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Light and electron microscopic demonstration of hypothalamic projections to the parabrachial nuclei in the cat
Neuroscience Letters, 46 (1984) 53-58
53
Elsevier Scientific Publishers Ireland Ltd. NSL 02657
LIGHT AND ELECTRON MICROSCOPIC DEMONSTRATION OF HYPOTHALAMIC PROJECTIONS TO THE PARABRACHIAL NUCLEI IN THE CAT
YOSHIKI TAKEUCHI* and DAVID A. HOPKINS**
Department of Anatomy, Dalhousie University, Halifax, N.S., B3H 41t7 (Canada) (Received August 30th, 1983; Revised version received December 7th, 1983; Accepted January 25th, 1984)
Key words: hypothalamus - paraventricular nucleus - parabrachial nuclei - horseradish peroxidase ultrastructure - degeneration
Hypothalamic connections with the parabrachial nuclei in the cat were studied at light and electron microscopic levels following wheat germ agglutinin-horseradish peroxidase injections into the parabrachial nuclei and electrolytic lesions in the hypothalamus. The greatest concentration of retrogradely labeled neurons occurred in the paraventricular nucleus. Labeled neurons were also seen within the preoptic, anterior, lateral, dorsomedial and ventromedial hypothalamic nuclei. Hypothalamic lesions resulted in the degeneration of terminals forming axosomatic and axodendritic synapses in the parabrachial nuclei, particularly its lateral division. These findings support the idea that hypothalamic connections to specific regions of the parabrachial nuclei may underlie the topographical functional organization demonstrated for these brainstem nuclei.
Regions in and near the parabrachial nuclei (PBN) in the cat have been implicated in behavioral, autonomic and endocrine functions [1, 9, 11, 16]. Anatomical studies which have demonstrated amygdaloid, hypothalamic and medullary afferents [3-5, 12, 15] to the PBN are consistent with suggestions that the PBN play an important role in the integration of visceral responses [8, 10]. Since the major descending afferent projections may pass through the PBN to the lateral tegmental field and dorsal vagal complex at more caudal levels of the brainstem [3, 12], the synaptic organization of these afferents to the PBN needs to be examined. The purpose of the present study was to elucidate the direct hypothalamic connections to the PBN in the cat using light and transmission electron microscopic methods. Four cats weighing 2.0-2.9 kg were anesthetized with sodium pentobarbital (25 mg/kg i.v.) and injected with 0.02-0.04 #1 of 5 % wheat germ agglutinin-horseradish *Present address: Department of Oral Anatomy, Hiroshima University, Hiroshima, Japan. **Author for correspondence. 0304-3940/84/$ 03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd.
54 peroxidase conjugate (WGA-HRP, Sigma) dissolved in distilled water. The injections were made stereotaxically into the PBN via an infratentorial approach at an angle o f 40 ° to the frontal plane by means of a glass micropipette (tip diameter 50-80/~m) attached to a 1 /~1 Hamilton microsyringe. After two days the animals were anesthetized and perfused transcardially with 0.15 M phosphate buffer (pH 7.4) followed by 1.0070 paraformaldehyde-l.25070 glutaraldehyde in 0.15 M phosphate buffer (pH 7.4). The brains were removed and processed for H R P histochemistry according to the method of Mesulam [7]. Three cats weighing 2.5-3.1 kg were anesthetized as above and electrolytic lesions (2 mA, 20 sec) were placed stereotaxically in the paraventricular nucleus via a vertical approach. After 2-4 days, the animals were anesthetized and perfused with 0.15 M phosphate buffer (pH 7.4) followed by 0.5070 paraformaldehyde-2.5070 glutaraldehyde in 0.15 M phosphate buffer (pH 7.4). The brains were removed and the PBN were processed for electron microscopy as in a previous study [15]. Transverse 40 #m-thick frozen sections of the diencephalon were stained with cresyl violet to confirm the lesion sites. Ultrathin sections of the PBN (see Fig. 1A) were examined with a Zeiss EM 10A electron microscope. The W G A - H R P injections into the PBN were concentrated near the middle of their rostrocaudal extent (Fig. 1A). Spread of the enzyme to adjacent areas including the subcoeruleus and Koelliker-Fuse nuclei did, however, occur. Retrogradely labeled neurons in the hypothalamus were located primarily ipsilateral to the injected PBN. Many labeled cells were present in the parvocellular paraventricular nucleus (PVH), the preoptic area, anterior, posterior and lateral hypothalamic areas, as well as the ventromedial and dorsomedial hypothalamic nuclei. O f particular interest was the concentration of labeled neurons in and lateral to the PVH. These neurons formed a striking and characteristic aggregation near the third ventricle (Fig. 1C). Heavy labeling, again primarily ipsilateratly, was also seen in the bed nucleus of the stria terminaiis and in the central nucleus of the amygdala. A substantial number of labeled neurons was present ipsilaterally in the region of the Edinger-Westphal nucleus, the central gray matter, substantia nigra and the ventral tegmental area, while only a few scattered labeled neurons were observed in the globus pallidus, entopeduncular nucleus and zona incerta. These W G A - H R P studies confirm that many neurons of the hypothalamus and of other subcortical areas project to or through the PBN and adjacent nuclei. In the present study, all electrolytic lesions involved a substantial part of the P V H and encroached on adjacent hypothalamic areas previously shown to project to the PBN (Fig. 1B). Although the site and size of the lesions varied slightly, each involved the PVH or its lateral extension. Degenerating axon terminals with a long axis usually of 1-2 t~m and characterized by a dark matrix and swollen mitochondria were observed in all lesioned animals (Fig. 2) and were most abundant ipsilaterally within the rostral half of the lateral division of the PBN. The medial division of the PBN contained fewer and more dif-
