- Email: [email protected]
Cardiovascular responses elicited by fastigial and hypothalamic stimulation in conscious cats
Brain Research, 60 (1973) 439--447
439
© Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands
C A R D I O V A S C U L A R RESPONSES ELICITED BY F A S T I G I A L A N D HYPOT H A L A M I C S T I M U L A T I O N IN CONSCIOUS CATS
N. K. ACHARI*, S, AL-UBAIDY** AND C. B. B. DOWNMAN Department of Physiology, Royal Free Hospital School of Medicine, London I¥C1N 1BP (Great Britain)
(Accepted March 10th, 1973)
SUMMARY The cardiovascular responses to stimulation within a fastigial nucleus of the cerebellum and the defence reaction area of the hypothalamus were tested in 3 conscious Cats in whom chronic indwelling electrodes had been inserted stereotaxically into the brain at previous aseptic operation. At the same time, indwelling catheters had bean inserted into the aorta for recording arterial pressure, and into the superior vena cava for intravenous injections of phenyl diguanide a n d other drugs. Rise of aortic pressure and tachycardia occurred When stimulating' either site at strengths of stimulation below the threshold of somatic movements. Bradycardia of reflex origin (phenyl diguanide, noradrenaline) was inhibited during the stimulations and this was not due to the onset of an equal and opposite tachycardia of sympathetic origin. These results of stimulation are the same in the anaesthetized animal, but were obtained at lower stimulusthreshold.They are not an artefact of anaesthesia.
INTRODUCTION Zanchetti and Zoccolini i6 reported outbursts of sham rage in acute thalamic cats when a cerebellar fastigial nucleus was stimulated. These responses, both somatic and autonomic, were lost after precollicular decerebration, suggesting that the cerebellum was acting via the hyPothalamus. Fastigial stimulation causes a rapid rise0~ arterial pressureS,e, la which persists after midcollicular decerebration6, showing that the cerebellum and hypothalamus can act independently. Together with other auto* Present address: Department of Neurosurgery, University of Birmingham, Queen Elizabeth Hospital, Birmingham B15 2TH, Great Britain. ** Present address! InStitute of Dental Surge~, Eastman Dental Hospital, Gray's Inn Road, London, WCIX 8LD, Great Britain.
440
N . K . ACHAR! et al.
nomic responses to fastigial stimulation there is strong inhibition of reflex bradycardia of various origins 6. Similar results have been described following stimulation in the hypothalamus. Vasomotor and cardioacceleratory responses and other autonomic responses have been recorded when stimulating the peri-fornical area in the ventral hypothalamus 8,1°,14. Stimulating these areas in conscious, unrestrained cats elicits the somatic and behavioural changes of the defence reaction. At the same time there is inhibition of reflex bradycardia of baroreceptor or other origin 4-n,9,u. Whereas some of the observations on hypothalamic stimulations have been made on conscious cat 1,1°, the experiments on cerebellar controls of autonomic function have been done in animals which were anaesthetized or had major transections of the neuraxis. The purpose of the present study was to determine whether fastigial stimulation is similarly effective in the intact conscious animal and whether the cardiovascular changes are an inseparable part of some pattern of response of the whole animal. The responses to stimulation within the defence reaction area of the hypothalamus were elicited alone or in conjunction with fastigial stimulation to test whether there is interaction between the descending pathways from the two regions. METHODS
At initial operation with full aseptic precautions, indwelling catheters were inserted into the aorta and the superior vena cava, and stimulating electrodes were implanted into the deep cerebellar nuclei and the hypothalamic defence reaction area. Cats were anaesthetized with sodium pentobarbitone (Veterinary Nembutal, Abbott) 40 mg/kg intraperitoneally, and the trachea was intubated during the operation. Through a small incision in the right-hand side of the neck, the common carotid artery was exposed and ligated and a stoppered polythene catheter was inserted centrally into the artery to bring its tip into the descending aorta. A similar catheter was inserted into the ligated external jugular vein so that its tip lay in the lower part of the superior vena cava. The catheters were filled with heparin-0.9 ~ NaC1 before insertion. Electrodes for unipolar stimulation within the brain were made from dental broaches with extra fine tips, insulated except at the tip with Tufnol varnish. These electrodes tapered from a shoulder of 0.75 mm diameter to a shaft of 100 #m diameter with an exposed tip of 50-75/~m diameter when varnished; total length of tapered shaft was 25 mm. Tip resistance in situ in the anaesthetized cat was about 25 kf~ at 1.5 V DC. Electrode length was adjusted so that when the tip lay in the chosen site for stimulation about 3 mm of the broach would project outside the skull. Coloured wire leads were soldered at 3--4 mm from the outer end of the broach and the junction insulated with an epoxy resin (Araldite). The electrodes were inserted vertically into the cranium through small holes drilled through the skull, the head being held in a stereotaxic frame. Hypothalamic placement was done with the head in the Horsley-Clarke horizontal position, and cerebellar placement with the head tilted 45 ° nose-down. During insertion, each electrode was advanced dorsoventrally in 0.5 mm steps, and the brain was stimulated with 50 Hz sine wave currents at 0.3 V r.m.s, for 10-30 sec. When
FASTIGIAL AND HYPOTHALAMIC STIMULATION
441
