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ACTH-related peptide containing neurons within the medulla oblongata of the rat
Brain Research, 276 (1983) 351-356 Elsevier
351
ACTH-related peptide containing neurons within the medulla oblongata of the rat* DANIEL G. SCHWARTZBERG**and PAUL K. NAKANE Department of Pathology, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver, Colorado 80262 (U.S.A.)
(Accepted May 31st, 1983) Key words: ACTH - - fl-endorphin-- nucleus tractus solitarius - - reticular formation- - brainstem - - immunohistochemistry
Neurons containing antigenic determinants of 16 Kdalton fragment, ACTH, 7-LPH, and fl-endorphin have been identified in the nucleus traetus solitarius and lateral reticular formation of the rat brainstem. Immunoreactivefibers extend longitudinally in dorsal and ventral midline tracts throughout the length of the brainstem, and are also concentrated in lateral reticular regions known to contain catecholamine nuclei. ACTH-containingneurons could participate with brainstem catecholamine cell groups in controllingvisceral function. The presence of antigenic determinants of proACTH/endorphin in brain has been shown immunohistochemically by a number of investigators 20. The consensus of these investigators is that these antigenic determinants coexist within a population of neurons whose perikarya exist only within the posterior basal hypothalamus and give rise to the widespread ACTH-related peptide (ACTH-RP)-containing fibers throughout the brain 7. Contrary to this consensus, however, another population of perikarya with ACTH-RP may exist in the brainstem, since neither surgicall9 nor chemical2, 5 destruction of arcuate nucleus areas with ACTH-RP-containing perikarya totally ablates A C T H - R P immunoreactivity (IR) in the brainstem. We have investigated the distribution of ACTHRP antigenic determinants in the rat brainstem immunohistochemically, and have identified another population of neurons containing ACTH-RP antigenic determinants within the nucleus tractus solitarius (nTS), and a widespread immunoreactive fiber distribution in midline raphe and lateral reticular areas. Male and female albino CDF rats (Charles River Breeding Labs, Wilmington, MA) weighing 175-225 g were maintained on standard laboratory diet with a 12-h light-dark cycle. In addition, neonatal rats aged 3, 6 and 18 days were used. Forty-eight h prior to sac-
rifice, some adult animals received 50/zg colchicine in 50/A sterile saline in the lateral cerebral ventricle. Adult animals were injected i.p. with 65 mg of sodium pentobarbital, perfused transaortically with 0.05 M phosphate buffered saline, pH 7.2 (PBS) for 1 min and perfused with paraformaldehyde-picric acid fixative 21 for 20 min. Brain and pituitary from adult and neonatal animals were dissected en bloc, and placed in fixative for an additional period of at least 24 h followed by washing with several changes in PBS with 5% sucrose for at least 24 h. Tissues were then processed for immunohistochemical studies by the indirect peroxidase-labeled antibody method as described previously 15. Both sagittal and transverse serial sections (12-16/~m) were made from rostral diencephalon to spinal cord regions of the brains. Sections were taken in groups of 3 to a slide and approximately every fifth slide was immunostained. Antisera to ACTH, fl-endorphin, ~,-LPH, and 16 Kdalton fragment used in this study were gifts of Drs. Mains and Eipper (University of Colorado Health Sciences Center, Denver, CO). The specificity of the antisera was established as described previouslylS. Medulla. Within the medulla a population of cell bodies immunoreactive for 16 Kdalton fragment exists within the commissural nucleus and rostrally within the nTS up to the level of the area postrema. These are oval 10--15 ~m bipolar cells with their ma-
* This study was presented at the llth Annual Meeting of the Societyfor Neuroscience, Los Angeles, CA 1981. ** Present address for correspondence: Dept. of Radiology, Brigham and Women's Hospital, 75 Francis Street, Boston, MA 02115, U.S.A. 0006-8993/83/$03.00(~ 1983Elsevier Science Publishers B.V.
