under halothane-N20 anaesthesia. 5. ACh release due to depolarization of the cortex with KCl was

22 Pages·2005·2.36 MB·English

J. Phy8iol. (1969), 203,pp. 741-762 741
With 9 text-figure
Printed in Great Britain
THE EFFECT
OF TOPICALLY APPLIED ATROPINE ON RESTING AND
EVOKED CORTICAL ACETYLCHOLINE RELEASE
BY J. D. DUDAR AND J. C. SZERB
From the Department ofPhysiology and Biophysics,
Dalhousie University, Halifax, Nova Scotia, Canada
(Received 14 April 1969)
SUMMARY
1. Cortical acetylcholine (ACh) output was measured in cats anaesthe-
tizedeither with Dial compound (0-6 ml./kg) orwith halothane-N20. ACh
output was found to be 1 67 ng/cm2.min under Dial anaesthesia, and
0 30ng/cm2.min under halothane-N20.
2. Addition of atropine sulphate (1 ,tg/ml.) to the collection fluid in-
creased ACh output fourfold under Dial anaesthesia but had no effect
under halothane-N20 anaesthesia.
3. Isolation of the cortex, lesions in the mesencephalon and topical
application of tetrodotoxin (TTX) reduced ACh output under Dial
anaesthesia to about 0*8ng/cm2.min. The effect of atropine on ACh out-
put was somewhat reduced by isolation and completely abolished by
mesencephalic lesions or TTX.
4. ACh release evoked by reticular formation stimulation under halo-
thane-N20 anaesthesia was increased fourfold by atropine but evoked
release due to direct stimulation ofthe cortexwasincreasedonlytwofold.
5. ACh release due to depolarization of the cortex with KCl was not
increased by atropine.
6. Dihydro-fl-erythroidine (DHE) or D-tubocurarine failed to affect
ACh output even in a concentration of 100jzg/ml.
7. It is concluded that atropine does not increase spontaneous ACh
release and only ACh release evoked by trans-synaptic stimulation of
cholinergic neurones is potentiated by atropine.
8. These findings are fully consistent with the hypothesis that atropine
increases ACh output by blocking cortical cholinergic synapses which are
a part ofa circuit inhibiting cholinergic neurones.
742 J. D. DUDAR AND J. C. SZERB
INTRODUCTION
Increased acetylcholine (ACh) release from the cerebral cortex in vivo
has been reported by many workers following the administration of atro-
pine either systemically (Mitchell, 1963; MacIntosh, 1963; Szerb, 1964;
Phillis & Chong, 1965; Celesia&Jasper, 1966;Beani, Bianchi, Santinoceto
& Marchetti, 1968) or topically at the site ofACh collection (Szerb, 1964;
Phillis & Chong, 1965; Collier & Mitchell, 1966; Beani et al. 1968; Phillis,
1968). Hyoscine hasa similareffectonAChrelease (Polak, 1965; Bartolini
& Pepeu, 1967). Several mechanisms have been suggested to explain the
effect ofthese drugs on ACh release: interruption ofa negative feed-back
circuit which controls activity in cholinergic neurones (MacIntosh, 1963),
preventionoftheinactivation ofreleasedtransmitterbyreceptorblockade
(Szerb, 1964; Celesia & Jasper, 1966; Collier & Mitchell, 1966; Bartolini &
Pepeu, 1967), blockade of ACh reuptake (Creese & Taylor, 1965; Schu-
berth & Sundwall, 1967), facilitation of presynaptic release of ACh
(Bertels-Meeuws & Polak, 1968). However, no in vivo experimental
evidence has been available to choose between any of these hypotheses.
Thisstudywasundertakentodeterminethemechanismbywhichatropine
increases cortical ACh output in vivo. The results to be presented give
experimental evidence for the hypothesis suggested by MacIntosh (1963)
thattheincrease inAChoutputfollowing atropine istheresultofaninter-
ruption ofcortical cholinergic synapses which form a part ofan inhibitory
circuit controlling the activity ofcholinergic neurones. Some oftheresults
have been communicated in a preliminary form (Dudar & Szerb, 1968).