the best stimulation site was reached, identified by the cardiovascular and other responses being elicited at lowest threshold, the electrode was fixed to the skull with a quick-setting cold-cure acrylic cement. When this cement was hardened, more was spread over the top of the electrode to ensure insulation. Electrode tips in interpositus and dentate nuclei were located by insertion in more lateral planes with the tips in the same antero-posterior and dorsoventral planes as the fastigial electrode. Two indifferent electrodes of stainless steel wire were sewn separately under the reflected temporal muscle. The wounds were closed with separate sutures after the catheters and wires had been brought out through stab wounds through the skin at the back of the neck and anchored to the skin with sutures. The cats were nursed postoperatively in a warm room, and were given antibiotics subcutaneously. The responses to brain stimulation were tested after 3-4 days when the cats had recovered and freely mobile. Arterial pressure was recorded from the intra-aortic catheter with a Statham B.P. transducer and Grass polygraph with 5PI preamplifier. Heart rate was recorded by connecting a signal output from the driver amplifier of the polygraph to a Grass 5P4 tachograph unit to record intervals between the pressure pulses. In some experiments breathing was recorded from a Devices mercury-in-rubber strain gauge round the thorax. Sessions of recording lasted 30-60 min daily for 4--6 days. Finally the animals were killed by intravenous injection of excess Nembutal, and the head was perfused with 10% formaldehyde-0.9 ~o NaCI fixative via the aorta. Subsequently histological sections were cut in the plane of the electrode insertions and stained by Weil's method 15. Electrode tip positions were marked on enlarged drawings of the brain according to the stereotaxic coordinates and the visible tracks. RESULTS
Stimulation of the fastigial nucleus and the hypothalamic defence area in the conscious unrestrained cat evoked comparable cardiovascular responses at one-third to one-half of the voltage needed when the same animal was anaesthetized. Stronger stimuli which produced somatic responses were not used unless these latter were tested deliberately. It was also observed that in conscious animals smaller amounts of phenyl diguanide produced reflex bradycardia when injected intravenously into the superior vena cava. Stimulating within the dentate nuclei at similar strengths did not produce cardiovascular responses. Stimulation within a fastigial nucleus
This consistently elicited increase of mean arterial and pulse pressures and tachycardia (Fig. 1). Stimulus thresholds for these responses were always lower than for somatic movements. Stronger stimuli produced additionally a characteristic posturing of the neck together with slow flexion of the ipsilateral foreleg and sometimes either abduction or adduction. There was also pupil dilation, more rapid breathing and intermittent quiet mewing. Injecting single doses of phenyl diguanide (15-20/zg) through the indwelling
442
N. K. A C H A R I et
1
2
P, D
3
al.
4
P,D
2OO
A
30SOC
_
F
_.iNA
I:~A
,NA
2OO
B l_1-2110
Fig. 1. Cardiovascular responses and inhibition of reflex bradycardia during stimulation of a fastigial nucleus. Cat conscious and unrestrained. Tracings from above downward: stimulus marker; aortic pressure (BP), mm Hg; heart rate (HR) tachographic recording, in beats/min. Series A and B show the responses to stimulation of the fastigial nucleus at 0.6 V, 50 Hz sine wave currents (F). Single intravenous injections of 15/~g of phenyl diguanide (PD) and 1 #g noradrenaline (NA) were given alone and also during a 20 sec period of stimulation of the fastigial nucleus (F + PD) and (F + NA). Intervals of 1 rain between recordings. catheter in the superior vena cava caused the expected reflex bradycardia, hypotension and bradypnoea 7. Stimulating the fastigial nucleus at strengths which produced cardiovascular but not somatic responses, partially or completely inhibited the reflex bradycardia caused by phenyl diguanide when this substance was injected during the period of fastigial stimulation. Thereflex bradycardia which followed an intravenous injection of noradrenaline (1.0 # g ) w a s also inhibited during a period of fastigial stimulation despite the rise of arterial pressure now being greater than with either fastigial stimulation or noradrenaline alone. Stimulating within a dentate nucleus did not cause cardiovascular responses up to stimulusstrengths which produced change of posture of the neck and forelimbs. Stimulation in the interpositus nucleus caused no, or only small rise of, arterial pressure and heart rate; small responses have been seen also in the anaesthetized animal 6 and may be explained by some fastigio-bulbar fibres passing through the nucleuslL
Stimulation in the defence reaction area of the hypothalamus This always elicited an increase in mean arterial and pulse pressure and tachycardia (Fig. 2) at low stimulus strengths. Stronger stimuli caused dilation of the pupils, widening of the palpebral fissures, retraction o f the ears, piloerection and characteristic movement of the neck. Still stronger stimuli caused hyperpnoea and posturing and attempts at flight or fight,
FASTIGIAL AND HYPOTHALAMIC STIMULATION 1
H BP
I
443
2
3
NA
H*NA
4
, i
NA
'too°° E
A
0
HR
150E 2so
,oo
PD
,I-I*PD
PD
BP too 0
HR
150E
.
B
2so
Fig. 2. Cardiovascular responses and inhibition of reflex bradycardia during stimulation of the hypothalamic defence reaction area. Cat conscious and almost unrestrained. Tracings from above downwards: stimulus marker; aortic pressure (BP) in mm Hg; heart rate (HR) tachograph recording in beats/min. Series A and B show the response s to stimulation in the hypothalamic defence reaction area at 0.75 V, 50 Hz sine wave currents (H). Single injections of 1/~g noradrenaline (NA) or 15/~g phenyl diguanide (PD) into the superior vena cava were given alone and then during a period of 20 sec stimulation of the hypothalamus (H d- NA and H d- PD). Intervals of 1 min between each recording.
