Potentiation of a cardioinhibitory reflex by hypothalamic stimulation in the rabbit

Brain Research, 154 (1978) 331-343 D Elsevier/North-Holland Biomedical Press

P O T E N T I A T I O N OF A CARDIOINHIBITORY H Y P O T H A L A M I C S T I M U L A T I O N IN T H E RABBIT

331

REFLEX

BY

M. H. EVANS The A. R.C. Institute of Animal Physiology, Babraham, Cambridge CB2 4A T (Great Britain)

(Accepted January 26th, 1978)

SUMMARY Repetitive stimulation of an area within the lateral hypothalamus, near the mammillothalamic tract, evoked pressor responses with bradycardia in anaesthetized rabbits. With weak stimulation (cathodal pulses below 75-150/tA, 1 msec ciuration, 60-100 per sec for 5-9 sec) the pressor responses were accompanied by bradycardia similar in intensity to that evoked by i.v. administration of noradrenaline. Stronger stimulating currents evoked an intense bradycardia that could not have arisen solely through the baroreceptor reflex. With these stronger currents the heart rate sometimes fell, transiently, to less than 20 ~ of the resting rate. After denervation of the 4 main buffer nerves (sinus and aortic nerves), hypothalamic stimulation could not readily evoke bradycardia, although the pressor, respiratory and other effects remained. When the baroreceptor afferents were activated, either by evoked pressor responses in rabbits whose buffer nerves were intact or by electrical stimulation of the central ends of divided aortic nerves, strong hypothalamic stimulation augmented the bradycardia evoked reflexly from these baroreceptor afferents. This evidence suggests that electrical stimulation of this area in the hypothalamus may facilitate the cardioinhibitory component of the baroreceptor reflex in the rabbit.

INTRODUCTION Electrical stimulation of a zone in the hypothalamus evokes either the aggressive 'defence reaction' or flight in the conscious cat4, 22 and this part of the hypothalamus is now sometimes referred to as the 'defence area'. In the anaesthetized carnivore (cat, dog) stimulation causes increases in arterial blood pressure and heart ratO2,e9, 3o accompanied by vasodilatation in muscle blood vessels, and vasoconstriction in skin and viscera 13 together with respiratory and other effects1. Hilton showed that the baroreceptor reflex was inhibited during stimulation of

332 the 'defence area' in the anaesthetized cat, thus explaining the presence of tachycardia with pressor responses z3, and his findings have been confirmed on the conscious cat by Achari et al. z. There has been some disagreement as to whether the inhibition is restricted to the cardioinhibitory component of the baroreceptor reflex :~,19,~6 and similar reflexes originating from cardiac receptors2, St, or whether the vascular component of the reflex can also be inhibited 6,44. When a similar region in the rabbit hypothalamus is stimulated under anaesthesia, many of the effects resemble those seen in the carnivore 1~,49. In the rabbit, however, there is a striking difference: the pressor response is accompanied by bradycardia and not by tachycardia 14,16,47. The experiments described in the present paper were performed to determine the role played by the baroreceptor reflex:~z in this bradycardia. The results suggest that in the rabbit stimulation of the hypothalamus facilitates the cardioinhibitory component of the baroreceptor reflex. Some of the results reported here have been published in an abstract ~5. MATERIALS AN D METHODS The experiments were performed on 53 adult rabbits of the New Zealand White strain, weighing 2.6-5.4 kg, mostly females. All were anaesthetized, usually with a mixture of ethyl carbamate (urethane) 25 g/100 ml and a-chloralose 1.5 g/100 ml, given intravenously. The initial doses lay in the ranges 0.92-1.22 g/kg and 55-73 mg/kg respectively. Further small doses were given later if required. The surgical preparations for stereotaxic stimulation of the brain stem and for recording respiratory and cardiovascular responses were similar to those described in previous publications14,16. The hypothalamus was stimulated through pointed steel monopolar electrodes, 0.30 or 0.35 mm diameter, insulated with several coats of epoxy resin. The uninsulated tip was not usually more than 0.1 mm in length. These electrodes were lowered into the brain after craniotomy and reflection of the dura, using the stereotaxic coordinates of Sawyer et al. 48 as a guide. In practice, the best effects were obtained when the electrode was lowered from a point 4 mm caudal to bregma and 1.5 mm lateral to the midline, so that the tip lay within the lateral hypothalamic area in the P.2 plane. The stimulus was a train of constant current cathodal pulses, the anode being an indifferent electrode under the nearby skin. Pulse duration was 1 msec, amplitude in the range 10-1000/zA (usually 100-330 ttA), their frequency was usually 100 (but sometimes 60-70) per sec and the train continued for 5-9 sec. Eight animals were immobilized with a neuromuscular blocking drug and ventilated artificially, usually at 23-35 breaths/min. The ventilatory volume was adjusted to maintain end-tidal CO2 at about 4.5 ~ . Gallamine triethiodide (Ftaxedil; May and Baker) 1.5-2 mg/kg, i.v., with small supplementary doses as required, was found to be a satisfactory immobilizer. Alcuronium chloride and o-tubocurarine were unsatisfactory, as they interfered with the cardioinhibitory effects of stimulation at doses comparable with those required for full immobilization. In 17 experiments the 4 major baroreceptor nerves were divided. The sinus

