Peptidergic regulation in neuroendocrine and autonomic systems

Peptides. Vol. 5, Suppl. 1, pp. 101-107, 1984. ~ Ankho International Inc. Printed in the U.S.A.

0196-9781/84 $3.00 + .00

Peptidergic Regulation in Neuroendocrine and Autonomic Systems R. E L D E , I V. S E Y B O L D , R. L . S O R E N S O N , S. C U M M I N G S , V. H O L E T S , " D. O N S T O T T , C. S A S E K , D. E. S C H M E C H E L * A N D W. H . O E R T E L ?

Department of Anatomy, University of Minnesota Division of Neurology, *Duke University Medical Center and Neurologisehe Klinik, ~Technische Universitaet Munehen

ELDE, R., V. SEYBOLD, R. L. SORENSON, S. CUMMINGS, V. HOLETS, D. ONSTOTT, C. SASEK, D. E. SCHMECHEL AND W. H. OERTEL. Peptidergic regulation in neuroendocrine and autonomic systems. PEPTIDES 5: Suppl 1, 101-107, 1984.--Neuropeptides are found in dense networks of neuronal perikarya, fibers and terminals within numerous brain regions. Among the more striking of these collections are sites within the central nervous system that are presumed to regulate either endocrine or autonomic function. A recent example of a neuropeptide which is likely to play a significant role in endocrine regulation is cortocotropin releasing factor (CRF). Immunohistochemical studies revealed that CRF immunoreactivity was found in many brain regions, including the paraventriculo-infundibular pathway. CRF released from nerve terminals belonging to this pathway presumably regulates ACTH release. Treatment of rats with reserpine depletes CRF as well as vasopressin from the external layer of the median eminence, suggesting tonic, monoaminergic inhibition of CRF and vasopressin containing neurons. CRF antisera were found which stain urotensin I immunoreactivity within the caudal neurosecretory system of fish. Numerous putative neurotransmitters impinge upon preganglionic sympathetic neurons within the intermediolateral cell column of the spinal cord. Preganglionic sympathetic neurons which innervate the adrenal medulla appear to have a specific input from somatostatin immunoreactive fibers. In addition, binding sites for serotonin and alpha-2 adrenergic ligands are more highly concentrated over sympathoadrenal neurons. Finally, the pancreatic islet contains peptide producing endocrine cells which possess several neuron-like properties. Some of these properties are reviewed, especially the finding that the insulin producing cells contain glutamate decarboxylase immunoreactivity, the biosynthetic enzyme for GABA. Further studies revealed that GABA agonists inhibit somatostatin release from islet cells. Neuropeptides Corticotropin releasing factor Urotensin I Neuroendocrine Adrenal medulla Pancreatic islets Caudal neurosecretory system Autonomic nervous system Monoamines

GABA

such as those within the pancreatic islet and adrenal medulla, are at least partially regulated by the n e r v o u s system through input from the autonomic nervous system. Interestingly, these and other peptide-secreting endocrine cells express many neuron-like properties, and therefore, may not differ greatly from n e u r o s e c r e t o r y neurons, e x c e p t that they lie outside of the central nervous system.

S E V E R A L architectural arrangements have e v o l v e d to enable neural regulation of endocrine tissues. Perhaps the most primitive of these arrangements is exhibited by invertebrates in which certain neurons function as the basic endocrine unit. T h e s e neurons synthesize, store and secrete a peptide hormone in response to appropriate stimuli [ 14]. Vertebrates have retained this arrangement in the form of hypothalamic n e u r o s e c r e t o r y neurons which deliver their h o r m o n a l products to capillary networks in the posterior lobe o f the pitui° tary or the external layer of the median eminence. An additional n e u r o s e c r e t o r y system is found in most species of fishes. This system has been termed the " c a u d a l neurosecretory s y s t e m " because the neuronal cell bodies are found within caudal segments of the spinal cord and the neurohemal a r r a n g e m e n t of axon terminals, the urophysis, is found in the most caudal segment of the cord [11]. Other peptide and m o n o a m i n e - p r o d u c i n g endocrine cells,

NEUROSECRETORY NEURONS, CORTICOTROPIN RELEASING FACTOR AND UROTENSIN I N e u r o s e c r e t o r y neurons within the vertebrate hypothalamus can be divided into two broad categories: those which regulate distant target organs such as the kidney and gravid uterus; and those which regulate a m o r e proximal target organ, the anterior lobe of the pituitary. The former neurons are known to release vasopressin and o x y t o c i n from nerve terminals in the posterior lobe of the pituitary, whereas the

~Requests for reprints should be addressed to Dr. Robert Elde, Department of Anatomy, University of Minnesota, 321 Church Street S.E., Minneapolis, MN 55455. 2Present Address: Department of Psychobiology, University of California, Irvine.

