mesencephalon, the ratio of the temperature-induced change in ACh

22 Pages·2005·3.37 MB·English

J. Phyaiol. (1973), 230,pp. 273-293 273
With 1plateand 7 text-figurew
Printed in GreatBritain
DIFFERENTIAL RELEASE OF ACETYLCHOLINE
FROM THE HYPOTHALAMUS AND MESENCEPHALON OF THE
MONKEY DURING THERMOREGULATION
BY R. D. MYERS AD M. B. WALLER
From the Laboratory ofNeuropsychology, Purdue University,
Lafayette, Indiana 47907, U.S.A.
(Received 13 June 1972)
SUMMARY
1. In unanaesthetized monkeys acclimated to primate chairs, 101 iso-
lated sites in the hypothalamus and mesencephalon were perfused at a
rate of 30-50,sl./min by means of push-pull cannulae. The perfusate,
which contained an anticholinesterase, was assayed for acetylcholine
(ACh) activity on the guinea-pig ileum in the presence of neostig-
mine.
2. The body temperature of each animal was monitored continuously
during an experiment by colonic and brain thermistors. To alter the
ambient temperature by 15-20' C, either a stream ofwarm air was passed
over the monkey's trunk or containers of ice were placed in its chair
chamber to cool the same region.
3. Assays ofthe effluentrevealedthattherelease ofAChvaried accord-
ingto the ambient temperature as follows: elevated only during cooling;
elevated only during warming; elevated by both thermal stimuli; sup-
pressedonly by cooling; suppressed onlybywarming; suppressed byboth
thermal stimuli; elevated during cooling but suppressed bywarming; and
elevated by warming and suppressed by cooling.
4. A composite anatomical 'mapping' of all perfusion sites revealed
that in response to either peripheral cooling or warming, the output of
ACh varied at only 36% of all sites anterior to the mid-hypothalamic
plane, but at 65% ofthose loci caudal to this coronal plane.
5. In the anterior, preoptic area, cooling enhanced the output ofACh
at 88% ofthe active releasing sites, whereas warming reduced the release
ofAChat 80% oftheseperfusionloci. Posteriortothisregion, AChrelease
was elevated by cooling at about half of the active releasing sites, but
lowered by warming at nearly every active perfusion locus. Within the
mesencephalon, the ratio of the temperature-induced change in ACh
release was similar but in an opposite direction, since the level ofACh in
274 B. D. MYERS AND M. B. WALLER
theeffluentcollectedfromtwooutofthreesiteswasaugmentedby cooling,
but diminished by warming.
6. These results provide additional evidence for the neurochemical
model of Myers & Yaksh (1969), which suggests that a cholinergic path-
way originating in the anterior, preoptic region transmits efferent signals
for heat production. Further, within the posterior hypothalamic area as
well as in the mesencephalon of the monkey, the characteristics of the
ACh releasing sites reflect a function delegated primarily to heat gain,
although evidenceofa cholinergic pathwayfortheheatlosssystem is also
presented.
INTRODUCTION
Withinthe context ofan amine theory ofthermoregulation, it has been
hypothesized that separate cholinergic pathways originate in the hypo-
thalamusoftheprimateforheatproductionandheatloss (Myers& Yaksh,
1969). The evidence for this hypothesis is twofold. First, 5-hydroxytrypt-
amine (5-HT)andnoradrenalineexerttheirhyper-orhypothermicactions,
respectively, only when they are applied locally to the anterior, preoptic
region. Since neither of these monoamines exerts a thermal effect when
micro-injected into the posterior hypothalamus, the classical 'heat main-
tenance centre' (Hardy, 1961; Bligh, 1966; Benzinger, 1969), a third
neurohumoral factor is presumed to transmit the efferent signals arising
from the more rostral 'thermostat' (Myers, 1969b). Second, when ACh
or a cholinomimetic substance is micro-infused into sites caudal to the
preoptic area, a sharp rise in temperature ensues. However, within the
mammillary region at the junction between the posterior hypothalamus
and mesencephalon, there are two cholinoceptive zones-one ostensibly
delegated to heat production whilst the other would subserve heat dis-
sipation (Myers & Yaksh, 1969).
