Cortical acetylcholine release and electroencephalographic arousal

15 Pages·2006·1.77 MB·English

J. Physiol. (1967), 192,pp. 329-343 329
With 6text-ftgures
Printedin GreatBritain
CORTICAL ACETYLCHOLINE RELEASE AND
ELECTROENCEPHALOGRAPHIC AROUSAL
BY J. C. SZERB
From the Department ofPhysiology and Biophysics,
Dalhousie University, Halifax, Nova Scotia, Canada
(Received 17 February 1967)
SUMMARY
1. In cats anaesthetized with N20-halothane acetylcholine (ACh)
release from the parietal cortex was measured. In addition, low and
high-frequency electroencephalographic (e.e.g.) activity was recorded
quantitatively.
2. Stimulation of the mesencephalic reticular formation at 30, 60 and
100/sec produced an identical increase in cortical ACh output, while
300/sec stimulation was about as effective and 10/sec stimulation failed
to increase ACh output.
3. Reticular formation stimulation at 60 and 100/sec reduced the
low-frequencyandincreasedthehigh-frequencye.e.g.activity.Stimulation
at 30 and 300/sec was less effective, while 10/sec stimulation had no effect
on e.e.g.
4. Acute undercutting of the cortex did not affect the resting output
of ACh but greatly reduced the increase due to reticular formation
stimulation as compared to the contralateral intact side. Cutting the
cortex aroundthe collection area didnot affecttheincrease inACh output
due to reticular formation stimulation.
5. Stimulation of the hypothalamus, medial thalamus and septum at
100/sec also increased cortical ACh output while stimulation ofthe dorsal
hippocampus and caudate nucleus failed to do so.
6. Low frequency cortical e.e.g. activity was reduced by stimulating
the reticular formation, the hypothalamus, the medial thalamus and
slightly by septum stimiulation. High-frequency e.e.g. activity was in-
creased by stimulating the reticular formation and the hypothalamus.
7. It is concluded that the ACh measured originates from the neural
tissue underlying the collection area. The increasedrelease is concomitant
to e.e.g. activation but the pathways involved in cortical e.e.g. activation
and increased ACh release are distinct, since the two phenomena do not
vary in a parallel fashion when the reticular formation is stimulated at
different frequencies or when different subcortical areas are stimulated.
330 J. C. SZERB
8. The effectiveness of septal stimulation in increasing ACh release
indicates that at least part of the cortical cholinergic fibres traverse this
area on their way to the cortex.
INTRODUCTION
It is now well established that acetylcholine (ACh) diffuses into physio-
logical saline solution placed on the pial surface ofthe brain after topical
inhibition ofcholinesterase (Elliott, Swank & Henderson, 1950; MacIntosh
& Oborin, 1953; Mitchell, 1963). The amountofAChreleasedfromsensory
areas of the cortex can be increased by stimulating appropriate sensory
pathways (Mitchell, 1963; Collier & Mitchell, 1966) while stimulation of
the mesencephalic reticular formation leads to an enhanced ACh output
in all areas ofthe cerebral convexity tested (Kanai & Szerb, 1965; Celesia
& Jasper, 1966). Since reticular formation stimulation also leads to an
e.e.g. activation, this study was undertaken to establish the relation
between these two phenomena and to delineate pathways involved in
evoking such an increased ACh output.
METHODS
Cats weighing between 2-5 and 3-5kg were anaesthetized throughout the experiment
with amixture of 80%N20 and 20% 02 and 05% halothane administered by means of
arespiratory pump. Artificial respiration assured an even gas flow through the halothane
evaporator(Fluotec),resultinginaconstantconcentrationofhalothanevapour.Toprevent
over-ventilation, the C02 content of the expired air was monitored (Beckman LB1 CO2
Analyser) and the ventilation was adjusted so that the C02 content of the end-tidal air
was 4%. The rectal temperature of the animals was kept at 37.50C throughout the
experiment with aheatingdevice describedbyKrnjevi6 & Mitchell (1961).
The ACh output wasmeasured as describedpreviously (Szerb, 1964). Briefly, a Perspex
cylinder withadiameter of 10mmwas placed lightly onthe parietal cortex. The cylinder
contained 0x25ml. ofLocke solution. In the first 30min 0-2mg/ml. echothiophate iodide
inLockesolutionwasappliedfollowedbyaLockesolutioncontaining 1jug/ml. ofatropine
sulphate and 1 ug/ml. eserine salicylate which was applied throughout the experiment.
