FACIUTATORY TNTERACTTON BETWEEN PHARMAce-Leemw ‘
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Thesis for the Degree of M. S.
MICHIGAN STATE UNIVERSITY
DAVID WELLS SNYDER
1969

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. ABSTRACT ‘
FACILITATORY INTERACTION BETWEEN
PHARMACOLOGICALLY .DISTINCTIVE
GANGLIONIC CHOLINOCEPTIVE SITES

by David Wells Snyder

Two pharmacologically distinct postganglionic
cholinoceptive sites have been identified in the mammalian
sympathetic ganglion. The two sites will be referred to as
the C6-sensitive site and.the atrOpine-sensitive site
since the discharges evoked at these receptors are blocked
by hexamethonium (C6) and atr0pine respectively. This
study deals with the mechanisms involved with the disparity
between the ability of single injections and infusions of
acetylcholine (ACh) and tetramethylammonium (TMA) to evoke
C6-sensitive and atropine-sensitive discharges.

Neurogenic- or drug-evoked potentials were recorded
fromacutely or chronically denervated superior cervical
‘ganglion of cats. Drugs were administered either intra-
tvenously or directly into the blood supply of the ganglion.

A single injection of TMAevoked a postganglionic
discharge that was blocked only by hexamethonium. In con-
trast, the discharge evoked during the infusion of TMA was
blocked by either C6 or atr0pine. Transmission was not
altered by the-infusion rates of TMA employed.

The data suggest that infusion of TMA initiated an
interaction between the C6-sensitive site and the atrOpine-
sensitive site. The infusion of TMA evoked a well maintained'

C6-sensitive depolarization, the amplitude of which was

David W. Snyder

25 to 50% of control spike height. Hexamethonium
simultaneously repolarized the ganglion and blocked the post-
ganglionic discharge evoked by infused TMA. AtrOpine
blocked the discharge but failed to repolarize the ganglion.
In a few animals a small atropine-sensitive discharge was
observed following the administration of C6’

Thus it is prOposed that the Spread of depolarization
from the Cs-sensitive site to the atropine-sensitive site
greatly facilitated the weak muscarinic stimulating pro-
perties of TMA. The postganglionic discharge evoked during
the infusion of TMA appeared to emanate mainly from the
atrOpine-sensitive site. It is prOposed that atr0pine
blocked the discharge at the site of initiation of the
asynchronous action potentials whereas C6 blocked the
-discharge by eliminating the spreading facilitatory depo-
larization.

Nicotine, administered either by single injections or
constant infusion evoked.postganglionic activity that=wa3'
blocked by C6 and unaffected by atrOpine. Depolarization
comparable to that evoked by infused TMA was observed with
nicotine infusion. The failure to demonstrate a muscarinic
stimulating action of nicotine indicates that continuous
depolarization of theCG-sensitive site cannot alone evoke
action potentials from the atrOpine-sensitive site.

'Single injections of ACh evoked only C6-sensitive
‘ firing in an unconditioned control ganglion. A constant

infusion of the compound usually elicited a discharge which

David W. Snyder

was blocked by atr0pine and unaffected by C6' The threshold
of activation of the two cholinoceptive sites appeared to.be
reversed by infusion of ACh in an unconditioned ganglion.

The possible mechanisms underlying the changes in sensitivity
of the two cholinoceptive sites to TMA and ACh during

infusion of these compounds were discussed.

FACILITATORY INTERACTION BETWEEN
PHARMACOLOGICALLY DISTINCTIVE

GANGLIONIC CHOLINOCEPTIVE SITES

BY

iDavid Wells Snyder

A THESISv
Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of

MASTER OF SCIENCE
Department of Pharmacology
1969

ACKNOWLEDGMENTS

The author wishes to eXpress his sincere appreciation
to Dr. G. L. Gebber for his interest, valuable assistance
and the constructive criticism offered throughout this
investigation.

He wishes to thank Drs. T. M. Brody, R. A. Fax and
J. B. Hook for their helpful and constructive assistance
in the preparation of this thesis.

He is very grateful for the competent technical

assistance of Mrs. Phyllis A. Whetter.

ii

TABLE OF CONTENTS

INTRODUCTION

A. Acetylcholine and Ganglionic Transmission
B. Ganglionic Cholinoceptive Sites

METHODS
RESULTS

1. Characterization of PostgangliOnic Activity
Evoked by TMA, ACh and Nicotine
A. TMA Infusion
B. Nicotine Infusion
C. ACh Infusion
D. Single Injections of TMA and Nicotine

II. Pharmacological Blockade of Postganglionic
, Activity Induced by TMA, ACh and Nicotine
A. TMA Infusion
B. Nicotine Infusion
C. ACh Infusion
D. Single Injections of Nicotine and TMA

III. Ganglionic Depolarization Evoked During the
Infusion of TMA '

IV. Ganglionic Response to Infusion of TMA
T Following.Repetitive Preganglionic
Stimulation

V. 'Bffects of Chronic Denervation on Ganglionic
ReSponse Evoked by Infusion of TMA

DISCUSSION
SUMMARY_

BIBLIOGRAPHY_

.111

39

4.9
54

'67

69

Figure

10

11

LIST OF FIGURES

Schematic drawing of the electrophysiological
recording set Up of the superior cervical
ganglion.

Comparison of the action of C and atrOpine on
postganglionic discharge evoked by infusions of
TMA and nicotine.

Comparison of the ganglionic response evoked by
infusion of TMA and preganglionic stimulation.

Comparison of the action of C and atrOpine on
changes in transmission and tge asynchronous
postganglionic discharge evoked by the i.v.
infusion of TMA (100 ug/kg/min).

Postganglionic discharge evoked by the infusion
of ACh.

Comparison of the action of atrOpine (4 pg,
i.a.) and C (1 mg, i.a.) on the postganglionic
discharge egoked by nicotine (5 ug, i.a.) and
TMA (5 pg, i.a.).

Comparison of the action of C (1 mg, i.a.) and
atrOpine (2 ug, i.a.) on changes of the ganglionic
demarcation potential and postganglionic discharge
evoked by the i.v. infusion of TMA (100 ug/kg/
min).

Effect of repetitive preganglionic stimulation on
postganglionic discharge evoked by infusion of
TMA o '

Postganglionic discharge evoked by i.v. infusion
of TMA (67 ug/kg/min) in ganglion conditioned
with repetitive preganglionic stimulation

(30 cps for 60 sec) and a transmission-blocking
dose of C6 (5 mg/kg, i.v.).

Postganglionic discharge evoked by infusion of
TMA in ganglia denervated for 7 and 16 days.

Diagram of the prOposed facilitatory interaction
between the cholinoceptive sites.

iv

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24

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30

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41

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4'7
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60

INTRODUCTION

A. Acetylcholine and Ganglionic Transmission

The first significant evidence that acetylcholine
might be a chemical mediator for transmission was demon-
strated by Otto Loewi (1921; cited by Brown, 1937) in the
frog heart muscle. Loewi demonstrated that the effluent
collected from a perfused heart during vagal stimulation
mimicked the reSponse ekaed by vagal stimulation when
injected into a second frog heart. The substance released
during vagal stimulation was pharmacologically identified
as acetylcholine (ACh). Studies in other areas of the
nervous system were undertaken in the 1930's and it was
concluded that transmission in the mammalian sympathetic
,ganglion was chemically mediated. Kibjakow (1933; cited
by Brown, 1937) develOped the technique for perfusing the
.superior cervical ganglion of the cat. During preganglionic
stimulation the venous effluent from the superior cervical
.ganglion could be collected and assayed for acetylcholine-
like activity by the bioasSay technique develOped by Chang
and Gaddum (1933). Using these techniques, an acetylcholine-
like substance was Shown to be released_from-the gangliOn
during stimulation of the preganglionic nerve trunk
(Peldberg and Gaddum, 1934; Feldberg and Vartiainen, 1934).
The quantity of ACh liberated during preganglionic stimula-
tion was Shown to be Sufficient to elicit a postgangliOnic

discharge (Feldberg and Gaddum, 1934). Reinjection of the

effluent collected during preganglionic stimulation evoked
a similar response (contraction of the nictitating membrane)
in the resting ganglion. . a

The prOposal that the site of liberation of ACh during
preganglionic stimulation was the terminals of the pre-
ganglionic sympathetic trunk and not the postganglionic _
elements was supported by the following: 1) Liberation of
ACh during preganglionic stimulation continued unabated
when transmission was blocked by physostigmine, nicotine,
curare, or excess potassium (Feldberg and Vartiainen, 1934;
Brown and Feldberg, 1936). 2) Acetylcholine was not
detected in the venous effluent of the perfused ganglion
during vagal stimulation or during ganglionic activation
induced by stimulation of the postganglionic nerve (Feldberg
and Vartiainen, 1934). Nicotine- or potassium-induced
‘ganglionic activation failed to release ACh from the
'ganglion (Brown and Feldberg, 1936). 3) Chronic degenera-
tion of the preganglionic trunk significantly reduced the
.ganglionic content of ACh (Brown and Feldberg, 1936). From;
these eXperiments it can be concluded that ACh was contained
within and.was released from the preganglionic nerve.

