Afferent projections to the hypothalamic area controlling emotional responses (HACER)

Brain Research, 252 (1982) 213-226 Elsevier Biomedical Press

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Afferent Projections to the Hypothalamic Area Controlling Emotional Responses (HACER) J. L. DEVITO and ORVILLE A. SMITH Regional Primate Research Center and Departments of Physiology and Biophysics and Neurological Surgery, University o f Washington, Seattle, WA 98195 (U.S.A.) (Accepted May 18th, 1982) Key words: horseradish peroxidase - - hypothalamic afferents - - cardiovascular responses - - emotion - - baboon

The Mesulam technique for horseradish peroxidase was used to study the subcortical afferent projections to a location in the hypothalamus that has been shown to control the complete cardiovascular (CV) response accompanying a specific emotional behavior. Major projections common to all baboons injected included the lateral septal nucleus; medial, cortical and basal amygdala, the anteroventral third ventricle area; the preoptic areas; the subiculum; the paraventricular nucleus of the thalamus; periventricular gray and the central gray of the midbrain; the midbrain tegmentum; locus ceruleus, parabrachial and raphe cells in the pons, and in the medulla, raphe nuclei, the nucleus of the solitary tract, in and around the dorsal motor nucleus of the vagus, and in the region of the nucleus ambiguus. Other projections in some but not all baboons included the subfornical organ and the midline and dorsomedial nuclei of the thalamus. The nucleus of the diagonal band of Broca was labeled to some degree with all injections but was most heavily labeled with the injection extending more laterally in the hypothalamus. These results fit well with physiological and behavioral studies dealing with neural control of emotional and CV responses and support the concept of an integrative area in the hypothalamus concerned specifically with the control of CV response accompanying emotion. INTRODUCTION Efforts to u n d e r s t a n d the neural basis for e m o tional b e h a v i o r a n d efforts to u n d e r s t a n d the o r g a n i z a t i o n o f the central nervous system ( C N S ) in its c o n t r o l o f the c a r d i o v a s c u l a r (CV) system have frequently o v e r l a p p e d . This c o n g r u i t y o f interest has resulted f r o m b o t h the introspective a n d the objectively m e a s u r e d r e l a t i o n between e m o t i o n a l behavior a n d the occurrence o f large CV changes. T h e a n a t o m i c a l focus for these lines o f investigation has b e e n the h y p o t h a l a m u s , beginning with m e a s u r e ments o f h e a r t rate a n d b l o o d pressure changes in response to electrical s t i m u l a t i o n o f the h y p o t h a l a mus 33. This was followed some years later by B a r d ' s a classical w o r k o n s h a m rage in the ' t h a l a m i c p r e p a r a t i o n , ' which p i n p o i n t e d the h y p o t h a l a m u s as critical in the expression o f e m o t i o n a l behavior. The N o b e l prize-winning w o r k o f Hess 27 defined a beh a v i o r a l 'defense' r e a c t i o n with a c c o m p a n y i n g a u t o 0006-8993/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press

n o m i c responses as a result o f stimulating the h y p o t h a l a m u s o f unanesthetized cats. I n the 1950s the discovery o f the cholinergically m e d i a t e d v a s o d i l a tion o f skeletal muscle c o n t r o l l e d b y the h y p o t h a l a mus 21 led to the w o r k o f A b r a h a m s et al. 1, w h o related 'defense' b e h a v i o r to this specific CV response. I n 1964, S m i t h a n d N a t h a n 59 bilaterally a b l a t e d a restricted p o r t i o n o f the h y p o t h a l a m u s in M a c a c a nemestrina a n d t h e r e b y e l i m i n a t e d the b l o o d flow a n d the h e a r t rate responses p r o d u c e d b y classical c o n d i t i o n i n g to a n o x i o u s stimulus (i.e., the CV responses associated with e m o t i o n a l behavior). This has been e l a b o r a t e d m o r e recently in b a b o o n prep a r a t i o n s in which b l o o d pressure, h e a r t rate, renal b l o o d flow, t e r m i n a l aortic b l o o d flow a n d 02 c o n s u m p t i o n were m e a s u r e d d u r i n g a series o f controlled behaviorsSO, 57. T h e CV responses to an acute e m o t i o n a l s i t u a t i o n in unanesthetized, chair restrained b a b o o n s included elevations in h e a r t rate, b l o o d

214 pressure and terminal aortic flow, and a complex biphasic reduction in renal flow. The same CV responses were produced by stimulating an area in the hypothalamus of chloralose-anesthetized baboons. In addition, bilateral ablation of the hypothalamic area eliminated the CV responses to the emotional behavior, while responses to exercise, free-feed and sleep remained the same. The somatic response to the emotional situation (suppression of bar-pressing) was not affected, which indicates that memory impairment was not responsible for the loss of the CV response. This identification of a precise neuroanatomical location responsible for producing the CV responses specifically associated with emotional behavior provided an entree for determining what other parts of the CNS are involved in this complex response. This location has been designated with the acronym H A C E R (hypothalamic area controlling emotional responses). Retrograde transport techniques such as cell labeling with horseradish peroxidase (HRP) make it possible to identify the structures that provide afferents to this hypothalamic region. Autoradiographic techniques allow the efferents from this area to be traced, providing information about how the CNS is organized to produce integrated CV re.sponses associated with emotional behavior. The present research addresses the problem of the subcortical afferents to this hypothalamic area. A description of the efferents from this area will be reported separately. Although HRP is taken up by severed axons as well as by fiber terminals, this is not a drawback in the present situation because the CV changes following hypothalamic lesions may result from either cell loss or damage to passing fibers. Obviously, the retrogradely labeled regions are only potential sites for CV regulation. First they will be tested in acute electrophysiological experiments to determine the degree of interaction with the hypothalamic stimulation. Sites that show positive effects in the anesthetized animal will be tested further by neural ablation in chronic preparations. MATERIALS AND METHODS Subjects were 4 juvenile male baboons (Papio cynocephalus) (8-12 kg). Three were used for injections