It has been shown that ACh or carbamylcholine injected in the rostral
hypothalamus of the rat may evoke a transient hyper- or hypothermia
(Avery, 1970; Beckman & Carlisle, 1969; Lomax & Jenden, 1966). Avery
(1971) has extended these findings by demonstrating that carbamyl-
choline produced arise inthe temperature ofthis species also whenmicro-
infused in the lateral and medial preoptic area. On the basis of other
micro-injection experiments, Hall and Myers recently proposed that
nicotinic receptors which mediate hypothermia are located in the anterior
hypothalamus of the monkey, whereas, in the posterior area, nicotinic
synapses serve ahyperthermic pathway (Hall & Myers, 1972). From these
findings, it now would appear essential that the hypothalamic release of
ACh must be demonstrated in order to elucidate its possible role as a
HYPOTHALAMIC ACh AND TEMPERATURE 275
transmitter inthe centralthermoregulatory mechanism (Bligh & Maskrey,
1969; Bligh, Cottle & Maskrey, 1971).
In previous studies, theresting release ofACh in subcortical structures
of the cat has been altered by electrical stimulation (McLennan, 1964;
Phillis, Tebecis & York, 1968), and in the unanaesthetized monkey, the
spontaneousreleaseofAChinsitesscatteredthroughoutthehypothalamus
and mesencephalon has been detected (Beleslin & Myers, 1970; Myers &
Beleslin, 1970). If cholinergic synapses should in fact transmit impulses
for the simultaneous activation and reciprocal inhibition ofeither hyper-
or hypothermia, then ACh should be released differentially at sites in the
longitudinal plane extending from the anterior hypothalamus through
the mesencephalon.
Inthepresentexperimentswe attempted to determine whetherchanges
in the resting release ofACh would occur when an animal was regulating
either against a warm or cold stimulus applied peripherally. So thateach
animalcouldbeusedasitsowncontrol,isolatedsitesinthebrainstemofthe
unanaesthetized monkeywere perfused repeatedly bymeans ofpush-pull
cannulae before and after the animal was warmed or cooled.
METHODS
Malerhesusmonkeys(Macacamulatta)weighingfrom4-5to8-5kgwereacclimated
to primate restraining chairs for 7-14 days before surgery. Throughout this period,
and during the experiments, each animal was maintained on Purina monkey chow
andwater,whichwerealwaysfreelyavailable. Theambienttemperaturewasmain-
tainedat 24-260C.
SurgicaZprocedures
Each of twenty-four animals was anaesthetized with pentobarbitone sodium
(25-35mg/kg) injected into the external saphenous vein or one of its superficial
branches. Under aseptic conditions and following the general surgical procedures
described earlier (Myers, 1967), four 17-gauge stainless-steel tubes were implanted
stereotaxically to serve as the guides for push-pull cannulae (Myers, 1970a). The
array oftubes was positioned above the anterior or posterior portions ofthe hypo-
thalamus or above structures in the mesencephalon. After each tube was affixed to
the calvarium by Cranioplast cement, apolystyrene pedestal whichsurrounded the
array was screwed to the skull and capped so that a sterile preparation was main-
tainedthroughouttheexperiments. Torecordbraintemperature, athermistorbead
wasalsoinsertedthrough aburhole drilled inthe calvarium andpositioned against
the posterior portion ofthe sagittal sinus.
Perfusionprocedures
Beforeanexperimentbegan,thebase-linetemperatureoftheanimalwasobtained
for at least 1 hr by means ofthe intracranial thermistor. As a cross-check, colonic
temperature was often monitored simultaneously with a YSI 401 thermistor probe
(Yellow Springs Instrument Co., Yellow Springs, Ohio, U.S.A.) inserted into the
colon to a depth of7-10cm as described elsewhere (Myers & Yaksh, 1969).