55
Fig. 1. Light photomicrographs showing results o f a W G A - H R P injection into the PBN and of an electrolytic lesion o f the hypothalamus. A: the site of a single W G A - H R P injection into the PBN. The large and small rectangles indicate the areas in the lateral and medial parabrachial nuclei, respectively, from which ultrathin sections were cut. B: an electrolytic lesion o f the hypothalamus. The arrow contralateral to the lesion site indicates the paraventricular nucleus. C: retrogradely labeled neurons in the right paraventricular nucleus. The location.of these labeled neurons is similar to the area indicated by the arrow and to the lesion site in B. Calibration bars = l m m in A and B and 200 ~m in C. Abbreviations: BC, brachium conjunctivum; LC, locus coeruleus; III, third cerebral ventricle.
56
Fig. 2. Electron photomicrographs showing degenerating axosomatic and axodendritic terminals in the lateral subdivision of the PBN. A: degenerating axosomatic terminal synapsing (framed area) on a neuron. B: high magnification of the terminal shown in A. C: degenerating axodendritic terminal containing round vesicles and forming asymmetrical synaptic contact. D: three degenerating axodendritic terminals (DAT), Note that the two degenerating terminals on the right side contain pleomorphic vesicles and make symmetrical synaptic contact with the dendritic shaft. Asterisks in B and C indicate glial cell cytoplasm. Survival period - 4 days. Calibration bars 5 #m in A, 0.5 j~m in B and C and 1 t~m in D,
57 fusely distributed degenerating terminals throughout its entire rostral-caudal extent. Degeneration in the PBN due to extra-hypothalamic sites such as the amygdala was likely to be negligible because the bulk of amygdalotegmental fibers descends in the lateral hypothalamus [3] rather than in the medial hypothalamus where the lesions were located. A few degenerating terminals, usually containing pleomorphic vesicles, formed symmetrical axosomatic synapses with the plasmalemma of small neurons (Fig. 2A, B). These neurons typically had a nucleus with one or more invaginations, a prominent nucleolus and a relatively well-developed cytoplasm. Axodendritic terminals were common in the PBN. The degenerating axodendritic terminals contained either round or pleomorphic vesicles and made asymmetrical or symmetrical synaptic contact with dendritic profiles or shafts (Fig. 2C, D). In many synapses forming asym-, metrical contacts clear pre- and post-synaptic densities and the synaptic cleft were observed (Fig. 2C). The results of the present study establish that hypothalamic neurons make axosomatic and axodendritic synaptic contact with neurons in the PBN and do not simply pass through the nuclei on the way to more caudal structures. Although light microscopic studies indicate that both lateral and medial divisions of the PBN receive equally heavy descending projections from the amygdala [3] and hypothalamus [12], in the present study substantially more degenerating terminals were observed in the lateral division of the PBN. This difference between lateral and medial divisions at the light and electron microscopic level could imply that more descending fibers traverse the medial than the lateral division of the PBN. Interestingly, reciprocal connections with the amygdala have been reported particularly in the lateral division of the PBN [15] and in the rat, degeneration and histochemical studies indicate that both dopaminergic and serotonergic terminals within the PBN are highly concentrated in its lateral division [2, 13]. A topographical organization within the PBN has also been reported for ascending projections to the PBN in the cat [4, 5] as well as for efferent ascending and descending PBN projections in the cat and other species [6, 14] (for further references see ref. 15). This evidence for morphological differences in the connectivity of the PBN is in keeping with the topographical organization reported for a variety of physiological functions [1, 9, 11, 16]. The present and previous results [15] also provide an initial description of a synaptic basis for descending hypothalamic and amygdaloid modulation of activity within the PBN. The authors thank Dr. J.H. McLean, G.V. Allen and P. Wilkinson for technical assistance and L. Boylan and D. Amirault for typing the manuscript. Drs. R.E. Clattenburg and J. McLean provided many helpful comments on the manuscript. This work was supported by the Medical Research Council of Canada (Grant MT7369), and Postdoctoral Fellowship support to Y.T. from the Killam Foundation and the Dalhousie Medical Research Foundation.