Samples were removed every 10 or 15min and assayed on the longitudinal dorsal muscle
ofthe leechsuspendedinamicrobath (Szerb, 1961).Ithasbeenshownpreviouslythatthe
ACh-like activity released from the cortex cannot be distinguished from ACh by paper
chromatography or by parallel assay (Szerb, 1963). Areas of the cortex not under the
cylinderwere covered withliquidparaffin.
The e.e.g. wasmonitoredeithermonopolarly bymeans ofawick-electrode placedinside
the cylinder and using the stereotaxic instrument as ground, or bipolarly by means of
two stainless-steel electrodes 4mm apart, and insulated except at the tip, which were
introduced through the Perspex cylinder. In order to obtain quantitative measurements
of e.e.g. activity at low and high frequencies, the signals derived from the cortex were
amplifiedbymeansofaSanborne.e.g.preamplifier. Formeasuringlow-frequencyactivity,
the output from thepreamplifierwasledintoalow-passfilterwhichpassed 3-S5c/s waves
maximally and reduced 15c/s waves by 6db. For measuringhigh-frequency activity, the
output from the preamplifier was passed through a variable band-pass filter (Krohn-Hite
Corp.) setinsuchawaythat25-40c/sactivitywaspassedthroughmaximally,attenuating
CORTICAL ACh RELEASE AND E.E.G. 331
15c/s frequency by 11db. The outputs from the two filters were integrated by means of
two Sanbornintegratingpreamplifiers. Changesine.e.g. due tostimulationwereexpressed
as the fraction of the activity during 10sec following stimulation over activity during
10sec immediatelybefore stimulation. E.e.g. activityduringstimulationwasnotincluded
because stimulus artifacts appeared inthe integrated tracings.
Inseveralexperiments, lesionswereplacedunderoraroundthePerspexcylinder. Tocut
under the cylinder, a thin, blunt spatula was introduced 2-3cm deep, 5-10mm from the
edge ofthe cylinder and the tip moved around in an arc in a plane parallel to the base
ofthecylinder. Carewastakennotto interruptthepialbloodvesselsanywherebutatthe
site ofintroduction ofthe spatula. To cutaround the cylinder a thinwire was introduced
3-5mmdeepandthecorticalgreymatterseparatedinacircle2-3cmindiameter,avoiding,
however, major blood vessels. Bleeding following the sections usually lasted for only a
short time, but ifit persisted, the experiment was discarded. In two instances, India ink
was given intravenously at the end of the experiment but no difference in the rate of
darkening between the sectioned and the control side was observed. This showedthat the
circulation wasnot seriously impairedbythesections.
Concentricelectrodeswithanexternaldiameterof09mmandwithadistanceof1-5mm
between the inside and outside electrode were used forstimulation. Theywere introduced
stereotaxically to the following sites: reticular formation, A 30, L 3.5, H -1*0; dorsal
hippocampus, A6-0,L35,H +6-5;medialthalamus,A7-5,L2-0,H +0-5;hypothalamus,
A 100, L 30, H -30; septum, A 16-0, L 1 0, H +1 0; caudate nucleus, A 17-0, L 4-0,
H +4-5. The location of the electrodes was checked in every instance by means of the
frozen sectiontechnique ofGuzman,Alcaraz&Fernandez(1958)andstructureswereidenti-
fied by comparison with the stereotaxic atlas of Snyder & Niemer (1961). The stimuli
delivered from Grass S4 stimulators through a stimulus isolation unit were of 0-3msec
duration withanominal voltage of3-8V.
RESULTS
Stimulation ofthe reticularformation atvariousfrequencies. Ina previous
paper (Kanai & Szerb, 1965) it was shown that 100/sec stimulation ofthe
mesencephalic reticular formation caused increased ACh release from
various areas of the cerebral cortex. It was of interest to find out what
frequency of reticular formation stimulation would give maximal ACh
release from the cortex. Since continuous stimulation of the reticular
formation during a 10-15 min collection period results in a rapid at-
tenuation of e.e.g. activation, intermittent stimulation using trains of
stimuli delivered at different frequencies had to be employed. To make
results obtained at different frequencies comparable, 100 stimuli were
delivered at various frequencies every 10 sec during the 10min collection
period, e.g. trains of 100 stimuli at a rate of 100/sec for 1 sec or at 30/sec
for 3-3 sec, etc. Stimulation at 10/sec was delivered continuously during
the 10 min collection period. At least three collection periods without
stimulation separated any two stimulation periods in order to establish
thelevel ofspontaneousAChrelease. In orderto compare theresults from
different preparations, one or two stimulation periods with 100/sec
stimulation were included in every experiment and the increases due to
332 J. C. SZERB
other frequencies were expressed as a percent of the increase due to
100/sec stimulation. Figure 1 shows the result of one such experiment,
while Fig. 2(A) summarizes all experiments on the effect of reticular
30
-
25
20-
15
5
100 300 60 100
Frequency ofstimulation (sec-')
Fig. 1. Therelease ofAChfromthe cortexduringreticularformationstimulation
atvariousfrequencies. CatanaesthetizedwithN20-halothane. 100pulsesdelivered
every 10secatindicatedfrequenciesduringthe 1Omincollectionperiodsindicated
byhorizontalbars.