Stimulation which evoked only cardiovascular changes inhibited the reflex bradycardia occurring after an intravenous injection of phenyl diguanide. This was shown by giving the injection during a period of electrical stimulation in the defence reaction area (Fig. 2). In some cats where the stimulation site was found to be more caudal than was intended, the post-injection hypotension was also abolished. The secondary bradycardia following an intravenous injection of noradrenaline was also inhibited during a period of hypothalamic stimulation (Fig. 2) although the hypertension caused by the stimulation and the injection together was greater than with either alone.
Effects of beta-adrenergic receptor blockade Propranolol, 2 mg/kg, was injected intravenously in solution during 15 min. This caused a decrease in resting heart rate and pulse pressure (Fig. 3). Stimulation within the hypothalamus or fastigial nucleus still caused marked rise of mean arterial pressure, but there was no, or only small, increase in heart rate or pulse pressure. The slight tachycardia was abolished after intravenous administration of atropine (1 rag/ kg); this indicates that both sites of stimulation can cause a small inhibition of vagal tone. The bradycardia following intravenous injections of phenyl diguanide or noradrenaline was completely or partially inhibited when the injections were given during a 20-25 sec period of stimulation of the fastigial or hypothalamic site.
N . K . ACHARI et aL
444 1
2 H
H
3 NA
4
H.~A
o j.NA
/00
B P
loo
A
0 150
H R
250
I--=--I 30see
_,,F
. _ . r a m~ . . . .
B P
. tNA
. F~NA
+NA
,oo o
H R
_
B
~oo
Fig. 3. Persistence of inhibition of reflex bradycardia after beta-adrenergic blockade of cardiac accelerator nerves. Cat conscious and unrestrained. Tracings from above downwards: stimulus marker; aortic pressure (BP) in m m Hg; heart rate (HR) tachograph recording in beats/min. Series A shows the effect of stimulation within the hypothalamic defence area with 0.5 V 50 Hz sine wave currents (H); series B shows fastigial nuclear stimulation with the same parameters of stimulus. In each series between recordings 1 and 2 propranolol (2 mg/kg) was injected i.v. slowly. Subsequently the effect of 1/.tg noradrenaline i.v. was tested alone (NA) or during brain stimulation (H + NA, or F + NA). Intervals of 1 min between recordings.
1
2
3
F
H
F*H
200
BP
lOO 0 100
HR
2oe
1----t 3osec
Fig. 4. Summation of cardiovascular responses when fastigial and hypothalamic stimulations are combined. Cat conscious and unrestrained. Tracings from above downwards: stimulus marker; aortic pressure (BP) in m m Hg; heart rate (HR) in beats/min. Using 50 Hz sine wave currents, in record 1 the fastigial nucleus was stimulated (F) at 0.4 V; in record 2 the defence reaction area was stimulated (H) at 0.5 V; in record 3 these two stimulations were combined. Intervals of 1 min between recordings.
FASTIGIAL AND HYPOTHALAMIC STIMULATION
445
Fastigial-hypothalamicsynergism Certain cardiovascular responses following stimulation within a fastigial nucleus or the defence reaction area of the hypothalamus are similar, as described above. Stimulation within both areas at the same time showed that these responses would summate (Fig. 4). Furthermore, stimulating a fastigial nucleus at just below the threshold of changes of arterial pressure and heart rate reduced the threshold of the hypothalamic stimulation which elicited the same responses. The facilitation was found to be reciprocal between the two stimulated regions, since hypothalamic stimulation lowered the threshold of effective fastigial stimulation. However, the same mutual facilitation was not found when behavioural responses were elicited. Stimulation which was at or above the threshold for eliciting these responses from the one site did not change the threshold for eliciting the same responses from the other site. DISCUSSION
It has been shown in these experiments that stimulating a fastigial nucleus in the conscious cat produces the same cardiovascular responses as in the anaesthetized animal. The threshold is lower but the pattern is the same. The threshold of these responses was always lower than the thresholds of somatic responses, indicating that the circulatory responses may be independent of the somatic responses even when the latter are not suppressed by anaesthesia. The present study confirms that fastigial inhibition of some patterns of reflex bradycardia is a feature of the intact unanaesthetized nervous system. It also shows that the hypothalamus can inhibit reflex bradycardia without invoking somatic activity. This recalls Hilton's early demonstration of hypothalamic inhibition of carotid sinus reflexes in the anaesthetized cat, and confirms similar demonstrations in the anaesthetized animal4, 9. It was confirmed histologically that the tips o f the stimulating electrodes were inserted into, and had not penetrated beyond, the deep cerebellar nuclei and the hypothalamus. In a previous paper 8 detailed evidence has been presented that stimulation with unipolar electrodes using 50 Hz sine wave stimulating currents at less than 3 V does not activate structures more than 0.5 mm from the electrode tip. Vasomotor centres in the brain stem are involved indirectly, probably via fastigio-bulbar pathways 12 and not by spread of current from the electrode tip. In the conscious cat marked cardiovascular responses were evoked by stimuli of less than 1 V, generally 0.5-0.6 V. Both hypothalamic and fastigial stimulation produce tachycardia in the conscious cat. This result of fastigial stimulation was seen less often in the anaesthetized animal 6. The inhibition of reflex bradycardia is not simply the algebraic cancellation of bradycardia by a stimulus-evoked tachycardia. In the anaesthetized animal the inhibition can occur when fastigial stimulation alone produces no tachycardia 6. The present study confirms this in the unanaesthetized cat. After beta-adrenergic blockade, when the central stimulation failed to have effect on the heart, because of the blocking of the sympathetic terminals, bradycardia was still abolished although tachycardia was no longer available. The observations of Zanchetti and Zoccolini 16 show that there may be inter-
446
N . K . ACHARI e t al.
