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Immunocytochemical localization of ACTH perikarya in nucleus tractus solitarius: evidence for a second opiocortin neuronal system
Naroscience Letters, 38 (1983) 221-225
221
Elsevier Scientific Publishers Ireland Ltd.
NUCLEUS S : E V I D E N ~ FOR A SECOND OPlOCOgglN NEURONAL SYSTEM
SHIRLEY A. JOSEPH, WEBSTER H. PILCHER and CAROL BENNETI'-CLARKE
The Nt~roendocrine Unit, Universit7 o f Rochester, School of Medicine and Dentist~, 601 Elmwood A~enue, Rochester, NY 14042 (U.S.A.) (Rectived March Sth, 1983; Revised version received May 4th, 1983; Accepted May 6th, 1983)
Key words: ACTH - opiocortin peptides - nucleus tractus solitarius - cardiovascular center brainstem - immunocytochemistry - anatomical localization
Immunucytochemical localization of ACTH.opioconin perikarya was demonstrated in the medulla of o;Ichicine-troated rat. Neuronal cell bodies and fibers containing ACTH-immunoractivity were abunc am in the caudal region of the nucleus tractus solitarius, spedf'gally within pars commisuralis. Location o; these opiocortin neurons within the nucleus tractus solitarius prmides additional evidence for a role of these peptides in cardiovascular functions.
The endogenous opioid peptides are represented in the central nervous system by several distinctly separate neuronal systems. These include the opiocortin [7, 12], enkephalin [9, 10] and 'dynorphin [8, I I] systems. Recognized components of the opiocortin neuronal organizations include: (a) the system where ACTH and/~endorphin, a-MSH containing pedkarya are confined to the arcuate, periarcuate region of medial basal hypothalamus [I 3] and (b) the a-MSH specific (a-2) neuronal pool residing in the dorsal and lateral hypothalamus 122]. in this study we report that existence of an additional group of opioconin neurons located in caudal medulla of the brainstem and spinal cord. Ten Sprague-Dawley rats ranging in weight from 180 to 250 g were used for this study. Forty-eight hours prior to sacrifice 100/tg of colchicine was infused stereotaxically into the lateral ventricle or "/$/~g into the 4th ventricle. Animals were pe,~fused intracardially with ice-cold 4o/0 paraformaldehyde or with Bouin's fixative. Brains were cut serially from mid-hypothalami¢ region through the entire spinal cord either on a freezing micTotome or on a vibratome at 50/~m and collected into wells containing phosphate-saline buffer, pH 7.4. lmmunostaininig procedures using the
peroxidase-antiperoxidase unlabeled antibody method and the avidin-biotin complex technique and antisera specificity have been described previously [12, 20]; antisera used for these studies included ACTH (531) and 16K ('Georgie', Mains and 0304-3940183/$ 03.00 @ 1983 Elsevier Scientific Publishers Ireland Ltd.