into the hypothalamus, and one for injection into the nucleus ventralis anterior of the thalamus, thus serving as a control. Two weeks before injection, an electromagnetic flow probe was aseptically implanted on the renal artery for measurement of blood flow to the kidney. On the day of the injection the animal was anesthetized with alpha chloralose, and an arterial cannula was placed in the femoral artery for measurement of arterial blood pressure and heart rate. The animal was maintained on chloralose anesthesia and placed in a restraining chair. The baboon's head was secured in a stereotaxic apparatus fitted with raised eye and ear bars to allow for ventriculography. A cannula was lowered into the lateral ventricle, and frontal and lateral Xray views were taken during the injection of metrizamide 56. The desired hypothalamic locus was calculated from the outlines of the third ventricle, which revealed the major anatomical structures such as anterior commissure, optic chiasm, mammillary bodies and posterior commissure. The injection cannula was used as a stereotaxic reference. Verification of the desired neural site was achieved by lowering a stimulating electrode through a guide cannula and recording the CV responses to electrical stimulation (100 Hz, 0.2 ms, 0.5-1.0 mA). When a response was obtained that mimicked the CV response to the emotional situation in chronic animals, the electrode was withdrawn and replaced by a needle attached to a microliter syringe. The syringe needle protruded 4 mm below the tip of the guide cannula. A volume of 0.15~0.2 #1 of a mixture of 3H-isotopes and 33 ~ H R P (Worthington) in 0.1 M phosphate buffer was injected over 10-15 min. The syringe needle was removed 5 min after completion of the injection and the cannula was left in place for an additional 20 min. The animals survived for 2 or 3 days, then were perfused with a pre-rinse of 0.1 M phosphate buffer (pH 7.4) followed by mixture of 1 ~ paraformaldehyde and 1.25 ~ glutaraldehyde in phosphate buffer. The brains were blocked in the stereotaxic apparatus, removed from the skull and placed in cold phosphate buffer and 10 ~ sucrose for about 7 h. They were transferred to cold phosphate buffer and 30 ~o sucrose overnight. Frozen sections were cut at 52 # m for H R P histochemistry and 26 # m for autoradiography. The course of the efferent

215 pathways revealed by autoradiography will be reported separately. For HRP histochemistry, sections were processed according to the tetramethyl benzidene procedure of Mesulam 41. RESULTS The effective uptake area around the injection site was difficult to define precisely because of a widespread, faint-blue reaction product. When a perimeter was drawn around the zone of dark-blue reaction product, two of the hypothalamic injections seemed to involve the same structures, whereas a third one was centered more laterally and extended somewhat more caudally. On the assumption that the effective uptake site was confined to the dark blue deposit, we cont,luded that the injections in animals 78160 and 77310 involved the perifornical area, dorsomedial hypothalamic nucleus, ventromedial hypothalamic nucleus and lateral hypothalamic area. The volume of 33 ~o HRP injected in animal 78160 was 0.15/~1 and in animal 77310, 0.2 #1. The sites at which retrogradely labeled cells were recorded were essentially the same in these two animals, with the number of labeled cells being generally greater in animal 77310 than in animal 78160. The more lateral injection of 0.15 /zl in animal 78081 involved the perifornical area, ventromedial hypothalamic nucleus, and the lateral hypothalamic area. There were some differences in the sites of retrograde labeling following the different hypothalamic injections, which would tend to uphold the validity of the borders placed around the dark blue stain and suggest that the areas stained a faint blue did not contribute to the retrograde label. The results from the smaller of the medial injections (78160) and from the more lateral injection (78081) will be described in detail. The nuclear classifications conform to those used for M. fuscata by Kusama and Mabuchi aT. Baboon 78160. The subcortical sites of retrogradely labeled cells following the hypothalamic injection of HRP are shown in Figs. 1 and 2. Rostral to the hypothalamus, labeled cells were numerous in the lateral septal nucleus and scant in the nucleus of the diagonal band of Broca (NDBB). There were a few labeled cells in the bed nucleus of the anterior commissure and cells were scattered in the medial and lateral preoptic areas. Labeled cells were numerous