Evidence_is available for the presende of both enzyme
systems involved in the synthesis and metabolism of ACh in
the sympathetic ganglion. Physostigmine, an anticholines-
-terase agent, was Shown to potentiate the effects of
repetitive submaximal stimulation of the preganglionic

nerve in the superior cervical ganglion (Feldberg and Gaddum,

1934), _This eXperiment demonstrated the presence of a
system capable of inactivating ACh. BrOwn and Feldberg .
(1937) reported that the superior cervical ganglion has the
capability of synthesizing ACh. _This was later confirmed
by Kahlson and MacIntosh (1939) and Birk and MacIntosh
(1961). Thus, these data provided some reasonable evidence
to establish ACh as the mediator Of neurohumoral trans-

mission in the sympathetic ganglion.

B. Ganglionic Cholinoceptive Sites

Acetylcholine releaSed during preganglionic stimulation
was thought to diffuse across the synaptic cleft to excite
the postsynaptic cholinoceptive Site. The classical
cholinoceptive site involved in transmission is blocked by
hexamethonium (C6), a competitive ganglionic blocking agent
(Paton and Perry, 1953). However, cholinoceptive sites
other than the classical CGSSensitive site have been pro-
posed in the superior cervical ganglion. Koppanyi (1932)
demonstrated that the sympatheticganglion possesses more
than one type of cholinergic receptOr site. -Koppanyi
reported that mydriasis was observed following application
of pilocarpine to the surface of the cat's superior cervical
.ganglion. Pretreatment of the ganglion withatrOpine
(abolished the reSponSe. Twenty years elapsed before
significant evidence for the existence of multiple
cholinoceptive Sites in the ganglion was reported.

R. M. Eccles (1952a, b) investigated the response

elicitedby preganglionic stimulation in the curare

pretreated superior cervical ganglion of the rabbit.
Pollowing blockade of spike generation, preganglionic
stimulation evoked a triphasic reSponse. A depolarizing
synaptic potential (N wave) was followed by a hyper-
polarizing potential (P wave). In turn, the P wave was
followed by a second and more prolonged depolarizing
potential, late negative, LN wave, (Eccles, 1952b). Eccles
reasoned that these were synaptic potentials-and not
afterpotentials since spike initiation was blocked by
curare.

Eccles and Libet (1961) used the same preparation to
determine the nature of these three postsynaptic potentials.
They reported that after the administration of botulinum
toxin the potentials elicited by pregangliOnic stimulation
. were progressively blocked. They concluded that the N, P
and LN slow waves were mediated by ACh released from the
preganglionic terminals. AtrOpine was administered to
determine if more than one type of chOlinergic receptor was
activated by endogenously releasedACh. Doses of atrOpine
which failed to reduce the N potential, abolished the P ’
and LN waves. Following the administration of N,N-
dibenzyl-B-chloroethylamine (dibenamine), Scales and Libet
demonstrated that the slow P wave was more Sensitive to
blockade, suggesting the ganglionic release of catechol-
amines. They also reported that high concentration of
curare blocked Specifically the N potential, leaving a

large P and LN slow waves. In a ganglia pretreated with a

high concentration of curare, anticholinesterase was shown
to suppress the LN wave while the P Wave increased.

To explain these results, Eccles and Libet postulated
that the ganglion contained multiple cholinoceptive Sites.
The scheme they prOposed is as follows: three cholino-
ceptive Sites are contained in the mammalian sympathetic
'ganglion. l) Acetylcholine initiated a depolarizing
synaptic potential, N wave at the N receptor site. The N
potential was blocked by curare and little affected by
atrOpine. 2) Acetylcholine evoked a slow lOng lasting
depolarization, LN wave, at the LN receptor site. The LN
potential was Specifically blocked by atrOpine. 3) The
slow hyperpolarizing P wave was initiated by the actions of
catecholamines at a P receptor site. Eccles and Libet
suggested that ACh released from the preganglionic nerve
activated an atropine-sensitive site on ganglionic'
chromaffin cells that affected the release of catechol-
amines. The catecholamines released from the chromaffin
cells were believed to diffuse to the P receptor Sites on
the ganglion to elicit the slow hyperpolarizing potential.
The actions of the catecholamines on the P receptor sites
were prevented by dibenamine.

The experiments Of Vblle and his colleagues'have
demonstrated that spike initiation can occur as the result
of activation of the atrOpine-senSitive cholinoceptive'
_ganglionic sites. Volle (1962) described the actions of

ganglionic blocking agents on the postganglionic discharge

elicited after the administration of an anticholinesterase,
diisoprOpyl phosphorofluoridate, DFP, (Volle and Koelle,.
1961). AtrOpine blocked this asynchronous discharge.
Classical ganglionic blocking agents, hexamethonium and
d-tubocurare, did not alter the discharge. Physostigmine
and neostigmine were Shown to produce the same characteris-
tic asynchronous firing (Takeshige_and Volle, 1962; 1963a).
Volle reasoned that an atrOpine-sensitive site in the
ganglion had been unmasked by an action of the anti- 4
cholinesterase. VOlle postulated that the discharge
resulted from the accumulation of endogenously released
ACh which activated this previously masked atropine-
sensitive site. ‘

In-addition, VOlle and his associates demonstrated
that the intraarterial administration of ACh directly to
the superior cervical ganglion of the cat activated two
excitatory cholinoceptive sites; Takeshige and VOlle
(1962) demonstrated that following the conditioning
procedures of either high frequency preganglionic
stimulation (30 ops for 30 sec.) or physostigmine ,
pretreatment, a bimodal reSponse to exogenously adminisf
tered ACh was recorded postganglionically. The two
component-discharge consisted of an "early” reSponse which
was blocked by C6 or curare and a ”late” response which
’was unaffected by C6 and abolished by small doses of

atrOpine. AtrOpine had no effect on the postganglionic I

action potential elicited by preganglionic stimulation and-
. did nOt alter the "early" response to ACh.-

- The bimodal response could be elicited in an uncon-
ditioned ganglion with a high dose of ACh (Takeshige and
VOlle, 1962). These experiments demonstrated that more than
one type of cholinergic postsynaptic receptor site was '
activated by exogenously administered as wellas
endogenously released ACh.

volle and his asSociates demonstrated that exogenously
administered ACh evoked a characteristic complex change in
the ganglionic demarcation-potential which was similar to
that elicited by pregangliOnic stimulation in a ganglion
pretreated with curare (Takeshige gt 31., 1963; Takeshige
and Volle, 1964; Eccles and Libet, 1961). A triphasic slow
potential was elicited following the administration of
ACh. Initially a depolarization (D potential) which coin-
_ cided with a postganglionic discharge was observed. This .
was followed by a slow hyperpolarization (H potential)
which correSponded to a depression of transmission. A late
Slow depOlarizing wave (LD pOtential) followed the H
potential. The D potential and the postganglionic discharge

were blocked by C The H potential.and the LD potential

6'
were blocked by atrOpine. -

The response was dose dependent since a smaller dose
of ACh evoked only the H and LD potentials while a large
dose elicited a prolonged D potential. The prolonged -

depolarization coincided with blockade of transmission but

was not related to the blockade (Takeshige and VOlle,
1964):" ' ' y ' * .

In contrast to the triphasic potential evoked by ACh,-
Takeshige st 31., (1963) reported a biphasic change in the
demarcation potential following the intraarterial injection
of acetylee—methylcholine (methacholine, MCh) directly to
the ganglion.. A hyperpolarizing wave (H potential) waS‘
followed by a late occurring depolarizing wave (LD poten- .
.tial). The hyperpolarization of the ganglion cells was
associated with a depression of transmission. No change or
perhaps an increase in transmission was associated with the
yLD potential. A postganglionic discharge occurred during
the LD potential after the administration of methacholine.
The slow potentials, postganglionic discharge and effects
on transmission were abolished following the administration
of a small dose of atrOpine. .

Nicotine on the other hand, has been shown to evoke a
brief depolarizing potential and a pOStganglionic discharge
then administered directly to the ganglion (Lundberg and
Thesleff, 1953; Paton and Perry, 1953). The depolarizing
wave paralleled exactly the initial depolarization evoked
by ACh. Hexamethonium abolished the depolarization and ,
the postganglionic discharge evoked by nicotine. AtrOpine'
did not alter the response. 9 . .

