The Effects of Facilitatory Drugs at the Skeletal Neuromuscular Junction Using Intracellular End-Plate Recordings

Home , Ambenonium chloride, Edrophonium, Neostigmine

Loyola University Chicago Loyola eCommons

Dissertations Theses and Dissertations

1969

Daryl Dean Christ Loyola University Chicago

Follow this and additional works at: https://ecommons.luc.edu/luc_diss

Part of the Medicine and Health Sciences Commons

Recommended Citation Christ, Daryl Dean, "The Effects of Facilitatory Drugs at the Skeletal Neuromuscular Junction Using Intracellular End-Plate Recordings" (1969). Dissertations. 975. https://ecommons.luc.edu/luc_diss/975

This Dissertation is brought to you for free and open access by the Theses and Dissertations at Loyola eCommons. It has been accepted for inclusion in Dissertations by an authorized administrator of Loyola eCommons. For more information, please contact [email protected].

AT THE SKELETAL NEUROMUSCULAR JUNCTION

USING INTBACELLULAH END-PLATE RECORDINGS

.A thesis presented by I DARYL DEAN CHRJfJ'f I for the degree of I IOCTOR OF PHILOSOPHY at the

DEPAHTMENT OF PHARMACOLOGY

LOYOLA. Ul\lIVERSITY STlUTCH SCHOOL OF MEDICINE

February, 1969

LIBRARY LOYOLA UNIVliRSITY MEDICAL CENTER 2

ABSTRACT

The effects of facilitatory drugs (neostigmine, edrophonium, amben-

~: , ' onium and methoxyambenonium) at the skeletal neuromuscular junction have been studied using intracellular microelectrodes in the isolated tenuissimus muscle of the cat. These drugs produced small changes in miniature end-plate potential (m. e. p. p.) frequency. The m. e. p. p. s were prolonged by neostigmine, ambenonium and methoxyambenonium, but not by edrophonium. High concentrations of neostigmine and edrophonium depressed the m. e. p. p. amplitude concurrent with a decrease in the resting mc.i..n.b!'ane potential. High c-0nccntr:::1.tions cf ::>cmb•=""'0l"li 11 rn ~ni4 methoxyambenonium depressed the m. e. p. p. amplitude with no change in the resting membrane potential.

The amplitude of (+)-tubocurarine end-plate potentials (e. p. p. s) was increased without change in time-course by all four drugs at a conc~ntra: tion which had no effect on m. e. p. p. s. The half-decay of (+)-tubocurar.i.ne e. p. p. s was prolonged at the same concentration which prolonged the m. e. p. p. s. On the other hand the facilitatory drugs produced nearly equivalent changes in (+)-tubocurarine e. p. p. s and iontophoretic acetylcholine-potentials. The drugs did not increase the quanta! release in

.. ma aa - 3

deficient calcium and high magnesium ion concentrations. Benzoquinon ium e. p. p. s were not affected by the facilitatory drugs. These results are discussed in conjunction with the current theories of neuromuscular pharmacology. 4

LIFE

Daryl Deq.n Christ was born in Buffalo Center; Iowa on November 3,

1942. He graduated from Lakota Consol~dated High School, Lakota, Iowa in 1960. He attepcied Wartb~rg Colleg~~ Waverly, Iowa and gra.

In September, 1964, the author becante a graduate student i.n the

Department of Pharmacology, Loyola University Stritch School of Meai ci,11e. Tn Aupst, 1966 he was married to Bonnie Jean:. Grabow. During the g:radu~te program he has been a grad11ate assistant and a trainee Grf the National In,titu.te of Health. 5

ACKNOWLEDGEMENTS

I wish to express my utmost appreciation to Dr. L. C. Blaber for his inspiration, guidance and extreme patience, thereby making this disser tation possible. I am also indebted to many others for their moral support.

Dr. John Goode

Robert Jacobs

Joel Gallagher

Bonnie Christ

Mr. and Mrs. George Christ 6

ABBR.EVIA TIONS

A Ch acetylcholine amp ampere Bz benzoquinonium oc · degrees centigrade ChAc choline acetyl transferase 3-CH3-PTMA · 3-methylphenyltrimethylammonium cm centimeter DFP diisopropylphosphofluorodate e. p. end-plate e. p. p. end-plate potential EXP experiment hr. hour I. D. inside diameter kg kilogram m quantal content M molar m. e. p. p. . miniature enJ-plate polential mg milligram pg microgram min minute ml milliliter mm millimeter mM millimolar msec millisecond psec microsecond mV millivolt N normal O.D. outside diameter 3-0H-PTEA 3-hydroxylphenyltriethylammonium 3-0H-PTMA 3-hydroxylphenyltrimethylammonium p.a.p. post-activation potentiation PTEA phenyltriethylammonium PTMA phenyltrimethylammonium p. t. p. post-tetanic potentiation sec second Tc ( + )-tubocurarine TEA tetraethylammonium TEPP tetraethylpyrophosphate 7

TABLE OF CONTENTS

INTRODUCTION • • . • . · .

Anatomy of the neuromuscular junction 9

Physiology of the neuromuscular junction . . . 11

Drug-induced facilitation of neuromuscular transmission . 28

METHODS . 48

RESULTS...... -. .

Section 1 - Effects of facilitatory drugs on m. e. p. p. s and (+)-tubocurarine e. p. p. s ...... • . . . . 63

Section 2 - Effects of facilitatory drugs on (+)-tubocurarine e. p. p. s and iontophoretic acetylcholine and carbachol potentials ...... • . . 87

Section 3 - Effects of facilitatory drugs on quantal content of the e. p. p...... 101

Section 4 - Effects of benzoquinonium at thP neuromuscular junction ...... 110

GENERAL DISCUSSION 129

BIBLIOGRAPHY . . .• 149 8

INTRODUCTION

I \ \ ~ ------~------~------':"""""'----. 9

~~tomy of the neuromuscular junction

Motor nerve axons originate in the ventral root of the spinal. cord and

extend to the periphery where they make synaptic contact with skeletal

muscle fibers (Sherrington, 1925 and Tiegs, 1932). The motor nerve

axon is a large diameter neuron surrounded by layers of myelin, produced

by an associated Schwann ·cell {Geren, 1954 and Robertson, 1955). Because

a single motor nerve branches in the nerve trunk (Adrian, 1925) and near

the muscle fiber, it can innervate between 100 and 200 muscle fibers

(Fulton, 1926 and Clark, 1931). The nerve, together with its muscle fibers~.

is called a 'motor unit' (Sherrington, 1925). The muscle fibers of a motor

unit are arranged in a longitudinal or chain-like manner (Adrian, 1925 and

Cooper, 1929).

Until the development of electronmicroscopy, there was much debate

concerning the anatomy of the neuromuscular junction. The nerve loses

its myelin sheath at the terminal. The nerve terminal, which has a

diameter of 2 to 3 microns lies in a synaptic trough on the surface of the ! \ muscle fiber (Anderson-Cede.rgren, 1959 and DeHarven and Coers, 1959).

A Schwann cell always encloses the surface of the nerve terminal away

from the synapse (Birks, :fluxley and Katz, 1960). The axoplasm does not

make cont;l.ct with the sarcoplasm (Reger, 1954) but is separated by a

five-layered membrane, the synaptolemma. The synaptolemma is com- 10 posed of neurilemma and sarcolemma which combine in the area of the

neuromuscular junction (Robertson, 1954; Couteaux, 1958 and Reger, 1958).

The nerve terminal and muscle end-plate (e. p.) are separated by a primary

synaptic cleft which is 200 Angstrom units wide {Palay, 1958). In the

region of the nerve terminal the muscle membrane is thrown into a series of junctional folds- -secondary synaptic clefts (Pa.lade and Palay, 1954;

Reger, 1954 and -Robertson, 1954). They are approximately one micron long and one-tenth micron wide, being narrower toward the axonal surface

(Robertson, 1956). The junctional folds probably are the "subneural apparatus" which is so prominent "in Couteaux's (1955) light microscope

::=ections.

The axoplasm which doe$ not dip into the junctional folds is distin guished by the presence of mitochondria {Robertson, 1956). Another distin guishing feature of the axoplasm is the occurrence of "synaptic vesicles"

(300 to 500 Angstrom units in diameter) which are concentrated near dense presynaptic membra11e areas directly opposite a junctional fold (Birks,

Huxley and Katz, 1960 and Hubbard and Kwanbunbumpen, 1968).

Birks (1966) made an unusual observation. By using acrolein as a fixing agent, rather than osmium tetraoxjde, a tubular system appeared in tt;ie nerve terminal a.xoplasm. Thus vesicles may be a fixation artifact. 11

~~_!:_ology of the neuromuscular junction

In a quiescent nerve-muscle preparation, small depolarizations of the membrane occur at the end-plate region of the muscle fiber. These miniature end-plate potentials (m. e. p. p. s)-initially called "end-plate noise" (Fatt and Katz, 1950)-have been recorded in several species, including frog (Fatt and Katz, 1950), rat (Liley, 1956a). guinea pig (Brooks,

1956b), cat (Boyd and Martin, 1956a) and human (Elmqvist, Johns and

Thesleff, 1960). Each rh. e. p. p. is due to the spontaneous release of a single quantum, which contains many molecules of transmitter (Fatt and

Katz, 1952; Liley, 1956a and Boyd and Martin, 1956a}. These quanta are r tlta>:>c:d froiu the nerve tern1inal (Fati. and Katz, 18 52), c..1-::hvi..i.~1:.. they i110.y originate from other structures (Birks, Katz and Miledi, 1960).

Upon activation of the motor nerve an action potential invades the motor nerve terminal, where it initiates the release of transmitter (Hubbard and

Schmidt, 1962 and Katz and Miledi, 1965c). In the unmyelinated terminal the action potential has a slow conduction velocity and long after-potentials. i These characteristics are typical of an unmyelinated C-fiber (\Verner, 1960a; ·

Hubbard and Schmidt, 1961; Blaber and Bowman, 1963b; Katz and Miledi, 1965

When transmitter release is measured in the presence of deficient calicum ions or excess magnesium ions, the end-plate depolarization

(end-plate potential- -e. p. p. ) becomes quite small. If the calcium ion concentration is sufficiently low, the e. p. p. a.m·plitude fluctuates considerably

with many of the e. p. p. s having exactly the same amplitude and time

·course as m. e. p. p. s. The smallest e. p. p. s which occur are essentially

m. e. p. p. s and the larger e. p. p. s are multiple m. e. p. p. s (Fatt and Katz,

1952}. The e. p. p. amplitude in deficient calcium follows a Poisson

distribution indicating an independent release of quanta from the activated

terminal (del Castillo and Katz, 1954b; Martin, 1955; Boyd and Martin;

1956b; and Liley, 1956b). On the basis of these results Liley (1956c) and

Katz (1962) concluded that a nerve stimulus merely increases the rate

of spontaneous release of transmitter.

On first analysis there appeared to be a correlation between the

amplitude of the nerve terminal depolarization and the release of trans-

mitt er. This relationship was arrived at by correlating the calculated

depolarization induced by various extracellular potassium ion concentra-

tions with the changes of m. e. p. p. frequency induced by the corresponding

I._' potassium ion concentration. Liley (1956c) stated that a 15 millivolt (mV)

depolarization produces a ten-fold increase in m. e. p. p. frequency.

According to his hypothesis, the nerve terminal action potential increases

the release of transmitter by increasing the m. e. p. p. frequency in a

logarithmic fashion. No other steps were considered necessary for

release. On the basis of Liley' s results it was possible to account for 13 all the quanta released by an action potential. Due to the logarithmic na.:. ·· ture of the release, most of the release would occur at the peak of the

action potential, when the m. e. p. p. frequency would be extremely high

(Eccles and Liley.. 1959). This theory also accounted for the observation

that depolarization of the nerve terminal decreased the amplitude of the

e. p. p. (Hubbard and w·illis, 1962b and 1968 and Vladimirova, 1964) and

hyperpolarization of the terminal increased the amplitude of the e. p. p.

(del Castillo and Katz, 1954b: Hubbard and Willis, 1962a and 1962c and

Vladimirova. 1964). On the basis of Lloyd's results {1950) in the spinal

cord, Eccles and Liley (1959) concluded that depolarization decreased the

amplitude of the terminal spike and thereby, decreased transmitter release;

while hy-perpolarization increased the amplitude of the terminal spike and

increased transmHter release.

Although this simple theory was quite attractive, data began to

accumulate indicating that transmitter release involves much more than

just depolarization of the nerve terminal (Hubbard and Schmidt, 1963;

Katz and MilecU, l965d, 1965e and 1967a and Hubbard, Jones and Landau,

1967). It has been shown that calcium ions are eosential for neurornuscular

transmission (F:-. ~, 1936; Cowan, 1940; Kuffler, 1944; del Castillo aud

Stark, 1952; Fatt and Katz, 1952; del Castillo and Katz, 1954b; Boyd and

Martin, 1956b and Liley, 1956c) and for spontaneous release of transmitter 14

(Boyd and Martin, 19 56a; Elmqvist and Feldman, 19 65a; Hubbard, 19 61 and

Hubbard,Jones and Landau, 1968a). Lowering the calcium ion concentration

decreases transmitter release with no effect on the nerve terminal action

potential (Katz and Miledi, 1965d, 1965e and 1967a). Thus Liley's theory had to be modified to include a calcium step. The exact mechanism by which calcium ions affect release of transmitter is not known. Del

Castillo and Katz (1954a and 1954c) hypothesized that calcium ,acts with an

external component of the nerve terminal (X). The complex (CaX) moves intraterminally during the terminal action potential and causes the release of a quantum of transmitter (Gage, 1967; Hubbard, Jones and Landau, 1968a;

RahamimofI, 1968 and Katz and Miledi, 1968). Raharn.imuff (1963) and D0dg~ and Rahamimoff (1967) have further hypothesized that four calcium ions are necessary to release one quantum of transmitter. This action of calcium is quite rapid and can be shown to occur even during the falling phase of the terminal action potential (Katz and Miledi, 1967a and 1967c). Calcium can be replaced in this 9.CtTun by strontium or barium ions (Elmqvist and

Feldman, 1965a and Miledi, 1966).

All the actions of calcium ions which have been previously described at the neuromuscular junction are antagonized by magnesium ions. The mag nesium ions decrease release of transmitter by competitively inhibiting the action of calcium ions (Del Castillo and Engback, 1954; Del Castillo and 15

Katz, 1954a; Huiier and Kostial, 1954; Liley, 1956c; Jenkinson, 1957;

Hubbard, 1961; Hubbard and Willis,1962c and 1968; Hubbard and Schmidt,

1963 and Hubbard,, Jones and Landau, 1968a). Magnesium ions may augment release, except their activity in this regard is much less than the activity of calcium ions ' (Hubbard, Jones and Landau, 1968a).

Some workers also involve sodium ions, directly, in the release of transmitter. rather than through any effects on the sodium conductance of the terminal action potential. Changing the sodium ion concentration has no effect on release in a normal ionic solution {Del Castillo and Katz, 1955b and Co1omo and Rahamimoff, 1968). When the muscle is immersed in tetrodotoxin, which blocks sodium conductance changes in nerve or muscle, release is normal (Elmqvist and Feldman, 1965c and Katz and Milcdi, J967h)

But in a deficient calcium ion concentration, . a decrease in the sodium ion concentration '\Vill increase m. e. p. p. frequency, augment the e. p. p. and increase the quantal content of the e. p. p. (Gage and Quastel, 1965b and

1966; Kelly, 1965; Rahamimoff and Colomo, 1967 and Colomo and Rahami moff1 1968). It has been hypothesized that sodium ions compete extrater minally with the calcium ions for the receptor (X).

Digoxin and ouabain inhibit the sodium-potassium pump by inhibiting

ATPase. They cause an inc~e::e :ease =nsmitter whlch must J. ~------~ 16

be due to a build up of sodium ions intraterminally (Birks, 1962 and 1963

and Elmqvist s.nd Feldman,1965b). Thus it has been further hypothesized

that sodium ions compete with calcium ions intraterminally, as well as

extraterrninally (Birks. and Cohen, 1965, 1968a and 1968b).

Gage and Quastel (1965a) and Parsons, Hofmann and Feigen (1965) have

involved potassium ions in the release of transmitter; although it is quite

difficult to distinguish between indirect effects on the nerve terminal

membrane potential and direct effects on transmitter release.

Chloride ions are not involved in the release of transmitter (Elm.qvist

and Feldman. 1966).

Hypoxia, stretch, increased osmotic pressure and increased

temperature all increase m. e. p. p. frequency and all, except hypoxia.,

·increase the release of transmitter. Hypoxia decreases release, and prob

_ably does so by blocking the sodium-potassium pump, thereby depolarizing

the terminal membrane (Hubbard and Loyning, 1966). Stretch, increased

osmotic pressure and increased temperature act by different mechanisms

to increase release (Fatt and Katz, 1952; Boyd and Martin, 1956a and

1956b; Furshspan, 1956; Hutter and Trautwein, 1956; Liley, 1956a;

Takeuchi, 1958b and Hubbard, Jones and Landau, 1968b). 17

Since the ·work of Dale and his co-workers, it is generally accepted th2.t

acetylcholine (ACh) is the transmitter at the neuromuscular junction.

ACh has been isolated from a neuromuscular preparation following

indirect stirnulationJDale, Feldberg and Vogt, 19~i6) and this ACh arises

from the nerve (Straughan, 1960 and Krnjevic and Mitchell, 1961).

Inj ected P.Ch mimics the effects of indirect stimulation (Brown, Dale

and Feldberg, 1936 and Brown, 1937). Also, the synthesizing and

degradating enzymes for A Ch are present (Marneymd Nachmansohn, 19311

and Nachmansohn and Machado, 1943}.

ACh is synthesized from choline and acetyl co-enzyme A in the pre.scEc.e of the enzyine, choline acetyl transfercise-ChAc (Nachmansohn •

and Machado; 1913 and Korkes, Del Campillo, Korey, Stern, Nachmansolm

and Ochoa, 1952). The site of synthesis is not established. rt has been

suggested that synthesis may take place in the terminal axoplasm

(V./hitt8ker, Michaelson and Kirkland, 1964 and Michaelson, 1967), mitochondria (Bodian, 1942 and Hebb and Smallnian, 1956) or synaptic vesicles (Hebb and Whittak2r, 19~18; Vvhittakcr, 1959 and DeRobertis,

Arnaiz, S2.lganiccff, Iraldi and Zieher, 1963). The ACh is stored in a bound form (Elliot and Henderson, 1951 and Brodkin and Elliot, 1953).

Many workers feel that the vesicles in the electronrnicrograph represent the 'bour1d for'm of ACh (DeRobertis and Bennett, 1955; Del Castillo and 18

Katz, 1955b; Macintosh, 1959 and Hubbard and Kwanbunbumpen, · 1968).

The transmitter whie:h is present in the nerve terminal can be

divided into two functionally, distinct stores- -"the available store" which

is readily releaseable, and the "unavailable store" which is not readily

releaseable. An indirect stimulus depletes the available store. This

store is rapidly refilled {Gage and. Hubbard, 1965) from the uriavailable

store. The unavailable store is comparatively, slowly filled; as it depends

on synthesis for its transmitter (Perry, 1953; Birks and Macintosh, 1957

and 1961 and Thies, 1960). The available store may be represented by the

vesicles which are located near the region of the presynaptic membrane,

while the unavailable store may be the vesicles which are further from the

membrane (Hubbard and Kwanbunbumpen, 1968). Elmqvist and Quastel

(1965} and Elmqvist (1965) estimate the total releaseable store to be

around 200, 000 quanta.

The mechanism whereby the vesicle extrudes its contents from the nerve terminal is unknown, but probably involves contact of the vesicle with the presynaptic membrane. In this way the vesicle ejects the transmitter directly into the synaptic gap (Katz, 1962; Bass and Moor~,

1966 and Hubbard, Jones and Landau, 1967). Hubbard and Kwanbunbumpen

(1968) were able to observe a few vesicles which were fused with the : .

19 terminal membrane. Once the transmitter is released, it rapidly diffuses throughout the synaptic gap (Eccles and Jaeger, 1958) and comes into contact with at least two types of receptors -- acetylcholinesterase and the junctional receptor. The neuromuscular junction contains a high concentration of the degrading enzyme for ACh, acetylcholinesterase

(Feng and Ting, 1938; Marney and Nachmansohn, 1938; Augustinsson and Nachmansohn, 1949; Koelle and Friedenwald, 1949 and Koe11e,

1950 and 1951). Most of the acetylcholinesterase occurs in the post synaptic regions; although it is present at four sites in the junction:

1) the plasma membrane of muscle covering the junctional folds,

2} the prlrnal1 y and secondary synaptic cl~fts, 3) the plasma me.mhra;ic covering the terminal '· and 4) the vesicles of the terminal (Barrnett,

1962 and 1966 and Miledi, 1964). The acetylcholinesterase catalyzes the hydrolysis of ACh to choline and acetate, (Matthes. 1930 and Stedman,

Stedman and Easson, 1932). thereby reducing the activity of the trans mitter at the cholinergic receptor by a factor of 1000 (Bacq and Brown,

H}37).

The ACh also acts at the nicotinic, cholinergic receptors {Langley,

190'/ and Bacq and Brown, 1937) which are located in the e. p. region of the muscle fiber {Nastuk, 1953a; Del Castillo and Katz, 1955a and Ki:i.tz and Miledi, 196la). As the muscle membrane is impermeable to ACh 20.

(Del Castillo and Katz, 19~Sa), the receptors must be locate.cl on the

external surface of the muscle membrane. ' When ACh· activates ~ receptor;

r . " the membrane resistance .d.ecreases (Katz; 1942 ·and Takeuchi.and Takeuchi,

-1960a), -dne to t..lle-simultaneous.-increase in-.permeability to both·sodiurn

and potassium ions~ but not to chloride ions (Del Castillo and KatzJ 1954e;

Takeuchi and Takeuchi, 1960b and Takeuchi, 1963). ACh does not carry

the current (Nastuk, 1953b and Del Castillo and Katz, 1955a). Due to this

flo\v of sodium and potassium ions, the e. p. membrane potential

approaches the e~uilibrium potential of the muscle fiber -- 10 to 20

millivolts, negative (Del Castillo and Katz, 195~e and Takeuchi and

Takeuchi., 1959). ·when the membrane potential reaches a c.ri.tieal depo-

larization--threshold--sufficient current flow is e.stablisb~o to activate

· the all or none processes of the muscle fiber (Ecclts, Katz an.ti Kufflef",

1941; Kuffler, 1942 and Fatt and Katz, 1951). The action of the tra.nsm.itter

is very sho:ti and irr1pulsive (Eccles, Katz and Kuffler, 1941) and the level

of depolarization is determ1ned by the concentration of transtnilter in the

area of the choli11ergic receptor (Eccles and .Jaeger, 1958). When the

transmitter release is complete, the concentration of transmitfor falls,

rapidly, as it is inactivated by acetylcholinesterase (Marney and Nae htnan-

sohn, 1938) or diffuses from the region of the e. p. (Ogston, 1955 and

Eccles and Jaeger, 1958). Then the e. p. p. passively decays in a logarithmic ------·;··------21

fashion, until it returns to the resting membrane potential (Eccles, Katz

and Kuffler, 1941; Katz, 1942 and F'att and Katz, 1951). Although the

e. p. is cbemically excitable, it is probably electrically inexcitable

_ . _(Werrnan,_ 1960).

Nor-mally, the e. p. p. is quite large and always initiates a muscle

action potential (KuffJ.er, 1942). In order to record a pure e. p. p., it

i.s necessary to block the initiation of the muscle action potential. This ·

can be accomplished by stimulating the nerve during a refractory state

or adrr1.·inistering (+)-tubocurarine (Gopfert and Schaefer, 1938 and Eccles

amt O'Connor, 1939 and 1941 ). In this manner it is possible to study

nearomuscular transmission by studying changes in the extracellular

or intracellular e. p. p. (Fatt and Katz, 1951; Nastuk, 1953b and Del

Castillo and Katz, 1956).

The e. p. p. follows the stimulus after a period of latency -- the

synaptic delay. The synaptic delay is not due to diffusion of transmitter

across the junction or to activation of the receptor substance. It must

be due to a delay in the release of transmitter, probably involving the

electro·-secretory coupling process (Katz and Miledi, 1965a, 1965b,

1965d, 1967a, 1967c and.1967d). 22

Stimulation of the nerve sets up conditions in the motor nerve terminal such that after the stimulus the probability of release is greater than in the contro1 state (Feng, 1940; Eccles, Katz and Kuffler, 1941 and 1942;

Hutter, 1952; Liley and North, 1953; Lundberg and Quilisch, 1953a and

1953b; Brooks ,1956a and 1956b; Liley, 1956a and 1956b and Hubbard, 1959 and 1963). This is referred to as post-activation potentiation (p.a. p.) or post ... tetanic potentiation (p. t. p.) and occurs independently of release from the conditioning impulse (Del Castillo and Katz, 1954c and Hubbard, 1963).

