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Distribution of FMRF-NH2-like immunoreactivity in rat and cat area postrema
Brain Research Rullcrin, Vol.
13, pp. 391-399, I984. *
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Distributionof FMRF-NH,-Like Immunoreactivity in Rat and Cat Area Postrema BRUCE W. NEWTON,’ BRUCE MALEY, CATHRII’JE SASEK* AND HAROLD TRAURIG Departments
of Anatomy, University of Kentucky Medical Center, Lexington, KY 40536 and *University of Minnesota, Minneapolis, MN 55455 Received
26 March 1984
NEWTON, B. W., B. MALEY, C. SASEK AND H. TRAURIG. Distribution of FMRF-N&-like immtmoreactivity in rat and cat area postrema. BRAINRBS BULL 13(3) 391-399, 1984.-m distribution ofFMRP-NH&e immunoreactivity, visualized with immunohistochemical techniques, was @otted in a range &om very dense to none. The rat area postrema had differential ids with the greatest amounts occun4ng at its ventral and ventrokteral bordem by the nucieus of the solitary tract. Immunoreactive cell bodies were located mainly in this i-e&m. Throughout the central region of the rat area postrema scattered immunostaining was consistently observed. The cat area postrema bad a different, greater, yd more compkx pattern of immunostaining,than the rat. Very dense to dense accumulations of immunostaining Occun’ed In the ventromedial region of the area postrema bordering the solitary tract and dorsal vagal nucki, while the central region possessed scattered amounts of immunorcactivity. Following colchicine treatment, no visible FMRF-NH&e immunoreactive cell bodies were observed in the cat area postrema. Areapostrema
Cat
FMRF-NH,
Irnm~o~~he~s~
THE area postrema (AP), present in all mammalian species, is the most caudal of the circumvent&u& organs and lies outside of the blood-brain barrier. It is a highly vascular&d om containing fenestrated capillaries that are permeable to small peptides and proteins firs] and are surrounded by large perivascuhu spaces [ 12,251.Because of its unique location at the caudal aspect of the IV ventricle the elements within the AP are in contact with cerebrospinal fluid (CSF) from both the ventricular and subarachnoid spaces with the tela choroidea partitioning the two. Recent investigations have demonstrated that the AP is mutely can be
involved
in the emetic reflex, and that this reflex
abolished in animals whose AP has been ablated [6]_ In the rat, lesions of the AP have most frequently induced enhanced saline intake [27], a decrease in body weight to a new lower set point [21], and increased consumption of preferred foods [is]. In species that do not exhibit the emetic reflex, the AP has been associated with the acquisition of taste aversions induced by gamma radiation [32], histamine 1343,and lithium chloride [35]. While there are increasing data concerning possible functional roles of the AP, little is known of the neuronal circuitry or transmitters involved in these mechanisms. Several putative neurotransmitters and catecholamines have been studied in the AP of the rat and cat 12, 3, 4, 16, 31, 421. Recently, FhiRF-NH:! ~~Met-~-~~NH~ immunoreactive cell bodies have been localized in the
NPY
PAP technique
PYY
Rat
brainstem of the rat in areas such as the nucleus and tractus solitarius @ITS) [38] and the ventrolateral portion of the caudal brainstem [20]. FMRF-NH, was first isolated from ganglii of bivalve molluscus in 1977 1331,and was found to be a molluscan ~i~xci~ or -~~~~~ peptide depending on the species [18]. In addition, Fh4RF-NH, evokes the excitation of neurons within the brainstem of the rat [ 171, and is structurally similar [ 131to bovine pancreatic polypeptide (BPP), avian pancreatic polypeptide (APP), peptide YY (PYY), and neuropeptide Y (NW). This present study is the first known report describing FIbiRF-NH&e immunoreactive (FMRF-LI) fibers and cell bodies in the AP of the rat and cat. METHOD
Animals For the present ~ves~on I2 Sprague-Dawley rats (6 normal; 6 colc~c~e-t~a~d), and 10 random bred cats (6 normal; 4 cdkhicine-treated) were ob@ined from the Division of Animal Care Services at the University of Kentucky. Forty-eight hours prior to sacrifice animals treated with colchicine were anesthesized with sodium pentobarbital (50 m&kg/rat; 38 -cat) and -ted with colchicine intracistemally (6.5 #/rat and 2.5 j&at; both 10 &g&l in 0.9% saline). On the day of sacrifice all animals were anesthesized with sodium pentobarbital, and injected with
‘Requests for reprints should be addressed to B. W. Newton, Department of Anatomy, A. II. Chandler Medical Center, 800 Rose Street, Lexington, KY 40536.
391
NEWTON,
392
---AP cc Gr IV NTS V X XII
ABBREVIATIONS -__--_-_----we--
Area Postrema Central Canal Gracile Nucleus Fourth Ventricle Nucleus Tractus Solitarius Vestibular Nucleus Dorsal Motor Nucleus of the Vagus Hypoglossal Nucleus
heparin intracardially (0.1 ml/rat and 1.0 ml/cat; 100 units/ml). The animals were then perfused intracardially with a pre-wash of 0.9% saline followed by 4% pamformaldehyde in 0.1 M Sorenson’s phosphate buffer, pH 7.2 (650 ml/rat; 2.0 l/cat) at room temperature. Immunohistochemistry Transverse 50 pm sections of the brainstem containing the AP were cut on a freezing microtome and subsequently, the sections were placed in wells containing phosphate buffered saline (PBS). The sections were placed for 15 minutes in PBS containing 0.3% Triton X-100 and 3% normal sheep serum (diluent), followed by overnight incubation with antiserum to FMRF-NH2 (1/4ooo in diluent). All subsequent antisera used in this study were diluted with the same diluent used for the primary antibody and exposed to the tissue at room temperature on a rotating stage. The next morning, the tissue was rinsed three times in PBS and incubated in sheep anti-rabbit IgG (11600) for one hour. After another series of rinses, the sections were then incubated for one hour with rabbit peroxidase, antiperoxidase (PAP; l/1000). Following additional rinses, the sections in each well were preincubated with 3,3’diaminobenzidine tetrahydrochloride (DAB; 0.5 mg/l.O ml of 0.1 M Tris buffer; pH 7.6) for 15 minutes. Fresh DAB (containing 0.3% H,O, at 3.3 pl/l.O ml DAB) was then placed in the wells and the sections were reacted for 15-20 minutes. The reaction was stopped by rinsing the sections with excess buffer. To test for specificity of the FMRF-NH2 antiserum adjacent sections of the tissue containing the AP were incubated with the antiserum that was pretreated with the FMRF-NH, peptide (10 pg/ml) 48 hours prior to its use. The tissue exposed to this “absorbed” antiserum was processed in parallel to the remainder of the tissue and in all cases this homologous antigen prevented any immunostaining in the adjacent absorbed control sections. The FMRF-NH, antibody was produced and characterized in the laboratory of Dr. R. Elde at the University of Minnesota. Its production and characterization has previously been reported and was shown to cross react with NPY, PYY, and APP [39]. The significance of these cross reactivities of the FMRF-NH, antiserum will be considered in the discussion. Evaluation. To map the distribution of FMRF-Ll, sections containing the AP of the brainstem were projected onto paper and the outlines of representative caudal to rostra1 levels of the AP and neighboring nuclei were traced. The density of FMRF-LI was subjectively evaluated and rated on a scale of very dense, dense, moderate, scattered, and no visible immunoreactivity by several investigators. lmmuno-
MALEY,
SASEK AND I’RAURIG
stained cell bodies, which were visible after enhancement by colchicine treatment, were mapped onto the left half of the rat AP line drawings according to their location within the AP. The distribution of FMRF-LX fibers and cell bodies in the AP line drawings was gathered from one animal in each species, but is representative of immunoreactivity in all animals examined. RESULTS
Rut
