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Facilitation of baroreflex-induced bradycardia by stimulation of specific hypothalamic sites in the rat
Brain Researclt. 384 ( 1980~ 274-28 I Elsevic~
274
BRE 12050
Facilitation of Baroreflex-Induced Bradycardia by Stimulation of Specific Hypothalamic Sites in the Rat B.J. PARDINI, K.P. PATEL, P.G. SCHMID and D.D. LUND
Veterans Administration Medical Center, Cardiovascular Center, and Department of Internal Medicine, University of lowa, Iowa City, 1,4.52240 (U.S.A.) (Accepted 3 April 1986)
Key words: Baroreflex - - Anterior hypothalamic area - - Ventromedial nucleus of the hypothalamus -Parasympathetic nervous system - - Heart rate
Hypothalamic stimulation generally inhibits baroreflex-induced bradycardia. However. we have noted discrete areas of the rat hypothalamus which facilitate reflex bradycardia. The effects of hypothalamic stimulation on baroreflex-induced changes in heart rate were investigated in urethane-anesthetized rats f 1.2 g/kg, i.p. : n = 6) instrumented with femoral arterial and venous catheters. Bipolar electrodes/250 ~m diameter) were implanted stereotaxically in the hypothalamus. Baroreflex-induced bradycardia was elicited by phenylephrine (PE) injection (8-20~g/kg). Responses to stimulation (STIM) (50-150/~A. 80 Hz. 0.5 ms). PE. and Stim - PE were studied for 1 min. In the ventral medial and anterior hypothalamus, STIM caused transient increases in blood pressure and no changes in heart rate. Peak blood pressure was lower during STIM - PE than during PE (144 + 5 vs 164 _+ 3 mm Hg; P < 0.05). However. STIM + PE resulted in a lower heart rate compared to PE (194 ___22 vs 270 + 17 bpm" P < 0.05). At l min. the heart rate in STIM + PE rats remained lower than in PE rats (205 + 37 vs 319 _+ 16 bpm: P < 0.05). Atropine administration indicated that the facilitation was primarily parasympathetic in nature These results identify specific hypothalamic regions which facilitate baroreflex-induced bradycardia by parasympathetic mechanisms. cleus or the a n t e r i o r h y p o t h a l a m i c a r e a in rats causes
INTRODUCTION
p r o f o u n d facilitation of p h e n y l e p h r i n e - i n d u c e d bradCardiovascular
effects of h y p o t h a l a m i c stimula-
tion are g e n e r a l l y b e l i e v e d to inhibit n o r m a l b a r o r e -
ycardia that is m e d i a t e d
by the p a r a s y m p a t h e t i c
n e r v o u s system.
flexes. M o s t n o t a b l y , s t i m u l a t i o n of the h y p o t h a l a m ic d e f e n s e a r e a inhibits b a r o r e f l e x - i n d u c e d b r a d y c a r dia 3'1°'11. H o w e v e r . early r e p o r t s by G e i l h o r n 7 and
MATERIALS AND METHODS
later by K l e v a n s and G e b b e r ~2. h a v e d e m o n s t r a t e d
Male S p r a g u e - D a w l e y rats ( 3 0 0 - 3 5 0 g) w e r e an-
facilitation of r e f e x - i n d u c e d b r a d y c a r d i a by stimula-
e s t h e t i z e d i n t r a v e n o u s l y with u r e t h a n e (1.2 g/kg, i.p.) and i n s t r u m e n t e d with a r t e r i a l and v e n o u s cath-
tion of specific h y p o t h a l a m i c sites in the cat. In contrast, electrolytic lesions of specific h y p o t h a l a m i c
eters. A r t e r i a l pulsatile and m e a n b l o o d p r e s s u r e
sites h a v e y i e l d e d o p p o s i t e results, R e c e n t l y , Miyaji-
w e r e m e a s u r e d with a p r e s s u r e t r a n s d u c e r and dis-
m a and Bufiag r e p o r t e d that d e s t r u c t i o n of the ante-
played on a p a p e r chart r e c o r d e r . T h e E C G was re-
rior h y p o t h a l a m u s
c o r d e d : h e a r t rate was d e r i v e d f r o m a t a c h o m e t e r
in the rat c a u s e d inhibition of
p h e n y l e p h r i n e - i n d u c e d b r a d y c a r d i a t3 W e h y p o t h e s i z e d that s t i m u l a t i o n of specific h y p o t h a l a m i c sites w o u l d facilitate reflex b r a d y c a r d i a in
d r i v e n by e i t h e r t h e Q R S c o m p l e x o f the E C G or the pulsatile p r e s s u r e signal. T h e E C G was also disp l a y e d at high r e s o l u t i o n on an o s c i l l o s c o p e to accu-
the rat. T h e p r e s e n t d a t a d e m o n s t r a t e that unilateral
rately d e t e r m i n e the p r e s e n c e of a p - w a v e and e n s u r e
electrical s t i m u l a t i o n o f e i t h e r the v e n t r a l m e d i a l nu-
that cardiac a c t i v a t i o n was initiated t h r o u g h n o r m a l
Correspondence." B.J. Pardini. Cardiovascular Center, Room 10W20, Veterans Administration Medical (.'enter. Iowa City, [A 52240. U.S.A. 0006-8993/86/$03.50 ~ 1986 Elsevier Science Publishers B.V. t Biomedical Division
275 pathways. When a large degree of bradycardia was observed, it was important to verify that a - v nodal block had not occurred and that the recorded rate was of ventricular origin. After placement in a stereotaxic apparatus, the skull was exposed. Concentric bipolar electrodes (250 ,urn outer diameter, Rhodes Medical Instruments) were used to stimulate hypothalamic regions with constant current (50-150 ktA, 80 Hz, 0.5 ms). Stimulus sites were verified histologically in 10-25 /~m sections after marking the area of interest with electrolytic lesions made by passage of DC current. Reflex-induced bradycardia was elicited by intravenous injection of phenylephrine (8-20 ug/kg, in 500 ul of saline). The blood pressure and heart rate responses to electrical stimulation, phenylephrine injection, or phenylephrine injection during electrical stimulation were recorded. Multiple hypothalamic sites in each rat were tested for their ability to facilitate phenylephrine-induced bradycardia. The areas tested were within the following stereotaxic boundaries according to Pellegrino~4: from bregma to 1.20 mm rostral to bregma, from the midline to 1.5 mm lateral to the midline, and from approximately 7.0 mm ventral to the surface of the brain to the base of the brain. The following hypothalamic sites were included within the designated boundaries: dorsomedial nucleus, ventromedial nucleus, arcuate nucleus, paraventricular nucleus, anterior hypothalamic area, fornix, and zona incerta. Additionally, the supraoptic nucleus was also stimulated. Statistical significance was determined by an independent analysis of variance followed by a least significant difference test for group differences. A probability level of less than 0.