276 R. D. MYERS AND M. B. WALLER
The methods for altering the ambient temperature of the monkey before a per-
fusion was begun were the same as those described previously (Myers & Sharpe,
1968; Myers & Beleslin, 1971). A Plexiglass cover was fitted over the front ofthe
primate chair so as to enclose the region just between the neck and pelvic girdle.
Tocoolthe airinthis enclosedspacetwo sealedwire-meshboxescontainingdryice
were placed against the inside walls of the chamber. The top ofthe chamber was
thensealedsothatthemonkeycouldnotinhaleexcessC02.Toraisetheairtempera-
ture surrounding the trunk ofthe animal, a stream ofhot air was blown into the
chairchamber,whichhadbeensealedinanidenticalmanner.Thus,thetemperature
oftheairinthechambercouldberaisedorloweredby 15-200Caboveorbelowthe
ambienttemperature, within 6-8min. Althoughtheairbreathedbytheanimalwas
at ambient temperature, these thermal stimuli were sufficient to evoke normal
thermoregulatory responses. During cooling these included shivering, vasoconstric-
tion, huddling behaviour or lever-pressing for heat-lamp reinforcement. During
warming ofthe trunk, the respiratory rate increased,theearvesselsbecamedilated
and the monkey sometimes drank water. No signs ofdistress such as vocalization,
hyperactivity, biting or struggling occurred. In fact, the animal responded to a
gesture by the experimenterwiththe typical threat behaviour in a normal fashion.
To perfuse an isolated region ofthe brain stem, the concentric cannulae ofeach
push-pull assembly (Myers, 1970a) were lowered through the guide tubes. The
outer or pull cannula consisted of20-gauge stainless-steel thin-wall tubing and the
inner or push cannula was cut from 27-gauge tubing. The tip ofthe push cannula
waspassedthrough asiliconerubberdiaphragmplacedinthecannulacapto alevel
1-0mmbeyondthetipofthepullcannula. Thedepthtowhichthecannulaassembly
couldbeloweredintothebraintissuewascontrolledsimplybythelengthofastain-
less-steel spacer which fitted snugly over the pull cannula. Each cannula assembly
wasconnectedwithpolyethylenetubing (PE-50) toacalibratedpushorpullsyringe
mounted on a multi-channel Harvard infusion-withdrawal pump. In most experi-
ments the perfusions were done bilaterally although not necessarily at homologous
sites.
The perfusate wasamodifiedLocke solution, withthe bicarbonate omitted, con-
taining the following salts in m-mole: Na, 154-9; K, 5-6; Ca, 1-7; C1, 162-9; and
glucose, 11-1 m-mole/l. Inaddition, neostigmine methylsulphate 0-3-1-0,g/ml. was
added, but in a few experiments the anticholinesterase 0 1 or 5-0flg/ml. was used.
Each perfusion solution was prepared just before an experiment began in ion-
exchange, glass-distilled water andpassed through a sterile 0-45#u Millipore filter.
The PE tubing and push-pull cannula assembly were stored in 70% ethanol and
flushed repeatedly with pyrogen-free saline just before use. The rate ofperfusion
was 50#sl./min, the duration usually 30min, and the temperature ofthe perfusate
at the tips ofthe cannulaewas always equilibrated with braintemperature (Myers
& Veale, 1970). A control perfusion was always carried out at room temperature
3-4hr either before or after the warmingor coolingsequence was begun.
A88ayfor acetylchotine
Each sample ofperfusate was assayed immediatelyforACh or in a few instances
stored overnight at pH 7-0 at -100C. The guinea-pig ileum was isolated and sus-
pendedaccordingtothemethodofPaton (1957) inTyrodesolutionwhichcontained
neostigminemethylsulphate(lOesg/l.),morphinesulphate(lOmg/l.) andmethysergide
(20,ug/l.) and was bubbled constantly with 5% C02 in 02. To obtain maximum
sensitivitytoACh, themusclestripwaskeptintheTyrodesolutionforatleast 2hr.