333 nerves were exposed on the medial side of the internal carotid artery, identified by the bradycardia and depressor effect resulting from their stimulation, and then divided. The aortic nerves were dealt with in a similar manner, near the rostral end of the trachea 46. To test that these procedures had deafferented the baroreceptors, noradrenaline was given i.v. before and after dividing the nerves. Denervation was deemed satisfactory if, after division, pressor responses were no longer accompanied by bradycardia. In 4 animals denervation was unsatisfactory, and this aspect of the experiment was abandoned. In a few experiments denervation was tested by allowing the animal to breathe a gas mixture low in 02 for 10-30 sec, to stimulate the chemoreceptors. In 6 experiments, baroreceptor afferents were prepared for electrical stimulation by inserting the central cut ends of one or both aortic nerves into indwelling electrodes which were sewn to the longus cervicis or scalenus muscles of the neck. The stimulus was a train of constant-voltage pulses, 5-20 V (usually 10 V) amplitude, 0.2~0.3 msec duration, in trains lasting 3-9 sec (usually 5 or 6 sec) at a frequency of 70 or 100 per sec. After every experiment the brain was fixed in situ by perfusing the carotid arteries with saline followed by 20% formol-saline, after which the head was immersed in formol-saline for several days. The relevant block of brain tissue was imbedded in celloidin, sectioned and stained with cresyl fast violet. The tracks of the electrodes were marked on diagrams made by enlarging these sections to 10 × life size, defined by the known distances in vivo between the tracks. The stimulation loci were marked along these tracks by reference to small electrolytic lesions, or deposits of Prussian blue developed by inclusion of ferrocyanide in the perfusion fluid. Statistical analysis of the results included the calculation of linear regressions, with analysis of variance (Anovar programme) giving confidence limits. RESULTS

Effects of hypothalamic stimulation in rabbits with functioning baroreceptor reflexes Some examples of increases in blood pressure (BP) and reductions in heart rate (HR) caused by hypothalamic stimulation are shown in Fig. 1. These responses were recorded from an anaesthetized rabbit, whose buffer nerves were intact, during stimulation of the lateral hypothalamic area with currents of 50-200/zA. It can be seen that the weakest stimuli (Fig. la) evoked mild pressor effects with hardly any bradycardia. The strongest stimuli (Fig. le) caused the BP to rise from 125/80 to about 180/130 mm Hg, while the H R fell from 305 to 120 beats/min during stimulation, rising through a short phase of mild tachycardia after stimulation ceased. During these periods of stimulation respiratory changes occurred, consisting of a shallow tachypnoea superimposed on an inspiratory shift of the baseline, and often preceded by a transient apnoea when the stimulus strength was greater than 100/zA. These respiratory effects have not been shown in Fig. 1, since they were typical of those described in detail in an earlier publication 16. Later in this experiment the animal was immobilized and artificially ventilated at a constant rate and amplitude. Stimulation during paralysis again evoked cardiovascular effects that were very similar to those shown in Fig. 1.