The quanta of transmitter (m) which a nerve impulse releases depends on the number of quanta available for release (n) and the probability that a single quahta will be released (p).

m = np (Martin, 1965)

During the period of post-activation potentiation· each quantum has a greater probability of being released (Mallart and Martin,19£7 and 1968) and under appropiate conditions will result in an increase in the quanta released and a larger e. p. p. (Lundberg and Quilisch, 1953a and 1953b;

Del Castillo and Katz, 1954c and Liley, 1956b).

There are several mechanisms by which this change of m can occur.

The nerve terminal action potential has a positive (or hyperpolarizing) after-potentia1 (Hubbard and Schmidt, 1961 and 1963) due to a prolonged increase of potassium conductance (Gage and Hubbard, 1964 and 1966a). 23

According to Liley' s theory. this hyperpolc:rization would increase the

amplitude of the succeeding terminal spikes and increase the release of

transmitter (Eccles and Liley. 1959). But there is not complete corre

lation between th~_tim~ course of effects of hyperpolarization and the

increased release (Hubbard. 1959 and 1963; Hubbard and Willis, 1962a

and 1962b and Hubbard and Schmidt. 1962 and 1963). Hubbard (1963)

observed two phases of facilitation, each due to increased release of

transmitter; and hypothesized that the early phase was due to mobilization

of transmitter to the release sites, while the secondary phase was the

result of a larger presynaptic spike from hyperpolarization. Katz and Mil

edi (1967b) observed that facilitation did not depend on a change in the terminal action potential. Gage and Hubbard {l966b) concluded that post-tetanic potentiation was :1ot due to mobilization in the terminal.

They also eliminated several other possibilities, including presynaptic

swelling. increased intracellular sodium and decreased intracellular potassium.

Changing the extracellula'r calcium ion concentration directly affects the _r:Glcase of transmitter, as described previously. Due to this pro nounced effect of calcium ion on release. it was suggested that residual

calcium intraterminally may account for facilitation {Katz and Miledi,

,' 24

1965e and Gage and Hubbard, 1966b). Katz and Miledi (1967a and 1967c)

observed that the process of transmitter release continues to operate and

even increases during the falling phase of the- terminal action potential.

Calcium utilization occurs during the depolarization of the nerve terminal

(Katz and Miledi, 1967d) and facilitation is increased only when calcium

ions are present during the conditioning stimulus (Katz and Miledi, 1968).

Rahamimoff (1968) suggested that four calcium ions act at a receptor to

release one quanta of transmitter. Facilitation would be expected to occur

if one or more calcium ions remain attached to the receptor until the

next impulse arrives.

Facilitation of the e. p. p. does not occur in all situations involving

more than one indirect stimulus. When the muscle is blocked by

(+)-tubocurarine, the second e. p. p. of mammalian preparations-arid

subsequent e. p. p. s of amphibian preparations become depressed (Eccles,

Katz and Kuffler, 1941 and 1942 and Lundberg and Quilisch, 1953a). This

depression has been shown to be due to a decreased quanta! content of the test e. p. p. {Hutter, 1952; Perry, 1953; Del Castillo and Katz, 1954c;

Takeuchi, 1958a; Thies, 1960 and 1965; Brooks and Thies, 1962 and

Elrnqvist and Quastel, 1965). Thesleff has shown some evidence for a postsynaptic desensitizing action from accumulated ACh, as a result of 25 the tetanus (Axelsson and Thesleff, 1958 and Thesleff, 1959); but these results have been directly countered by others who show no evidence for a desensitizing action during a tetanus (Otsuka and Endo, 1960a; Lilleheil and Naess, 1961 and Otsuka, Endo and Nonomura, 1962). It is possible that a tetanic depression may be due to a propagation failure in the fine nerve terminals (Krnjevic and Miledi, 1958 and 1959); but this type of failure occurs only at high frequencies of stimulation and would be quite obvious with unicellular recording techniques, if it did occur.

Evidence indicates that the decreased quantal content is due to a depletion of the available stores of ACh (Perry, 1953 and Hubbard and

Kwanbunbumpen, 1968), even though there is an increased probability of release during the depression (Elmqvist and Quastel, 1965 and Mallart and Martin, 1968). The results from the deficient calcium experiments are different as they do not involve a depletion of the available store.

Thus they show only the increased probability of release (Mallart and

Martin, 1868).

In the in vivo experiments the muscle contracts supramaximally when tetanically stimulated. The tetanic contraction reaches a plateau and be gins to relax from the supramaximal contraction. Upon cessation of the 26 tetanus, the twitch again becomes supramaximal-- p. t. p. of the twitch

(Guttman, Horton and Wilber, 1937; Rosenhlueth and Morison. 1937;

Feng. Lee, Meng and Wang. 1938 and Von Euler and Swank. 1940).

_The m1usual _aspect of this observation is the fact that the muscle can contract greater than maximally. This has been shown to occur upon direct stimulation of denervated or curarized muscles with no change in the gross muscle action potential (Brown and Von Euler. 1938 and

Von Euler and Swank. 1940). Thus the supramaximal contraction involves the active state of the muscle fibers and not the neuromuscular junction

(Hill, 1949; Macpherson and Wilkie, 1954; Ritchie and "Wilkie, 1955 and

Close and Hoh, 1968). In the frog part of the p. t. p. of the twitch can be explained by recruitment of quiescent muscle fibers (Grumback and Wilber.

1940), but this is probably due to the low safety factor of transmission in some muscle fibers of the frog (Wakabayashi and Iwasaki, 1962). This would not be expected to occur in mammals which have a high safety factor of neuromuscular transmission (Paton and Waud. 1967).

The tctanic depression is due to a decreased release of ACh. as a result of depletion. Thus many of the e. p. p. s are below threshold and the gross muscle twitch becomes smaller (Dale. Feldbert and Vogt,

1936; Brown and Von Euler. 1938 and Li and Ting, 1941). !""'.·

When the motor nerve to a skeletal muscle is cut. the muscle becomes

inactive for a period of 12 to 15 days {Hines. Thomson and Lazere, 1942).

The distal nerve degenerates and loses its ability to initiate musde activity.·

At the same time there is a decrease in the ACh and ChAc activities

(Feldberg, 1943; Bannister and Scrase. 1950 and Berry and Rossiter, 1958).

The nerve completely degenerates leaving only the Schwann cells (Birks.

Katz and Miledi, 1960). The m. e. p. p. s which had completely disappeared

shortly after sectioning the nerve. reappear after several days and must

originate from a site other than the nerve -- possibly the Schwann cell

(Birks, Katz and Miledi, 1960). During degeneration the whole muscle

fiber becomes sensitive to ACh (Luco and Eyzaguirre. 1955; Axelsson

and Thesleff, 1959; Miledi, 1959 and 1960a; Frank. 1959; Jenkinson. 1960

and Katz and Miledi, l96lb) and there is a decrease in the acetylcholin

esterase concentration around the original e. p. (Snell and Mcintyre, 1955

and Frank. 1959).

The distal stump of the regenerating neuron contains high concentration ;

of acetylcholinesterase and choline acetyl transferase, indicating that

these enzymes are produced by the nerve cell body (Sawyer, 1946; Hebb

and Waites, 1956 and Hebb and Silver, 1961). The regenerating stump 28 approaches the muscle fiber and induces the formation of an e. p. Also, there is a sensitivity decrease to ACh at the non-e. p. regions of the

t muscle ·and an increase in the acetylcholinesterase concentration at the e...p. .It has been concluded that this effect of the nerve on the .muscle is due to a neural factor (probably not ACh) which is released and can induce profound changes in the muscle fiber (Buller, Eccles and Eccles, 1960a and 1960b and Miledi, 1960b and 1962). 29

Drug-induced facilitation of neuromuscular transmission

Facilitation is defined as an increase in the twitch tension of an

indirectly stimulated skeletal muscle or antagonism of (+)-tubocurarine paralysis. Drugs which produce facilitation are termed 'facilitatory

drugs'. The majority of these drugs have been shown to produce some inhibition of cholinesterase.

As early as 1900, it was shown that physostigmine is capable of antag onizing neuromuscular paralysis induced by curare and this observation has since been reported many times (Pal, 1900; Rothberger, 1901;

Rosenblueth, Lindsley and Morison, 1936 and Koppanyi and Vivino, 1944).

Also, physostigmine can facilitate the maximal indirect twitch of the cat gastrocnemius muscle. It has no effect on nerve conduction and is ineffective when the muscle is stimulated directly in a curarized or denervated preparation. The facilitation is associated with repetitive action potentials in the muscle fibers, which occur at intervals near the absolute refractory period of the muscle fiber. This repetition acts as a tetanus and induces a tetanic-like contraction of the muscle fiber, resulting in facilitation of the muscle contraction (Brown, Dale and

Feldberg, 1936 and Brown, 1937). ACh, administered close-arterially, also elicits a brief, asynchronous tetanus. Thus the drug-induced repetitive firing is probably due to maintenance of transmitter 30 at the muscle e. p. As physostigmine is well known for its ability to prolong the action of ACh by inhibition of cholinesterase and as it also slightly increases' the ACh contraction(Englehart and. Loewi, 1930; Matthes,

1930 and Bacq and Brown, 1937), it seemed reasonable for the early workers to conclude that physostigmine maintains transmitter levels at the e. p. by inhibiting junctional cholinesterase.

Later studies using extracellular recording techniques in frog and cat muscle, have shown that tubocurarine diminishes the a.mplitude of the e. p. p. by competing with the transmitter at the cholinergic receptor

(.Jenki.rnrnn, 1960) but h:Js no effect on the time-course of the e. p. p.

·when the e. p. p. is decreased beJ.ow threshold by tubocurarine, it is poss1.bh: to rec.ord a pure e. p. p. with no distortion from muscle action potentials. Aclr..:1inistration of physostigmine or neostigmine inereases the amplitude and duration of the curarized e. p. p. (Feng, 1940; Eccles,

Katz and Kuffler, 1942 and Eccles and MacFarlane, 1949). Physostigmine a.lso prolong:.:> the active phase of the e. p. p. by a factor of two or three.

Thus the t:r:ansrnittcr is acting for a longer period of time at the e. p. when pbysostigmine is present (Katz, 1942).

In a non-curarized preparation (refractory) physostigmine increases ~·

------• 31

the amplitude and time-course of the e. p. p. When the e. p. p. is longer

than the refractory period of the muscle fiber, repetitive discharges are

initiated in the muscle fiber. Even in the presence of physostigmine the

fast e. p. p. is quite rapid, indicating that a physostigmine-resistant

mechanism of halting the action of the transmitter is occurring-probably

diffusion.

These results on the fast e. p. p. are compatible, and indeed broaden,

the theory of cholinesterase inhibition. Thus it was hypothesized that

physostigmine and neostigmine by inhibiting cholinesterase prolong the

e. p. p .• such that repetitive activity is induced in the muscle. The

repetitive activity gives rise to a tetanic muscle contraction.

Physostigmine also sets up a long secondary negative potential

change. The secondary potential has a maximal amplitude of 40% of the

spike height and it may persist for a second or more. This wave is

readily abolished by sub paralytic doses of curare which have little effect

on the fast e. p. p. As these effects occur with no change in the muscle

membrane electric time constant, and as tubocurarine has no effect on

cholinesterase inhibition in vitro, it is difficult to explain this action of

tubocurarine (Eccles. Katz and Kuffler, 1942). 32

Although most of the previous results were compatible with the theory that facilitatory drugs acted solely by inhibiting cholinesterase, n(me of the experiments provided conclusive evidence that prolongation of trans mitter action was due to cholinesterase inhibition. Thus many workers searched for evidence which would establish that cholinesterase activity at the neuromuscular junction was. decreased during the action of the facilitatory drugs. The most common study involved correlating inhibition of cholinesterase (such as, serum cholinesterase or electric eel cholin esterase) with the anticurare potency of these drugs (Bacq and Brown,

1937; EccJes, Katz and Kuffler, 1942; Bulbring and Chou, 19·17;

Blascho, Bulbring and Chou, 1949; Randall and Lehmann, 1950; Hobbigcr,

1952 and Karc~mar and Howard, 1955) The results of these experiments were quite conflicting, and the probable reason for the discrep<-.ncies was brought Otit in a statement by Augustinsson (1959), "Plasma cholinesterases arc regarded as a group of esterases with nrnch divergent properties with regard to specificity against various choline and non-choline esters, se:v:1sitivity to inhibitors and activity-substrate relationships. · · ·It is not possible to apply results obtained with the plasma of one species to any other species." This statement was well illustrated with muscle cholinesterases by Blaber and Bowman (1962). They reported that ambenoniurn wcis 30 times as potent as neostigrnine in inhibiting cat muscle 33 cholinesterase, but 1/200 as potent as neostigmine in inhibiting hen muscle cholinesterase. Thus conclusive experiments must involve a study of the acetylcholinesterase of the neuromuscular junction and facilitatory or anticurare.aGtion of a drug at a neuromuscular junction of the same species.

If cholinesterase is previously inhibited, any drug which acts by inhibiting cholinesterase should be ineffective. Hobbiger (1952) observed that previous inhibition of cholinesterase blocked the anticurare action of the facilitatory drugs. Neostigmine, applied iontophoretically, blocked pote!ltiation of the ACh-potential by edrophonium (Katz and

Thesleff, 1957a}. Koelle (1957), using the observation that physostigmine pretreatment (Koster, 1946) or neostigmine pretreatment (Koelle, 1946) protected cholinesterase from irreversible inhibition by DFP, observed by histochemical methods that ambenonium and methoxyambenonium in concentrations, which were pharmacologically active, protected the junctional cholinesterase from DFP inhibition. These results indicated that ambenonium and methoxyambenonium were inhibiting junctional cholinesterase when they antagonized curare; but as stated by Koelle (1957) this does not show that cholinesterase inhibition was the important mechanism of action. 34

The facilitatory drugs cause not only potentiation of the neuromuscular

~. -· - . twitch, but also depression at high doses (Bacq and Brown, 1937; Rosen- blueth and".l\1orison, 1937;· Van Der Meer and Meeter, ·r956b arid Riker,

--Roberts, Standaert and Fujimori, .1957) .. In toxicity .studies-this dep:r·ession

appeared to be due to cholinesterase inhibition {Bodansky and Mazur, 1946 ·

and Nachmansohn and Field, 1947). TEPP, tetraethylpyrophosphate, cause

a depolarization of the e. p. at high doses, but was not capable of eliciting

a twitch ~t any dose level. Thus Douglas and Paton 0 9 54) concluded that

TEPP induced depression of the e. p. by inhibiting junctional cholinesterase.

Yet many workers have shown that total inhibition of cholinesterase did not block the indirecrr.vitch (Berry and·Ev:ons, H!~l; Barnes and Duff, 1953;

Barstad, 1956b and Van Der Meer and Meeter, 1956a). Douglas and

Matthews (1952) observed that TEPP blockade was readily reversible, even though TEPP was an irreversible cholinesterase inhibitor. On the basis of this type of data Barstad (19 56a) concluded that DFP, in addition to its cholinesterase inhibiting properties, also exhibited a curare-like adion at the e. p.

The theory of cholinesterase inhibition by facilitatory drugs was generally accepted until 1946. Then Riker and Wescoe (1946) observed that neostigmine produced a contraction of skeletal muscle, even though the acetylcholine.sterase was previously inhibited by DFP- - 35

diisopropylphosphofl.uorddatc;, a very potent. irreversible cholinesterase

inhibitor (Mazur and Bodansky. 1946; Mackworth and Webb, 1948 and

Michel and Krop. 1951). They concluded the primary effect of neostigmine

. ~was a direct, depolarizing action (like ACh) at the e. p. and did not

involve cholinesterase inhipition. Several analogues of neostigmine.

including 3-hydroxy phenyltrimethylammonium (3-0H-PTMA). which

facilitated the neuromuscular twitch and antagonized tubocurarine block

ade of the twitch. also stimulated denervated muscle. As 3-0H-PTMA

lacked the carbamate grouping which was responsible for cholinesterase

inhibition {Stedman. 1926). Riker concluded these compounds acted via a

direct, stimulatory action and that other facilitatory drugs had the same

mechanism of action (Randall, 1950; Randall and Lehmann, 1950 and

Riker and Wesco<:,1950).

Edrophonium {3-hydroxy phenyldimethylethylammonium) was more

specific than neostigmine in its anticurare action at the neuromuscular

jundion--it had fewer cholinergic side effects. Randall and Jampolsky

(1953) hypothesi?.ed that neostigmine acted by cholinesterase inhibition,

while edrophonium acted by a direct, stimulatory action on skeletal muscle.

Smith, Cohen, Pelikan and Unna {1952) observed that neostigmine,

physostigmine and edrophonium exhibited direct stimulatory effects on the

----·~· ____ ..______,_,,_.. 36

frog rectus preparation which were 1/ 560, 1/ 75, 000 and 1/216 as effective. --·- respectively. as ACh. These drugs were at least one-thousandtih1.es as

effective in potentiating an·ACh contracture. as in producing a direct,

. stimulatory effect. Furthermore there_ was no correlation between the

stimulatory effect and the potentiation of ACh contractures. They conclude9

that the effects of the facilitatory drugs on the frog rectus could not be

due to a direct, stimulatory actiori.

Edrophonium did not depolarize the muscle e. p. except at concen

trations which were much higher than those necessary to produce

facilitation of the muscle twitch in the frog (Nastuk and Alexander, 1954).

Katz and Thesleff (1957a}, using microelectrode iontophoretic

techniques, applied A Ch to a frog muscle e. p. If minimal doses of

edrophonium were applied, the ACh-potential was augmented. This same

dose of edrophonium, alone, produced no depolarization. Only at much

highc.t" doses was edrophonium cap2.ble of depolarizing the e. p. Also,

ech ophonium did not potentiate a depolarization due to carbachol { a

stable choEne ester}, as would be expected if edrophonium was acting

by a direct, depolarizing action. These results could only be explained by

t.be theory that edrophonium inhibited cholinesterase. Although it was not

possible to determine what concentration of edrophonium was necessary 37

to inhibit cholinesterase, these results were the first to show conclusively

that acetylcholinesterase inhibition at the neuromuscular junction .

produced a physiological effect- -prolongation of the action of A Ch.

Randall (1950) and Riker (1953) suggested that displacement of tubo-

curarine from the e. p. receptor could explain the antagonistic action of

edrophonium. Yet when tubocurarine was displaced from the receptor

by rinsing the muscle in non-curarizedRinger, the amplitude of thee. p. p.

was augmented with no change in time-course. Edrophonium increased

both the amplitude and time course of the e. p. p. (Nastuk and Alexander,

1954).

Succinylcholine, like decamethonium, acted at the mammalian neuro-

muscular junction by depolarizing the muscle e. p. (Burns and Paton,

Initially this depolarization increased the excitability of the

muscle, such that facilitation of the muscle twitch occurred. After

prolonged depolarization, there was a secondary depression of the

muscle twitch (Zaimis, 1951). Methoxyambenonium was capable of

antagonizing depolarization block by succinylcholine and decamethonium

(Karczmar, 1957) and competitive block by tubocurarine (Lands, Karczmar,

Howard and Arnold, 1955). These results seemed to contradict the

. ·classical theory of neuromuscular pharmacology- -blocking drugs must

act by depolarization or competition, and depolarizers and competitors 38 are mutually antagonistic. In. addition pretreatment with methoxyambenoff:- ium increased the succinylcholine facilitation, but decreased the secondary

, . . blockade. Karczmar (1957 and 1961) stated that the underlying mechanism of neuromuscular facilitation was more complex than originally thought and "may depend on more sites of action. "

ACh was known to have a desensitizing action in isolated amphibian preparations (Thesleff, 1955a, 1955b and 1958 and Axelsson and Thesleff,

1958) and methoxyambenonium readily restored the sensitivity of muscles which had been desensitized by ACh (Kim and Karczmar, 1967}.

Thus a sensitizing action which had been hypothesized for other facilitatory drugs (zaimis, 1951; Cohen and Posthumus, 1955 and Van Der Meer and Meeter, 1956b} was hypothesized by Karczmar, Kim and Koketsu

(1961) to be involved in the mechanism of action of methoxyambenonium.

This theory was countered by other evidence. Edrophonium was not capable of potentiating the depolarization from an iontophoretic application of carbachol (Katz and Thesleff, 1957a). Blaber (1960) observed that· methoxyambenonium and ambenonium exhibited curarimimetic properties at doses higher than were necessary to antagonize tubocurarine. Thus it was concluded that antagonism to depolarizing block was due to this 39 curarimimetic activity and not due to sensitization. This was further supported by the observation that succinylcholine blockade was an,tagonized by appropriate mixtures of neostigmine and tubocurarine (Blaber and

Karczmar,-'1967a and1967b).

Masland and Wigton (1940) investigated the origin of spontaneous twitching which occurred after administration of facilitatory drugs (Langley and Kato, 1915). Upon administration of neostigmine, spontaneous antidromic action potentials and antidromic potentials after an indirect stimulus were recorded in the ventral root of the cat spinal cord. Masland and Wigton concluded that the antidromic impulses originated in the region of the motor n.e:rve endings. As ACh also produced the spontaneous activity in the nerve it seemed likely that neostigmine acted by causing an accumulation of ACh at the neuromuscular junction, thus allowing ACh to act at the nerve endings (Eccles, 1964 and Hubbard, 1965).

Physostigmine also produced antidromic activity in the cat. Feng and Li (19,11) co:1clnded that physostigmine acted directly on the presynaptic nerve endings in the mammalian preparation. Yet there was not complete correlation between repetitive firing of the nerve and repditive firing of the muscle. Due to this inconsistancy, it seemed likely that the nerve terminal was not the site of drug action, but was 40

merely stimulated by the prolonged e. P~ current in the presence of physostigmine (Eccles, Katz and Kuffler, 1942). Antidromic activity was obvious in the nerve following direct stimulation of the muscle.

After indirect stimulation the repetitive activity of the muscle appeared to occur earlier than the repetitive activity in the nerve. Also, antidromic activity occurred in.nerves which were not stimulated and was· decreased when the, nerve stimulus was desynchronized. These results indicated that muscle action potentials were capable of depolarizing the nerve terminals, such that antidromic activity was initiated. This depolarization· of the nerve terminal in turn induced repetitive activity in the muscle, resultjng in a tetanic-like twitch (Lloyd, 1941 and 1942 and Brown and

Matthews, 1960). According to these theories all the effects of physostigmine can be explained on the basis of in'hibition of junctionaJ cholinesterase.

Barstad (1962} observed that antidromic nerve discharges could be elicited t:ven though the muscle fibers were cut and could not respond to a stimulus. Upon more careful examination, two types of antid~omic

11crve activity were observed by Werner (1960a, · 1960b and 1961) in the presence of the facilitatory drugs. The early phase had a very short latency and was due to the ephaptic backfiring from the muscle action potentials as described by Lloyd and Brown and Matthews. But there was also a secondary phase of antidromic activity which had a longer latency.

This secondary phase could only be explained by the theory that a long generator. potential occurred in the .motor -nerve terminal (Werner, 19 60b).

This generator potential probably was the negative afterpotential of the nerve terminal spike. Thus paired stimuli with a short interval increased the antidromic activity in the nerve and paired sUmuli of a long interval depressed antidromic activity in the nerve (Werner, 1960a and Blaber and Bowman, 1963b}. This theory was supported by the observation that antidromic activity which occurred in unstimulated nerve fibers had a short latency. The secondary activity, which had a longer latenc:y, occurred only in nerve fibers which had been stimulated ·(Werner, 1961).

There was one discrepancy in these results. Werner (1960a) by using paired stimuli concluded that the negative afterpotential of the motor nerve terminal had a duration of only 5 msec. Yet the antidromic activity after an orthodromic stimulus in the presence of a facilitatory drug had a much longer duration; thus Blaber and Bowman (1963b) concluded that the negative afterpotential was also longer. The short duration observed by \Verner (1960a) was probably due to extinction of orthodromic stimuli by antidromic stimuli (Blaber and Bowman, 1963a}. Hubbard, Schmidt and> ' Yokota (1965) also observed a longer duration for the negative 42

afterpotential of the motor nerve terminal.

Pentobarbital, procaine and cyclopropane blocked antidromic nerve

activity induced by 3-0H-PTEA (3 hydroxyphenyltriethyla...mmonium), but

these anesthetics had no effects on the facilitation of neuromuscular twitch

which was produced by 3-0H-PTEA {Riker, Werner, Roberts and

Kuperman, 1959). Thus the nerve terminal generator was distinguished

from the muscle generator of repetitive activity. Tubocurarine blocked

the nerve terminal generator potential at lower doses than it blocked

the ephaptic activity. On the basis of these results it was concluded

that the safety factor for axonal excitation by the terminal generator was

narrow·er than the safety factor for transmission to postSynaptic units

(Werner, 1959 and 1961).