101

102 latter neurons are known to release hypophysiotropic hormones from n e r v e terminals in the external layer of the median eminence. The isolation and chemical characterization of the elusive corticotropin releasing factor (CRF) has finally been realized by Vale and colleagues ([36,56]; see also Vale, this volume). The amino acid sequence of the biosynthetic precursor to CRF, p r e - p r o C R F has also been predicted by c D N A analysis [13]. These findings have enabled studies which, in turn, have expanded our notions of n e u r o e n d o c r i n e mechanisms. Firstly, the hypothalamic paraventricular nucleus now seems even more important in n e u r o e n d o c r i n e as well as autonomic regulation and more c o m p l e x l y organized [43, 50, 57]. This nucleus contains a striking n u m b e r of C R F iramunoreactive neurons ([1, 5, 6, 7, 8, 10, 23, 31, 39, 40, 51, 53]; see also Paull, Fellman, Palkovits and Merchanthaler, this volume) which project, in all likelihood to the external layer of the median eminence [53]. Previous work had shown neurons of this nucleus to contain vasopressin [24, 39, 43, 59, 61, 63], oxytocin [24, 43, 59, 63], dynorphin [40,61], enkephalin [24], cholecystokinin octapeptide [58], and neurotensin ([18,55]; see also lbata, this volume) immunoreactivities. Thus, it was likely to be established that C R F immunoreactivity co-existed within one or more of these previously identified populations of paraventricular neurons. Indeed, serial section analysis has suggested the co-existence o f C R F and vasopressin [39], C R F and dynorphin [40] and C R F and oxytocin [8] in rat paraventricular neurons. Work from our laboratory indirectly indicates that under some circumstances vasopressin and C R F are likely to be released concomitantly from nerve terminals in the median e m i n e n c e , since treatment of rats with reserpine, a potent stimulus of A C T H release, selectively depletes vasopressin [44] as well as C R F i m m u n o r e a c t i v i t y from the external layer [9]. These m a n e u v e r s do not deplete oxytocin. Thus, we hypothesize that C R F and vasopressin neurons are under a tonic inhibition mediated by m o n o a m i n e r g i c neurons. The functional significance of the possible co-release of vasopressin and C R F may be related to the p h e n o m e n o n reported by Turkelson and colleagues who found an augmentation of the action of C R F on the anterior pituitary by vasopressin ([54]; see also Vigh, this volume). Certain physiological stimuli, such as stress, may also cause simultaneous release of vasopressin and C R F (see [35]). Secondly, the study of C R F i m m u n o r e a c t i v i t y has also shown, as for other hypophysiotropic h o r m o n e s , a significant distribution of C R F - p o s i t i v e neurons outside of the hypothalamic n e u r o e n d o c r i n e system. We [10] and others ([23,51]; see also Merchanthaler, this volume) are in substantial a g r e e m e n t concerning the e x t r a h y p o t h a l a m i c occurrence of C R F in rat brain. The most prominent sites containing C R F - p o s i t i v e perikarya are the bed nucleus of the stria terminalis, the central nucleus of the amygdala, the region of the dorsal raphe, locus coeruleus, the external cuneate nucleus and the medullary reticular formation. Thus, C R F , as for n u m e r o u s other neuropeptides, should also be considered a putative neurotransmitter within the central nervous system. Thirdly, since the isolation of ovine C R F and more recently rat C R F ([36]; see Vale, this volume), analogs of C R F have been discovered in lower vertebrates (Table 1). For a number of years Lederis and colleagues have methodically purified peptide fractions from extracts of the caudal neurosecretory system of fish. One peptide, urotensin I, was charac-

E L D E El" A L . TABLE 1 AMINO ACID SEQUENCES OF CRF AND R E L A T E D PEPTIDES*

CRF ovine" 1 2 3 4 5 6 7 8 9 l0 I1 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

Ser Gin Glu Pro Pro Ile Ser Leu Asp Leu Thr Phe His Leu Leu Arg Glu Val Leu Glu Met Thr Lys Ala Asp Gln Leu Ala Gin Gin A la His Ser Asn Arg Lys Leu Leu Asp lie Ala-N H_,