353
Fig. 2. Neurons within the nucleus tractus solitarius immunostained for y-LPH, fl-endorphin and ACTH. Scale bar = 14 /tm.
clei and the pyramidal decussation, and also were localized within the lateral reticular region in areas known to contain the A1 and A 3 noradrenergic cell populations (Fig. 3a). The cells of origin of these fibers could not be ascertained although these fibers a p p e a r e d to arise from the nTS in arching projections up to 100 g m long. These lateral A C T H - R P fibers were furthermore observed to be concentrated discretely over certain cell bodies and their processes and not r a n d o m l y over intervening areas (Fig. 3b). In addition, occasional neurons were stained strongly in the lateral reticular formation of pons and medulla (Fig. 4). There was a weak diffuse diaminobenzidine staining over the cell bodies of the large hypogiossal motor neurons and large reticular neurons but not their fibers when the specific sera, the specific sera preabsorbed with antigen, or normal rabbit sera were used (Fig. l b ) . In neonatal rats, the A C T H - R P - c o n taining neurons were present within the nTS in the same anatomic distribution as in the adult as early as day 3 of neonatal life, although their fibers stained with less intensity than in the adult. Pons and mesencephalon. No immunoreactive cell groups were found within the pons. In coronal sections, as in the medulla, dense collections of immunoreactive fibers were present in the midline raphe areas dorsal and lateral to the corticospinal tract. Fi-
Fig. 3. Coronal section of the ventral medulla of the rat at the level of the nTS, immunostained for 16 Kdalton fragment, a: immunoreactive fibers are contained within a portion of the lateral reticular formation (-,). Scale bar = 400gm. b: immunoreactive fibers and varicosities ( 1' ) are clustered over the perikarya (p) and processes (open arrows) of some large reticular neurons and not randomly within the surrounding neuropil. Scale bar = 20/~m.
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Fig. 4. Medullary reticular formation, immunostained for 16 Kdalton fragment. A single neuron is apparent (~); a slight diffuse non-specificstaining of the perikarya of large reticular neurons may be seen. Scale bar = 100~m
bers were also apparent laterally over reticular areas which contain the A5 and A7 noradrenergic cell groups as well as dorsally at the lateral tips of the fourth ventricle in the region of the parabrachial nuclei adjacent to the locus coeruleus. At the level of the inferior colliculus, heavy fiber densities were identified in the periaqueductal gray and appeared to extend out laterally toward lateral reticular areas. The immunoreactive fibers were also seen in the midline and lateral to the corticospinal tracts. At the pontomesencephalic junction, fibers were found in ventrolateral periaqueductal gray and in midline areas including the dorsal raphe nuclei and the region of the A8 cell group. Further rostrally, immunoreactive fibers were present on the dorsal and lateral aspects of the interpeduncular nucleus, within the periaqueductal gray, dorsal to the crus cerebri and medial to the medial geniculate nucleus but not within the substantia nigra. In paramedian sagittal sections, longitudinal fibers hundreds of microns long were present at pontine and medullary levels beneath the ventricle and near the ventral surface. As in the transverse sections, a heavy ACTH-RP fiber distribution was apparent within the nTS both rostral and caudal to the cell group. Long A C T H - R P fibers also extended into the spinal cord.
Diencephalon. The distribution of ACTH-RP differs little from that reported by other authors20. Midline fibers were seen in ventral regions dorsal to the crus cerebri and surrounding the mammillary bodies, and rostrally surrounding but not penetrating the suprachiasmatic and supraoptic nuclei. The basal ganglia did not contain ACTH-RP structures. This report demonstrates the existence of a population of neurons containing 4 antigenic determinants of the pro-ACTH/endorphin precursor, located in the nucleus tractus solitarius. This finding is contrary to the current understanding that only one ACTH-RP-containing neuronal population exists and is confined to the hypothalamus6. 7.20. The nTS is a region in the dorsal medulla and is the initial site of integration of visceral inputs ~0~7. Cells of the nTS project to preganglionic sympathetic neurons in the intermediolateral cell column, to lateral reticular adrenergic groups, and to phrenic and thoracic ventral horn neurons. In addition, the cells of nTS reciprocally innervate and are innervated by a number of forebrain structures including the basal hypothalamus-arcuate region 12.18. Indeed, reciprocal pathways between brainstem noradrenergic cell groups including the A2 cell group and anterior hypothalamic vasopressin cell groups have been demonstrated histochemically TM.