58 1 Coote, J.H., Hilton, S.M. and Zbrozyna, A.W., The ponto-medullary area integrating the defensc reaction in the cat and its influence on muscle blood flow, J. Physiol. (Lond.), 229 (1973) 257-274. 2 Hedreen, J., Terminal degeneration demonstrated by the Fink-Heimer method following lateral ventricular injection of 6-hydroxydopamine, Brain Res. Bull., 5 (1980) 425-436. 3 Hopkins, D.A. and Holstege, G., Amygdaloid projections to the mesencephalon, pons and medulla oblongata in the cat, Exp. Brain Res., 32 (1978) 529-547. 4 King, G.W., Topology of ascending brainstem projections to nucleus parabrachialis in the cat, .I. comp. Neurol., 191 (1980) 615-638. 5 Loewy, A.D. and Burton, H., Nuclei of the solitary tract: efferent projections to the lower brain stem and spinal cord of the cat, J. comp. Neurol., 181 (1978) 421-450. 6 Mehler, W.R., Subcortical afferent connections of the amygdata in the monkey, J. comp. Neurol., 190 (1980) 733-762. 7 Mesulam, M.-M., Tetramethyl benzidine for horseradish peroxidase neurohistochemistry: a noncarcinogenic blue reaction-product with superior sensitivity for visualizing neural afferents and efferents, J. Histochem. Cytochem., 26 (1978) 106-117. 8 Mizuno, N., Nomura, S. and Takeuchi, Y., The parabrachial nucleus as an intermediate relay station of the visceral afferent pathways in the cat. In M. Ito, N. Tsukahara, K. Kubota and K. Yagi (Eds.), Integrative Control Functions of the Brain, Vol. 3, Elsevier/North-Holland, Amsterdam, 1980, pp. 51-64. 9 Mraovitch, S., Kumada, M. and Reis, D.J., Role of the nucleus parabrachialis in cardiovascular regulation in cat, Brain Res., 232 (1982) 57-75. 10 Nauta, W.J.H. and Domesick, V.B., Ramification of the limbic system. In S. Matthysse (Ed.), Psychiatry and the Biology of the Human Brain, Elsevier/North-Holland, Amsterdam, 1981, pp. 165-188. 11 Saito, H., Sakai, K. and Jouvet, M., Discharge patterns of the nucleus parabrachialis lateralis neurons of the cat during sleep and waking, Brain Res., 134 (1977) 59-72. 12 Saper, C.B., Loewy, A.D., Swanson, L.W. and Cowan, W.M., Direct hypothalamo-autonomic connections, Brain Res., 117 (1976) 305-312. 13 Steinbusch, H.W.M., Distribution of serotonin-immunoreactivity in the central nervous system of the rat-cell bodies and terminals, Neuroscience, 6 (1981) 557-618. 14 Takeuchi, Y., Uemura, M., Matsuda, K., Matsushima, R. and Mizuno, N., Parabrachial nucleus neurons projecting to the lower brain stem and the spinal cord. A study in the cat by the Fink-Heimer and the horseradish peroxidase methods, Exp. Neurol,, 70 (1980) 403-413. 15 Takeuchi, Y., McLean, J.H. and Hopkins, D.A., Reciprocal connections between the amygdala and parabrachial nuclei: ultrastructural demonstration by degeneration and axonal transport of horseradish peroxidase in the cat, Brain Res., 239 (1982) 583-588. 16 Ward, D.G., Grizzle, W.E. and Gann, D.S., Inhibitory and facilitatory areas of the rostral pons mediating ACTH release in the cat, Endocrinology, 99 (1976) 1220-1228.