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Fig. 1. Records of pressor responses (above) and bradycardia (below) to hypothalamic stimulation in a rabbit anaesthetized with urethane 1.06 g/kg and chloralose 64 mg/kg. The buffer nerves were intact. The signal marks indicate the periods of stimulation, all of which lasted 9 sec and consisted of constantcurrent pulses at t 00/sec. Intensities were: (a) 50 #A; (b) 75 t~A; (c) 100 ttA; (d) ! 50 k~A;and (e) 200/~A. The locus of stimulation was in the lateral hypothalamic area at P2.5, 0.4 mm lateral to the border of the mammillothalamic tract and about 2 mm dorsal to the lateral mammillary nucleus.

Comparison of the bradycardia induced by hypothalamic stimulation with that induced by i. v. noradrenaline It was obviously possible that the falls in H R seen during hypothatamic stimulation could have resulted from reflex cardioinhibition, arising from activation of baroreceptors by the pressor responses evoked by stimulation. To test this possibility, a range of pressor responses was evoked by graded stimulation of the hypothalamus and also by graded intravenous doses of noradrenaline (NA) given in the same animal, and the accompanying bradycardial responses were measured and compared. In some animals the procedure was repeated after immobilization and artificial ventilation. The results of 4 of these experiments are shown in Fig. 2, in which falls in H R are plotted against the corresponding rises in BP. The BP responses are expressed as a percentage change in systolic pressure, compared with the resting value during the few seconds preceding the stimulus. Systolic pressure, rather than diastolic or mean pressure, was chosen because it would be the most significant in terms of baroreceptor activation. It can be seen from Fig. I that the diastolic and systolic pressures rose almost in parallel during hypothalamic stimulation. In the experiments whose results are included in Fig. 2 and Table I, the pulse pressure increased only slightly when pressor responses were evoked either by stimulation or by administration of NA. When the changes in pressure evoked by administration of 10 or 15/~g NA were compared with a similar range of pressure changes evoked by stimulation, it was found that the N A h a d caused the pulse pressure to rise by 6.6 ~ (mean, n == 28), whereas hypothalamic stimulation had caused an increase of 13.6 % (mean, n == 27). There did,