In view of these results Volle (1966) has classified
cholinomimetic agents that stimulate Sympathetic ganglia

on the basis of their susceptibility to blockade either by

9

C6 orgatrOPine. ”Drugs related to nicotine (e.g.,
tetramethylammonium) evoked ganglionic depolarization and
firing that was immediate in onset and blocked by C6’ Those
substances related to muscarine (e.g., pilocarpine and
methacholine) evoked responses that were delayed in onset
and prevented by atrOpine. By contrast, acetylcholine wast
capable of activating both atropine- and C6-sensitive
~cholinoceptive Sites in the ganglion.

To eXplain the actions of these various drugs in the
superiOr cervical ganglion Takeshige gt al,(1963) and
Takeshige and Volle (1964) have presented the following
model of heterogeneity of cholinoceptive ganglionic sites.
Acetylcholine activated a C6-sensitive receptor site to
elicit the initial depOlarization and postganglionic dis-
charge corresponding with this depolarization. This'same
site was assumed to mediate transmission Since the trans-
mission process was blocked by C6 (Paton and Perry, 1953).
An atrOpine-sensitive receptor Site activated by ACh‘

. evoked a hyperpolarization and a corresponding decrease in
transmission. ”A Second atrOpine-sensitive receptor site
evoked the late occurring depolarization (LD potential)
follOwing the administration of ACh or MCh. This second
atrOpineésensitive site elicited a pOstganglionic discharge
following the administration of an anticholinesterase agent
or NCh.

This model was very Similar to the one prOposed by

Eccles and Libet (1961). The D potential appeared to be

10

identical with the N wave, the depolarizing Synaptic
,potential. Both responses were sensitive to hexamethonium.
The H potential corre5ponded with the P wave, both'hyper-
polarizing and atrOpine-sensitive. However, Takeshige gt_al.
(1963) demonstrated that the H potential could be elicited
in'a ganglion pretreated with-reserpine.- This demonstrated
that one type of cholinergic receptor could, upon
activation by ACh, elicit a hyperpolarizing SlOWpotential.
Therefore ganglionic releaSe of catecholamines, as prOpOSed
by Eccles and Libet (1961) would not necessarily be

_ involved in the hyperpolarization. The late-occurring
depolarization (LD potential) can be equated to the LN wave
of Eccles and Libet. AS was the case with the LN potential,
the LD potential was blocked by atrOpine.

Libet and Tosaka (1966, 1969) have demonstrated that
three different kinds of cholinoceptive Sites are located
on one sympathetic ganglion cell. They reported a tri-
phasic Slow potential in the rabbit superior cervical
ganglion during preganglionic stimulation, while recording
intracellularly'from a Single neuron. An initial depolar-‘
ization was followed by a slow hyperpolarizing potential
and a Slow depolarizingpotential. The initial depolar-
ization was blocked by C6' The Slow potentials following
the initial depolarization were selectively blocked by
atrOpine and unaffected or increased slightly by C6'

In summary, three different types of postsynaptic

cholinergic receptor Sites have been demonstrated to exist

11

in a Single mammalian sympathetic ganglion cell (Libet and
Tosaka, 1966, 1969). The one ekaing the.hyperpolarizing'
potential, which is abolished by atrOpine, can be
considered as an inhibetory site, since transmission is
depressed during this hyperpolarization. The other two,
therefore, are excitatory in nature Since they evoke a
depolarizing potential when activated by ACh. .The
excitatory receptOr Site whose response is blocked by
'hexamethonium will be referred to as the Cé-sensitive
site. Correspondingly the other excitatory site, blocked

by atrOpine will be designated the atrOpine-sensitive site.

METHODS

-

All experiments were performed on the superior cervical
ganglion of cats of both sexes weighing 2-4 kg. .The cats
were anesthetized by the intraperitoneal administration
of a mixture of sodium diallybarbiturate (70 mg/kg),
urethane (280 mg/kg) and monoethylurea (280 mg/kg).

-_The cat was placed in the supine pOsition andwas
secured to a dissecting table. To keep the head and neck
"stationary, a mouth clamp was attached to the lower jaw
and secured to the metal frame of the cat board. A midline
.incision was made from the symphysis of the lower jaw to
the sternal notch. To insure the patency of the respira-
tory pathway, the trachea was cannulated at the level of
the clavicle. ’

"‘The superior cervicalganglion and associated struc-
tures were expoSed by inverting the upper portions of the
-trachea, larynx and eSOphagus into the animal's mouth. The
_left_superior cervicalganglion and the external carotid
postganglionic nerve were prepared for recording following
removal of the surrounding connective tissue. Care was
taken so that the small blood vessels supplying the
ganglion were not disturbed. A silk ligature, soaked in
saline (0.9% NdCl) was tied to the poStganglionic nerve
near its junction with the external carotid artery. The
nerve was then sectioned between the silk tie and the

artery.

12

13

The cervical sympathetic trunk was dissected free from
the carotid sheath approximately 3 centimeters from the . '
gangliOn. The cervical trunk was tied with a silk
ligature and cut at the level of the clavicle. A deep
cervical well was formed by tying skin flaps to the metal
framework and the eXposed area was covered with mineral
- oil. LOOpS were formed in the Silk ligatures and suspended
in the oil on glass hooks which were fastened to the metal
framework. 9 N

Bipolar electrodes of 26 guage platinum wire were used
for stimulating the decentralized ganglion.‘ They were
positioned on the isolated preganglionic trunk approxi-
mately 2 centimeters from the gangliOn (fig. 1). Electrical
Istimulation was provided by a Grass model 3-8 square wave
generator led through a Grass model SID-4678 stimulus
isolation unit to the bipolar electrodes. Supramaximal
stimuli, 15 volts, of constant duration (0.1 msec.) were
employed with a frequency nOted for the particular
SXperiment. - - .

' Drug- or neurogenically-induced changes in the
demarcation potential of the decentralized ganglion were
recorded with silver-silver chloride bipolar electrodes.
These electrodes were prepared from bright silver wire by
electrolytic deposition of chloride from an acidified
0.1N KCl solution. The electrodes were replated after
visible damage to the silver chloride precipitate had

occurred. One pole of the electrode was placed in direct

14

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16

contact with the surface of the ganglion and the other pole
was placed on the crushed end of the external carotid nerve
(fig. 1). The recorded potentials were led to a cathode
follower circuit that in turn led to a resistance-coupled
preamplifier (Grass Model P-612, DC preamplifier). The
preamplifier's low and high amplitude frequency controls
were set at DC and 2000 KC reSpectively. The evoked
potentials were displayed on a dual beam'oscilloSOOpe
(Tektronix Type 502).

The demarcation potential or surface potential was a
crude extracellular recording of the resting membrane
potential.' Drug- or neurogenically-induced changes in the
'potential of the ganglion cells were monitored using the
crushed end of the postganglionic nerve as the reference
point. An upward deflection of the demarcation potential
tracing indicated ganglionic negativity (i.e., depolariza-
tion) in all records. Similarly a sudden downward shift in
the tracing denoted repolarization.

To record drug-induced postganglionic action potentials
platinum electrodes were used. One pole of the bipolar
electrode was placed on the crushed end of the external
carotid nerve. The other pole was positioned on an intact
portion of the external carotid nerve.- The asynchronous
burst of action potentials were amplified by a capacitance—
coupled preamplifier (Grass Model P-Sll, AC preamplifier).
The half amplitude frequency controls of the amplifier's

low and high coupling filters were set at 30 cps and

17

,1000 KC, respectively. The potentials were visualized on
the dual beam_oscillosc0pe.' Changes in the demarcation .
potential of the ganglion and the asynchronous ganglionic
discharge were monitored simultaneously in some experi-
ments. Permanent records were made on moving photographic
paper (Kodak Kind 1732) with a Kymograph camera (Grass

A Model C4L). ‘

In.a number of eXperiments, movement of the left
nictitating membrane was monitored in conjunction with
neural recordings from the left external carotid nerve.
Initial tension of the membrane was set at 7 grams and

1

-was recorded with a force-displacement transducer. Neural
) . -

recording from the external carotid nerve did not compro-
mise the innervation to the nictitating membrane. In these
experiments nerve action potentials and tone of the
nictitating membrane were recorded with a Grass Model 7
pen-writing polygraph. The half amplitude response of the
prnamplifier was set at 10 and 75 cps for the neural
recordings. .
Chronically denervated ganglia were studied in one
Series of experiments. Resection of the left vago-
sympathetic trunk, approximately 2 centimeters from the
I gangliOn was performed under near sterile conditions. "
These cats were anesthetized with pentobarbital Sodium

(30 mg/kg, i.p.) prior to surgery.‘ One centimeter of the

vagosympathetic trunk was removed. The animals were

18

allowed to recover and the experiments were run 7-54 days
following resection of the preganglionic nerve.