(15 was accepted as a significant difference. RESULTS A typical procedure consisted of electrode placement at the most dorsal site of a track followed by a bolus injection of phenylephrine. The blood pressure and heart rate response to phenylephrine was recorded; this response was used to compare with other responses elicited within the same electrode track. Hypothalamic sites were investigated at 0.5 mm intervals; the effect of electrical stimulation alone was
followed by electrical stimulation initiated approximately 2 s after phenylephrine injection. Electrical stimulation of an area was considered to facilitate phenylephrine-induced bradycardia when electrical stimulation plus phenylephrine (STIM + PE) caused a greater bradycardia than the sum of heart rate decreases induced by individual phenylephrine injection (PE) and electrical stimulation alone (STIM). Fig. 1 illustrates the pulsatile and mean arterial pressure and the heart rate recorded from a rat during investigation of a site located in the anterior hypothalamic region. Electrical stimulation alone caused a biphasic response in blood pressure: an "on response', a small hypertensive effect at the start of the stimulation, and an 'off response', a small hypotensive effect when the stimulation was terminated (Fig. 1A). A small degree of bradvcardia was also observed. Phenylephrine injection caused a typical increase in blood pressure followed bv a bradycardia of approximately 50 beats per minute (bpm). However, when a bolus injection of phenylephrine was administered concomitantly with electrical stimulation the magnitude of the bradveardia was markedly increased, and lasted for the duration of the stimulation (Fig. 1A). Lesions made by passage of DC current completely eliminated the stimulation-induced facilitation of the phenylephrine-induced bradycardia, but did not affect the normal cardiowtscular responses to phenylephrine injection. Additionally, the cardiovascular alterations to electrical stimulation alone were eliminated by prior lesion of the area. If the facilitation is due to discrete stimulations, it should be eliminated or reduced by small movements of the electrode. Fig. 1B illustrates the responses obtained 0.5 mm dorsal to the site of Fig. 1A. All electrode penetrations were made dorsal to ventral; thus, the stimulus of Fig. 1B was made prior to that of Fig. 1A and was not made on previously penetrated tissue. Although the biphasic blood pressure response to electrical stimulation alone was present, the degree of bradycardia elicited by simultaneous electrical stimulation and phenylephrine injection was markedly decreased compared to the results observed 0.5 mm away. The elimination of most of the response bv a small shift in the electrode demonstrates the highly localized area in which the facilitation can be elicited.
276 A.
STIM
PE
STIM + PE
200 PULSATILE
ARTERIAL PRESSURE
I00
(ram Hg)
0 200
MEAN ARTERIAL PRESSURE
I00
(ram Hg)
0 sec
HEART RATE (bpm)
500 I
250I
f
t
0 L i
i
g.
STIM
PE
S T I M + PE
2O0 PULSATILE ARTERIAL PRESSURE
I00
(ram H(J)
200 MEAN ARTERIAL
I00
PRESSURE (ram Hg)
0
I---7 3 0 sec
500 HEART RATE
250
(bpm)
-
-
r
1
Fig. 1. Panels depict pulsatile and mean arterial pressure and heart rate of rats during electrical stimulation (STIM), phenylephrme rejection (PE), or both interventions simultaneously (STIM + PE). At the bottom of each panel the bar indiCates the duration of electrical stimulation and the arrow indicates the time of phenylephrine injection. In panel A, the tip of the stimulating electrod e was in the anterior hypothalamus. STIM produced small 'on' pressor and 'off' depressor responses, but little Change in heart rate. STIM + PE produced profound facilitation Of the bradycardia associated with either STiM or PE. The results shown in panel 13were recorded just prior to the interventions of panel A. The tip of the stimulating electrode wasO.5 mm dorsal to its placement in p a n e f A . The results indicate that the response is localized to a small area and that current spread was not excessive.
277
MEAN ARTERIAL PRESSURE (ram Hg)
iii t .... 1
2
0
80 t
÷
/
~
÷
.
*
÷
,el 0
I
o
::::::
;;,.
- -
PE+$TIM
'6 72 1'8 2'4 3'0 3'6 4'2 48 5'. 6'o TIME (see)
400 • .....
HEART RATE (bpm)
.....
3oo i ......... +.........t.........
..... !----t
.........t ........ t
.....
t
........
250 200" 150 .
.
.
.
.
.
.
.
.
.
.
.
.
" 0
PE .....
STIM
- -
PE + S TIM
,~ ,'~ ~'4 3'o 3'6 ;~ ,;8 5'4 6'o TIME (see)
Fig. 2. Summary diagram illustrates the effect of stimulation (STIM), phenylephrine (PE), and stimulation plus phenylephrine (STIM + PE) on mean arterial pressure and heart rate in 6 rats (mean + S.E.M.). Even though the increase in blood pressure was less in STIM + PE than in PE rats. the reduction in heart rate was greater. Blood pressure: + = P < 0.05 vs PE + STIM. Heart rate: + = P < 0.05vs PE + STIMorPE;o = P < 0.05 vs STIM.