222 Eipper). Specif'gity controls consisted of (a) deletion of primary antisera and Co) absorption of ami-ACTH antisera and synthetic human ACTHt-j9 (2-10 tqg/ml diluted antiserum). Immunoreactive ACTH-opiocortin perikaryn were observed in caudal medulla from a level caudal to area postrema to the medullary-cord junction and within the first 2-3 segments of spinal cord. The greatest concentration of cell bodies was observed caudal to the level of the area postrema (AP) within the commissural nucleus (CnTS) dorsal to the central canal (Figs. I and 2). in Fig. I, cell bodies can be ~ at their rostral extent medial and ventral to tractus solitarius within CnTS; at this level scattered ceil bodies ~'ere observed, along a ventro-lateral axis through the nucleus reticularis medullae oblongata to the dorsal border of nucleus ambiguus and Aj catecholamine cell region. Sparsely scattered cell bodies were also observed more caudally within the first 2-3 segments of cervical spinal cord surrounding the central canal. This group of opiocortin-containing neurons were visualized only in animals in which colchieine was infused into the lateral or 4th ventricle and were immunorcactive with antisera generated against ACTHj_.~v and the 16K fragment of the proopiocortin molecule. Within the medullary regi~:t fiber immunostainmg was .~'en within all the divisions of the nucleus tractus solitarius and the tractns .~litarius throughout its caudal-rostral extent, within and surrounding the area postrema. Extending in a ventro-lateral direction from the nucleus commissuralis of nucleus tractus ~litariu.~ (NTS) and within this ventro-lateral trajectory, fibers could be traced through the nucleus reticularis medullae oblongata, nucleus ambi~uu.~ and the nucleus reticularis lateralis which corresponds to the At cateeholaminergic cell region. A few fibers were aim observed within the nucleus reticulari.~ gigantocellularis and throughout the spinal cord surrounding the central canal. In thi~ study we have described a previously unidentified group of opiocortinimmunoreactive neurons located in the caudal region of the nucleus tractus solitaflus. Although a rich innervation of ACTH and ~-endorphin immunoreactivity has been shown to distribute within this nuclear complex and its faseiculus, as well as to other as.~m:iated areas of brainstem, for example the parabrachial nucleus and raphe systems [7, 20|, this is the first report of opiocortin immunoreactive perikarya in the brainstem. The NTS has for some time been regarded as a major integration center for the regulation of cardiovascular reflexes [14|. Myriad neuroanatomical and electrophysiologic studies have contributed to the origin and identification of vi.~eral afferent projections to NTS as well as its vast reciprocal efferent projections with other regions known to influence cardiovascular homeostasis (for review see refs. I and 16). Within minutes following bilateral lesions of NTS dramatic and sustaining elevations in blood pressure ensued due to the loss of the baroreceptor reflex [5, 61 and, possibly, certain catecholaminergic influences at this site [3, 19, 23]. Escorting these reports are electrophysiological studies which demonstrate a significant fall in blood pressure and vagal bradycardia following electrical stimulation in various regions of NTS [4, 211.
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Fig. I. Photomicrograph of the dorsal medial region of lower medulla approximately tO0/Am caudal to area postrcma. Nmnerus ACTH-immunoreactive perikarya are demonstrated in the commissural nucleus of tractus solitarius (CnTS) and extend ventro-latefally to the dorsal region of nucleus ambiguu~. Cells (:an be observed ventral, medial and lateral to the tractus solitarius (TS) at this level. X, dorsal motor nucleus of the vagus; XII, hypolgiossalnucleus.
