Raphe region mediates changes in cutaneous vascular tone elicited by stimulation of amygdala and hypothalamus in rabbits

Brain Research 891 (2001) 130–137 www.elsevier.com / locate / bres

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Raphe region mediates changes in cutaneous vascular tone elicited by stimulation of amygdala and hypothalamus in rabbits Eugene Nalivaiko*, William W. Blessing Departments of Physiology and Medicine, Center for Neuroscience, Flinders University, Bedford Park, SA 5042, Australia Accepted 31 October 2000

Abstract Raphe pallidus / parapyramidal neurons control cutaneous vasoconstriction induced by noxious stimuli. To determine whether they mediate forebrain-induced cutaneous vasoconstriction, we assessed changes in ear pinna blood flow elicited by electrical stimulation of amygdala and hypothalamus before and after injection of muscimol into the raphe / parapyramidal region. We compared ear flow with simultaneously recorded mesenteric flow. Experiments were performed in rabbits anesthetized with urethane (1.25–1.5 g / kg), paralysed and mechanically ventilated. Amygdala stimulation reduced skin conductance from 0.3260.06 to 0.1060.02 cm / s per mm Hg (P,0.05, n59), without effect on mesenteric conductance. Hypothalamic stimulation caused vasoconstriction in both cutaneous and mesenteric beds (conductances fell from 0.2760.05 to 0.0560.02 cm / s per mm Hg and from 0.2760.06 to 0.1460.04 cm / s per mm Hg (P,0.05, n59), respectively). Muscimol microinjection (5 nmol in 100 nl) to raphe / parapyramidal region eliminated amygdala- and hypothalamusinduced skin vasoconstriction (pre-stimulus conductance 0.4260.13 and 0.4160.11 cm / s per mm Hg, post-stimulus 0.4160.12 and 0.3960.10 cm / s per mm Hg, respectively), but not hypothalamically-induced mesenteric vasoconstriction (pre-stimulus 0.2960.06, post-stimulus 0.1660.03 cm / s per mm Hg, P,0.05, n58). The latter was strongly attenuated by bilateral injection of muscimol to the rostral ventrolateral medulla. Data suggest that descending hypothalamo–spinal and amygdala–spinal pathways constricting the cutaneous vascular bed relay in the raphe / parapyramidal area. A relay in the rostral ventrolateral medulla contributes substantially to mesenteric vasoconstriction elicited from the hypothalamus.  2001 Elsevier Science B.V. All rights reserved. Theme: Endocrine, autonomic regulation Topic: Cardiovascular regulation, central control Keywords: Alerting response; Skin blood flow; Stress

1. Introduction Cutaneous vasoconstriction is an integral part of the cardiovascular response pattern initiated by emotionally charged salient stimuli, as demonstrated by appropriate regional blood flow measurements in conscious humans [1,14] and conscious rabbits [22,34]. A corresponding selective cutaneous vasoconstriction is also seen in response to normally painful stimuli in anesthetized humans [30] and in anesthetized rabbits [22,35]. These major changes in cutaneous blood flow are largely sympathetically-mediated and they occur without major increases

*Corresponding author. Tel.: 161-8-8204-4107; fax: 161-8-82045450. E-mail address: [email protected] (E. Nalivaiko).

in arterial pressure. Thus, brain neuronal circuitry controls cutaneous vessels independently of the general circulation. The question arises as to the particular brain neural circuitry responsible for this selective regulation of the cutaneous circulation. In our previous studies in rabbits we have identified two important brain centers. In conscious rabbits, bilateral inactivation of the amygdaloid region eliminates the sudden falls in ear pinna blood flow which normally occur when the animal detects a salient environmental event [34]. In anesthetized rabbits, inactivation of neuronal function in the medullary raphe / parapyramidal area eliminates the sudden falls in ear pinna blood flow which occur when the animal is subjected to a normally painful stimulus [3]. This latter observation is consistent with our earlier demonstration [3,4] that the raphe / pyramidal region selectively regulates cutaneous blood flow in the rabbit.