Fig. 2 illustrates the mean arterial pressure and heart rate data obtained from 6 rats in which stimulation was carried out for at least 60 s. The heart rate during electrical stimulation plus phenylephrine is significantly reduced c o m p a r e d to phenylephrine injection alone. It is significant to note that the peak blood pressure response to phenylephrine plus electrical stimulation is significantly less than to phenylephrine alone. Thus, even though a smaller baroreceptor stimulus is present in the stimulation plus phenylephrine group, the magnitude of the bradycardia is significantly greater. A t r o p i n e (1 mg/kg, i.v. bolus) was administered in some rats to determine if the facilitation of the bradycardia was parasympathetic in nature. Fig. 3A and 3B demonstrate the results of one such experiment. Before atropine injection, electrical stimulation plus phenylephrine caused a maximal decrease in heart
rate of 150 bpm that was much greater than the maximal bradycardia elicited by either electrical stimulation (45 bpm decrease) or phenylephrine alone (30 bpm decrease). However, atropine abolished the heart rate response to phenylephrine and electrical stimulation plus phenylephrine. A d d i t i o n a l l y , the small bradycardia associated with electrical stimulation alone was eliminated. Fig. 4 demonstrates the hypothalamic sites at which the facilitation of bradycardia was observed. The facilitation was localized primarily to the region which corresponds to the ventromedial hypothalamic nucleus and the anterior hypothalamic area. No facilitation was observed in the other m a j o r nuclei of the hypothalamus that were studied, including the paraventricular nucleus, the supraoptic nucleus, and the dorsomedial nucleus. DISCUSSION The present experiments d e m o n s t r a t e that electrical stimulation of the ventromedial or anterior hypothalamic region in the rat greatly facilitates the bradycardia associated with phenylephrine-induced increases in arterial pressure. Such facilitation is not observed during stimulation of the paraventricular nucleus, supraoptic nucleus or dorsomedial nucleus of the hypothalamus. A t r o p i n e administration eliminates the facilitation as well as the b r a d y c a r d i a associated with phenylephrine alone. Thus, the efferent pathway of the facilitation is p r e s u m e d to act through the parasympathetic nervous system. Two criteria used in the present experiments to maximize stimulation of discrete sites and minimize activation of fibers of passage were utilization of low stimulus currents, and the absence of the response by stimulation a short distance from the site of interest. Typically, current intensities were 50-75 /~A. In those experiments where greater than 100 # A were used, the facilitation was not present when the stimulus site was moved 0.5 mm away (Fig. 1, stimulus intensity: 150HA ). It is expected that the stimulations activated cell somata to a greater extent than fibers of passage. H o w e v e r , the possibility that fibers of passage were also activated can not be eliminated by the present methodology. Gellhorn and associates first r e p o r t e d that stimulation of the anterior hypothalamus in cats facilitated
278 BEFORE
ATROPINE
STIM PULSATILE ARTERIAL PRESSURE ( m m Hg)
PE
STIM+PE
200 I00 0 200
MEAN ARTERIAL
I00
PRES SURE (ram H g )
0
3b HEART RATE (bpm)
Zllf OL
AFTER
PULSATILE
2o0[
STIM
ATROPINE PE
STIM + PE
ARTERIAL PRESSURE (ram Hg)
200 MEAN ARTERIAL
I00
PRESSURE ( m m Hg)
0 500
50 se c
HEART RATE
250
(bpm)
Fig. 3. Same configuration as in Fig, 1. The top panel illustrates a typical response obtained from stimulations in the ventromedial hypothalamus, The effects of atropine (1 mg/kg, Lv.) administered prior to repetition of the same interventions is illustrated in the lower panel. Virtually all of the bradycardia associated with any of the interventions was eliminated.
279
I
--/PVN ~--AHA ~ ~ 7 0 . 4 0 ~MA
+1.20 mm
J +0,80 mm
mm lilll 2ram
0.00 mm
Fig. 4. Diagrams of hypothalamic cross-sections illustrate regions 0.0-1.2 mm rostral to bregma. The lateral ventricles are indicated by the stippled area; the optic tract is indicated by the cross-hatching. Asterisks indicate the central location of the stimulating electrode in which facilitation of reflex bradycardia was elicited. Abbreviations: PVN, paraventricular nucleus; AHA, anterior hypothalamic area; SON, supraoptic nucleus: VMH, ventromedial nucleus of the hypothalamus.
the bradycardia associated with norepinephrine injection 7. Electrolytic lesions or injection of barbiturates into the anterior hypothalamic area inhibited norepinephrine-induced bradycardia s. Investigations by Klevans and Gebber extended the work of Gellhorn by demonstrating that the facilitation could also be produced by stimulation of the area septalis and area preoptica, as well as the anterior hypothalamic area 6'12. Furthermore, the facilitation: (1) affected only the vagal component of the baroreflex; (2) was mediated by the central nervous system; and (3) was dependent on baroreceptor input for initial activation of the cardiac vagus. A similar potentiation of the cardioinhibitory reflex was demonstrated by stimulation of the caudal regions of the lateral hypothalamic area in the rabbit l'a'5. The bradycardia evoked by raising blood pressure in response to hypothalamic stimulation was greater than that evoked by norepinephrine. Most recently, Miyajima and Bufiag, using the opposite paradigm of the present experiments, have demonstrated that electrolytic lesions of the anterior hypothalamic area selectively
impaired reflex bradycardia in the conscious and anesthetized rat ~3. One general characteristic of the above reports is that facilitation of reflex bradycardia by hypothalamic stimulation depends upon baroreceptor input. In the cat, facilitation of reflex bradycardia was only observed when carotid sinus pressure was at least 100 mm Hg or when mean arterial pressure was transiently increased to levels far above control with norepinephrine 12. In the rabbit, section of the carotid sinus and aortic nerves eliminated the stimulation-induced bradycardia4; the magnitude of the fall in heart rate was directly correlated with the initial mean arterial pressure 1. In the present study, atropine injection did not affect baseline heart rate suggesting absence of tonic vagal activity; hypothalamic stimulation alone (Fig. 3) also did not affect heart rate. Thus, facilitation of reflex bradycardia produced by stimulation in these hypothalamic areas appears to: (1) be dependent upon a threshold level of baroreceptor input; and (2) modulate ongoing reflex bradycardia rather than invoking a bradycardic response. These findings are not surprising since it is well documented that various hypothalamic structures are known to participate in the baroreflex. Electrophysiological studies suggest that cardiovascular afferents influence the hypothalamus 2'm. Spyer reported that single unit activity in the anterior hypothalamus could be affected by electrical activation of the carotid sinus nerve 16. Calaresu and Ciriello have electrophysiologically demonstrated connections between carotid sinus afferents activated by pressure increases and the supraoptic and paraventricular nuclei of the hypothalamus 2. Taken together, these data provide evidence for direct functional connections between the baroreceptor afferents and the hypothalamus. These studies have been supported by neuroanatomical evidence of ascending projections from the nucleus of the tractus solitarius, the site of primary synapses of baroreceptor afferents, to the hypothalamus Is. Additionally, the lateral and periventricular hypothalamus receive fibers from other locations in pontine and medullary regions I~. While neural connections between the baroreceptors and the hypothalamus have been demonstrated, their significance is still highly speculative. One functional consequence of these connections appears to be facilitation of baroreflex-induced bradycardia.