355 In sagittal sections of the brainstem, A C T H - R P cells in the ffTS~glve rise to fibers projecting anteriorly, and long ACTH-RP-containing fibers extend rostrocaudally in the dorsal periventricular bundle. This distribution of cells and of fibers closely parallels that of the A2 cell group s suggesting that A C T H - R P nTS cells may also innervate forebrain structures via a fiber system ascending in the periaqueductal region. In addition to the dorsal periventricular system, two other patterns of fiber distribution were present in the ventral areas. In one pattern, A C T H - R P fibers extended laterally from the NTS cells and appeared to arch across the lateral reticular formation and to terminate on the large reticular neurons in the region of the A1 cell group as well as on other neurons situated in more rostral areas which contain the lateral reticular catecholamine groups. Whether an association of A C T H - R P fibers with noradrenergic brainstem neurons represents a synaptic relationship needs to be clarified by immunoelectron microscopic techniques. In the other pattern, A C T H - R P fibers were seen in sagittal section extending hundreds of microns in the ventral midline throughout the length of the brainstem. Although neither the source nor the termination point of individual ventral fibers was systematically defined, one possibility is that this system originates from A C T H - R P hypothalamic neurons. Another possible source for these fibers is the strongly-stained cells occasionally found in the medial reticular formation, which may correspond to fl-endorphin-IR cells noted by others 3. However, as these cells were infrequent and not localized to a particular nucleus, it was not possible to trace the fiber projections from these cells. Whether the A C T H - R P nTS cells contain the same peptides and play a role similar to the ACTHRP hypothalamic cells remains to be established. Although their morphology, antigenic specificity and the reactivity to antibodies are identical, there is a possibility that these two cell groups may elaborate different products, just as the A C T H - R P cells in the anterior and intermediate lobes of the pituitary gland stain similarly immunohistochemically, yet have different peptide processing 1. The A C T H - R P hypotha-
lamic cells arise early in the developing embryo15; whether the nTS cells similarly appear early in embryogenesis is unknown, although we have found them in their adult location as early as day 3 in the neonatal rat. The anatomic location of A C T H - R P cells and fibers within the nTS may give a clue as to their physiological role since the nTS is the initial site for integration of visceral afferents, in particular afferents from the cardiovascular system4,9A°. A C T H - R P cells in this region may also be involved in the processing of these inputs. If the A C T H - R P cells in the nTS contain opiate-active peptides, as the corticotrophs do, then these nTS cells may represent an anatomic substrate for the well-described opiate effects on visceral function, particlarly in view of the recent demonstration of opiate receptors within the nTS 16. A C T H - R P fibers are in close proximity as to the A1 and A2 cell groups which are known to be involved in blood pressure control and cardiovascular responses 11. If ACTH-RP fibers do make synaptic connections with A1 and A2 cells, it is possible that A C T H - R P cells may exert effects on visceral function through their actions on the A1 or A2 cell groups. Two previous reports have suggested that fl-endorphin antigenic determinants exist in other brainstem cells, particularly within the visceral motor nucleP, 13. We were unable to reproduce these findings using our antisera. We observed diffuse non-specific staining over these cells using both preimmune and specific antisera which had been preabsorbed with their respective antigens. The existence of a second population of ACTHRP immunoreactive neurons broadens the potential functional significance of these peptides in brain, and requires modification of conclusions drawn from experiments which are based upon the assumption of a single anatomic source for such peptides in brain. Supported in part by National Institutes of Health Fellowship Grant AM-06569, Grants AI-09109 and CA-15823 from the National Institutes of Health, and a departmental gift from R. J. Reynolds Indistries, Inc.