The facilitatory drugs produced facilitation of the mammalian

neuromuscular twitch, rather easily. The amphibian muscle was much

less respon_sive to the actions of these drugs (Raventos, 1937), although

it was possible to produce facilitation of the amphibian muscle twitch

with physostigmine or edrophonium, when a stimulus rate no greater

than one per minute was used {Feng, 1937; Hodes and Steiman, 1939

·and Nastuk and Alexander, 1954). This difficulty in obtaining facilitation 43 was paralleled by a lack of antidromic activity in the presence of the facilitatory drugs (Dun and Feng, 1940). These results may point to a fundamental difference in the pharmacological action of the facilitatory drugs at the mammalian as compared to the· amphibian neuromuscular junction {Blaber and Karczmar, 1967b).

Riker and co-workers (1957) began an investigation of the nerve terminal activity of a series of phenolic compounds. These compounds · wer~ structurally similar to neostigmine. except they were considerably less potent than neostigmine in inhibiting acetylcholinesterase. 3-0H-P'IMA ·. facilitated the neuromuscular twitch at low doses and blocked the neuro muscular twitch at high doses. When the hydroxyl group was eliminated, as in PTMA (phenyltrimethylammonium) or PTEA {phenyltriethylammonium , the ability to facilitate the twitch was lost. As 3-CH3-PTMA (3 methyl phenyltrimethylammonium) was ineffective. the ability to facilitate the twitch seemed to require the presence of a hydroxyl or carbamyl grouping in the meta position. The meta grouping was also necessary to initiate antidromic activity in the motor nerve. On the basis of these results

Riker, Roberts, Standaert and Fujimori (1957) concluded that

3-0H PTMA and its analogues which also facilitated the twitch produced their facilitatory effects by an action on the motor nerve terminal

(Kuperman, Gill and Riker, 1961). As the ethyl analogues showed 44

no depression, they further concluded that the depressing activity was due

to depolarization and was related to the number of methyl groups on the

quaternary nitrogen' ' (Randall, 19 50 and Smith, Cohen, Pelikan and Unna,

1952).

Edrophonium, ambenonium and neostigmine facilitated the neuro-

muscular twitch and induced antidromic activity in the ventral root.

Mcthoxyambenonium depressed the sensitivity of the e. p. and nerve termina

and probably, for this reason could not facilitate the muscle twitch or

induce antidromic nerve activity (Blaber, 1960). Goode, Blaber and

Karczmar (1965), using Koelle's (1957) technique, observed that it was

possible to facilitate neuromuscular transmission with doses of edrophon-

ium and neostigmine which did not protect cholinesterase from inhibition

by DFP. This may indicate another mechanism of action for these drugs

although such a conclusion, at least with edrophonium was not completely

justified as kinetic studies have shown that inhibition of cholinesterase by

edrophonium was very rapidly reversed, and therefore could not be

expected to protect from DFP (Wilson, 1955 and Katz and Thesleff, 1957a).

Intravenous edrophonium and close-arterial edrophonium and

neo~~tigmine facilitated the neuromuscular twitch but did not increase the

contracture from close-arterially injected ACh. Methoxyambenonium 45

(intravenously or close-arterially) antagonized tubocurarine but did not

augment the ACh-contracture. If the facilitatory d:rugs acted by inhibiting

junctional cholinesterase, the ACh-contracture and indirect muscle twitch

should be increased at the same dose. Furthermore, there was rto corre-

lation between facilitation or anticurare action• and the cholinesterase inhibiting acUvity of these drugs. On the basis of these results Blaber

and Bowman (1959 and 1963a) and Blaber (1960 and 1963) concluded that the

facilitatory drugs produced their effects by a direct action on the motor nerve terminal.

The development of unicellular recording techniques opened a new realm in which to study the effects of drugs at the neuromuscular junction.

Fatt and Katz (1950 and 1952) in their early experiments in the frog observed that neostigmine increased the amplitude and half-decay time, but not the frequency of m. e. p. p. s. As hypothesized by Katz (1958 and

1962) experimental changes of m. e. p. p. time-course involve a postsy- napUc rnechanism while experimental changes of m. e. p. p. frequency involve a presynaptic mechanism. Liley (1956a) reported similar observations vvith neostigmine in the rat diaphragm, but Boyd and Martin (1956a) observed small increases in the frequency of m. e. p. p. s in the cat tenuissimus. 46

Neostigmine augmented the amplitude of a curarized e. p. p. (Fatt and Katz, 1951 and Boyd and Martin, 1956b) but produced relatively small changes in the time-course. Neostigmine greatly prolonged the time-course of an e. p. p. which was recorded in a ringer solution that had

20% of the control sodium ion concentration (Fatt and Katz, 1951). IncreasN of the e. p. p. were accompanied by proportional increases in e. p. current

(Takeuchi and Takeuchi, 1959).

Several drugs have been shown to produce effects on the e. p. p. which are very similar to those reported for neostigmine, but these drugs do not act by inhibiting junctional cholinesterase. TEA(Stovner, 1958 and

Koketsu, 1958), guanidine (Otsuka and Endo, 1960b) and phenol (Otsuka and

Nonomura, 1963) have been s_hown to increase the release of transmitter.

Thus the present investigation will be undertaken to try to determine the mechanism by which the facilitatory drugs produce their effects at the . neuromuscular junction. The drugs which will be used are: l)ambenonium, a very potent acetylcholinesterase inhibitor; 2) neostigmine, a classical facilitatory drug; 3) edrophonium, a potent facilitatory drug which has been reported to be a very weak acetylcholinesterase inhibitor and 4) methoxyambenonium, a potent antagonist of tubocurarine blockade that is not able to facilitate the maximal muscle twitch. It also has weak acetyl-

. ' cholinesterase inhibiting properties. 47

-CH 0-CO-N CH 3 OH I I CH- 3

I 3 yH3 -N-CH3 -'J1-CHz-CH3 CH3 0 CH3

NEOSTIGMINE EDROPHONIUM

CHz-CH3 'CHz-CH3 I I CH3-CHz-N-CHz-CHz-NH-CO-CO-NH-CHz-CHz-N-CHz-CH3 I . I CHz . CHz ~Cl ~-.Cl v 0

AMBENONIUM

CHz-CH3 CHz-CH3 I I CH3-CHz-N-CHz-CHz-NH-CO-CO-NH-CHz-CHz-N-CHz-CH3 I I CHz CHz I I ~-OCH3 ~-OC:E-~3 v 0

METHOXYAMBENONIUM 48

METHODS 49

I. Anatomy of the Tenuissirnus Muscle of the Cat

All experiments were performed on the tenuissimus muscle of the cat.

This muscle was chosen as it. is very similar pharmacologically to the gastrocnemius and tibialis muscles which have been used in in vivo studies

(MacLagen. 1962). The tenuissimus muscle originates from the trans verse process of the second caudal vertebra. It lies between the biceps femoris and semimembranous muscles in the sciatic notch and inserts on fascia of the shank (Hyman. 1962). As the tenuissimus muscle is quite long ()5 cm.) and thin (1 mm) it makes a very good isolated tissue preparation. Ringer solution can reach most of the fibers in the muscle, whiclJ. i::> :o.ot ~.i:ue of such large muscles as the tibialis o.ntcrior a;.-1d gastrocnemius. The tenuissimus nerve arises from the sciatic nerve.

Just before entering the tenuissimus muscle. the nerve trunk divides -- one branch passing anteriorly and the other branch passing posteriorly -- and enters the muscle. Each branch passes superficially down the middle of the muscle giving off nerve fibers to the muscle fibers.

It. Removal of Muscle

Cats were anesthetized with 70 mg/kg alpha-chloralose intraperitoneall .

5 mg/kg pentobarbital sodium facilitated induction and 1. 5 mg/kg atropine aided in blocking mucous secretions. The sciatic notch was 50 opened to expose the tenuissimus muscle and nerve. The tenuissi.mus nerve was dissected free from the sciatic nerve so that a segment of nerve about 3 cm long could be obtained. The muscle was then dissected free.

In general, the distal portion of the mu~cle (below the entry of the nerve) was used in the experimental procedure. The muscle was placed in a muscle bath .. Removal of connective tissue from the muscle made it possible to see the superficial nerve fibers with the aid of a microscope.

Pinning the muscle securely to the bottom of the bath eliminated extra neous movements. The ends of the muscle were then cut distal to the pins.

The nerve was placed over the bipolar platinum electrodes (5mm apart) in a separate compartment of the bath (except in m. e. p. p. studies when the nerve was not removed from the animal).

III. Perfusion of Muscle

Krebs solution was used to supcrfuse the isolated tissue. All reagents except NaCl,. D-glucose, and NaHC03 were kept in stock solutions. Thus addition of 2 ml of each of the stock solutions to a liter of distilled water gave the proper concentrations (Table 1) (Boyd and Martin, 1956a). The

NaCl, D-glucose, and NaHC03 were measured as dry weight. This solution was aerated with 95% o2 and 5% co2 through a sintered glass aerator in a 50 ml bath before passing into the isolated muscle bath. 51

Table 1

·· · -········Composition of the Kreb's solutfon superfusing

the isolated tenuissimus muscle.

NaCl 115 mM

KCl 4. 60 mM

1.15 mM KH2P04 24.1 mM NaHC0 3

Cr·d. C'l, 2 2. 46 mlVI

JVIgS04 1.15 mM

Glucose 8. 85 mM

I l~.__:·x..: ...~~~~-~·.~:,~ ...~~+-..~ .. ~~..cm:::z.&:..... ~~~-~~~~" 52

(Another bath was used for drug-Krebs solution. ) The 50 ml bath had a

wann water jacket, controlled by a Haake constant temperature c ircu-

f' lation pump. so that the temperature of the Krebs solution was raised to

.. zpproximately 40°G•... The Krebs .solution then flow_e.d ___int.o tn_e __ m~s_cle

11 bath via a narrow diameter (PE 90 - I. D. 0. 034 , O. D. 0.. 050") poly-

ethylene tubing of approximately 16 inches in length. The tubing also ran

through a warm water jacket again to reach a constant temperature before

entering the muscle bath. The polyethylene tubing was placed in the

muscle bath and the Krebs solution was allowed to run freely into the

muscle bath. The flow was constant for any experiment (200-250 ml/hr)

and was determined by the length of polyethylene tubi.ng and height of the

50 ml bath. The temperature was constant {it depended mainly oi: flow

rate) but could. be varied by a temperature control on the circulation

pump such that the final temperature in the muscle bath was constant at

approximately 37°C (36-38°C).

The muscle bath had the following dimensions -- 0. 6 cm x 8. 0 cm

x O. 5 cm. The nerve compartment measured 1. 0 cm x 1. 0 cm x 0. 5 cm.

Thu_s the total volume of the bath was approximately 3 ml. This allowed

for rapid turnover of solutions { 2x volume in 1. 5 min. ) with a minimal

amount of movement. The solution was removed by overflow into a drain. 53

The only adjustment necessary to change to new solution was to move a

clamp from one tubing to another. The rate and final temperature were

constant throughout the experiment. A muscle could be maintained for

at least 10 hours without significant decay of membrane potential or neuromuscular transmission.

IV. The Making and Filling of Electrodes

Microelectrodes were made from pyrex capillary tubing (0. 1 cm diameter - 7 cm length) The tubing was pulled to a narrow tip with a

David Kopf #700 vertical pipette puller using nichrome heater coils. The electrodes were floated on distilled water and examined under a micro scope (lOOx magnification factor) to obtain a rough estimate of the tip size (determined by distance water passes up electrode) and general contour of the tip and shank of the electrode .. Only those electrodes with narrow tips (little water passing in) and narrow shanks were filled. A 11 others were discarded. The electrodes were then boiled in filtered methyl or ethyl alcohol for 3 minutes using a Precision Scientific vacuum pump to fill the electrodes with alcohol. The alcohol was then replaced with distilled water by immersing the alcohol filled electrodes in filtered distilled water for 30 minutes. Finally, the electrodes were immersed in filtered 3M KCl until the day of use (at least 24 hours). .54

When electrodes were made for iontophoretic studies, they were immersed

in ACh or carbachol (0. 3 M to 3M), rather than KCl. By storing in a

refrigerator, these electrodes were usable for periods up to several months

V. Recording Appartus

Recordings were made using the intracellular technique developed

by Ling and Gerard (1949) and Nastuk and Hodgkin (1950). The electrode.

was supported in a glass tubing (I. D. 0. 2 cm) which was filled with an

agar - 3 M KCl column. The agar - 3 M KCl column made connection

with a silver chloride coated-silver wire spiral to eliminate a junction

· potential. Ail indifferent electrode which made contact with Krebs

solution consisted of an agar ,.. Krebs column in contact· with a silver

silver chloride spiral wire. (The silver wire spiral was coated with

silver chloride by electrolysis in 0. 1 N HCl. (2Cl- + Ag ----+

AgC12 + 2 e:-.)) The.-two-inputs (one from each agar bridge) were

connected to a Nikkon hi-input impedance amplifier. The output of this

pre-amplifier led into a Tektronics Type 502 dual beam oscilloscope.

This trace was used to monitor the membrane potential of the muscle cell.

A second output from the preamplifier led into a Tektronics Type 122

low-level preamplifier. This output led into two Tektronics Type 502

dual. beam oscilloscopes. r_r:he first oscilloscope which was monitoring ..•. t>C AC PreQ.tv\f'lifler Preamplifier

Recordin3 Osei llor.cope

J.f7 K.Q i WQ.rM Lc.la.ter JQ.cket

DIAGRAM OF PERFUSION AND RECORDING APPARATUS

.______,__ ,...______, ______._l 56 the membrane potential (low gain) was also used as a monitoring scope for the end-plate phenomena (high gain). The second oscilloscope had only the end-plate phenomena and was used for recording purposes.

Permanent records were made with an Analab oscilloscope camera. Thirty five millimeter still or continuous motion pictures could be taken of the potentials depending on the particular experiment.

VI. Selecting an Electrode

Electrodes were selected on the basis of their resistance and abiiity to penetrate the muscle fiber without causing considerable damage. [n general, a resistance of less than 20 megohms was necessary to make:: the noise level sufficiently small to record miniature potentials. If the resistance was less than 5 megohms, the electrode tip was usually too large and therefore damaged the muscle fiber. After an electrode of t11e correct resistance had been obtained, the electrode was placed in several rr:uscle fibers. If the electrode gave a deflection equivalent or greater than 60 m V and there was no significant decay of the resting membrane potential, ihe electrode was considered usable. If there were no deflections of greater than 60 mV for resting membrane potentials, or the resting membrane potenticil decayed constantly, the electrode was discarded. An AC square wave signal was passed through the electrode ------·------. 57

and by means of a capacity compensator in the Nikkon hi-input impedance

amplifier, it was possible to keep the time-constant of the system below

70 µsec.

VII. Locating an End-Plate and Recording M. e. p. p. s

Connective tissue on the surface of the muscle was removed to expose

the nervous structure of the muscle (small superficial branches come

off a large central nerve). The superficial nerve branches were located

with the aid of a binocular stereo microscope (magnification - 15x). The

microelectrode and indifferent electrode were carried on a Narishige

micromanipulator. The microelectrode was generally inserted in the

area of a small superficial nerve. As a result, the prob.ability of

locating an e. p. was quite high. The presence of m. e. p. p. s indicated

that the electrode was near the e. p. region of the muscle. 'The electrode

was usually reinserted into the muscle until a point was found where the

m. e. p. p. s were of maximal amplitude and shortest time-course. 'The

resting membrane potential was also recorded to show that the muscle

fiber was in good condition and not decaying over a long time period.

Pictures of them. e. p. p frequency were taken on a slow sweep (O. l cm/sec)

and tirne course were taken on a rapid sweep ( 2 msec/ cm). 58

VIII. Locating an End-Plate and Recording (+)-Tubocurarine, High

Magnesium and Benzoquinonium E. p. p. s

The nerve to the tenuissimus muscle was placed over bipolar platinum electrodes (0. 5 cm apart) in a separate compartment of the bath. The nerve was stimulated at 2 x maximal voltage with a pulse of 50psec at a rate of 1/2. 5 to 1/10 stimulus per second. The muscle twitch was blocked by tbe lowest possible concentration of (+)-tubocurarine (2 to 5 pg/ml), benzoquinonium (1. 5 to 3 pg/ml) or decreased calcium and increased magnesium. The blocking agent was added to the Ringer solution and infused through a second 50 ml bath system. An ~- p. p. was localized in a manner similar to that for the m. e. p. _µ. s. Amplitude was the major criteria. An amplitude of greater than 3 mv was genera1ly used (except in quantal content studies). The half-decay was usually below 2 msec

(rise to one-half fall) and the rise-time was less than 1 msec. This e. p. p. was then used as control for drug-induced effects.

IX. Iontophoretic Application of Drug§

In this series of experiments the recording apparatus was the same as previously described, except only DC-amplification was used. The muscle twitch was always blocked with (+)-tubocurarine (4 pg/ml). The iontophoretic electrode (with A Ch or carbachol) was supported in a

~ccond mj cromanipulator. Two stimulators were necessary, one to 59

eject the drug and ci.notber to retard diffusion of the drug from the tip of the

eiectrode. Each stimulator was connected to an.isolation unit. The two

isolation units were connected in series, with one lead from the isolators

_pas.~ ing_ thr9_ugt~ __ _a. 2_q_ m~_g()hI? r_~Ej~S-~~~c~_ _in!o_ t?e.. iontophoretic electrode._ ------·--- , _____ , ·------·· . -

The other lead passed through 47 kohm resistance into the bath .. The end

of each lead was coated with silver chloride. By recording the voltage

change across the 47 koh:in resistance it was possible to determine the

amount of current {as ACh or carbachol) which passed from the electrode.

This current recording was used as a monitor to be certain that equal

dosE:s of drug i.,vere ejected with each pulse.

After an e. p. p. had been located, the iontophoretic electrode \V8.S

moved into the region of the e. p. By careful manuevering of this

electrode, it was possible to produce a rapid depolarization of the e. p.

from application of ACh or carbachol. These drug-induced potentials

remainea relatively constant fri amplitude and time-course. Rapid

iontophoretic potential.s {half-decay less than 100 msec) required precise

localization at the e. p. Any movement produced large changes in the

iontophoretic potential. As these experiments required long infusion times,

it seemed desireable to use slower potentials {100 to 500 rnsec half-decay).

Then movements were not so critical. The pulse voltage and duration for 60

drug ejection were varied such that the amplitude of the iontophoretic

potential was similar to the amplitude of the e. p. p. The braking current

(opposite polarity) was adjusted, such that no decay of the membrane was

obvious from diffusing drug.

X. Infusion of Facilitatory Drugs

The agents used were neostigmine bromide, edrophonium chloride,

ambenonium chloride, and methoxyambenonium chloride. The facili-

tatory drug was added to a volume of the control Krebs-Ringer solution

and infused through a second 50 ml bath. The initial concentration of the

facilitatory drug was 10 -B M (except in the case of ambenonium where -10 -1'> due to its high potency, the initial concentration was 10 ~M or 10 ""M)

and the concentration was increased by IO times every 15 - 20 minutes until a predominate postsynaptic action occurred - twitching, depolarizing

(decreased amplitude and decreased resting membrane potential) or

competitive blockade (decreased amplitude but no effect on the resting membrane potential). The muscle was then returned to a control Krebs-

Ringer solution to determine if the drug was reversible or irreversible.

Drugs were infused for a period of ten minutes before recordings were mad

XI. Measuring the End-Plate Phenomena

The film which had been exposed to the e. p. phenomena was projected on a Durs.t, 606 Enlarger such that the potentials were magnified 2. 5x in 61

__ .order to make the necessary measurements.

M. e. p. p. frequency was determined from a series of traces which were

fihned at a sweep speed of 0.1 sec/ cm or I sec/ sweep for a period of 25

seconds. Five such samples were taken at 2 minute intervals for each

experimental situation. The standard error of the five samples was

calculated as well as t-values for significant changes due to the drugs.

M. e. p. p. time-course was recorded on fast multiple sweeps (2

msec/ cm) by holding the camera shutter open until a m. e. p. p. occurred

on the monitor scope. This resulted in a thick base line. In each

experimental situation, 10 m. e. p. p. s were measured for 1) amplitude,

2) rise-time, and 3) half-decay time and the average was calculated.

E. p. p. s were also recorded on a fast sweep (2 msec/ cm) which

could be triggered to give a single sweep. Again the 1) amplitude, 2)

rise-time and 3) half-decay time were measured from ten samples in

each experimental situation.

In iontophoretic studies e. p. p. s and iontophoretic potentials were

recorded on single, slow sweeps (100 to 500 msec/ cm). 1) E. p. p. ampli

tude and iontophoretic potential 2) amplitude 3} rise time and 4) half

decay time were measured from five records for each experim;ental 62

condition.

Standard deviation and t-values were calculated for each group of

samples.

The effect of facilitatory drugs on quantal content was calculated from

the number of e. p. p. failures during 200 nerve stimuli with decreased

c~lcium (O. 9:?nM) and increased magnesium (5-7 mM) ion concentrations

or by comparing the amplitudes of 50 m. e. p. p. s and 50 e. p. p. s with increased magnesium ions (12-15 mM) (Del Castillo and Katz, 1954c). 63

RESULTS SECTION 1

EFFECTS OF FACILITATORY DRUGS ON

M. E. P. P. SAND (+)-TUBOCURARINE E. P. P. S. 64

INTRODUCTION

' Several microelectrode techniques are commonly used to dis - tinguish presynaptic and postsynaptic ~echanisms of drug action. The most direct technique involves the effects of drugs on m. e. p. p. frequency and time-course (Katz, 1958, and 1962). Also, a comparison of the ampl~- tude and time-course of m. e. p. p. s and e. p. p. s, and observation of the e. p. membrane potential at various drug concentrations should enable a distinction between: 1) a direct, depolarizing action, 2) sensitization of the e. p. receptor, 3) cholinesterase inhibition and 4) a pre-junctiona1 action. 65

RESULTS

Effects of neostigmine on m. e. p. p. s

The effect of neostigmine on m. e. p. p frequency was to produce a small but significant increase in three out of four experiments. The effects on frequency were not readily reversible and the frequency re mained at the increased level after the muscle had been returned to

Krebs solution for 90 minutes.

No effects on amplitude or rise-time were noted when the perfusion 7 solution contained 10-8M neostigmine. With 10- M neostigmine. the increase in both the amplitude and the time course became very significant

(P'= O. 001) and the parameters were further increased by 10-6M neostig-· mine. With 10-5M the amplitude and time course of m. e. p. p. s. decreased 6 as the membrane potential fell from 60 to 39 mV. At 10- M the amplitude increased by 67%. the rise-time by 86% and the greatest increase was the half-·decay which increased by 188%. The effects on amplitude and time course, and the depolarization of the end-plate membrane were all reversed after returning the muscle to Krebs solution for 90 min. An example of the effects of the neostigmine on m. e. p. p. s. is given in

Table 2A. TABLE 2A

Effects of Neostigmine on M. e. p. p. s.

Frequency ' I Membrane Concentration of .. i of m. e. f,· p. s Amplitude Rise-time Ealf-decw potential neostigmirie (M) (sec- )a: (mV)b (msec)b (msec) (mV)

Control 1. 9+0. 4 . o. 6+0. 1 0.7+0.l 0.8+0.2 T 61

10-8 c 0.7+0.2 o. 7+0. 1 o. 9_:!:0. 3 i 61 7 10- 2. 4,+0. 2* 0.9+0.2** 1. 0+0. 2** 1. 6+0. 4** 65 ' 10-6 2.5+0.3** 1. o+o. 2** 1. 3+0. 3** 2.3+0.7**' 60

10-5 39 a + Standard error of the mean. b + Standard deviation. c Film recording poor with this observation for accurate measurement, but no change in frequency was apparent.

* Diff e!'ence statistically significant at 1% level.

>!<>:< Difference statistically si.gnificant at O. l % level. M. e. . n. s too small to me::i.sure accuratel . TABLE 2B

Effects of Neostigmine on E. p. p. s. (2 ug/ml Tc)

Concentration Membrane of Ampli~de Rise-time Half-decay potential neostigmine (M) (mV) (msec)b (msec)0 (mV)

Control 5.4+0.2 0.6+0.l o. 9+0. 1 68 8 10- 6.9+0.3** o. 6+0. 1 0.9+0.l 68 7 10- 8.5+0.3** o. 8+0 . .1* 1. 3+0. l** 68

10-6 Muscle began to twitch

a + Standard error of the mean. b + Standard deviation. c Jfilm recording poor with this observation for accurate measurement, but no change in frequency was apparent. >:< Difference statistically significant at 1% level. 0 •: :c Difference statistically significant at 0. 1% level. --- M. e. p. p. s too small to measure accurately. 08

Effects of neostigmine on (+)-tubocurarine e. p. p. s.