actions between cerebellar and hypothalamic nuclei, although Achari and Downman 6 and Miura and Reis la have given p r o o f of independence of action during anaesthesia. In the unanaesthetized animal the threshold of a fastigial nucleus was lowered when the hypothalamic site was stimulated; similarly fastigial stimulation lowered the hypothalamic threshold. When the threshold stimulations of the two sites were combined there was summation of the peripheral effects. This was a feature of the elements controlling cardiovascular responses but not of those controlling somatic responses. The observation that fastigial stimulation did not lower the threshold of the somatic responses of the defence reaction area contrasts with the finding of Zanchetti and Zoccolini in the thalamic cat. The mutual facilitation between fastigiat and hypothalamic nuclei controlling cardiovascular activity could represent summation of descending impulses at the lower motor level. It has been shown that there is early facilitation and later inhibition of the discharges in the inferior cardiac and renal nerves evoked by single-shock stimulation in the ipsilateral hypothalamus and in the fastigial nucleus ~,a. However, the mutual facilitation between the fastigial and hypothalamic sites was shown by using stimuli which caused no response of the effectors and so presumably did not activate the spinal sympathetic neurones. It is possible that the facilitation depends upon altered excitability of neurones round the tip of the electrode which is mediated by pathways connecting the fastigial and the hypothalamic nuclei. Stimulating the dentate nucleus in the intact unanaesthetized cat caused no cardiovascular changes within the range of voltage used. Stimulation within the adjacent nucleus interpositus caused small changes which might be explained by some fastigio-bulbar fibres being stimulated as they course through the nucleus lg. The results of stimulating the deep cerebellar nuclei in the conscious cat confirm the evidence from the anaesthetized cat 6 that the fastigial nuclei have particular control of sympathetic outflow to the cardiovascular system, and demonstrate that this is not an abnormal consequence of anaesthesia or damage to the brain stem.
REFERENCES 1 ABRAHAM,V. S., HILTON,S. M., AND ZBROZYNA,A., Active muscle vasodilation produced by stimulation of the brain stem; its significance in the defence reaction, J. Physiol. (Lond.), 154 (1960) 491-513. 2 ACHAm,N. K., AL-UBAIDY,S., AND DOWNMAN, C. B. B., Cerebellar initiation of discharges in sympathetic nerves, J. Physiol. (Lond.), 215 (1971) 21-22P. 3 AeriAl, N. K., AL-UBAIDY,S., ANDDOWNMAN,C. B. B., Fastigial influence on autonomic and somatic spinal reflexes, Indian J. Physiol. Pharmacol., 16 (1972) 251-252. 4 ACHAm,N. K., ANDDOWNMAN,C. B. B., Inhibition of chemoreflexesby stimulation of the defence reaction area in the cat, J. Physiol. (Lond.), 201 (1969) 100-101P. 5 ACHAm,N. K., AND DOWNMAN,C. B. B., Autonomic responses evoked by stimulation of the fastigial nucleus in the anaesthetized cat, J. Physiol. (Lond.), 204 (1969) 130-131P. 6 ACnARI,N. K., AND DOWNMAN, C. B. B., Autonomic effector responses to stimulation of the fastigial nucleus, J. Physiol. (Lond.), 210 (1970) 637-650. 7 DAWES,G. S., MOTT,J. C. A., ANDWn)DICOM~,J. G., Respiratory and cardiovascular reflexes from the heart and lungs, J. Physiol. (Lond.), 115 (1951) 258-291. 8 FOLKOW,B., ANDRUBINSTE1N,E. H., Behavioural and autonomic patterns evoked by stimulation of the lateral hypothalamic area in the cat, ,4cta physiol, scand., 65 (1965) 292-299.
FASTIGIAL AND HYPOTHALAMIC STIMULATION
447
9 GEBBER, G. L., AND SNYDER, D. W., Hypothalamic control of baroreceptor reflexes, Amer. J. Physiol., 218 (1970) 124-131. 10 HEss, W. R., Diencephalon Autonomic and Extrapyramidal Functions, Grune and Stratton, New York, 1954, Ch. 3, pp. 12-24. 11 HILTON, S. M., Inhibition of baroreceptor reflexes on hypothalamic stimulation, J. Physiol. (Lond.), 165 (1963) 56-57P. 12 JANSEN,J., AND JANSEN,JR., J., On the efferent fibres of the cerebellar nuclei in the cat, J. comp. NeuroL, 102 (1955) 607-632. 13 MIURA, M., AND REIS, D. J., Cerebellum: A pressor response elicited from fastigial nucleus and its efferent pathway in the brain stem, Brain Research, 13 (1969) 595-599, 14 WANG, S. C., AND RANSON, S. W., Descending pathways from the hypothalamus to the medulla and spinal cord. Observations on blood pressure and bladder reflexes, J. comp. Neurol., 71 (1939) 457-472. 15 WEIL, A., Rapid method for staining myelin sheaths, Arch. Neurol. Psychiat. (Chic.), 20 (1928) 392-393. 16 ZANCHETTI, A., AND ZOCCOLINI, A., Autonomic hypothalamic outbursts elicited by cerebellar stimulation, J. NeurophysioL, 17 (1954) 475-483.