Urotensin I rat ~'

Glu

sucker ~ Asn Asp Asp

Ite

Ash Met lie

Ala Arg Glu

Ala Arg lie Glu Ash Glu Arg Glu

Gly Leu

Tyr Met Glu IIe-NH,

Glu VaI-N H.,

carp 'j Asn Asp Asp

1 2 3 4 5 6 7 lle 8 9 10 1I 12 13 14 15 16 Asn 17 Met 18 Ile 19 20 21 Ala 22 Arg 23 Asn 24 Glu 25 Ash 26 Gin 27 Arg 28 Glu 29 30 31 Gly 32 Leu 33 34 35 36 Tyr 37 38 39 Glu 40 Val-N H., 41

*All sequences are compared to ovine CRF. Thus, residues are listed if they are different from ovine CRF. Data obtained from the following sources: a, [56]; b, [36]; c, [20]: d, 117].

terized for its hypotensive properties in a mammalian superior mesenteric artery preparation. U p o n isolation to homogeneity and chemical characterization, urotensin I was found to be a single chain polypeptide equal in length to C R F [17,20]. M o r e o v e r , this peptide was shown to exhibit a strong seq u e n c e h o m o l o g y to ovine C R F ([17,20]; also Table l). T h e r e f o r e , we tested our antisera raised to synthetic ovine C R F for their ability to crossreact with synthetic urotensin I (kindly provided by Dr. Jean Rivier, Salk Institute). When tested on formaldehyde-fixed blots of peptides on filter paper

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FIG. 1. Immunofluorescence photomicrographs of cryostat sections of spinal cord of catfish demonstrating elements of the caudal neurosecretory system after incubation with antiserum directed against ovine CRF which crossreacts with urotensin I. (A) CRF/urotensin I immunoreactive cell bodies are prominent in the dorsal portion of the caudal spinal cord. A dense zone of immunostaining is present in the ventral portion of the spinal cord at this level, representing fibers directed caudally toward the urophysis. (B) CRF/urotensin l immunoreactive fibers and terminals are seen to fill the urophysis and are especially prominent in the outer regions of this structure. Calibration bar= 100 (A), 50 (B) microns.

we found CRF antisera which crossreact completely with urotensin I, but not with other neuropeptides [29]. Large numbers of neuronal perikarya with the caudal spinal cord of fish are stained after application of CRF antisera ([29]; Fig. I A). The axons of these neurons appear to gather in the ventral funiculus of the caudal spinal cord and enter the urophysis in the caudalmost segment of the spinal cord, where they terminate in a palisade fashion in apposition to a rich capillary network (Fig. 1B). The architecture of this system appears to be analagous to the hypothalamic magnocellular neurosecretory system whose terminals end in the posterior pituitary. These images suggest that massive quantities of urotensin I are available for release to peripheral circulation, but the physiological role for this putative hormone remains to be established. Functional analysis of this system should prove to be intriguing, since the only mechanisms for control of secretion of urotensin I are likely to be either local mechanisms or descending pathways through the spinal cord. Concerning the latter, two descending pathways to the caudal neurosecretory system have been identified [26,27]. CENTRAL AUTONOMICREGULATIONAND SYMPATHOADRENAL NEURONS A number of sites within the central nervous system are known to regulate the autonomic nervous system. High densities of neuropeptide immunoreactive neurons have been found in such regions including the nucleus of the solitary tract [22], the dorsal motor nucleus of the vagus [21] and the sacral parasympathetic nucleus [42]. Using retrograde

tracing techniques we have identified sympathetic preganglionic neurons within the intermediolateral cell column of the thoracic spinal cord which project to the adrenal medulla [15,16]. In the rat and kitten these "sympathoadrenal neurons" are most abundant in thoracic segments T~,.,, where more than 70% of the total sympathoadrenal neurons are found. Striking networks of serotonergic and peptidergic fibers are found in the vicinity of retrogradely labeled sympathoadrenal neurons. Substance P and enkephalin immunoreactive varicosities were the most prominent peptidergic elements. All of the systems studied were of descending and probably supraspinal origin, since spinal cord transection at Cz led to a complete disappearance of peptidergic and serotonergic fibers in the intermediolateral cell column [15]. Spatial analysis indicated that substance P, enkephalin and serotonin immunostained fibers did not preferentially occupy the area surrounding sympathoadrenal neurons but that these fibers were uniformly dense within the interrnediolateral cell column. However, somatostatin immunoreactive fibers seemed to preferentially occupy the neuropil surrounding sympathoadrenal neurons whereas oxytocin immunoreactive fibers seemed to preferentially avoid sympathoadrenal neurons [15,16]. Thus, one might hypothesize a generalized sympathetic regulation by substance P, enkephalin and serotonin neurons; a specific role regulating the adrenal medulla for somatostatin containing fibers: and no role for oxytocin containing fibers in regulating the adrenal medulla. Recently, we have extended these studies to discrete neurotransmitter binding sites within the intermediolateral cell