Fig. 2. Photomicrosraph of medullary-cord junction. ACTH-immunoreactive perikarya are seen within CnT$ and surrounding the central canal (C)-..
224
The effects of opioid substances and opiate ~ on cardiovascular function have been known for some time. F ~ e , it has recently been shown that depressor effects of ~,ndmphln" and ~ _ n e might inlu'l~t sympathetic tone at the levd of the IqTS. For example ~ ~'ndOt'l~in ~fusiom 11~ or infusions directly into NTS [171dick a naloxone-mvcm'lge fall in mean arterial pressure and valgal ~ c a r d i a without a s~gnificant change in respbmory rate. In addition, the dcprc~ror response was prevented and bradycardia reduced by prior injections of ~ antisem directly into N'I'S, whereas microinjection of ~t~'mlorphin antisera alone resulted in a rise in blood p r e m ~ . Even thoush ¢n~ephalincontainitqg neurons ate localized in Jq'rs [9, IOI, antisera to enkephalin had little or no effect on cardiovascular parmnetcrs when injected with identical protocol 1171. However, in the dog and at the level of nucleus mbilNus, ¢nkephalinerl~c involv¢mclllt in Val~l responses has ~ denlonstralred 1151. These studies and those of Bolme et al. 121 arc compellin$ in lisht of our new finclinl~ and s u l ~ that specific medullary centers may intclp'at¢ opioid/autonomi¢ mechanisms via a specific opiate reccptor/opioid peptide interaction. In previous studies [12, 13, 20] we described the distribution of ACTH iramunoreactiv¢ opiocortin fibers in the brainstcm of normal, non colchicine treated rats; opiocortin perikarya were not obsc~ed in the medulla and it was postulated that these brainstem fibers orilginated from perikarya of the hypothalamic arcuate opiocortin bed nuclens. The presence of a medulL'u'y UonP of pcrikarya clearly indicates a more complex origanization of the opiocortin distribution to brainstem centers. Further study is needed to elucidate the detailed distribution of these forebrain and hindbrain components and how they function in the myriad interactions of the opiocortin peptides in central autonomic functions. S.A.J. is recipient of Grant RCDA 5 K04 HD(Kg230. I Abboud, F.M., The sympathetic sygem in hypertension: gate-of-the.art review. Hypertension, 4. Suppl. II. (19112)~0S-225. 2 Bolme. P., Fuxe, K., Algnali, L.F., Bradley, R. and Smythies, J., Cardio~'ascular effects of morphine and opioid peptides followitqg inir~-istcrnal administration in chloralosc-ancsthetized rats, Europ. J. Pharmix-ol., 48 (1928) 319-324. 3 13¢ Jonlg, W., Noradrenalin¢: central inhibitory control of blood pressure and heart rate, Europ. J. Pharmacol.. 29 (1974) 179-181. 4 De Jong, W., Nijkamp, F.P. and Bohus, B., Role of noradrenalin and .qcrotonin in the central control of blood pressure in normotensive and spontaneously hypertensive rats, Arch. int. Pharmacodyn., 213 (1975) 272-284. 5 De .tong, W., Zandberg, P., Palkovits, M. and Bohus, B., Acute and chronic hyl~-rtension after lesions and transcctions of rat braimtem. In W. De .long, A.P. Provoost and A.P. Shapiro (Ed$.), lbTcrtension and Brain Mechanisms, Progress in Brain Research, Vol. 47, Elsevier Biomedical, Amsterdam, 1977, pp. 189-197. 6 Doba, N and Rots, D.J.. Acute fulminating nmrogenic hypertension produced by brainstcm lesions in the cat, Circular. Res., 32 (1973) 584-593.
225 7 Finley, J.C.W., Lindstr6m, P. and Petruz. P., lmmunocytochemical localization of l~'ndorphincomainin8 neurom in the rat brain. Neuroendocrinology. 33 (1981) 28-42. 8 Cmaldstei~A., Tachibana, S., Lowney, L.J., Hunkapiller, M. and Hood, L., Dynorphin (I-13), an c~Jraordinary pt~,pnt opioid peptide, Proc. nat. Acad. Sci. U.S.A., 76 (1979) 6666-6670.