METHODS
Cats ofeither sex weighing 2-0-5-0kg were anaesthetized either with Dial com-
pound (0-6ml./kg I.P.) or with a gas mixture consisting of 0-25-1-0% halothane,
70%N20 and 30 02administeredthroughaHarvardrespiratorypump.Artificial
respiration was also administered when Dial anaesthesia was used and the minute
ventilation was adjusted so that the end-tidal C02 concentration was 4% as moni-
tored by means ofa Beckman LB-1 C02 analyzer. Aheating device similar to that
described by Krnjevic' & Mitchell (1961) maintained the rectal temperature at
370 C.
The head ofthecat was fixedin astereotaxic holder, the skullwasopenedabove
theparietalcortexandthedurareflectedformeasuringAChrelease.Forstimulating
the medullary pyramids, the cat was placed on its back, and much of the basal
occipital bone wasremoved afterreflectingthecervicalmuscles andremovingparts
ofthetracheaandoesophagus. Isolatedcorticalslabswerepreparedfromthelateral
and suprasylvian gyri by means of a spatula bent to form a longitudinal trough
25mmlongand12mmwide.Thiswasintroducedintotheposteriorgyrusandpushed
gently forwarduntil the desired area was undercut. With the spatula still in place,
a fine probe was passed along the anterior and lateral borders ofthe spatula com-
pleting the cut from the spatula to the pia mater.
ATROPINE AND CORTICAL ACh RELEASE 743
Electrolytic lesions inthemid-brainwereplaced stereotaxically inplane A6-0by
means oftwo stainless-steel electrodes 3mm apart and bared for 7mm above the
tip. First, the electrodes were introduced at 1-0 and 4-0mm laterally with the tips
atH-4-0andcurrentwaspassedbetweenthemfor30sec.Thenthepairofelectrodes
wasretractedandmoved3mmlaterally,placedinthesameverticalpositionandthe
lesion made. The same procedure was repeated on the opposite side. This way the
aquaduct was spared, and no brain oedema occurred. The source ofcurrent was a
Grass model LM3 lesion maker and the intensity of current was the same as that
requiredto coagulate egg white between the electrodes. Anesthesia was continued
after the lesionswere made.
The reticular formation was stimulated at A3-0, L3-5, H-1-0 with concentric
electrodes 1 mm apart at the tips. The duration ofpulses was 0-3msec at a rate of
100/sec for 1 sec repeatedevery 10sec with nominal voltages of4-10V. The cortex
was stimulated with apair offlat-footed electrodes 4mm apart insulated except at
thelowersurface ofthefoot andplaced on the surface ofthebrain. Thecortexwas
stimulatedat 30/secwithpulsesof1 msecdurationandwitheither20or 70nominal
voltage settings. The pyramids were stimulated with needle electrodes at 50/sec
with0-1msecpulsedurationandvoltagesettingsof30-80V.Thepotentialsrecorded
inthecortexasaresultofmedullarypyramidstimulationremainedunchangedwhen
the frequency ofstimulation was increased up to 150/sec.
The electrical activity ofthe cortexwasmonitoredonan oscilloscope throughout
the experiment and occasionally bothunipolar andbipolar (between theflat-footed
electrodes placed inside the collection cup) electrocorticograms were recorded on a
Beckmandynograph.Thestereotaxicframeservedasground.Electrodeplacements,
location oflesions and isolation ofthe cortex were verified on fixed frozen sections
150g thick prepared according to the method of Guzman, Alcaraz & Fernandez
(1958).
AChwascollectedinLockesolutioncontainedinPerspexcylindersplacedlightly
onthecortex.Perspexcylindersoftwosizeswereused;thesmallerones(i.d. 10mm)
containing 0-25ml. Locke solutionforAChassayontheleechmuscleandthelarger
ones (i.d. 14mm) containing 0-5ml. Locke solution forAChassayon the rat blood
pressure. After inhibiting cholinesterase atthe site ofACh collectionwith echothio-
phate iodide (0-5mg/ml.) applied for 30min, Locke solution containing eserine
salicylate (1jug/ml.) was applied. After discarding the first 10min sample, ACh
release was followed in samples collected for 10 or 15mi.