The distribution of FMRF-Ll fibers and cell bodies in the AP of the rat is summarized in a series of schematic drawings (Figs. lA-F). FMRF-LI fibers (Figs. 3a and e) were differentially distributed in the rat AP with the greatest accumulations of immunostaining found along the periphery of the AP. The density of FMRF-LI fibers decreased in a caudal to rostral direction in all regions of the AP, with the exception of the central part where consistent scattered amounts of immunoreactivity was present (Figs. 3a-c). In caudal sections (Figs. IA-C) a dense accumulation of immunoreactive staining was present along the AP’s ventrolateral border djacent to the NTS (Fig. 3a and b). In rostra1 sections (Figs. ID-F) the immunostaining of the ventrolateral border of the AP was moderate or scattered with one section (Fig. 1E) possessing dense immunoreactivity. Caudally, in the region of the AP’s ventral border neighboring the NTS (Figs. IA-C), immunostaining increased from scattered amounts medially to dense accumulations laterally (Fig. 3b). Rostrally, this same region of the AP (Figs. 1D and D exhibited moderate to scattered amounts of immunostaining. The caudal two-thirds of the dorsal border of the AP by the IV ventricle mainly had moderate amounts of immunoreactive fibers (Figs. 3a and b). In more rostral sections, the dorsal AP’s moderate immunoreactivity was replaced with scattered immunostaining extending from the midline. While a number of FMRF-Ll cell bodies could be visualized in the AP of the rat using the PAP procedure without colchicine, a greater number of more intensely stained FMRF-Ll cell bodies could be detected following colchicine treatment. Although some immunoreactive cell bodies were found in central regions of the AP, most FMRF-LI cell bodies were located near the NTS at both the ventral (Figs. 3c and d; 4d) and ventrolateral boundaries (Fig. 4b and c). The greatest numbers of immunoreactive cell bodies in the rat AP were observed at intermediate levels. The FMRF-LI cell bodies appeared most often as small bipolar cells (Figs. 3c and d; 4a-d) with occasional dendritic arborizations (Figs. 3d and 4b). The fusiform cell bodies averaged 15x8 pm, but the length of their visible immunostained processes varied from 10 pm to 75 pm (Figs. 3d and 4a). ln many sections neuronal processes crossed the ventroiateral AP-NTS border while their cell bodies were located on either side of the ventrolateral border (Figs. 4b and c). Many of the FMRF-LI cell bodies lying on the AP-NTS border (Fig. 4d) sent their processes to the AP, NTS, or into both nuclei. Cat The distribution of FMRF-Ll fibers in the AP of the cat is summarized in a series of schematic drawings (Fig. 2A-F). The FMRF-Ll fibers were found to be differentially distributed in the cat in a manner different than that faund in the rat (Figs. 5a-e).
FMRF-LI
393
IN RAT AND CAT AREA POSTREMA
PMRC-LI
RAT
FIG. 1. Line drawings (A-F, caudal to rostrai) of the dorsal portion of the rat caudal brainstem at the level of the area postrema. The densities of immunoreactive fibers within the AP following incubation with FMRF-NHz antiserum are indicated on the right half of the line drawing. Densities were classified as very dense (V), dense (D), moderate (M), scattered (S), or none (IQ FWRF-LI cell bodies in the AP are shown on the leff half of the line drawing with each point corresponding to one cell body.
FMRF-LI fibers were most abundant at intermediate levels of the cat AP, with lesser amounts of immunostaining in the rostraI third, and the least amount in the caudal third of the AP. The central region of the cat AP consistently possessed scattered amounts of immunoreactive fibers, except for the most caudal level (Figs. 2A and Sa) where a moderate amount of immunostaining was present. The caudal thii of the AP’s lateral border exhibited moderate to dense amounts of immunostaining, while the rostraI two-thuds possessed scattered to moderate accumulations. The ventromedial
border of the cat AP contained the greatest densities of immunoreactivity observed. This very dense immunostaining was at intermediate and rostral levels of the AP (Figs. 2C-E; 5b and e) and represented the most medial portion of a tongue of diminishing immunoreactivity that swept laterally through nearly one-half of the central region of the AP. Figures 2D and 2E demonstrate the very dense imnmnoreactivity which decreased laterally to scattered amounts. The ventromedial border at caudal AP levels had moderate immunoreactivity. At intermediate levels the lateral portion of
NEWTON,
MALEY.
SASEK
AND TRAURIG
”
\
FIG. 2. Line drawings (A-F, caudal to rostral) of the right half of the dorsal portion of the cat caudal brainstem at the level of the area postrema. The densities of immunoreactive fibers within the AP following incubation with FMRF-NH, antiserum are indicated and classified as very dense (V), dense (D), moderate (M), scattered (S), or none (N). There were no FMRF-LI cell bodies in the AP of
untreated or colchicine-treated cats.
the ventral border of the AP possessed moderate and scattered amounts of immunostaining, while more rostral levels contained only moderate immunoreactivity and the more caudal sections had only scattered FMRF-LI staining. The dorsal border of the AP contained scattered to dense immunostaining (Figs. 5b and c). The only exception was the most caudal section (Figs. 2A and 5a) where a dense level of immunoreactive staining comprised the majority of the dorsal surface. The FMRF-LI puncta along the dorsal surface of the AP often approached and appeared to be incorporated
into part of the ependymal layer next to the IV ventricle (Fig. 5~). The cat, unlike the rat, contained no FMRF-LI cell bodies in the AP of either untreated or colchicine-treated animals. DISCUSSION
The present study confirms and extends reports by other investigators [20, 38, 461 concerning the immunohistochemical localization of FMRF-LI structures in rat brain, and
FMRF-LI
IN RAT AND CAT AREA POSTREMA
395
FIG. 3a-e. Photomicrographs of the rat area postrema cut transversely and immunostained for FMRP-LI with the PAP precedure. (a) FMRF-LI varicose fibers (arrowheads) and puncta (arrows) in the caudal AP. Level of section corresponds to A in Pii. 1. x 135. (b) FMRF-LI at an intermediate level of the AP. Note the dense immunostaining at the ventrolateral border, moderate immunostaining near the dorsal surface of the AP, and scattered immunostaining in the center of the AP. Dashed line indicates the AP border. Level of section corresponds to C in Fig. 1. x 120. (c) FMRF-LI puncta and cell bodies in the rostral AP of a cokhicine-treated rat. Note the. PMRF-LI cell bodiis (arrows) in the NTS and along the border of the AP. x80. The cell body indicated by the arrowhead is enlarged in Pi. 3d. (d) FMRF-LI cell body enlarged from Fii. 3c with a bifurcating process that approaches the NTS. x550. (e) FMRF-LI in rat AP. Most _._2____ CL___...__- _I’._~.> ____ .1! ~.I__A. _* I- _... . . -2. ..,.C.. vancosc aocrs were onenrea pcrpcnatcurar to me aorsar Ar sunace. x 1,2w.
demonstrates the presence of FMRF-LI in the brainstem of the cat. FMRF-LI fibers and cell bodies were differentially distributed in the AP of the rat with the greatest degree of immunostaining occurring at the ventrolateral and ventral borders of the AP. FMRF-LI cell bodies were most frequently found clustered at the ventrolateral border, or scat-
tered along the ventral aspect of the AP. FMRF-LI fibers in the AP of the cat showed greater amounts and more complex immunostaining than found in the rat AP. The greatest numbers of immunoreactive fibers could be found in the ventromedial region of the AP next to the boundaries of the NTS and dorsal motor nucleus of the vagus. In contrast to findings
NEWTON,
MALEY,
SASEK
AND ‘I‘KAUKl~;
FIG. k-d. Photomicrographs of colchicine-treated rat AP cut transversely and immunostained for FMRF-Ll with the PAP procedure. (a) An PMRF-LI cell body in rat AP. x 1090. (b) Several FMRF-LL ceti bodies located on the AF-NTS-Gr boundary. Note that the mm dorsal cell body (arrow) has a process that bifurcates as it enters the AP. Dashed tine imiicates the AP border. x335. (c> Ventrolateral region of the AP containing an FMRF-LI cell body (arrow) with a process extending into the NTS. A large number of FMRF-LI cell bodies are present in the NTS. Dashed line indicates the AP border. x390. (d) FMRF-LI cell bodies on the AP-NTS ventral boundary. Dashed fine indicates the AP-NTS border. x 365.