280 A second general characteristic of the facilitated bradycardia is that it is m e d i a t e d p r e d o m i n a n t l y by the p a r a s y m p a t h e t i c nervous system. In the cat 12, rabbit 1. and rat 13 (and present study), the facilitation of bradycardia was eliminated by atropine, vagotomy, or both. A d r e n e r g i c r e c e p t o r b l o c k a d e failed to eliminate the response in cats 12 and rats 13, Thus, these experiments support the concept of Gellhorn that the anterior h y p o t h a l a m u s is a vagal center s. Recent anatomical investigations provide evidence that descending pathways which originate in hypothalamic regions terminate in m e d u l l a r y parasympathetic sites ~5'19. R e t r o g r a d e tracer experiments d e m o n s t r a t e d both direct and indirect pathways from h y p o t h a l a m i c nuclei, including the ventromedial and anterior hypothalamic areas, that terminate in the dorsal m o t o r nucleus of the vagus ( D M X ) and the nucleus ambiguus ( N A ) 19. Both the D M X and N A are sites of origin of cardiac vagal preganglionic neurons 17. In addition, experiments which utilized anterogradely t r a n s p o r t e d r a d i o l a b e l e d amino acids indicate that hypothalamic fibers pass through or terminate in the dorsal m o t o r nucleus of the vagus and the nucleus of the tractus solitarius, the latter being the primary synaptic site of b a r o r e c e p t o r afferents 15. Thus, n e u r o a n a t o m i c a l tracer studies support the physiological observations of p a r a s y m p a t h e t i c mediation of the facilitated bradycardia. Although both afferent and efferent pathways which terminate in and originate from the regions of the anterior and v e n t r o m e d i a l h y p o t h a l a m u s have been d e m o n s t r a t e d , it is possible that stimulation of fibers which pass through these areas may play a role in the observed response. In this regard, the antero-
REFERENCES 1 Buss, Y. and Evans. M,H., Bradycardia evoked by hypothalamic stimulation in the rabbit: dependence upon the arterial blood pressure, Neuroscience, 12 (1984) 489-493. 2 Calaresu, F.R. and Ciriello, J., Projections to the hypothalamus from buffer nerves and nucleus tractus solitarius in the cat, Am. J. Physiol., 239 (1980) R100-R109. 3 Djojosugito, A.M., Folkow, B., Kylstra, P.H., Lisander, B. and Tuttle, R.S., Differentiated interaction between the hypothalamic defence reaction and baroreeeptor reflexes. I. Effects on heart rate and regional flow resistance, Acta Physiol. Scand., 78 (1970) 376-385. 4 Evans, M.H., Potentiation of a cardioinhibitory reflex by hypothalamic stimulation in the rabbit, Brain Research, 154 (1978) 331-343.
ventral third ventricular region {AV3V), which includes the subfornical organ, the o r g a n u m vasculosum of the lamina terminalis, and the preoptic area. has been shown to elicit changes in autonomic outflow The effects are m e d i a t e d primarily through the medial forebrain bundle, which contains n u m e r o u s ascending and descending pathways, and a m o r e medial. periventricular pathway that passes through the anterior h y p o t h a l a m u s and v e n t r o m e d i a l nucleus '~. In summary, the present experiments d e m o n s t r a t e that hypothalamic stimulation in the rat elicits facilitation of b a r o r e c e p t o r - i n d u c e d bradycardia The facilitation is: (1) d e p e n d e n t upon b a r o r e c e p t o r afferent input: (2) elicited bv discrete stimulation of the ventromedial or anterior h y p o t h a l a m i c areas: and (3) mediated through efferent p a r a s y m p a t h e t i c nerves. This study provides functional evidence for the abilitv of hypothalamic regions to m o d u l a t e the baroreflex control of arterial pressure: and thus complements n e u r o a n a t o m i c a l and electrophysiological evidence that d e m o n s t r a t e s a relationship between medullary cardiovascular sites and supramedullary structures. F u r t h e r m o r e , these results represent the first d e m o n s t r a t i o n of baroreflex modulation in the rat bv stimulation of supramedullar> structures.
ACKNOWLEDGEMENTS The authors would like to thank P,G. Schmid. I l i . for his expert technical assistance. This work was s u p p o r t e d by the V . A . IVied. Ctr., N _ I . H . N . R , S . A . GM09568. and N . I . H . G r a n t s HL35484. HL14388, HL24246. and HL20768.