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ELDE ET A L .

column [451. The same paradigm was used for revealing sympathoadrenal neurons, namely retrograde transport of the fluorescent dye Fast Blue from the adrenal medulla. The position of the Fast Blue-positive sympathoadrenal neurons was recorded using digital mapping methods and the tissue was further processed for the autoradiographic demonstration of neurotransmitter binding sites by the emulsion-coated coverslip method [62]. The density of binding sites in relation to Fast Blue-positive, sympathoadrenal neurons was determined by counting silver grains within a 40x40 micron square centered over a Fast Blue-positive cells. Nearby preganglionic sympathetic neurons, negative for Fast Blue and therefore presumed to innervate structures other than the adrenal, were also targets for the counting square. Of the ligands investigated thus far, para-aminoclonidine and lysergic acid diethylamide, ligands for the alpha-2-adrenergic and serotonin binding sites, respectively, have been found to preferentially occupy the neuropil surrounding sympathoadrenal neurons. Muscarinic and mu opiate ligands demonstrated no preferential binding over sympathoadrenal neurons. These findings suggest that noradrenergic and serotonergic input to the intermediolateral cell column may preferentially regulate the adrenal medulla [45]. Thus it appears as if the input to the sympathetic nervous system is at least partially organized on a viscerotopic principle. Furthermore, this organization is expressed at the level of neural circuits, the putative transmitters they utilize and transmitter binding sites in the intermediolateal cell column in the vicinity of sympathoadrenal neurons. PANCREATIC ISLETS Numerous studies over several decades have brought attention to the important role of serum glucose and amino acid concentrations in regulating the hormonal output of the endocrine portion of the pancreas. It has also become clear that the integrated hormonal output of pancreatic islets is largely responsible for glucose homeostasis. Taken together, these findings suggest that the pancreatic islet may function as an autonomous unit in nutrient homeostasis. In recent years, additional mechanisms have been identified which contribute significantly to regulation of islet hormone output. These include input from the autonomic nervous system, intra-islet influences of one islet hormone upon islet cells producing other hormones, and intercellular communication among islet cells via gap junctions. The latter is beyond the scope of this summary. A variety of data suggest neuronal mechanisms are important in control of islet hormone release. Morphological studies suggest a rather deliberate interaction of nerve terminals with cells of the pancreatic islet [25]. Neurotransmitters dramatically perturb islet hormone release. For example, norepinephrine, whether derived from nerve fibers or the adrenal medulla, potently counteracts the stimulatory effect of glucose upon beta cell electrical activity [41]. Norepinephrine also inhibits glucose-stimulated insulin and somatostatin release [49]. Under similar conditions, norepinephrine augments the release of glucagon, especially as glucose concentrations fall [46,49]. Moreover, several aspects of the structure and function of islet cells resemble some features of neurons. For example, "secretion granules" of the cells of islets resemble " n e u r o s e c r e t o r y " or "'large granular" vesicles found within numerous cells of the nervous system. The release of hormones from cells of the pancreatic islet is calcium dependent