335 therefore, appear to be a tendency for the pulse pressure to have increased more during stimulation than during NA administration; but, when these results were subjected to Student's t-test it was found that they were not significantly different even at the P = 0.1 level (S.E. of combined means: 4.3344; t -- 1.6548). Furthermore, when all the changes in pulse pressure evoked by all doses of NA administration (2.5-15 #g, i.v.) were compared with the changes evoked by different strengths of stimulation in the same experiments (25-250/zA), and analyzed as linear regressions against the systolic pressor responses over comparable ranges, each regression line was found to overlap throughout its range with the 9 5 ~ confidence limits of the other line. Therefore, it is concluded that differential effects on diastolic or pulse pressures can be discounted as possible causes of any differences in the bradycardias to be described. In Fig. 2 the responses induced by various doses of NA are shown as open circles, and the responses to hypothalamic stimulation are shown either as squares if the animals was allowed to breathe normally, or as diamonds if it was immobilized and ventilated. With weak stimulation the percentage falls in HR, for any given percentage increase in BP, did not seem to differ from those evoked by small doses of NA, and could therefore have been due solely to reflex cardioinhibition. Larger doses of NA produced stronger bradycardia and pressor responses, and presumably the bradycardia was entirely reflex in origin. The maximum dose given in these experiments was 15 /~g (4.4-5.1 g/kg), which resulted in pressor responses ranging from 4-23.3 to 4-54.6 ~ (the mean of 11 trials in these 4 animals was +36.6 -~ 9 . 9 ~ S.D.). The accompanying falls in H.R. ranged rather widely from --9.4 to --34.4 ~ (mean: --19.4% 4- 7.5 ~ S.D.). Three out of the 4 animals whose responses to NA are shown in Fig. 2 had calculated regression lines with slopes in the range 4 . 2 2 to --0.36 beats/rain/ram Hg, but the animal whose results are shown in Fig. 2b had an unusually steep response, with a slope o f - - 0 . 6 8 ( ± 0.20 S.E.) beats/min/mm Hg. These regression lines, with their 9 0 ~ confidence limits, are shown in Fig. 2 and further statistical details are given in Table I. Stronger hypothalamic stimuli evoked stronger pressor responses with intense bradycardia, and as the stimulus strength was raised above a value, that varied between about 75 and 150 /zA in different experiments, the graph of H R against pressor response deviated strongly downwards, diverging well away from that of the NA-induced responses in 3 out of the 4 animals. This divergence can be seen in Fig. 2a, c and d: when the BP rose by more than about + 3 0 ~ the H R usually fell well below the ,,,onO//oconfidence limit of the NA-induced response. Only in Fig. 2b can the effect not be seen clearly, because of the unusually vigorous reflex bradycardia; even in this animal the regression line of the stimulation-induced responses is steeper (--1.30 40.22 S.E. beats/rain/ram Hg) than that of the NA-induced responses. The responses to hypothalamic stimulation shown in Fig. 2 also show that immobilization and artificial ventilation of the animal made no apparent difference to the effects of stimulation. These results suggest that the falls in HR during moderate-to-strong stimulation were too intense to be accounted for solely by baroreceptor reflex inhibition from the pressor effects of the stimulation. The smaller falls which accompanied weak stimulation could have resulted from reflex cardioinhibition.

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Fig. 2. Graphs of the relationship between increases in systolic blood pressure and the accompanying decreases in heart rate, on abscissa and ordinate respectively, expressed as percentages o f the resting values. Each graph, a-d, is from a different rabbit. The responses were evoked either by i.v. administration of noradrenaline (2.5-15 #g) ( © ) or by hypothalamic stimulation (25-500 #A) in rabbits that were either breathing spontaneously ( I ) or immobilized and artificially ventilated ( 0 ) . Each point indicat~ the peak values following a single administration orstimulation. The continuous lines show the calculated linear regressions for the NA-induced responses, with the 90% confidence limits indicated as dotted lines on either side. The dashed lines are the regressions for those responses to stimulation inwhich the systolic BP rose by more than 30 %.

Effects of hypothalamic stimulation after section of the buffer nerves In 13 animals the 4 major baroreceptor inputs (sinus and aortic nerves on each side) were divided and tests showed that baroreceptor denervation was complete. When the hypothalamus was stimulated in these animals it was at best difficult, and often impossible, to evoke the expected bradycardia, although typical pressor and other responses were still seen. Most of the deafferented animals were unresponsive. failing to show a n y b r a d y c a r d i a even to 500 # A currents, though it was sometimes found possible to slow the heart by using stronger stimuli. A slight tachycardia was often seen in response to stimuli in the 100-500 y A range. In a few animals, such as the example shown in Fig. 3, stimulating currents

337 TABLE I Slopes and intercepts ( + S.E.) in beats~rain~ram Hg, o f the regression lines shown in Fig. 2 F and R are the conventional variance ratio and correlation coefficients for the linear regressions, and *, ** (or *** in the case of R only) indicate whether the fit is significant at the 5 ~ , 1% or 0.1% levels respectively. Due to the scatter of individual points the regression line fit was not significant (n.s.) even at 10 ~ level in the case of the stimulation responses in Fig. 2a, although l0 of the 14 points fell below the lower 90% confidence limit of the NA-induced responses. Slope ± S.E. N A -induced responses 2a --0.22 ± 0.08 2b 4 . 6 8 ± 0.20 2c ~0.33 ~ 0.05 2d 0.36 ~- 0.07