439
© Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands
C A R D I O V A S C U L A R RESPONSES ELICITED BY F A S T I G I A L A N D HYPOT H A L A M I C S T I M U L A T I O N IN CONSCIOUS CATS
N. K. ACHARI*, S, AL-UBAIDY** AND C. B. B. DOWNMAN Department of Physiology, Royal Free Hospital School of Medicine, London I¥C1N 1BP (Great Britain)
(Accepted March 10th, 1973)
SUMMARY The cardiovascular responses to stimulation within a fastigial nucleus of the cerebellum and the defence reaction area of the hypothalamus were tested in 3 conscious Cats in whom chronic indwelling electrodes had been inserted stereotaxically into the brain at previous aseptic operation. At the same time, indwelling catheters had bean inserted into the aorta for recording arterial pressure, and into the superior vena cava for intravenous injections of phenyl diguanide a n d other drugs. Rise of aortic pressure and tachycardia occurred When stimulating' either site at strengths of stimulation below the threshold of somatic movements. Bradycardia of reflex origin (phenyl diguanide, noradrenaline) was inhibited during the stimulations and this was not due to the onset of an equal and opposite tachycardia of sympathetic origin. These results of stimulation are the same in the anaesthetized animal, but were obtained at lower stimulusthreshold.They are not an artefact of anaesthesia.
INTRODUCTION Zanchetti and Zoccolini i6 reported outbursts of sham rage in acute thalamic cats when a cerebellar fastigial nucleus was stimulated. These responses, both somatic and autonomic, were lost after precollicular decerebration, suggesting that the cerebellum was acting via the hyPothalamus. Fastigial stimulation causes a rapid rise0~ arterial pressureS,e, la which persists after midcollicular decerebration6, showing that the cerebellum and hypothalamus can act independently. Together with other auto* Present address: Department of Neurosurgery, University of Birmingham, Queen Elizabeth Hospital, Birmingham B15 2TH, Great Britain. ** Present address! InStitute of Dental Surge~, Eastman Dental Hospital, Gray's Inn Road, London, WCIX 8LD, Great Britain.
440
N . K . ACHAR! et al.
nomic responses to fastigial stimulation there is strong inhibition of reflex bradycardia of various origins 6. Similar results have been described following stimulation in the hypothalamus. Vasomotor and cardioacceleratory responses and other autonomic responses have been recorded when stimulating the peri-fornical area in the ventral hypothalamus 8,1°,14. Stimulating these areas in conscious, unrestrained cats elicits the somatic and behavioural changes of the defence reaction. At the same time there is inhibition of reflex bradycardia of baroreceptor or other origin 4-n,9,u. Whereas some of the observations on hypothalamic stimulations have been made on conscious cat 1,1°, the experiments on cerebellar controls of autonomic function have been done in animals which were anaesthetized or had major transections of the neuraxis. The purpose of the present study was to determine whether fastigial stimulation is similarly effective in the intact conscious animal and whether the cardiovascular changes are an inseparable part of some pattern of response of the whole animal. The responses to stimulation within the defence reaction area of the hypothalamus were elicited alone or in conjunction with fastigial stimulation to test whether there is interaction between the descending pathways from the two regions. METHODS
At initial operation with full aseptic precautions, indwelling catheters were inserted into the aorta and the superior vena cava, and stimulating electrodes were implanted into the deep cerebellar nuclei and the hypothalamic defence reaction area. Cats were anaesthetized with sodium pentobarbitone (Veterinary Nembutal, Abbott) 40 mg/kg intraperitoneally, and the trachea was intubated during the operation. Through a small incision in the right-hand side of the neck, the common carotid artery was exposed and ligated and a stoppered polythene catheter was inserted centrally into the artery to bring its tip into the descending aorta. A similar catheter was inserted into the ligated external jugular vein so that its tip lay in the lower part of the superior vena cava. The catheters were filled with heparin-0.9 ~ NaC1 before insertion. Electrodes for unipolar stimulation within the brain were made from dental broaches with extra fine tips, insulated except at the tip with Tufnol varnish. These electrodes tapered from a shoulder of 0.75 mm diameter to a shaft of 100 #m diameter with an exposed tip of 50-75/~m diameter when varnished; total length of tapered shaft was 25 mm. Tip resistance in situ in the anaesthetized cat was about 25 kf~ at 1.5 V DC. Electrode length was adjusted so that when the tip lay in the chosen site for stimulation about 3 mm of the broach would project outside the skull. Coloured wire leads were soldered at 3--4 mm from the outer end of the broach and the junction insulated with an epoxy resin (Araldite). The electrodes were inserted vertically into the cranium through small holes drilled through the skull, the head being held in a stereotaxic frame. Hypothalamic placement was done with the head in the Horsley-Clarke horizontal position, and cerebellar placement with the head tilted 45 ° nose-down. During insertion, each electrode was advanced dorsoventrally in 0.5 mm steps, and the brain was stimulated with 50 Hz sine wave currents at 0.3 V r.m.s, for 10-30 sec. When
FASTIGIAL AND HYPOTHALAMIC STIMULATION
441