AChwas assayed in one oftwo preparations. In experiments where atropine was
used,AChwasassayedonthedorsallongitudinalmusclestripoftheleechsuspended
inamicrobathasdescribedbefore (Szerb, 1964). TTXintheconcentrationsuseddid
notinterferewiththeassayofAChontheleechmuscle. Solutions containingahigh
concentration of KCI were diluted for assay purposes with a sufficient volume of
KCl-free Locke solution to give the usual KCI concentration of 5-8mm. Whenever
D-tubocurarine (curare)orDHEwereused,AChwasassayedontheratbloodpressure
accordingtoamethodsimilartothatdescribedbyStraughan (1958). Ratsweighing
150-180gwereanaesthetizedwith amixtureof 20% urethaneand0-8% chloralose
(0.6ml./kg). Bloodpressurewasrecordedinthecarotidarterybymeans ofapressure
transducer connected to a Sanborn carrier preamplifier operated in an averaging
mode.TheratwasartificiallyventilatedwithaPalmersmallanimalpumpattherate
of180/minwithastrokevolumeof15-2ml. Samplesof0-1ml.wereinjectedintothe
femoral veinandthepreparations were regularly sensitive to 05ngACh after pre-
treatment with eserine salicylate (0-1 mg/kg). The antinicotinic agents in the con-
centrations employeddid not interfere with ACh assay nor did substances, such as
histamineorserotonin, whichmighthaveoccurred inthesamples,haveanyeffectin
744 J. D. DUDAR AND J. C. SZERB
concentrations up to 0-1 mg/ml. A few samples collected from the brain and not
containing any drugs were cross-assayed on the leech muscle and the rat's blood
pressure and gave estimates ofthe ACh content that agreed closely. Acetylcholine
chloride (Hoffmann-LaRoche) was used as the standard and results are expressed
in terms ofthis ACh salt.
RESULTS
Release ofACh under Dial and halothane-N20 anaesthesia. The release
ofACh undereitherDial orhalothane-N20 anaesthesia was quite uniform
in the same cat from one sample tothenext and samples collected simul-
taneously from the left and right hemispheres had almost identical ACh
contents. However, the amount of ACh released under Dial anaesthesia
was considerably greater than that released under halothane-N20
anaesthesia. ThemeanreleaseunderDialwas1-67(S.D. + 1.29) ng/cm2.min
(325 observations, fifty-nine cats), while underhalothane-N20 anaesthesia
themeanreleasewasonly0-30 (s.D. +0-13)ng/Cm2.min (118observations,
thirty-six cats), a highly significant difference. The large variation in ACh
outputwasmostly duetothe differences between cats, since an analysis of
variance showedthatthe variance ofAChoutput between cats was highly
significantly greater than the variance of ACh release in the same cats.
Under Dial anaesthesia, the e.e.g. showed frequent spindles interrupting
a high amplitude, slow wave pattern, while under halothane-N20 anaes-
thesia spindles were absent from the e.e.g. trace.
EffectofatropineonAChrelease.Theeffectofatropinesulphate(1 sug/ml.)
added to the collection fluid on the amount ofACh released under both
anaesthetics is shown in Fig. 1. Data for ACh release have been converted
to % in order to make comparison easier since the mean release ofACh
under Dial in these experiments was nine times greater than that released
under halothane-N20. Atropine was without effect on ACh output under
halothane-N20, even in % terms, whereas the high output under Dial
anaesthesia wasincreasedfourfold bythe application ofatropine. If, how-
ever, ACh output was increased under halothane-N20 anaesthesia by
stimulating the reticular formation, atropine greatly enhanced the evoked
release as shown in Fig. 7.
Effect ofcurare andDHEonACh release. To ascertain that the increase
inAChreleasewasspecifically duetothe antimuscarinic effectofatropine,
two antinicotinic drugs, curare and DHE, were tested on the resting
release of ACh under Dial anaesthesia and on the evoked release due to
reticular formation stimulation under halothane-N20 anaesthesia. As
shown in Fig. 2, curare had no effect on the resting release ofACh under
Dial anaesthesia whether it was applied in the same concentration as
atropine or in one hundred times greater concentration. DHE similarly
failedtohavean effectonAChoutputunderDial anaesthesia. As shownin
ATROPINE AND CORTICAL ACh RELEASE 745
Table 1, curare and DHE, in a concentration one hundred times greater
thanthatfoundeffective foratropine, failedtoincreasetheevokedrelease
ofACh under halothane-N20 anaesthesia.
L
500 Atropine1,fg/ml. added to cup
= a;---;-;---;-;-''-!--;--;-----**--a-,~~~~~~~~~~~~~~~~~~~~~~
-
4.
e-i 300-
r
0
.'E 200-
100-
r_.
