0.9
0.8
- * 0.7
>
E
IT0- 6
•a
3
= 0.5
a
E
< 0 .4
Q .
a.
“ 0.3
0.2
0.1
0
-5 0 5 1 0 1 5 20 25 30 35 40 45 50 55 6 0 6 5 70 75
Time (min)
Figure ”. CGRP did not affect EPP amplitude in
newborn rats
This sample recording o f the acute application o f 2 pM CGRP (at time 0) to a P2 rat
hemidiaphragm muscle does not show any effect exerted by the neuropeptide. Each data point
is an average of 24 consecutive EPPs recorded from the same junction.
23
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.
1.6
1.4
> 12
0 1
■ c
M 0.8
a
E
< 0.6
a.
Q .
UJ0.4
0.2
0
-20 -10 0 10 20 30 40 50
Time (min)
5
ill
1
0.5
0
-10 -5 0 5 10 15 20 25 30 35
Time (min)
Figure 8. Substance P decreased EPP
amplitude tn regenerating but not in normal
frog NMJs.
100 fx iV l SP reduced EPP amplitude in 14 day reinnervated frog CP muscle (A), but not in
normal CP (B). Each data point is an average of 100 consecutive EPPs recorded from the
same junction.
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.
>
E .
0
T3
a
E
<
CL
a.
UJ
0
O )
re
> _
0
>
<
4
3.5
3
2.5
2
1.5
1
0.5
0
Before Substance P After Substance P
Figure 9. Substance P reduced EPP
amplitude in regenerating frog NMJs.
Substance P significantly reduced EPP amplitude in 14 day reinnervating frog CP muscle (P <
0.04). Shown are average values and SEM o f five experiments. The average values were
obtained by taking the mean o f several data points during a stable state before and after drug
application.
25
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A
1.2
>
£ - j . H.F. Stun
Replace bath
3 0.8
a.
3 0.6
10 pM Nif.
0.2
-30 -20 -10
2.5
10 20 30 40
Time (min)
50 60 70 80
>
E
©
■ o
3
a
E
<
a.
o.
U i
1.5
10 pM Nif.
0.5
B
0 • -
-10 0 10 20 30 40 50 60 70 80 90
Time (min)
Figure 10. High frequency stimulation
prevented the potentiation effect of
nifedipine.
10 pM nifedipine, an L-type VSCC blocker, did not potentiate EPP amplitude in a
P0 rat hemidiaphragm muscle (A) after 10 minutes of high frequency stimulation
(100 Hz) and replacement o f recording bath, while it potentiated a similar muscle
under normal condition (B). Each data point is an average of 24 consecutive EPPs
recorded from the same junction.
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.
I
demonstrated in earlier studies, 10 pM nifedipine readily potentiated EPP
amplitude in a similar subject (Fig. 10B).
To examine whether the chronic blockade of P/Q-type VSCCs would
induce L-type VSCCs to overtake the mediation role of P/Q-type VSCCs in
synaptic transmission or interfere with the G-protein signal transduction
pathway, various amounts of SNX-230 were injected into the soleus muscles
of neonatal rats. Unfortunately, the toxicity of SNX-230 resulted in several
deaths of neonatal rats (Fig. 11). Apparently SNX-230 was diffused and
circulated throughout the body from the site of injection, and possibly
obstructed the respiratory system by paralyzing the diaphragm. The surviving
pups did not show any signs of paralysis in the leg where SNX-230 was
injected, suggesting insufficient dosage levels. The original goal was to have a
localized blockade of P/Q-type VSCCs in the NMJs of the soleus muscle, it
now appeared that SNX-230 was not the proper blocking agent.
3.3 Activity and Synaptic Dynamics
The functionality of L-type VSCCs is not unique to neonatal NMJs.
Sugiura and Ko (1997) demonstrated that L-type functionality returned after
nerve crush and regeneration in adult mice and frogs. To explore the
possibility that inactivity after nerve crush might have instigated the return of
L-type VSCC modulation in regenerated synapses, the effects of L-type VSCC
blockers were further investigated. Soleus muscles were inactivated by TTX
blockade of the sciatic nerve for two or four weeks, and intracellular
recording was performed before and after the application of various L-type
blockers. Unlike the regenerated synapses, these inactivated muscles did not
display any synaptic enhancement by L-type blockers (Fig. 12 A & B). Thus,
27
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Days of SNX-230 Injection
Figure 11. SNX-230 injection w as lethal to
neonatal rats.
Shown are SNX-230 injection dosages in pg/day. For the lethal dosages, paralysis o f the
hindlimb was observable within 2 to 3 hours after injection. The other dosages did not cause
any signs o f paralysis or activity blockade, and their injections were terminated on day 2 of
treatment.
28
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1.2
1
>
£.0.8
®
T3
i 0.6
a
E
<
CL 0.4
a.
U l
1 p i\t
0.2
-5 10 15
1
0.9
0.8
■0.7
U J
0.2
0.1
0
20 25
Time (min)
30 35 40 45 50
B
3 0.6
= 0.5
a.
3 0.4
0.3
-20 -10 0 10 20 30 40 50 60 70 80 90 100
Time (min)
Figure 12. Inactivation o f muscle did not
induce L-type VSCC functionality.
Unlike the reinnervation study (see Sugiura and Ko 1997), 1 pM isradipine. an L-type VSCC
blocker, did not have any potentiation effect on EPPs from rat soleus muscles inactivated with
FI X for 2 weeks (A), and 4 weeks (B). Each data point is an average o f 100 consecutive EPPs
recorded from the same junction.
29
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inactivity alone might not have been a sufficient paradigm in the study of L-
type VSCC functionality in newly formed and regenerated synapses.
30
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C h a p t e r 4
DISCUSSION
Although generally believed to be the most simple and understood