FMRF-LI
IN RAT AND CAT AREA POSTREMA
397
FIG. 5a-e. Photomicrographs of the cat AP cut transversely and immunostained for PMRF-LI with the PAP procedure. (a) Caudal level of the cat AP with a dense distribution of PMRF-LI puncta on the dorsal border. This dense distribution of FMRF-LI decreases ventrally to scattered amounts. Many of the fibers lie out of the plane of focus. Note that the AP in the cat crosses the midline caudaUy. This section corresponds to level A in Fig. 2. x500. (b) Rostrai AP of cat showing the medial third of the AP next to the NTS and X. Note the dense distribution of immunoreactivity in the ventromedial region of the AP (*), and the scattered immunostaining along the AP’s ventral border next to the NTS. Dashed line indicates the AP border. Level of section corresponds to F in Fig. 2. x200. (c) Dorsal surface of the cat AP demonstrating a moderate distribution of FMRF-LI (arrow) in the ependymal layer and the dorsal region of the AP. These numbers are equivalent to scattered immunostainhtg. x 235. (d) Several FMRF-LI fibers in the cat AP located near the lateral border of the AP by the Gr. x 520. (e) Medial third of the cat AP at an intermedii level. Note the very dense distribution of immunostaining in the ventrolaterai resipn _C.L_ .D I., .L... -I_^___^__ ,_.___ll.. I_-_.___\ r\__L_J II__ :_Af__r__ .L_ .TBL__*__ . __._1 _* ___.I__ _________I_I_ m 1_lx_ ,a ..-*
NEWTON,
398
in the rat, FMRF-LI cell bodies were not present in the AP of the cat. The FMRF-NH, antibody used in the present study cross reacts with APP, NPY, and PYY [39]. However, APP is not found in the rat brain [ 1,201 and PYY apparently has a limited CNS distribution [20]. NPY, on the other hand, has been found extensively throughout the CNS of the rat [ 1,201 and it was accordingly concluded that the FMRF-NH, antibody was most likely recognizing this neuropeptide or PYY. Therefore, the use of this antibody may provide another means of visualizing NPY or PYY in the vertebrate nervous system. FMRF-Ll fibers may represent the terminals of neurons projecting to the AP from surrounding structures such as the NTS, the dorsal motor nucleus of the vagus and the nodose ganglion which are known to project to the AP [I 1,26, 301. It is interesting to note that in the rat large numbers of FMRF-Ll cell bodies lie within the NTS near its border with the AP. Numerous FMRF-Ll fibers were seen to cross the AP-NTS boundary in this study and many other investigators have noted an exchange of fibers between the AP and the NTS [23, 26, 43, 441. It may be possible that a large number of the FMRF-Ll fibers within the rat AP may be originating from neurons immediately adjacent to the AP, although conformation of this would depend upon experiments using immunocytochemical and tract-tracing techniques. The distribution of FMRF-Ll afferents, from any source, to the AP would enable these terminals to penetrate its neuropil providing access to the vasculature and the CSF of the IV ventricle and subarachnoid space. Many terminals, some demonstrated to be immunoreactive for tyrosine hydroxylase, have been found within the large perivascular spaces of the AP [3]. Other terminals have been observed in this region contacting the epithelial cells of the fenestrated capillaries [24,41]. These observations suggest that the putative neurotransmitters contained within varicosities of these nerve fibers may be released into the blood stream or into the CSF. and act as neurohumoral agents altering the internal milieu of the CNS. However, the actual morphological location of FMRF-Ll structures within the AP requires further elucidation with the use of immunocytochemistry at the ultrastructural level before a possible neurohumoral role can be proposed. The only established function for the AP of vomiting animals, e.g., the cat, is that of a chemoreceptive trigger zone sampling noxious blood-borne substances for the emetic center lying within the parvicellular reticular formation [7,10]. In the rat, a non-vomiting species, the AP has been associated with numerous other functions including epinephrine-induced hyperglycemia [36], serotonin-induced
MALEY. SASEK AND I’RAlJRlG
synchronization of the electroencephalogram 1371. and the cardiovascular effects of angiotensin [22,401. Many of thcsc AP functions have not been fully established in the rat [5, 8. 191. The only AP function that has been definitely demonstrated in the rat is taste aversion learning which can he abolished with a lesion of the AP 1321. In a recent investigation [9] in which numerous neurotransmitters and peptides were applied onto AP neurons of the dog, a strong correlation was demonstrated between agents that excited neurons within the AP for long periods of time after application, and their ability to cause emesis when injected intravenously. These substances were histamine. dopamine, apomorphine, angiotensin 11, neurotensin. leucine-enkephalin, vasoactive intestinal polypeptide, gastrin, vasopressin. and substance P. Conversely, the three agents they applied to canine AP neurons that were not excitatory, acetylcholine, somatostatin, and cholecystokinin, did not evoke emesis when injected into the vascutar system. These results strongly suggested that the similar response of the AP neurons to so many substances relied upon a common mechanism, and the prolonged nature of the response would subserve the sensation of nausea that precedes emesis. It has been demonstrated that FMRF-NH, ih excit+ tory in the brainstem of the rat 1171 therefore, FMRF-NH, may also act as an emetic agent on AP neurons. Differences in FMRF-Ll between the rat and the cat may form the morphological basis for the differences in the emetic reflex displayed by these two species. Functionally it has been found that NPY and PYY are potent vasoconstrictors 01’blood vessels in the salivary glands of cats 128,291. Other investigator\ have observed a dense network of NPY immunoreactive fibers surrounding cerebral arteries in cats. These NPY fiber\, which can initiate strong contractions in cerebral arteries, are abolished when the superior cervical ganglion is ablated [ 141. if this same vasoconstrictive effect ic present in the CNS of rats, then the functional significance of FMRF-LI ceil bodies in the rat AP may retate to control of choroidal blood flow. The large numbers of FMRF-t-1 fibers observed in the present study suggest that the NPY or PYY released into the AP may be acting as vasoconstrictive agents locally. or that they may be carried to other CNS areas via the V~SCIIlature andlor CSF flow.
ACKNOWLEDGEMENTS
This work was supported in part by NIH grant lR2H130702 to Bruce Maley. The authors would like to thank Kimberly A. Howes for her technical assistance and Aiita Anderson for her secretarial aid. The helpful comments by Drs. M. B. Nikitovitch-Winer and H. Parks are gratefully acknowledged.
REFERENCES 1. Allen, Y. S., T. E. Adrian, J. M. Allen, K. Tatemoto, T. J. Crow, S. R. Bloom and J. M. Polak. Neuropeptide Y distribution in the rat brain. Science 221: 877-879, 1983. 2. Armstrong, D. M., V. M. Pickel, T. H. Joh, D. J. Reis and R. J. Miller. Immunocytochemical localization of catecholamine synthesizing enzymes and neuropeptides in area postrema and medial nucleus tractus solitarius of rat brain. J Camp Neural 1%: 505-517, 1981.
3. Armstrong, D. M., V. M. Pickel, T. J. Joh and D. J. Reis. Electron microscopic immunocytochemical localization of tyrosine hydroxylase in the area postrema of rat. J Camp Neural 206: 259-272, 1982. 4. Armstrong, D. M., V. M. Pickel and D. J. Reis. Electron microscopic immunocytochemical localization of substance P in fhe area postrema of rat. Brain Rrs 243: 141-146, 1982. 5. Borison, H. L. Area postrema: chemoreceptive trigger zone for vomiting-is that all? L.Ue Sci 14: 1807-1817, 1974.