UO
U 60 120
min
Fig. 1. AverageACh output under Dial (filled columns) orhalothane-N20
(open columns) anaesthesia and the effect oftopically applied atropine on
this output. To normalize results, in each experiment the values for ACh
output before atropinewerepooled andindividual outputswere expressed
as %ofthesepooledvalues.InallsubsequentFigs.showingaverageoutput,
this method ofcalculation was followed. The average andthes.E ofmean
of these percentages are shown as bars (in this and subsequent Figs.)
for every collection period. The over-all averages before atropine for each
anaesthetic are for halothane-N20, 100% = 0-26ng/cm2.min (n = 3) and
for Dial, 100% = 2 23ng/cm2.min (n = 8).
A Curare 1fig/mt. B Curare 1OX g/ml.
100 100
a-
._ 50-o
C
._0
60 120 60 120
min
Fig. 2. Effect of topically applied curare on average ACh output under
Dial anaesthesia. A, curare (1,ug/ml.); 100% = 1-35ng/cm2.min (n = 2).
B, curare (100,ug/ml.); 100% = 1 53ng/cm2.min (n = 2).
I
746 J. D. DUDAR AND J. SZERB
C.
4 C>
1 + +1
0 *.0.
OD -P0(tiD
+1 +1
10
4Q
D
o - co
4a cq eO -
0Z
+1 +1
.,4z
i
COeD0o - cq
0 Iw
+1 +1
1v4
S
C4- 5
oQ-D+- 2Q mCO4-1 e10 t0o
+1 +1
00
0 b0 ~PtO
a- II c~d 1* q00
C-P
+1 +1
o
m
14 C to 10
+q)o
+1 +1
H.
1-1
1-:
-C
fH m
40-i CO
w
0
z
1~4 P
ATROPINE AND CORTICAL ACh RELEASE 747
Effect ofatropine on AChoutputfrom isolated cortex. Atropine was found
to be without an effect on the low output of ACh under halothane-N20
anaesthesia but was effective in increasing the high output under Dial
anaesthesia. However, atropine increased ACh output under halothane-
N20 anaesthesia whenever the cholinergic neurones were activated by
stimulation of the reticular formation. These findings suggested that
activity in the cholinergic fibres was necessary for the action ofatropine.
AtropineI#g/mi.
300
-
200
0
'100
0
Fig. 3. Effect ofatropine on average ACh output collected simultaneously
from intact (dotted columns, 100% = 2-08ng/CM2.min, n = 4) and
isolated (hatched columns, 100% = 0-85ng/CM2.min, n = 4) parietal
cortex under Dial anaesthesia.
To test this hypothesis, preparations were required in which neural
activity had been intentionally eliminated. Three methods were employed
to achieve this: isolation ofanareaofthe cortex, destruction ofthemedial
mesencephalon, and the application ofTTX to the site ofACh collection.
Data from experiments in which ACh was collected simultaneously from
the undercut and contralateral intact cortices are shown in Fig. 3. It can
be seen that the amount of ACh released from the isolated cortices was
about 40% ofthat ofthe intact side. Atropine increased the release from
748 J. D. DUDAR AND J. C. SZERB
both cortical areas although the % increase was not quite as large in the
isolated cortices as in the intact ones. The bipolar e.e.g. recorded from
the isolated cortices was flat, showing a total isolation which was also con-
firmed histologically.
Effectofatropine after electrocoagulation ofthe mesencephalon. The results
ofexperiments in which the medial mesencephalon was electrocoagulated
under Dial anaesthesia are shown in Fig. 4. Before coagulation the resting
Bilateral R.F. destruction
200
150
0 60 ~~~~~m~~i~120
Fig. 4. Effect of bilateral electrolytic lesion in the mesencephalon and
subsequent application of atropine on average ACh output under Dial
anaesthesia (100%O = l53ng/cm2.min, n = 3).
Output continuously increased, a phenomenon not observed in other
experiments. This increase in ACh output could have been due to injury
discharges inthe mesencephalon since the electrodes to be used for electro-
coagulation had been placed before collection started. The ACh content
of the sample after electrocoagulation contained even more ACh but
following this the output fell quickly to reach a steady level of approxi-
mately Ou7ng/cm2.tmm. Topical application of atropine did not increase
this low ACh output. Following the placement of the lesion the cortical
e.e.g. showed either a decline in the frequency ofspindling or a decrease in
the amplitude of slow waves or both. Histological examination of the
ATROPINE AND CORTICAL ACh RELEASE 749
mesencephalon showed large lesions extending about 5 mm above and
below the coronal plane ofthe electrodes.