the best stimulation site was reached, identified by the cardiovascular and other responses being elicited at lowest threshold, the electrode was fixed to the skull with a quick-setting cold-cure acrylic cement. When this cement was hardened, more was spread over the top of the electrode to ensure insulation. Electrode tips in interpositus and dentate nuclei were located by insertion in more lateral planes with the tips in the same antero-posterior and dorsoventral planes as the fastigial electrode. Two indifferent electrodes of stainless steel wire were sewn separately under the reflected temporal muscle. The wounds were closed with separate sutures after the catheters and wires had been brought out through stab wounds through the skin at the back of the neck and anchored to the skin with sutures. The cats were nursed postoperatively in a warm room, and were given antibiotics subcutaneously. The responses to brain stimulation were tested after 3-4 days when the cats had recovered and freely mobile. Arterial pressure was recorded from the intra-aortic catheter with a Statham B.P. transducer and Grass polygraph with 5PI preamplifier. Heart rate was recorded by connecting a signal output from the driver amplifier of the polygraph to a Grass 5P4 tachograph unit to record intervals between the pressure pulses. In some experiments breathing was recorded from a Devices mercury-in-rubber strain gauge round the thorax. Sessions of recording lasted 30-60 min daily for 4--6 days. Finally the animals were killed by intravenous injection of excess Nembutal, and the head was perfused with 10% formaldehyde-0.9 ~o NaCI fixative via the aorta. Subsequently histological sections were cut in the plane of the electrode insertions and stained by Weil's method 15. Electrode tip positions were marked on enlarged drawings of the brain according to the stereotaxic coordinates and the visible tracks. RESULTS
Stimulation of the fastigial nucleus and the hypothalamic defence area in the conscious unrestrained cat evoked comparable cardiovascular responses at one-third to one-half of the voltage needed when the same animal was anaesthetized. Stronger stimuli which produced somatic responses were not used unless these latter were tested deliberately. It was also observed that in conscious animals smaller amounts of phenyl diguanide produced reflex bradycardia when injected intravenously into the superior vena cava. Stimulating within the dentate nuclei at similar strengths did not produce cardiovascular responses. Stimulation within a fastigial nucleus
This consistently elicited increase of mean arterial and pulse pressures and tachycardia (Fig. 1). Stimulus thresholds for these responses were always lower than for somatic movements. Stronger stimuli produced additionally a characteristic posturing of the neck together with slow flexion of the ipsilateral foreleg and sometimes either abduction or adduction. There was also pupil dilation, more rapid breathing and intermittent quiet mewing. Injecting single doses of phenyl diguanide (15-20/zg) through the indwelling
442
N. K. A C H A R I et
1
2
P, D
3
al.
4
P,D
2OO
A
30SOC
_
F
_.iNA
I:~A
,NA
2OO
B l_1-2110
Fig. 1. Cardiovascular responses and inhibition of reflex bradycardia during stimulation of a fastigial nucleus. Cat conscious and unrestrained. Tracings from above downward: stimulus marker; aortic pressure (BP), mm Hg; heart rate (HR) tachographic recording, in beats/min. Series A and B show the responses to stimulation of the fastigial nucleus at 0.6 V, 50 Hz sine wave currents (F). Single intravenous injections of 15/~g of phenyl diguanide (PD) and 1 #g noradrenaline (NA) were given alone and also during a 20 sec period of stimulation of the fastigial nucleus (F + PD) and (F + NA). Intervals of 1 rain between recordings. catheter in the superior vena cava caused the expected reflex bradycardia, hypotension and bradypnoea 7. Stimulating the fastigial nucleus at strengths which produced cardiovascular but not somatic responses, partially or completely inhibited the reflex bradycardia caused by phenyl diguanide when this substance was injected during the period of fastigial stimulation. Thereflex bradycardia which followed an intravenous injection of noradrenaline (1.0 # g ) w a s also inhibited during a period of fastigial stimulation despite the rise of arterial pressure now being greater than with either fastigial stimulation or noradrenaline alone. Stimulating within a dentate nucleus did not cause cardiovascular responses up to stimulusstrengths which produced change of posture of the neck and forelimbs. Stimulation in the interpositus nucleus caused no, or only small rise of, arterial pressure and heart rate; small responses have been seen also in the anaesthetized animal 6 and may be explained by some fastigio-bulbar fibres passing through the nucleuslL
Stimulation in the defence reaction area of the hypothalamus This always elicited an increase in mean arterial and pulse pressure and tachycardia (Fig. 2) at low stimulus strengths. Stronger stimuli caused dilation of the pupils, widening of the palpebral fissures, retraction o f the ears, piloerection and characteristic movement of the neck. Still stronger stimuli caused hyperpnoea and posturing and attempts at flight or fight,
FASTIGIAL AND HYPOTHALAMIC STIMULATION 1
H BP
I
443
2
3
NA
H*NA
4
, i
NA
'too°° E
A
0
HR
150E 2so
,oo
PD
,I-I*PD
PD
BP too 0
HR
150E
.
B
2so
Fig. 2. Cardiovascular responses and inhibition of reflex bradycardia during stimulation of the hypothalamic defence reaction area. Cat conscious and almost unrestrained. Tracings from above downwards: stimulus marker; aortic pressure (BP) in mm Hg; heart rate (HR) tachograph recording in beats/min. Series A and B show the response s to stimulation in the hypothalamic defence reaction area at 0.75 V, 50 Hz sine wave currents (H). Single injections of 1/~g noradrenaline (NA) or 15/~g phenyl diguanide (PD) into the superior vena cava were given alone and then during a period of 20 sec stimulation of the hypothalamus (H d- NA and H d- PD). Intervals of 1 min between each recording.