At 10-8M neostigmine, a very significant increase in amplitude was produced with no change in-time course: -~he amplitude and time course both increased at 10~ 7 M neostigmine; however, the increase in amplitude· was greater (57%) than the increase in rise-:-time (33%) or half-decay 6 (44%). At l0- M neostigmine the muscle began to twitch and the record ings were lost. Table 2B illustrates the effects of neostign1ine on single e.p.p.s.

~ffects of edropJ:i_?.nium on m. e. p. p. s

In four experiments infusion of concentrations of edrophonium of

10-8M or greater produced a very significant increase in m. e. p. p. fre quency (Figure 1). This effeCt was reversed after the muscle had been

-returned to Krebs solution for 60 min. (Table 3A).

There were no significant changes in amplitude or time course of the m. e. p. p. s. with edrophonium concentl·ations up to 10-5M. At 10-4M the e. p. . membrane potential was depolarized to 4lm V and the m. e. p. p. s. were no longer visible. The control readings were again obtained 60 min after the infusion of drug solution had been replaced by Krebs solution. 69

Figure 1. The effect of edrophoniµm on m. e. p. p. frequency. (a) Control (b) In the presence of edrophonium 10-8M. Voltage calibration, 1 m V; time calibration, 0. 2 sec. ..------..-..·\----....·, I ' TABLE 3A

Effects of Edrophonium on M. e. p. p. s

Frequency

Concentration of i Membrane of m. e. P·R· s Amplitude Rise-time Half-decay potential edrophonium {M) (sec- )a (mV)b {msec)b (msec)b (mV)

Control 1. 7+0. 1 0.6+0.2 1. l+O. 2 1. 4+0. 3 70

10-8 2.7+0.4** o. 6+0.1 1. 2+0. 2 1. 3+0. 3 70

10-7 2.8+0.5** o. 6+0.1 1. o+o. 2 1. 5+0. 3 71 -6 10 2. 5+0. 2** 0.7+0.2 1. 3+0. 2 1. 5+0. 3 70 5 10- 2.5+0.2** 0.6+0.2 1. 2+0. 1 1. 5+0. 2 66

10-4 48

For symbols see Table 2A. .------~\----c,.\ I,. TABLE 3B

Effects of Edrophonium on E. p. p. s (4 µg/ml Tc)

Concentration Membrane of Amplitude Rise-time Half-decay potential edrophonium (M) (mV)b (msec)b (msec)b (mV)

Control· 3.5+0.2 0.7+0.l 0.9+0.l 65

10-8 3.6+0.l 0.7+0.l 0.9+0.l 65

10-7 4.0+0.2** 0.7+0.l 1. 0+0, 1 65

10-6 5,5+0.2** o. 8+0. 1 1. 2+0, l** 65

10-5 9,3+0,3** 1. 2+0. l** 2.4+0,l** 65

Muscle began to twitch

For symbols see Table 2A. 72

Effects of edrophonium on (+)-tubocurarine e. p. p. s.

In three experiments 10-SM edrophonium produced no significant

changes in the amplitude or time course of the e. p. p. s; l0-7M increased

the amplifude with no-·changein time cour::re~--ct(J'"- 6M and10-=5M increased

both amplitude and time course. At 10-GM the increases were:amplitude

57%, rise-time 14%, half-decay 33%, and at 10-5M, amplitud~ 165%,

rise-time 71% ancl half-decay 167%. Ten minutes after perfusion with

10-5M edrophonium, the muscle began to twitch and the recording was

lost (Table 3B).

Effects of ambenonium on m. e. p. p. s

In four experiments ambenonium did not produce any significant

increase in m. e. p. p. frequency. In the experiment illustrated by Table -9 -8 4A, the frequency decreased at 10 M and 10 M but there was no change

at 10-7M. The amplitude and time course were very significantly increased

at concentrations of 10-6M and 10-7M At l0-7M the amplitude was

increased by 71%, the rise-time 57%, and the half-decay 1 70%. The half- -6 -5 decay decreased at 10 M and the m. e. p. p. s disappeared in 10 M

ambenonium. The m. e. p. p. s returned when the drug solution was replaced

by Krebs solution at the increased amplitude and time course which did not

return to control values after three hours perfusion.

~~--=_....._,.,,__,._ ___..,,....._.,.... __ ..., __ _.._.._.~-=-•nm,___...... , _____,, ______....,._,.. ____ ,_., 73

P."'igure 2. The effect of ambenonium on e. p. p. and m. e. p. p. amplitude and time course. (a) .E. p. p. s. recorded in the presence of 5 µg/ml tubocu rarine; A--control; B-- with 10-9M ambenonh:_rf; C--with 10-8M ambenon ium. (b) M. e. p. p. s.: A--control; B--with 10 M ambenonium. The thick base line is due to the record being photographed from a recurrent sweep, the camera shutter being left open until a m. e. p. p. occurred. Voltage calibration, (a) --·5 m V, (b) O. 5 m V; time calibration, (a) I msec. {b) 1 msec. TABLE 4A

Effects of Ambenonium on M. e. p. p. s

Frequency Concentration of Membrane of m. e. p. p. s Amplitude Rise-time Half-dec~y potential ambenonium (M) (sec-l)a (mV)b (msec)b (msec)b (mV)

Control 1. 5+0. 1 0.7+0.l o. 7+0.1 1. O+O. 3 66

10-10 1. 3+0. 3 0.7+0.l 0.7+0.l 1. l+O. 2 65

10-9 1. l+O. 3** 0.7+0.l o. 7+0. 1 1. o+o. 2 61

10-8 1. 2+0. l** 0.8+0.2 o. 8+0.1 1. 3+0. 3 58

10-7 1. 5+0. 2 1. 2+0. l** 1.1+0. 2** 2.7+0.4** 56 6 10- 1. 3+0. 2* 1. 2+0. l** 1. l+O. 2** 2.3+0.6** 60

10-5 65

Note: Changes in m. e. p. p. frequency show a statistically significant decrease. 1

For symbols see Table 2A. TABLE 4B

Effects of Ambenonium on E. p. p. s. (5 µg/ml Tc)

Concentration Membrane of Amplitude Rise-time Half-deca:b potential ambenonium (M) (mV)b (msec)b (msec) (mV)

Control 5.8+0.3 0.7+0.l 1. O+O. 1

10-10 6.3+0.2** 0.7+0.l 1. o+o. 1 78

10-9 6.5+0.3** 0.7+0.l 1. o+o. 1 78

10-8 13. 8+0. 7** 1. l+O. l** 1. 8+0. l** 80

10-7 Muscle began to twitch

For symbols see Table 2A · 76

When using ambenonium, it was difficult to maintain a stable end- plate membrane resting potential throughout a complete experiment due to the muscle twitching at concentrations between 10-9M and 10-7M. The shift in membrane potential in the experiment illustrated in Tab le 4A was not due to depolarization but due to movements of the electrode caused by the muscle twitching. In some experiments it was possible to observe a stable resting potential up to a concentration of 10-8M. In these experiments there was no change in amplitude or time course of the m. e. p. p. s with 10-10M or 10-9M. However, there were significant changes in amplitude, rise-time and half-decay at 10-8M and, therefore, the change in membrane potential shown in Table 4A did mask changes occurring with 10-8M ambenonium.

Effects of ambenonium on (+)-tubocurarine e. p. p. s

In three experiments 10-lOM and 10-9M ambenonium produced a very significant increase in e. p. p. amplitude with no change in time· course

(Figure 2). At 10-8M the amplitude was increased by 138%, the rise-time -7 by57% and the half-decay by 80%. At 10 M the muscle began to twitch and no further recordings were possible. (Table 4B). 77

Effects of methoxyambenonium on m. e. p. p. s

' In three experiments methoxyambenonium produced a significant jrtcrease in m. e. p. p. -frequency at-10-:~M.---No-significant increase in amplitude or rise-time was observed. The half-decay increased at 10-7M · and 10-BM. At 10-5M them. e. p. p. s. disappeared with no change in end:- plate membrane potential. The drug solut"ion was replaced by Krebs solution and them. e. p. p. s. returned at an increased amplitude and time course which did not completely return to control values after 150 min. In one experiment the m. e. p. p. frequency was greatly increased in the final rinse solution. (Table 5A).

Effects of methoxyambenonium on (+)-tubocurarine e. p. p. s

Methoxyambenonium, at a concentration of 10-8M, increased the amplitude of the e. p. p. s without change of time course. At 10-7M and

10-6M both amplitude and time course were increased; 10-6M increased the amplitude by 219%, the rise-time by 114% and the half-decay by 211%.

After 15-min perfusion with 10-6M methoxyambenonium, the muscle began to twitch and the recording was lost. (Table 5B).

Effects of acetylcholine

In four experiments acetylcholine (ACh) produced no consistent TABLE 5A

Effects of Methoxyambenonum on _M. e. p. p. s.

! Concentration Frequency I

of of I Membrane methoxy- m. e. P·f· s Amplitude Rise-ti~e Half-deck.y potential ambenonium (M) (sec- )a (mV)b (msec) (msec)b (mV)

Control 2. l+O. 3 0.7+0.l 0.6+0.l 1. o+o. 2 73

10-8 2.7+0.4* 0.6+0.l o. 5+0. 1 1. o+o. 2 73

10-7 2. l+O. 4 o. 8+0. 1 0.7+0.l 1. 5+0. 2** 71

10-6 2.0+0.2 o. 6+0,l 0.7+0.l 1. 5+0.1** 73

10-5 71

For symbols see Table 2A. i TABLE 5B ' I Effects of Methoxyamenonium on E. p. p. s. (3µg/ml Td)

Concentration

of I Membrane methoxy- Amplitude Rise-time Half-decay potential ambenonium (M) (mV)b '. (msec)b (msec)b (mV).

Control 3. l+O. 2 0.1+0.1 0.9+0.l 66

10-8 3.6+0.2** 0.8+0.l 0.9+0.l 66

10-7 6.6+0.4** 1. o+o. l** 1. 3+0. l** 68

10-6 9.9+0.6** 1. 5+0. l** 2.8+0.l** 68

For symbols see Table 2A bO

changes in m. e. p. p. frequency, either as the concentration was increased during the sarv.e experiment or at the same concentration in different experiments. The frequency varied throughout the experiment and it is unlikely that any changes were drug-induced. ACh did not increase the amplitude or time course of the m. e. p. p. s, but at concentrations greater than I0-4M, the amplitude diminished as the end-plate membrane was depolarized. (Table 6).

Effects of tubocurarine

Experiments using tubocurarine alone showed no significant changes of amplitude ur time course of e. p. p. s over a period of 2 hr. In experiments using 5-10 µg/ml tubocurarine, similar changes tO those recorded above were observed but higher concentrations of the facilitatory drugs were required. Concentrations of tubocurarine of 10-15 µg/ml completely prevented any increases in amplitude and time course in the presence of the facilitatory drugs.

DISCUSSION

.All the facilitatory drugs used produced statistically significant changes in m. e. p. p. frequency. However, the changes were small and assuming that a ten fold increase in frequency of the m. e. p. p. s repre- TABLE 6

Effects of Acetylcholine on M. e. p. p. s

Cone entration Frequency of of Membrane acetyl m. e. p. p. s Amplitude Rise-t~e Half-decw potential choline (M) (sec-l)a . (mV)b (msec) (msec) (mV)

Control 1. 2+0. 2 0.8+0.2 0.6+0.l 1. o+o. 2 70

10-8 1. l+O. 1 0.9+0.2 o. 7+0. 1 I. o+o. 2 71

10-7 1. 3+0. 4 0.9+0.2 o. 7+0.1 0.9+0.2 70

10-6 1. 5+0. 3* 0.8+0.2 o. 6+0. 1 1. o+o. 2 71

10-5 1. 5+0. l** o. 8+0. 1 0.6+0.2 1. l+O. 1 71

10-4 1. 5+0. 2** 0.7+0.l o. 6+0.1 1. o+o. 2 70

10-3 49

For symbols see Table 2A. 82

sented a 15 mV depolarization of the nerve terminal (Liley, 1956b),

depolarization of the terminal could only have been in the order of 1 to 3 m V. Also, m. e. p. p. frequency varies over an extended period of time

(Boyd and Martin, 1956a) thus some of the changes noted may have been

spontaneous and not drug-induced.

Boyd and Martin (19 56a) also. noted a change in m. e. p. p. frequency using 3. 3 X 10-7M neostigmine and a decrease at 3. 3 X 10-6M, but as in the present results, the changes recorded were only small. The reduction of frequency at higher concentrations may simply reflect the difficulty in observing the m. e. p. p. s which were greatly decreased in amplitude when the end-plate was depolarized. No change in m. e". p. p. frequency has been observed in the frog sartorius using neostigmine (Fatt and Katz,

1952) or iri the rat diaphragm using neostigmine (Liley, 1956a).

It is difficult to assess the importance of the small changes in nerve terminal membrane potential that these increases in m. e. p. p. frequency reflect. The change in spontaneous release of ACh indicated by the change in m. e. p. p. frequency is probably not physiologically significant.

However, this small e.ffect on the terminal may be indicative of a pre synaptic action which do.es have significant effects on the release of A Ch due to nerve action potentials. 83

The changes in time course of them. e. p. p. s were much more easily

observed and more consistent than the changes in amplitude. Since the

changes in a.~plitude were small_<:~?__o_n~y_()_':~ur:~:?_i1:_~~e p:r~s_ence of

changes of time course, it can be concluded that the drugs produced no significant changes in the sensitivity of the end-plate. This conclusion confirms that of Blaber (1963), and the changes in end-plate sensitivity produce.cl by methoxyambenonium in frog muscle (Karczmar et al., 1961) point to a species difference in the effect of these drugs at the neuromuscular junction. A logarithmic plot of amplitude during the decay of the e. p. p. was found to be linear with time, both in the control recordings and in the presence of facilitatory drugs; thus an increase of the time to half-decay indicates prolongation of time course. The degree of prolongation gives a rough estimate of the anticholinesterase activity of these compounds. Thus ambenonium is the most potent cholinesterase inhibitor, while neostigmine, methoxyambenonium and edrophonium exhibit decreas - ; ing potencies. Edrophonium produces no effects on m. e. p. p. amplitude or half-decay. This indicates that edrophonium does not inhibit cholin -6 esterase up to a concentration of 10 M. Changes in amplitude and time course of m. e. p. p. s have been reported in other species previously, neostigmine (3. 3 X 10-6M) in frog sartorius (Fatt and Katz, 1952}, neo- 84

6 stigmine (3. 3 X 10- M) in rat diaphragm (Liley l956a.) and also in the cat

tenuissimus muscle,, neostigminefa. 3 X 10-7M) (Boyd and Martin, l956a).

Edrophonium (lo-4M) and neostigmine (10-5M) produced depolarization

of the end-plate,, confirming the conclusions of Nastuk and Alexander

{1954).. and Katz and Thesleff (1957). These concentrations are higher

than those necessary to facilitate transmission and would indicate that

depolarization of the end-plate is not a factor in the production of

facilitation of neuromuscular transmission by these drugs .. confirming the

conclusion of Blaber and Bowman (1959).

5 Ambenonium (l0- M) and methoxyambenonium {lo-5M) produced a

decrease in the amplitude and time course of the m. e. p. p. s without

any change in the resting potential of the e. p. membrane. These drugs have been shown previously to produce neu1·omuscular paralysis resembling that of {+)-tuobcurarine (Blaber, 1960). This potent curare like action may account for the unusual observation that methoxyam benonium increased the half-decay time but not the amplitude of the m. e. p. p. s.

The facilitatory drugs also increased and prolonged the {+)-tubocura rine e. p. p. but the effects on e. p. p. s occurred at concentrations which hac I 85 no effect on the m. e. p. p. s. This fact, together with an increase in amplitude and no change in time-course of the . e. p. p. indicates a pre synaptic action .for these drugs.

Masland and Wigton (1940) and Eccles (1964) have suggested that

ACh may be mediating in the action of the facilitatory drugs on the motor nerve terminal after inhibition of cholinesterase. However, the present results show that the time course of the e. p. p. was not prolonged at the time it was augmented, showing cholinesterase was not inhibited. Also,

A Ch was found not to produce any change in frequency of m. e. p. p. s, thus confirming the results of Hubbard, Schmidt and Yokota (1965). These results, although not eliminating A Ch as a mediator, would suggest that nerve terminal effects are not produced by a previous inhibition of cholinesterase, though the drugs may sensitize the nerve terminal to an action of ACh. The action of ACh would not be to depolarize the terminal, since no change in m. e. p. p. frequency was produced.

Because the amplitude of the m. e. p. p. s was not increased in the absence of a prolonged time course, it can be assumed that the size of the quantum is not increased by facilitatory drugs. The increase in ACh release for each stimulus mu~t. therefore. be due to an increase in the quantal content of the e. p. p. 86

Further work is required to support the present conclusions since

they are based upon the assumption that m. e. p. p. s and e. p. p. s di.ffer

only in their mechanism of release. But m. e. p. p. s are well kr10wn for

their small and highly variable amplitude (Fatt and Katz, 1952). E. p. p. s

are comparatively large and very constant in amplitude. It is possible that the present results show no significant changes of the m·. e. p. p. s

only because of this high variability. This can be illustrated by the

results with methoxyambenonium (Table 5). 10-8M methoxyambenonium increased the e. p. p. amplitude by 16%. The same percentage increase of the m. e. p. p. amplitude (O. 7 to O. 8mv) would not have been significant.

Thus the initial conclusion that the facilitatory drugs act by a pre-junc tional action is weakened and must be tested by further experiments. 87

RESULTS SECTION 2

EFFECTS OF FACILITATORY DRUGS ON

(+)-TUBOCURARINE E. P. P. SAND IONTOPHORETIC

ACETYLCHOLINE AND CARBACHOL POTENTIALS. 88

INTRODUCTION

From the prev-ious experiments it was concluded that the e. p. p.

amplitude-was augmented at a concentration of facilitatory drug which was

approximately one-tenth the concentration necessary to produce an effect

on m. e. p. p. amplitude or time-course. Since m. e. p. p. s have a high variability and low amplitude, further experiments were performed to

provide additional information regarding the mechanism of action of the facilitatory drugs. Therefore experiments were performed with the

same drugs, using a microelectrode drug application technique (Nastuk,

1953a and Del Castillo and Katz, 1955a). This technique was used for the following reasons. First, the membrane potential change resulting from iontophoretic application of ACh is not as V8.riable as m. e. p. p. s.

Second, the ACh-potential can be adjusted to any desired amplitude, simply by increasing the current which passes through the electrode. Finally. it is possible to record both the e. p. p. and ACh-potential at the same muscle. e. p., thereby eliminating any variability of drug response which may occur between different muscle fibers.

RESULTS

It proved to be quite difficult to maintain ACh-potentials at a 89

constant amplitude for long periods of time. Thus both ACh-potential

amplitude and half-decay time were observed to ·determine the presence

of drug-induced changes to the response of ACh at the e. p. membrane.

-- ~-- ---·· ·- - (The half-decay seemed very constant·for long periods of time, even though the amplitude decreased considerably.)

Effects of the facilitatory drugs on (+)-tubocurarine e. p. p. s and A Ch-

potentials.

Neostigmine.

Table 7 shows the results obtained from two experiments with neostigmine. In EXP #1 neostigmine produced no signi~icant changes at

10-8M, although there were ·small effects {less than 10%) on the ACh potential. At 10-7M there were large changes in both the e.p.p. and

A Ch-potential. In EXP #2 the e. p. p. was augmented by 10-8M neostigmine. The increase was 28% of control and was highly significant. Also, the ACh-potential was prolonged by 17%, although this increase was not as significant as the effect on the e. p. p .

.E_~drophonium.

It was quite difficult to produce the e. p. p. changes at 10-7M edrophonium which had been observed in the prmous section. However, 90 TABLE 7

Effects of Neostigmine on E. p. p. s

, .. and ACh-potentials (4 µg/ml. Tc)

· Concentration ·-·E. P. P. . --Ach -potential A Ch-potential of amplitude amplitude halfideca:b neostigmine (M) (mV)b (mV)b {msec)

EXP #1

Control 2.6+0.2 1. 4+0. 1 480+30 8 10- M 2.5+0.1 1. 5+0. 2 520+30 (0) (7%) -(8%) -7 10 M 3.6+0.2** 3.3+0.2** 760+50*ii~ (38%) (i::j6%) (58%)

EXP #2

Control 1. 8+0. 1 I. 3+0. 1 120+10 8 I0- M 2. 3+0. Iii~* I. 3+0. 1 140+10* (28%) (0) 0.7%)

~~ Difference statistically significant at the 1% level.

** Difference statistically significant at the 0.1% level.

b + Standard deviation.

Values in parentheses indicate the percentage increa.se over control. 91 TABLE·a

Effects of Edrophonium on E. p. p. s

. and ACh-potentials (4 µg/mlTc}

Concentration E. P. P. A Ch-potential ACh-potential of amplitu.ge amplitude half-decay edrophonium (M) (mV) . {mV)b {msec)b

EXP #1

Control I. o+o. 1 I. 9+0. I 110+10

I0-7M 1. 5+0. I 110+10 1. l+O. 1 - (10%) (0) (0)

EXP #2

Control 1. 4+0. 1 1. 8+0. 3 280+10

10-6M 2.2+0.2** 2. 1+0. 2 350+20** (57%) (17o/o) (25%)

EXP #3

Control 1. 5+0. 2 1. 7+0. I 550+30

10-BM 1. 4+0. 2 I. 4+0. I 500+60 (O) (0) (O)

I0-7M 1. 5+0. 2 1. 8+0. 1 620+60 (O) (6%) (20%)

6 10- M 2. o+o .. 2~~* 2. 7+0. 2::.;;* 710+40>:•* - (33%) (59%) (29%)

For symbols see Table 7

L-..------...---...------..---~~"'""""'--d 92

there were large changes in both the e. p. p. and ACh-potential at 10-6M

edrophonium (Table 8).

· Ambenonimn.

The experiments with ambenonium resulted in almost identical

increases in e. p. p. amplitude and ACh-potential half-decay (Table 8 and

Figure 3). The only difference occurs in EXP #2 at io-9M ambenonium

when the e. p. p. was augmented, significantly, by 16% while the ACh

potential half-decay was increased by 8%. which was insignificant.

Methoxyarnbenonium.

EXP #1 shows large increases in e. p. p. amplitude and ACh-potential 8 amplitude at 10- M methoxyambenonium. 10-8M was much less effective

in EXP #2, although the small increase of e. p. p. amplitude was sign: f

'icant (Table 10).

Time-course of anti-Tc action.

In a series of four experiments effects of the facilitatory drugs were

observed continuously from the beginning of drug infusion. In this way it

was possible to determine if effects on e. p. p. amplitude followed the

same time-course as effects on ACh-potential amplitude or half-decay .

. The results of one experiment are illustrated in Figure 4, Records were 93

A -

Figure 3. The effect of ambenonium on e. p. p. s and ACh-potentials recorded in the presence of 4 µg/ml (+)-tubocurarine:A--control e. p. p. and ACh-potential; B-~with 10-8M ambenonium. Voltage calibration, 5mV; time calibration, 100 msec; current, 2. 5 x 10-7 amps. 94 TABLE 9

Effects of Ambenonium on E. p. p. s

• and ACh-potentials (4 µg/ml Tc) e oncentration --E. P. P. ACh-potential ACh-potential of amplitude ampli~de half-decay: ambenonium (M) (mV)b (mV) (msec)b

EXP#l

Control 1. 7+0. 1 2. ·4+0. 2 80+10

10-9M 1. 7+0. 1 1. 4+0.1 90+10 -(0) (O) (13%) -8 10 M 3.0+0.2** 2.8+0.3 l40+10:\0 l: (76%) (17%) (75%)

EXP #2

Control 1. 9+0. 1 2.0+0.2 130+10 9 l0- M 2. 2+0. l*:\( I. 9+0. 1 140+10 06%) (O) (8%)

10-8M 5. l+O. 5*:\( 2.2+0.1 350+10:\(:\C (l68%) 00%) (169%)

For symbols see Table 7 TABLE 10 95

Effects of Methoxyambenonium on E. p. p. s

and .AC:h-potentials (4 µg/ml Tc) ,

Concentration of E. P. P. AGh-potential AGh-potential methoxy- amplitude amplitude half-decal ambenonium (M) (mV)b (mV )b (msec)

EXP #1

Control 6.6+0.3 4. 2+0. 1 60+10

10-BM 11. 4+0. 2*:>i'< 6.6+0.8** 50+10 (73%) (57%) (0)

EXP #2

Control 2. 1-10. 1 I. 7+0. 1 140+10

10-BM 2. 4-IO. J>:c I. 5+0. 1 150+10 (14%) (0) (7%)

10-7M 3. 8+0. 3*>:c I. 2+0. I 180+10** (81%) (O) (29%)

For syi.nbols see Table 7 96

______5 - •

x -> 4 - E ••llX• - • x X X>C•>C >C w 3 - x •.• l x • • • 0 xx x • • •• xxl ::) • xx • • I- ,. xx.••>

0 I 1 J I 5 10 15 0 2 Az TIME (min)

Figure 4. The effect of ambenonium on e. p. p. (•) and ACh-potential (x). amplitude recorded in the presence of 4 µg/ml (+)-tubocurarine versus time: A --beginning of 10-8M ambenonium infusion; A2-- beginning of rn- 7M 1 ambenonium infusion. 97

made every thirty seconds throughout the experiment. The controls were

relatively constant. 10-SM ambenonium produced parallel increases in

both e. p. p. amplitude and ACh-potential amplitude. Two minutes after -7 -beginning-to -infuse 10 M ambenonium,- both-potentials.increased very

rapidly in amplitude.