356 1 Eipper, B. A. and Mains, R. E., Structure and biosynthesis of proadrenocorticotropin/endorphin and related peptides, Endocrine Rev.. 1 (1980) 1-27. 2 Eskay, R. L., Brownstein, M. J. and Long, R. T., a-melanocyte-stimulating hormone: reduction in adult rat brain after monosodium glutamate treatment of neonates, Science, 205 (1979) 827-829. 3 Finley, J. C. W., Lindstrom, P. and Petrusz, P., Immunocytochemical localization of/3-endorphin-containing neurons in the rat brain, Neuroendocrinology, 33 (1981) 28-42. 4 Kalia, M. and Mesulam, M.-M., Brain stem projections of sensory and motor components of the vagus complex in the cat. II. Laryngeal, tracheobronchial, pulmonary, cardiac, and gastrointestinal branches. J. cornp. Neurol., 193 (1980) 467-508. 5 Krieger, D. T., Liotta, A. S., Nicholsen, G. and Kizer, J. S., Brain ACTH and endorphin reduced in rats with monosodium glutamate-induced arcuate nuclear lesions, Nature tLond.), 278 (1979) 62-63. 6 Krieger, D. T., Liotta, A. S., Brownstein, M. J. and Zimmerman, E. A., ACTH, fl-lipotropin, and related peptides in brain, pituitary, and blood, Recent Progr. Horm. Res., 36 (1980) 277-344. 7 Krieger, D. T. and Martin, J. B., Brain peptides, N. Engl. J. Med., 304 (1981) 876-885. 8 Lindvall, O. and Bj6rklund, A., Organization of catecholamine neurons in the rat central nervous system. In L. Iversen, S. Iversen and S. H. Snyder (Eds.), Handbook of Psychopharmacology, Plenum Press, Amsterdam, 1978, pp. 139-231. 9 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. 10 Loewy, A. D. and McKellar, S., The neuroanatomical basis of central cardiovascular control, Fed. Proc., 39 (1980) 2495-25O3. 11 Reis. D. J., The nucleus tractus solitarius and experimental neurogenic hypertension: evidence for a central neural imbalance hypothesis of hypertensive disease. In J. B. Mar-
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tin, S. Reichlin and K. L. Bick (Eds.), Neurosecretion and Brain Peptides, Raven Press, New York, 1981, pp. 409-420. Richardo, 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. Salih, H., Panerai, A. E. and Friesen, H. G., Cellular distribution of fl-endorphin-like substance in the rat pituitary and brain, Life Sci., 25 (1979) 111-118. Sawchenko, P. E. and Swanson, L., Central noradrenergic pathways for the integration of hypothalamic neuroendocrine and anatomic responses, Science. 214 (1981) 685-687. Schwartzberg, D. G. and Nakane, P. K., Ontogenesis of adrenocorticotropin-related peptide determinants in the hypothalamus and pituitary gland of the rat, Endocrinology, 110 (1982) 855-864. Simantov, R., Kuhar, M. J., Uhl, G. R. and Snyder, S. H., Opioid peptide enkephalin: immunohistochemical mapping in rat central nervous system, Proc. nat. Acad, Sci. U.S.A.. 74 (1977) 2167-2171. Spyer, K. M., Neural organization and control of the baroreceptor reflex, Rev. Physiol. Biochem. Pharmacol., 88 (1981) 23-124. Swanson, L. W. and Sawchenko, P. E., Paraventricular nucleus: A site for the integration of neuroendocrine and anatomic mechanisms, Neuroendocrinology, 31 (1980) 410-417. Watson, S. J., Akil, H., Richard, C. W., III and Barchas, J. D., Evidence for two separate opiate peptide neuronal systems, Nature (Lond.), 275 (1978) 226-338. Watson, S. J. and Akil, H., Opioid peptides and related substances: immunocytochemistry. In J. B. Martin, S. Reichlin and K. L. Bick (Eds.), Neurosecretion and Brain Peptides, Raven Press, New York, 1981, pp. 77-86. Zamboni, L. and DeMartino, C., Buffered picric acid-formaldehyde: a new rapid fixative for electron microscopy, J. Cell Biol., 35(1967) 148A.
351
ACTH-related peptide containing neurons within the medulla oblongata of the rat* DANIEL G. SCHWARTZBERG**and PAUL K. NAKANE Department of Pathology, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver, Colorado 80262 (U.S.A.)