Stimulation which evoked only cardiovascular changes inhibited the reflex bradycardia occurring after an intravenous injection of phenyl diguanide. This was shown by giving the injection during a period of electrical stimulation in the defence reaction area (Fig. 2). In some cats where the stimulation site was found to be more caudal than was intended, the post-injection hypotension was also abolished. The secondary bradycardia following an intravenous injection of noradrenaline was also inhibited during a period of hypothalamic stimulation (Fig. 2) although the hypertension caused by the stimulation and the injection together was greater than with either alone.
Effects of beta-adrenergic receptor blockade Propranolol, 2 mg/kg, was injected intravenously in solution during 15 min. This caused a decrease in resting heart rate and pulse pressure (Fig. 3). Stimulation within the hypothalamus or fastigial nucleus still caused marked rise of mean arterial pressure, but there was no, or only small, increase in heart rate or pulse pressure. The slight tachycardia was abolished after intravenous administration of atropine (1 rag/ kg); this indicates that both sites of stimulation can cause a small inhibition of vagal tone. The bradycardia following intravenous injections of phenyl diguanide or noradrenaline was completely or partially inhibited when the injections were given during a 20-25 sec period of stimulation of the fastigial or hypothalamic site.
N . K . ACHARI et aL
444 1
2 H
H
3 NA
4
H.~A
o j.NA
/00
B P
loo
A
0 150
H R
250
I--=--I 30see
_,,F
. _ . r a m~ . . . .
B P
. tNA
. F~NA
+NA
,oo o
H R
_
B
~oo
Fig. 3. Persistence of inhibition of reflex bradycardia after beta-adrenergic blockade of cardiac accelerator nerves. Cat conscious and unrestrained. Tracings from above downwards: stimulus marker; aortic pressure (BP) in m m Hg; heart rate (HR) tachograph recording in beats/min. Series A shows the effect of stimulation within the hypothalamic defence area with 0.5 V 50 Hz sine wave currents (H); series B shows fastigial nuclear stimulation with the same parameters of stimulus. In each series between recordings 1 and 2 propranolol (2 mg/kg) was injected i.v. slowly. Subsequently the effect of 1/.tg noradrenaline i.v. was tested alone (NA) or during brain stimulation (H + NA, or F + NA). Intervals of 1 min between recordings.
1
2
3
F
H
F*H
200
BP
lOO 0 100
HR
2oe
1----t 3osec
Fig. 4. Summation of cardiovascular responses when fastigial and hypothalamic stimulations are combined. Cat conscious and unrestrained. Tracings from above downwards: stimulus marker; aortic pressure (BP) in m m Hg; heart rate (HR) in beats/min. Using 50 Hz sine wave currents, in record 1 the fastigial nucleus was stimulated (F) at 0.4 V; in record 2 the defence reaction area was stimulated (H) at 0.5 V; in record 3 these two stimulations were combined. Intervals of 1 min between recordings.
FASTIGIAL AND HYPOTHALAMIC STIMULATION
445
Fastigial-hypothalamicsynergism Certain cardiovascular responses following stimulation within a fastigial nucleus or the defence reaction area of the hypothalamus are similar, as described above. Stimulation within both areas at the same time showed that these responses would summate (Fig. 4). Furthermore, stimulating a fastigial nucleus at just below the threshold of changes of arterial pressure and heart rate reduced the threshold of the hypothalamic stimulation which elicited the same responses. The facilitation was found to be reciprocal between the two stimulated regions, since hypothalamic stimulation lowered the threshold of effective fastigial stimulation. However, the same mutual facilitation was not found when behavioural responses were elicited. Stimulation which was at or above the threshold for eliciting these responses from the one site did not change the threshold for eliciting the same responses from the other site. DISCUSSION
It has been shown in these experiments that stimulating a fastigial nucleus in the conscious cat produces the same cardiovascular responses as in the anaesthetized animal. The threshold is lower but the pattern is the same. The threshold of these responses was always lower than the thresholds of somatic responses, indicating that the circulatory responses may be independent of the somatic responses even when the latter are not suppressed by anaesthesia. The present study confirms that fastigial inhibition of some patterns of reflex bradycardia is a feature of the intact unanaesthetized nervous system. It also shows that the hypothalamus can inhibit reflex bradycardia without invoking somatic activity. This recalls Hilton's early demonstration of hypothalamic inhibition of carotid sinus reflexes in the anaesthetized cat, and confirms similar demonstrations in the anaesthetized animal4, 9. It was confirmed histologically that the tips o f the stimulating electrodes were inserted into, and had not penetrated beyond, the deep cerebellar nuclei and the hypothalamus. In a previous paper 8 detailed evidence has been presented that stimulation with unipolar electrodes using 50 Hz sine wave stimulating currents at less than 3 V does not activate structures more than 0.5 mm from the electrode tip. Vasomotor centres in the brain stem are involved indirectly, probably via fastigio-bulbar pathways 12 and not by spread of current from the electrode tip. In the conscious cat marked cardiovascular responses were evoked by stimuli of less than 1 V, generally 0.5-0.6 V. Both hypothalamic and fastigial stimulation produce tachycardia in the conscious cat. This result of fastigial stimulation was seen less often in the anaesthetized animal 6. The inhibition of reflex bradycardia is not simply the algebraic cancellation of bradycardia by a stimulus-evoked tachycardia. In the anaesthetized animal the inhibition can occur when fastigial stimulation alone produces no tachycardia 6. The present study confirms this in the unanaesthetized cat. After beta-adrenergic blockade, when the central stimulation failed to have effect on the heart, because of the blocking of the sympathetic terminals, bradycardia was still abolished although tachycardia was no longer available. The observations of Zanchetti and Zoccolini 16 show that there may be inter-
446
N . K . ACHARI e t al.