FMRF-LI IN RAT AND CAT AREA F’OSTREMA 6. Borison, R. and H. Borison. Postnatal development of the area postrema with relation to digitalis-induced vomiting in the cat. Exp Neural 16: 138-152, 1973. 7. Brizzee, K. R. and L. M. Neal. A re-evahation of the cellular morphology of the area postrema in view of recent evidence for a chemoreceptor function. J Camp Neurol l@Oz41-61, 1954. 8. Brooks, J. M, J. I. Hubbard and N. E. Sirett. Extracelhdar recording in rat area postre_ma in vitro and the effects of cholinergic drugs, serotonin and angiotensin II. Brain Res 261: 85-90,1983. 9. Carpenter, D. O., D. B. Briggs and N. Strominger. Responses of neurons of canine area postrema to neurotransmitters and peptides. Cell Mol Biol3: 113-126, 1983. 10. Chinn, H. I. and S. C. Wang. Locus of emetic action following irradiation. Proc Sot Exp Biol Med 85: 472474, 1954. 11. Clemente, C. D. and V. L. van Breemen. Nerve fibers in the area postrema of cat, rabbit, guinea pig and rat. Anaf Ret 123~ 65-79, 1955. 12. Dempsey, E. W. Neural and vascular ultrastructure of the area postrema in the rat. J Comp Neural 150: 177-200, 1973. im13. Dockray, G. J. and R. G. Williams. FMRFamide-like munoreactivity in rat brain: development of a radioimmunoassay and its application in studies of distribution and chromatographic properties. Brain Res 266: 295-303, 1983. 14. Edvinsson, L., P. Emson, J. McCulloch, K. Tatemoto and R. Uddman. Neuropeptide Y: cerebrovascular innervation and vasomotor effects in the cat. Neurosci Lert 43: 79-84, 1983. 15. Edwards, G. L. and R. C. Ritter. Ablation of the area postrema causes exaggerated consumption of preferred foods in the rat. Bruin Res %S: 265-276, 1981. 16. Fuxe. K. and C. Owman. Cellular localization of monoamines in the &a oostrema of certain mammals. J Comp Neural 125: 337-354, i%s. 17. Gayton, R. J. Mammalian neuronal actions of FMRFamide and the structurallv related ooioid met-enkenhalin-ar&de’. Nature - _ 2% 275-276, -1982. 18. Greenberg, M. J. and D. A. Price. Cardioregulatory peptides in molluscs. In: Peptides: Integrators of Cell and Tissue Function, edited by F. R. Bloom. New York: Raven Press, 1980, pp. 107-126. 19. Haywood, J. R., G. D. Fink, J. Buggy, M. 1. Phillips and M. J. Brody. The area postrema plays no role in the pressor action of angiotensin in the rat. Am J Physiol 239: HlO8-H113, 1980. 20. Hokfelt, T., J. M. Lundberg, K. Tatemoto, V. Mutt, L. Terenius, J. Polak, S. Bloom, C. Sasek, R. Elde and M. Goldstein. Neuropeptide Y (NPY)-and FMRFamide neuropeptidelike immunoreactivities in catecholamine neurons of the rat medulla oblongata. Acta Physiol Stand 117: 315-318, 1983. 21. Hyde, T. M. and R. R. Miselis. Effects of area postrema/caudal medial nucleus of solitary tract lesions on food intake and body weight. Am J Physiol244: R5774587, 1983. 22. Joy, M. D. The intramedullary connections of the area postrema involved in the central cardiovascular response to angiotensin II. Cfin Sci 41: WlOO, 1971. 23. Kalia, M., K. Fuxe, T. Hokfelt, 0. Johansson, R. Lang, D. Ganten, C. Cuello and L. Terenius. Distribution of neuropep tide immunoreactive nerve terminals within the subnuclei of the nucleus of the tractus solitarius of the rat. J Comp Neural 2U: 409-444,1984. 24. Karasawa, N., M. Yoshida, Y. Kondo, T. Nagatsu and I. Nagatsu. Immunohistocytochemical studies on neurons in the hamster area wstrema. Acta Histochem Cytochem 16: 138-147, 1983. 25. Klara, P. M. and K. R. Brizzee. Ultrastructure of the feline area postrema. J Comp Neurol 171: 404-432, lY77.
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26. van der Kooy, D. and L. Y. Koda. 0-n of the pmtions of a circumventricular 0~: the area postrema in the rat. J Comp Neural 219: 328-338,1983. 27. Kosten, T., R. J. Contmras, P. W. Stetson and M. J. Ernest. Enhancedsabeintakeanddecreasedheartrateafterarea posbwua ablations in rat. Physiol Behav 31: 777-785, 1983. 28. Lundberg, J. M. and K. Tatemoto. Vascular effects of the pep tides PYY and PHI: comparison with APP and VIP. Eur J Pharmacol83: 143-146,1982. 29. Lundberg, J. M. and K. Tatemoto. Paocreatic polypeptide family (APP, BPP, NPY and PYY) in relation to sympathetic vasoconstriction resistant to alpha-adrenoceptor blockade. Acta Physiol Stand 116: 3-, 1982. 30. Lundberg, J. M., L. Terenius, T. Hokfelt and M. Goldstein. High levels of neuropeptide Y in peripheral noradrenergic neurons in various mammals including man. Neurosci Lett 42: 167-172, 1983. 3 1. Newton, B. W., B. Maley and H. H. Traurig. The distribution of metenkephalin (ME), serotonin (5-HT) and substance P (SP) immunoreactivities in the area postrema (AP) of the rat and cat. Sot Neurosci Abstr 9: 293, 1983. 32. Ossenkopp, K. Taste aversions conditioned with gamma radiation: attenuation by area postrema lesions in rats. Eehav Brain Res 7: 297-305, 1983. 33. Price, D. A. and M. J. Greenberg. Structure of a molluscan cardioexcitatory neuropeptide. Science 141: 67&671, 1977. 34. Rabin, B. M., W. A. Hunt and J. Lee. Studies on the role of central histamine in the acquisition of a radiation-induced conditioned taste aversion. Radiat Res 99: 604-620, 1982. 35. Ritter, S., J. J. McGlone and K. W. Kelley. Absence of lithiuminduced taste aversion after area postrema lesion. Bruin Res 201: 501-506, 1980. 36. Rosenberg, F. J. and V. DiStefano. A central nervous system component of the hyperglycemic response to exogenous epinephrine. Fed Proc 20: 314, l%l. 37. Roth, G. I., P. L. Walton and W. S. Yamamoto. Area postrema: abrupt EEG synchronization following close &a-arterial perfusion with seiotonin. Brain Res 23~ 223-233, 1970. 38. Sasek. C. A.. R. Elde and V. S. Seybold. Localization of FMRF-NH,-like immunoreactivity in a&onomic regions of the brainstem and spinal cord of the rat. Sot Neurosci Abstr 8: 809, 1982. 39. 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. Neuroscience 12: 855-873, 1984. 40. Simpson, J. B. The circumventricular organs and the central action of angiotensin. Neuroendocrinology 32: 248-256, 1981. 41. Spacek, J. and J. Parizek. The fine structure of the area postrema of the rat. Acta Morphol Acad Sci Hung 17: 17-34, 1%9. 42. Takeuchi, Y. and Y. Sano. Serotonin distribution in the circumventricular organs of the rat. Anat Embryof 167: 311-319, 1983. 43. Vigier, D. and P. Portalier. Efferent projections of the area postrema demonstrated by autoradiography. Arch Itol Biol 117: 30%324, 1979. 44. Vigier, D. and A. Rouviere. Afferent and efferent connections ofthe area postrema demonstrated by the horseradish peroxidase method. Arch Ztal Biol 117: 325-339. 1979. 45. Weindl, A. Neuroendocrine aspects of &umventricular organs. In: Frontiers in Neuroendocrinology 1973, edited by W. F. Ganong and L. Martini. New York: Oxford University Press, 1973, pp. 3-32. 46. Williams, R. G. and G. J. Dockray. Immunohistochemical studies of FMRFamide-like immunoreactivity in rat brain. Brain Res 276: 213-229, 1983.