Effects of the facilitatory drugs on (+)-tubocurarine e. p. p. s and

carbachol-potentials.

Non~ of the drugs studied (neostigmine, edrophonium, ambenonium

and methoxyambenonium) produced changes in the carbachol-potential at

doses which caused considerable augmentation of e. p. p. amplitude. This

is illustrated in Figure 5. After control records, l0-6IVJ methoxy·-

ambenonium was administered. The e. p. p. and carbachol-potential were

recorded every thirty seconds. After three minutes of methoxyambenonium

infusion there was a rapid augmentation of the e. p. p. amplitude, but no

change in the carbachol-potential. After four minutes, the e. p. p. was not

recorded to avoid any subsequent muscle twitches. Even after ten

minutes there was no change in the carbachol-potential.

DISCUSSION

In five of seven experiments there was a statistically significant

\' 'ft 98

A - l BJ.._ . .. L -

Figure 5. The effect of methoxyambenonium on e. p. p. s and carbachol potentials recorded in the presence of 4 µg/ml (+)-tubocurarine: A--control e. p. p. and carbachol-potential; B--with 10-6M methoxyambenonium. _ 8 Voltage calib.1;~~tion, 5 m V; time calibration, 500 msec; current, 3. 2xl0 amps. 99 increase in ACh-potential amplitude or half-decay at the same minimal concentration as the increase in e. p. p. amplitude. Two experiments in which there was no correlation (ambenonium EXP #2 and methoxyambenonium EXP #2) showed small, but significant, increases in the e. p. p. amplitude at concentrations which did not significantly affect the ACh-potential.

Yet the changes of the A ch-potential are 50% as large as the changes in the e. p. p. at the low concentrations. In fact, in ambenonium EXP #2 -8 10 M ambenonium produced exactly the same percentage changes (168% and 169%) in e. p. p. amplitude and ACh-potential half-decay. On the basis of these results it seems likely that there is a good correlation between the effects of facilitatory drugs on the e. p. p. and ACh-potential in a curarized mammalian neuromuscular preparation.

This conclusion is substantiated even further by the observation that augmentation of the e. p. p. and ACh-potential amplitudes follow approximately the same time course, as shown in Figure 4. These conclusions are very similar to those of Goldsmith (1963) and Otsuka and Nonomura

(1963). Both papers gave results showing the effects of edrophonium on e. p. p. s and ACh-potentials at the frog neuromuscular junction.

Correlation between effects of facilit:i.tory drugs on e. p. p. s and

A Ch-potentials can be explained by several postsynaptic mechanisms of 100

action. The most likely mechanism involves the cholinesterase inhibiting properties of the facilitatory drugs. This theory receives strong support from the observation that the action of a stable choline ester (carbachol) is not affected by facilitatory drugs. This would eliminate the other possibilities, sensitizing and depolarizing actions, which have been hypothesized (Riker and Wescoe, 1946 and Karczmar, 1957). · If sensitizing or depolarizing actions were occurring, an increase in the carbachol potential should have been observed. 101

RESULTS SECTION 3

EFFECTS OF FACILITATORY DRUGS ON

QUANTAL CONTENT OF THEE. P. P. 102

INTRODUCTION

Calcium ions are essential for transmitter release at the neuromuscular junction. By decreasing the calcium ion concentration or increasing the magnesium ion concentration in the Ringer solution, release of transmitter can be decreased to any level. At appropriate

concentrations of calcium and magnesium the average quanta in a single

e. p. p. is near one {equivalent to a m. e. p. p. ). At this level of blockade it is possible to quantify transmitter release per indirect stimulus (Del

Castillo and Katz. 1954c). ·

The facilitatory drugs were tested in a deficient calcium preparation to determine if they were capable of increasing the quanta! content of the e. p. p ..

RESULTS

The effects of neostigmine. edrophonium, ambenonium and methoxyambenonium are illustrated in Tables 11 to 14, respectively. The quanta! content was measured by two methods:

I) ave. e. p. p. amplitude of 50 samples ave. m. e. p. p. amplitude of 50 samples and 2) loge no. of e. p. p. s in 200 trials (Figure 6). no. of failures 103

- •

------f"---e""'"'

-1l ...----""-'""•""'""·""'"'"""-4""'•-" i ! l~ ·'

Figure 6. Effect of edrophonium on quanta! release. Indirect stimulation at the artifact. (a) control; (b) with I0-6M edrophonium. Voltage calibra tion, 1 m V; time calibration, 5 msec. ) 04

TABLE 11

Effect of Neostigmine on Quanta! Content

Quanta! Content

Concentration Mg (10-15mM)a Ca (O. 92 mM) of Mg (5-? mM)b neostigmine (M) Muscle 1 Muscle 2 Muscle 3 Muscle 4

Control 2.1 1. 9 0.54 o. 71 -8 10 2.1 2. 0 0.42 0.87

10-7 2.1 1. 9 0.68 0.70

io- 6 1. 7 0.64 0.73

10-5 . 1. 8 o. 82*

Average amplitude of 50 e. p. p. s a - Average amplitude of 50 m. e. p. p. s _ number of stimuli b Log en·1mber of failures

* indicates a significant increase of 33% or a significant decrease of 25% (Edwards and Ikeda, 1962). 105

TABLE 12

Effect of Edrophonium on Quantal Content

Quantal Conte~_!

Concentration Mg{l0-15 mM)a Ca (O. 92 mMb of Mg (5-7 mM) edrophonium (M) Muscle 1 Muscle 2 Muscle 3 Muscle 4

Control 1. 2 2.4 0.36 o. 87 8 10- I. 2 2. 5 0.45 0.96 -7 10 1. 2 2. 9 0.34 0.90

10-6 1. 6* 2. 6 0.44 1. 4* 5 10- 2. 4 1. 3*

For symbols see Table 11 106

TABLE 13

Effect of Ambenonium on Quanta! Content

Quanta! Content

Concentration Mg (10-15 mM)a Ca (0. 92 mM) of Mg (5-7 · mM)b ambenonium (M) Muscle 1 Muscle 2 Muscle 3 Muscle 4

Control 1. 6 1. 5 0.67 1. 9

10-10 1. 8 1. 6 o. 81 1. 8 10-9 1. 8 1. 7 0.66 1. 9

10-8 1. 8 2. o* 0.53 1. 7

10-7 2.0 2.2* 0. 33::!c 2. 0

10-6 1. 8 1. 4 o.2s* 1. 7

~ ""'-5 10 1. 5

For symbols see Table 11

', 107

TABLE 14

Effect of Methoxyambenonium on Quanta! Content

Quanta! Content a Concentration Mg (10-15 mM) Ca (O. 92 mMb of methoxy- Mg (5-7 mM) ambenonium (M) Muscle 1 Muscle 2 Muscle 3 Muscle 4

Control 2. 5 3. 6 0.79 1. 04 -8 10 2. 5 3. 6 o. 89 o. 83

10-7 3.4* 3. 9 0.59 0.74*

10-6 3. 2 0.92 o. 82

For symbols see Table 11 108

With either of these techniques the facilitatory drugs did not produce consistent coonges in the quantal content of the e. p. p. at any concentration.

DISCUSSION

The quantal content of the e. p. p. in deficient calcium or increased magnesium ions is not increased significantly by facilitatory drugs.

This is similar to the conclusion of Hubbard. Schmidt and Yokota (1965). except they observed a small decrease in quantal content of the e. p. p. in the presence of neostigmine.

M. e. p. p. s and e. p. p. s in high magnesium or low calcium are augmented at.the same concentrations. Also. both are depressed at high concentrations. indicating the e. p. p. s and m. e. p. p. s are affected by the same postsynaptic mechanisms.

Augmentation of the e. p. p. s and m. e. p. p. s is compatible with the theory of cholinesterase inhibition by facilitatory drugs. Depression at higher concentrations is probably due to direct competitive or depolarizing . actions. ·

Thus these results can be explained completely on the basis of postsynaptic actions and they indicate that in a calcium deficient solution .. '

109 facilitatory drugs do not increase release of transmitter from presynaptic nerve terminals. 110

RESULTS SECTION 4

EFFECTS OF BENZOQUINONIUM

AT THE NEUROMUSCULAR JUNCTION 111

INTRODUCTION

Benzoquinonium {Bz} produces competitive blockade of neuromuscular

-transmission,,-and in this respect acts much like-(+)-tubocurarine. The

distinguishing feature of benzoquinonium blockade is that it is not _antag-

onized by facilitatory drugs (Hoppe,, 1950; . Hougs and Johansen,, 1957;

Bowman,, 1958 and Blaber and Bowman,, 1962}. As Blaber and Bowman

(1963a and 1963b} concluded facilitatory drugs act at the motor nerve

terminal, Blaber and Bowman {1962 and 1963a} further concluded that

benzoquinonium prevents the action of facilitatory drugs at the motor

nerve terminal.

Alternatively,, it is possible that the potent anticholinesterase activity

of benzoquinonium (Hoppe, 1951 and Blaber and Bowman,, 1962) prevents

any subsequent inhibition of cholinesterase by facilitatory drugs.

The following experiments attempt to elucidate the sites of action

of benzoquinonium and thereby obtain presumptive evidence for the

mechanism of action of facilitatory drugs. 112

CH2-CH3 CH2-CH3 CH 3-CH2-~ - CH2-CH2-CH 2-NH- -NH- CH 2- CH2-CH 2- ~ - CH 2-CH 3 I . I CH2 0 CH2 0 . 0 ~

BENZOQUINONIUM 113

RESULTS

Benzoquinonium control

- The e. p. p. recorded when the m~scle was blocked by Bz progres- · sively decreased in size as a function of time. This was especially evident·

in the amplitude, which decreased by approximately 50% after one hour ~f

recording (Figure 7). There were much s'maller decreases in rise-time

and half-decay (Figure8).

Effects of the facilitatory drugs on the benzoquinonium e. p. p.

Infusion of concentrations of neostigmine, edrophonium, ambenonium,

or methoxyambenonium which increased the amplitude and/ or time course

of e. p. p. s recorded during Tc blockade, produced little change in the

rise-time, half-decay, or amplitude of the Bz e. p. p. (Figure 9). There

was a continuous decrease of the amplitude; but the rate of decline resem-1;

bled closely that observed in the control e. p. p. with Bz alone. Since the

drug concentrations were changed at 15 minute intervals, the slopes of

Figure 7 and 9 can be compared. At high concentrations of the facilitatory drugs, effects on the e. p. p. were observed. Concentrations

of 10-4M edrophonium caused a facilitation of all parameters. 10-5M neostigmine increased the time to half-decay. Ambenonium prolonged the . -7 -6 -5 half-decay at concentrations of 10 M and 10 M. Also, 10 M amben- ' 114

6 CONTROL

-> 5 E - 4 LLJ 0 ....:::> 3 -_J a. 2 ~ <( I 0 .- 0 15 30 45 60

TIME (min)

Figure 7. E. p. p. amplitude recorded in a muscle fiber blocked by Bz (2 µg/ml). Zero time represents the time at which an e. p. p. was located and the first record taken. .:- 115

Figure 8. End-plate potentials recorded in a muscle fiber blocked by Bz (1. 5 pg/ml). E. p. p. s recorded at 30 mins (upper trace}, 60 mins {center trace, 90 mins (lower trace) after beginning of the Bz infusion have been superimposed. Voltage calibration 5 m V; time calibration 1 msec. 116

o...___,-~~-~~- 0 .J...-.--~-~-~-~~- 9 7 6 5 c !0°8 10·1 !0°6 10~ 5 c 10·10 I0° 10·• I0° 10" I0°

LOG CONCENTRATION (M)

Figure 9. The effects of facilitatory drugs on the amplitude of e. p. p. s recorded in a muscle blocked by Bz. C represents the control reading with Bz alone. The facilitatory drugs were added 15 min after the control reading and the concentrations increased at 15 min intervals. onium and methoxyambenonium depressed the amplitude and rise-time.

Comparison of the benzoquinonium e. p. p. with the (+)-tubocurarine e. p. p.

It was observed that the values for the rise-time and half-decay of the_

Bz e. p. p. were prolonged when compared to a Tc e. p. p. In three experi-

ments a muscle was initially blocked. with the lowest possible concentration

of Tc. ,After a rapid e. p. p. was found (rise-time < 1 msec and half-

decay< 1. 5 msec), the Tc was removed by washing until the m. e. p. p. s

became visible. Then the $ame end-plate was blocked with Bz. This

gave the results in Figure 10. When e. p. p. s of the same amplitude were

compared for any effect on time course, it was found that the rise-time

and half-decay of the e. p. p. s in Bz were about twice the duration when

compared to the rise-time and half-decay of the e. p. p. s in Tc. When the

Bz was washed out and replaced by Tc, the time course returned to that

of the Tc control level. Each change-over from Tc to Bz, or from Bz to

Tc, took approximately one hour. If a facilitatory drug was added to

Ringer solution of the final Tc e. p. p., the facilitating action of these agents was considerably less potent than in the control Tc e. p. p. This

decreased potency is obvious in the increased latency of action for ambenonium. Normally, 10-7M ambenonium produced its anticurare

action in about five minutes, but in the final Tc solution, the anticurare ~ ~--c ------~------. f~.: , ~:; ::)' 118

Bz L

. ''!''

Figure 10. Comparison of the time course of e. p. p. s recorded in a muscle fiber blocked by Tc (2 µg/ml) and Bz (1. 5 µg/ml). Voltage calibration 5 m V; time calibration l msec. 119

action of ambenonium was delayed to about 15 minutes. In addition, the

augmented amplitude of the e. p. p. occurred without any increase in the .. ·,

rate of rise (~igure 11). An action of the facilitatory drugs increasing

the rate of rise of the e. p. p. during Tc blockade has been previously

reported (Kuperman and Okamoto, 1964).

Effect of benzoquinonium on m. e. p. p. frequency and time course

Bz had little effect on m. e. p. p. frequency. There was only a slight 8 increase which occurred at 10- M. There was no significant increase at

-7 Concentrations of Bz up to 10 M produced no effect on the m. e. p. p.

amplitude and time course. At 10-7M Bz, the amplitude of m. e. p. p. s

was depressed without any change in the resting membrane potential, thus

confirming that Bz does not produce any depolarization of the end-plate.

At 10-61\II the m. e. p. p. s were too small to measure accurately. These

effects are illustrated in Table 15A.

Effect of benzoquinonium on Tc e. p. p. s

In three of four experiments Bz produced a facilitation of the Tc 7 e. p. p. As shown in Table 15B, this effect occurred at 10-8M and 10- M

Bz. The response was very slow to develop and the level of facilitation 120

-:;~~".~;.~~<~<~?'_l'}•f-«.~-:~"7"!'·,~--~·~~-~~; 1· ·i• I .i.!::

Figure 11. Comparison of the effects of facilitatory drugs in a muscle fiber blocked by Tc before Bz (a) and after Bz(b). In a, A is a control e. p. p. re corded in a muscle blocked by 3 µg/ml Tc; B is an e. p. p. recorded when a concentration of 10-6M edrophonium was added to the perfusion solution. Between a and b the Tc was removed and the muscle blocked with Bz (1. 5 µg/ml). The Bz was then removed and the muscle re-infused with Tc. In b A is a control e. p. p. recorded in a muscle blocked by 3 µg/ml Tc; B is an e. p. p. recorded when a concentration of 10-6M edrophonium was added to the perfusion solution, and C when .I0-5M edrophonium was added to the per fusibn solution. Voltage calibration 5 m V; time calibration 1 msec. · '·-·,,,,.·~=as~ ' '· ,. ':\"' ·1'.~,~~~~~~ .

' '

TABLE 15A .. Effects of B·enzoquinonium on M. e. p. p. s

Frequency Concentration of Membrane ' of m. e. p. p. s Amplitude Rise-time Half-de~y potential benzoquinonium (M) (sec-l)a (mV)b (msec)b (msec) (mV)

Control 3.0+0.4 1. 4+0. 3 o. 7+0. 1 1. 2+0. l 62 -10 10 3.0+0.7 1. 5+0. l o. 7+0. l 1. 3+0. 2 60

10-9 3. l+O. 3 1. 5+0. 3 o. 8+0. l 1. 3+0. 2 60

10-8 3.7+0.5* 1. 4+0. 2 o. 7+0.1 1. 2+0. 1 60

10-7 3.3+0.6 0.9+0.2** o. 7+0. 1 1. 2+0. l 60

10-6 60 i '.

* Differenc~ statistically significant at the 1% level. ** Difference statistic.ally significant at the 0. 1% level. --- M. e. p. p. s too small to measure accurately. a + Standard error of the mean. b + Standard deviation. . ...,.,

TABLE 15B

Effects of Benzoquinonium on E.-p. p. s

R.ecorded in the Presence of 4 µg/ml Tubocurarine

Concentration Membrane of Amplitude Hise-time Half-decay potential benzoquinonium (M) (mV)b (msec)b (msec)b (mV)

Control 5. l+O. 4 0.7+0.l 1. 3+0. 1 73

10-10 5. l+O. 3 o. 8+0. 1 1, 4+0.1 75

10-9 4.9+0.3 o. 8+0.l 1. 4+0. 1 77

10-8 5. 6+0. 4* 0.8+0.l 1. 3+0. 1 75 7 10- 7. o+o. 3>.'<* o. 9+0. l** 1. 4+0. 1 75

10-6 Muscle began to twitch i ·-- For symbols see table 15A . 123

-7 varies greatly between muscles. At concentrations above 10 M:, the

three muscles in which facilitation occurred began to twitch; in the other,

the level of blockade was increased. Bz, therefore, appeared to have a

weak facilitatory action which occurred at 10-SM and 10 - 7 M. However, 7 Bz significantly depressed the amplitude of them. e. p. p. s at 10- M;

therefore, the weakness of the facHitatory action may have been due to

the presence of both facilitatory and curare-like actions at this concenc.· tration.

Effect of benzoquinonium on quantal release

Bz, at concentrations which did not decrease the amplitude of the

m. e. p. p. s and e. p. p. s, produced no significant effect ori the quantal

release in the presence of a high magnesium ion concentration (12-15mM)

(50 m. e. p. p. amplitude/ 50 e. p. p. amplitude) or in low calcium (O. 92 mM)

and high magnesium (5-7 mM) concentrations, based on the number of

failures in 200 stimuli (Table 16). At concentrations where the m. e. p. p. s

and e. p. p. s were very small, due to the curare-like action of Bz, a just

significant reduction in the quantal content was observed. ~------1-2_4______;.• >.

TABLE 16 , Effect of Benzoquinonium on Quanta! Content

Quanta! Content a Concentration -Mg (10-15 mM) Ca (0. 92 m~ of benzo Mg (5-7 mM) quinonium (M) Muscle 1 Muf?cle 2 Muscle 3 Muscle 4

Control 1. 6 2.3 o. 7 1. 2

10-10 o. 6 I. 4:

10-9 0.5* 1. 1 -8 10 1.4 2.5 0.4* 1. 4 10-7 1. 7 1. 9 0.7*

Average amplitude of 50 e. p. p. s a Average amplitude of 50 m. e. p. p. s number of stimuli b Loge number of failures

* Indicates a significant decrease (25% or more; Edwards and Ikeda, 1962). ~------, ''.·r;l 125 DISCUSSION

The potency of Bz in blocking neuromuscular transmission in the

isolated tenuissimus of the cat is approximately twice that of Tc, agreeing

· with the potency reported by Bowman il958) for ·the anterior tibialis

muscle of the cat in vivo.

The e. p. p. observed during Bz paralysis had a longer time course

than that observed during Tc paralysis; the rise-time and half-decay being

approximately double. These effects can be explained on the basis of

cholinesterase inhibition by Bz. At the concentration used to block the

muscle, 2. 4 - 4. 8 x 10-6M, cholinesterase would be inhibited approximately

70-90% {Blaber and Bowman, 1962). The e. p. p. in the presence of Bz

decreased in amplitude as a function of time. The reason for this failure

to reach an equilibrium may have been due to a progressive decrease in

quantal content, however, the reduction in quantal content was only

observed at concentrations of Bz which reduced the amplitude of the e. p. p. s

and, therefore, the accuracy with which the number of failures could be

counted was also reduced. Quantal release at the concentrations of Bz

used for blockade could not be measured by the techniques used.

Facilitatory drugs, at concentrations which increase the amplitude

of e. p. p. s in the presence of Tc produced no effect on the e. p. p. s in the

presence of Bz, thus confirming the lack of effect of facilitatory drugs 126

in antagonizing Bz paralysis (Hoppe, 1950; Randall, 1951; Bowman, 1958;

Blaber and Bowman, 1962).

Ambenonium (10-7M) produced a prolongation of the time course of the

Bz e. p. p. thus showing the possibility of the further inhibition of cholin-

esterase in the presence of Bz. Bowman {1958) showed that TEPP would produce a small amount of ·antagonism against Bz; ambenonium is also a powerful cholinesterase inhibitor (Blaber, 1960) comparable in potency to 5 TEPP. 10- M ambenonium and methoxyambenonium produced depression of the e. p. p., and high concentrations of these two drugs have been shown to have a curare-like action (Blaber, 1960).

Neostigmine at 10-5M increased the time to half-decay, and slowed the rate of decline in the amplitude of the Bz e. p. p. s. Edrophonium at 4 a concentration of l0- M increased the amplitude and time course of the

Bz e. p. p. At these concentrations edrophonium and neostigmine depolarize the end-plate and also antagonize the bfocking action of Bz in vivo in the cat (Blaber and Bowman, 1959). The depolarizing action produced at high concentrations of edrophonium and neostigmine is not thought to contribute to the facilitatory a.ction (Nastuk and Alexander, 1954;

Katz and Thesleff, 1957a; Blaber, 1963). Thus it must be concluded that the facilitatory action of the drugs has ·been prevented by Bz. 127

_Bz (lo-SM) increased the amplitude of the Tc e. p. p. without any

significant change in the time course, also there was no· effect on the

m. e. p. p. s at this concentration. No increase in quanta! release has been

- shown for-Bz bythe-·techniques···used-in.--the ·p-resent study. In this respect,

it resembles the facilitatory drugs used in this study which produced

similar effects. At 10-7M , Bz also prolongs the rise-time of the Tc

e. p. p. Higher concentrations of Bz produced only depression of the Tc.

e. p. p. due to its curare-like effect. Bz has been previously shown to

antagonize the paralysis due to Tc in vivo in soleus and tibialis anterior

muscles (Hougs and Johansen, 1958).

Most of the effects of benzoquinonium can be explained on the basis

of its ability to inhibit junctional cholinesterase. Thus benzoquinonium

inhibits cholinesterase as it produces competitive blockade of the muscle.

This cholinesterase inhibition accounts for the slight augmentation by

benzoquinonium of the (+)-tubocurarine e. p. p. Also the facilitatory drugs

are not able to augment the benzoquinonium e. p. p. except at concentrations

which produce higher levels of cholinesterase inhibition (e.g. ambenonium)

or produce direct, depolarizing actions (e.g. edrophonium and neostigmine)

Although most of the evidence supports the theory of cholinesterase

inhibition, this theory is not supported by the experiment illustrated in 128

Figure 11. Edrophonium was not able to antagonize (+)-tubocurarine after

previous administration of benzoquinonium. This is similar to the

'. ability of (+)-tubocurarine to decrease a drug-induced prolongation of an

e. p. p. (Karczmar, Koketsu and Soeda, 1968). This prolongation is

generally considered to be due to cholinesterase inhibition. Yet it is

decreased by tubocurarine, even ·though tubocurarine has no· effect on

cholinesterase inhibition, in vitro (Eccles, Katz and Kuffler, 1942 and

Mcintyre and King, 1943). This may be explained somewhat by the fact

that (+)-tubocurarine is capable of slightly decreasing the half-decay of a

normal e. p. p. (Beranek and Vyskocil, 1968). 129

GENERAL DISCUSSION 130

The experimental results indicate that facilitatory drugs have several actions at the mammalian neuromuscular junction. Some of these results implicate a presynaptic mechanism of drug action for facilitatory drugs. The (+)-tubocurarine e. p. p. amplitude is augmented at a concentration which has no statistically significant effect on m. e. p. p. s. This is illustrated in Table 17. E. p. p. amplitude is augmented at 1/10 the concentration necessary to affect m. e. p. p. amplitude or time-course.