Effect of atropine after TTX. The third method of eliminating neural
activity at the area ofACh collection was the topical application ofTTX.
This drugis known to block action potentials bypreventing an increase in
sodium conductance (Kao, 1966). When TTX was applied in a concen-
tration of 10,ug/ml., the bipolarly recorded electrical activity in the area
A Leftside B
differential>
Leftside \
>by
monopolar
Right side
ht differential
Lfl
Rightside monopolar
i sec
Fig. 5. E.e.g. tracing under Dial anaesthesia from the left and right
parietal cortex before (A) and 30min after (B) the application of TTX
(10jug/ml.) to the right parietal cortex. Differential recordings taken at
twice the gain ofmonopolar records.
exposed diminished or disappeared entirely in 20-30 min (Fig. 5). As
shown in Fig. 6A, ACh outputprogressively declinedfollowingthe appli-
cation ofTTX to a steady level of about 0-8 ng/cm2.min. When atropine
was appliedinthe presence ofTTX, ACh output not only didnot increase
but slightly declined. The output on the opposite side, treated with TTX
only, remained at the same level.
Since there was apossibility that the failure ofatropine toincrease ACh
release following TTX might have been due to a low ACh output and not
to an absence ofaction potentials, the effect ofatropine was tested after
reducing ACh output by a non-specific method, namely, by increasing the
depth of anaesthesia. Figure 6B shows the effect of Dial compound (0.1
ml./kg) injectedi.v. onbilateralACh output. Here, too, therelease ofACh
dropped progressively to a level similar to that seen with TTX but the
application of atropine to one side still increased ACh output from that
side. This showed that the absence of neural activity is specifically
responsible for the lack ofan effect ofatropine.
Effect ofatropine with different types of neural stimulation. The absence
ofaneffect ofatropine onACh outputfollowing mesencephalic lesions and
TTX indicated that neural activity had to be present in cholinergic
750 J. D. DUDAR AND J. C. SZERB
neurones in order for atropine to increase ACh output. The next question
to be answered was whether activity ofthe whole neurone, including the
soma, was necessary for the effect of atropine or whether neural activity
in the cholinergic nerve endings alone was sufficient. Since the cell bodies
of the great majority of corticocholinergic fibres are found subcortically
(Hebb, Krnjevic & Silver, 1963; Krnjevic & Silver, 1965; Shute & Lewis,
A
Tetrodotoxin 10,ag/ml. on both sides
4, 100 E
Atropine1 pgjml. on rightside
n-i5
I~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
C
Ao _wz_ _:
0 60 120 180 min
B 0-1 ml.lkg Atropine I 4ugfml. on right side
1 k Fiji ~Dial l.v.
.............
0.
10
0 60 120 180 min
Fig. 6. Average ACh output collected simultaneously from left and right
parietal cortex under Dial anaesthesia A, effect ofbilateral application of
TTX (10 ug/ml.) followed by atropine on the right side only (right side
100% = 2 96ng/cm2.min, n = 4; left side 100% = 3 09ng/cm2.min,
n = 4); B, effect ofDial (0.1 ml./kgi.v.) followed byatropine ontheright
side only (right side 100% = 1V89ng/cm2.min, n = 3; left side 100 =
1.60ng/cm2.min,n = 3).
1967), stimulation ofthe cortex bysurface electrodes shouldpreferentially
activatethe cholinergicnerveendings. Ontheotherhand, thereisevidence
to showthatreticularformation stimulation increases cortical AChrelease
through a pathway involving at least one synapse (Szerb, 1967). There-
fore, reticular formation stimulation would activate orthodromically
the whole cholinergic neurone including the soma. For this reason, the
effect ofatropine on ACh release due to local cortical and reticular forma-
tion stimulation was compared in halothane-N20 anaesthetized cats
where atropine has no effect on resting output.
One such experiment is shown in Fig. 7. Before atropine, local stimu-
lation produced an increase in ACh release similar to that produced by
stimulation of the reticular formation. However, following the topical