5 Evans. M.H., Facilitation of reflex bradycardia by hypothalamic stimulation in the anaesthetized rabbil. J. Physiol. ~London J. 265 {1977~ 33P-34P. 0 Gebber G.L. and Klevans. L.R.. Central nervous system modulation of cardiovascular reflexes, Fed, Proc. bed. Am. Soc. Exp. Biol., 31 (1972) 1245-1252. - Gellhorn. E., Autonomic Imbalance and the Hypothalamus. Univ. of Minnesota Press. Minneapolis. 1957, pp. 77- 118. 8 Gellhorn. E.. Nakao, H and Redgate, E.S., The influence of lesions in the anterior and posterior hypothalamus on tonic and phasic autonomic reactions, J. Physiol. t.Londonl. 13I (1956) 402-423. 9 Hartle. D.K and Brody, M.J.. The angiotensin II pressor system of the rat forebrain. Circ. Res.. 54 (1984) 355-366, 10 Hilton. S.M., Inhibition of baroreceptor reflexes on hypo-
281
11
12
13
14
15
thalamic stimulation, J. Physiol. (London). 165 (1963) 56-57. Hilton, S.M.. Hypothalamic control of the cardiovascular responses in fear and rage. In The Scientific Basis of Medicine Annual Reviews. The Athlone Press, London, 1965, pp. 217-238. Klevans, L.R. and Gebber, G.L., Facilitatory forebrain influence on cardiac component of baroreceptor reflexes. Am. J. Physiol.. 219 (1970) 1235-1241. Miyajima. E. and Bufiag, R.D., Anterior hypothalamic lesions impair reflex bradycardia selectively in rats, Am. J. Physiol., 248 (1985") H937-H944. Pellegrino. L.J., Pellegrino, A.S. and Cushman. A.J., A Stereotaxic Atlas of the Rat Brain, Plenum Press, New York, 1979. Saper, C B . , Loewy, A.D., Swanson, L.W. and Cowan,
16
17
18
19
W.M., Direct hypothalamo-autonomic connections, Brain Research, 117 (1976) 305-312. Spyer, K.M., Baroreceptor sensitive neurons in the anterior hypothalamus of the cat, J. Phvsiol. ¢London). 224 (1972) 245-257. Stuesse, S i . , Origins of cardiac vagal preganglionic fibers: a retrograde transport study, Brain Research, 236 (1982) 15-25. Swanson, L.W. and Sawchenko, P.E., Hypothalamic integration: organization of the paraventricular and supraoptic nuclei, Annu. Rev. Neurosci.. 6 (1983) 269-324. Ter Horst. G.J., Luiten, P.G.M. and Kuipers. F., Descending pathways from hypothalamus to dorsal motor vagus and ambiguus in the rat, J. Autonotn. Nerv. Svst.. l I (1984) 59-75.
274
BRE 12050
Facilitation of Baroreflex-Induced Bradycardia by Stimulation of Specific Hypothalamic Sites in the Rat B.J. PARDINI, K.P. PATEL, P.G. SCHMID and D.D. LUND
Veterans Administration Medical Center, Cardiovascular Center, and Department of Internal Medicine, University of lowa, Iowa City, 1,4.52240 (U.S.A.) (Accepted 3 April 1986)
Key words: Baroreflex - - Anterior hypothalamic area - - Ventromedial nucleus of the hypothalamus -Parasympathetic nervous system - - Heart rate
Hypothalamic stimulation generally inhibits baroreflex-induced bradycardia. However. we have noted discrete areas of the rat hypothalamus which facilitate reflex bradycardia. The effects of hypothalamic stimulation on baroreflex-induced changes in heart rate were investigated in urethane-anesthetized rats f 1.2 g/kg, i.p. : n = 6) instrumented with femoral arterial and venous catheters. Bipolar electrodes/250 ~m diameter) were implanted stereotaxically in the hypothalamus. Baroreflex-induced bradycardia was elicited by phenylephrine (PE) injection (8-20~g/kg). Responses to stimulation (STIM) (50-150/~A. 80 Hz. 0.5 ms). PE. and Stim - PE were studied for 1 min. In the ventral medial and anterior hypothalamus, STIM caused transient increases in blood pressure and no changes in heart rate. Peak blood pressure was lower during STIM - PE than during PE (144 + 5 vs 164 _+ 3 mm Hg; P < 0.05). However. STIM + PE resulted in a lower heart rate compared to PE (194 ___22 vs 270 + 17 bpm" P < 0.05). At l min. the heart rate in STIM + PE rats remained lower than in PE rats (205 + 37 vs 319 _+ 16 bpm: P < 0.05). Atropine administration indicated that the facilitation was primarily parasympathetic in nature These results identify specific hypothalamic regions which facilitate baroreflex-induced bradycardia by parasympathetic mechanisms. cleus or the a n t e r i o r h y p o t h a l a m i c a r e a in rats causes
INTRODUCTION
p r o f o u n d facilitation of p h e n y l e p h r i n e - i n d u c e d bradCardiovascular
effects of h y p o t h a l a m i c stimula-
tion are g e n e r a l l y b e l i e v e d to inhibit n o r m a l b a r o r e -
ycardia that is m e d i a t e d
by the p a r a s y m p a t h e t i c
n e r v o u s system.
flexes. M o s t n o t a b l y , s t i m u l a t i o n of the h y p o t h a l a m ic d e f e n s e a r e a inhibits b a r o r e f l e x - i n d u c e d b r a d y c a r dia 3'1°'11. H o w e v e r . early r e p o r t s by G e i l h o r n 7 and
MATERIALS AND METHODS
later by K l e v a n s and G e b b e r ~2. h a v e d e m o n s t r a t e d
Male S p r a g u e - D a w l e y rats ( 3 0 0 - 3 5 0 g) w e r e an-
facilitation of r e f e x - i n d u c e d b r a d y c a r d i a by stimula-
e s t h e t i z e d i n t r a v e n o u s l y with u r e t h a n e (1.2 g/kg, i.p.) and i n s t r u m e n t e d with a r t e r i a l and v e n o u s cath-
tion of specific h y p o t h a l a m i c sites in the cat. In contrast, electrolytic lesions of specific h y p o t h a l a m i c
eters. A r t e r i a l pulsatile and m e a n b l o o d p r e s s u r e
sites h a v e y i e l d e d o p p o s i t e results, R e c e n t l y , Miyaji-
w e r e m e a s u r e d with a p r e s s u r e t r a n s d u c e r and dis-
m a and Bufiag r e p o r t e d that d e s t r u c t i o n of the ante-
played on a p a p e r chart r e c o r d e r . T h e E C G was re-
rior h y p o t h a l a m u s
c o r d e d : h e a r t rate was d e r i v e d f r o m a t a c h o m e t e r
in the rat c a u s e d inhibition of
p h e n y l e p h r i n e - i n d u c e d b r a d y c a r d i a t3 W e h y p o t h e s i z e d that s t i m u l a t i o n of specific h y p o t h a l a m i c sites w o u l d facilitate reflex b r a d y c a r d i a in
d r i v e n by e i t h e r t h e Q R S c o m p l e x o f the E C G or the pulsatile p r e s s u r e signal. T h e E C G was also disp l a y e d at high r e s o l u t i o n on an o s c i l l o s c o p e to accu-
the rat. T h e p r e s e n t d a t a d e m o n s t r a t e that unilateral
rately d e t e r m i n e the p r e s e n c e of a p - w a v e and e n s u r e
electrical s t i m u l a t i o n o f e i t h e r the v e n t r a l m e d i a l nu-