All major branches of the common carotid artery except
those directly supplying the ganglion were tied. Single
doses of drugs were administered directly to the ganglion
through a 27 guage needle inserted into the common carotid
artery. The injection apparatus was clamped to the metal
‘framework. ClOtting in the.needle was preventedby
adminiStering heparin (300 units/kg, i.v.). The intra-
arterial injection volume for a single dose was 0.1 ml. A
catheter was placed in the femoral vein to infuse various
‘drugs. Drugs were infused by the intraarterial or intra-
venous route with the aid of a constant rate infusion pump.
The infusion volume was 0.1 to 1.0 ml/min because the
concentration of drugs-to evoke a postganglionic discharge
varied from animal to animal. The criteria used in
determining the concentration of the ganglionic stimulants
employed in each experiment was such to produCe a minimal
'effect on transmission.

To avoid movement of the recording electrodes during
the experiment, the animals were paralyzed with
decamethonium bromide (0.5-0.75 mg/kg, i.v.) and placed on.
(artificial respiration. This prevented any Spontaneous)
muscle twitches. Additional doses of decamethonium were
administered as required throughout the experiment. This
neuromuscular blocking agent had little effect on the

responses of the ganglion evoked by drugs or preganglionic

.19

stimulation., Blood pressure was monitored from the femoral
artery with a Statham P-23 series pressure transducer and

recorded on the Grass Polygraph.

.The following drugs were used: acetylcholine chloride
(ACh), tetramethylammonium chloride (TMA), nicotine
salicylate, hexamethonium chloride (C6), and atrOpine
sulfate. All drugs were dissolved in 0.9% saline.- All

doses are expressed in terms of the salt.

RESULTS

-CHARACTERIZATION OF POSTGANGLIONIC ACTIVITY EVOKED BY

TMA, ACh AND NICOTINE

A. TMA Infusion

 

Infusions of TMA (SO-200 ug/kg/min. i.v,; 5-20 ug/
min. i.a.) elicited a low amplitude, asynchronous post--
ganglionic discharge. In 25 experiments, the discharge
rapidly reached and was maintained at or near peak
amplitude for the duration of the infusion. A typical
postganglionic response induced by the infusion of TMA
is illustrated in the TMA record of fig. 2. Line 1 of

fig. 2 illustrates the control background of the acutely

"denervated, unstimulated ganglion. The low amplitude,

asynchronous ganglionic activity evoked by infused TMA
is demonstrated by the increased width of the record in

line 2 of fig. 2. The functional significance of the

low amplitude discharge was demonstrated by monitoring'

contractions of the nictitating membrane. In fOur
experiments, the amplitude of the contractiOn of the
nictitating membrane during the infusion of TMA
equalled that evoked by supramaximal stimulation of the
preganglionic nerve at frequencies from 1-3 impulses
per SecOnd. A comparison of the drug; and
neurogenically—induced contraction of the nictitating
membrane is illustrated in fig. 3. Crushing the

ganglion eliminated 80-95% of the reSponse of the

20

§

21

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22

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Figure 3.

23

Comparison of the ganglionic response evoked by

p infusion of TMA and preganglionic stimulation.

.Upper record, contraction of the nictitating

membrane (top) and asynchronous postganglionic
discharge (bottom) evoked by the i.v. infusion’
of TMA (67 ug/kg/min). Lower record, contraction
of the nictitating membrane (tep) and compound
ganglionic action potentials (bottom) evoked by
supramaximal stimulation of the preganglionic
nerve (2 cps). Nerve action potentials were
amplified with a Grass 7P3A preamplifier and
displayed on a pen-writing polygraph with half-
amplitude response at 10 and 75 ops. .Vertical
calibrations referring‘to neural activity are
25 uV (upper record) and 1 mV (lower record).

‘ Vertical calibration referring to contraction of .

nictitating membrane is 10 grams. .Time base is
1 sec per division. Downward deflection of time
base in upper record indicates start of TMA
infusion.

24

 

 

 

 

 

 

 

 

  
 

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Comparison of the ganglionic response evoked by

infusion of TMA and preganglionic stimulation.

25

nictitating membrane. Thus, the‘contraction evoked by
TMA was primarily of ganglionic origin.

The characteristics of the postganglionic discharge
could be studied during the period of time (10-15 min.)
before TMA began to block ganglionic transmission. As
illustrated in fig. 4, the amplitude of the action .
potential was only slightly depressed at the indicated
.times during the infusion of TMA. This is one of four
experiments in which transmission was monitored during

the infusion of TMA.

B. Nicotine Infusion

 

Intravenous infusion of nicotine (SO-100 ug/kg/
7min.) evoked a low amplitude, asynchronous postganglionic
discharge in four eXperiments. The amplitude of the
nicotine induced postganglionic discharge was comparable

to that evoked by the TMA infusion as shown in fig. 2.

C. ACh Infusion

 

In nine eXperiments,'ACh was infused into the
arterial circulation of the superior cervical ganglion.
Even though the intraarterial route of administration
helped to minimize the systemic effects produced by-
_ACh there was a marked fall in blood pressure during
the infusion. To limit this pronounced depressor
effect, ACh was never infused for more than 5-6 minutes
and the dose required to produce a postganglionic

discharge was kept to a minimum (40-80 ug/min.). Blood

26

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27

 

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.28

pressure quickly returned to control level upon
cessation of.the infusion.

The postganglionic discharge evoked during the
intraarterial infusion of ACh followed one of two
characteristic patterns. In six of nine eXperiments
the asynchronous discharge was continuous and of low'
amplitude. The amplitude of the discharge was similar
to the postganglionic discharge evoked by the infusion
of TMA or nicotine. A typical ACh induced discharge
‘ of this type is illustrated in fig. 5A.

In the remaining three.eXperiments, the post-
.ganglionic discharge evoked during the infusion of ACh
followed a second pattern. The ganglionic activity
'appeared in characteristic bursts (fig. SB). The bursts
'were of higher amplitude and shorter duration when com-
3pared with the continuous low amplitude discharge

discussed previously.

 

D. Single Injections of TMA and Nicotine'

' In contrast to the low amplitude asynchronous-
discharge evoked by an infusion of_TMA or nicotine,
single intraarterial injections of TMA (l-lO ug, i.a.)
and nicotine (l-lO ug, i.a.) evoked a brief discharge
of relatively high amplitude in eighteXperiments.
The discharge was rapid in onset and gradually
'dissipated to background level within 20 seconds as
illustrated in fig.6. In two emperiments the post-‘

ganglionic discharge evoked by single injections of TMA

Figure 5.

29

Postganglionic discharge evoked by the infusion of
ACh. .

A: I, control background. II, administration of

C (1 mg, i.a.) 2 min after initiation of post-
ggnglionic discharge by ACh (60 ug/min, i.a.).

III, effect of atropine (2 pg, i.a.) administered

2 min after C . B: I, control background. II,
effect of C6 fil mg, i.a.) on discharge evoked by
ACh (80 ug/min, i.a.). III, effect of atrOpine 3
(4 pg, i.a.) on discharge. AtrOpine was administered
45 sec after C . Vertical calibration is 10 uV.
Horizontal calibration is 4 sec. Dots under records
indicate administration of C6 or atrOpine.

3O

ACh
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Figure 5. Postganglionic discharge evoked by the infusion

of ACh.

31

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32

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2:82

33

and nicotine were enhanced following repetitive pre-

__ganglionic stimulation. This observation has been
previously reported by Takeshige §E_gl. (1963);
Trendelenburg and Jones (1965) and Gebber and Volle
(1966).

II. PHARMACOLOGICAL BLOCKADE 0F POSTGANGLIONIC ACTIVITY
INDUCED BY TMA, ACh AND NICOTINE ‘-

A. TMA Infusion

 

The postganglionic discharge evoked by the infusion
of TMA (so-zoo ug/kg/min.; 5;2o ug/min., i.a.) in an
,acutely decentralized ganglion was markedly reduced or
abolished by either C6 or atrOpine. As illustrated in
line 2 of the TMA record of fig. 2, C6 (0.5—2 mg, i.a.)
abolished the discharge evoked by the infusion of TMA.
This was observed in each of eight eXperiments. These
doses of C6 completely blocked transmission. A small

yu._but perceptible postganglionic discharge reappeared
2-3 minutes after the administration of‘CG.‘ Additional
doses of C6 (1 mg) failed to alter the residual dis-
charge. AtrOpine (1-4 ug, i.a.) abolished the C6--
resistant discharge. A .
i In two additional eXperiments, C6 was administered
intraarterially to the ganglion, in amounts that failed
to completely block ganglionic transmission. Doses of
C6 (l-S ug, i.a.) which did not alter transmission,

failed to affect the postganglionic discharge evoked

34

during the infusion of TMA. Similarly, doses of C6
.(20-50 pg, i.a.) which produced partial blockade of -
transmission produced a parallel reduction in the post-
ganglionic discharge evoked by TMA infusion.