in the median preoptic nucleus, the periventricular region of the anteroventral third ventricle (AV3V), and the supraoptic nucleus (see Fig. 5B). In the amygdala, HRP-positive cells occurred in the medial, cortical and basal nuclei. At the level of the injection, there were additional labeled cells in the subfornical organ (See Fig. 5C), internal segment of the globus pallidus, and substantia innominata. In the hypothalamus itself, some labeling was obscured by the blue deposit around the injection site. Labeled cells could be discerned in the paraventricular nucleus of the hypothalamus, infundibular nucleus, tuberomamillary nucleus, and in the periventricular gray. There were scattered cells in the lateral and posterior areas. The paraventricular nucleus of the thalamus was labeled throughout its course. Heavy labeling in the subiculurn was present at all rostrocaudal levels. Caudal to the hypothalamus, retrogradely labeled neurons were plotted in the ventral tegmental area, the periventricular gray, and the dorsal tegmentum. At some levels, a continuous band of cells extended across the dorsal tegmentum to join with the medial periventricular gray labeling. Further caudally, cells were labeled in the interpeduncular nucleus and its paramedian region, in the central tegmentum, and the peripeduncular nucleus. Occasional cells were labeled in the substantia nigra. At collicular levels, cells were labeled in and around the superior cerebellar peduncle, in the periaqueductal gray and in the dorsal and superior central raphe nuclei. At pontine levels, cells were labeled in the medial parabrachial nucleus and in and around the lateral parabrachial nucleus (see Fig. 5A). The locus ceruleus was extensively labeled. In the rostral medulla, a few cells were labeled in the nucleus of the raphe. Labeled cells were also scattered in the reticular formation and, in the caudal medulla, formed a bridge between the region of the nucleus ambiguus and HRP-positive cells in and around the dorsal motor nucleus of the vagus. There were also labeled cells in the nucleus of the solitary tract. All retrograde labeling in the medulla was bilateral, as was that in the locus ceruleus, lateral parabrachial nucleus, and periventricular gray. There was also bilateral labeling in hypothalamic and preoptic areas.

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Fig. 1. Rostral subcortical regions of baboon 78160. HRP injection site in HACER shown by shaded areas. Sites of retrograde transport are shown as filled circles on tracings of coronal sections.

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Fig. 2. Sites of retrograde transport (filled circles) in brain stem of baboon 78160.

Baboon 78081. Figures 3 and 4 show the sites of retrograde labeling following the slightly more lateral hypothalamic injection in baboon 78081. Rostral to the hypothalamus, cell labeling extended more ventrally in the NDBB and the cells were more numerous than in animal 78160, while the lateral septal labeling was lighter. There were also a few cells in nucleus accumbens at this level. In the preoptic region, the supraoptic nucleus was free of label and only a few cells were labeled in the caudal hypothalamic part of this nucleus. The subfornical organ was free of label, but the median preoptic nucleus and the AV3V region (Fig. 5E) were extensively

labeled. The subiculum, amygdala, preoptic area, substantia innominata and globus pallidus showed similar labeling in baboons 78081 and 78160. The tuberomamillary nucleus contained fewer cells in animal 78081. A striking difference between the two animals was the presence of retrograde labeling in the midline thalamic nuclei (nucleus reuniens and central medial nucleus) and in the medial and dorsal parts of the dorsomedial nucleus (Fig. 5D) in baboon 78081. A few cells in the lateral habenular nucleus and pretectum were also labeled in baboon 78081.

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1 Fig. 3. Rostral subcortical regions of baboon 78081 showing injection site (shaded areas) and sites of retrograde transport (filled circles).

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Fig. 4. Sites of retrograde transport (filled circles) in brain stem of baboon 78081.

In the midbrain, the interpeduncular nucleus showed less labeling than in baboon 78160. The dorsal raphe nucleus was labeled in its caudal region but not rostrally at the level of the fourth nerve nucleus as it was in animal 78160. The medial parabrachial nucleus contained considerably more labeled cells in this animal. In the caudal pons and rostral medulla, the reticular formation and the nucleus of the raphe were more heavily labeled following this more lateral injection. In the caudal medulla, however, cell labeling was lighter in the reticular formation, the region of the dorsal motor nucleus of the vagus, and the nucleus of the solitary tract.

Regions showing bilateral labeling were the same in baboons 78160 and 78081. Control injection. Following the control injection into the nucleus ventralis anterior of the thalamus, labeled cells were found in the globus pallidus, lateral preoptic area, bed nucleus of the stria terminalis, central medial nucleus, thalamic reticular nucleus, substantia nigra, dorsomedial thalamic nucleus, pedunculopontine nucleus, lateral habenular nucleus, dorsal raphe and medial parabrachial nucleus. No HRP-positive cells were noted caudal to the pons. Although some of the sites were the same as those labeled by the hypothalamic injections, the

220

Fig. 5. HRP-positive neurons in the lateral parabrachial nucleus (A), supraoptic nucleus (B), and subfornical organ (C) of baboon 78160. In baboon 78081, HRP-positive neurons are shown in the dorsal part of the dorsomedial nucleus (D) and the AV3V region (E). Calibration bars, 50 pro.