Furthermore drug-induced changes in m. e. p. p. s, when they occur, are much smaller than drug-induced changes in e. p. p. s. This differential effect of facilitatory drugs on m. e·. p. p. s and e. p. p. s suggests that facilitatory c:trugs have a presynaptic mechanism of action. 1'his conclusion is based on the assumption that m. e. p. p. s and e. p. p. s are identical with exception that m. e. p. p. s do not require a nerv-e stimulus for release. This is not completely valid, as there are several limitations in the technique.

1) M. e. p. p. s and e. p. p. s were recorded in different experiments.

2) M. e. p. p. s were recorded in normal Ringers solution but e. p. p. s

were recorded in the presence of (+)-tubocurarine.

3) The amplitude of m. e. p. p. s averaged less than one m V while

e. p. p. s averaged more than three m V.

4) M. e. p. p. s involved one quantum of transmitter while e. p. p. s 131

TABLE 17 ,

Minimal Molar Concentration of Drugs Producing ;.,._ __ . Significant Changes in M. e~ p. p. s and E. p. p. s

M. e. p. p. E~ p. p.

Amplitude Half-decay Amplitude Half-decay 7 7 8 7 Neostigmine 10- 10- 10- 10- 6 Edrophonium 10-7 10- 8 Ambenonium 10-8 10- 10-10 10-8 7 Methoxyambenonium 10-7 10-8 10- 132

involved more than one-hundred quanta.

5) M. e. p. p. s were more variable in amplitude and time-course

than e. p. p. s.

These considerations, especially the greater variability of m. e. p. p. s, make it questionable whether the effects summarized in TJ.ble 17 are attributable to a presynaptic action of the facilitatory drugs. It is possible that these results are due to a general technical inability to detect small changes in m. e. p. p. amplitude or time-course.

These complications are overcome by the method of iontophoretic application of ACh to the e~ p. However, this method also has limitations.

1) Iontophoretic applications of ACh are not physiological. They may

not be acting at the same receptors and do not exhibit the same

time-course of receptor activation as the normal release of ACh.

2) Prolonged application of ACh may affect neuromuscular trans

mission.

3) The A Ch-potentials are not always stable for the duration of an

experiment.

Facilitatory drugs produce much larger changes in ACh-potentials than in m. e. p. p. s; thus the ACh-potentials seem more responsive tothe &...-..--...... ------__,~------..... 133

effeds of facilitatory drugs than the m. e. p. p. s. At high drug concen-

trations there are no significant differences between the magnitude of l augmentation of (+)-tubocurarine e. p. p. s and the magnitude of augmen f- ~,. tation of ACh-potentials. On the basis of this observation it would appear

that most of the changes of the e. p. p. which are induced by facilitatory

drugs can be explained on the basis of postsynaptic actions. In order to

explain all the changes observed with facilitatory drugs on the basis of

postsynaptic actions it is necessary to show augmentation of the ACh-

potential and (+)-tubocurarine e. p. p. at the same minimal concentration of

facilitatory drug. These results are summarized in Tuble 18. There is

very good correlation between minimal concentrations 9f drug necessary

to augment (+)-tubocurarine· e. p. p. s and ACh-potentials. There is a

discrepancy in the results of the iontophoretic studies. The minimal drug

concentrations necessary to augment (+)-tubocurarine e. p. p. s in ion-

tophoretic studies are consistently higher than minimal concentrations

necessary to augment (+)-tubocurarine e. p. p. s in m. e. p. p. -e. p. p.

studies. At these higher concentrations facilitatory drugs are also

capable of prolonging m. e. p. p. s. The only difference in experimental

technique is the presence of an ACh-electrode in the iontophoretic studies.

Thus this electrode must be depressing the effects of facilitatory drugs. 134

TABLE 18

Minimal Molar Concentration of Drugs Producing

·- ·-·-Significant Changes in E. p~p. s and :A-Ch-Potentials

E. P. P. ACH-POTENTIAL -7 Neostigmine Exp #1· 10-7 10

Exp #' 2 10-8 10-8 6 Edrophonium Exp # 1 10- · 10-6

Exp # 2 8 8 Ambenonium Exp # 1 10- 10- -9 -8 Exp # 2 10 10 -8 8 Methoxyamb enonium Exp # 1 10 10- 10-8 10-7 Exp # 2 135

There are several possible explanations for this depressant effect.

1) A depressant action of A Ch at the neuromuscular junction~

ACh depolarizes the motor nerve terminal (Hubbard and Yokota,

1964 and Hubbard. Schmidt and Yokota, 1965), and this action

may depress the presynaptic actions of facilitatory drugs in

iontophoretic experiments.

2) Damage to the nerve terminal.

Placement of an ACh-electrode involves considerable manipu-

lation. Such movement may damage the terminal such that it

does not respond normally to pharmacological tests.

· 3) Use of low amplitude e. p. p. s.

Facilit~tory drugs are relatively ineffeclive in augmenting low

amplitude e. p. p. s in the presence of high concentrations of

(+)-tubocurarine. This is generally explained by a second action

of (+)-tubocurarine, but it is possible that the facilitatory drugs

are just not as effective in augmenting low amplitude e. p. p. s

as high amplitude e. p. p. s.

It is difficult to determine which of the above effects is responsible for a

lack of drug action in the iontophoretic experiments. As the ACh-elec- trocle had little effect on e. p. p. amplitude, it is unlikely that nerve termina 136

damage can explain these results. Any damage which would decrease the pharmacological effectiveness of a drug should also decrease physiological

_rel.ease of transmitte~•.. Any_d.epola_:r:Jzatio11 ol_a._n_erve tt;?r:i:ninal b_y A Ch would also be expected to decrease physiological release; although ACh may depress actions of facilitatory drugs by another mechanism.

In spite of this discrepancy high concentrations of facilitatory drugs are very effective in augmenting .or prolonging A Ch-potentials. In fact the ability of facilitatory drugs to affect ACh-potentials is nearly equivalent to their ability to affect e. p. p. s. On the basis of this observation it is necessary to conclude that most of the anti-Tc action of. facilitatory drugs on isolated curarized mammalian muscle preparations is due to postsynaptic actions. To determine conclusively that there is not a small presynaptic action by these drugs in the presence of (+)-tubocurarine, it would be necessary to augment e. p. p. s and A Ch-potentials at lower concentrations in iontophoretic studies.

Low concentrations of a facilitatory drug produce an increase in the rate of rise of (+)-tubocurarine e. p. p. s without prolongation of time to half-dE'.cay. This effect can be explained by increased release of transmitter, sensitization of the e. p. to the transmitter or possibly, depolarization of the e. p. by facilitatory drugs (Otsuka and Endo, 1960b). It is 137

unlikely that facilitatory drugs have sensitizing or depolarizing actions,

as these drugs are not capable of augmenting carbachol-potentials. This

appears to leave only a presynaptic mechanism.

~· It is generally assumed that cholinesterase inhibition will augment and ~- prolong the e. p. p. with no c~ange in the rate of rise. ~et in _several

experiments facilitatory drugs did increase the rate of rise of ACh-poten-

tials without prolongation. Although not reported as such, the records of

Katz and Thesleff (1957a) especially figure 1 -..,. give similar results with

edrophonium in frog muscle. This increase in ACh-potential amplitude

with no prolongation indicates that cholinesterase affects only the quantity

of ACh reaching the e. p. In other words cholinesterase inhibition, espe- ·

cially low levels of inhibition, produces no prolongation of ACh action.

Diffusion must be the major mechanism of termination of the action of

iontophoretic ACh. It is difficult to determine if the same mechanism holds

true for the involvement of cholinesterase in the generation of the e. p. p.

It has been hypothesized that diffusion is quite important in the decay of

transmitter action (Ogston, 1955), but it is unknown how important

cholinesterase is in determining the initial quantity of transmitter reaching.

the e. p. This observation on the effects of facilitatory drugs does not

support the idea that cholinesterase inhibition can not increase the rate of l 38 rise of (+)-tubocurarine e. p. p..s. This increase in the rate of rise j.s most prominent at concentrations of facilitatory drug, that do not prolong the e. p. p. At higher drug concentrations there is prolongation of the e. p. p •• indicating that cholinesterase inhibition does lengthen the action of the neuromuscular transmitter.

The primary effect of facilitatory drugs on (+)-tubocurarine e. p. p. s in isolated mammalian muscle is cholinesterase inhibition. This conclu sion is supported by studies on quantal content.. None of the drugs tested produce increases in the quanta! release of the e. p. p. at concentrations whid1 produce large increases in (+)-tu.bocurarine e. p. p. s. Thus changes in amplitude of e. p. p. s in a high magnesium ion concentration parallel changes in amplitude of m. e. p. p. s. This result indicates a mechanism of action for the facilitatory drugs which is postsynaptic, and this .action may well be cholinesterase inhibition.

Benzoquinonium blocks neuromuscular transmission much like

(+)-tubocurarine, but blockade by benzoquinonium is not antagonized by facilitatory drugs (Bowman, 1958). As benzoquinonium e. p. p. s are prolonged (compared to (+)-tubocurarine e. p. p. s). it is likely that cholinesterase is at least partially inhibited by benzoquinonium. In vitro ~·; '

] 39

results indicate that benzoquinonium is nearly as potent as neostigmine

in inhibiting cholinesterase {Blaber and Bowman, 1962). This inhibitio~

may explain the ability of benzoquinonium to augment the (+)-tubocurarine

e. p. p. amplitude. Also, previous inhibition of cholinesterase by benzo-

quinonium limits the in..'1ibition of cholinesterase produced by the facilita-

tory drugs.

The theory of cholinesterase inhibition by facilitatory drugs is support-•

ed by experiments on neuromuscular depression produced by paired stimuli

to isolated curarized frog muscle preparations. Neuromuscular depression

··, .. is not affected by edrophonium or physostigmine (Takeuchi, 1958a; Otsuka,

Endo and Nonomura, 1962 and Otsuka and Nonomura, 1963). If the facili-

tatory drugs increase the release of transmitter. they should increase the

depletion-induced depression resulting from paired stimuli. Although

this work was performed in frogs it is probably applicable to mammalian

preparations.

Although cholinesterase inhibition appears to be the primary action

of facilitatory drugs on e. p. p. s, this action does not explain the sponta -

neous twitching observed in the presence of ambenonium. Originally, it

was thought that m. e. p. p. s are augmented to such an extent by facilitatory 140

drugs that they give rise to propagated muscle action potentials. But Katz

and Thesleff (1957b) concluded that even in small muscle fibers, cholin- ·

, esterase inhibition could not augment m. e. p. p. s sufficiently to induce

muscle action potentials. It is possible that spontaneous twitching is due to

spontaneous giant potentials (Liley, 1957). except giant potentials have been

observed only in the rat diaphz:agm. Furthermore if they can give rise to

fibrillations, they can not cause fasciculations. Fasciculations must be

induced by motor unit a~tivity.

A possible origin of drug-induced fasciculations is suggested by the

experiments of Hubbard, Schmidt and Yokota (1965). Neostigmine ·(3. 3 x

10-6M) reduces the stimulus threshold of a nerve terminal in the presence

of (+)-tubocurarine or excess magnesium and decreased calciµm.Ringers.

This apparent depolarization may initiate propagated activity in the motor

nerve, which could spread throughout the motor unit giving rise to a

fasciculation and antidromic activity (Masland and Wigton, 1940).

Neostigrnine, edrophonium and ambenonium initiate spontaneous

fasciculations and spontaneous antidromic nerve action potentials in vivo

(Blaber and Goode, 1968). As neostigmine and edrophonium do not

produce spontaneous twitching in isolated muscle, it is likely that in vitro

preparations are not as responsive to facilitatory drugs as in vivo

preparations. 141

Methoxyambenonium is as potent as neostigmine and edrophonium in

augmenting the (+)-·tubocurarine e. p. p., but is unable to facilitate an

indirect, maximal twitch (Blaber, 1960). If the sole mechanism of action

_ .. ...of facilitatory drugs is cholinesterase inhibition, methoxyambenonium

should al so produce facilitation of the maximal twitch. Blaber and Goode

(1968) observed that methoxyambenonium is incapable of initiating.

spontaneous antidromic nerve activity. Initiation of spontaneous activity

may involve the same mechanism of action as facilitation of transmission.

If this is the case, methoxyambenonium would not be expected to facilitate

the muscle twitch.

Repetitive muscle activity after an orthodromic stimulus lasts for c::.

period of approximately 50 msec in the presence of a facilitatory drug

(Blaber and Bowman, 1963a). The longest (+)-tubocurarine e. p. p. observed

in the presence of high concentrations of a facilitatory drug, has a

half fall of only 2. 8 msec. This degree of prolongation would not giv·e

rise to prolonged repetitive muscle activity. It is necessary to assume

that (+)-tubocurarine drastically decreases the prolongation induced by

the facilitatory drugs (Eccles, Katz and Kuffler, 1942 and Karczmar,

Koketsu and Soeda, 1968). Only in the presence of a deficient sodium ion

concentration do facilitatory drugs pro long e. p. p. s to such an extent 142

(Fatt arid Katz, 1951 and Kordas, 1968). This discrepancy between e. p. p.

half-decay time and length of repetitive activity suggests that a mechanism

other than cholinesterase inhibition is involved in facilitation. ;---

The occurrence of spontaneous twitching makes it seem likely that a

pre synaptic mechanism is involved in the action of facilitatory drugs. The

explanation, as to why it does not occur in the e. p. p. studies, may be

involved in the depressant action of tubocurarine on the motor nerve

terrninal. Small doses of tubocurarine depress the supernormal period

of the nerve terminal spike (Hubbard and Schmidt, 1961 and Hubbard,

Schmidt and Yokota, 1965) and drug-induced antidromic nerve activity

(Werner, 1961; Blaber and Bowman, 1963b and Standaert, 1964). As the

concentration of (+)-tubocurarine used in these experiments was well over

the concentration necessary to produce 100% block of the muscle twitch, it

is quite possible that the nerve terminal action of facilitatory drugs was

completely depressed in these experiments.

Defici_ent calciurn ions and benzoquinonium ;:ire even n1ore effective

in blocking the terminal act ions of facilitatory drugs. For this reason a

terminal effect would not be expected to occur in their presence either

(Hubbard, Schmfdt and Yokota, 1965 and Blaber and Bowman, 1963a:). 143

This depressant effect of blocking drugs may be amplified by the abnormal

environment of the isolated muscle.

THEORY OF THE MECHANISM OF DRUG-INDUCED FACILITATION AND

ANTI-Tc ACTION AT THE SKELETAL NEUROMUSCULAR JUNCTION

The mechanisms of drug-induced facilitation and anti-Tc action by

facilitatory drugs are at present in hypothetical stages. All facilitatory

drugs appear to act at at least three sites:

1) cholinesterase,

2) directly at the e. p. , and

3) presynaptically.

The relative importance of each site of action depends on the particular

drug being considered.

· -- The presynaptic action of neostigmine, ed·rophonium, ambenonium and rr1ethoxyambenonium has not been clearly elucidated. It does not involve

depolarization of the motor nerve terminal in the same manner as potassium ions. Yet neostigmine, edrophonium and ambenonium do depo larize the terminal at another site, possibly the first node, as hypothesized by Hubbard, Schmidt and Yokota (1965) and Blaber and Goode (1968). This ..------v_.. ______....., ______..,., 144

initiates spontaneous twitching. Methoxyambenonium blocks at this site,

thus it is not capable of inducing spontaneous antidromic activity. · This is

probably a direct action (not mediated by ACh)(Hubbard, 1965) as ACh did

not produce spontaneous twitching. Also edrophonium and neostigmine

should have produced spontaneous twitching at some concentration if the

effect was due to ACh accumulation.

Neo$tigmine increases the duration of the nerve terminal action

potential and negative after potential (Hubbard and Schmidt, 1961 and

Hubbard, Schmidt and Yokota, 1965). A longer duration of the terminal

action potential should lead to increased release of transmitter. The

prolonged negative afterpotential may also increase release somei..~hat;

although according to Liley's theory (1956c), this release would be

insignificant. To hypothesize that an augmented negative afterpotential

increases the release of transmitter, it is necessary to hypothesize that an

additional factor is involved (maybe calcium is accumulated,

intraterminally).

The augmented negative afterpotential probably generates antidromic

nerve activity. This antidromic activity passes into the ventral root where

it is recorded. Also, when it reaches a nerve terminal branch, it may 145 pass orthodromically into another terminal. This reverberation could lead to a continuous release of transmitter.

, . In addition to a presynaptic action, facilitatory drugs inhibit junctional cholinesterase. _It is .ctifficJlll.to as_se_ss'Jhe r:o1e_Qf.cb_olineste.rase inhibition in facilitation. Yet this appears to be the primary mechanism involved in antagonism of high concentrations of (+)-tubocurarine.

The direct actions of facilitatory drugs have only a very minor role in facilitation and antagonism of (+)-tubocurarine but they may be responsible for blockade of neuromuscular transmission at high concentrations.

These same mechanisms probably are involved in the action of every drug.at the neu.r.omus cular junction. The importance of each action is determined by the chemical structure of the drug being studied.

Depolarizing drugs act primarily by a direct, depolarizing action, but they also act at the nerve terminal and inhibit cholinesterase. Competitive blockers have a potent competitive blocking action, but may act at the terminal and inhibit cholinesterase (e.g. benzoquinonium). TEA and guanidine act primarily at the nerve terminal, but also exhibit direct actions. Thus the effect of a drug depends not on any one site of action but upon the sum of actions at many sites. ·•. 146

SUMMARY

1) The facilitatory drugs (neostigmine, edrophonium, ambenonium and methoxyambenonium) produced only small changes in m. e. p. p. frequency. As this effect was small compared to the effect of potassium

ions, it was concluded that the facilitatory drugs did not significantly

change the nerve terminal membrane potential.

2) In all experiments in which no blocking drugs were present ambenonium produced spontaneous twitching.

3) Each of the facilitatory drugs augmented but did not prolong the Tc e. p. p. Furthermore this augmentation of c. p. p. amplitude occurred ai low concentrations of facilitatory drug which had no effect on m. e. p. p. amplitude or time-course. This lack of correlation implicated a presynaptic site of drug action.

4) When A Ch-potentials and Tc e. p. p. s were recorded, simultaneously; both were augmented by the same minimal concentration of facilitatory drug. However, the concentration of drug necessary to augment the Tc e. p. p. was higher when A Ch was applied to the e. p. by iontophoresis than in the experiments summarized in 3) above.

5) The facilitatory drugs did not augment the carbachol-potential at any concentration. 147

6) The facilitatory drugs did not increase the quantal content of -the e. p. p. recorded in the presence of a low ·calcium ion and high magnesium ion concentrations.

7) Benzoquinonium administration, unlike Tc, caused a continuous decline in e. p. p. amplitude. The benzoquinonium e. p. p. had a longer half-decay time, which is indicative of its ability to inhibit junctional cholinesterase. The facilitatory drugs were ineffective in augmenting the benzoquinonium e. p. p.

8) The correlation between the :minimal concentrations necessary to augment the Tc e. p.p. and ACh-potential indicates that most of the effects of the facilitatory drugs in antagonizing Tc are due to a postsynaptic action. This postsynaptic action is probably cholinesterase inhibition, as the facilitatory drugs had no effect on the carbachol potential. The lack of correlation between effects on m. e. p. p. s and Tc e. p. p. s indicates that these drugs might have a weak presynaptic action in the antagonism of Tc.

9) The conclusion that the primary effect of the facilitatory drugs is due to cholinesterase inhibition is supported by the lack 0f effect of these drugs on quantal release. Also most of the actions of benzo quinonium can be explained by it cholinesterase inhibiting properties. 148

10) Cholinesterase inhibition can not account for the production o.f spontaneous twitching by ambenonium. Thus a presynaptic effect must occur in the absence of Tc. li) On the basis of these results it seems that a presynaptic action of the fa~ilitatory drugs is minimal in the presence of high concentrations of Tc which are necessary for use of microelectrode recording techniques. ,,. .. r' .-...------149.

BIBLIOGRAPHY r ______,

150 BIBLlOGRAPHY

Adrian, E. D. (1925). The spread of activity in the tenuissimus of the

cat and in other complex muscles. J. Physiol. 60:301-315.

Anderson-Cedergren, E. (1959). Ultrastructure of motor end-plate and

sarcoplasmic components of mouse skeletal muscle fiber. J.

Ultrastruct. Res.,· Suppl. 1.

Augustinsson, K. B. (1959). Electrophoresis studies on blood plasma

esterases III. Conclusions. Acta Chemica Scand. 13:1097-1105.

Augustinsson, K. B. and Nachmansohn, D. (1949). Distinction between

acetylcholine-esterase and other choline ester-splitting enzyrnes.

Science 110:98-99.

Axelsson, J. and Thesleff, S. {1958). The 'desensitizing' effect of acetyl-

choline on the mammalian motor end-plate. -Acta Physiol.

Scand. 43:15-26.

Axelsson, J. and Thesleff, S. (1959). A study of supersensitivity iu

denervated mammalian skeletal muscle. J. Physiol. 149:178-193,

Bacq, Z. M. and Brown, G. L. (1937). Pharmacological experiments on r~.· ______..., 151

mammalian voluntary muscle in relation to the theory o.f chem-

ical transmission. J. Physiol. 89:45-60.

Bam1ister, J .• and Scrase, M. (1950). Acetylcholine synthesis in normal

and denervated sympathetic ganglia of the cat. J. Physiol. 111:

437-444.

Barnes,1 J. M. and Duff, J. I. (1953). The role of cholinesterase at the

myoneural junction. Brit. J. Pharmacol. 8:334-339.

Barrnett, R. J. (1962). The fine structural localization of acetylchol-

inesterase at the myoneural junction. J. Cell. Biol. 12:247-262.

Barrnett, R. J. (1966). Ultrastructural histochemistry of normal neuro-

muscular junctions. Ann. N. Y. Acad. Sci. 135:27-33.

Barstad, J. A. B. (1956a). The effect of d-tubocurarine on the neuro-

muscular blocks caused by diisopropylfluorophosphate and

acetylcholine. Arch. Int. Pharmacodyn. 107:4-20.

Barstad, J. A. B. (1956b). The effect of diisopropylfluorophosphate on

the neuro-muscular transmission and the importance of cholin-

esterase for the transmission of single impulses. Arch. Int.

Pharmacodyn. 107:21-32. 152

Barstad, J. A. B. (1962). Presynaptic effect of the neuromuscular

transmitter. Experientia 18: 579-580. , Bass, L. and Moore, W. J. (1966). Electrokinetic mechanism of

miniature postsynaptic potentials. Proc. Natn. Acad. Sci. U.S. A.

55:1214-1217.

Beranek, R. and Vyskocil, F .(1968). The effect of atropine on the frog

sartorius neuromuscular junction. J. Physiol. 195:493-503.

Berry, W. J(. and Evans, C. L. (1951). Cholinesterase and neuromuscular

block. J. Physiol. · 115:46P - 47P.

Berry, J. F. and Rossiter, R. J. (1958). Chemical studies of peripheral

nerve during Wallerian degeneration-VIII. Acetic thiokinase and

choline acetylase. J. Neurochem. 3: 59-64.

Birks, R. I. (1962). The effects of a cardiac glycoside on subcellular

structures within nerve cells and their processes in sympathetic

ganglia and skeletal muscle. Canad. J. Biochem. Physiol. 40:

303-315.

Birks, R. I. (1963). The role of sodium ions in the metabolism of

acetylcholine. Canad. J. Biochem. Physiol. 41:2573-2597. 153

Birks, R. I. (1966). The fine structure of motor nerve endings at frog

myoneural junctions. Ann. N. Y. Acad. Sci. 135:9-19.

Birks, R. I. artd Cohen;- M: W. {1965). Effects ofS'.odiuin oh transmitter

release from frog motor nerve terminals. Muscle. Pergamon

Press pp. 403-420.

Birks. R. I. and Cohen, M. W. (1968a). The action of sodium pump inhib-

1 itors on neuromuscular transmission. Proc. Roy. Soc. (B)

170:381-399.

Birks. R.. I. and Cohen, M. W. (1968b). The influence of internal sodium

on the behavior of motor nerve endings. Proc. Roy. Soc. (B)

170:401-421.

Birks, R. I., Huxley, H. E. and Katz, B. (1960). The fine structure of

the neuromyal junction of the frog. J. Physiol. 150:134-144.

Birks, R. I., Katz, B. and Miledi, R.. (1960). Physiological and struc-

'tural changes at the amphibian myoneural junction, in the course oj

nerve degeneration. J. Physiol. 150:145-168.

Birks, R. I., and Macintosh, F. C. (1957). Acetylcholine metabolism at

nerve endings. Brit. Med. Bull. 13:157-161. 154

Bi:cks, R. I., and Macintosh, F. C. (1961). Acetylcholine metabolism of

a sympathetic ganglion. Canad. J. Biochem. Physiol. 39:

787-327.