formation stimulation at various frequencies on cortical ACh output.
It can be seen that 10/sec stimulation increased ACh output very little,
while 30, 60and100/secstimulationhadaboutidenticaleffects. Stimulation
at 300/sec caused an increase approximately i. of that of 100/sec. The
increases due to 10 and to 300/sec stimulation were significantly less than
those due to either 30, 60 or 100/sec stimulation.
The effect ofstimulating the reticular formation at various frequencies
on high and low-frequency e.e.g. activity was tested by delivering a train
of 100 stimuli at the same five frequencies. However, experiments on
e.e.g. activity differed from those on ACh output in two respects: (1) no
cholinesterase inhibitor or atropine was applied, (2) only single trains of
stimuli were delivered every 4 or 5 min, allowing the e.e.g. to return to
the sleep pattern in between. The results shown in Fig. 2(B) and (C) are
the averages of five sets of stimulations at various frequencies delivered
inrandomorder. Itcanbeseenthatincreasingthefrequencyofstimulation
from 10 to 100/sec increased the effect ofstimulation on e.e.g. gradually,
CORTICAL ACh RELEASE AND E.E.G. 333
in contrast to the increase in ACh output which had already reached its
maximum at 30/sec stimulation. The changes in high and low-frequency
activities due to 30/sec stimulation were significantly less than those due
to 100/sec stimulation. At the same time, the effect of300/sec stimulation
B
A
100
2-
75
10 30 60100 300
Stim./seStim/sec
o~~ ~~~~~co
qwUU=
~~~~~~~25~~~~~~.
10 30 60 100 300
Stima./sec
Fig. 2.(A). IncreaseinAChreleasefromcortexduringstimulationofthereticular
formationatdifferentfrequencies.Summaryofexperimentsoneightcatsanaesthe-
tizedwithN20-halothane. Eachobservationconsistsofthedifferencebetweenthe
outputduringstimulationandtherestingoutputimmediatelyprecedingit.Ineach
experiment, the increase at 100/sec stimulation was compared with the effect of
1-3 other frequencies and the increase due to 100/sec stimulation was made
equalto 100%.EachresultisanaverageoffourorfiveobservationswiththeS.E.
ofthe meanindicated byverticallines.
(B). Changes in high-frequency e.e.g. activity due to reticular formation
stimulation at different frequencies. Each bar represents an average of five
observations onone catanaesthetizedwithN20-halothane. Atrainof100 stimuli
were delivered every 4-5min at indicated frequencies, the order offrequencies
selectedatrandom. The vertical axis shows theratio ofactivity 10sec following
stimulation over the activity 10sec immediately before stimulation. Vertical
linesrepresent S.E. ofthemeans.
(C). Changesinlow-frequencye.e.g. activityduetoreticularformationstimula-
tion. Results were obtained simultaneously with those shown in (B). Symbols
asin (B).
was significantly lessthanthatof 100/sec onlyincase ofthelow-frequency
activity.
Effect ofsubcorticalandcorticallesionsonACChoutput. Stimulationofthe
reticular formation results in an increase in ACh output in all cortical
areas tested (Kanai & Szerb, 1965). There was a possibility that the
increased ACh output was not neural in origin but came from non-neural
tissues, such as blood vessels. If the increased ACh output originated in
334 J. C. SZERB
non-neural tissue, lesions in the cortex, not interrupting the vascular
supply, should not have any effect on this increase. The effect of two
kinds of lesions on ACh release was tested: (a) cutting the grey matter
around the cup, and (b) cutting the white matter under the cup, keeping
the cortical circulation as intact as possible in both kinds of lesions. In
these experiments, the reticular formation was stimulated bilaterally and
cylinders were placed in symmetrical locations on the parietal cortices on
bothsides,theleftservingastheexperimental,therightasthecontrolside.
Figures 3 and 4 show individual experiments with cut-around and
cut-under cortices and Table 1 shows the results ofall such experiments.