lack of label in any circumventricular organ makes it unlikely that uptake of H R P from the ventricles occurred in the experimental animals. DISCUSSION At the present time, the site subserving the conditioned emotional response (CER) cannot be described in terms of a specific hypothalamic nucleus. The lesions that are effective in eliminating the CV component of the C E R always involve the perifornical area. In addition, varying amounts of the dorso- and ventromedial hypothalamic nuclei and the lateral hypothalamic area have been involved. A lesion would also be expected to interrupt fibers

passing to other hypothalamic regions as well as fibers traversing the hypothalamus. The H R P injection sites appear to involve the same regions that were destroyed by electrolytic lesions in chronic behavior experiments. Furthermore, H R P is taken up and transported by cut fibers as well as by fiber terminals. This labeling method should, therefore, reveal all sites that might influence the CER. Whether or not a retrogradely labeled region is involved in CV control can be determined only by physiological or behavioral methods. One of the three H R P injections involved more of the lateral hypothalamic area and less of the dorsoand ventromedial hypothalamic nuclei than did the other two injections. The results of these injections

221 showed differences in numbers of labeled cells in certain regions as well as some differences in retrogradely labeled sites. Testing the CV effects of the differentially labeled sites should aid in delimiting the hypothalamic structures involved in the CER, i.e., whether the system is mainly periventricular or whether connections are mainly via the medial forebrain bundle. It is apparent, however, that certain structures were equally well labeled following all three injections. In the following discussion the more medial hypothalamic injection in animal 78160 will be called the medial injection and the more laterally placed hypothalamic injection in animal 78081 will be called the lateral injection.

Septum and amygdala Differential labeling from the two injection sites was evident in the septai region. The NDBB was well labeled and the lateral septal area lightly labeled by the lateral injection, whereas the reverse was true for the medial injection. Retrograde labeling of septal neurons from the hypothalamus has been reported in rats with some diversity in the results. The lateral septal nucleus has been reported to project to the medially placed paraventricular nucleus s2 and NDBB efferents have been found to run in the medial forebrain bundle through the lateral hypothalamic area 16. Retrograde labeling of only the lateral septal nucleus has been reported following an injection into the lateral hypothalamic area 40, while other investigators labeled the medial and lateral septal nuclei and the NDBB from the same area 29. Retrograde labeling of the NDBB from the posterior hypothalamus alone has also been reported 5. In the amygdala, the same nuclear subdivisions (medial, cortical and basal) were labeled in all animals. These same three subnuclei have been found to project to the hypothalamus in cat a9 and ratS,52. Of the afferents to the HACER, the amygdala and the lateral septal area are most directly implicated in the control of emotional behavior. Since the classic work of Klfiver and Bucya6, the amygdala has been studied repeatedly and the general conclusion is that the removal of critical portions of the structure results in an abnormal placidity20,23,51, while stimu-

lation results in a defense reaction is. In contrast, lesions of the septal area produce explosive, hyperreactive, aggressive behavior 10. Studies of the interactive effects of lesions in these structures suggest that the two areas act reciprocally: the septal area may 'dampen' the hypothalamic activity associated with emotional states while the amygdala may facilitate it 84. Whether stimulation of these structures elicits CV effects is less clear. Amygdalar stimulation produces muscle vasodilation that has been associated with the 'defense' reaction 2s, and it has also produced hypotension in ratsZL The CV effects from septal stimulation depend on stimulation frequency, with tachycardia following high-frequency stimulation and bradycardia resulting from low-frequency stimulation11. Cardiac slowing during septal self-stimulation in rats has also been reported as. The NDBB has received relatively little attention. Lesions of NDBB induce mouse-killing by rats that otherwise do not kill mice s, and stimulation of NDBB inhibits hypothalamically elicited attack by cats on rats a0. The structure thus has been implicated in modulation of emotional behavior, but its exact role is unclear. No CV effects have been reported from manipulation of NDBB.

Preoptic area The medial and lateral preoptic areas contained scattered HRP-positive cells in all animals in this study. These areas have long been implicated in CV control: particularly dramatic is the fatal pulmonary edema that results from bilateral preoptic ablation 24. The preoptic area also has been implicated as having a role in emotional behavior3°. The median preoptic nucleus and the AV3V region were well labeled in all animals. It is not certain that the organum vasculosum lamina terminalis (OVLT) itself was visualized in the frontal plane sections. The median preoptic nucleus and the OVLT are anatomically connected to the supraoptic nucleus and subfornical organ according to Miselis et al. 42, and have been found to play a role in water balance. Both the subfornical organ and supraoptic nucleus were labeled following the medial injection. The lateral injection, however, labeled only a few cells in the caudal supraoptic nucleus and none in the subfornical organ. A supraoptic projection to the hypothalamus has been reported sS, and injec-

222 tions of H R P into the paraventricular nucleus of the hypothalamus produce labeling in the subfornical organ 52. The projections from the AV3V region and the subfornical organ are most interesting on the basis of their role as sites of angiotensin (AII) receptors31,sL Plasma renin activity is significantly increased during stress situations 6, and it would seem reasonable that some kind of feedback from the renin-angiotensin system to H A C E R should be occurring during emotional situations.