Blaber, L. C. (1960). The antagonism of muscle relaxants by ambenoniuni

and methoxyambenonium in the cat. Brit. J. PharmacoL

15:476-484.

Blaber, L. C. (1963). Facilitation of neuromuscular transmission by

anticholinesterase drugs. Brit. J. Pharmacol. 20:63-73.

Blabt::r, L. C. and Bowman, V./. C. {1959). A comparison betweer. the

effects of edrophonium and choline in the skeletal muscles of

the cat. Brit. J. Pharmacol. 14:456-466.

Blaber, L. C •• and Bowman, W. C. (1962). The interaction between

benzoquinonium and anticholinesterases in skeletal muscle.

Arch. Int. Pharmacodyn. 138:90-104.

Blaber, L. C. and Bowman, \V. C. (1963a). The effects of some drugs on

the repetitive discharges produced in nerve and muscle by

anticholinesterases. Int. J. Neuropharmacol. 2:1-16.

Blaber, L. C. and Bowman, W. C. (1963b). Studies on the repetitive 155

- . discharges evoked in motor nerve and skeletal muscle after

inje

20:326-344.

-- ~------~-· ------~------·------....,,.--,,--~--- .

,·l Blaber, L. C; and Goode, J. W. (1968). A comparison of the action of

facilitatory and depolarizing drugs at the mammalian motor nerye

terminal. Int. J. Neuropharmacol. 7:429-440.

Blaber, L. C. and Karczmar. A.G. (1967a). Interaction between facili-

tating and depolarizing drugs at the neuromyal junction of the cat.

J. Pharmacol. Exp. Ther. 156:55-62.

Blaber, L. C. and Karczmar, A.G. (1967b). Multiple cholinoceptive

and related sites at the neuromuscular junction. Ann. N. Y.

Acad. Sci. 144:571-583.

Blaschke, H., Bulbring, E. and Chou, T. C. (1949). Tubocurarine ant8g-

Jnism and inhibition of cholinesterases. Brit. J. Pharmacol.

4:29-32.

Bodansky, 0. and Mazur, A. (1946). Mechanism of in vitro and in vivo

inhibition of cholinesterase activity by diisopropyl fluorophosphate

Fed. Proc. 5:123. 156

Bodian, D. (1942). Cytological aspects of synaptic function. Physiol.

Rev. 22:146-169.

Bowman;-w. C. (1958). - ThEfheuromuscular blocking action of benzo.;..

quinonium chloride in the cat and the hen. Brit. J. Pharmacol.

13: 521-530.

Boyd, I. A. and Martin, A. R. {1956a). Spontaneous subthreshold activ

ity:- at mammalian neuromuscular junctions. J. Physiol. 132:

61-73.

Bo,Yd, I. A. and Martin, A. H. (1956b). The end-plate potential in mam

malian muscle. J. Physiol. 132:74-91.

Brodkin, E. and Elliot, A. C. (1953). Binding of acetylcholine. Amer .

. J. Physiol. 173:437-442.

Brooks, V. B. (W56a). Action of repetitive nerve volleys and of botulinum

toxin on miniature end-pl3.te potentials. Fed. Proc. 15:25.

Brooks, V. B. (1956b). An intracellular study of the action of repetitive

nerve volleys and of botuUnum toxin on miniature end-plate

potentials. J. Physiol. 134:264-277.

Brooks, V. B. and Thies, R. E. (1962). Reduction of quantum content 157 I

during neuromuscular transmission. J. PhysiOI. 162:2·98-310. , Brown, G. L. (1937). Action potentials of normal mammalian muscle.

~ffects of acetylcholine and e.serine~ J. ·-Physiol. --89:220-237.

Brown, G. L., Dale, H. H. and Feldberg, W. (1936). Reaction o:f the.

normal mammalian muscle to acetylcholine and to eserine. J.

Physiol. 86:394-424.

Brown, G. L. and Von Euler, V. S. (1938). The after effects of a tetanus

on mammalian muscle. J. Physiol. 93:39-60.

Brown, M. C. and Matthews, P. B. C. (1960). The effect on a muscle

twitch of the back-response of its motor nerve fibers. J. Phys-

ioL. 150:332-346.

Bulbr1ng, E. and Chou, T. C. (1947). The relative activity of prostigmine

homologues and other substances as antagonists to tubocurarine.

Brit. J. Pharmacol. 2:8-22.

Buller, A. J., Eccles, J. C. and Eccles, R. M. (1960a). Differentiation

of fast and slow muscles in the cat hind limb. J. Physiol 150;

399··416. 158

Buller, A. J., Eccles, J. C. and Eccles, R. M. (1960b). Interactions

between motoneurones and muscles in respect to the character

istic speeds of their responses. J. Physiol. 150:417-439.

Burns, B. D. and Paton, W. D. M. (1951). Depolarization of the motor

end-plate by decamethonium and acetylcholine. J. Physiol.

115:41-73.

Clark.• D. A. (1931). Muscle counts of motor units: a study in innervation

ratios. Amer. J. Physiol. 96:296-304.

Close, H. and Hoh, J. F. Y. (1968). The after-effects of repetitive stim

ulation on the isometric twitch contraction of rat fast skeletal

muscle. .T. Physiol. 197:461-477.

Cohen, J. A. and Posthumus, C. H. (1955). The mechanism of action of

anticholinesterases. Acta Physiol. Pharm. Neerl. 4:17-36.

Colomo, F. and Rahamimoff, R. (1968). Interaction between sodium and

calcium ions in the process of transmitter release at the neuro

muscular junction. J. Physiol. 198:203-218.

Cooper, S. {1929). The relation of active to inactive fibers in fractional

contraction of muscle. J. Physiol. 67:1-13. 159

Couteaux, R. (1955). Localization of cholinesterases -at neuromuscular

junctions. Int. Rev. Cytol. 4:335-375.

---couteaux1 -R:-(l958-): -Morphological-and cytochemical observations on--the

post-synaptic membrane at motor end-plates and ganglionic

synapses. Exp. Cell Res. Suppl. 5:294-322.

Cowan, s. L. (1940). The actions of eserine-like compounds upon frog's

nerve-muscle preparations, and conditions in which a single

shock can evoke an augmented muscular response. Proc. Roy.

Soc. {B) 129:356-391.

Dale, H. H., Feldberg, W. and Vogt, M. {1936). Release of acetylcholine

at vo!untary motor nerve endings. J. Physiol. 86:353-380.

Deharven, E. and Coers, C. (1959). Electron microscope study of the

human ne1ffomuscular junction. J. Biophys. Biochem. Cytol.

6:7-10.

Del Castillo, J. and Engbaek, L. {1954). The nature of the neuromuscular

block produced by magnesium. J. Physiol. 124:370-384.

Del Castillo, J. and Katz, B. {1954a). The effect of magnesium on the

a•::tivity of :motor nerve endh1gs. J. Physiol. 124:553-559. 160

Del Castillo, J. and Katz, B. (1954b). Quanta! components of the end

plate potential. J. Physiol. i24:560-573.

Del Castillo, ·3. and Katz-; B.--{1954c) .. Statistical factors involved in

neuromuscular facilitation and depression. J. Physiol. 124:

574-585.

Del Castillo, .T. and Katz, B. (1954d). Changes in end-plate activity

produced by pre-synaptic polarization. J. Physiol.. 124:586-

604.

Del Castillo, ..:r. and Katz, B. (l954e). The membrane change produced by

the neurornuscular transmitter, J. Physiol. 125: 546 ... 5-65.

Del Castillo, J. and Katz, B. (1955a}. On the localization of acetylcholine

receptors. J. Physiol, 128:157-181.

Del Castillo, S. and Katz, B. (1955b). Local activity at a depolarized

nerve-muscle junction. J. Physiol. 128:396-411.

Del Castillo, ,J. and Katz, B. (195G). Localization of active spots within

the neuromuscular junction of the frog. J. Physiol. 132:630-649.

Del Castillo, J. and Stark, L. (1952). The effect of calcium ions on the

...... 161

motor end-plate potentials. J. Physiol. 116:507-515.

DeRobcrtis, E. D. P. and Bennett, H. S. (1955). Some features of the

--subm1cros-c6plc morphoiogy of synapEfes rn.- frog- and ea:1"'thworm. - -- ·

J. Biophys. Biochem. Cytol. 1:47-58.

DeRobertis, E. D. P., Rodriquez de Lores Arnaiz, G., Salganicoff, L .•

Pellegrino de Iraldi, A. and Zieher, L. N. (1963). Isolation of

synaptic vesicles and structural organization of the acetylcholine

system within brain nerve endings. J. Neurochem. 10:225.

Dodge, F. A. a.11d Rahamimoff, R. (1967). Co-operative action· of calcium

ions in transmitter release at the neuromuscuiar junction. J.

Physiol. 193:419-432.

Douglas, W. V./. and Matthews, P. B. C. {1952). Acute tetraethylpyro

phosplrnte pDisoning in cats with its modification by atropine or

hyosc:i.ne. J. Physiol. 116:202-218.

Do1Jg1.as, \V. \V. and Paton, W. D. M. (1954). The mechanisms of mot or

end-plate depolarization due to a cholinesterase-inhibiting drug.

,T. Physiol. 124:325-344.

-- 162

Dun, R. T. and Feng, T. P. (1940). Studies on the neuromuscular junction

The site of origin of the junctional afterdischarge in muscles

treated with guanidine, barium or eserine. Chin. J. Physiol.

15:433-444.

Eccles, J.C. (1964). The:physiology of Synapses. Springer-Verlag

(Berli.n) p. 127.

Eccles, J. C. and Jaeger,. J. C. '; {1958). The relationship between the

mode of operation and the dimensions of the junctional regions

at synapses and motor end-organs. Proc. Roy. Soc. (B)

148:38-56.

Eccles, .T. C., Katz, B. and Kuffler, S. W. (1941). Nature of the 'end-

plate potential' in curarized muscle. J. Neurophysiol. 4:362-387.

Eccles, J.C., Katz, B. and Kuffler, S. W. (1942). Effect of eserine on

neuromuscular transmission.

Eccles, ,J. C. and Liley, A. W. (1959). Factors controlling the liberation

of acetylcholine at the neuromuscular junction. Arner. J. Phys. ·

Med. 38:96-103.

Eccles, J, C. and MacFarlane, W. V. (1949). Actions of anti-cholinester-

------... ·~· ..... ------.1 163 ..

ases on endplate potential of frog muscle. J. Neurophysiol.

12:5il-80.

Eccles, J. -C. arid orconnor,-·-w.- J: -(~939)~ - Res:l:Yorises-whichnerve

impulses evoke in mammalian striated muscles. J. Physiol.

97:44-102.

Eccles, J. C. and O'Connor, W. J. (1941). Abortive impulses at the neuro

muscular junction. J. Physiol. 100:318-328.

Edwards, C. and Ikeda, K. (1962). Effects of 2-PAM and succinylcholine

.. on neuromuscular transmission in the frog. J. Pharmacol. Exp.

Ther. 138:322-328.

Elliott, K. A. C. and Henderson, N. (1951). Factors affecting acetyl-

choline found in excised rat brain. Amer. J. Physiol. 165:

365-374.

Elmqvist .. D. (1965). Potassium induced release of transmitter at the

human neuromuscular junction. Acta Physiol. Scand. 64:

340-344.

Eimqvist, D. and Feldman, D. S. (1965a). Calcium dependence of spon-

taneous acetylcholine release at mammalian motor nerve termi- 164

nals. J. Physiol. 181:487-497.

Elmqvist, D. and Feldman, D. S. (l965b). Effects of sodium pump inhibi

-- tors on -spontaneous -acetylcholine release at the neuromuscular -

junction. J. Physiol. 181:498-505.

Elmqvist~ D. and Feldman, D. S. (1965c). Spontaneous activity at a

mammalian neuromuscular junction in tetrodotoxin. Acta

Physiol. Scand. 64:475-476.

Elmqvist, D. c;_nd FeJdn~an, _D. S. (1966). Influence of ionic environment

on acetylc.:holine release from the motor nerve terminals. Acta

Physio1. Scand. 67 ·: 34-42.

Elrn.qvist. D., Johns, T. R. and Thesleff, S. (1960). A study of some

electrophysiological properties of human intercostal muscle. J.

PhysioL 154: 602-607.

Elmqvist, IJ. and Quastel, D. M. J. (1965). A quantitative study of end

plate potentials in isolated human muscle. J. Physiol. 178:

505-529.

Engelhart, E. and Loewi, 0. (1930). Fermentative acetylcholinspaltung

im blut und ihre hemmung durch physostigmin. Arch Exp. 165

Path. Pharmak. 150:1-13.

Fatt, P. and Katz, B. (1950). Some observations on biological noise.

. . . ---.--· ------•- e••7 -

Fatt, P. and Katz, B. (1951). An analysis of the end-plate potential re--

corded with an intra-cellular electrode. J. Physiol. 115:320-370.

: J . ~--·: Fatt, P. and Katz, B. (1952). Spontan~ous subthreshold activity at motor

·''.,, ' l. net've endjngs. J. Physiol. 117:109-128.

Feldbe:cg, W. (1943). Synthesis of acetylcholine in sympathetic ganglia

and cholinergie nerves. J. Physiol. 101:432-~45.

· Feng, T. P. (191_6). Studies on the netlromuscular junction U. The

universal antagonism between caldum and curarizing agencies.

Chin, J. Physiol. 10:513-528.

Feng, T. P. (1937). Studies on the neuromuscular junction. VI. Poten-

tiation by eserine of response to single indirect stimulus in

aniphibian nerve-muscle preparations. Chin. J. Physiol. 12:

51-58 .

.Feng, T. P. (1940). Studies on th.e neuromuscular junction. XVIII. The 166

local potentials around N - Mjunctions induced by single and

muUiple volleys. Chin. J. Physiol. 15:367-404•

. Feng, "T. ·p. I ·Lee, -L. -y.- I· Meng; -c.~·w:-·and·wang.-s~--- e~- {1938) .. ·-Studies

on the neuromuscular junction. IX. The after effects of tetani

zation on N-M transmission in cat. Chin. J. Physiol. 13:79-108.

Feng, T. P. and Li, T. H. (1941). Studies on the neuromuscular junction.

XXIII. A new aspect of the phenomena of eserine potentiation

and post-tetanic facilit9.tion in mammalian muscles. Chin. J.

Physiol. 16:37-56 ..

Feng, T. P. and Ting, Y. (1938). Studies on the neuromuscular junction.

XI. A note on the local concentration of cholinesterase of motor

nerve endings .. Chin. J. Physiol. 13:141-144.

Frank, G. B. (1959). Increased sensitivity of an end-plate region to its

cherni cal mediator following denervation of another end-plate

region in the same cell. Canad. J. Biochem. Physiol. 37:

1239-1246.

Fulton, J. F. (1926). Muscu]ar Contraction and the Reflex Control of

Movement. Williams and Wilkins (Baltimore} p. 369. 101

Furshpan, E. J. (1956). The effects of osmotic pressure changes on the

spontaneous activity at motor nerve endings. J. Physiol. 134:

689-697.

Gage, P. W. {1967). Depolarization and excitation-secretion coupling in

presynaptic terminals. Fed. Proc. 26:1627-1632.

Gage, P. W. and Hubbard, J. I. {1964). Ionic changes responsible for

· post-tetanic hyperpolarization. Nature 203:653-654.

Gage, P. w·. and Hubbard, J. I. (1965). Evidence forapoisson distribution

of miniature end-plate potentials and some implications. Nature

208:395-396.

Gage, P. V/. and Hubbard, J. I. (1966a). The origin of the post-tetanic

hyperpolarization of mammalian motor nerve terminals. · J.

Physiol. 184:335-352.

Gage, P. W. and Hubbard, J. I. (1966b). An investigation of th_e post-

teto.rdc potentiation of end-plate potentials at a mammalian neuro-

muscular junction. J. Physiol. 184:353-375.

Gage, P. W. and Quastel, D. M. J. (1965a). Dual effect of potassium on

transmitter release. Nature 206:625-626. 168

Gage, P. W. and Quastel, · D. M. J. {1965b). Influence of sodium ions

on transmitter release. Nature 206:1047-1048.

Gage, P. W. and Quastel, D. M. J. (1966). Competition between sodium

and calcium· ions in transmitter release at mammalian neuro-

muscular junctions. J. Physiol. 185:95-123.

Geren, B. B. (1954). The formation from the schwann cell surface of

myelin in the peripheral nerves of chick embryos. Exp. Cell

Res. 7:558-562.

Goldsmith, T. H. (1963). Rates of action of bath-applied drugs at the I neuromuscular junction of the frog. J. Physiol. 165:368-386.

Goode, J. W., Blaber, L. C. and Karczmar, A.G. (1965). Inhibition of

cholinesterase by facilitatory drugs at neuromyal junction. The

Pharmacologist 7:188.

Gopfert, H. and Schaefer, H. (1938). Uber den direkt und indirekt

erregten aktionsstrom und die funktion der motorischen endplatte.

Pflugers Arch .Ges. Physiol. 239:597-619.

Grumbach, L. and Wilber, D. T. _(1940). Post-tetanic potentiation and

suppre.'3sion in muscle. Amer . .J. Physiol. 130:433-444. lo9

Guttman, S. A •• Horton, R. G. and Wilber. D. T. (1937). Enhancement of

muscle contraction after tetanus. Amer. J. Physiol. 119:

463-473.

Hebb, C. O. and Silver, A. (1961). Gradient of choline acetylase activity.·

Nature 189:123-125.

Hebb, C. O. and Smallman, B. N. (1956). Intracellular distribution of

choline acetylase. J. Physiol. 134:385-392.

Hebb, C. O. and Waites, G. M. H. (1956). Choline acetylase in antero

and retrograde degeneration of a cholinergic nerve. J. Phy&lol.

132:667-671.

Hebb. c. O. and V/hittaker, V. P. (1958). Intracellular distribution of

acetylcholine and choline acetylase. J. Physiol. 142:187-196.

Hill, A. V. (1949). The abrupt transition from rest to activity in muscle.

Proc. Roy. Soc. (B) 136:399-420.

Hines, H. M., Thomson, J. D. and Lazere, B. {1942). Quantitative studies

on muscle and nerve regeneration in the rat. Amer. J. Physiol.

137:527-532.

Hobbiger, F. (1952). The mechanism of anticurare action of certain 170

neostigmine analogues. Brit. J. Pharmacol. 7:223-236.

Hodes, R. and Steiman, S. E. (1939). The effects of acetylcholine and

· - -eserine ort frog muscle. Amer. J. Physiol. 127:470-479.

Hoppe, J. O. (1950). A pharmacological investigation of 2, 5 bis- (3-

diethylaminopropylamino) benzoquinone -bis -b enzy1 ·chloride·

(WIN 2747): a new curarimimetic drug. J. Pharmacol. Exp.

Ther. .100:333-345.

Hoppe, J. 0. (1951). A new series of synthetic curare-like compounds.

Ann. N. Y. Acad. Sci. 54:395-406.

Hougs, V.l. and Johansen, S. (1957). Relation of benzoquinonium to other

neuromuscular blocking agents. Acta Pharmacol. Tox. 13:

410-419.

Hougs, W. and Johansen, S. (1958). The interaction of benzoquinonium

and other neuromuscular blocking agents. Acta Pharmacol.

Tox. 14:385-389.

Hubbard, J. I. (1959). Post-activation changes at the mammalian neuro

muscular junction. Nature 184:1945. 171

Hubbard, J. I. {1961). The effect of calcium and magnesium on the spon-

tanecus release of transmitter from mammalian motor rierve

endings. J. Physiol. 159:507-517. rh:.bbard, J. I. (1963). Repetitive stimulation at the mammalian neuro-

muscular junction, and the mobilization of transmitter. J.

Physiol. 169:641-662.

Hubba1·d, J. I. (1965). Studies in Physiology. Springer-Verlag (Berlin)

pp. 85-92.

Hub0ard, J. I., Jones, S. F. and Landau, E. M. (1967). The relatiunship

between the state of nerve-terminal polarization and Hberation of

acetylcholine. Ann. N. Y. A cad. Sci. 144:459-469.

Huhbard, J. I., ~Tones, S. F. and Landau, E. M. (1968a). On the mechanisn

by which calcium and magnesium affect the spontaneous release

of t:t·Hnsrnitter from mammalian motor nerve terminals. J.

Physiol. 194:355-380.

Hubbard, J. J., Jones, S. F. and Landau, E. M. (1968b)~ An examination

of the effects of osmotic pressure changes upon transmitter re-

lease from mammalian motor nerve terminals. J. Physiol. 172

Hubbard, J. I. and Kwanbunbumpen (1968). Evidence for the vesicle

hypothesis. J. Physiol. 194:407-420.

Hubbard, -J. I. a.nd Loyning,-y: (1966). The effects of hypoxia on neuro

muscular transmission in a mammalian preparation. J. Physiol. ·

185:205-223.

Hubbard, J. I. and Schmidt, R. F: (1961). Stimulation of motor nerve

terminals. Nature 191:1103-1104.

Hubbard, J. I. and Schmidt, R. F; (1962). Repetitive activation of motor

nerve t:ndings. Nature 196:378-379.

Hubbard, J. I. and Schmidt, R. F. (1963). An electrophysiological

investigation of mammalian motor nerve terminals. J. Physiol.

166:145-167.

Hubb:=u:d, J. I., Schmidt, R. F. and Yokota, T. {1965). The effect of

aceiylcholine upon mammalian motor nerve terminals. J.

Physiol. 181:810-820.

Hubbard .J. I. and 'Willis, W. D. {1962a). Mobilization of transmitter by

hyperpolarization.. Nature 193:174-175.

Hubbard, J. I. and Willis, W. D. (19 62b). Reduction of transmitter output 173

by depolarization. Nature 193:1294-1295.

Hubbard, J. I. and Willis, W. D." (1962c). Hyperpolarization of mammalian

·· motor nerve terminals. -J :- ~hysiol. --1.63:115:..;137.

Hubbard, J. I. and Willis, W. D. {1968). The effects of depolarization of

motor nerve terminals upon the r_elease of transmitter by nerve

impulses. J. Physiol. 194:381-405.

Hubbard, J. I. and Yokota, T. {1964). Direct evidence for an action of

aceiylcholine on motor nerve terminals. Nature 203:1072-1073.

Hutter, O. F. {1952). Post-tetanic restoration of neuromuscular trans-

mission blocked by d-tubocurarine. J. Physiol. 118:216-227.

Hutter, O. F. and Kostial, K. {1954}. Effect of magnesium and calcium ion

·- -on the release of acetylcholine. J. Physiol. 124:234-241.

Hutter, O. F. and Trautwein, V./. {1956}. Neuromuscular facilitation by

stretch of motor nerve-endings. J. Physiol. 133:610-625.

Hyman, L. H. (1962). Comparative Vertebrate Anatomy•. The University

of Chicago Press {Chicago} p. 244 .

.Jenkinson, D. H. {195'7). The nature of the antagonism between calcium 174

and magnesium ions at the neuromuscular junction. J. · Physiol.

138:434-444.

Je-rikinson, D:"I·L -(1960).- -The antagoriism-0-etweeri tubociirarine- ancrsuo::..

stances which depolarize the motor end-plate. J. Physiol.

152:309-324.

Karczmar, A. G~' (1957). Antagonism between a bis-quaternary oxamide,

"WIN 8078, and depolarizing and competitive blocking drugs. J.

Pharmacol. Exp. Therap. 119:39-47.

Karczmar. A.G. (1961). Pharmacologic effects of newer bis-quaternary

anticholinesterases. particularly at the neuromyal junction.

Bioeledrogenesis. Elsevier Publishing (New York) pp. 320-340.

Karczmar, A. a;· and Howard, S;W. (1955). Antagonism of d-tubocura

rine and other pharmacologic properties of certain bis-quater -

rary salts of basically substituted oxamides (WIN 8077 and

analogs). J. Pharmacol. Exp. Ther. 113:30.

Karczmar, A. G;', Kim, K. C. and Koketsu. K. (1961). Endplate effects

and antagonism to d-tubocurarine and decamethonium of tetra

ethylammonium and meth.oxyarn.benonium' ("WIN8078). J. Phar- 175

col. Exp. 'Ther. 134:199-205. , Karczmar, A. G;, Koketsu, K. and Soeda, S. (1968). Possible reactiva-

ti.ng and sensitizing action of ·neuromyally acting agents. Int.

J .. Neu.ropharmacol. 7:241-252.

Katz, B. (1842). Impedance changes in frog's muscle associated with

electrotonic and "endplate" potential. J. Neurophysiol. 5:

169-184.

< ' TI (-.···i:.,O) I "a·i.:z, 1 .• ~ ..t~tuc.·. 1\tlic:rophysiology of the neuro-muscular junction. A

phys:i.clogical 'quantum of act:i.on: at the myoneural junction. Bull.