Then, the organ bath was washed repeatedly every 10min. The contractions were
HYPOTHALAMIC ACh AND TEMPERATURE 277
registered on a single-channel recorder, and the value for each contraction was
determined interms ofthe chloride salt ofACh. The amount ofACh contained in a
sample ofeffluent was expressed in terms ofng/30min perfusion period only ifthe
criteria for ACh activity were met as described previously (Myers &Beleslin, 1970)
including the blockade ofthe contractile response by atropine, ortheelimination of
ACh activity by boiling the effluent in an alkaline medium (Feldberg, 1945).
Histological analysis ofperfusion sites
After a series ofexperiments had been completed, either 10#I. of05% bromo-
phenol blue or 25% Indian ink in 0.9% saline was microinjected at each perfusion
site to verify its locus. Then, the monkey was killed by an overdose ofpentobarbi-
tone sodium given i.P., and 10% buffered neutral formalin was perfused through
the thoracic aorta after the heart had been clamped. The brain was washed and
blocked, and sections taken on a freezing microtome at 30,t were stained for cells
and fibres following a method modified after Klhver & Barerra (1953).
RESULTS
In order to analyse the results, a series of anatomical maps was con-
structedofthe 101 discreteperfusionsitesdistributedinthehypothalamus
and mesencephalon of the group of twenty-four monkeys during normal
aswellasaltered ambienttemperatures. Eachmapwasbasedonadetailed
morphological analysis under light microscopy of a series of histological
sections prepared for every monkey. The criterion that was selected to
designate a change in the output ofACh was either a 20% increase above
or a 20% decrease below the resting level ofACh release which was de-
tected in the effluent during a control perfusion. Thus, a shift in ACh
output could be identified independent of the magnitude of ACh in the
sample. A site at which such a change in ACh output was observed is
referred to hereafter as an active releasing site.
Of all the loci examined, it was found that the release of ACh varied
within seventy-three of these sites in response to a change in ambient
temperature. That there seemed to be a functional overlapping of the
morphological systems which were sampled by individual perfusions is
illustrated by the fact that the alteration in the resting output of ACh
could be classified in one of eight ways: (1) elevated during cooling;
(2) elevated during warming; (3) elevated during cooling and warming;
(4) elevatedduringwarmingbutsuppressedbycooling; (5)elevatedduring
cooling but suppressed during warming; (6) suppressed during cooling;
(7) suppressed during warming; (8) suppressed during either cooling or
warming.
Anatomical mapping ofACh release during cooling
Acompositeanalysis ofthehistologicalsectionsobtainedforallmonkeys
showedthata changeintherelease ofAChoccurredatsixtysitesfollowing
278 R. D. MYERS AND M. B. WALLER
0
AP18-0 AP17-0
AP19-0
AP140
AP16-0 API50
API4-0
Va
R
AP13-0 AP12-0 AP11-0
AP10-0 AP90 AP8-0
AP7-0 AP6-0
Cooling ACh
-
Text-fig. Anatomical mappingatfourteencoronal levelsextendingfrom
1.
AP 6-0 to AP 19-0 ofsites distributed throughout the hypothalamus and
mesencephalon of the unanaesthetized rhesus monkey at which isolated
push-pull perfusions were carried out. As aresult ofcooling, therelease of
ACh either increased (A), decreased (V), or remained unchanged (0).
Anatomicalabbreviationsare: a, anteriorhypothalamus; ac,anteriorcom-
missure; b, branchiumpontis; c, caudatenucleus; d, dorsomedial nucleus;
f,fornix;ff,fieldsofForel; g,globuspallidus; i, internalcapsule;Is,lateral
septum;m,mammillarybody;ms,medialseptum;mt,mammillo-thalamic
tract; na, nucleus accumbens; nc, nucleus centralis; nr, nucleus reuniens;
nv,ventrolateralnucleusofthethalamus; oc,opticchiasm; ot,optictract;
p,paraventricularnucleus;pn,nucleuspontis;po,preopticarea;pp,cerebral
peduncle;r,reticularnucleus;rn,nucleusruber; nucleussubthalamicus;
a,
sn,substantianigra;v,ventromedialnucleus;va,anteroventralnucleusof
the thalamus.