Thalamus In the thalamus, the paraventricular nucleus was labeled in all animals, while the midline thalamic nuclei and the medial and dorsal parts of the dorsomedial nucleus were labeled only by the lateral injection. Smith and Flynn54, 5~ noted paraventricular labeling and inconsistent labeling of midline nuclei in cats, but label in the dorsomedial nucleus following hypothalamic injection has not been reported before. In our experiments, a few cells were labeled in the lateral habenular nucleus after the lateral injection, a finding also reported in the ratS, 29. The dorsomedial nucleus has long been implicated as a potentially important structure in the elaboration of emotional behavior because of its strong connections with the prefrontal lobe. Nathan and Smith 43 reported that lesions of this structure inhibit the suppression of lever pressing during a CER. Tegmentum Hypothalamic afferents arising in the periventricular gray and the periaqueductal gray have also been reported for cat and ratS,13,26,54,55, as have ventral tegmental area projections to the lateral hypothalamus in the cat 54. We observed a band of cells stretching laterally from the periventricular gray across the dorsal tegmentum in all animals. This source of afferent fibers to the hypothalamus has not been reported previously. The peripeduncular projection has been described before in monkey 32. Scattered cells in the central tegmentum form a cluster at the lateral margin of the superior cerebellar peduncle, similar to the region that McBride and Sutton a9 found to project to the ventromedial hypothalamic nucleus. Hypothalamic projec-

tions from the interpeduncular nucleus and the dorsal and superior central raphe nuclei have also been reported in rat and catV,t5,49,54. It has long been known that the hypothalamus and the central gray of the midbrain are intimately related. It was generally believed that the central gray was largely an efferent station from the hypothalamus, so the rather large afferent component is somewhat surprising. Stimulation of the central gray produces very large CV changes, and the area is believed to play an important role in pain perception. Stimulation of the midbrain dorsal raphe has been demonstrated to reduce fear produced by excitation of the central gray aS.

Pons In the pons, labeled cells were observed in the lateral and medial parabrachial nuclei, with the medial parabrachial labeling being heavier after the lateral injection. Sakumoto et al. 49 also noted differential labeling with the medial parabrachial nucleus projecting to the lateral hypothalamus in rat. The locus ceruleus was extensively labeled and this projection has been confirmed by several investigatorsg,Zg,~4,55. As reported for the rat ~, the nucleus of the raphe and adjoining reticular formation were labeled in the caudal pons continuous with that in the rostral medulla. The locus ceruleus is known to provide noradrenergic fibers that traverse or possibly terminate in the region of HACER, so H R P labeling in this nucleus was expected. Whether this nucleus functions to influence CV or emotional responses is controversial at present. Behavioral responses have been reported 47, and both pressor 60 and cardiac 62 effects have been associated with the structure. However, a recent report 17 questions the essential role of locus ceruleus neurons in generating such responses. Of significance for the afferent connections demonstrated in the current report are data showing a reduction in the pressor effect in response to stimulation of the locus ceruleus after lesions of the hypothalamus 46. A salient feature of both electrical stimulation of the H A C E R in the anesthetized animal and of the CER in the awake animal is the profound effect on renal blood flow. The effect is an immediate, large decrease in renal flow, which is neurally mediated.

223 This is followed by a recovery to almost normal flow levels, which then reverses into a long-duration decrease produced by the renal constrictor effects of circulating epinephrine 5a. In this regard, the HRP label in the parabrachial region of the pens is particularly interesting because of evidence that pathways mediating renal vasoconstriction in response to hypothalamic stimulation pass through the parabrachial region 3. Medulla In the medulla, labeled cells occurred in the nucleus of the solitary tract, in and surrounding the dorsal motor nucleus of the vagus, and stretching laterally and ventrally in the reticular formation to the region of the nucleus ambiguus. The labeled regions were highly bilateral and cells were more numerous following the medial injection. The projection from the rostral medullary nucleus of the raphe and adjoining reticular formation was heavier following the lateral injection. Similar projections have been described in the raO4, 4s and some of the ceils were identified as belonging to the A1 and A2 noradrenergic cell groups49, 50. The bridging arrangement of cells between the vagal and ambiguus nuclei that we observe6 in the baboon was not apparent in the rat studies, although a similar arrangement has been described for catecholamine cells in M. speciosa 25. The labeling of cells in the medulla oblongata is intriguing in that the preponderance of label is found in the nuclei associated with neural control of circulation. The nucleus of the solitary tract is the major CNS relay for the baroreceptor reflex, and it is reasonable to expect information to flow from this blood pressure control system to the HACER, whose activity will have an important effect on blood pressure. More surprising are the labeled cells found in close association with the nucleus ambiguus and dorsal motor nucleus of the vagus. These two nuclei are the source of the efferent fibers to the heart that are concerned with control of heart rate. It may be that feedback information to the HACER is provided from those motor areas directly con-

ABBREVIATIONS Ab AB

nucleusambiguus basal amygdaloidnucleus

trolling cardiac function. Stimulation of the medullary raphe has been shown to cause both an increase and a decrease in blood pressure in cats 2. Other areas It is not surprising, in view of the relatively large injections, that most of the hypothalamic nuclei showed some uptake of HRP. The presence of HRPpositive cells in the internal segment of the globus pallidus can be explained by the uptake of HRP by fibers looping through the hypothalamus before passing into the fields of Forel. Several studies describing these fibers have failed to show terminals within the hypothalamus 12,19,4a. The subiculum was heavily labeled throughout its extent in all animals. Swanson and Cowan 61 have shown that the postcommissural fornix arises entirely from the subicular region of the hippocampal formation. Because the fornix was invariable damaged by the injection needle, at least some of the label in the subieulum can be attributed to uptake of HRP by cut fibers. The hippocampal formation is also known to contribute fibers to the perifornical area 45. There has been speculation about the role of the hippocampal formation in emotional behavior, but there is no firm evidence for such a function, nor is there evidence about a CV role for the structure. It is evident from these observations that the major neural areas showing HRP label after injection of the HACER are also areas that have been previously implicated in either emotional behavior or directly in CV control. Our data reinforce the idea that the HACER serves an integrative function in the relation between emotional behavior and its associated CV effects.