I0 ~ f e l t i T , Tetmius~ L~, g ~ H!G!j~M~ i five neurons in the tnedulla o b l o n ~ projecting to t k s p i ~ cord, Seurosci, ~ t ~ , 14(! 979) 5$-60. I I HOIIt, V., Hammann, I., Bovermann, K., Jerlicz. M, and Herz, A., D ~ ~ n - r e l a t e d immunoreactive peptides in rat brain and pituitary, Neurosci, ~ , , 18(1980) 149-153. 12 Joseph, S.A., Immunoreactive adrenoconicotropin in rat brain: a neuroanatomical study using antberum Igenerated agaimt synthetic ACTH ' . ~ , Amer. J. Anat., 158 (1980) $33-$48. 13 Kniluge,K.M., Joseph, S.A. and Nocton, J., Topography of the ACTH-immunoreactive neurons in the basal hypothalamus of the rat brain, Brain Res., 216 (1981) 333-341. 14 Korner, P., Central nervous control of autonomic cardiovascular function, Handbook of Physiology, Sect. 2: The Cardiovascular System. Vol. I: The Heart, Amer. Physiol. Soc., Baltimore, MD, 1971, pp. 691-739. 15 Laubie. M. and Sehmitt, H., Indication for central vagal eudorphinergic control of heart rate in dogs, Europ. J. Pharmacol., 71 (1981) 401-409. 16 Palkovits, M. and Zaborszky, L., Nenrnanatomy of central cardiovascular control. Nucleus traetus solitarii: afferent and efferent neuronal connections in relation to the baroreceptor reflex arc., In W. De Jon$, A.P. Provoost and A.P. Shapiro (Eds.). Hypertension and Brain Mechanisms, Progress in Brain Research, Vol. 47, Elsevkr Biomedical, Amsterdam, 1977, pp. 9-34. 17 Petty, M.A. and De Jong, W., Cardiovascular effects of ~.x,ndorphin after microinjection into the nucleus tractus solitarii of the anesthetized rat, Europ. J. Pharmacol., 81 (1982) 449-457. 18 Petty, M.A., De Jons, W. and De Wk,d, D., An inhibitory role of ~-endorphin in central cardiovascular rqgulation, Life Sci., 30 (19S2) 18)5-1~10. !9 Reis, D.J., Doha, N., Snyder, D.W. and Nathan, M.A., Brain lesions and hypertension: Chronic lability and elevation of ~rterial pressure produ~d by electrolytic lesions and 6-hydro~ydopamine treatment of nucleus tractus solitarius (NTS) in rat and cat. In W. De Jong, A.P. Provoost and A.P. Shapiro (Eds.), Hypertension and Brain Mechanisms, Progress in Brain Re,arch, Vol. 47, El~vier Biomedical, 1977, pp. 169-188. 20 Romagnano, M.A. and Joseph, S.A.0 Immunocytochemical localization of ACTH j'J9 in the braiustm of the rat, Brain Reg., in press. 2t Seller, H. and IIIm, M., Localization of the first synapse in the carotid baroreceptor reflex pathway and its alteration of the afferent input, Pfliigers Arch., 306 (1969) 1-19. 22 Watson, S.J. and Akil, H., aMSH in rat brain: Occurrence within and outside of ~-endorphin neurons, Brain Res., 182 (1980) 217-223. 23 Zandherg, P. and De Jong, W., a-Melhylnoradrenaline-induced hypo~nsion in the nucleus traclus solitarii of the rat: a localization study, NenropharmacololD', 16 (1977) 219-222.
221
Elsevier Scientific Publishers Ireland Ltd.
NUCLEUS S : E V I D E N ~ FOR A SECOND OPlOCOgglN NEURONAL SYSTEM
SHIRLEY A. JOSEPH, WEBSTER H. PILCHER and CAROL BENNETI'-CLARKE
The Nt~roendocrine Unit, Universit7 o f Rochester, School of Medicine and Dentist~, 601 Elmwood A~enue, Rochester, NY 14042 (U.S.A.) (Rectived March Sth, 1983; Revised version received May 4th, 1983; Accepted May 6th, 1983)
Key words: ACTH - opiocortin peptides - nucleus tractus solitarius - cardiovascular center brainstem - immunocytochemistry - anatomical localization
Immunucytochemical localization of ACTH.opioconin perikarya was demonstrated in the medulla of o;Ichicine-troated rat. Neuronal cell bodies and fibers containing ACTH-immunoractivity were abunc am in the caudal region of the nucleus tractus solitarius, spedf'gally within pars commisuralis. Location o; these opiocortin neurons within the nucleus tractus solitarius prmides additional evidence for a role of these peptides in cardiovascular functions.