AtrOpine (1-4 ug, i.a.) was shown in four
experiments to abolish or markedly reduce the discharge
evoked by the infusion of TMA in a ganglion which had
not been previously treated with C6' These doSes of
atrOpine failed to alter transmission in the absence
of TMA. However, during the infusion of TMA, atrOpine
produced a transient (5-20 sec.) and partial block
(10-20%) of transmission. This was determined from the
results of two eXperiments. However, it should be
stressed that the complete blockade of the asynchronous
discharge far outlasted the transient and partial
blockade of transmission produced by atropine. Following
the administration of atrOpine, the postganglionic
vdischarge induced by the TMAinfusion couldnot be
' elicited for 2-3 hours. ‘

These observations suggested that most or all of
the postganglionic discharge initiated by the infusion
of TMA could be blocked by either C6 or atrOpine in any
particular ganglion. Figure 2 compares the actions of
atrOpine and C6 on the discharge evoked by TMA in the
same ganglion. The discharge was initially blocked by
C6 (1 mg, i.a.) (line 2). Following the administration

of C6 the infusion was discontinued until the effects of

35

C6 had dissipated ‘30-60 min.) and transmission returned
to control level. The amplitude of the postganglionic
‘discharge evoked during the second TMA infusion .
approximated that observed during the initial period of
infusion (line 3). Administration of atrOpine (2 ug,
i.a.) abolished the discharge (line 3). The same
pattern of results were observed in seven additional
ekperiments. In three of the eight eXperiments per-
formed, however, a small component of the postganglionic
discharge was resistant to blockade by atropine. The
small, but perceptible, atrOpine-resistant discharge
was abolished by‘C6 and unaffected by additional doses
of atropine. Although increasing the infusion rate of
TgA-§§00-l000 ug/kg/min._i.v.) quickly blocked trans-
mission, it did not enhance this small atrOpine-
resistant discharge. .

A comparison of the actions of C6 and atrOpine on
.transmission during the TMA infusion is illustrated in
fig. 4. AtrOpine abolished or markedly reducedthe_
postganglionic discharge but produced little effect on
the TMA-induced ganglionic action potential evoked by
preganglionic stimulation (line 4). C6, on the other
hand, abolished both the postganglionic discharge and

ganglionic transmission.

B. Nicotine Infusion

 

C6 abolished the postganglionic discharge evoked
during the infusion of nicotine (30-100 ug/kg/min. i.v.).

36

In contrast to its action on the postganglionic dis-

_ charge induced by infused TMA, atrOpine (1-4 ug, i.az)
failed to block the postganglionic discharge evoked by
the infusion of nicotine. A comparison of the actions
of atrOpine and C6 on the discharge evoked by the
infusion of nicotine is illustrated in fig. 2 which is

one of four exPeriments performed.

C. ACh Infusion

 

As previously noted the intraarterial infusion of
ACh (40-80 ug/min.) either evoked a continuous low
amplitude postganglionic discharge (pattern one) or
the postganglionic activity occurred mainly in bursts
(pattern two). A comparison of the actions of C6 and
atrOpine on the continuous low amplitude discharge
~ evoked during the infusion of ACh is illustrated in
fig. SA. CS (1 mg, i.a.) had_1ittle effect on the
postganglionic discharge (line 2). AtrOpine (2 ug, i.a.)
however abolished the discharge (line 3). The results
‘were consistent in the six eXperiments in which ACh
evoked a discharge similar to pattern one. After
dissipation of the transmission blocking effects of C6
”(30‘60 min.) an-additional dose of atrOpine was admin-
istered (0.5 mg/kg, i.v.) to prevent the systemic depres-
sor actions of ACh. The infusion of ACh was
re-initiated at a much higher rate (200-500 ug/min,
i.a.). In the presence of atrOpine, ACh failed to evoke

a postganglionic discharge. Transmission quickly failed

37

at the higher infusion rates.

Figure SB illustrates the second pattern of
discharge evoked during the infusion of ACh observed in
the three remaining experiments. The activity occurred
mainly in bursts. C6 (1 mg, i.a.) abolished the dis-
charge (line 2). However, within seconds a post-
ganglionic discharge reappeared. The discharge was'
abolished_by atropine (4 pg, i.a.) as shown in line 3
of fig. SB. An additional dose of C6 failed to alter

the atropine-sensitive discharge.

D. Single Injections of Nicotine and TMA

 

The actions of C6 and atrOpine on the postganglionic
activity evoked by single injections of TMA and nicotine
followed that of the nicotine infusion. As illustrated
in fig. 6 the postganglionic discharge evoked by
single injections of TMA (1-10 ug, i.a.) and nicotine
‘(1-10 pg, i.a.) were blocked‘byC6 (025-1 mg, i.a.).
This confirms earlier reports (Takeshige gt_gl., 1963;
Takeshige and Volle, 1964). The dose of hexamethonium
used to block the drug-induced discharge also blocked
ganglionic transmission. AtrOpine (1-4 ug, i.a.) was
tested on the postganglionic discharge evoked by single
’doses of TMA and nicotine. The results of four
eXperiments demonstrated that atropine failed to pro-
duce consistent changes in the postganglionic responses
evoked by TMA and nicotine. These doses of atrOpine

did not alter transmission. However, in two

\

III.

38

experiments, larger doses of atrOpine (10-20 ug, i.a.)

_which caused a perceptible blockade of transmission '

produced a parallel reduction in the postganglionic
discharge evoked by TMA and nicotine. Takeshige g£_gl.
(1963) previously reported a lack of specific effect of
atrOpine on the postganglionic discharge evoked by

nicotine.

GANGLIONIC DEPOLARIZATION EVOKED DURING THE INFUSION OF
TMA

Because of the long infusion period and the
inherent prOperties of the resistance coupled pre-

amplifier to drift during this time, the changes in the

_ganglionic demarcation potential could not be measured

directly: However, an approximate measure of ganglionic
depolarization induced by an infusion of TMA could be

gained from the immediate relief from depolarization

m~produced by the administration of CG‘ In a control

.ganglion, C6 blocked transmission without altering the

demarcation potential. Administering C6 to a drug-
induced depolarized ganglion will show a positive shift
in the demarcation potential recording, demonstrating
repolarization. -
In five eXperiments repolarization of the ganglion
by C6 amounted to 25 to 50% of the amplitude of the
control compound ganglionic action potential. Relief

from depolarization coincided with the blockade of the

Iv.

39

asynchronous postganglionic discharge evoked by the

_ infusion of TMA-as illustrated in fig. 7.

In contrast to the actions of C6' atropine blocked
the asynchronous postganglionic discharge induced by the
TMA infusion but failed to alter the ganglionic
demarcation potential. Thus, atropine failed to block
TMA-induced ganglionic depolarization. One of the five
experiments performed is illustrated in fig. 7.

Rapid ganglionic depolarization was demonstrated
following single injections of TMA into the blood
supply of the superior cervical ganglion (Gebber and)
Volle, 1966). However, a period of rapid depolarization
was not observed at the initiation of the TMA infusion.
Thus, it appeared that the C6-sens1tive depolarization

gradually reached peak amplitude.

GANGLIONIC RESPONSE TO INFUSION OF TMA FOLLOWING
REPBTITIVE PREGANGLIONIC STIMULATION

Repetitive preganglionic stimulation has been
demonstrated to potentiate the stimulating actions of
ACh, nicotine, THA and methacholine in the superior
cervical ganglion (Volle, 1962; Takeshige gt al.,_.
1963; Trendelenburg and Jones, 1965; Gebber and VOlle,
1965). Post-tetanic potentiation at both the C6-
sensitive site and the atrOpine-sensitive site endures
for several hours. In view of these reports and the

marked effect of preganglionic tetanus on drug-induced

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42

activation of the ganglion, it was considered important
to test the effects of repetitive preganglionic

istimulation on the postganglionic discharge induced by
the infusion of TMA.

High frequency preganglionic stimulation (30 ops
for 30 sec.) was performed during TMA infusion in
eight experiments.. The amplitude of the postganglionic
_discharge initiated by the infusion of TMA was not
substantially altered by the preganglionic tetanus.
However, repetitive preganglionic stimulation altered
the blocking effects of both C6 and atrOpine on the
postganglionic discharge evoked by TMA.