HYPOTHALAMIC ACh AND TEMPERATURE 279
the lowering of ambient temperature. The anatomical distribution is
presented in Text-fig. 1 of those perfusion sites at which ACh release
increased (A), decreased (v) or remained unchanged (0). Peripheral
cooling enhanced the release of ACh at thirty-five of the sites but sup-
pressed it at twenty-five other loci. Within the morphological boundaries
of the anterior, preoptic area from coronal planes AP 16-0 through 18-0
and excluding a site adjacent to the globus pallidus and one in the optic
chiasm, cooling increased the output ofACh at seven sites and decreased
it at one. Within the mid-hypothalamic area from AP 13-0 to 15-0, the
ACh level was elevated at four of the ten active releasing sites. In the
posterior hypothalamic, mammillary region that included coronal planes
AP 10.0 to AP 12*0 the pattern of output was nearly identical, since
cooling of the monkey stimulated the ACh output at seven of thirteen
sites. A similar enhancement or suppression of ACh release was again
found within mesencephalic structures extending caudally from coronal
planes AP 10.0 to AP 6-0.
Ofspecial importance was the finding that the direction in the change
ofthe ACh release was in some instances opposite at sites 0 5 mm ofone
another. In other experiments the output ofACh failed to vary at certain
perfusion loci virtually adjacent to those sites at which ACh release was
altered when the monkey was cooled. Examples of these were found at
diencephalic and mesencephalic sites within a single animal or at homo-
logous loci in different monkeys.
Anatomical mapping ofACh release during unarming
The morphological distribution of the fifty-one sites at which ACh
release changed as a result of raising the ambient temperature of the
monkeyispresentedinText-fig. 2.Anincrease (A) inAChoutputoccurred
at only thirteen perfusion sites, whereas a decrease (7) was evoked at
thirty-eight loci. Within the region rostral to AP 15-0, anelevationinthe
ambient temperature suppressed the output of ACh at four out of five
active sites. In the mid-hypothalamic region, extending from AP 13-0 to
15.0 as well as in the more caudal plane ofAP 12-0, the level ofACh in
the effluent failed to increase and in fact was reduced at every active
releasing site. However, in the coronal planes ranging from AP 6-0 to
AP 11 0, the pattern ofACh outputwas quite different. Inthese posterior
and mesencephalic regions, peripheral warming evoked a release which
was higher at twelve sites and lower at twenty-five loci. As in the experi-
ments in which the ambient temperature of the monkey was lowered,
some sites located less than 0 5 mm from one another released ACh in an
opposing fashion in response to warming. This again reflects the close
280 B. D. MYERS AND M. B. WALLER
AP19X0 AP18.0 AP17-0
AP16-0 AP1S-0 AP,14-0
AP6130 AP120 AP1140
V. Va~~3
AP100*' AP90 AP8-0
APA7A0 APA6-0
WarmingP0 ACh
Text-fig. 2. Anatomical mappingatfourteen coronal levelsfromAP 6-0to
AP 19-0 of sites distributed throughout the hypothalamus and mesen-
cephalonoftheunanaesthetizedrhesusmonkeyatwhichisolatedpush-pull
perfusions were carried out. As a result of warming, the release of ACh
either increased (A), decreased (V), or remained unchanged (0). Ana-
tomical abbreviations are the same as in Text-fig. 1.
HYPOTHALAMIC ACh AND TEMPERATURE 281
anatomical proximity of the ACh mechanism underlying the heat-loss
pathway.
ACh release according to the region ofperfusion
Because ofthediffusenature oftheanatomicalsitesatwhichthe output
ofACh was observed to change following cooling or warming, the results
were also analysed in such a way as to relate the temperature-evoked
release ofACh to a given region ofthe brain stem. When active releasing
sitesinthe sevenrostral planes including AP 13-0 to 19-0 were compared
withthosecontainedwithinthesevencaudalplanesextendingfromAP 6X0
to 12X0, distinct patterns ofcholinergic activity emerged.