ACKNOWLEDGEMENTS This study was supported by Grants RR00166 and HL 16901 from the National Institutes of Health, U.S. Public Health Service. We thank Kate Schmitt for editorial assistance, Cliff Astley for surgical assistance and Kathleen Walsh for histological preparations. Ace nucleus accumbens ACe cortical amygdaloidnucleus AM medial amygdaloid nucleus AV3V anteroventral third ventricle

224 Cd CeM CM CT Dm DM DR DSCP DT DV F GP Hb Hp In Ip LC LM LS MLF MM NCA NCS NDBB NRa NTS n III N IV n VII

caudate nucleus central medial nucleus centromedian nucleus central tegmentum dorsomedial nucleus of hypothalamus dorsomedial nucleus of thalamus dorsal raphe nucleus decussation of superior cerebellar peduncle dorsal tegmentum dorsal motor nucleus of vagus fornix globus pallidus habenula posterior hypothalamic area infundibular nucleus interpeduncular nucleus locus ceruleus medial lemniscus lateral septal nucleus medial longitudinal fasciculus medial mamillary nucleus bed nucleus of anterior commissure superior central nucleus nucleus of diagonal band of Broca nucleus of the raphe nucleus of the solitary tract third nerve fourth nerve nucleus facial nerve

N VII Pa PAG PbL PbM PL Pm PM Pp Put Pv PVG Re RF RL RN SCP SFO SNc SNr SI So Sub TM TS TSV VA Ves Vm VTA

REFERENCES 1 Abrahams, V. C., Hilton, S. M. and Zbrozyna, A. W., Active muscle vasodilation by stimulation of the brain stem: its significance in the defense reaction, J. Physiol. (Lond.), 154 (1960) 491-513. 2 Adair, J. R., Hamilton, B. L., Scappatici, K. A., Helke, C. J. and Gillis, R. A., Cardiovascular responses to electrical stimulation of the medullary raphe area of the cat, Brain Research, 128 (1977) 141-145. 3 Adair, J. R., Ward, D. G., Schramm, L. P. and Gann, D. S., Parabrachial ports mediates hypothalamically induced renal vasoconstriction, Amer. J. Physiol., 234 (1978) R223-R228. 4 Bard, P., A diencephalic mechanism for the expression of rage with special reference to the sympathetic nervous system, Amer. J. PhysioL, 84 (1928) 490-515. 5 Berk, M. L. and Finkelstein, J. A., Afferent projections to the preoptic area and hypothalamic region in the rat brain, Neuroscience, 6 (1981) 1601-1624. 6 Blair, M. L., Feigl, E. O. and Smith, O. A., Elevation of plasma renin activity during avoidance performance in baboons, Arner. J. PhysioL, 231 (1976) 772-776. 7 Bobillier, P., Seguin, S. Petitjean, F., Salvert, D., Touret, M. and Jouvet M., The raphe nuclei of the cat brain stem: a topographical atlas of their efferent projections as revealed by autoradiography, Brain Research, 113 (1976) 449--486. 8 Bouder, R. and Vergnes, M., Interspecies aggression in the rat: the role of the diagonal band of Broca, Brain Research, 175 (1979) 327-333. 9 Bowden, D. M., German, D. C. and Poynter, W. D.,

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facial nerve nucleus paraventricular nucleus of thalamus periaqueductal gray lateral parabrachial nucleus medial parabrachial nucleus lateral preoptic area median preoptic nucleus medial preoptic area peripeduncular nucleus putamen paraventricular nucleus of hypothalamus periventricular gray nucleus reuniens reticular formation lateral reticular nucleus red nucleus superior cerebellar peduncle subfornical organ substantia nigra, pars compacta substantia nigra, pars reticulata substantia innominata supraoptic nucleus subiculum tuberomamillary nucleus solitary tract spinal tract of trigeminal nerve anteroventral nucleus vestibular nucleus ventromedial nucleus of hypothalamus ventral tegmental area