53
Elsevier Scientific Publishers Ireland Ltd. NSL 02657
LIGHT AND ELECTRON MICROSCOPIC DEMONSTRATION OF HYPOTHALAMIC PROJECTIONS TO THE PARABRACHIAL NUCLEI IN THE CAT
YOSHIKI TAKEUCHI* and DAVID A. HOPKINS**
Department of Anatomy, Dalhousie University, Halifax, N.S., B3H 41t7 (Canada) (Received August 30th, 1983; Revised version received December 7th, 1983; Accepted January 25th, 1984)
Key words: hypothalamus - paraventricular nucleus - parabrachial nuclei - horseradish peroxidase ultrastructure - degeneration
Hypothalamic connections with the parabrachial nuclei in the cat were studied at light and electron microscopic levels following wheat germ agglutinin-horseradish peroxidase injections into the parabrachial nuclei and electrolytic lesions in the hypothalamus. The greatest concentration of retrogradely labeled neurons occurred in the paraventricular nucleus. Labeled neurons were also seen within the preoptic, anterior, lateral, dorsomedial and ventromedial hypothalamic nuclei. Hypothalamic lesions resulted in the degeneration of terminals forming axosomatic and axodendritic synapses in the parabrachial nuclei, particularly its lateral division. These findings support the idea that hypothalamic connections to specific regions of the parabrachial nuclei may underlie the topographical functional organization demonstrated for these brainstem nuclei.
Regions in and near the parabrachial nuclei (PBN) in the cat have been implicated in behavioral, autonomic and endocrine functions [1, 9, 11, 16]. Anatomical studies which have demonstrated amygdaloid, hypothalamic and medullary afferents [3-5, 12, 15] to the PBN are consistent with suggestions that the PBN play an important role in the integration of visceral responses [8, 10]. Since the major descending afferent projections may pass through the PBN to the lateral tegmental field and dorsal vagal complex at more caudal levels of the brainstem [3, 12], the synaptic organization of these afferents to the PBN needs to be examined. The purpose of the present study was to elucidate the direct hypothalamic connections to the PBN in the cat using light and transmission electron microscopic methods. Four cats weighing 2.0-2.9 kg were anesthetized with sodium pentobarbital (25 mg/kg i.v.) and injected with 0.02-0.04 #1 of 5 % wheat germ agglutinin-horseradish *Present address: Department of Oral Anatomy, Hiroshima University, Hiroshima, Japan. **Author for correspondence. 0304-3940/84/$ 03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd.
54 peroxidase conjugate (WGA-HRP, Sigma) dissolved in distilled water. The injections were made stereotaxically into the PBN via an infratentorial approach at an angle o f 40 ° to the frontal plane by means of a glass micropipette (tip diameter 50-80/~m) attached to a 1 /~1 Hamilton microsyringe. After two days the animals were anesthetized and perfused transcardially with 0.15 M phosphate buffer (pH 7.4) followed by 1.0070 paraformaldehyde-l.25070 glutaraldehyde in 0.15 M phosphate buffer (pH 7.4). The brains were removed and processed for H R P histochemistry according to the method of Mesulam [7]. Three cats weighing 2.5-3.1 kg were anesthetized as above and electrolytic lesions (2 mA, 20 sec) were placed stereotaxically in the paraventricular nucleus via a vertical approach. After 2-4 days, the animals were anesthetized and perfused with 0.15 M phosphate buffer (pH 7.4) followed by 0.5070 paraformaldehyde-2.5070 glutaraldehyde in 0.15 M phosphate buffer (pH 7.4). The brains were removed and the PBN were processed for electron microscopy as in a previous study [15]. Transverse 40 #m-thick frozen sections of the diencephalon were stained with cresyl violet to confirm the lesion sites. Ultrathin sections of the PBN (see Fig. 1A) were examined with a Zeiss EM 10A electron microscope. The W G A - H R P injections into the PBN were concentrated near the middle of their rostrocaudal extent (Fig. 1A). Spread of the enzyme to adjacent areas including the subcoeruleus and Koelliker-Fuse nuclei did, however, occur. Retrogradely labeled neurons in the hypothalamus were located primarily ipsilateral to the injected PBN. Many labeled cells were present in the parvocellular paraventricular nucleus (PVH), the preoptic area, anterior, posterior and lateral hypothalamic areas, as well as the ventromedial and dorsomedial hypothalamic nuclei. O f particular interest was the concentration of labeled neurons in and lateral to the PVH. These neurons formed a striking and characteristic aggregation near the third ventricle (Fig. 1C). Heavy labeling, again primarily ipsilateratly, was also seen in the bed nucleus of the stria terminaiis and in the central nucleus of the amygdala. A substantial number of labeled neurons was present ipsilaterally in the region of the Edinger-Westphal nucleus, the central gray matter, substantia nigra and the ventral tegmental area, while only a few scattered labeled neurons were observed in the globus pallidus, entopeduncular nucleus and zona incerta. These W G A - H R P studies confirm that many neurons of the hypothalamus and of other subcortical areas project to or through the PBN and adjacent nuclei. In the present study, all electrolytic lesions involved a substantial part of the P V H and encroached on adjacent hypothalamic areas previously shown to project to the PBN (Fig. 1B). Although the site and size of the lesions varied slightly, each involved the PVH or its lateral extension. Degenerating axon terminals with a long axis usually of 1-2 t~m and characterized by a dark matrix and swollen mitochondria were observed in all lesioned animals (Fig. 2) and were most abundant ipsilaterally within the rostral half of the lateral division of the PBN. The medial division of the PBN contained fewer and more dif-