In five eXperiments, C6 (1 mg, i.a.) initially
blocked the postganglionic discharge induced by the
I TMA infusion in a ganglion which had been conditioned
with repetitive preganglionic stimulation (fig. 8A).
In contrast to an unconditioned ganglion, however, the
duration of blockade was Shortened. .As is illustrated
I in fig. 8A, the postganglionic discharge reappeared
within one minute following the administration of CG'
failed to alter the discharge

6
even though transmission was blocked. Administration

'~Additional doses of C

-of atrOpine (4 ug, i.a.) abolished the C6-resistant
discharge for the duration of the infusion.

In three experiments, the ganglionic blocking
agents were administered in the reverse order in ganglia

conditioned with repetitive preganglionic stimulation.

Figure 8.

43

Effect of repetitive preganglionic stimulation on
postganglionic discharge evoked by infusion of TMA.

A: I, control background; between I and II infusion
of TMA (60 ug/kg/min, i.v.) Was initiated and supra-
maximal preganglionic stimulation (30 ops for 60 sec)
was performed. II, effect of C (0.5 mg, i.a.) on
discharge evoked by TMA in tetagized ganglion; C

was administered 4 min after preganglionic tetanSs.
III, effect of atrOpine (2 ug, i.a.) on discharge
which returned 70 sec after administration of C“.

IV, background recorded in the continued presenge

of TMA infusion and 5 min after administration of
atrOpine. B: I, control background. II, effect of
atrOpine (4 ug, i.a.) administered 3 min after
preganglionic tetanus (30 ops for 60 sec) performed
during the infusion of TMA (80 ug/kg/min, i.v.).,
III, effect of C (1 mg, i.a.) which was administered

’1 min after atrOpine. Vertical calibration is 10 uV.

Horizontal calibration is 4 seo. Calibrations refer
to both eXperiments. Dots below records indicate
time of administration of C6 and atrOpine.

44

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Figure 8. Effect of repetitive preganglionic stimulation on

postganglionio'diSCharge evoked by infusion of TMA.

45

The duration of blockade of the TMA-induced discharge

by atrOpine (4 pg, i.a.) was also attenuated (fig. 8B).
The postganglionic discharge recovered within seconds

and additional doses of atrOpine (4 ug, i.a.) failed to
alter the response.. C6 (1 mg, i.a.) abolished the dis-
charge for the duration of the infusion. Transmission
returned within 20-40 minutes following the administra-
tion of C6 and only then could a postganglionic discharge
be re-initiated by the TMA infusion.

Another series of eXperiments were performed to
further study the effects of repetitive stimulation on
the TMA discharge. In five exPeriments the ganglion
was conditioned with repetitive preganglionic stimula-
.tion and a large dose of C6 (5 mg/kg, i.v.) was
administered which blocked transmission. The TMA
infusion was then initiated. As shown in fig. 9, even
though transmission was blocked, the amplitude of the
postganglionic discharge evoked by the infusion of TMA
fell within the range of that elicited in an uncon-
ditioned ganglion. Additional doses of C6 (1 mg, i.a.)
did not alter the postganglionic discharge. AtrOpine
(4 pg, i.a.) abolished the discharge. Thus the
atrOpine-sensitive discharge which was just perceptible
2-3 minutes after the administration of C6 in a control
.ganglion appeared to be enhanced in ganglia conditioned

with repetitive preganglionic stimulation.

46

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48

.Three other eXperiments were performed in a similar
manner to those previously described. Following pref
:ganglionic tetanus, atropine (0.5 mg/kg, i.v.) was
administered prior to the infusion. During the
infusion of TMA a postganglionic discharge was elicited
whose amplitude fell within the range observed in
ganglia which had not been preconditioned with
preganglionic tetanus and atropine. Additional doses
of atrOpine (4 ug, i.a.) injected directly into the
blood stream of the ganglion failed to alter the dis-
charge. C6 (1 mg, i.a.) abolished the postganglionic
discharge. Again repetitive preganglionic stimulation
appeared to enhance the small but perceptible C6-
sensitive discharge which was observed following the
administration of atrOpine in three of eight control
‘ganglion.

Single doses of TMA (l-lO ug, i.a.) and nicotine
(l-lO pg, i.a.) were administered directly into the
circulation of the ganglion conditioned with
repetitive preganglionic stimulation (30 cps for
60 sec.). The preganglionic tetanus appeared to have
enhanced the postganglionic discharge to single
injections of TMA and nicotine. ‘This.confirms earlier
reports (Takeshige 93 al., 1963; Trendelenburg and
Jones, 1965; Gebber and VOlle, 1966). In two
experiments atrOpine (1-4 pg, i.a.) failed to reverse

the enhancement of the discharges evoked by TMA and i

49

nicotine after repetitive stimulation. Hexamethonium

(1 mg, i.a.) abolished the discharge evoked by single

‘doses of nicotine and IMA in tetanized ganglia.

EFFECTS OF CHRONIC DENERVATION _ON GANGLIONIC RESPONSE
EVOKED BY INFUSION or TMA.

It has been reported that the atrOpine-sensitive
postganglionic discharge evoked by single injections of
ACh was not dependent upon the integrity of presynaptic
terminals of the superior cervical ganglion (Takeshige
and Volle, 1963b). However, the postganglionic discharge

evoked_by certain anticholinesterase agents was

— dependent on the presence of a functional presynaptic

terminal (Takeshige and VOlle, 1962). In addition it

has been reported that the cholinergic stimulating

‘ agent, carbachol, released ACh from the preganglionic

nerve terminal (McKinstry and Koelle, 1967a, b). In
view of these reports, it was considered important to
determine if the discharge evoked by the infusion of
TMA was dependent on the integrity of the presynaptic
terminal in the ganglion.

The characteristics of the postganglionic discharge
evoked by TMA (504200 ug/kg/min.) were-studied 7-54 days
following section of the cervical vagosympathetic
trunk. In all experiments performed with chronically

denervated ganglia, the amplitude of the postganglionic

50

discharge evoked during the infusion of TMA, was within
the range observed in acutely decentralized_ganglia.-

TMA was infused (SO-200 ug/kg/min, i.v.) in four
eXperiments which were performed 7—11 days following
nerve section. Degeneration of the preganglionic nerve
lappeared complete since a compound postganglionic action
potential could not be elicited by stimulating the pre-
ganglionic trunk rostal‘to the site of resection. The
discharge evoked by TMA in these experiments were very
similar to those observed in the acutely decentralized
ganglion. Figure 10, upper record, illustrates a
_ typical eXperiment. AtrOpine (1-4 pg, i.a.) or C6
(0.5-l mg, i.a.) abolished or markedly reduced the
postganglionic discharge evoked during an infusion of
TMA. The duration of blockade by these ganglionic
blocking agents was the same as in an unconditioned
. acutely decentralized ganglion.

Atropine was less effective :n blocking the dis-
charge evoked bnyMA in ganglia which were denervated
for 16-23 days. Typical of the four experiments per-
formed is the one illustrated in fig. 10, lower record.
As Shown in line 3 of the record of a ganglion
denervated for 16 days, a major component of the.
postganglionic discharge was resistant to blockade by
atropine (1-4 ug, i.a.). C6 (0.5-l mg, i.a.) abolished
the discharge when administered before atrOpine and

blocked the residual discharge when administered after

I Figure 10.

51

Postganglionic discharge evoked by infusion of TMA
in ganglia denervated for 7 and 16 days.

7 days: I, control background. II, effect of C

(1 mg, i.a.) on discharge elicited by TMA (100 ug/
kg/min, i.v.); C was administered 3 min after the
discharge was initiated. III, effect of atropine

(4 ug, i.a.) on the discharge which was reinitiated
40 min after the administration of C . 16 days:

I, control background. II, effect of C (1 mg, i.a.)
on discharge evoked by TMA (100 ug/kg/min, i.v.).
III, effect of atrOpine (4 ug, i.a.) on discharge
whiCh was reinitiated 40 min after the administration
of C . Vertical calibration is 10 uV. Horizontal.
caligration is 4 sec. Dots below records indicate’
time of administration of C6 and atrOpine.