[2-4] as is transmitter release from neurons. The presence ot " n e u r o p e p t i d e s " within cells of the islet, somatostatin being a prime example, further strengthens the notion that islet cells are similar to neurons. Islet cells have long been known to take up and decarboxylate biogenic amines [32] and have been shown to at least transiently express a catecholaminergic phenotype [52]. At the same time, it must be emphasized that direct proof of islet cell origin from neural tissue is lacking [12,33]. The discovery of somatostatin within islet cells bearing dendrite-like processes (Fig. 2C) similar to those found within the gastrointestinal tract [ 19], and the inhibitory effect of exogenous somatostatin on insulin and glucagon release provoked formulation of the paracrine hypothesis which suggests a local, intra-islet action for somatostatin. This concept is essentially analogous to the notion of the local circuit neuron in which dendrites may release neurotransmitters and thereby affect membrane potential in neighboring dendrites I34]. Immunohistochemical studies from our laboratory several years ago [38] using antiserum to glutamate decarboxylase [28] have shown that the insulin producing B cells (Fig. 2A) are also GABAergic in that they contain glutamate decarboxylase immunoreactivity (Fig. 2B), the biosynthetic enzyme for GABA. Somatostatin (Fig. 2C) and glucagon immunoreactive cells (not shown) occur at the periphery of the islet, in contrast to the more centrally disposed insulin and glutamate decarboxylase positive cells. More recent studies have confirmed this finding [60]. Moreover, we have shown that G A B A agonists specifically inhibit the release of somatostatin without altering insulin or glucagon release profiles [37,38]. Since neither insulin nor glucagon release was altered by the significant, GABA-mediated decrease in somatostatin output, it is difficult to invoke somatostatin as a tonic, local inhibitor of insulin and glucagon release. Various other experiments have also failed to demonstrate a local role for somatostatin in regulation of insulin and glucagon release [46-49]. Thus, the site of action of islet somatostatin remains to be determined. Although substances regarded as neurotransmitters (norepinephrine, G A B A and acetylcholine) have been shown to alter islet hormone release, their contribution to integrated islet hormone output under strict physiological circumstances has not been clarified. It may be that they serve to alter the threshold or magnitude of the response of various islet cell types to glucose concentration. Experiments are in progress which are designed to test this hypothesis. Finally, the presence of glutamate decarboxylase in islet beta cells, which to date is the only known site of production of this enzyme outside of the central nervous system, is noteworthy in adding strength to the suggestion that the peptideproducing endocrine cells of the pancreatic islet possess many neuron-like properties.

ACKNOWLEDGEMENTS The authors thank M. Frick and T. Mullett for expert technical assistance. Studies from our laboratories and reviewed herein were supported in part by USPHS grants DA 02148 (RE) and AM 24006 (RLS) and the lmmuno Nuclear Corporation.

PEI:rI'IDERGIC R E G U L A T I O N IN N E U R O E N D O C R I N E

IIII

105

III1!

A

i

|! ? ~

ii ~ ~

FIG. 2. Immunoperoxidase photomicrographs of cryostat sections of normal rat pancreas after incubation with antisera to insulin (A), glutamate decarboxylase (B) and somatostatin (C). (A) Insulin immunoreactivity is prominent in most cells within the central mass of the islet. (B) Glutamate decarboxylase immunoreactivity is found within a similar population of cells that were stained with antiserum to insulin (A). (C) Somatostatin immunoreactivity is confined to angular cells at the periphery of the islet. Calibration bar= 100 microns.