0
Cutaround
*Control
20
'
0
04
30 60 90 120min
ST ST
Fig. 3. Release of ACh from normal and cut around cortices during reticular
formation stimulation. Cat anaesthetized with N20-halothane. White bars show
AChoutputfromcutaround (left) cortex, hatchedbarsACh outputfromnormal
(right) cortex. Samples from the two sides were collected simultaneously during
15mincollectionperiods. STindicatesbilateralstimulationofreticularformation
at 100/secfor 1/secevery 10sec throughoutthe collectionperiod.
Itcanbeseenthataverageincreasesduetoreticularformationstimulation
were about equal on the two sides, when no lesions were placed on either
side. Furthermore, the maximal increase occurred at the same time on
both sides. Cutting the cortex around the cup did not have any marked
effect on the increase in ACh output. By contrast, cutting under the cup
reduced the increase in ACh output a great deal, and the increase that
was observed on the undercut side frequently reached its maximum one
collection period later than on the control side.
By expressing the increase in ACh release due to stimulation on the
left side as percent of the simultaneous increase on the right side, no
significant differences were foundbetweenthe twointact sides or between
335
CORTICAL ACh RELEASE AND E.E.G.
the cut-around and the intact side. However, on the undercut side, the
increase in ACh output during stimulation amounted to only 25% of
that on the normal side in the samples during and 30% in the samples
following stimulation. Both of these reductions were very highly sig-
0Undercut
35
IControl
30
25
420
20
04
0
115
10
5
30 60 90 120mmni
ST ST
Fig. 4. Release of ACh from normal and undercut cortices during reticular
formation stimulation. Cat under N20-halothane anaesthesia. White bars show
ACh output from undercut (left) cortex, hatched bars ACh output from normal
(right) cortex. Experimental procedure andsymbolsidenticalto Fig. 3.
nificant (t = 12-86 and 8.36). A similar paired comparison between pre-
stimulation samples fromthenormal andundercut sideshowedtheoutput
from the undercut side to be 94% of that from the control side, which
was a non-significant reduction.
Monopolar recordings from the undercut side before and during stimu-
lationdidnotdiffervisibly fromthat ofthe control side. However, bipolar
recordings revealed a complete electrical silence on the undercut side.
Effect of stimulating various subcortical sites on cortical e.e.g. activation
andAChoutput. Itisknownthathigh-frequencystimulationofsubcortical
structures other than the reticular formation, such as the hypothalamus
(Green & Morin, 1953; Longo, 1956; Torii & Wikler, 1966) and the
intralaminar thalamic nuclei (Moruzzi & Magoun, 1949) produces cortical
336 J. C. SZEIRB
e.e.g. activation. It was of interest, therefore, to see whether arousal
resulting from stimulating these centres would also lead to an increased
ACh output from the cortex. In addition, some centres, the stimulation
of which is known to produce only a weak or no e.e.g. activation, were
also included in order to see whether stimulation of any subcortical
area, irrespective of an effect on e.e.g., would result in an increased
TABLE 1. The effect ofcuttingaroundandcuttingunderthecollectionareaoncorticalACh
output before, during and after reticular formation stimulation. L, left cortex; R, right
cortex
ACh outputng/15minsamples
Sections 1st prestimul. 2ndprestimul. Duringstimul. Afterstimul.
Exp. A ,
no. L R L R L R L R __L____ R
1 None None 3-9 40 4-8 6-0 19.5 27-0 14-0 20*4
2 25-0 17-5 25-0 24-0 38-4 32-5 32-4 24*5
3 - - 3-0 2-7 3-5 3-0 22-0 24-0 25*2 24-0
4 - 14-0 110 12-0 9.0 20-0 22*0 15-0 20-0
Av. 11.6 8-8 11.3 10-5 25-0 26-4 21-7 22.2
5 Cut None 4-8 3-3 4-5 3-6 16-8 14-4 6-0 10.8
around
6 - - 6-0 6.0 14-4 19-2 10-8 12-6
7 3-3 2.7 3.4 2-2 6-0 8-4 12*6 14-4
8 9-8 14-0 99 15-0 750 75-0 37-5 40-0
9 22-5 25-0 15.0 15-0 450 42-0 26-3 37-5
Av. 10.1 11-3 7-8 8-4 31-4 31-8 18-6 23-1
10 Cut None 3-0 3-6 3-1 3-5 13-2 36-0 21-6 32-4
under
9*0
11 13-2 8-4 7-5 12-0 36-0 19-2 35-1
12 - 5*0 4-8 4-8 4-8 5-3 19-2 60 12-0
13 3-0 6-0 3-0 4-5 4-0 8-4 4-2 80
14 - 1-8 1.0 1V8 1-5 4-2 8-3 2-5 8 0
15 1-3 3-8 1*7 1*4 4-2 7.5 2-3 60
Av. 3-9 5-4 3-8 3-9 7-2 19-2 9-3 16-9
ACh output. In each preparation three subcortical sites were stimulated,
one ofwhichwas the reticular formation. Changes in e.e.g. orACh output
resulting from stimulating the reticular formation were made equal to
100% and the effect of stimulating other structures was expressed as
apercent ofthe effect ofstimulating thereticularformation. The stimula-
tion frequency was 100/sec for 1 sec delivered in a single train for e.e.g.