actions between cerebellar and hypothalamic nuclei, although Achari and Downman 6 and Miura and Reis la have given p r o o f of independence of action during anaesthesia. In the unanaesthetized animal the threshold of a fastigial nucleus was lowered when the hypothalamic site was stimulated; similarly fastigial stimulation lowered the hypothalamic threshold. When the threshold stimulations of the two sites were combined there was summation of the peripheral effects. This was a feature of the elements controlling cardiovascular responses but not of those controlling somatic responses. The observation that fastigial stimulation did not lower the threshold of the somatic responses of the defence reaction area contrasts with the finding of Zanchetti and Zoccolini in the thalamic cat. The mutual facilitation between fastigiat and hypothalamic nuclei controlling cardiovascular activity could represent summation of descending impulses at the lower motor level. It has been shown that there is early facilitation and later inhibition of the discharges in the inferior cardiac and renal nerves evoked by single-shock stimulation in the ipsilateral hypothalamus and in the fastigial nucleus ~,a. However, the mutual facilitation between the fastigial and hypothalamic sites was shown by using stimuli which caused no response of the effectors and so presumably did not activate the spinal sympathetic neurones. It is possible that the facilitation depends upon altered excitability of neurones round the tip of the electrode which is mediated by pathways connecting the fastigial and the hypothalamic nuclei. Stimulating the dentate nucleus in the intact unanaesthetized cat caused no cardiovascular changes within the range of voltage used. Stimulation within the adjacent nucleus interpositus caused small changes which might be explained by some fastigio-bulbar fibres being stimulated as they course through the nucleus lg. The results of stimulating the deep cerebellar nuclei in the conscious cat confirm the evidence from the anaesthetized cat 6 that the fastigial nuclei have particular control of sympathetic outflow to the cardiovascular system, and demonstrate that this is not an abnormal consequence of anaesthesia or damage to the brain stem.
REFERENCES 1 ABRAHAM,V. S., HILTON,S. M., AND ZBROZYNA,A., Active muscle vasodilation produced by stimulation of the brain stem; its significance in the defence reaction, J. Physiol. (Lond.), 154 (1960) 491-513. 2 ACHAm,N. K., AL-UBAIDY,S., AND DOWNMAN, C. B. B., Cerebellar initiation of discharges in sympathetic nerves, J. Physiol. (Lond.), 215 (1971) 21-22P. 3 AeriAl, N. K., AL-UBAIDY,S., ANDDOWNMAN,C. B. B., Fastigial influence on autonomic and somatic spinal reflexes, Indian J. Physiol. Pharmacol., 16 (1972) 251-252. 4 ACHAm,N. K., ANDDOWNMAN,C. B. B., Inhibition of chemoreflexesby stimulation of the defence reaction area in the cat, J. Physiol. (Lond.), 201 (1969) 100-101P. 5 ACHAm,N. K., AND DOWNMAN,C. B. B., Autonomic responses evoked by stimulation of the fastigial nucleus in the anaesthetized cat, J. Physiol. (Lond.), 204 (1969) 130-131P. 6 ACnARI,N. K., AND DOWNMAN, C. B. B., Autonomic effector responses to stimulation of the fastigial nucleus, J. Physiol. (Lond.), 210 (1970) 637-650. 7 DAWES,G. S., MOTT,J. C. A., ANDWn)DICOM~,J. G., Respiratory and cardiovascular reflexes from the heart and lungs, J. Physiol. (Lond.), 115 (1951) 258-291. 8 FOLKOW,B., ANDRUBINSTE1N,E. H., Behavioural and autonomic patterns evoked by stimulation of the lateral hypothalamic area in the cat, ,4cta physiol, scand., 65 (1965) 292-299.
FASTIGIAL AND HYPOTHALAMIC STIMULATION
447
9 GEBBER, G. L., AND SNYDER, D. W., Hypothalamic control of baroreceptor reflexes, Amer. J. Physiol., 218 (1970) 124-131. 10 HEss, W. R., Diencephalon Autonomic and Extrapyramidal Functions, Grune and Stratton, New York, 1954, Ch. 3, pp. 12-24. 11 HILTON, S. M., Inhibition of baroreceptor reflexes on hypothalamic stimulation, J. Physiol. (Lond.), 165 (1963) 56-57P. 12 JANSEN,J., AND JANSEN,JR., J., On the efferent fibres of the cerebellar nuclei in the cat, J. comp. NeuroL, 102 (1955) 607-632. 13 MIURA, M., AND REIS, D. J., Cerebellum: A pressor response elicited from fastigial nucleus and its efferent pathway in the brain stem, Brain Research, 13 (1969) 595-599, 14 WANG, S. C., AND RANSON, S. W., Descending pathways from the hypothalamus to the medulla and spinal cord. Observations on blood pressure and bladder reflexes, J. comp. Neurol., 71 (1939) 457-472. 15 WEIL, A., Rapid method for staining myelin sheaths, Arch. Neurol. Psychiat. (Chic.), 20 (1928) 392-393. 16 ZANCHETTI, A., AND ZOCCOLINI, A., Autonomic hypothalamic outbursts elicited by cerebellar stimulation, J. NeurophysioL, 17 (1954) 475-483.