that cardiac a c t i v a t i o n was initiated t h r o u g h n o r m a l
Correspondence." B.J. Pardini. Cardiovascular Center, Room 10W20, Veterans Administration Medical (.'enter. Iowa City, [A 52240. U.S.A. 0006-8993/86/$03.50 ~ 1986 Elsevier Science Publishers B.V. t Biomedical Division
275 pathways. When a large degree of bradycardia was observed, it was important to verify that a - v nodal block had not occurred and that the recorded rate was of ventricular origin. After placement in a stereotaxic apparatus, the skull was exposed. Concentric bipolar electrodes (250 ,urn outer diameter, Rhodes Medical Instruments) were used to stimulate hypothalamic regions with constant current (50-150 ktA, 80 Hz, 0.5 ms). Stimulus sites were verified histologically in 10-25 /~m sections after marking the area of interest with electrolytic lesions made by passage of DC current. Reflex-induced bradycardia was elicited by intravenous injection of phenylephrine (8-20 ug/kg, in 500 ul of saline). The blood pressure and heart rate responses to electrical stimulation, phenylephrine injection, or phenylephrine injection during electrical stimulation were recorded. Multiple hypothalamic sites in each rat were tested for their ability to facilitate phenylephrine-induced bradycardia. The areas tested were within the following stereotaxic boundaries according to Pellegrino~4: from bregma to 1.20 mm rostral to bregma, from the midline to 1.5 mm lateral to the midline, and from approximately 7.0 mm ventral to the surface of the brain to the base of the brain. The following hypothalamic sites were included within the designated boundaries: dorsomedial nucleus, ventromedial nucleus, arcuate nucleus, paraventricular nucleus, anterior hypothalamic area, fornix, and zona incerta. Additionally, the supraoptic nucleus was also stimulated. Statistical significance was determined by an independent analysis of variance followed by a least significant difference test for group differences. A probability level of less than 0.(15 was accepted as a significant difference. RESULTS A typical procedure consisted of electrode placement at the most dorsal site of a track followed by a bolus injection of phenylephrine. The blood pressure and heart rate response to phenylephrine was recorded; this response was used to compare with other responses elicited within the same electrode track. Hypothalamic sites were investigated at 0.5 mm intervals; the effect of electrical stimulation alone was
followed by electrical stimulation initiated approximately 2 s after phenylephrine injection. Electrical stimulation of an area was considered to facilitate phenylephrine-induced bradycardia when electrical stimulation plus phenylephrine (STIM + PE) caused a greater bradycardia than the sum of heart rate decreases induced by individual phenylephrine injection (PE) and electrical stimulation alone (STIM). Fig. 1 illustrates the pulsatile and mean arterial pressure and the heart rate recorded from a rat during investigation of a site located in the anterior hypothalamic region. Electrical stimulation alone caused a biphasic response in blood pressure: an "on response', a small hypertensive effect at the start of the stimulation, and an 'off response', a small hypotensive effect when the stimulation was terminated (Fig. 1A). A small degree of bradvcardia was also observed. Phenylephrine injection caused a typical increase in blood pressure followed bv a bradycardia of approximately 50 beats per minute (bpm). However, when a bolus injection of phenylephrine was administered concomitantly with electrical stimulation the magnitude of the bradveardia was markedly increased, and lasted for the duration of the stimulation (Fig. 1A). Lesions made by passage of DC current completely eliminated the stimulation-induced facilitation of the phenylephrine-induced bradycardia, but did not affect the normal cardiowtscular responses to phenylephrine injection. Additionally, the cardiovascular alterations to electrical stimulation alone were eliminated by prior lesion of the area. If the facilitation is due to discrete stimulations, it should be eliminated or reduced by small movements of the electrode. Fig. 1B illustrates the responses obtained 0.5 mm dorsal to the site of Fig. 1A. All electrode penetrations were made dorsal to ventral; thus, the stimulus of Fig. 1B was made prior to that of Fig. 1A and was not made on previously penetrated tissue. Although the biphasic blood pressure response to electrical stimulation alone was present, the degree of bradycardia elicited by simultaneous electrical stimulation and phenylephrine injection was markedly decreased compared to the results observed 0.5 mm away. The elimination of most of the response bv a small shift in the electrode demonstrates the highly localized area in which the facilitation can be elicited.
276 A.
STIM
PE
STIM + PE
200 PULSATILE
ARTERIAL PRESSURE
I00
(ram Hg)
0 200
MEAN ARTERIAL PRESSURE
I00
(ram Hg)
0 sec
HEART RATE (bpm)
500 I
250I
f
t
0 L i
i
g.
STIM
PE
S T I M + PE
2O0 PULSATILE ARTERIAL PRESSURE
I00
(ram H(J)
200 MEAN ARTERIAL
I00
PRESSURE (ram Hg)
0
I---7 3 0 sec
500 HEART RATE
250
(bpm)
-
-
r
1
Fig. 1. Panels depict pulsatile and mean arterial pressure and heart rate of rats during electrical stimulation (STIM), phenylephrme rejection (PE), or both interventions simultaneously (STIM + PE). At the bottom of each panel the bar indiCates the duration of electrical stimulation and the arrow indicates the time of phenylephrine injection. In panel A, the tip of the stimulating electrod e was in the anterior hypothalamus. STIM produced small 'on' pressor and 'off' depressor responses, but little Change in heart rate. STIM + PE produced profound facilitation Of the bradycardia associated with either STiM or PE. The results shown in panel 13were recorded just prior to the interventions of panel A. The tip of the stimulating electrode wasO.5 mm dorsal to its placement in p a n e f A . The results indicate that the response is localized to a small area and that current spread was not excessive.
277
MEAN ARTERIAL PRESSURE (ram Hg)
iii t .... 1
2
0
80 t
÷
/
~
÷
.
*
÷
,el 0
I
o
::::::
;;,.
- -
PE+$TIM
'6 72 1'8 2'4 3'0 3'6 4'2 48 5'. 6'o TIME (see)
400 • .....
HEART RATE (bpm)
.....
3oo i ......... +.........t.........
..... !----t
.........t ........ t
.....
t
........
250 200" 150 .
.