52

- ‘ TMA , . -
Chronic - 7 days

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- .| ‘ L ‘ ,‘5‘ A, . , ‘ ‘ A‘. ..." r ‘r {4.1. f3'5u 4.255}. ‘ ,1_..!& .V.‘ ’rw?" ‘,Ii.‘g.\ J‘f‘"? v‘ ‘.
< \ . -‘ . . o ‘ '. e ., .,- t . L. , » . ' " ‘-
- ' " 7 ": ~ - H" “ hi ‘- « '- ~I' 3 ‘ 'r "5'. ..n."* -‘ . .‘ ‘..¢
. -,_ ."‘ « - “‘.4'..l' .Ag‘.." ”‘3“:‘4 ‘ " -"t'; "‘ n" 'I' , ' ‘ ' 5 '

Figure 10. Postganglionic discharge evoked by infusion of TMA

in ganglia denervated for 7 and 16 days.

53

atrOpine. -As in the ganglia denervated for 7—11 days,
a postganglionic action potential could not be elicited
by stimulation of the remanents of the preganglionic
nerve trunk rostal to the site of resection.

Two eXperiments were performed on cats where the
ganglia had been chronically denervated for 53 and 54
days. In these experiments, a small compound post-
ganglionic action potential was elicited during
preganglionic stimulation of the residual sectioned
nerve. Thus it appeared that partial reinervation had
occurred. The blocking actions of atrOpine and C6 on
the postganglionic discharge evoked during the infusion
of TMA was essentially the same as observed in the
acutely decentralized ganglion. The discharge was

blocked by either atrOpine or C6'

IDISCUSSION

,It is surprising that the infusion of TMA evoked a
postganglionic discharge that was markedly reduced or
abolished by either C6 or atropine. Yet TMA is usually
considered as a nicotinic, ganglionic, stimulating agent
(Vblle, 1966) and its actions should therefore be similar to
those of nicotine. However, the infusion of nicotine evoked,
a discharge that was blocked only by C6 and unaffected by
atrOpine. The question to be answered then, is "What
possible mechanisms may be involved to eXplain the discharge
evoked during the TMA infusion?“

The mechanisms involved in the initiation of the dis-
charge evoked by TMA appeared to be of postganglionic origin.
This was suggested by the fact that atrOpine and/or C6
'markedly reduced or abolished the postganglionic activity
evoked by infused TMA in a ganglion which had been dener-
vated for 7-11 days. The preganglionic terminals of the
superior cervical ganglion disappear within this time after
resection of the cervical sympathetic trunk (Koelle and
Koelle, 1959). .

One possible explanation of the action of both
ganglionic blocking agents during the TMA infusion is that
atrOpine was acting non-specifically at the C6-sensitive
site to block the discharge. However, from the following
observations this does not appear to be the case.

.1) Blockade of the discharge evoked during the infusion of

TMA far outlasted the transient and partial block of

54

55

of transmission following the administration of atrOpine
(see-fig. 4). 2) AtrOpine blocked the postganglionic
discharge without producing a concomitant repolarization
of the ganglion (fig. 7). 3) The postganglionic discharge
evoked by infused nicotine was unaltered by doses of atrOpine
that abolished the discharge induced by infused TMA.
4) Atropine failed to alter the postganglionic activity
evoked by single injections of TMA or nicotine.
Alternatively, the ability of both C6 and atrOpine to
block the discharge evoked during the infusion of TMA
suggests that infusion of TMA initiated an interaction
between the two excitatory ganglionic cholinoceptive sites.
In this case, the interaction can be defined as the process
whereby the action of TMA at one cholinoceptive site
~facilitates the initiation of action potentials at the
second excitatory cholinergic receptor on the same ganglion
cell. This, then, implies that the discharge of many of the
individual cells participating in the recorded population

response could be abolished by either atrOpine or C The

6’
recent reports of Libet and Tosaka (1966, 1969) demonstrated
.theexistence of both excitatory cholinoceptive sites on the
same individual ganglion cells in the mammalian sympathetic
ganglion.

At least three possibilities concerning the nature of
the interaction between the two sites can be considered.

1) The weak muscarinic stimulating prOperty of TMA may have

been facilitated by the more familiar C6-sensitive action

56

of the compound. Trendelenburg (1966a) has observed a weak.
muscarinic ganglionic stimulating property of TMA in the
presence of nicotine-induced transmission block in the
superior cervical ganglion while recording from the
nictitating membrane. 2) Initiation of action potentials at
the C6-sensitive site could have been facilitated by the
weak muscarinic stimulating property of the compound.

3) A combination of the above two possibilities may have
occurred. In this case the postganglionic discharge would
have emanated from both cholinoceptive sites.

No direct evidence was obtained to suggest that the
initiation of asynchronous activity at the CS-sensitive site
by TMA was facilitated by an atrbpineésensitive prOperty of
the drug. The administration of atrOpine failed to produce
-a consistent change in the demarcation potential of the
ganglion even though it abolished the postganglionic dis-
charge evoked during the TMA infusion. However, extra-
cellular recording techniques may have limited the detection
of atropine—induced repolarization of'a few cells. In this.
regard, it should be noted that Libet (1964) observed that
atrOpine at times prevented the gradual increase in the!
amplitude of the compound ganglionic action potential evoked
by the first four or five volleys cf-a train of preganglionic
stimuli.

The following observations suggest that the depolari-
zation evoked at the C6-sensitive site during the infusion

of TMA facilitated the initiation of asynchronous action

57

potentials at the atrOpine-sensitive site. 1) Hexamethonium
simultaneously repolarized the ganglion and blocked the-
asynchronous discharge evoked by infused TMA. 2) AtrOpine
blocked the discharge evoked by the TMA infusion but failed
to repolarize the ganglion. 3) A small atropine-sensitive
discharge was observed in some animals following the
administration of C6' Thus it is probable that the weak
muscarinic effect of TMA was greatly facilitated by the
simultaneously occurring depolarization at the C6-sensitive
site in an unconditioned ganglion. The fact that atrOpine
had no affect on the nicotine—induced discharge (Trendelen-
burg, 1966a) further suggests that the spread of depolari-
zation from the C6-sensitive site cannot alone initiate
action potentials from the-atropine-sensitive site, but must
-be accompanied by a concomitant direct activation or change
in sensitivity of the atropine-sensitive site. This,
observation is also consistent with the lack of a
ganglionic, muscarinic-stimulating prOperty of nicotine. '
Thus, it is prOposed thatthe facilitatory interaction
_ is the result of the depolarization initiated at the C6-
sensitive site which spreads via local current flow to the
atrOpine-sensitive site to enhance the weak muscarinic
stimulating action of TMA. Under this condition the post?
ganglionic discharge would be emanating mainly from the
atropine-sensitive site. Thus atrOpine blocks the post-
ganglionic discharge directly at the site of initiation

of the asynchronous discharge whereas C6 blocks it

58

indirectly by eliminating the facilitatory spreading
depolarization initiated at the C6-sensitive site. The pro-
posed facilitatory interaction is illustrated in fig. 11 in
which E1 and E2 represent the C6-sensitive site and the
atrOpine-sensitive site respectively.

This proposal is supported by the reports of Trendelen-
burg (1966a, b) and is in agreement with the facilitatory
action of nicotine described by Gebber (1968). While ‘
monitoring the movement of the nictitating membrane
Trendelenburg (1966a, b) noted that ganglionic stimulation
of angiotensin and other non-nicotinic agents was enhanced
in the presence of nicotine. Hexamethonium blocked the
facilitatory effects of nicotine on the ganglionic responses
evoked by these agents. Gebber (1968) suggested that C6-
'sensitive depolarization evoked by nicotine was reSponsible
for the enhancement of non-nicotinic postganglionic dis-
charges produced by ACh, MCh and serotonin. Blockade of
_depolarization by C6 abolished the facilitatory effectsof
nicotine. Thus facilitation of the non-nicotinic discharge
lasted only as long as ganglionic depolarization evoked by
nicotine. It was proposed that the spread of depolarization
from the C6-sensitive site to the sites activated by non-
nicotinic agents accounted for the facilitation (Gebber,
1968).

. The reasons for the disparity between the ability of
infusions and single doses of TMA to evoke atrOpine-sensitive

firing are not clear. It is difficult to understand why a

59

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60

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61

single injection of TMA which evoked C6-sensitive depolariza-
tion of considerable amplitude and duration (Gebber and
vo11e, 1966) failed to evoke a discharge which was sensitive
to atrOpine as well as C6' Thus, other actions of TMA must
have been involved in the initiation of the discharge which
occurred during the infusion of TMA.