13, pp. 391-399, I984. *
Ankho in~ernationai Inc. Prin&din the U.S.A.
0361~%m/84 $3.00 + .oo
Distributionof FMRF-NH,-Like Immunoreactivity in Rat and Cat Area Postrema BRUCE W. NEWTON,’ BRUCE MALEY, CATHRII’JE SASEK* AND HAROLD TRAURIG Departments
of Anatomy, University of Kentucky Medical Center, Lexington, KY 40536 and *University of Minnesota, Minneapolis, MN 55455 Received
26 March 1984
NEWTON, B. W., B. MALEY, C. SASEK AND H. TRAURIG. Distribution of FMRF-N&-like immtmoreactivity in rat and cat area postrema. BRAINRBS BULL 13(3) 391-399, 1984.-m distribution ofFMRP-NH&e immunoreactivity, visualized with immunohistochemical techniques, was @otted in a range &om very dense to none. The rat area postrema had differential ids with the greatest amounts occun4ng at its ventral and ventrokteral bordem by the nucieus of the solitary tract. Immunoreactive cell bodies were located mainly in this i-e&m. Throughout the central region of the rat area postrema scattered immunostaining was consistently observed. The cat area postrema bad a different, greater, yd more compkx pattern of immunostaining,than the rat. Very dense to dense accumulations of immunostaining Occun’ed In the ventromedial region of the area postrema bordering the solitary tract and dorsal vagal nucki, while the central region possessed scattered amounts of immunorcactivity. Following colchicine treatment, no visible FMRF-NH&e immunoreactive cell bodies were observed in the cat area postrema. Areapostrema
Cat
FMRF-NH,
Irnm~o~~he~s~
THE area postrema (AP), present in all mammalian species, is the most caudal of the circumvent&u& organs and lies outside of the blood-brain barrier. It is a highly vascular&d om containing fenestrated capillaries that are permeable to small peptides and proteins firs] and are surrounded by large perivascuhu spaces [ 12,251.Because of its unique location at the caudal aspect of the IV ventricle the elements within the AP are in contact with cerebrospinal fluid (CSF) from both the ventricular and subarachnoid spaces with the tela choroidea partitioning the two. Recent investigations have demonstrated that the AP is mutely can be
involved
in the emetic reflex, and that this reflex
abolished in animals whose AP has been ablated [6]_ In the rat, lesions of the AP have most frequently induced enhanced saline intake [27], a decrease in body weight to a new lower set point [21], and increased consumption of preferred foods [is]. In species that do not exhibit the emetic reflex, the AP has been associated with the acquisition of taste aversions induced by gamma radiation [32], histamine 1343,and lithium chloride [35]. While there are increasing data concerning possible functional roles of the AP, little is known of the neuronal circuitry or transmitters involved in these mechanisms. Several putative neurotransmitters and catecholamines have been studied in the AP of the rat and cat 12, 3, 4, 16, 31, 421. Recently, FhiRF-NH:! ~~Met-~-~~NH~ immunoreactive cell bodies have been localized in the
NPY
PAP technique
PYY
Rat
brainstem of the rat in areas such as the nucleus and tractus solitarius @ITS) [38] and the ventrolateral portion of the caudal brainstem [20]. FMRF-NH, was first isolated from ganglii of bivalve molluscus in 1977 1331,and was found to be a molluscan ~i~xci~ or -~~~~~ peptide depending on the species [18]. In addition, Fh4RF-NH, evokes the excitation of neurons within the brainstem of the rat [ 171, and is structurally similar [ 131to bovine pancreatic polypeptide (BPP), avian pancreatic polypeptide (APP), peptide YY (PYY), and neuropeptide Y (NW). This present study is the first known report describing FIbiRF-NH&e immunoreactive (FMRF-LI) fibers and cell bodies in the AP of the rat and cat. METHOD
Animals For the present ~ves~on I2 Sprague-Dawley rats (6 normal; 6 colc~c~e-t~a~d), and 10 random bred cats (6 normal; 4 cdkhicine-treated) were ob@ined from the Division of Animal Care Services at the University of Kentucky. Forty-eight hours prior to sacrifice animals treated with colchicine were anesthesized with sodium pentobarbital (50 m&kg/rat; 38 -cat) and -ted with colchicine intracistemally (6.5 #/rat and 2.5 j&at; both 10 &g&l in 0.9% saline). On the day of sacrifice all animals were anesthesized with sodium pentobarbital, and injected with
‘Requests for reprints should be addressed to B. W. Newton, Department of Anatomy, A. II. Chandler Medical Center, 800 Rose Street, Lexington, KY 40536.
391
NEWTON,
392
---AP cc Gr IV NTS V X XII
ABBREVIATIONS -__--_-_----we--
Area Postrema Central Canal Gracile Nucleus Fourth Ventricle Nucleus Tractus Solitarius Vestibular Nucleus Dorsal Motor Nucleus of the Vagus Hypoglossal Nucleus
heparin intracardially (0.1 ml/rat and 1.0 ml/cat; 100 units/ml). The animals were then perfused intracardially with a pre-wash of 0.9% saline followed by 4% pamformaldehyde in 0.1 M Sorenson’s phosphate buffer, pH 7.2 (650 ml/rat; 2.0 l/cat) at room temperature. Immunohistochemistry Transverse 50 pm sections of the brainstem containing the AP were cut on a freezing microtome and subsequently, the sections were placed in wells containing phosphate buffered saline (PBS). The sections were placed for 15 minutes in PBS containing 0.3% Triton X-100 and 3% normal sheep serum (diluent), followed by overnight incubation with antiserum to FMRF-NH2 (1/4ooo in diluent). All subsequent antisera used in this study were diluted with the same diluent used for the primary antibody and exposed to the tissue at room temperature on a rotating stage. The next morning, the tissue was rinsed three times in PBS and incubated in sheep anti-rabbit IgG (11600) for one hour. After another series of rinses, the sections were then incubated for one hour with rabbit peroxidase, antiperoxidase (PAP; l/1000). Following additional rinses, the sections in each well were preincubated with 3,3’diaminobenzidine tetrahydrochloride (DAB; 0.5 mg/l.O ml of 0.1 M Tris buffer; pH 7.6) for 15 minutes. Fresh DAB (containing 0.3% H,O, at 3.3 pl/l.O ml DAB) was then placed in the wells and the sections were reacted for 15-20 minutes. The reaction was stopped by rinsing the sections with excess buffer. To test for specificity of the FMRF-NH2 antiserum adjacent sections of the tissue containing the AP were incubated with the antiserum that was pretreated with the FMRF-NH, peptide (10 pg/ml) 48 hours prior to its use. The tissue exposed to this “absorbed” antiserum was processed in parallel to the remainder of the tissue and in all cases this homologous antigen prevented any immunostaining in the adjacent absorbed control sections. The FMRF-NH, antibody was produced and characterized in the laboratory of Dr. R. Elde at the University of Minnesota. Its production and characterization has previously been reported and was shown to cross react with NPY, PYY, and APP [39]. The significance of these cross reactivities of the FMRF-NH, antiserum will be considered in the discussion. Evaluation. To map the distribution of FMRF-Ll, sections containing the AP of the brainstem were projected onto paper and the outlines of representative caudal to rostra1 levels of the AP and neighboring nuclei were traced. The density of FMRF-LI was subjectively evaluated and rated on a scale of very dense, dense, moderate, scattered, and no visible immunoreactivity by several investigators. lmmuno-
MALEY,
SASEK AND I’RAURIG
stained cell bodies, which were visible after enhancement by colchicine treatment, were mapped onto the left half of the rat AP line drawings according to their location within the AP. The distribution of FMRF-LX fibers and cell bodies in the AP line drawings was gathered from one animal in each species, but is representative of immunoreactivity in all animals examined. RESULTS
Rut