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Responses to sthnulation where B P rose 30 % or more 2a --0.87 5z 0.59 6.03 " 27.95 2b 1.30 ± 0.22 8.81 ± 9.92 2c 0.37 ± 0.14 --22.55 ± 6.49 2d --2.17 ± 0.49 41.18 ± 2 1 . 0 1

stronger than usual did slow the heart, but the effect was weak and could be obtained only from an unusually restricted part of the hypothalamus. The brain section in Fig. 3 shows that 3 electrode penetrations were made on each side of the brain in this animal. At least 5 of these tracks would have been expected to pass through the responsive area of the hypothalamus, yet in this example the rather strong 250/~A stimulus evoked no

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,m~, Fig. 3. Transverse section of the brain stem to show 6 microelectrode tracks into the hypothalamus. The 4 main buffer nerves had been divided. The tracings on either side show, from above downwards, the respiratory, pressor and bradycardial responses to stimulation of the hypothalamus at the points marked on the section by black spots Stimu ation, in each case, was with a 9 sec train of 250/~A pulses at 100/sec during the period indicated by the signal marks below the responses. Rabbit: 4.2 kg, anaesthetized with urethane 1.2 g/kg and chloralose 72 mg/kg. Abbreviations: Hipp, hippocampus; M, mammillary bodies ; TMT, mammillothalamic tracts ; ZI, zona incerta.

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Fig. 4. Records obtained during stimulation of aortic nerves and/or hypothalamus in an anaesthetized rabbit whose 4 main buffer nerves had been divided. The sets of tracings in the top row show the depressor responses and bradycardia to stimulation of both aortic nerves for 6 sec, at 5 V and 100/sec during the periods shown by the signal marker below the 0 HR line. The tracings inthemlddlorow show the respiratory and presser effects, with slight tachycardia, to hypothalamic stimulation for 6 sec at 65/ sec, at 200, 300 and 500 uA during the periods shown by the signal marker below the respiratory traces. The bottom row shows the changes in respiration, blood pressure and heart rate when tl~e~ hypothalamic stimulations were given together with the aortic nerve stimulation. The records were obtained with hypothalamic stimulation of a locus slightly lateral to the mammillothalamic tract at H-4, about 1 ram above the ventral surface of the brain at the margin of the mammillary bodies. There are 30 sec time calibration bars under each column of records.

more t h a n the m o d e r a t e bradycardia shown, from the two points m a r k e d by filled circles near the b o t t o m of two of the tracks. A weaker b r a d y c a r d i a was also o b t a i n e d from a p o i n t near the b o t t o m of the medial track o n the left, at the lateral border o f the m a m m i l l o t h a l a m i c tract. Such b r a d y c a r d i a as was obtained, was lost if t h e electrode was m o v e d 0.5 m m dorsal or ventral to the responsive " p o i n t or if the stimulus strength was reduced to more n o r m a l levels. Presser, respiratory, ocular a n d facial responses of n o r m a l a p p e a r a n c e could still be evoked from each of these 6 tracks t h r o u g h o u t the usual part of the h y p o t h a l a m u s . In these partially responsive animals, use of very

339 strong stimulus currents of 500 #A or more evoked a more powerful bradycardia, with falls of 30% or more in heart rate. Therefore, although it had been shown that reflex cardioinhibition by baroo receptors was too weak to account for the intensity of the bradycardia seen when the hypothalamus was stimulated by currents stronger than 75-150/~A, these experiments on deafferented animals made it clear that this bradycardia was nevertheless largely dependent upon a baroreceptor input. Intense bradycardia could not be evoked by stimulating this area of the hypothalamus in the absence of a baroreceptor input.