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REFERENCES

I. Antoni, F. A., M. Palkovits, G. B. Makara, E. A. Linton, P. J. Lowry and J. Z. Kiss. Immunoreactive corticotropin-releasing hormone in the hypothalamoinfundibular tract. Neuroendocrinology 36: 415-423, 1983. 2. Atwater, I., A. A. Goncalves and E. Rojas. Electrophysiological measurement of an oscillating potassium permeability during the glucose-stimulated burst activity in mouse pancreatic B-cell. B i o m e d Res 3: 645-648, 1982. 3. Atwater, I., B. J. Frankel, E. Rojas and G. M. Grodsky. B-cell membrane potential and insulin release; role of calcium and calcium:magnesium ratio. Q J Exp Physiol 68: 233-245, 1983. 4. Beigelman, P. M. and B. Ribalet. Beta-cell electrical activity in response to high glucose concentration. Diabetes 29: 263-265, 1980. 5. Bloom, F. E., E. L. F. Battenberg, J. Rivier and W. Vale. Corticotropin releasing factor (CRF): immunoreactive neurons and fibers in rat hypothalamus. Regul Pept 4: 43-48, 1982. 6. Bugnon, C., D. Fellmann, A. Gouger and J. Cardot. Etude immunocytochemique de Fontogenese du systeme neuroglandulairea CRF chez le rat. (" R S e a n c e s A('ad Sci (111) 294: 599604, 1982. 7. Bugnon, C., D. Fellmann, A. Gouget and J. Cardot. Ontogeny of the corticoliberin neuroglandular system in rat brain. Nature 298: 159-161, 1982. 8. Burlet, A., M.-C. Tonon, P. Tankosic, D. Coy and H. Vaudry. Comparative immunocytochemical localization of corticotropin releasing factor (CRF-41) and neurohypophysial peptides in the brain of Brattleboro and Long-Evans rats. Neuroendo¢'rinology 37: 64-72, 1983. 9. Cummings, S., R. Elde and V. Seybold. Reserpine induces depletion of CRF-4[ immunoreactivity from the external layer of the median eminence. S o ( N e u r o s c i Abstr 9: 417, 1983. 10. Cummings, S., R. Elde, J. Ells and A. Lindall. Corticotropinreleasing factor immunoreactivity is widely distributed within the central nervous system of the rat: An immunohistochemical study, ,I N e u r o s c i 3: 1355-1368, 1983. 11. Fridberg, G. and H. A. Bern. The urophysis and the caudal neurosecretory system of fishes. Biol Rev 43: 175-199, 1968. 12. Fontaine, J., C. LeLievre and N. M. LeDouarin. What is the developmental fate of the neural crest cells which migrate into the pancreas of the avian embryo'? Gen ( ' o m p Endocrim~l 33: 394-404, 1977. 13. Furutani, Y., Y. Morimoto, S. Shibahara, M. Noda, H. Yakahashi, T. Hirose, M. Asai, S. lnayama, H. Hayashida, T. Miyata and S. Numa. Cloning and sequence analysis of cDNA for ovine corticotropin-releasing factor precursor. Nature 301: 537-540, 1983. 14. Highnam, K. C. and L. Hill. The Comparative Endo('rinoh,~,,y q[" the Invertebrates. New York: American Elsevier, 1969. 15. Holets, V. and R. Elde. The differential distribution and relationship of serotonergic and peptidergic fibers to sympathoadrenal neurons in the intermediolateral cell column of the rat: A combined retrograde axonal transport and immunofluorescence study, Nem'oscience 7: 1155-I 174, 1982. 16. Holets, V. and R. Elde. Sympathoadrenal preganglionic neurons: their distribution and relationship to chemically-coded fibers in the kitten intermediolateral cell column. J A//ton N e r v Svst 7: 14%163, 1983. 17. Ichikawa, T. D., D. McMaster, K. Lederis and H. Kobayaski. Isolation and amino acid sequence of urotensin l, a vasoactive and ACTH-releasing neuropeptide from the carp (Cyprinus carpio) urophysis. Peptides 3: 85%867, 1982. 18. Kahn, D., A. Hou-Yu and E. A. Zimmerman. Localization of neurotensin in the hypothalamus. A n n N Y A~'ad Sci 400: 117131, 1982. 19. Larsson, L.-I., N. Goltermann, L. de Magistris. J. F. Rehfeld and T. W. Schwartz. Somatostatin cell processes as pathways for paracrine secretion. Science 205: 1393-1395, 1979.