evaluation, or every 10 sec when ACh output was measured. Both e.e.g.
activity and ACh output were determined on the side ofstimulation.
Figure 5 shows theresult ofan experiment inwhich the effect ofstimu-
lating three structures on cortical ACh output was measured. The e.e.g.
monitored before and during stimulation of these structures is also
shown. It can be seen that while stimulation ofthe septum (B) had only
asmalleffectonthee.e.g., itgreatlyincreasedtheAChoutput. Stimulation
CORTICAL ACh RELEASE AND E.E.G. 337
of the medial thalamic nuclei (C) and of the reticular formation (D)
resulted in both e.e.g. activation and increased ACh output.
Figure 6 summarizes the quantitative result obtained on ACh release
and e.e.g. activation, resulting from the stimulation of six subcortical
A C
VsN%^s
Parietal A
cup
VAY
Occipital M* Wv ^4AhN9 \y4 {vY\i ¢4
mvL
01
B D 2sec
Parietal -V.01_4MAA*A*&4_ _j6jL._ I
cup
Occipital -.M1uk soh-Il.-. 'I_.-14-01%Pm-l-1--lAl,, A"_OJTdI-PA".r`6
.5
0
-
150mi.
S.E. M.T. R.F.
Fig. 5. E.e.g. and cortical ACh output during stimulation ofvarious subcortical
sites. Cat anaesthetized with N20-halothane. Horizontal bars below records
indicatestimulationat100/secfor1 secevery10secthroughouta10mincollection
period. S.E.-sptum, M.T.-medial thalamus, R.F.-reticular formation stimu-
lation. Lettersabovee.e.g. tracings refertothe collection periodswhenthee.e.g.
tracingswere obtained. Sampleswere collectedande.e.g. wasrecordedipsilateral
to stimulation.
22 Physiol. I92
338 J. C. SZERB
sites. It can be seen that the effects ofreticular formation stimulation on
low-frequency e.e.g. were the greatest, followedbythe hypothalamus, the
medial thalamic nuclei and the septum in decreasing order. Stimulation
a
o | ACh output
.F150 0 flLeo.we.-gf.requency
AHigh-frequency
125quency e.e.g.
100
C"
0
o75
0
O 25
D.H. M.T. HY. SE. CA. R.F.
Fig.-6. ee.g. and increase in cortical ACh output during stimulation of six
subcortical sites. Summary ofexperiments on seventeencats anaesthetized with
N20-halothane. AChrelease,hatchedbars; low-frequencye.e.g., white bars; high-
frequency e.e.g., stippled bars; vertical lines represent S.E. ofthe means; D.H.-
dorsalihippocampus, M.T.-medial thalamus, H1Y.-hypothalamus, SE.--septum,
CA.--caudatenucleus,R.F.-reticularformation. Increa8einACYhrelea8e.In each
experiment, the effect ofreticular formation stimnulation was compared with the
effect ofstimulating one or two other subcortical sites using the same electrode
and the same parameters of stimulation. The increase resulting from reticular
formation stimulation was made equal to 100%. Stimulation frequency 100/sec
for 1sec every 10sec during 10min collection period. Each bar represents the
average of3-4experiments. E.e.g. activity. Oneachside ofthebrainthereticular
formation was stimulated and its effect on low and high-frequency e.e.g. was
compared with the effect ofstimulating one or two other ipsilateral subcortical
sites. The average change producedbyreticular formation stimulationwasmade
equal to 100%. In each cat, each site was stimulated 3 times every 4-5min for
1 sec at 100/sec. Each barrepresents theaverage of9-15 observations.
of the dorsal hippocampus and of the caudate nucleus was without an
effect on the e.e.g. An increase in high-frequency activity was observed
only following the stimulation of the reticular formation and the hypo-
thalamus.