synapse, this study revealed that NMJ at its newly developed stage, possesses
tremendous plasticity and complexity. Unlike matured NMJs, synaptic
transmission in developing NMJs involves a modulatory mechanism activated
by the influx of Ca~ through L-type VSCCs in the presynaptic nerve terminal.
Further evidence is now provided for the possibility of neuropeptide or
neuromodulator participation that supports the original model (Fig. 1), despite
the fact that activity and inactivity alone may not be sufficient to explain L-
type VSCC functionality in developing NMJs. In addition, as in studies in
vitro, synaptic transmission in developing NMJs can be influenced by
neurotrophic factors in vivo, though there exists a definite difference in terms
of scale, potency, and characteristics.
Neurotrophic Factors
Neurotrophic factors such as NT-3 have been shown to dramatically
influence synaptic transmission and neuronal development in vitro. Lohof et
al. (1993) reported the application of 50 ng/ml NT-3 into cell culture of
Xenopus spinal neurons and myotomal myocytes rapidly increased the
frequency o f spontaneous synaptic currents (almost 8 times control) and the
amplitude o f evoked synaptic currents (almost 2 times control). However in
this study, in vivo experiments were not effective for the range of NT-3
concentrations of 20 to 300 ng/ml. This may indicate that even at 300 ng/ml,
the amount o f NT-3 is not enough to be an effective dosage in vivo considering
31
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the fact that unlike the in vitro environment where there are mainly pre and
postsynaptic components, other components of the NMJ are now present in
relatively predominant proportions in vivo. These components, such as ECM
and PSCs are known scavengers in the neuromuscular synapse, and they may
preferentially uptake or trap the free-flowing exogenous NT-3 thus reducing
its availability to the motor neurons. In fact, a few experiments were
conducted in this study to closely model the culture setting by recording
MEPPs from the tail muscle o f Xenopus tadpoles at stage 42. The tail muscle
at this developmental stage is very similar to that in cell culture, yet unlike the
isolated system in culture, MEPPs recorded from the tail muscle were not
affected by NT-3 as well (data not shown). The lack of effect could also be
attributed to the age of the subjects examined. Although NT-3 is expressed
postnatally, its abundance occurs in embryonic stages (Maisonpierre et al.
1990; Griesbeck et al. 1995).
Similar to acute experiments, chronic application of NT-3 via injection
showed no effect in synaptic maintenance. It was hoped that a continuous
supply of NT-3 to the NMJs would be more effective than acute application,
but the result turned out to be negative. However, the interpretation of the data
in Figure 4 warrants the following considerations. First, the amount o f NT-3
injected might not have been enough to be effective; second, the method used
to assess the number of multiple innervations might have been insensitive.
Indeed, toward the termination of this experiment, a similar study conducted
by Kwon and Gurney (1996) was published. They demonstrated that NT-3
stabilized silent synapses and retained multiple innervations, but it was at a
dosage much higher than what was used in the current study (5000 ng/g/day to
15000 ng/g/day vs. 100 ng/g/day). Thus, obtaining the effective dosage of
neurotrophic factors in vivo could be elusive; it was evident again in the case
of GDNF. The current result on GDNF confirms its higher potency level as
32
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I
reported previously (Yan et al. 1995), but in a more direct manner in terms of
the NMJs. The ability for neurotrophic factors to influence synaptic
transmission and maintenance in vivo is significant because it hints their
possible roles in synaptic plasticity, and therefore deserves further study.
Neuropeptides and VSCCs
One of the key elements in our model (Fig. 1) that explains the
potentiation of synaptic transmission in developing NMJs by L-type VSCC
blockers is the involvement of neuropeptides or neuromodulators. CGRP is a
very strong candidate because it is developmentally regulated; its expression
diminishes as neural tissues develop, up-regulates after axotomy and
inactivity, then diminishes again as nerve returns and regenerates (Hassan et
al. 1994; Sala et al. 1995; Tarabal et al. 1996; Meunier et al. 1996). These
properties seem to fit into the model seamlessly as the functionality of L-type
VSCC also diminishes as NMJs develop, increases and then diminishes as
nerve regenerates (Sugiura and Ko 1997). This study however, showed that
rather than inhibiting, exogenous application of CGRP did not affect synaptic
transmission in developing NMJs. On the other hand, exogenous SP showed a
dramatic reduction in synaptic transmission in regenerating frog NMJs,
suggesting SP could well be a neuropeptide which activates the inhibitory G-
protein in frogs. Since no SP immunoreactivity has been shown in the motor
nerve terminals in mammals (Andreose et al. 1994), the neuropeptide
responsible for G-protein activation in newborn rats remains in obscurity.
However, it is certain that such G-protein activation pathway exists assuming
high frequency stimulation effectively removed neuropeptides from nerve
terminals (Sala et al. 1995). The high frequency study here supports this