0006-8993 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 00 )03210-8

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The amygdala does not have direct descending projections to the thoracic spinal cord, so that activation of sympathetic preganglionic neurons regulating the ear pinna bed must occur via a relay in one of the brainstem / hypothalamic presympathetic neuronal groups [7]. In the present experiments in anesthetized rabbits we have determined whether electrical stimulation of the amygdaloid region causes vasoconstriction in the cutaneous and / or the mesenteric bed, and whether blockade of neuronal function in the raphe / parapyramidal region prevents the vasomotor effects which occur. We compared amygdala stimulation with hypothalamic stimulation, since both these regions are involved in regulating the cardiovascular responses associated with alerting / defence reactions [12,13].

2. Materials and methods Experiments were conducted in 11 male New Zealand White rabbits (2.5–3.5 kg). All experimental procedures were approved by the Flinders University of South Australia Animal Ethics Committee. For measurement of skin and mesenteric blood flow, Doppler ultrasonic flow probes (Iowa Doppler Products, Iowa, USA) were implanted around the ear pinna artery and the superior mesenteric artery, respectively, under midazolam / hypnorm anesthesia (2 mg / kg i.m., 0.3 mg / kg i.m., respectively) at least 1 week prior to the experiment. On the day of the experiment animals were anesthetized with urethane (1.5 g / kg i.v. over 20–30 min), paralysed (vecuronium bromide 0.5 mg / kg i.v.) and mechanically ventilated. End-expiratory CO 2 was monitored and maintained at 35–40 mm Hg. Body temperature was maintained at 38.5–39.58C. For stereotaxic access to the amygdala and the hypothalamus, the rabbit’s head was held in a Kopf apparatus, and an appropriate burrhole was made using the stereotaxic approach described in our previous experiments [36]. Electrical stimulation of the amygdala was made 11.5 to 12.5 mm ventral to the dura, 4.5 to 5 mm lateral to the midline at the level of bregma, in the region containing the central nucleus of the amygdala and the descending outflow pathway [27]. Electrical stimulation of the hypothalamus was made 13 to 14 mm ventral to the dura, 1.5 mm lateral to the midline, 1.5 mm caudal to the level of bregma, just ventral and medial to the mammillothalmic tract, in the region of the dorsomedial nucleus of the hypothalamus [9]. Parameters of electrical stimulation via insulated monopolar stainless steel electrodes were 50 Hz, 1 ms, 50–400 mA, 10 s train. Electrical stimulation sites were marked by an anodal current (50 mA DC for 20 s) and the brains were histologically processed for staining with the Perl’s Prussian blue reaction. The medulla oblongata was exposed by incision and retraction of the atlanto–occipital membrane. Microinjections of muscimol (5 nmol in 100 nl) into the raphe / parapyramidal region in the midline and bilaterally into the

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RVLM were made through a glass capillary micropipette (tip diameter 30–50 mm). Forebrain stimulation was repeated 10 min after the muscimol injections. Injection sites were marked by including horseradish peroxidase (for raphe / parapyramidal region) and b-galactosidase (for RVLM) in the vehicle used for the injections. At the end of the experiment animals were perfused with fixative and the brain prepared for histological examination after the appropriate histochemical reaction. Muscimol, horseradish peroxidase and b-galactosidase were obtained from Sigma Chemical Company, USA. The Doppler frequency signal was relayed to a Triton Technologies Flowmeter (San Diego, California, USA). Arterial pressure (AP) was measured via a catheter in the femoral artery. Analogue signals were digitized (40 Hz) with MacLab (ADInstruments, Australia) and displayed and stored on a G3 Apple Macintosh computer. Ear and mesenteric conductances were computed on-line by dividing corresponding flow signal by the arterial pressure signal. Pupillary size was recorded on a video tape using a SONY Hi8 Handicam camera and measured off-line. Mean AP and mean blood flow were calculated offline from selected 5 s recording periods using Chart software. Mean vascular conductance for these 5 s periods was obtained by dividing mean flow velocity by mean AP. Analysis of variance with repeated measures and Fisher’s protected t-test were used to determine the significance of changes induced by stimulation of the amygdala or the hypothalamus before and after injection of muscimol agents into the medulla. Values in the tables are means6S.E.M.