Interactions between baroreceptors and hypothalamic stimulation For the investigation of possible interactions between the baroreceptor reflexes and the hypothalamus, the 4 buffer nerves were divided and the cut central ends of one or both aortic nerves were placed on electrodes for stimulation by pulses of 5-10 V, 0.2-0.3 msec duration, at 100/sec. A few seconds stimulation evoked reflex falls in BP and HR, such as those illustrated in the upper row of tracings in Fig. 4a-c. Stimulation of the hypothalamus in these deafferented rabbits evoked, as in the previous set of experiments, respiratory, pressor and other effects with little or no bradycardia. Typical sets of tracings are shown in the middle row of Fig. 4, and it can be seen that stimulation with 200-500/,A evoked respiratory responses, rises in BP to nearly 200 mm Hg systolic, and a slight tachycardia of about L~25 beats/rain. The stimulated point was later found by histology to have been in the lateral hypothalamic area, 1.0 mm dorsal to the margin of the mammillary bodies, 1.7 mm from the midline and about 0.5 mm lateral to the edge of the mammillothalamic tract. This locus corresponds to the centre of the region from which bradycardia can be evoked easily in the normal anaesthetized rabbit 14. When both the aortic nerves and the hypothalamus were stimulated concurrently, pressor and respiratory responses similar to those seen in normal anaesthetized rabbits were obtained, as shown in the bottom 3 sets of tracings of Fig. 4a-c. Now, with concurrent stimulation, the reflex bradycardia was potentiated and the degree of potentiation increased with increasing intensity of hypothalamic stimulation, from 200 to 500 /~A, as shown. The pressor responses were unchanged, so this part of the hypothalamus seemed also to be inhibiting or overriding the vasodepressor component of the bradycardial reflex. Potentiation of baroreceptor cardioinhibition was obtained only when this zone in the hypothalamus was stimulated. Other zones in the hypothalamus sometimes evoked pressor effects without potentiation of the reflex cardioinhibition. The hypothalamic elements responsible for this potentiation of the reflex cardioinhibition appeared to have a higher electrical threshold than those elements which mediated the pressor responses. Evidence for this is seen in responses illustrated in Fig. 1. Progressively stronger stimulation, with currents of 50, 75 and 100/~A, evoked progressively stronger pressor responses, accompanied by correspondingly greater falls in HR (Fig. la-c). When the stimulus strength was increased further, to 150 and 200/,A, the pressor effects were hardly any stronger than with 100/~A, but now there was a sudden increase in the bradycardia (Fig. 1d and e) in which an abrupt further fall

340 in H R was superimposed on the previously graded responses. This additional component had a sharply defined onset and termination, coinciding closely with the period of stimulation, and the total intensity of the bradycardia during th is phase w as about double the intensity of the response to 100 #A. It is suggested that the graded bradycardia seen with 50-100 #A stimulus strength was mainly due to simple reflex cardioinhibition from the baroreceptors, while the additional abrupt component seen with stronger stimulation arose through facilitation of this reflex. The records shown in Fig. 1 were obtained before the animal was immobilized, but almost identical results were obtrained later in this experiment, from a corresponding point in the hypothalamus on the opposite side. with the animal immobilized and artificially ventilated. Only some animals seemed to have sufficient difference in the electrical thresholds to show the additional abruptly potentiated bradycardia as clearly as shown in Fig. l, but the effect could be detected in the recordings from most animals whose buffer nerves were intact. DISCUSSION Hilton ~a demonstrated that stimulation of the hypothalamic 'defence area' in the anaesthetized cat inhibits the baroreceptor reflexes and similar inhibition has been demonstrated in the conscious cat 2. Stimulation of an apparently equivalent region in the hypothalamus of the anaesthetized rabbit evokes a set of responses broadly similar to those seen in the cat 14,16, except that bradycardia is evoked instead of tachycardia. With strong stimulation the H R may fall to less than 20 ~ o f the resting rate, when it may be followed by a mild poststimutation tachycardia. Many workers have in the past stimulated the rabbit hypothalamus electrically, and some have described a variety of autonomic effects5,17,41,49,50. It is surprising that the strong bradycardia has received little notice. Mentioned briefly by Cross and Silver s and described by Yuasa et al. who recorded changes in the ECG 52, it appears to have been investigated mainly by Schneiderman and his colleagues, who concluded that when the bradycardia accompanied pressor responses, the fall in H R was due mainly to operation of a normal baroreceptor reflex17,2°,~7. The present experiments have shown that. when the stimulus to the caudal part of the lateral hypothalamic area is weak. it is likely that the bradycardia is due to a simple baroreceptor reflex evoked by the rise in BP, but that the intense bradycardia obtained by stronger stimulation cannot be accounted for by the baroreceptor reflex. The stronger stimulation does not usually evoke much bradycardia in the absence of a baroreceptor input, but acts by potentiating the reflex. These results therefore suggest that relatively high-threshold elements present in this part o f the hypothalamus may be able to facilitate the cardioinhibitory component of the baroreceptor reflex in the rabbit. There is a brief mention by Gimpl et al. 2° that reflex bradycardia was enhanced in the rabbit when the aortic nerve and the posterior hypothalamus were stimulated concurrently, but the reflex was depressed some 10-t5 sec after stimulation of the hypothalamus. This late inhibition probably explains the poststimulation tachycardia seen in many of the present experiments.