20. Lederis, K., A. Letter, D. McMaster, G. Moore and D. Schlesinger. Complete amino acid sequence of urotensin I, a hypotensive and corticotropin-releasing neuropeptide from Catostomus. Science 218" 162-164, 1982. 21. Maley, B. and R. Elde. Localization of substance P-like immunoreactivity in cell bodies of the feline dorsal vagal nucleus. Neurosci Left 27: 187-191, 1981. 22. Maley, B. and R. Elde, Immunohistochemical localization of putative neurotransmitters within the feline nucleus tractus solitarius. Neuroscience 7: 246%2490, 1982. 23. Merchenthaler, 1., S. Vigh, P. Petrusz and A. V. Schally. lmmunocytochemical localization of corticotropin-releasing factor (CRF) in the rat brain .Am J Anat 165: 385-396, 1982. 24. Micevych, P. and R. Elde. Relationship between enkephalinergic neurons and the vasopressin-oxytocin neuroendocrine system of the cat. An immunohistochemical study. J ('omp Neurol 190: 135-146. 1980. 25. Miller, R. E. Pancreatic neuroendocrinology: Peripheral neural mechanisms in the regulation of the islets of Langerhans. Endocr Rev 2: 471-494, 1981. 26. O'Brien, J. P. and R. M. Kriebel. Brain stem innervation of the caudal neurosecretory system. ('ell Tissue Res 227: 153-160, 1982. 27. O'Brien, J. P. and R. M. Kriebel. Caudal neurosecretory system synaptic morphology following deafferentation: An electron microscopic degeneration study. Brain Res Bull 10: 8%95, 1983. 28. Oertel, W. H., D. E. Schmechel, M. L. Tappaz and 1. J. Kopin. Production of a specific antiserum to rat brain glutamic acid decarboxylase by injection of an antigen-antibody complex. Nem'os('ience 6: 268%2700, 1981. 29. Onstott, D. and R. Elde. Immunohistochemical localization ol urotensin I/corticotropin releasing factor immunoreactivity in neurosecretory neurons in the caudal spinal cord offish. Nem'oendocrim~/ok, y, in press. 30. Orci, L. and R. H. Unger. Hypothesis: the possible role of the pancreatic D-cell in the normal and diabetic states. Diabetes 26: 241-244, 1977. 31. Paull, W. K., J. Scholer. A. Arimura, C. A. Meyers, J. K. Chang, D. Chang and M. Shimizu. lmmunocytochemical localization of CRF in the ovine hypothalamus. Peptides 3: 183191, 1982. 32. Pearse, A. G. E. The cytochemistry and ultrastructure o! polypeptide hormone producing cells of the APUD series and the embryologic, physiologic and pathologic implications of the concept. ,I t t i s t o c h e m ('ytochem 17:303-313, 1969. 33. Pictet, R., L. Rall, P. Phelps and W. J. Rutter, Neural crest and origin of insulin-producing and other gastrointestinal hormoneproducing cells. Science 191: 191-192, 1976. 34. Rail, W. and G. M. Shepherd. Theoretical reconstruction o1 field potentials and dendrodendritic synaptic interactions in olfactory bulb. J Neurophysiol 31:884-915, 1968. 35. Rivier, C. and W. Vale. Modulation of stress-induced ACTH release by corticotropin-releasing factor, catecholamines and vasopressin. Nature 305: 325-327. 1983. 36. Rivier, J., J. Spiess and W. Vale. Characterization of rat hypothalamic corticotropin-releasing factor. Proc Natl A c a d Sci USA 80:4851-4855, 1983. 37. Robbins, M. S., L. H. Grouse, R. L. Sorenson and R. P. Elde. Effect of muscimol on glucose stimulated somatostatin and insulin release from the isolated perfused rat pancreas. Diabetes 30: 168-171, 1981. 38. Robbins, M. S., R. L. Sorenson, R. P. Elde, D. E. Schmechel and W. H. Oertel. Gamma-aminobutyric acid (GABA) inhibition of somatostatin secretion from the isolated perfused rat pancreas and immunohistochemical localiation of L-glutamate decarboxylase in the islet beta cell. In: Proceedin~,,s q['the Secured lnterm~tional Symposittm (1981) on Somatostatin. edited by S. Raptis and J. Gerich. New York: Academic Press, in prcss.

P E P T I D E R G I C R E G U L A T I O N IN N E U R O E N D O C R I N E

39. Roth, K. A., E. Weber and J. D. Barchas. lmmunoreactive corticotropin releasing factor (CRF) and vasopressin are colocalized in a subpopulation of the immunoreactive vasopressin cells in the paraventricular nucleus of the hypothalamus. Life Sci 31: 1857-1860, 1982. 40. Roth, K. A., E. Weber, J. D. Barchas, D. Chang and J.-K. Chang, Immunoreactive dynorphin-(l-8) and corticotropinreleasing factor in subpopulation of hypothalamic neurons. Science 219: 18%191, 1983. 41. Santana de Sa, S., R. Ferrer, E. Rojas and I. Atwater. Effects of adrenaline and noradrenaline on glucose-induced electrical activity of mouse pancreatic B-cell. Q J Exp Physiol 68: 247-258, 1983. 42. Sasek, C. A., V. S. Seybold and R. P. Elde. The immunohistochemical localization of nine peptides in the sacral parasympathetic nucleus and the dorsal gray commissure in rat spinal cord. Submitted. 43. Sawchenko, P. E. and L. W. Swanson. Immunohistochemical identification of neurons in the paraventricular nucleus of the hypothalamus that project to the medulla or to the spinal cord in the rat. J Comp Neurol 205: 260-272, 1982. 44. Seybold, V., R. Elde and T. Hokfelt. Terminals of reserpine sensitive vasopressin-neurophysin neurons in the external layer of the rat median eminence. Endocrinology 108: 1803-1809, 1981. 45. Seybold, V. S. and R. P. EIde, Receptor autoradiography in thoracic spinal cord: Correlation of neurotransmitter binding sites with sympathoadrenal neurons. Submitted. 46. Sorenson, R. L. and R. P. Elde. Dissociation of glucose stimulation of somatostatin and insulin release from glucose inhibition of glucagon release in the isolated perfused rat pancreas. Diabetes 32: 561-567, 1983. 47. Sorenson, R. L., D. V. Lindell and R. P. Elde. Glucose stimulation of somatostatin and insulin release from the isolated, perfused rat pancreas. Diabetes 29: 747-751, 1980. 48. Sorenson, R. L., L. H. Grouse and R. P. Elde. Cysteamine blocks somatostatin secretion without altering the course of insulin or glucagon release: A new model for the study of islet function. Diabetes 32: 377-379, 1983. 49. Sorenson, R. L., R. Elde and V. Seybold. Effect of norepinephrine on insulin, glucagon and somatostatin secretion in isolated perfused rat islets. Diabetes 28: 89%904, 1979. 50. Swanson, L. W. and H. G. J. M. Kuypers. The paraventricular nucleus of the hypothalamus: cytoarchitectonic subdivisions and organization of projections to the pituitary, dorsal vagal complex, and spinal cord as demonstrated by retrograde fluorescence double-labeling methods. J Comp Neurol 194: 555-570, 1980.