One such action may occur at the atrOpine-sensitive
site. An infusion of TMA occasionally evoked a low ampli-
tude atropine-sensitive discharge which was initiated 2-3
minutes following the administration of transmission
blocking dose of C6' Single doses of TMA failed to elicit
a discharge that was sensitive to blockade by atropine.
These observations indicate that the atrOpine-sensitive_site
on the ganglion was more sensitive to the direct stimulating
,actions of TMA when the compound was administered by con-
stant infusion rather than by single injections. In ganglia
conditioned with repetitive preganglionic stimulation the
direct atrOpine-sensitive stimulating action of TMA occurred
more frequently and was of greater amplitude. It should be
stressed that in these experiments as well as in those of
unconditioned ganglion the atrOpine-sensitive discharge was
initiated after the administration of transmission blocking
doses of C6' In this case, the atrOpine-sensitive discharge
was independent of any interaction hetween the two cholino-
ceptive sites. In contrast, repetitive preganglionic
stimulation failed to unmask an atrOpine-sensitive discharge

evoked by single injections of TMA. In view of these

62

observations, it appears that-both the facilitatory effect
of C6-sensitive depolarization and some other mechanism of
sensitization of the ganglion to the muscarinic stimulating
prOperties of TMA were necessary for the initiation and
maintenance of the largest component of the postganglionic
firing observed in unconditioned ganglion during the
infusion of TMA.

Equally puzzling is that, in contrast to the results
obtained with single injections of TMA, infusion of TMA
only occasionally evoked a discharge in unconditioned
ganglia which were blocked by C6 and resistant to atropine.
The extremely small amplitude and infrequent occurrence of
this component of firing is striking when one recalls that
the infusion of TMA produced a C6-sensitive depolarization
«whose amplitude was 25 to 50% of control Spike height.
Although increasing the infusion rate 5 to 10 times failed
to increase the amplitude of the atrOpine-resistant dis-
charge in control ganglia, this component of firing was
considerably larger in ganglia which had been denervated for
16-23 days and in those whichhad been conditioned with a
preganglionic tetanus.

One possible eXplanation for the small and infrequent
pure C6-sensitive discharge (atropine-resistant) evoked
during the infusion of TMA is that the threshold of depolari-
zation (Eccles, 1964) for the C6-sensitive site was not
obtained in most ganglion cells during the infusion. This

however, does not appear to be the case since the amplitude

63

of the C6-sensitive depolarization (25 to 50% of control
Spike height) evoked-during the TMA infusion was comparable
to that evoked by single injections of TMA which produced
good C6—sensitive firing (Takeshige and Volle, 1964; Gebber
and vo11e, 1966). The threshold of depolarization may play
an important role in the mechanism if accommodation of the
C6-sensitive site had occurred (see below).

The second explanation may deal with the difference in
the rate of depolarization following the administration of
TMA. Sasaki and Otani (1961) have reported that the
threshold of depolarization of the cat motoneuron may vary
according to the different time course of augmenting depolar-
ization. The slower the rate of depolarization (cathodal
current) the higher the threshold of depolarization. A
(similar mechanism may have been involved during the infusion
of TMA. The C6-sensitive site may discharge_on1y in the
face of a rapid depolarization provided the threshold of
Spike initiation is reached._ Gebber and VOlle (1966) have
demonstrated a sudden depolarization of the ganglion and a
simultaneOus CGesensitive postganglionic discharge following
an intraarterial injection of TMA. A gradual depolarization
' of the CG-sensitive site during the infusion of TMA to a
level equivalent to that following a single injection of TMA
might not produce equivalent C6-sensitive responses. A
rapid upward shift in the demarcation potential tracing
(denoting depolarization) was never observed following the

initiation of the TMA infusion. It was not possible to

64

accurately follow changes in the demarcation potential of the
_ganglion for periods longer than three minutes. Therefore

it was assumed that the infusion of TMA evoked a depolariza-
tion which gradually reached peak amplitude. Thus, during
the infusion of TMA, the C6¥sensitive site may have
accommodated to the gradual depolarization and thereby raised
the threshold of depolarization for spike initiation which
limited the pure C6-sensitive response.) However, in the
presence of the depolarization induced by infused TMA, trans-
mission was only slightly altered yet there was very little
pure C6-sensitive firing. This disparity may also be
explained in terms of the rate of depolarization. Rapid C6-
sensitive depolarization evoked by neurogenically released
ACh, (Eccles, 1963) initiated the postganglionic action
'potential. In this case, the rapid change in ganglionic '
depolarization would be the major factor in evoking the C6—
sensitive postganglionic spike.

However, the possibility exists that the infused TMA
was acting at C6-sensitive sites other than those involved
'in transmission.- This was suggested by the fact that trans-
mission was only slightly affected when the TMA infusion '
evoked a large Ca-sensitive depolarization and an infrequent,
low amplitude atrOpine-resistant discharge.) This is in
agreement with the reports of Riker (1967) and Gebber (1968)
which demonstrated that the subsynaptic CG-sensitive sites
involved in transmission may differ from the C6-sensitive'

sites activated by nicotine like drugs.

65

The ganglionic atrOpine-sensitive stimulating action
of ACh was also prominent when that compound was infused .
rather than injected in a single dose. The infusion of
ACh usually evoked a postganglionic discharge which was
sensitive to blockade by atrOpine and unaffected by C6' In
contrast, a single injection of ACh evoked a Cs-sensitive
discharge in an unconditioned ganglion. Takeshige and vo11e
(1962) reported that the threshold dose of a single injec-
tion of ACh required to activate the atrOpine-sensitive
'1ate' reSponse was much higher than that required for
activation of the C6—sensitive site. It is interesting that
the changes in the reactivity of the two cholinoceptive sites
observed during the infusion of ACh were similar to those
observed for single injection after repetitive preganglionic.
'stimulation and the administration of anticholinesterase
agents. As noted by Takeshige and velle (1962) following
these conditioning procedures, the threshold for activation
of the two cholinoceptive sites by a single injection of ACh
was reversed. Activation of the '1ate"atrOpine-sensitive
discharge required smaller doses of ACh than activation of
'the 'early' C64sensitive response.

The prominence of the atrOpine—sensitive discharge
elicited during the infusion of ACh may also be eXplained I
-as the result of the gradual rate of depolarization of the
cé—sensitive site. Sasaki and Otani (1961) reported that
due to the accommodation of the initial segment of the cat

motoneuron the site of spike initiation changed. Their

66

results indicated that the spikes are generated from the
initial segment when a rectangular current (rapid depolari-
zation)is applied and from the soma when the current rise
is slow enough. Similarly, the C6-sensitive site may have
accommodated to the gradual depolarization during the
infusion of ACh. This may have raised the threshold of the
C6-sensitive site above that of the atropine-sensitive site
so.that the initiation of asynchronous action potentials

was at the atrOpine-sensitive site.

summer

It has been shown that altering the mode of administra—
tion of TMA changed the pharmacologic prOperties of the
postganglionic discharge evoked by TMA. Single intra—
arterial injections of TMA evoked a brief burst of activity

that was blocked by C and unaffected by atropine. In

6
contrast, a constant infusion of TMA evoked a continuous e1
postganglionic discharge that was sensitive to blockade by
either C6 or atrOpine.

The results with infusions are explained on the basis
of a facilitatory interaction between the two pharmacologi—
cally distinct cholinoceptive sites. It is prOposed that
constant infusion of TMA evoked a depolarization at the C6-
‘sensitive site which spread via local current flow to the
atropine-sensitive site to enhance the responsiveness of
the muscarinic receptor. Thus, the weak muscarinic
stimulating prOperties of TMA are greatly facilitated during
' the infusion of TMA. The spread of depolarization to the
atrOpine-sensitive site and the concomitant direct
muscarinic stimulating action of TMA are necessary for the
facilitatory interaction. The actions of the ganglionic}
blocking agents can be eXplained in the following way:
AtrOpine blocked the postganglionic discharge evoked by TMA
directly at the site of initiation. C6 blocked the discharge
indirectly by abolishing the Spreading facilitatory

depolarization.

67

68

This proposal assumed that the two excitatory cholino-
ceptive sites are located on the same individual ganglion.
cell. The recent studies of Libet and Tosaka (1966, 1969)
indicate that this assumption can be made.

Similarly, this study showed that the mode of
administration of ACh is a critical factor as concerns the
relative participation of the two cholinoceptive sites in'
the discharge initiated by this compound. Constant infusion
of ACh usually evoked an atropine—sensitive discharge. In
contrast Takeshige and Volle (1962) have reported only a
C6-sensitive discharge in unconditioned ganglion following
single intraarterial injections of ACh. It appeared that
the threshold of activation of the two cholinoceptive sites
were reversed during the infusion of ACh._ This could also
be explained as facilitatory interaction between the .
cholinoceptive Sites together with a possible accommodation
of the C6-Sensitive site to the gradual depolarization
evoked by the ACh infusion.

The'role of the proposed facilitatory interaction in
the normal integrative functions of Sympathetic ganglia

is yet to be determined.

BIBLIOGRAPHY

BIBLIOGRAPHY

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