The endogenous opioid peptides are represented in the central nervous system by several distinctly separate neuronal systems. These include the opiocortin [7, 12], enkephalin [9, 10] and 'dynorphin [8, I I] systems. Recognized components of the opiocortin neuronal organizations include: (a) the system where ACTH and/~endorphin, a-MSH containing pedkarya are confined to the arcuate, periarcuate region of medial basal hypothalamus [I 3] and (b) the a-MSH specific (a-2) neuronal pool residing in the dorsal and lateral hypothalamus 122]. in this study we report that existence of an additional group of opioconin neurons located in caudal medulla of the brainstem and spinal cord. Ten Sprague-Dawley rats ranging in weight from 180 to 250 g were used for this study. Forty-eight hours prior to sacrifice 100/tg of colchicine was infused stereotaxically into the lateral ventricle or "/$/~g into the 4th ventricle. Animals were pe,~fused intracardially with ice-cold 4o/0 paraformaldehyde or with Bouin's fixative. Brains were cut serially from mid-hypothalami¢ region through the entire spinal cord either on a freezing micTotome or on a vibratome at 50/~m and collected into wells containing phosphate-saline buffer, pH 7.4. lmmunostaininig procedures using the
peroxidase-antiperoxidase unlabeled antibody method and the avidin-biotin complex technique and antisera specificity have been described previously [12, 20]; antisera used for these studies included ACTH (531) and 16K ('Georgie', Mains and 0304-3940183/$ 03.00 @ 1983 Elsevier Scientific Publishers Ireland Ltd.
222 Eipper). Specif'gity controls consisted of (a) deletion of primary antisera and Co) absorption of ami-ACTH antisera and synthetic human ACTHt-j9 (2-10 tqg/ml diluted antiserum). Immunoreactive ACTH-opiocortin perikaryn were observed in caudal medulla from a level caudal to area postrema to the medullary-cord junction and within the first 2-3 segments of spinal cord. The greatest concentration of cell bodies was observed caudal to the level of the area postrema (AP) within the commissural nucleus (CnTS) dorsal to the central canal (Figs. I and 2). in Fig. I, cell bodies can be ~ at their rostral extent medial and ventral to tractus solitarius within CnTS; at this level scattered ceil bodies ~'ere observed, along a ventro-lateral axis through the nucleus reticularis medullae oblongata to the dorsal border of nucleus ambiguus and Aj catecholamine cell region. Sparsely scattered cell bodies were also observed more caudally within the first 2-3 segments of cervical spinal cord surrounding the central canal. This group of opiocortin-containing neurons were visualized only in animals in which colchieine was infused into the lateral or 4th ventricle and were immunorcactive with antisera generated against ACTHj_.~v and the 16K fragment of the proopiocortin molecule. Within the medullary regi~:t fiber immunostainmg was .~'en within all the divisions of the nucleus tractus solitarius and the tractns .~litarius throughout its caudal-rostral extent, within and surrounding the area postrema. Extending in a ventro-lateral direction from the nucleus commissuralis of nucleus tractus ~litariu.~ (NTS) and within this ventro-lateral trajectory, fibers could be traced through the nucleus reticularis medullae oblongata, nucleus ambi~uu.~ and the nucleus reticularis lateralis which corresponds to the At cateeholaminergic cell region. A few fibers were aim observed within the nucleus reticulari.~ gigantocellularis and throughout the spinal cord surrounding the central canal. In thi~ study we have described a previously unidentified group of opiocortinimmunoreactive neurons located in the caudal region of the nucleus tractus solitaflus. Although a rich innervation of ACTH and ~-endorphin immunoreactivity has been shown to distribute within this nuclear complex and its faseiculus, as well as to other as.~m:iated areas of brainstem, for example the parabrachial nucleus and raphe systems [7, 20|, this is the first report of opiocortin immunoreactive perikarya in the brainstem. The NTS has for some time been regarded as a major integration center for the regulation of cardiovascular reflexes [14|. Myriad neuroanatomical and electrophysiologic studies have contributed to the origin and identification of vi.~eral afferent projections to NTS as well as its vast reciprocal efferent projections with other regions known to influence cardiovascular homeostasis (for review see refs. I and 16). Within minutes following bilateral lesions of NTS dramatic and sustaining elevations in blood pressure ensued due to the loss of the baroreceptor reflex [5, 61 and, possibly, certain catecholaminergic influences at this site [3, 19, 23]. Escorting these reports are electrophysiological studies which demonstrate a significant fall in blood pressure and vagal bradycardia following electrical stimulation in various regions of NTS [4, 211.