An autoradiographic, semistereotaxic mapping of major projections from locus coeruleus and adjacent nuclei in Macaca mulatta, Brain Research, 145 (1978) 257-276. Brady, J. V. and Nauta, W. J. H., Subcortical mechanisms in emotional behavior: affective changes following septal forebrain lesions in the albino rat, J. comp. physiol. Psychol., 46 (1953) 339-346. Calaresu, F. R. and Mogenson, G. J., Cardiovascular responses to electrical stimulation of the septum in the rat, Amer. J. Physiol., 223 (1973) 777-782. Carpenter, M. B., Anatomical organization of the corpus striatum and related nuclei. In A. Yahr (Ed.), The Basal Ganglia, Raven Press, New York, 1976, pp. 1-36. Chi, C. C., An experimental silver study of the ascending projections of the central gray substance and adjacent tegmentum in the rat with observations in the cat, J. comp. NeuroL, 139 (1970) 259-272. Ciriello, J. and Calaresu, F. R., Autoradiographic study of ascending projections from cardiovascular sites in the nucleus tractus solitarii in the cat, Brain Research, 186 (1980) 448~,53. Conrad, L. C. A., Leonard, C. M. and Pfaff, D. W., Connections of the median and dorsal raphe nuclei in the rat: an autoradiographic and degeneration study, J. comp. Neurol., 156 (1974) 179-206. Conrad, L. C. A. and Pfaff, D. W., Efferents from medial basal forebrain and hypothalamus in the rat. I. An autoradiographic study of the medial preoptic area, J. comp. Neurol., 169 (1976) 185-220. Crawley, J. N., Maas, J. W. and Roth, R. H., Evidence

225 against specificity of electrical stimulation of the nucleus locus coeruleus in activating the sympathetic nervous system in the rat, Brain Research, 183 (1980) 301-311. 18 De Molina, A. F. and Hunsperger, R. W., Central representation of affective reactions in forebrain and brain stem: electrical stimulation of amygdala, stria terminalis, and adjacent structures, J. Physiol. (Lond.), 145 (1959) 251-265. 19 DeVito, J. L. and Anderson, M. E., An autoradiographic study of efferent connections of the globus pallidus in Macaca mulatta, Exp. Brain Res., in press. 20 Dicks, D., Uncus and amygdala lesions: effects on social behavior in the free-ranging rhesus monkey, Science, 165 (1969) 69-71. 21 Eliason, S., Lindgren, P. and fdvnas, B., Representation in the hypotbalamus and the motor cortex in the dog of the sympathetic vasodilator outflow to the skeletal muscles, Acta physiol, scand., 27 (1952) 18-27. 22 Faiers, A. A., Calaresu, F. R. and Mogensen, G. J., Pathway mediating hypotension elicited by stimulation of the amygdala in the rat, Amer. J. PhysioL, 228 (1975) 1358-1366. 23 Fonberg, E., Brutkowski, S. and Mempel. E., Defensive conditioned reflexes and neurotic motor reactions following amygdalectomy in dogs, ,4cta bioL exp., 22 (1962) 51-57. 24 Gamble, J. E. and Patton, H. D., Pulmonary edema and hemorrhage from preoptic lesions in rats, Amer. J. PhysioL, 172 (1953) 623-631. 25 Garver, D. L. and Sladek, J. R., Jr., Monoamine distribution in primate brain. I. Catecholamine-containing perikarya in the brain stem of Macaca speciosa, J. comp. NeuroL, 159 (1975) 289-304. 26 Hamilton, B. L. and Skultety, F., Efferent connections of the periaqueductal gray matter in the cat, J. comp. NeuroL, 139 (1970) 105-114. 27 Hess, W. R. and Brfigger, M., Das subkortikale Zentrum der affektiven Abwehrreaction, Helv. physiol. Acta, 1 (1943) 37-52. 28 Hilton, S. M. and Zbrozyna, A. W., Amygdaloid region for defense reactions and its efferent pathway to the brain stem, J. Physiol. (Lond.), 165 (1963) 160173. 29 Hosoya, Y. and Matsushita, M., Cells of origin of the descending afferents to the lateral hypothalamic area in the rat, studied with the horseradish peroxidase method, Neurosci. Lett., 18 (1980) 231-236. 30 Inselman, B. and Flynn, J. P., Modulatory effects of preoptic stimulation on hypothalamically-elicited attack in cats, Brain Research, 42 (1972) 73-87. 31 Johnson, A. K. and Buggy, V., A critical analysis of the site of action for the dipsogenic effect of angiotensin II. In J. P. Buckley and C. M. Ferrario (Eds.), Central ,4ction of ,4ngiotensin and Related Hormones, Pergamon Press, New York, 1977, pp. 357-386. 32 Jones, E. G., Burton, H., Saper, C. B. and Swanson, L. W., Midbrain, diencephalic and cortical relationships of the basal nucleus of Meynert and associated structures in primates, J. comp. Neurol., 167 (1976) 385--420. 33 Karplus, J. P. and Kreidl, A., Gehirn und Sympathicus. II. Mitteilung. Ein Sympathicuszentrum im Zwischenhirn, Pfliigers Arch. PhysioL, 135 (1910) 401-416.