341 The bradycardia and other effects of stimulation of the caudal part of the rabbit's lateral hypothalamic area, as described here and elsewhere 14,16, are not comparable with effects obtained from the cat, including the bradycardia evoked by stimulation of more rostral regions of the brain stem. In the cat, arterial blood pressure usually fell, and when reflex bradycardia was facilitated the vasodepressor component of the baroreceptor reflex was also facilitatedZ5,33. This component does not appear to have been facilitated in the present experiments. In the rabbit the usual respiratory response to stimulation of this part of the hypothalamus is either apnoea in inspiration or a shallow tachypnoea superimposed on a baseline shift towards the inspiratory position 1~. Respiratory activity can modulate the activity of cardiac vagal efferents in some species. Hypoxia or hypercapnia causes vagally mediated bradycardia in the rabbit 37,34-36. Spontaneous activity in cardiac vagal efferents is, however, minimal during inspiratory movements, and reflex bradycardia occurs most strongly during the expiratory phase of the respiratory cycle 2s,31,42,43,45. Therefore, it is not logically possible that the bradycardia in the present experiments could have been evoked either by the inspiratory movements or by the inspiratory drive from hypothalamus to respiratory centre that occurred during stimulation. Furthermore, in a few of the present experiments, stimulation evoked intense bradycardia without much effect on natural ventilation, while in other experiments the cardiovascular responses were unchanged after immobilizing the animal with a neuromuscular blocking drug and ventilating the lungs at a steady rate which also maintained adequate oxygenation and a normal end-tidal pCO2. This region of the lateral hypothalamic area of the rabbit is known to contain neurones sensitive to anoxia or hypercapnia% with which it probably forms a part of the supramedullary cardiovascular regulating mechanism that has been extensively investigated by Korner and his colleagues 34-~°. This region would also appear to be homologous with the 'defence area' of the carnivore, except that tachycardia is obtained by stimulation of the 'defence area' in the latter 1,12,13,22,~9. Tachycardia is not, however, the invariable accompaniment of fear or anxiety in all mammals. Bradycardia has been reported in pigs and some other animals subjected to classical autonomic conditioning regimes10,~1, as a transient 'startle' response in man 27 and in young hares in the presence of a predator is. The differing cardiovascular responses of carnivore and rabbit may perhaps be related to their roles as predator and prey. ACKNOWLEDGEMENTS I am most grateful to Dr. E. A. Bower for his guidance on methods for denervating the buffer nerves in the rabbit. It is a pleasure to acknowledge the skill and conscientious assistance of Mrs. Vanessa A. Behrsin and Mrs. Jenny J. Rydzewski with the experimental work and that of Mr. Ian S. King with the histological processing of the brain stem material.

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