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Fig. I. Photomicrograph of the dorsal medial region of lower medulla approximately tO0/Am caudal to area postrcma. Nmnerus ACTH-immunoreactive perikarya are demonstrated in the commissural nucleus of tractus solitarius (CnTS) and extend ventro-latefally to the dorsal region of nucleus ambiguu~. Cells (:an be observed ventral, medial and lateral to the tractus solitarius (TS) at this level. X, dorsal motor nucleus of the vagus; XII, hypolgiossalnucleus.
Fig. 2. Photomicrosraph of medullary-cord junction. ACTH-immunoreactive perikarya are seen within CnT$ and surrounding the central canal (C)-..
224
The effects of opioid substances and opiate ~ on cardiovascular function have been known for some time. F ~ e , it has recently been shown that depressor effects of ~,ndmphln" and ~ _ n e might inlu'l~t sympathetic tone at the levd of the IqTS. For example ~ ~'ndOt'l~in ~fusiom 11~ or infusions directly into NTS [171dick a naloxone-mvcm'lge fall in mean arterial pressure and valgal ~ c a r d i a without a s~gnificant change in respbmory rate. In addition, the dcprc~ror response was prevented and bradycardia reduced by prior injections of ~ antisem directly into N'I'S, whereas microinjection of ~t~'mlorphin antisera alone resulted in a rise in blood p r e m ~ . Even thoush ¢n~ephalincontainitqg neurons ate localized in Jq'rs [9, IOI, antisera to enkephalin had little or no effect on cardiovascular parmnetcrs when injected with identical protocol 1171. However, in the dog and at the level of nucleus mbilNus, ¢nkephalinerl~c involv¢mclllt in Val~l responses has ~ denlonstralred 1151. These studies and those of Bolme et al. 121 arc compellin$ in lisht of our new finclinl~ and s u l ~ that specific medullary centers may intclp'at¢ opioid/autonomi¢ mechanisms via a specific opiate reccptor/opioid peptide interaction. In previous studies [12, 13, 20] we described the distribution of ACTH iramunoreactiv¢ opiocortin fibers in the brainstcm of normal, non colchicine treated rats; opiocortin perikarya were not obsc~ed in the medulla and it was postulated that these brainstem fibers orilginated from perikarya of the hypothalamic arcuate opiocortin bed nuclens. The presence of a medulL'u'y UonP of pcrikarya clearly indicates a more complex origanization of the opiocortin distribution to brainstem centers. Further study is needed to elucidate the detailed distribution of these forebrain and hindbrain components and how they function in the myriad interactions of the opiocortin peptides in central autonomic functions. S.A.J. is recipient of Grant RCDA 5 K04 HD(Kg230. I Abboud, F.M., The sympathetic sygem in hypertension: gate-of-the.art review. Hypertension, 4. Suppl. II. (19112)~0S-225. 2 Bolme. P., Fuxe, K., Algnali, L.F., Bradley, R. and Smythies, J., Cardio~'ascular effects of morphine and opioid peptides followitqg inir~-istcrnal administration in chloralosc-ancsthetized rats, Europ. J. Pharmix-ol., 48 (1928) 319-324. 3 13¢ Jonlg, W., Noradrenalin¢: central inhibitory control of blood pressure and heart rate, Europ. J. Pharmacol.. 29 (1974) 179-181. 4 De Jong, W., Nijkamp, F.P. and Bohus, B., Role of noradrenalin and .qcrotonin in the central control of blood pressure in normotensive and spontaneously hypertensive rats, Arch. int. Pharmacodyn., 213 (1975) 272-284. 5 De .tong, W., Zandberg, P., Palkovits, M. and Bohus, B., Acute and chronic hyl~-rtension after lesions and transcctions of rat braimtem. In W. De .long, A.P. Provoost and A.P. Shapiro (Ed$.), lbTcrtension and Brain Mechanisms, Progress in Brain Research, Vol. 47, Elsevier Biomedical, Amsterdam, 1977, pp. 189-197. 6 Doba, N and Rots, D.J.. Acute fulminating nmrogenic hypertension produced by brainstcm lesions in the cat, Circular. Res., 32 (1973) 584-593.