3. Results Electrical stimulation of the amygdala substantially reduced ear flow (Table 1, Fig. 1A), without consistently changing AP and with no change in mesenteric flow (Table 1). Ear flow commenced to fall 2–3 s after the onset of the stimulus and returned to the basal level during the subsequent 25–50 s. Administration of muscimol to the raphe / parapyramidal region totally abolished ear vasoconstriction in response to amygdala stimulation in every rabbit (Table 1 and Fig. 1B). Electrical stimulation of the hypothalamus increased AP and reduced blood flow in both the cutaneous and the mesenteric beds, so that vascular conductance was substantially reduced in both beds (Table 1 and Fig. 2A). Administration of muscimol to the raphe / parapyramidal region also totally abolished ear vasoconstriction elicited by hypothalamic stimulation, but did not significantly affect the mesenteric vasoconstriction elicited by this stimulation (Table 1, Fig. 2B). The rise in AP elicited by hypothalamic stimulation was slightly reduced by injection of muscimol to the raphe / parapyramidal region, but a substantial rise was still observed (Table 1). A summary of

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Table 1 Effects of raphe / parapyramidal muscimol injection on changes in cardiovascular parameters evoked by electrical stimulation of amygdala (A) and hypothalamus (B) Ear flow

Ear conductance

Mesenteric flow

Mesenteric conductance

AP

Heart rate

A. Response to amygdala stimulation Before Pre-stim 2564(9) muscimol Post-stim 761(9)**

0.3260.06(9) 0.1060.02(9)**

2066(8) 2066(8)

0.2560.08(8) 0.2860.08(8)

8664(9) 8365(9)

34166(9) 33466(9)

After muscimol

0.4260.13(8) 0.4160.12(8)‡‡

2467(7) 2467(7)

0.3360.09(7) 0.3460.09(7)

7566(8) 7666(8)

340610(8) 337610(8)*

B. Response to hypothalmic stimulation Before Pre-stim 2063(9) muscimol Post-stim 461(9)

0.2760.05(9) 0.0560.02(9)**

1963(8) 1263(8)*

0.2760.06(8) 0.1460.04(8)**

7966(9) 9968(9)**

34068(9) 33768(9)

After muscimol

0.4160.11(9) 0.3960.10(9)‡

1963(8) 1463(8)*

0.2960.06(8) 0.1660.03(8)**

7166(9) 8567(9)**‡

33668(9) 34469(9)**

Pre-stim Post-stim

Pre-stim Post-stim

2867(8) 2766(8)‡‡

2667(9) 3168(9)*‡‡

*P,0.05, **P,0.01, significantly different from pre-stimulation baseline level. ‡P,0.05, ‡‡P,0.01, significant response to stimulation, compared to control prior to muscimol injection. Pre-stim, pre-stimulation; post-stim, post-stimulation; AP, arterial pressure. The number of rabbits is indicated in parentheses.

the effects of muscimol on amygdala-evoked and hypothalamic-evoked changes in ear and mesenteric conductances is presented in Fig. 3. Following muscimol blockade of the raphe / parapyramidal area, we tested whether the hypothalamically-induced rise in AP and mesenteric vasoconstriction would be affected by subsequent additional bilateral injection of muscimol into the RVLM. After bilateral injection of

muscimol to the RVLM, AP fell to approximately 40 mm Hg (Fig. 4, Table 2). Bilateral microinjection of muscimol to RVLM substantially reduced, but did not completely abolish, the mesenteric vasoconstriction and the rise in AP normally elicited by hypothalamic stimulation. Hypothalamic stimulation also increased pupil diameter from 10.560.5 to 12.960.4 mm (P,0.01, n57). After

Fig. 1. Ear and mesenteric blood flows, mean vascular conductances and AP changes in response to electrical stimulation of the amygdala before (A) and after (B) injection of muscimol into the raphe / parapyramidal area.

Fig. 2. Ear and mesenteric blood flows, mean vascular conductances and AP changes in response to electrical stimulation of the hypothalamus before (A) and after (B) injection of muscimol into the raphe / parapyramidal area.

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combined raphe and RVLM muscimol injection, pupillary dilatation in response to this stimulation was unchanged (pupil diameter increased from 10.760.5 mm before stimulation to 13.260.3 mm after stimulation; n57, P, 0.01). Forebrain stimulation sites and medullary injection sites are shown in Figs. 5 and 6, respectively.