55
Fig. 1. Light photomicrographs showing results o f a W G A - H R P injection into the PBN and of an electrolytic lesion o f the hypothalamus. A: the site of a single W G A - H R P injection into the PBN. The large and small rectangles indicate the areas in the lateral and medial parabrachial nuclei, respectively, from which ultrathin sections were cut. B: an electrolytic lesion o f the hypothalamus. The arrow contralateral to the lesion site indicates the paraventricular nucleus. C: retrogradely labeled neurons in the right paraventricular nucleus. The location.of these labeled neurons is similar to the area indicated by the arrow and to the lesion site in B. Calibration bars = l m m in A and B and 200 ~m in C. Abbreviations: BC, brachium conjunctivum; LC, locus coeruleus; III, third cerebral ventricle.
56
Fig. 2. Electron photomicrographs showing degenerating axosomatic and axodendritic terminals in the lateral subdivision of the PBN. A: degenerating axosomatic terminal synapsing (framed area) on a neuron. B: high magnification of the terminal shown in A. C: degenerating axodendritic terminal containing round vesicles and forming asymmetrical synaptic contact. D: three degenerating axodendritic terminals (DAT), Note that the two degenerating terminals on the right side contain pleomorphic vesicles and make symmetrical synaptic contact with the dendritic shaft. Asterisks in B and C indicate glial cell cytoplasm. Survival period - 4 days. Calibration bars 5 #m in A, 0.5 j~m in B and C and 1 t~m in D,
57 fusely distributed degenerating terminals throughout its entire rostral-caudal extent. Degeneration in the PBN due to extra-hypothalamic sites such as the amygdala was likely to be negligible because the bulk of amygdalotegmental fibers descends in the lateral hypothalamus [3] rather than in the medial hypothalamus where the lesions were located. A few degenerating terminals, usually containing pleomorphic vesicles, formed symmetrical axosomatic synapses with the plasmalemma of small neurons (Fig. 2A, B). These neurons typically had a nucleus with one or more invaginations, a prominent nucleolus and a relatively well-developed cytoplasm. Axodendritic terminals were common in the PBN. The degenerating axodendritic terminals contained either round or pleomorphic vesicles and made asymmetrical or symmetrical synaptic contact with dendritic profiles or shafts (Fig. 2C, D). In many synapses forming asym-, metrical contacts clear pre- and post-synaptic densities and the synaptic cleft were observed (Fig. 2C). The results of the present study establish that hypothalamic neurons make axosomatic and axodendritic synaptic contact with neurons in the PBN and do not simply pass through the nuclei on the way to more caudal structures. Although light microscopic studies indicate that both lateral and medial divisions of the PBN receive equally heavy descending projections from the amygdala [3] and hypothalamus [12], in the present study substantially more degenerating terminals were observed in the lateral division of the PBN. This difference between lateral and medial divisions at the light and electron microscopic level could imply that more descending fibers traverse the medial than the lateral division of the PBN. Interestingly, reciprocal connections with the amygdala have been reported particularly in the lateral division of the PBN [15] and in the rat, degeneration and histochemical studies indicate that both dopaminergic and serotonergic terminals within the PBN are highly concentrated in its lateral division [2, 13]. A topographical organization within the PBN has also been reported for ascending projections to the PBN in the cat [4, 5] as well as for efferent ascending and descending PBN projections in the cat and other species [6, 14] (for further references see ref. 15). This evidence for morphological differences in the connectivity of the PBN is in keeping with the topographical organization reported for a variety of physiological functions [1, 9, 11, 16]. The present and previous results [15] also provide an initial description of a synaptic basis for descending hypothalamic and amygdaloid modulation of activity within the PBN. The authors thank Dr. J.H. McLean, G.V. Allen and P. Wilkinson for technical assistance and L. Boylan and D. Amirault for typing the manuscript. Drs. R.E. Clattenburg and J. McLean provided many helpful comments on the manuscript. This work was supported by the Medical Research Council of Canada (Grant MT7369), and Postdoctoral Fellowship support to Y.T. from the Killam Foundation and the Dalhousie Medical Research Foundation.