.
.
.
.
.
.
.
.
.
.
.
" 0
PE .....
STIM
- -
PE + S TIM
,~ ,'~ ~'4 3'o 3'6 ;~ ,;8 5'4 6'o TIME (see)
Fig. 2. Summary diagram illustrates the effect of stimulation (STIM), phenylephrine (PE), and stimulation plus phenylephrine (STIM + PE) on mean arterial pressure and heart rate in 6 rats (mean + S.E.M.). Even though the increase in blood pressure was less in STIM + PE than in PE rats. the reduction in heart rate was greater. Blood pressure: + = P < 0.05 vs PE + STIM. Heart rate: + = P < 0.05vs PE + STIMorPE;o = P < 0.05 vs STIM.
Fig. 2 illustrates the mean arterial pressure and heart rate data obtained from 6 rats in which stimulation was carried out for at least 60 s. The heart rate during electrical stimulation plus phenylephrine is significantly reduced c o m p a r e d to phenylephrine injection alone. It is significant to note that the peak blood pressure response to phenylephrine plus electrical stimulation is significantly less than to phenylephrine alone. Thus, even though a smaller baroreceptor stimulus is present in the stimulation plus phenylephrine group, the magnitude of the bradycardia is significantly greater. A t r o p i n e (1 mg/kg, i.v. bolus) was administered in some rats to determine if the facilitation of the bradycardia was parasympathetic in nature. Fig. 3A and 3B demonstrate the results of one such experiment. Before atropine injection, electrical stimulation plus phenylephrine caused a maximal decrease in heart
rate of 150 bpm that was much greater than the maximal bradycardia elicited by either electrical stimulation (45 bpm decrease) or phenylephrine alone (30 bpm decrease). However, atropine abolished the heart rate response to phenylephrine and electrical stimulation plus phenylephrine. A d d i t i o n a l l y , the small bradycardia associated with electrical stimulation alone was eliminated. Fig. 4 demonstrates the hypothalamic sites at which the facilitation of bradycardia was observed. The facilitation was localized primarily to the region which corresponds to the ventromedial hypothalamic nucleus and the anterior hypothalamic area. No facilitation was observed in the other m a j o r nuclei of the hypothalamus that were studied, including the paraventricular nucleus, the supraoptic nucleus, and the dorsomedial nucleus. DISCUSSION The present experiments d e m o n s t r a t e that electrical stimulation of the ventromedial or anterior hypothalamic region in the rat greatly facilitates the bradycardia associated with phenylephrine-induced increases in arterial pressure. Such facilitation is not observed during stimulation of the paraventricular nucleus, supraoptic nucleus or dorsomedial nucleus of the hypothalamus. A t r o p i n e administration eliminates the facilitation as well as the b r a d y c a r d i a associated with phenylephrine alone. Thus, the efferent pathway of the facilitation is p r e s u m e d to act through the parasympathetic nervous system. Two criteria used in the present experiments to maximize stimulation of discrete sites and minimize activation of fibers of passage were utilization of low stimulus currents, and the absence of the response by stimulation a short distance from the site of interest. Typically, current intensities were 50-75 /~A. In those experiments where greater than 100 # A were used, the facilitation was not present when the stimulus site was moved 0.5 mm away (Fig. 1, stimulus intensity: 150HA ). It is expected that the stimulations activated cell somata to a greater extent than fibers of passage. H o w e v e r , the possibility that fibers of passage were also activated can not be eliminated by the present methodology. Gellhorn and associates first r e p o r t e d that stimulation of the anterior hypothalamus in cats facilitated
278 BEFORE
ATROPINE
STIM PULSATILE ARTERIAL PRESSURE ( m m Hg)
PE
STIM+PE
200 I00 0 200
MEAN ARTERIAL
I00
PRES SURE (ram H g )
0
3b HEART RATE (bpm)
Zllf OL
AFTER
PULSATILE
2o0[
STIM
ATROPINE PE
STIM + PE
ARTERIAL PRESSURE (ram Hg)
200 MEAN ARTERIAL
I00
PRESSURE ( m m Hg)
0 500
50 se c
HEART RATE
250
(bpm)
Fig. 3. Same configuration as in Fig, 1. The top panel illustrates a typical response obtained from stimulations in the ventromedial hypothalamus, The effects of atropine (1 mg/kg, Lv.) administered prior to repetition of the same interventions is illustrated in the lower panel. Virtually all of the bradycardia associated with any of the interventions was eliminated.
279
I
--/PVN ~--AHA ~ ~ 7 0 . 4 0 ~MA
+1.20 mm
J +0,80 mm
mm lilll 2ram
0.00 mm
Fig. 4. Diagrams of hypothalamic cross-sections illustrate regions 0.0-1.2 mm rostral to bregma. The lateral ventricles are indicated by the stippled area; the optic tract is indicated by the cross-hatching. Asterisks indicate the central location of the stimulating electrode in which facilitation of reflex bradycardia was elicited. Abbreviations: PVN, paraventricular nucleus; AHA, anterior hypothalamic area; SON, supraoptic nucleus: VMH, ventromedial nucleus of the hypothalamus.