4. Discussion

Fig. 3. Summary of ear and mesenteric conductance changes in response to stimulation of the amygdala and the hypothalamus before (A) and after (B) injection of muscimol to raphe / parapyramidal region. Data represent percentage to which corresponding conductance fell from the pre-stimulus level (100%) after electrical stimulation. Numbers indicate number of experimental animals.

Fig. 4. Mesenteric flow, mean vascular conductance and AP changes in response to electrical stimulation of the hypothalamus before (A) and after (B) injection of muscimol into the RVLM. Data obtained after inhibition of raphe / parapyramidal area by muscimol.

Stimulation of the amygdaloid area clearly caused a robust fall in ear blood flow, without changing the mesenteric flow, and with little or no change in AP. Similar selective falls in cutaneous blood flow occur when conscious rabbits detect salient environmental events eliciting an alerting response [22,34] and these falls are entirely abolished by bilateral pharmacological inactivation of the amygdala region [36]. Thus, it may be that our present amygdala stimulation activated neuronal circuitry which normally links the amygdala with the appropriate spinal sympathetic vasomotor neurons mediating the cutaneous vasoconstriction associated with the alerting response. Inhibition of raphe / parapyramidal neurons entirely abolished the falls in ear blood flow produced by amygdala stimulation. It must be therefore, that the neural pathway from the amygdala to the spinal cord includes a synaptic relay in the raphe / parapyramidal region. The connection from the amygdala to the raphe / parapyramidal region could be direct. So far there are very few neuroanatomical reports investigating this possibility. A study in the rabbit noted sparse labelling in the central nucleus of amygdala following administration of retrograde tracer into the medullary raphe area, but few details were provided [10]. A more recent study confirmed the existence of such a projection in the rat [11]. We have also observed a direct projection from the central nucleus of the amygdala to the raphe / parapyramidal region in the rabbit and found it to be quite substantial (unpublished results). In contrast to amygdala stimulation, hypothalamic stimulation caused a significant rise in AP with vasoconstriction in the mesenteric bed, as previously demonstrated in rabbits [8,9,29]. Hypothalamic stimulation also caused

Table 2 Effects of RVLM muscimol injection after pharmacological block of raphe / parapyramidal area on changes in cardiovascular parameters evoked by electrical stimulation of hypothalamus Ear flow

Ear conductance

Mesenteric flow

Mesenteric conductance

AP

Heart rate

Before muscimol

Pre-stim Post-stim

2365(8) 3868(8)*

0.4760.14(8) 0.4760.12(8)

1863(6) 1563(6)*

0.3360.09(6) 0.1860.04(6)*

6367(8) 9968(8)**

327612(8) 348610(8)*

After muscimol

Pre-stim Post-stim

1564(7) 1965(7)

0.5660.17(7) 0.6060.21(7)

1462(6) 1462(6)‡

0.4760.11(6) 0.4260.09(6)‡

4068(8) 4369(8)**‡‡

280618(8) 308614(8)**

*P,0.05, **P,0.01, significantly different from pre-stimulation baseline level. ‡P,0.05, ‡‡P,0.01, significant response to stimulation, compared to control prior to muscimol injection. Pre-stim, pre-stimulation; post-stim, post-stimulation; AP, arterial pressure. The number of rabbits is indicated in parentheses.

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Fig. 5. Stimulation sites (filled circles) in amygdala (A) and hypothalamus (B) as defined by the Prussian Blue reaction (see examples in insets). Abbreviations: Ca, nucleus caudatus; CeA, central nucleus of the amygdala; DMH, dorsomedial nucleus of hypothalamus; ic, internal capsula; f, fornix; GP, globus pallidus; Hi, hippocampus; mtt, mamillothalamic tract; ot, optic tract; PVH, paraventricular nucleus of hypothalamus; RF, rhinal fissura; III, third ventricle.