225 7 Finley, J.C.W., Lindstr6m, P. and Petruz. P., lmmunocytochemical localization of l~'ndorphincomainin8 neurom in the rat brain. Neuroendocrinology. 33 (1981) 28-42. 8 Cmaldstei~A., Tachibana, S., Lowney, L.J., Hunkapiller, M. and Hood, L., Dynorphin (I-13), an c~Jraordinary pt~,pnt opioid peptide, Proc. nat. Acad. Sci. U.S.A., 76 (1979) 6666-6670.
I0 ~ f e l t i T , Tetmius~ L~, g ~ H!G!j~M~ i five neurons in the tnedulla o b l o n ~ projecting to t k s p i ~ cord, Seurosci, ~ t ~ , 14(! 979) 5$-60. I I HOIIt, V., Hammann, I., Bovermann, K., Jerlicz. M, and Herz, A., D ~ ~ n - r e l a t e d immunoreactive peptides in rat brain and pituitary, Neurosci, ~ , , 18(1980) 149-153. 12 Joseph, S.A., Immunoreactive adrenoconicotropin in rat brain: a neuroanatomical study using antberum Igenerated agaimt synthetic ACTH ' . ~ , Amer. J. Anat., 158 (1980) $33-$48. 13 Kniluge,K.M., Joseph, S.A. and Nocton, J., Topography of the ACTH-immunoreactive neurons in the basal hypothalamus of the rat brain, Brain Res., 216 (1981) 333-341. 14 Korner, P., Central nervous control of autonomic cardiovascular function, Handbook of Physiology, Sect. 2: The Cardiovascular System. Vol. I: The Heart, Amer. Physiol. Soc., Baltimore, MD, 1971, pp. 691-739. 15 Laubie. M. and Sehmitt, H., Indication for central vagal eudorphinergic control of heart rate in dogs, Europ. J. Pharmacol., 71 (1981) 401-409. 16 Palkovits, M. and Zaborszky, L., Nenrnanatomy of central cardiovascular control. Nucleus traetus solitarii: afferent and efferent neuronal connections in relation to the baroreceptor reflex arc., In W. De Jon$, A.P. Provoost and A.P. Shapiro (Eds.). Hypertension and Brain Mechanisms, Progress in Brain Research, Vol. 47, Elsevkr Biomedical, Amsterdam, 1977, pp. 9-34. 17 Petty, M.A. and De Jong, W., Cardiovascular effects of ~.x,ndorphin after microinjection into the nucleus tractus solitarii of the anesthetized rat, Europ. J. Pharmacol., 81 (1982) 449-457. 18 Petty, M.A., De Jons, W. and De Wk,d, D., An inhibitory role of ~-endorphin in central cardiovascular rqgulation, Life Sci., 30 (19S2) 18)5-1~10. !9 Reis, D.J., Doha, N., Snyder, D.W. and Nathan, M.A., Brain lesions and hypertension: Chronic lability and elevation of ~rterial pressure produ~d by electrolytic lesions and 6-hydro~ydopamine treatment of nucleus tractus solitarius (NTS) in rat and cat. In W. De Jong, A.P. Provoost and A.P. Shapiro (Eds.), Hypertension and Brain Mechanisms, Progress in Brain Re,arch, Vol. 47, El~vier Biomedical, 1977, pp. 169-188. 20 Romagnano, M.A. and Joseph, S.A.0 Immunocytochemical localization of ACTH j'J9 in the braiustm of the rat, Brain Reg., in press. 2t Seller, H. and IIIm, M., Localization of the first synapse in the carotid baroreceptor reflex pathway and its alteration of the afferent input, Pfliigers Arch., 306 (1969) 1-19. 22 Watson, S.J. and Akil, H., aMSH in rat brain: Occurrence within and outside of ~-endorphin neurons, Brain Res., 182 (1980) 217-223. 23 Zandherg, P. and De Jong, W., a-Melhylnoradrenaline-induced hypo~nsion in the nucleus traclus solitarii of the rat: a localization study, NenropharmacololD', 16 (1977) 219-222.