(Accepted May 31st, 1983) Key words: ACTH - - fl-endorphin-- nucleus tractus solitarius - - reticular formation- - brainstem - - immunohistochemistry
Neurons containing antigenic determinants of 16 Kdalton fragment, ACTH, 7-LPH, and fl-endorphin have been identified in the nucleus traetus solitarius and lateral reticular formation of the rat brainstem. Immunoreactivefibers extend longitudinally in dorsal and ventral midline tracts throughout the length of the brainstem, and are also concentrated in lateral reticular regions known to contain catecholamine nuclei. ACTH-containingneurons could participate with brainstem catecholamine cell groups in controllingvisceral function. The presence of antigenic determinants of proACTH/endorphin in brain has been shown immunohistochemically by a number of investigators 20. The consensus of these investigators is that these antigenic determinants coexist within a population of neurons whose perikarya exist only within the posterior basal hypothalamus and give rise to the widespread ACTH-related peptide (ACTH-RP)-containing fibers throughout the brain 7. Contrary to this consensus, however, another population of perikarya with ACTH-RP may exist in the brainstem, since neither surgicall9 nor chemical2, 5 destruction of arcuate nucleus areas with ACTH-RP-containing perikarya totally ablates A C T H - R P immunoreactivity (IR) in the brainstem. We have investigated the distribution of ACTHRP antigenic determinants in the rat brainstem immunohistochemically, and have identified another population of neurons containing ACTH-RP antigenic determinants within the nucleus tractus solitarius (nTS), and a widespread immunoreactive fiber distribution in midline raphe and lateral reticular areas. Male and female albino CDF rats (Charles River Breeding Labs, Wilmington, MA) weighing 175-225 g were maintained on standard laboratory diet with a 12-h light-dark cycle. In addition, neonatal rats aged 3, 6 and 18 days were used. Forty-eight h prior to sac-
rifice, some adult animals received 50/zg colchicine in 50/A sterile saline in the lateral cerebral ventricle. Adult animals were injected i.p. with 65 mg of sodium pentobarbital, perfused transaortically with 0.05 M phosphate buffered saline, pH 7.2 (PBS) for 1 min and perfused with paraformaldehyde-picric acid fixative 21 for 20 min. Brain and pituitary from adult and neonatal animals were dissected en bloc, and placed in fixative for an additional period of at least 24 h followed by washing with several changes in PBS with 5% sucrose for at least 24 h. Tissues were then processed for immunohistochemical studies by the indirect peroxidase-labeled antibody method as described previously 15. Both sagittal and transverse serial sections (12-16/~m) were made from rostral diencephalon to spinal cord regions of the brains. Sections were taken in groups of 3 to a slide and approximately every fifth slide was immunostained. Antisera to ACTH, fl-endorphin, ~,-LPH, and 16 Kdalton fragment used in this study were gifts of Drs. Mains and Eipper (University of Colorado Health Sciences Center, Denver, CO). The specificity of the antisera was established as described previouslylS. Medulla. Within the medulla a population of cell bodies immunoreactive for 16 Kdalton fragment exists within the commissural nucleus and rostrally within the nTS up to the level of the area postrema. These are oval 10--15 ~m bipolar cells with their ma-
* This study was presented at the llth Annual Meeting of the Societyfor Neuroscience, Los Angeles, CA 1981. ** Present address for correspondence: Dept. of Radiology, Brigham and Women's Hospital, 75 Francis Street, Boston, MA 02115, U.S.A. 0006-8993/83/$03.00(~ 1983Elsevier Science Publishers B.V.
353
Fig. 2. Neurons within the nucleus tractus solitarius immunostained for y-LPH, fl-endorphin and ACTH. Scale bar = 14 /tm.
clei and the pyramidal decussation, and also were localized within the lateral reticular region in areas known to contain the A1 and A 3 noradrenergic cell populations (Fig. 3a). The cells of origin of these fibers could not be ascertained although these fibers a p p e a r e d to arise from the nTS in arching projections up to 100 g m long. These lateral A C T H - R P fibers were furthermore observed to be concentrated discretely over certain cell bodies and their processes and not r a n d o m l y over intervening areas (Fig. 3b). In addition, occasional neurons were stained strongly in the lateral reticular formation of pons and medulla (Fig. 4). There was a weak diffuse diaminobenzidine staining over the cell bodies of the large hypogiossal motor neurons and large reticular neurons but not their fibers when the specific sera, the specific sera preabsorbed with antigen, or normal rabbit sera were used (Fig. l b ) . In neonatal rats, the A C T H - R P - c o n taining neurons were present within the nTS in the same anatomic distribution as in the adult as early as day 3 of neonatal life, although their fibers stained with less intensity than in the adult. Pons and mesencephalon. No immunoreactive cell groups were found within the pons. In coronal sections, as in the medulla, dense collections of immunoreactive fibers were present in the midline raphe areas dorsal and lateral to the corticospinal tract. Fi-
Fig. 3. Coronal section of the ventral medulla of the rat at the level of the nTS, immunostained for 16 Kdalton fragment, a: immunoreactive fibers are contained within a portion of the lateral reticular formation (-,). Scale bar = 400gm. b: immunoreactive fibers and varicosities ( 1' ) are clustered over the perikarya (p) and processes (open arrows) of some large reticular neurons and not randomly within the surrounding neuropil. Scale bar = 20/~m.