the bradycardia associated with norepinephrine injection 7. Electrolytic lesions or injection of barbiturates into the anterior hypothalamic area inhibited norepinephrine-induced bradycardia s. Investigations by Klevans and Gebber extended the work of Gellhorn by demonstrating that the facilitation could also be produced by stimulation of the area septalis and area preoptica, as well as the anterior hypothalamic area 6'12. Furthermore, the facilitation: (1) affected only the vagal component of the baroreflex; (2) was mediated by the central nervous system; and (3) was dependent on baroreceptor input for initial activation of the cardiac vagus. A similar potentiation of the cardioinhibitory reflex was demonstrated by stimulation of the caudal regions of the lateral hypothalamic area in the rabbit l'a'5. The bradycardia evoked by raising blood pressure in response to hypothalamic stimulation was greater than that evoked by norepinephrine. Most recently, Miyajima and Bufiag, using the opposite paradigm of the present experiments, have demonstrated that electrolytic lesions of the anterior hypothalamic area selectively
impaired reflex bradycardia in the conscious and anesthetized rat ~3. One general characteristic of the above reports is that facilitation of reflex bradycardia by hypothalamic stimulation depends upon baroreceptor input. In the cat, facilitation of reflex bradycardia was only observed when carotid sinus pressure was at least 100 mm Hg or when mean arterial pressure was transiently increased to levels far above control with norepinephrine 12. In the rabbit, section of the carotid sinus and aortic nerves eliminated the stimulation-induced bradycardia4; the magnitude of the fall in heart rate was directly correlated with the initial mean arterial pressure 1. In the present study, atropine injection did not affect baseline heart rate suggesting absence of tonic vagal activity; hypothalamic stimulation alone (Fig. 3) also did not affect heart rate. Thus, facilitation of reflex bradycardia produced by stimulation in these hypothalamic areas appears to: (1) be dependent upon a threshold level of baroreceptor input; and (2) modulate ongoing reflex bradycardia rather than invoking a bradycardic response. These findings are not surprising since it is well documented that various hypothalamic structures are known to participate in the baroreflex. Electrophysiological studies suggest that cardiovascular afferents influence the hypothalamus 2'm. Spyer reported that single unit activity in the anterior hypothalamus could be affected by electrical activation of the carotid sinus nerve 16. Calaresu and Ciriello have electrophysiologically demonstrated connections between carotid sinus afferents activated by pressure increases and the supraoptic and paraventricular nuclei of the hypothalamus 2. Taken together, these data provide evidence for direct functional connections between the baroreceptor afferents and the hypothalamus. These studies have been supported by neuroanatomical evidence of ascending projections from the nucleus of the tractus solitarius, the site of primary synapses of baroreceptor afferents, to the hypothalamus Is. Additionally, the lateral and periventricular hypothalamus receive fibers from other locations in pontine and medullary regions I~. While neural connections between the baroreceptors and the hypothalamus have been demonstrated, their significance is still highly speculative. One functional consequence of these connections appears to be facilitation of baroreflex-induced bradycardia.
280 A second general characteristic of the facilitated bradycardia is that it is m e d i a t e d p r e d o m i n a n t l y by the p a r a s y m p a t h e t i c nervous system. In the cat 12, rabbit 1. and rat 13 (and present study), the facilitation of bradycardia was eliminated by atropine, vagotomy, or both. A d r e n e r g i c r e c e p t o r b l o c k a d e failed to eliminate the response in cats 12 and rats 13, Thus, these experiments support the concept of Gellhorn that the anterior h y p o t h a l a m u s is a vagal center s. Recent anatomical investigations provide evidence that descending pathways which originate in hypothalamic regions terminate in m e d u l l a r y parasympathetic sites ~5'19. R e t r o g r a d e tracer experiments d e m o n s t r a t e d both direct and indirect pathways from h y p o t h a l a m i c nuclei, including the ventromedial and anterior hypothalamic areas, that terminate in the dorsal m o t o r nucleus of the vagus ( D M X ) and the nucleus ambiguus ( N A ) 19. Both the D M X and N A are sites of origin of cardiac vagal preganglionic neurons 17. In addition, experiments which utilized anterogradely t r a n s p o r t e d r a d i o l a b e l e d amino acids indicate that hypothalamic fibers pass through or terminate in the dorsal m o t o r nucleus of the vagus and the nucleus of the tractus solitarius, the latter being the primary synaptic site of b a r o r e c e p t o r afferents 15. Thus, n e u r o a n a t o m i c a l tracer studies support the physiological observations of p a r a s y m p a t h e t i c mediation of the facilitated bradycardia. Although both afferent and efferent pathways which terminate in and originate from the regions of the anterior and v e n t r o m e d i a l h y p o t h a l a m u s have been d e m o n s t r a t e d , it is possible that stimulation of fibers which pass through these areas may play a role in the observed response. In this regard, the antero-
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ventral third ventricular region {AV3V), which includes the subfornical organ, the o r g a n u m vasculosum of the lamina terminalis, and the preoptic area. has been shown to elicit changes in autonomic outflow The effects are m e d i a t e d primarily through the medial forebrain bundle, which contains n u m e r o u s ascending and descending pathways, and a m o r e medial. periventricular pathway that passes through the anterior h y p o t h a l a m u s and v e n t r o m e d i a l nucleus '~. In summary, the present experiments d e m o n s t r a t e that hypothalamic stimulation in the rat elicits facilitation of b a r o r e c e p t o r - i n d u c e d bradycardia The facilitation is: (1) d e p e n d e n t upon b a r o r e c e p t o r afferent input: (2) elicited bv discrete stimulation of the ventromedial or anterior h y p o t h a l a m i c areas: and (3) mediated through efferent p a r a s y m p a t h e t i c nerves. This study provides functional evidence for the abilitv of hypothalamic regions to m o d u l a t e the baroreflex control of arterial pressure: and thus complements n e u r o a n a t o m i c a l and electrophysiological evidence that d e m o n s t r a t e s a relationship between medullary cardiovascular sites and supramedullary structures. F u r t h e r m o r e , these results represent the first d e m o n s t r a t i o n of baroreflex modulation in the rat bv stimulation of supramedullar> structures.
ACKNOWLEDGEMENTS The authors would like to thank P,G. Schmid. I l i . for his expert technical assistance. This work was s u p p o r t e d by the V . A . IVied. Ctr., N _ I . H . N . R , S . A . GM09568. and N . I . H . G r a n t s HL35484. HL14388, HL24246. and HL20768.
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13
14
15
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19
W.M., Direct hypothalamo-autonomic connections, Brain Research, 117 (1976) 305-312. Spyer, K.M., Baroreceptor sensitive neurons in the anterior hypothalamus of the cat, J. Phvsiol. ¢London). 224 (1972) 245-257. Stuesse, S i . , Origins of cardiac vagal preganglionic fibers: a retrograde transport study, Brain Research, 236 (1982) 15-25. Swanson, L.W. and Sawchenko, P.E., Hypothalamic integration: organization of the paraventricular and supraoptic nuclei, Annu. Rev. Neurosci.. 6 (1983) 269-324. Ter Horst. G.J., Luiten, P.G.M. and Kuipers. F., Descending pathways from hypothalamus to dorsal motor vagus and ambiguus in the rat, J. Autonotn. Nerv. Svst.. l I (1984) 59-75.