The distribution of FMRF-Ll fibers and cell bodies in the AP of the rat is summarized in a series of schematic drawings (Figs. lA-F). FMRF-LI fibers (Figs. 3a and e) were differentially distributed in the rat AP with the greatest accumulations of immunostaining found along the periphery of the AP. The density of FMRF-LI fibers decreased in a caudal to rostral direction in all regions of the AP, with the exception of the central part where consistent scattered amounts of immunoreactivity was present (Figs. 3a-c). In caudal sections (Figs. IA-C) a dense accumulation of immunoreactive staining was present along the AP’s ventrolateral border djacent to the NTS (Fig. 3a and b). In rostra1 sections (Figs. ID-F) the immunostaining of the ventrolateral border of the AP was moderate or scattered with one section (Fig. 1E) possessing dense immunoreactivity. Caudally, in the region of the AP’s ventral border neighboring the NTS (Figs. IA-C), immunostaining increased from scattered amounts medially to dense accumulations laterally (Fig. 3b). Rostrally, this same region of the AP (Figs. 1D and D exhibited moderate to scattered amounts of immunostaining. The caudal two-thirds of the dorsal border of the AP by the IV ventricle mainly had moderate amounts of immunoreactive fibers (Figs. 3a and b). In more rostral sections, the dorsal AP’s moderate immunoreactivity was replaced with scattered immunostaining extending from the midline. While a number of FMRF-Ll cell bodies could be visualized in the AP of the rat using the PAP procedure without colchicine, a greater number of more intensely stained FMRF-Ll cell bodies could be detected following colchicine treatment. Although some immunoreactive cell bodies were found in central regions of the AP, most FMRF-LI cell bodies were located near the NTS at both the ventral (Figs. 3c and d; 4d) and ventrolateral boundaries (Fig. 4b and c). The greatest numbers of immunoreactive cell bodies in the rat AP were observed at intermediate levels. The FMRF-LI cell bodies appeared most often as small bipolar cells (Figs. 3c and d; 4a-d) with occasional dendritic arborizations (Figs. 3d and 4b). The fusiform cell bodies averaged 15x8 pm, but the length of their visible immunostained processes varied from 10 pm to 75 pm (Figs. 3d and 4a). ln many sections neuronal processes crossed the ventroiateral AP-NTS border while their cell bodies were located on either side of the ventrolateral border (Figs. 4b and c). Many of the FMRF-LI cell bodies lying on the AP-NTS border (Fig. 4d) sent their processes to the AP, NTS, or into both nuclei. Cat The distribution of FMRF-Ll fibers in the AP of the cat is summarized in a series of schematic drawings (Fig. 2A-F). The FMRF-Ll fibers were found to be differentially distributed in the cat in a manner different than that faund in the rat (Figs. 5a-e).
FMRF-LI
393
IN RAT AND CAT AREA POSTREMA
PMRC-LI
RAT
FIG. 1. Line drawings (A-F, caudal to rostrai) of the dorsal portion of the rat caudal brainstem at the level of the area postrema. The densities of immunoreactive fibers within the AP following incubation with FMRF-NHz antiserum are indicated on the right half of the line drawing. Densities were classified as very dense (V), dense (D), moderate (M), scattered (S), or none (IQ FWRF-LI cell bodies in the AP are shown on the leff half of the line drawing with each point corresponding to one cell body.
FMRF-LI fibers were most abundant at intermediate levels of the cat AP, with lesser amounts of immunostaining in the rostraI third, and the least amount in the caudal third of the AP. The central region of the cat AP consistently possessed scattered amounts of immunoreactive fibers, except for the most caudal level (Figs. 2A and Sa) where a moderate amount of immunostaining was present. The caudal thii of the AP’s lateral border exhibited moderate to dense amounts of immunostaining, while the rostraI two-thuds possessed scattered to moderate accumulations. The ventromedial
border of the cat AP contained the greatest densities of immunoreactivity observed. This very dense immunostaining was at intermediate and rostral levels of the AP (Figs. 2C-E; 5b and e) and represented the most medial portion of a tongue of diminishing immunoreactivity that swept laterally through nearly one-half of the central region of the AP. Figures 2D and 2E demonstrate the very dense imnmnoreactivity which decreased laterally to scattered amounts. The ventromedial border at caudal AP levels had moderate immunoreactivity. At intermediate levels the lateral portion of
NEWTON,
MALEY.
SASEK
AND TRAURIG
”
\
FIG. 2. Line drawings (A-F, caudal to rostral) of the right half of the dorsal portion of the cat caudal brainstem at the level of the area postrema. The densities of immunoreactive fibers within the AP following incubation with FMRF-NH, antiserum are indicated and classified as very dense (V), dense (D), moderate (M), scattered (S), or none (N). There were no FMRF-LI cell bodies in the AP of
untreated or colchicine-treated cats.
the ventral border of the AP possessed moderate and scattered amounts of immunostaining, while more rostral levels contained only moderate immunoreactivity and the more caudal sections had only scattered FMRF-LI staining. The dorsal border of the AP contained scattered to dense immunostaining (Figs. 5b and c). The only exception was the most caudal section (Figs. 2A and 5a) where a dense level of immunoreactive staining comprised the majority of the dorsal surface. The FMRF-LI puncta along the dorsal surface of the AP often approached and appeared to be incorporated
into part of the ependymal layer next to the IV ventricle (Fig. 5~). The cat, unlike the rat, contained no FMRF-LI cell bodies in the AP of either untreated or colchicine-treated animals. DISCUSSION
The present study confirms and extends reports by other investigators [20, 38, 461 concerning the immunohistochemical localization of FMRF-LI structures in rat brain, and
FMRF-LI
IN RAT AND CAT AREA POSTREMA
395
FIG. 3a-e. Photomicrographs of the rat area postrema cut transversely and immunostained for FMRP-LI with the PAP precedure. (a) FMRF-LI varicose fibers (arrowheads) and puncta (arrows) in the caudal AP. Level of section corresponds to A in Pii. 1. x 135. (b) FMRF-LI at an intermediate level of the AP. Note the dense immunostaining at the ventrolateral border, moderate immunostaining near the dorsal surface of the AP, and scattered immunostaining in the center of the AP. Dashed line indicates the AP border. Level of section corresponds to C in Fig. 1. x 120. (c) FMRF-LI puncta and cell bodies in the rostral AP of a cokhicine-treated rat. Note the. PMRF-LI cell bodiis (arrows) in the NTS and along the border of the AP. x80. The cell body indicated by the arrowhead is enlarged in Pi. 3d. (d) FMRF-LI cell body enlarged from Fii. 3c with a bifurcating process that approaches the NTS. x550. (e) FMRF-LI in rat AP. Most _._2____ CL___...__- _I’._~.> ____ .1! ~.I__A. _* I- _... . . -2. ..,.C.. vancosc aocrs were onenrea pcrpcnatcurar to me aorsar Ar sunace. x 1,2w.
demonstrates the presence of FMRF-LI in the brainstem of the cat. FMRF-LI fibers and cell bodies were differentially distributed in the AP of the rat with the greatest degree of immunostaining occurring at the ventrolateral and ventral borders of the AP. FMRF-LI cell bodies were most frequently found clustered at the ventrolateral border, or scat-
tered along the ventral aspect of the AP. FMRF-LI fibers in the AP of the cat showed greater amounts and more complex immunostaining than found in the rat AP. The greatest numbers of immunoreactive fibers could be found in the ventromedial region of the AP next to the boundaries of the NTS and dorsal motor nucleus of the vagus. In contrast to findings
NEWTON,
MALEY,
SASEK
AND ‘I‘KAUKl~;
FIG. k-d. Photomicrographs of colchicine-treated rat AP cut transversely and immunostained for FMRF-Ll with the PAP procedure. (a) An PMRF-LI cell body in rat AP. x 1090. (b) Several FMRF-LL ceti bodies located on the AF-NTS-Gr boundary. Note that the mm dorsal cell body (arrow) has a process that bifurcates as it enters the AP. Dashed tine imiicates the AP border. x335. (c> Ventrolateral region of the AP containing an FMRF-LI cell body (arrow) with a process extending into the NTS. A large number of FMRF-LI cell bodies are present in the NTS. Dashed line indicates the AP border. x390. (d) FMRF-LI cell bodies on the AP-NTS ventral boundary. Dashed fine indicates the AP-NTS border. x 365.