107

51. Swanson, L. W., P. E. Sawchenko, J. Rivier and W. Vale. Organization or ovine corticotropin-releasing factor immunoreactive cells and fibers in the rat brain. An immunohistochemical study. Neuroendoerinology 36: 165-186, 1982. 52. Teitelman, G., T. H. Joh and D. J. Reis. Transformation of catecholaminergic precursors in glucagon (A) cells in mouse embryonic pancreas. Proc Natl Acad Sci USA 78: 5225-5229, 1981. 53. Tilders, F. J. H., J. Schipper, P. J. Lowry and I. Vermes. Effect of hypothalamus lesions on the presence of CRFimmunoreactive nerve terminals in the median eminence and on the pituitary-adrenal response to stress. Regul Pept 5: 77-84, 1982. 54. Turkelson, C. M., C. R. Thomas, A. Arimura, D. Chang, J. K. Chang and M. Shimizu. In vitro potentiation of the activity of synthetic ovine corticotropin-releasing factor by arginine vasopressin. Peptides 3: 111-113, 1982. 55. Uhl, G. R. Distribution of neurotensin and its receptor in the central nervous system. Ann N Y Acad Sei 400: 132-149, 1982. 56. Vale, W., J. Spiess, C. Rivier and J. Rivier. Characterization of a 41 residue ovine hypothalamic peptide that stimulates the secretion of corticotropin and B-endorphin. Science 213: 13941396, 1981. 57. Van den Pol, A. N. The magnocellular and parvocellular paraventricular nucleus of rat: Intrinsic organization. J Comp Neurol 206: 317-345, 1982. 58. Vanderhaeghen, J. J., F. Lofstra, F. Vandesande and K. Dierickx. Coexistence of cholecystokinin and oxytocinneurophysin in some magnocellular hypothalamo-hypophyseal neurons. Cell Tissue Res 221: 227-231, 1981. 59. Vandesande, F. and K. Dierickx. Identification of the vasopressin producing and of the oxytocin producing neurons in the hypothalamic magnocellular neurosecretory system of the rat. Cell Tissue Res 164: 153-162, 1975. 60. Vincent, S. R., O. Johansson, T. Hokfelt, B. Meyerson, C. Sachs, R. P. Elde, L. Terenius and J. Kimmel. Immunohistochemical studies of the GABA system in the pancreas. Neuroendocrinology 36: 197-204, 1983. 61. Watson, S. J., H. Akil, W. Fischli, A. Goldstein, E. Zimmerman, G. Nilaver and T. B. van Wimersma Greidanus. Dynorphin and vasopressin: Common localization in magnocellular neurons. Science 216: 85-87, 1982. 62. Young, III, W. S. and M. J. Kuhar. A new method for receptor autoradiography: [3H]-opioid receptors in rat brain. Brain Res 179: 255-270, 1979. 63. Zimmerman, E. A. Localization of hypothalamic hormones by immunocytochemical techniques. In: Frontiers in Neuroendocrinology, vol 4, edited by L. Martini and W. F. Ganong. New York: Raven Press, 1976, pp. 25-62.