58 1 Coote, J.H., Hilton, S.M. and Zbrozyna, A.W., The ponto-medullary area integrating the defensc reaction in the cat and its influence on muscle blood flow, J. Physiol. (Lond.), 229 (1973) 257-274. 2 Hedreen, J., Terminal degeneration demonstrated by the Fink-Heimer method following lateral ventricular injection of 6-hydroxydopamine, Brain Res. Bull., 5 (1980) 425-436. 3 Hopkins, D.A. and Holstege, G., Amygdaloid projections to the mesencephalon, pons and medulla oblongata in the cat, Exp. Brain Res., 32 (1978) 529-547. 4 King, G.W., Topology of ascending brainstem projections to nucleus parabrachialis in the cat, .I. comp. Neurol., 191 (1980) 615-638. 5 Loewy, A.D. and Burton, H., Nuclei of the solitary tract: efferent projections to the lower brain stem and spinal cord of the cat, J. comp. Neurol., 181 (1978) 421-450. 6 Mehler, W.R., Subcortical afferent connections of the amygdata in the monkey, J. comp. Neurol., 190 (1980) 733-762. 7 Mesulam, M.-M., Tetramethyl benzidine for horseradish peroxidase neurohistochemistry: a noncarcinogenic blue reaction-product with superior sensitivity for visualizing neural afferents and efferents, J. Histochem. Cytochem., 26 (1978) 106-117. 8 Mizuno, N., Nomura, S. and Takeuchi, Y., The parabrachial nucleus as an intermediate relay station of the visceral afferent pathways in the cat. In M. Ito, N. Tsukahara, K. Kubota and K. Yagi (Eds.), Integrative Control Functions of the Brain, Vol. 3, Elsevier/North-Holland, Amsterdam, 1980, pp. 51-64. 9 Mraovitch, S., Kumada, M. and Reis, D.J., Role of the nucleus parabrachialis in cardiovascular regulation in cat, Brain Res., 232 (1982) 57-75. 10 Nauta, W.J.H. and Domesick, V.B., Ramification of the limbic system. In S. Matthysse (Ed.), Psychiatry and the Biology of the Human Brain, Elsevier/North-Holland, Amsterdam, 1981, pp. 165-188. 11 Saito, H., Sakai, K. and Jouvet, M., Discharge patterns of the nucleus parabrachialis lateralis neurons of the cat during sleep and waking, Brain Res., 134 (1977) 59-72. 12 Saper, C.B., Loewy, A.D., Swanson, L.W. and Cowan, W.M., Direct hypothalamo-autonomic connections, Brain Res., 117 (1976) 305-312. 13 Steinbusch, H.W.M., Distribution of serotonin-immunoreactivity in the central nervous system of the rat-cell bodies and terminals, Neuroscience, 6 (1981) 557-618. 14 Takeuchi, Y., Uemura, M., Matsuda, K., Matsushima, R. and Mizuno, N., Parabrachial nucleus neurons projecting to the lower brain stem and the spinal cord. A study in the cat by the Fink-Heimer and the horseradish peroxidase methods, Exp. Neurol,, 70 (1980) 403-413. 15 Takeuchi, Y., McLean, J.H. and Hopkins, D.A., Reciprocal connections between the amygdala and parabrachial nuclei: ultrastructural demonstration by degeneration and axonal transport of horseradish peroxidase in the cat, Brain Res., 239 (1982) 583-588. 16 Ward, D.G., Grizzle, W.E. and Gann, D.S., Inhibitory and facilitatory areas of the rostral pons mediating ACTH release in the cat, Endocrinology, 99 (1976) 1220-1228.