FMRF-LI
IN RAT AND CAT AREA POSTREMA
397
FIG. 5a-e. Photomicrographs of the cat AP cut transversely and immunostained for PMRF-LI with the PAP procedure. (a) Caudal level of the cat AP with a dense distribution of PMRF-LI puncta on the dorsal border. This dense distribution of FMRF-LI decreases ventrally to scattered amounts. Many of the fibers lie out of the plane of focus. Note that the AP in the cat crosses the midline caudaUy. This section corresponds to level A in Fig. 2. x500. (b) Rostrai AP of cat showing the medial third of the AP next to the NTS and X. Note the dense distribution of immunoreactivity in the ventromedial region of the AP (*), and the scattered immunostaining along the AP’s ventral border next to the NTS. Dashed line indicates the AP border. Level of section corresponds to F in Fig. 2. x200. (c) Dorsal surface of the cat AP demonstrating a moderate distribution of FMRF-LI (arrow) in the ependymal layer and the dorsal region of the AP. These numbers are equivalent to scattered immunostainhtg. x 235. (d) Several FMRF-LI fibers in the cat AP located near the lateral border of the AP by the Gr. x 520. (e) Medial third of the cat AP at an intermedii level. Note the very dense distribution of immunostaining in the ventrolaterai resipn _C.L_ .D I., .L... -I_^___^__ ,_.___ll.. I_-_.___\ r\__L_J II__ :_Af__r__ .L_ .TBL__*__ . __._1 _* ___.I__ _________I_I_ m 1_lx_ ,a ..-*
NEWTON,
398
in the rat, FMRF-LI cell bodies were not present in the AP of the cat. The FMRF-NH, antibody used in the present study cross reacts with APP, NPY, and PYY [39]. However, APP is not found in the rat brain [ 1,201 and PYY apparently has a limited CNS distribution [20]. NPY, on the other hand, has been found extensively throughout the CNS of the rat [ 1,201 and it was accordingly concluded that the FMRF-NH, antibody was most likely recognizing this neuropeptide or PYY. Therefore, the use of this antibody may provide another means of visualizing NPY or PYY in the vertebrate nervous system. FMRF-Ll fibers may represent the terminals of neurons projecting to the AP from surrounding structures such as the NTS, the dorsal motor nucleus of the vagus and the nodose ganglion which are known to project to the AP [I 1,26, 301. It is interesting to note that in the rat large numbers of FMRF-Ll cell bodies lie within the NTS near its border with the AP. Numerous FMRF-Ll fibers were seen to cross the AP-NTS boundary in this study and many other investigators have noted an exchange of fibers between the AP and the NTS [23, 26, 43, 441. It may be possible that a large number of the FMRF-Ll fibers within the rat AP may be originating from neurons immediately adjacent to the AP, although conformation of this would depend upon experiments using immunocytochemical and tract-tracing techniques. The distribution of FMRF-Ll afferents, from any source, to the AP would enable these terminals to penetrate its neuropil providing access to the vasculature and the CSF of the IV ventricle and subarachnoid space. Many terminals, some demonstrated to be immunoreactive for tyrosine hydroxylase, have been found within the large perivascular spaces of the AP [3]. Other terminals have been observed in this region contacting the epithelial cells of the fenestrated capillaries [24,41]. These observations suggest that the putative neurotransmitters contained within varicosities of these nerve fibers may be released into the blood stream or into the CSF. and act as neurohumoral agents altering the internal milieu of the CNS. However, the actual morphological location of FMRF-Ll structures within the AP requires further elucidation with the use of immunocytochemistry at the ultrastructural level before a possible neurohumoral role can be proposed. The only established function for the AP of vomiting animals, e.g., the cat, is that of a chemoreceptive trigger zone sampling noxious blood-borne substances for the emetic center lying within the parvicellular reticular formation [7,10]. In the rat, a non-vomiting species, the AP has been associated with numerous other functions including epinephrine-induced hyperglycemia [36], serotonin-induced
MALEY. SASEK AND I’RAlJRlG
synchronization of the electroencephalogram 1371. and the cardiovascular effects of angiotensin [22,401. Many of thcsc AP functions have not been fully established in the rat [5, 8. 191. The only AP function that has been definitely demonstrated in the rat is taste aversion learning which can he abolished with a lesion of the AP 1321. In a recent investigation [9] in which numerous neurotransmitters and peptides were applied onto AP neurons of the dog, a strong correlation was demonstrated between agents that excited neurons within the AP for long periods of time after application, and their ability to cause emesis when injected intravenously. These substances were histamine. dopamine, apomorphine, angiotensin 11, neurotensin. leucine-enkephalin, vasoactive intestinal polypeptide, gastrin, vasopressin. and substance P. Conversely, the three agents they applied to canine AP neurons that were not excitatory, acetylcholine, somatostatin, and cholecystokinin, did not evoke emesis when injected into the vascutar system. These results strongly suggested that the similar response of the AP neurons to so many substances relied upon a common mechanism, and the prolonged nature of the response would subserve the sensation of nausea that precedes emesis. It has been demonstrated that FMRF-NH, ih excit+ tory in the brainstem of the rat 1171 therefore, FMRF-NH, may also act as an emetic agent on AP neurons. Differences in FMRF-Ll between the rat and the cat may form the morphological basis for the differences in the emetic reflex displayed by these two species. Functionally it has been found that NPY and PYY are potent vasoconstrictors 01’blood vessels in the salivary glands of cats 128,291. Other investigator\ have observed a dense network of NPY immunoreactive fibers surrounding cerebral arteries in cats. These NPY fiber\, which can initiate strong contractions in cerebral arteries, are abolished when the superior cervical ganglion is ablated [ 141. if this same vasoconstrictive effect ic present in the CNS of rats, then the functional significance of FMRF-LI ceil bodies in the rat AP may retate to control of choroidal blood flow. The large numbers of FMRF-t-1 fibers observed in the present study suggest that the NPY or PYY released into the AP may be acting as vasoconstrictive agents locally. or that they may be carried to other CNS areas via the V~SCIIlature andlor CSF flow.
ACKNOWLEDGEMENTS
This work was supported in part by NIH grant lR2H130702 to Bruce Maley. The authors would like to thank Kimberly A. Howes for her technical assistance and Aiita Anderson for her secretarial aid. The helpful comments by Drs. M. B. Nikitovitch-Winer and H. Parks are gratefully acknowledged.
REFERENCES 1. Allen, Y. S., T. E. Adrian, J. M. Allen, K. Tatemoto, T. J. Crow, S. R. Bloom and J. M. Polak. Neuropeptide Y distribution in the rat brain. Science 221: 877-879, 1983. 2. Armstrong, D. M., V. M. Pickel, T. H. Joh, D. J. Reis and R. J. Miller. Immunocytochemical localization of catecholamine synthesizing enzymes and neuropeptides in area postrema and medial nucleus tractus solitarius of rat brain. J Camp Neural 1%: 505-517, 1981.
3. Armstrong, D. M., V. M. Pickel, T. J. Joh and D. J. Reis. Electron microscopic immunocytochemical localization of tyrosine hydroxylase in the area postrema of rat. J Camp Neural 206: 259-272, 1982. 4. Armstrong, D. M., V. M. Pickel and D. J. Reis. Electron microscopic immunocytochemical localization of substance P in fhe area postrema of rat. Brain Rrs 243: 141-146, 1982. 5. Borison, H. L. Area postrema: chemoreceptive trigger zone for vomiting-is that all? L.Ue Sci 14: 1807-1817, 1974.