TABLE 1. The composite frequency of active ACh releasing sites for all monkeys
designated on the basis of the ambient temperature as well as two anatomical
regions. One site in AP 19*0 was not included because ofthe location of the per-
fusion locus
Number ofsites at
whichACh
r ~~~A
Ambient temperature Increased Decreased Total
Cold
Rostral (AP 13.0-19.0) 11 9 20
Caudal (AP 6.0-12.0) 24 15 39
Warm
Rostral (AP 13.0-19.0) 1 11 12
Caudal (AP 6.0-12-0) 12 27 39
Table 1 gives the number of active ACh releasing loci separated
according to the composite anatomical region and the subsequent
enhancement or suppression of ACh output in terms of the ambient
temperature ofthe animal. It can be seen that the frequency ofthe ACh
responses to a change in temperature in either direction was over twice
as great at all caudal sites (seventy-eight) in contrast to all rostral loci
(thirty-two). Takinginto account all oftherostral perfusion sites,itis also
clear that cooling enhanced the release ofACh at eleven oftwenty loci.
In contrast, warminghadthis effectinonlyone oftwelve active sites, and
suppressed the release in the other eleven. Considering all of the caudal
perfusionloci,approximatelytwothirdsoftheactivereleasingsitesshowed
anincreasedAChoutputinresponsetocooling (twenty-fourofthirty-nine)
and a reduced release to warming (twenty-seven ofthirty-nine).
As shown in Text-figs. 1 and 2, the output ofACh failed to change at a
very large number of sites, although many released ACh at a steady
resting level. While thiswould be expected because ofthe large number of
functions served by these distinct areas ofthe brain stem (Myers, 1969a),
282 R. D. MYERS AND M. B. WALLER
the regional distribution of inactive sites showed a relatively constant
pattern when the animal was cooled but not when warmed. Table 2
presents a frequency analysis of sites at which ACh release increased,
decreased orremained unchanged. Onthe basis ofthefourmajormorpho-
logical subdivisions, the frequency of inactive sites recorded during
warming is higher in the anterior, preoptic area (eighteen sites) and much
lowerwithinthe mesencephalon (eight sites) with a corresponding change
infrequencyinthetwointermediateregions(thirteenandten,respectively).
TARTi 2. The composite frequency ofsites in the diencephalon and mesencephalon
ofall monkeys at which the level ofACh detected in a given perfusate was altered
orremainedunchangedinresponsetoperipheralcoolingorwarmingofthemonkey.
One siteinAP 19-0wasnot included because ofthe location ofthe perfusionlocus
Number ofACh releasing sites
5~~~
t A-
Anatomical region Increased Decreased Unchanged
Aambient temperature cold
Anteriorpreoptic area 7 3 12
(AP 16-0-19-0)
Mid-hypothalamus 4 6 10
(AP 13.0-15.0)
Mammillary region 7 6 9
(AP 10-0-12-0)
Mesencephalon 17 9 9
(AP 6.0-9.0)
Totals 35 24 40
AInbient temperature warm
Anterior preoptic area 1 4 18
(AP 16-0-19-0)
Mid-hypothalamus 0 7 13
(AP 13-0-15-0)
Mammillary region 3 9 10
(AP 10-0-12-0)
Mesencephalon 9 18 8
(AP 6-0-9-0)
Totals 13 38 49
In terms ofthe over-all effects ofchanging the monkey's temperature, the
cooling stimulus caused a greater ACh release at the majority of active
releasing sites (thirty-five of fifty-nine) whereas raising the temperature
enhanced the release of ACh at only thirteen of fifty-one sites and sup-
pressed the output at the other thirty-eight loci.
Ofthose active sites at which a change in the output ofACh occurred,
cooling the animal evoked a more frequent release ofACh in all regions
exceptthemid-hypothalamic area. Onthe otherhand, Table 2 showsthat