354
Fig. 4. Medullary reticular formation, immunostained for 16 Kdalton fragment. A single neuron is apparent (~); a slight diffuse non-specificstaining of the perikarya of large reticular neurons may be seen. Scale bar = 100~m
bers were also apparent laterally over reticular areas which contain the A5 and A7 noradrenergic cell groups as well as dorsally at the lateral tips of the fourth ventricle in the region of the parabrachial nuclei adjacent to the locus coeruleus. At the level of the inferior colliculus, heavy fiber densities were identified in the periaqueductal gray and appeared to extend out laterally toward lateral reticular areas. The immunoreactive fibers were also seen in the midline and lateral to the corticospinal tracts. At the pontomesencephalic junction, fibers were found in ventrolateral periaqueductal gray and in midline areas including the dorsal raphe nuclei and the region of the A8 cell group. Further rostrally, immunoreactive fibers were present on the dorsal and lateral aspects of the interpeduncular nucleus, within the periaqueductal gray, dorsal to the crus cerebri and medial to the medial geniculate nucleus but not within the substantia nigra. In paramedian sagittal sections, longitudinal fibers hundreds of microns long were present at pontine and medullary levels beneath the ventricle and near the ventral surface. As in the transverse sections, a heavy ACTH-RP fiber distribution was apparent within the nTS both rostral and caudal to the cell group. Long A C T H - R P fibers also extended into the spinal cord.
Diencephalon. The distribution of ACTH-RP differs little from that reported by other authors20. Midline fibers were seen in ventral regions dorsal to the crus cerebri and surrounding the mammillary bodies, and rostrally surrounding but not penetrating the suprachiasmatic and supraoptic nuclei. The basal ganglia did not contain ACTH-RP structures. This report demonstrates the existence of a population of neurons containing 4 antigenic determinants of the pro-ACTH/endorphin precursor, located in the nucleus tractus solitarius. This finding is contrary to the current understanding that only one ACTH-RP-containing neuronal population exists and is confined to the hypothalamus6. 7.20. The nTS is a region in the dorsal medulla and is the initial site of integration of visceral inputs ~0~7. Cells of the nTS project to preganglionic sympathetic neurons in the intermediolateral cell column, to lateral reticular adrenergic groups, and to phrenic and thoracic ventral horn neurons. In addition, the cells of nTS reciprocally innervate and are innervated by a number of forebrain structures including the basal hypothalamus-arcuate region 12.18. Indeed, reciprocal pathways between brainstem noradrenergic cell groups including the A2 cell group and anterior hypothalamic vasopressin cell groups have been demonstrated histochemically TM.
355 In sagittal sections of the brainstem, A C T H - R P cells in the ffTS~glve rise to fibers projecting anteriorly, and long ACTH-RP-containing fibers extend rostrocaudally in the dorsal periventricular bundle. This distribution of cells and of fibers closely parallels that of the A2 cell group s suggesting that A C T H - R P nTS cells may also innervate forebrain structures via a fiber system ascending in the periaqueductal region. In addition to the dorsal periventricular system, two other patterns of fiber distribution were present in the ventral areas. In one pattern, A C T H - R P fibers extended laterally from the NTS cells and appeared to arch across the lateral reticular formation and to terminate on the large reticular neurons in the region of the A1 cell group as well as on other neurons situated in more rostral areas which contain the lateral reticular catecholamine groups. Whether an association of A C T H - R P fibers with noradrenergic brainstem neurons represents a synaptic relationship needs to be clarified by immunoelectron microscopic techniques. In the other pattern, A C T H - R P fibers were seen in sagittal section extending hundreds of microns in the ventral midline throughout the length of the brainstem. Although neither the source nor the termination point of individual ventral fibers was systematically defined, one possibility is that this system originates from A C T H - R P hypothalamic neurons. Another possible source for these fibers is the strongly-stained cells occasionally found in the medial reticular formation, which may correspond to fl-endorphin-IR cells noted by others 3. However, as these cells were infrequent and not localized to a particular nucleus, it was not possible to trace the fiber projections from these cells. Whether the A C T H - R P nTS cells contain the same peptides and play a role similar to the ACTHRP hypothalamic cells remains to be established. Although their morphology, antigenic specificity and the reactivity to antibodies are identical, there is a possibility that these two cell groups may elaborate different products, just as the A C T H - R P cells in the anterior and intermediate lobes of the pituitary gland stain similarly immunohistochemically, yet have different peptide processing 1. The A C T H - R P hypotha-