FMRF-LI IN RAT AND CAT AREA F’OSTREMA 6. Borison, R. and H. Borison. Postnatal development of the area postrema with relation to digitalis-induced vomiting in the cat. Exp Neural 16: 138-152, 1973. 7. Brizzee, K. R. and L. M. Neal. A re-evahation of the cellular morphology of the area postrema in view of recent evidence for a chemoreceptor function. J Camp Neurol l@Oz41-61, 1954. 8. Brooks, J. M, J. I. Hubbard and N. E. Sirett. Extracelhdar recording in rat area postre_ma in vitro and the effects of cholinergic drugs, serotonin and angiotensin II. Brain Res 261: 85-90,1983. 9. Carpenter, D. O., D. B. Briggs and N. Strominger. Responses of neurons of canine area postrema to neurotransmitters and peptides. Cell Mol Biol3: 113-126, 1983. 10. Chinn, H. I. and S. C. Wang. Locus of emetic action following irradiation. Proc Sot Exp Biol Med 85: 472474, 1954. 11. Clemente, C. D. and V. L. van Breemen. Nerve fibers in the area postrema of cat, rabbit, guinea pig and rat. Anaf Ret 123~ 65-79, 1955. 12. Dempsey, E. W. Neural and vascular ultrastructure of the area postrema in the rat. J Comp Neural 150: 177-200, 1973. im13. Dockray, G. J. and R. G. Williams. FMRFamide-like munoreactivity in rat brain: development of a radioimmunoassay and its application in studies of distribution and chromatographic properties. Brain Res 266: 295-303, 1983. 14. Edvinsson, L., P. Emson, J. McCulloch, K. Tatemoto and R. Uddman. Neuropeptide Y: cerebrovascular innervation and vasomotor effects in the cat. Neurosci Lert 43: 79-84, 1983. 15. Edwards, G. L. and R. C. Ritter. Ablation of the area postrema causes exaggerated consumption of preferred foods in the rat. Bruin Res %S: 265-276, 1981. 16. Fuxe. K. and C. Owman. Cellular localization of monoamines in the &a oostrema of certain mammals. J Comp Neural 125: 337-354, i%s. 17. Gayton, R. J. Mammalian neuronal actions of FMRFamide and the structurallv related ooioid met-enkenhalin-ar&de’. Nature - _ 2% 275-276, -1982. 18. Greenberg, M. J. and D. A. Price. Cardioregulatory peptides in molluscs. In: Peptides: Integrators of Cell and Tissue Function, edited by F. R. Bloom. New York: Raven Press, 1980, pp. 107-126. 19. Haywood, J. R., G. D. Fink, J. Buggy, M. 1. Phillips and M. J. Brody. The area postrema plays no role in the pressor action of angiotensin in the rat. Am J Physiol 239: HlO8-H113, 1980. 20. Hokfelt, T., J. M. Lundberg, K. Tatemoto, V. Mutt, L. Terenius, J. Polak, S. Bloom, C. Sasek, R. Elde and M. Goldstein. Neuropeptide Y (NPY)-and FMRFamide neuropeptidelike immunoreactivities in catecholamine neurons of the rat medulla oblongata. Acta Physiol Stand 117: 315-318, 1983. 21. Hyde, T. M. and R. R. Miselis. Effects of area postrema/caudal medial nucleus of solitary tract lesions on food intake and body weight. Am J Physiol244: R5774587, 1983. 22. Joy, M. D. The intramedullary connections of the area postrema involved in the central cardiovascular response to angiotensin II. Cfin Sci 41: WlOO, 1971. 23. Kalia, M., K. Fuxe, T. Hokfelt, 0. Johansson, R. Lang, D. Ganten, C. Cuello and L. Terenius. Distribution of neuropep tide immunoreactive nerve terminals within the subnuclei of the nucleus of the tractus solitarius of the rat. J Comp Neural 2U: 409-444,1984. 24. Karasawa, N., M. Yoshida, Y. Kondo, T. Nagatsu and I. Nagatsu. Immunohistocytochemical studies on neurons in the hamster area wstrema. Acta Histochem Cytochem 16: 138-147, 1983. 25. Klara, P. M. and K. R. Brizzee. Ultrastructure of the feline area postrema. J Comp Neurol 171: 404-432, lY77.
399
26. van der Kooy, D. and L. Y. Koda. 0-n of the pmtions of a circumventricular 0~: the area postrema in the rat. J Comp Neural 219: 328-338,1983. 27. Kosten, T., R. J. Contmras, P. W. Stetson and M. J. Ernest. Enhancedsabeintakeanddecreasedheartrateafterarea posbwua ablations in rat. Physiol Behav 31: 777-785, 1983. 28. Lundberg, J. M. and K. Tatemoto. Vascular effects of the pep tides PYY and PHI: comparison with APP and VIP. Eur J Pharmacol83: 143-146,1982. 29. Lundberg, J. M. and K. Tatemoto. Paocreatic polypeptide family (APP, BPP, NPY and PYY) in relation to sympathetic vasoconstriction resistant to alpha-adrenoceptor blockade. Acta Physiol Stand 116: 3-, 1982. 30. Lundberg, J. M., L. Terenius, T. Hokfelt and M. Goldstein. High levels of neuropeptide Y in peripheral noradrenergic neurons in various mammals including man. Neurosci Lett 42: 167-172, 1983. 3 1. Newton, B. W., B. Maley and H. H. Traurig. The distribution of metenkephalin (ME), serotonin (5-HT) and substance P (SP) immunoreactivities in the area postrema (AP) of the rat and cat. Sot Neurosci Abstr 9: 293, 1983. 32. Ossenkopp, K. Taste aversions conditioned with gamma radiation: attenuation by area postrema lesions in rats. Eehav Brain Res 7: 297-305, 1983. 33. Price, D. A. and M. J. Greenberg. Structure of a molluscan cardioexcitatory neuropeptide. Science 141: 67&671, 1977. 34. Rabin, B. M., W. A. Hunt and J. Lee. Studies on the role of central histamine in the acquisition of a radiation-induced conditioned taste aversion. Radiat Res 99: 604-620, 1982. 35. Ritter, S., J. J. McGlone and K. W. Kelley. Absence of lithiuminduced taste aversion after area postrema lesion. Bruin Res 201: 501-506, 1980. 36. Rosenberg, F. J. and V. DiStefano. A central nervous system component of the hyperglycemic response to exogenous epinephrine. Fed Proc 20: 314, l%l. 37. Roth, G. I., P. L. Walton and W. S. Yamamoto. Area postrema: abrupt EEG synchronization following close &a-arterial perfusion with seiotonin. Brain Res 23~ 223-233, 1970. 38. Sasek. C. A.. R. Elde and V. S. Seybold. Localization of FMRF-NH,-like immunoreactivity in a&onomic regions of the brainstem and spinal cord of the rat. Sot Neurosci Abstr 8: 809, 1982. 39. 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. Neuroscience 12: 855-873, 1984. 40. Simpson, J. B. The circumventricular organs and the central action of angiotensin. Neuroendocrinology 32: 248-256, 1981. 41. Spacek, J. and J. Parizek. The fine structure of the area postrema of the rat. Acta Morphol Acad Sci Hung 17: 17-34, 1%9. 42. Takeuchi, Y. and Y. Sano. Serotonin distribution in the circumventricular organs of the rat. Anat Embryof 167: 311-319, 1983. 43. Vigier, D. and P. Portalier. Efferent projections of the area postrema demonstrated by autoradiography. Arch Itol Biol 117: 30%324, 1979. 44. Vigier, D. and A. Rouviere. Afferent and efferent connections ofthe area postrema demonstrated by the horseradish peroxidase method. Arch Ztal Biol 117: 325-339. 1979. 45. Weindl, A. Neuroendocrine aspects of &umventricular organs. In: Frontiers in Neuroendocrinology 1973, edited by W. F. Ganong and L. Martini. New York: Oxford University Press, 1973, pp. 3-32. 46. Williams, R. G. and G. J. Dockray. Immunohistochemical studies of FMRFamide-like immunoreactivity in rat brain. Brain Res 276: 213-229, 1983.