34 King, F. A. and Meyer, P. A., Effects of amygdaloid lesions upon septal hyperemotionality in the rat, Science, 128 (1958) 655-656. 35 Kiser, S. R., Brown, C. A., Saughera, M. K. and German, P. C., Dorsal raphe nucleus stimulation reduces centrally (periaqueductal gray) elicited fearlike behavior, Brain Research, 191 (1980) 265-272. 36 Kliiver, H. and Bucy, P. C., Preliminary analysis of functions of the temporal lobes in monkeys, Arch. Neurol. Psychiat., 42 (1939) 979-1000. 37 Kusama, T. and Mabuchi, M., Stereotaxic Atlas of the Brain of Macaca fuscata, University of Tokyo Press, Tokyo, 1970. 38 Malmo, R. B., Slowing of heart rate after septal selfstimulation in rats, Science, 133 (1961) 1128-1130. 39 McBride, R. L. and Sutin, J., Amygdaloid and pontine projections to the ventromedial nucleus of the hypothalamus, J. comp. NeuroL, 174 (1977) 377-396. 40 Meibach, R. C. and Siegel, A., Efferent connections of the septal area in the rat: an analysis utilizing retrograde and anterograde transport methods, Brain Research, 119 (1977) 1-20. 41 Mesulam, M. M., Tetramethyl benzidine for horseradish peroxidase neurohistochemistry: a non-carcinogenic blue reaction-product with superior sensitivity for visualizing neural afferents and efferents, J. Histochem. Cytochem., 26 (1978) 106-117. 42 Miselis, R. R., Shapiro, R. E. and Hond, P. J., Subfornical organ efferents to neural systems for control of body water, Science, 205 (1979) 1022-1024. 43 Nathan, M. A. and Smith, O. A., Conditional cardiac and suppression responses after lesions in the dorsomedial thalamus of monkeys, J. comp. physiol. PsychoL, 76 (1971) 66-73. 44 Nauta, W. J. H. and Mehler, W. R., Projections of the lentiform nucleus in the monkey, Brain Research, 1 (1966) 3-42. 45 Poletti, C. E. and Creswell, G., Fornix system efferent projections in the squirrel monkey: an experimental degeneration study, J. comp. NeuroL, 175 (1977) 101-128. 46 Przuntek, H. and Philipu, A., Reduced pressor responses to stimulation of the locus ceruleus after lesions of the posterior hypothalamus, Naunyn-Schmiedeberg's Arch. Pharmacol., 276 (1973) 119-122. 47 Redmond, D. E. J., Huang, Y. H., Snyder, D. R. and Maas, J. W., Behavioral effects of stimulation of the nucleus locus eeruleus in the stump-tailed monkey Macaca arctoides, Brain Research, 116 (1976) 502-510. 48 Ricardo, J. A. and Koh, E. T., Anatomical evidence of direct projections from the nucleus of the solitary tract to the hypothalamus, amygdala, and other forebrain structures in the rat, Brain Research, 153 (1978) 1-26.

49 Sakumoto, T., Tohyama, M., Satoh, K., Kimoto, Y., Kinugasa, T., Tanizawa, O., Kurachi, K. and Shimizu, N., Afferent fiber connections from lower brain stem to hypothalamus studied by the horseradish peroxidase method with special reference to noradrenaline innervation, Exp. Brain Res., 31 (1978) 81-94. 50 Sawchenko, P. E. and Swanson, L. W., Central noradrenergic pathways for the integration of hypothalamic neuroendocrine and autonomic responses, Science, 214 (1981) 685-687.

226 51 Schreiner, L. and Kling, A., Behavioral changes following rhinencephalic injury in cat, J. NeurophysioL, 16 (1953) 643-659. 52 Silverman, A. J., Hoffman, D. L. and Zimmerman, E. A., The descending afferent connections of the paraventricular nucleus of the hypothalamus (PVN), Brain Res. Bull., 6 (1981) 47-61. 53 Simpson, J. B., Epstein, A. N. and Camardo, J. S., Jr., The localization of dipsogenic receptors for angiotensin II in the subfornical organ, J. comp. physiol. Psychol., 92 (1978) 581-608. 54 Smith, D. A. and Flynn, J. P., Afferent projections to quiet attack sites in cat hypothalamus, Brain Research, 194 (1980) 29--40. 55 Smith, D. A. and Flynn, J. P., Afferent projections to affective attack sites in cat hypothalamus, Brain Research, 194 (1980) 41-51. 56 Smith, O. A., Astley, C. A., DeVito, J. L., Stein, J. M. and Walsh, K. E., Functional analysis of hypothalamic control of the cardiovascular responses accompanying emotional behavior, Fed. Proc., 39 (1980) 24872494.

57 Smith, O. A., DeVito, J. L. and Astley, C. H., The hypothalamus in emotional behavior and associated cardiovascular correlates. In A. R. Morrison and P. L. Strick (Eds.), Changing Concepts of the Nervous System, Academic Press, New York, 1982, pp. 569-584. 58 Smith, O. A., Hohimer, A. R., Astley, C. A. and Taylor, D. J., Renal and hindlimb vascular control during acute emotion in the baboon, Amer. J. Physiol., 236 (1979) R193-R205. 59 Smith, O. A. and Nathan, M. A., Effect of hypothalamic and pre-frontal cortical lesions on conditioned cardiovascular responses, Physiologist, 7 (1964) 259. 60 Snyder, D. R., Huang, Y. H. and Redmond, D. E., Jr., Contribution of locus ceruleus-noradrenergic system to cardioacceleration in nonhuman primates, Neurosci. Abstr., 3 (1977) 828. 61 Swanson, L. W. and Cowan, W. M., Hippocampohypothalarnic connections: origin in subicular cortex, not Arnmon's horn, Science, 189 (1975) 303-304. 62 Ward, D. G. and Gunn, C. G., Locus coeruleus complex: elicitation of a pressor response and a brain stem region necessary for its occurrence, Brain Research, 107 (1976) 401-408.