lamic cells arise early in the developing embryo15; whether the nTS cells similarly appear early in embryogenesis is unknown, although we have found them in their adult location as early as day 3 in the neonatal rat. The anatomic location of A C T H - R P cells and fibers within the nTS may give a clue as to their physiological role since the nTS is the initial site for integration of visceral afferents, in particular afferents from the cardiovascular system4,9A°. A C T H - R P cells in this region may also be involved in the processing of these inputs. If the A C T H - R P cells in the nTS contain opiate-active peptides, as the corticotrophs do, then these nTS cells may represent an anatomic substrate for the well-described opiate effects on visceral function, particlarly in view of the recent demonstration of opiate receptors within the nTS 16. A C T H - R P fibers are in close proximity as to the A1 and A2 cell groups which are known to be involved in blood pressure control and cardiovascular responses 11. If ACTH-RP fibers do make synaptic connections with A1 and A2 cells, it is possible that A C T H - R P cells may exert effects on visceral function through their actions on the A1 or A2 cell groups. Two previous reports have suggested that fl-endorphin antigenic determinants exist in other brainstem cells, particularly within the visceral motor nucleP, 13. We were unable to reproduce these findings using our antisera. We observed diffuse non-specific staining over these cells using both preimmune and specific antisera which had been preabsorbed with their respective antigens. The existence of a second population of ACTHRP immunoreactive neurons broadens the potential functional significance of these peptides in brain, and requires modification of conclusions drawn from experiments which are based upon the assumption of a single anatomic source for such peptides in brain. Supported in part by National Institutes of Health Fellowship Grant AM-06569, Grants AI-09109 and CA-15823 from the National Institutes of Health, and a departmental gift from R. J. Reynolds Indistries, Inc.
356 1 Eipper, B. A. and Mains, R. E., Structure and biosynthesis of proadrenocorticotropin/endorphin and related peptides, Endocrine Rev.. 1 (1980) 1-27. 2 Eskay, R. L., Brownstein, M. J. and Long, R. T., a-melanocyte-stimulating hormone: reduction in adult rat brain after monosodium glutamate treatment of neonates, Science, 205 (1979) 827-829. 3 Finley, J. C. W., Lindstrom, P. and Petrusz, P., Immunocytochemical localization of/3-endorphin-containing neurons in the rat brain, Neuroendocrinology, 33 (1981) 28-42. 4 Kalia, M. and Mesulam, M.-M., Brain stem projections of sensory and motor components of the vagus complex in the cat. II. Laryngeal, tracheobronchial, pulmonary, cardiac, and gastrointestinal branches. J. cornp. Neurol., 193 (1980) 467-508. 5 Krieger, D. T., Liotta, A. S., Nicholsen, G. and Kizer, J. S., Brain ACTH and endorphin reduced in rats with monosodium glutamate-induced arcuate nuclear lesions, Nature tLond.), 278 (1979) 62-63. 6 Krieger, D. T., Liotta, A. S., Brownstein, M. J. and Zimmerman, E. A., ACTH, fl-lipotropin, and related peptides in brain, pituitary, and blood, Recent Progr. Horm. Res., 36 (1980) 277-344. 7 Krieger, D. T. and Martin, J. B., Brain peptides, N. Engl. J. Med., 304 (1981) 876-885. 8 Lindvall, O. and Bj6rklund, A., Organization of catecholamine neurons in the rat central nervous system. In L. Iversen, S. Iversen and S. H. Snyder (Eds.), Handbook of Psychopharmacology, Plenum Press, Amsterdam, 1978, pp. 139-231. 9 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. 10 Loewy, A. D. and McKellar, S., The neuroanatomical basis of central cardiovascular control, Fed. Proc., 39 (1980) 2495-25O3. 11 Reis. D. J., The nucleus tractus solitarius and experimental neurogenic hypertension: evidence for a central neural imbalance hypothesis of hypertensive disease. In J. B. Mar-
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