notion since the potentiation by L-type VSCC blockers did not occur after high
33
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frequency stimulation of the motor nerve and removal of neuropeptide
containing solution.
In addressing the relationship between L-type and P/Q-type VSCCs,
the choice of SNX-230 as the P/Q-type VSCC blocker injected into neonatal
rats was improper. The goal was to have just enough P/Q-type VSCC blocker
to hinder local transmitter release and examine its influence on L-type VSCCs.
However, the steep dose response curve o f SNX-230 as reported by Bowersox
et. al (1995) raises a challenge to balance this act. Identifying a P/Q-type
VSCC blocker that can produce a localized and long lasting blockade will be a
key to facilitate the study of this relationship in the early stages of
development.
Activity and L-type VSCC Functionality
Muscle inactivity induced by TTX blockade of sciatic nerve did not
have any apparent effects on the absence of L-type VSCC functionality in
adult rats, suggesting that muscle inactivity alone is not the criterion for the
presence of L-type VSCC functionality. Nonetheless, one should be cautious
about this interpretation because the degree of inactivity might not have
reached absolute certainty. Even though there was a lack of muscle activity
and response as assessed by stimulation or transection above the nerve cuff on
the sciatic nerve, it might not be fully representative and indicative of a
complete blockade of activity under TTX treatment. In fact, there exists the
potential that a small portion of axons of the sciatic nerve bundle might still be
active below or above threshold. Such activity level was either too small to
observe or the motor units recruited were minimal, respectively. Furthermore,
this experimental design did not address and discriminate the small activity via
34
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MEPPs on the NMJs, as distal nerve stems continue to release
neurotransmitters spontaneously.
Aside from the above concerns and assume that the activity block
obtained was truly complete, the disparity in the outcomes of regeneration and
activity blockade in terms of L-type VSCC functionality hints the existence of
some messengers in axonal transport critical to L-type VSCC activity. The
rationale is that axonal transport was interrupted initially during nerve crush
while it was maintained in the TTX blockade. These messengers might
suppress or deactivate the inhibitory pathway of L-type VSCC in a normal and
robust synapse, but an interruption in their transport results in the activation of
the inhibitory pathway to impose a self-limiting feature on transmitter release.
This makes logical sense as an interruption in axonal transport may well signal
trauma suffered on the distal nerve end, and that regeneration is required. By
activating the inhibition mechanism, regenerating nerve terminals can now
better preserve neurotransmitters until the link reconnects gradually.
Future Directions
The most interesting part of this study is in the functionality of L-type
VSCCs in developing and regenerating NMJs and the relationship between
activity and plasticity. So far there is no concrete evidence that the existence
of L-type VSCCs is activity dependent, but it is worthwhile for continued
effort because it can be used as a simple plasticity model. The fact that the
existence of L-type VSCC functionality is age and developmental dependent
provides another angle in the study of synapse elimination. Specifically, the
age dependency of the effects of L-type VSCC blockers parallels to the
timeline of synapse elimination in the first two weeks of development and
regeneration, there is a large potential that the two processes correlate and
35
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compliment each other. Traditional studies on synapse elimination have
focused on trophic agents and competition alone, L-type VSCCs could now
shed new lights on them.
Besides the presynaptic nerve terminals and receptors, other
components o f the NMJ also show signs of dynamics, and they deserve further
studies as well. ECM, postsynaptic junctional folds, and notably, PSCs all
show signs of transformation throughout synaptic development. K .o and Qiang
(1997) recently discovered PSCs actually lead nerve terminal sprouts during
nerve regeneration in the frog. This is an important breakthrough in the sense
that the once believed support cells can modify synapse formation and
synaptic structure, a big role indeed!
36
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Asset Metadata

Creator Li, Chuck Zhuo-Kang (author)

Core Title Dynamics of the newly formed neuromuscular synapse

School School of Engineering

Degree Master of Science

Degree Program Biomedical Engineering

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biology, animal physiology,biology, neuroscience,engineering, biomedical,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor [illegible] (committee chair), [illegible] (committee member), D'Argenio, David (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-19113

Unique identifier UC11337933

Identifier 1389989.pdf (filename),usctheses-c16-19113 (legacy record id)

Legacy Identifier 1389989.pdf

Dmrecord 19113

Document Type Thesis

Rights Li, Chuck Zhuo-Kang

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus, Los Angeles, California 90089, USA