Johns Hopk. Hosp. 102:275-295.

Katz, B. (1962). The transmission of impulses from nerve to muscle, and

the subcellular unit of synaptic action. Proc. Roy. Soc. (B)

155:455-4 79.

Kr.tz, B. and Miledi, R. (196la). The localized action of 'end-plate drugs'

in the twitch fibers of the frog. J. Physiol 155:399-415.

Katz, B. and Miledi, R. (196lb). The development of acetylcholine

sensitivity in nerve-free muscle segments. J. Physiol. 156: 176

Katz, B. and Miledi, R. (1965a). Release of acetylcholine from· a nerve

terminal by electric pulses of variable strength and duration.

Nature 207:1097-1098.

Katz, B. and Miledi, R. (1965b). The effect of temperature on the syn~ ·

aptic delay at the neuromuscular junction. J. Physiol. 181:

656-670.

Katz, B. and Miledi, R. (1965c). Propagation of electric activity in

motor nerve terminals .. Proc. Roy. Soc. (B) 161:453-482.

Katz, B. and Miledi, R. (1965d). The measurement of synaptic delay,

and the time course of acetylcholine release at the neuromuscular

junction. Proc. Roy. Soc. (B) 161:483-495.

Katz, B; and Miledi, R. (1965e). The effect of calcium on acetylcholine

release from motor ner:ve terminals. Proc. Roy. Soc. (B)

161:496-503.

Katz, B. and Miledi, R. (1967a). Modification of transmitter release

by electrical interference with motor nerve endings. Proc.

Roy. Soc. (B) 167:1-7.

Katz, B. and MHedi, R. (1967b). Tetrodotoxin and neuromuscular trans- ...... -----=------0..-...l! 177

mission. Proc. Roy. Soc. (B) 167:8-22.

Katz, B. and Miledi, R. (1967c).. The release of acetylcholine from nerve

. -.endings by graded electric.pulses.. -Proc•. Roy. Soc. (B) 167:-

23-38.

Kat7., B. and Miledi, R. (1967d) .. The timing of calcium action during

. neuromuscular transmission. J. Physiol. 189:535-544.

Katz, B. and M:iledi, R. (1968). The role of _calcium in neuromuscular

faci1itatior•. J. Physiol. 195:481··492.

Kat~. B. and Thesleff, S. (1957a). The interaction between edrophonium

(tensilon) and acetylcholine at the motor end plate. Brit.

J. Pharrnacol. 12:260-264.

Katz, B. and Thesleff, S. {1957b). On the factors which determine the

ampytude of the 'niiniature end .. plate potential'. J. Physiol.

137:26'1-278.

Kelly, J·. S. {18G5). Antagonism betv..-een Na+ and Ca++ at the neuromus-

cular junction. Nature 205:296-297.

Kim, K. C; and Karczmar, A.G. (1967). Adaptation of the neuromuscular

junction to constant cot1ccnb.'ation of Ach. Int. J. Neuropharrnacol. 178

6:51-61.

Koelle, G;B. (1946). Protection of cholinesterase against irreversible

inactivation by di-isopropyl fluorophosphate i.n vitro. J. Phar

macol. Exp. Ther. 88:232-237.

Koelle, G."'B. (1950). The histochemical differentiation of types of cho··

linesterases and their localizations in tissues of the cat. J.

Pharmacol. Exp. Ther. 100:158-179.

Koelle, G. B. (1951). The elimination of enzymatic diffusion artifacts

in the histochemical localization of cholinesterases and a survey

of their cellular distributions. J. Pharmacol. Exp. Ther.

103:153-171.

Koelle, G.''B. (1957). Histochemical demonstration of reversible anti

cholinesterase action at selective cellular sites in vivo. J.

Pharmacol. Exp. Ther. 120:488-503.

Koelle, G. B. and Friedenwald, J. S. (1949). A histochemical method

for localizing cholinesterase activity. Proc. Soc. Exp. Biol.

70: 617-622.

-Koketsu, K~ (1958). Action of tetraethylammonium chloride on neuro

muscular transmission in frogs. Amer. J. Physiol. 193: 179

213-218.

Koppanyi, T. and Vivino, A. E. (1944). Prevention and treatment of d-

- tubocurarine poisoning. - Science 100~474-475~

Kordos, M. (1958). A study of the end-plate potential in sodium deficient

solution. J. Physiol. 198:81-90.

Korkes, S., Del Campillo, A., Korey, S. R., Stern, J., Nachmansohn, D.

and Ochoa, S. {1952). Coupling of acetyl donor systems with

cho~_ine acetylase. J. Biol. Chem. 198:215-220.

Koster, R. {1946}. Synergisms and antagonisms between physostigmine - _, I and di-isopropyl fluorophosphate in cats. J. Pharrnacol. Exp.

'Ther. 88:39-46.

Krnjevic, K. and Miledi, R. (1958). Failure of neuromuscular propagation

in rats. J. Physiol. 140:440-461.

Krnjevic, K. and Miledi, R. (1959). Presynaptic failure of neuromuscular

propagation in rats. J. Physiol 149:1-22.

Krnjevic, K. and Mitchell, J. F: (1961). The release of acetylcholine in

the isolated rat diaphragm. J. Physiol. 155:246-262. 180

Kuffler, S. W.' (1942). · Electric potential changes at an isolated p.erve

muscle junction. J. Neurophysiol. 5:18-26.

Kuffler, s. w: (1944). The effect of calcium on the neuromuscular junc

tion. J. Neurophysiol. 7:17-26.

Kuperman, A. S;, Gill, E. and Riker, W. F~ (1961). The relationship

between cholinesterase inhibition and drug-induced facilitation

of mammalian neuromuscular transmission. J. Pharmacol.

Exp. Ther. 132:65-73.

Kuperman. A:s. and Okamoto, M. (1964). The relationship between

anticurare activity and time course of the endplate potential, a

structure-activity approach. Brit. J. Pharmacol. 23:575-591.

Lands, A;M., Karczmar, A. G~\ Howard, J. W. and Arnold, A. (1955).

An evaluation of the pharmacologic actions of some bis-quater

nary salts of basically substituted oxamides (\V1N 8077 and

analogues). J. Pharmacol. Exp. Ther. 115:185-198.

Langley, J. N; (1907). On the contraction of muscle, chiefly in relation

to the presence of "receptive" substances. Part I. J. Physiol.

36:347-384. 181

Langley, .T. N. and Kato, T. (1915). The physiological action of. physo-

stig:tnine and· its action on denervated skeletal muscle. J.

Physiol. 49: 410-431.

Li, T. H. anc Ting, Y. C. (1941). Studies on the neuromuscular junction.

XXL Responses of cat muscles to acetylcholine during Wedensky

inhibition and post-tetanic facilitation. Chin. J. Physiol. 16:1-8.

LUey, A. W. (1956a). An investigation of spontaneous activity at neuro-

muscular junction of the rat. J. Physiol. 132:650-666.

The quantnl components of the mammalian end-

plate potential. J. Physiol. 133:571-587.

Liley, A. \V. (i956c). The effects of pre synaptic polarization on the

spontaneous activity at the mammalian neuromuscular junction.

J. Physiol. 134:427-443.

I ;l·1 ey, _A... • '-er'V ~ (1., J.V "5'"') • " Spontaneous release of transmitter substance in

multiquantal units. J. Physiol. 136:595-605.

Liley, A. VI. and North, K. AK. (1953). An electrical investigation of

effects of repetitive stimulation on mammalian neuromuscular

junction. J. Neurophysiol. 16:509-527. 182

Lilleheil, G. and Naess, K. (1961). Note on the effect of increased NaCl-

concentration on the neuromuscular transmission. Acta Physiol.

Scand. 52:23-31.

Ling, G. and Gerard, R. W. (1949}. The normal membrane potential of

frog sartorius fibers. J. Cell. Comp. Physiol. 34:383-396.

Lloyd, D. P. C. (1941). Centripetal discharges in dorsal and ventral roots

following stimulation of muscle by ventral root volleys. Proc.

Soc. Exp. Biol. 47:44-47.

Lloyd, D. P. C. (1942). Stim·1lation of peripheral nerve terminations by

active muscle. J. Neurophysiol. 5:153-165.

Lloyd, D • .e. C. (1950). Post-tetanic potentiation of response in mono-

synaptic reflex pathways of the spinal cord. J. Gen. Physiol.

33:147-170.

Luco, J. V. and Eyzaguirre, C. (1955). Fibrillation and hypersensitivity

to ACh in denervated muscle: effect of length of degenerating

nerve fibers. J. Neurophysiol. 18:65-73.

Lundberg, A. and Quilisch, H. (1953a). Presynaptic potentiation and

depression of neuro-muscular transmission in frog and rat. 183

Acta Physiol. Scand. 30 (Supple. 111):111-120.

Lundberg, A. and Quilisch, H. (l953b) . On the effect of calcium on pre-

· ·· ··- synaptic potentiation and· depression at the neuromuscular junction

Acta Physiol. Scand. 30(Supple. 111):121-129.

Macintosh, F. C. (1959}. Formation, storage, and release of acetylcho-

: line at nerve endings. Canad. J. Biochem. Physiol. 37:343-356.

Mcintyre, A. R. and King, R. E. (1943}. d-tubocurarine chloride and

cholinesterase. Science 97:69.

Mackworth, J. F. and ·webb, E. c. (1948). The inhibition of serum cho-

linesterase by alkyl fluorophosphonates. Biochem. J. 42:91-95.

MacLagen, J. (1962}. A comparison of the responses of the tenuissimus

muscle to neuromuscular blocking drugs ~n vivo and in vitro.

Brit. J. Pharmacol. 18:?.04-216.

Macpherson, L. and Wilkie, D.R. (1954). The duration of the active

state in a muscle twitch. J. Physiol. 124:292-299.

Mallart, A. and Martin, A. R. (1967). An analysis of facilitation of

transmitter release at the neuromuscular junction of the frog.

J. Physiol. 193:679-694. 184

Mallart, A. and Martin, A. R. (1968). The relation between quantum con

tent and facilitation at the neuromuscular junction ~f the. frog. J.

Physiol. 196:593-604.

Marnay, A. and Nachmansohn, D. (1938). Cholinesterase in voluntary

muscle. J. Physiol. 92:37-47.

Martin, A. R. (1955). A further study of the statistical composition of the

end-·plate potential. J. Physiol. 130:114-122.

Martin, A. R. (1966). Quantal nature of synaptic transmission. Physiol.

Rev. 46:51-66.

Masland, R. L. and Wigton, R. S. (1940). Nerve activity accompanyjng

fasciculation produced by prostigmin. J. Neurophysiol.

3:269-275.

Matthes, K. (1930). The action of blood on acetylcholine. J. Physiol.

70:338-348.

Mazur, ,ti,., a~1d Bodansky, 0. (1946). The mechanism of in vitro and in

vivo inhibition of cholinesterase activity by diisopropyl fluorophos

phate. J. Biol. Chem. 163:261-276. ·

= 185

Michaelson, A. (1967). The subcellular distribution of acetylcholine,

choline acetyltransferase and acetyl cholinesterase in nerve

tissue. Ann. N. Y. Acad. Sci. 144:387-407.

Michel, H. O. and Krop, S. (1951). The reaction of cholinesterase with

di-isopropylfluorophosphate. J. Biol. Chem. 190:119-125.

Miledi, R. {1959). Acetylcholine sensitivity of partially denervated frog

muscle fibers. J. Physiol. 147:45P-46P.

Miledi, R. (l960a). The acetylcholine sensitivity of frog muscle fibers

after complete or partial denervation. J. Physiol. 151:1-23.

Miledi, R. (1960b). Properties of regenerating neuromuscular synapses

in the frog. J. Physiol. 154:190-205.

Miledi, R. (1962). Induced innervation of end-plate free muscle segments.

Nature 193:281-282. l\/liledi, R. (1964). Electron-microscopial localization of products from

histochemical reactions used to detect cholinesterase in muscle.

Nature 204:293-295.

Miledi, R. (1966). Strontium as a substitute for calcium in the process of 186

transmitter release at the neuromuscular junction. Nature 212:

1233-1234.

-Nachmansohn, -D. and Field, E. A~ (1947). Studies on cholinesterase. IV.

On the mechanism of diisopropyl fluorophosphate action in vivo.

J. Biol. Chem. 171:715-724.

Nachmansohn, D .. and Machado, A. L. (1943). The formation of acetyl

choline. A new enzyme: "choline acetylase". J. N euro -

physiol. 6:397-403.

Nastuk, Vv. L. (1953a). Membrane potential changes at a single muscle

end-plate produced by transitory application of acetylcholine with

an electrically controlled microjet.. Fed. Proc. 12:102.

Nastuk, W. L. (1953b). The electrical activity of the muscle cell membrane

at the neuromuscular junction. J. Cell. Comp. Physiol. 42:

249-272.

Nastuk, W. L. and Alexander, J. T. (1954). The action of 3-hydroxy

phenyldimethylethylammonium (tensilon} on neuromuscular trans

mission in the frog. J. Pharmacol. Exp. Ther. ll1:302-328.

Nastuk, \V. L. and Hodgkin, A. L. (1950). The electrical activity of single 187

muscle fibers. J. CelL Comp. Physiol. 35:39-74.

Ogston, A.G. (1955). Removal of acetylcholine from a limited volume by

diffusion. J. Physiol. 128:222-224.

Otsuka, M. and Endo, M. (1960a). Presynaptic nature of neuromuscular

depression in the frog. Nature 188:501-502.

Otsuka, M. and Endo, lVI. (1960b). The effect of guanidine on neuromuscu ..

la.r transmission. J. Pharmacol. Exp. Ther. 128:273-281.

Otsuka, M., Endo, M, and Nonom.ura, Y. (1962). Presynaptic nah1re of

neuro-m.uscula:c depression. Jap. J. Phyoiol. 12:573 -584.

Otsuka, M. ::1.11d Nonomura, Y. (1963). The action of phenolic substances

on motor nerve endings. J. Pharm::icol. Exp. Ther. 140:41-45.

Pal, J. {1'300). Physostigmin, ein gegengift des curare. Zbl. Physiol.

14:255-258.

Pa1ade, G. E:. and Palay, S. L. (1954). Eledron microscope observations

on interncuronal and neuromuscular synapses. Anat. Hee.

118:335.

Palay, S. L. (1958). The morphology of synapses in the central nervous

system. Exp. Cell Res. Supple. 5:2'75-293. Parsons .. R. L., Hofmann, W.W. and Feigen, G. A. (1965). Presynap-

tic effects of potassium ion on the mammalian neuromuscular

junction. Nature 208:590-591.

Paton, W. D. M. and Waud, D.R. (1967). The margin of safety, of neuro-

muscular transmission. J. Physiol. 191:59-90.

Perry, W. L. M. (1953). Acetylcholine release in the cats superior cer-

vical ganglion. J. Physiol. 119:439-454.

Rahami moff, R. (1968}. A dual effect of calcium ions on neuromuscular

facilitation. J. Physiol. 195:471-480.

Rrtham1moff, R. and Colomo, R. (1967). Inhibitory action of sodium ions

on transmitter rele a se at the motor end-plate. Nature 215:

11'74-1176.

H2ndall, L. O. (1950). Anticurare action of phenolic quaternary ammonium

salts. J. Pharmacol. Exp. Ther. 100:83-93.

Randall, L. o. (1951). Synthetic curare-like agents and their antagonists.

Ann. N. Y. A cad. Sci. 54:460-479 .

. Randall. L. O. and Jampolsky, L. M. {1953). Pharmacology.of drugs

affecting skeletal muscle. Amer. J. Phys. Med. 32:102-125. 189

Randall, L. O. and Lehmann, G. (1950). Pharmacological properties of

some neostigmine analogs. J. Pharmacol. Exp. Ther. 99:16-32.

Raventos, J .--'(1937). The effects of arteriarinjection of drugs on the

frog's gastrocnemius. J. Physiol. 90:8P-9P.

Reger, J. F. (1954). Electron microscopy of the motor end-plate in

intercostal muscle of the rat. Anat. Rec. 118:344.

Reger, J. F. (1958). The fine structure of neuromuscular synapses of

gastrocnemii from mouse and frog. Anat. Rec. 130:7-24.

Riker, W. F. (1953). Excitatory and anti-curare prope:i;ties of acetyl

choline and related· quaternary ammonium compounds at the

neuromuscular junction. Pharmacol. Rev. 5:1-86.

Riker, W. F •• Roberts, J •• Standaert, F. G. and Fujimori, H. (1957).

The motor nerve terminal as the primary focus for drug induced

facilitation of neuromuscular transmission. J. Pharmacol.

Exp. Ther. 121:286-312.

Riker, W. F., Werner, G .• Roberts, J. and Kuperman, A. (1959).

Pharmacologic evidence for the existence of a presynaptic event

in neuromuscular transmission. J. Pharmacol. Exp. Ther. 190

125:150-158.

' Riker, W. F. and Wescoe, W. C. (1946). The direct action of prostigmine

on- skeletal muscfeT-its relat~oriship to the cno1ine esters. J.

Pharmacol. Exp. Ther. 88:58-66.

Riker, W. F. and Wescoe, W. C. (1950). $tudies on the inter-relationship

of certain cholinergic compounds. V. The significance of the

actions of the 3-hydroxyphenyltrimethylammonium ion on

neuromuscular function. J. Pharmacol. Exp. Ther. 100:

454-464.

RJtchie, .J.M. and Wilkie, D.R. (1955). The effect of previous stimulation

on the active state of muscle. J. Physiol. 130:488-496.

Robertson, J. D. (1954). Electron microscope observations on a reptilian

myoneural junction. Anat. Rec. 118:346.

Robertson, J. D. (1955). The ultrastructure of adult vertebrate periph-

er a 1 · myelinated nerve fibers in relation to myelinogenesis.

J. Biophys. Biochem. Cytol. 1:271-278.

Robertson, J. D. (1956}. The ultrastructure of a reptilian myoneural

junction. J. Biophys. Biochem. Cytol. 2:381-394. 191

-- Rosenblueth, A •• Lindsley, D. B. and Morison, R. S. (1936). A study

·or some decurarizing substances. Amer. J. Physiol. 115:53-68. ,

Rosenblueth, A. and Morison, R~;S. (1937). Curarization, fatigue and

Wedensky inhibition. Amer. J. Physiol. 119:236-256.

Rothberger, J.C. (1901). Uber die gegenseitigen beziehunger zwischen

curare und physostigmin. Pfluge.rs Arch. Ges. Physiol. 87:

117-169.

Sawyer, C.H. (1946). Cholinesterases in degenerating and regenerating

nerves. Amer. J. Physiol. 146:246-253.

Sherrington, C. S. (1925). Remarks on some aspects of reflex inhibition.

Proc. Roy. Soc. (B} 97:519-545.

Smith, C. M •• Cohen, H. L., Pelikan, E.W. and Unna, K. R. (1952).

Mode of action of antagonists to curare. J. Pharmacol. Eip.

Ther. 105:391-399.

Snell, R. S. and Mcintyre, N. (1955). Effect of denervation on the histo-

chemical appearance of cholinesferase at the myoneural junction.

Nature 176: 884-885.

Standaert, F. G~ (1964). The a.ction of d-tubocurarine on the motor nerve . terminal. J. Pharmacol. Exp. Ther~ 143:181-186.

Stedman. E. (1926). Studies on the relationship between chemicar

constitution and physiologic action. Part I. Position isomerism

in relation to the miotic activity of sorrie synthetic urethanes.

Biochem. J. 20:719-734.

Stedman, E .• Stedman, E. and Easson, L. H. (1932). Choline-esterase.

:An enzyme present in the blood serum of the horse. Biochem.

J. 26: 2056-2066.

Stovner, J. (1958). The anticurare activity of tetraethylammonium (TBA).

Acta. Pharm. Tox. 14:317-322.

Straughan, D. W. (1960). The release of acetylcholine from mammalian

motor nerve endings. Brit. J. Pharmacol. 15:417-424.

Takeuchi, A. (1958a). The long-lasting depression in neuromuscular

transmission of frog. Jap. J. Physiol. 8:102-113.

Takeuchi, N. (1958b). The effect of temperature on the neuromuscular

junction of the frog. Jap. J. Physiol. 8:391-404.

Takeuchi, N. (1963). Some properties of conductance changes at the 193

end-plate memb!"_~me during the action of acetylcholine. J.

Physiol. 167:128-140.

Takeuchi, A. and Takeuchi, N. {1959). Active phase of frog's end-plate

potential. J. Neurophysiol. 22:395-411.

Takeuchi, A. and Takeuchi, N. (1960a). Further analysis of relationship

between end-plate potential and end-plate current. J.

Neurophysiol. 23:397-402.

Takeuchi, A. and Takeuchi, N. {1960b). On the permeability of the end-

plate mernbrccme during the action of transmitter. J. Physiol.

154: 52-6'1.

Thesleff, S. (1955a). The mode of neuromuscular block caused by

acetylcholine, nicotine, decamethonium and succinylcholine.

Acta Physiol. Sc and. 34: 218-231.

T.hesleff, S (1955b). The effects of acetylcholine, decamethonium, and

si_1ccinylcholi.ne on neuromuscular transmission in the rat. Acta

Physiol. Scand. 34: 386-392.

Thesleff, S. (1958). A study of the interaction between neuromuscular

blocking agents and acetylcholine at the mammalian motor end 194

plate. Acta Anaesth. Scand. 2:69-79.

Thesleff, S. (19 59}. Motor end-plate 'desensitization' by repetitive nerve

stimuli. J. Physiol. 148: 659-664.

Thies, R. E. (1960). Kinetics of acetylcholine release from single neuro

muscular junctions. The Physiologist 3:163.

Thies, R. E. (1965). Neuromuscular depression and the apparent deple..:

tion of transmitter in mammalian muscle. J. Neurophysiol.

28:427-442.

Tiegs, O. ~N. (1932). A study by denervation methods of the innervatic;n

of the muscle of a lizard (Egernia). J. Anat. 66:300-322.

Van der Meer, C. and Meeter, E. (1956a). The mechanism of action of

anticholinesterases. II. The effect of diisopropylfluoro

phosphonate (DFP} on the isolated rat phrenic nerve-diaphragm

preparation. Acta Physiol. Pharmacol. Neerl. 4:454-4'"11.

Van der lVIeer, C. and Meeter, E. (1956b}. The mechanism of action

of anticholinestel:·ases. II. The effect of diisopropylfluoro

phosphonate (DFP} in the isolated rat phrenic nerve-diaphragm

preparation. Acta Physiol. Pharmacol. Neerl. 4:472-481. 195

Vladimirova, I. A. (1964). Effect of electrical polarization of motor nerve

endings on transmission of single impulses. Fed. Proc. 23:

T1125-Tll26.

Von Euler, U.S. and Swank, H. L. (1940). Tension changes during tetanus·

in mammalian and avian muscle. Acta Physiol. Scand.. 1:203-2~9.

Wakabayashi, T. and Iwasaki., S. (1962). Neuromuscular facilitation and

asthenic synapses. Jap. J. Physiol. 12:1-13.

Werman, H. (1960). Electrical inexcitability of the synaptic membrane in

the frog skeletal muscle fibre. Nature 188:149-150.

Werner, G. (1959). The presynaptic action of d-tubocurarine and flaxedil

at the neuromuscular junction. Fed. Proc. 18:458.

Werner, G. (1960a). Neuromuscular facilitation and antidromic dis

charges in motor nerves: their relation to activity in motor nerve

terminals. J. Neurophysiol. 23:171-187.

Werner, G. (1960b). Generation of antidromic activity in motor nerves.

J. Neurophysiol. 23:453-461.

Werner, G. (1961). Antidromic activity in motor nerves and its relation 196

to a generator event in nerve terminals. J. Neurophysiol.

24:401-413.

-whittaker, V. P~ (1959). -Isolation and chara:cterization of acetylcholine

containing particles from brain. Biochem. J. 72:694-706.

Whittaker, V. P., Michaelson, I.A. and Kirkland, H.J. (1964). The

· separation of synaptic vesicles from nerve-ending particles

('synaptosomes'). Biochem. J. 90:293-303.

Wilson, I. B. (1955). The interaction of tensilon and neostigmin~ with

acetylcholinesterase. Arch. Int. Pharmacodyn. 104:204-213.

Zaimis, E. J. (1951). The action of decamethonium on normal and dener

vated mammalian muscle. J. Physiol. 112:176-190. APPROVAL SHEET

The dissertation submitted by Daryl Dean Christ has been read and approved by five members of the faculty of the Graduate School.

The final copies have been examined by the director of the dissertation and the signature which appears below verifies the fact that any necessary changes have been incorporated and that the dissertation is nov; given final approval with reference to content, form and mechanical II accuracy. I ! The dissertation is, therefore, accepted in partial fµHillment of the requirementf; for the Degree of Doctor of Philosophy.

0 --:AU}J~---·---~ l /1 Date Signature~ of Advisor L. C. Blaber, Ph.D.