vigorous cutaneous vasoconstriction, an observation not previously reported. Descending pathways responsible for changes in arterial pressure elicited by hypothalamic stimulation appear to include a relay in the RVLM [32], a finding consistent with electrophysiological and anatomical studies [6,28,31,37,38]. In agreement with this, we demonstrated that mesenteric vasoconstriction elicited by hypothalamic stimulation was unaffected by raphe / parapyramidal inactivation, but was substantially reduced by bilateral RVLM inactivation. Our hypothalamic findings provide strong evidence for the existence of separate, regionally specific vasoconstrictor pathways linking the hypothalamus with the relevant spinal sympathetic neurons. Cutaneous vasoconstriction elicited by hypothalamic stimulation was totally abolished by inhibition of neurons in the raphe / parapyramidal region. Thus, although hypothalamic neurons are known to

project directly to the spinal sympathetic centres, it appears that hypothalamically-induced cardiovascular changes involve a relay in the lower brainstem. In the case of mesenteric vasoconstriction the link is via the RVLM. In the case of cutaneous vasoconstriction, the link is via raphe / parapyramidal neurons, just as occurs in the case of the amygdala-induced cutaneous vasoconstriction (Fig. 7). In the case of cutaneous vasoconstriction, the direct hypothalamic–spinal pathway is not of great functional importance in our experimental preparation. Combined raphe / parapyramidal and RVLM neuronal blockade, a procedure which eliminates a substantial proportion of lower brainstem presympathetic neurons, did not affect the pupillary dilatation elicited by hypothalamic stimulation. Similar findings for the RVLM have previously been reported for the cat [20].The functional hypothalamo–spinal pathway for pupillary dilatation must

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Fig. 6. Muscimol injection sites in the raphe / parapyramidal area (A) and RVLM (B) in transverse sections of the medulla oblongata. In (A), the dark area indicated by arrowhead contains HRP reaction product. In (B) the corresponding area contains b-galactosidase reaction product. Abbreviations: icp, inferior cerebellar peduncle; IO, inferior olive; mVe, medial vestibular nucleus; nA, nucleus ambiguous; p, pyramidal tract; RVLM, rostral ventrolateral medulla; ts, tractus solitarius; Vsp, spinal nucleus of the trigeminal nerve; Vspt, spinal tract of the trigeminal nerve.

therefore be direct or via a relay in another brainstem presympathetic group. An impressive body of evidence indicates that neural circuitry, in the different subnuclei of the amygdala, integrate information indicating a potential threat to the well being of the individual [15,17,21]. Outputs from the amygdala produce integrated protective behavioural and physiological responses [16]. Vigorous constriction of cutaneous vessels is a robust and consistent cardiovascular component of these responses [34]. In rabbits, inactivation of the amygdala prevents this cutaneous response [36]. In rats, inactivation of neuronal function in either the amygdala or the lateral hypothalamus prevents increases in

AP associated with the conditioned fear response [18,25]. At present we do not know whether the pathway for cutaneous vasoconstriction evoked from the amygdala also involves an amygdalo–hypothalamic link [24,33]. The periaqueductal gray is another brain region which could link the amygdala with the raphe / parapyramidal neurons [2,5,19]. A direct pathway from the amygdala to the raphe / parapyramidal region also seems likely. Stimulation of the amygdala vigorously excites raphe–spinal neurons activated by noxious stimuli in anesthetized rabbits, with a latency of approximately 30 ms [23]. In conclusion, we have demonstrated that in anesthetized rabbits, cutaneous vasoconstriction evoked from both

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Fig. 7. Proposed neural connections linking the amygdala and hypothalamus with cutaneous (grey lines) and mesenteric (black lines) sympathetic vasomotor neurons. IML, intermediolateral column; RVLM, rostral ventrolateral medulla.

the amygdala and the hypothalamus is totally abolished by pharmacological blockade of raphe / parapyramidal neurons in the lower brainstem. In contrast, mesenteric vasoconstriction evoked from the hypothalamus is unaffected by raphe / parapyramidal blockade, but is substantially reduced by blockade of the RVLM, a region vital for the general regulation of arterial blood pressure. A diagram summarizing these findings is given in Fig. 7. Our findings add to the growing body of evidence that cutaneous vascular tone is controlled by raphe / parapyramidal neurons and that cutaneous vascular tone in many physiological situations is regulated independently of the general arterial pressure [3,4,22,26].

Acknowledgements Our research was supported by the National Health and Medical Research Council, the National Heart Foundation of Australia and the Neurosurgical Research Foundation of South Australia. We thank Robyn Hook, Kate Barber and Joseph Garcia for technical assistance.

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