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Proc. Nati. Acad. Sci. USA Vol. 74, No. 8, pp. 3424-3428, August 1977 Cell Biology Vasoactive intestinal polypeptide in brain: Localization in and release from isolated nerve terminals (synaptosomes//hypothalamic, cortical /gastrointestinal /K+) ANTONIO GIACHETTI*, SAMI I. SAID*tf, ROLLAND C. REYNOLDS§, AND FRANK C. KONIGESt Departments of * Pharmacology, * Internal Medicine, and § Pathology, University of Texas Health Science Center, Dallas, Texas 75235; and tVeterans Administration Hospital, Dallas, Texas 75216 Communicated by Berta Scharrer, May 19, 1977

ABSTRACT The vasoactive intestinal polypeptide was Labeled VIP was obtained from Nuclear International Corp. present in synaptosomal (nerve ending) preparations from ce- (Waltham, MA); DL-[3H]-NE (12 mCi/mmol) was from New rebral cortex, , and striatum of rat brain in higher concentrations than in these tissues as a whole. The total content England Nuclear Corp. (Boston, MA); a-endorphin, somatos- and relative specific activity of the increased with tatin, Met- and Leu-, and were pur- progressive purification of the synaptosomal fractions and chased from Peninsula Laboratories, Inc. (San Carlos, CA). All generally followed the distribution of known synaptosomal other chemicals were obtained from Sigma Chemical Co. (St. constituents-, , and lactate dehy- Louis, MO). Pargyline (N-methyl-N-2-propynylben- drogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27). The zylamine) was a gift of Abbot Labs (Chicago, IL). peptide was also released from synaptosomal pellets with in- creased K+ concentration, and this release was Ca2+-dependent. Preparation of Synaptosomes. Male rats (Sprague-Dawley, The findings suggest a role for vasoactive intestinal polypeptide 350 g) were killed by decapitation. Brains were removed, and as a transmitter or modulator of synaptic function. hypothalamic, striatal, and cortical areas were dissected ac- cording to the method of Glowinsky and Iversen (13). All pro- Vasoactive intestinal polypeptide (VIP), a 28-residue peptide cedures were carried out at 40. Brain areas were homogenized that is structurally related to and , has been in 10 volumes of cold 0.32 M sucrose containing pargyline (0.1 isolated from porcine (1, 2). Until recently, its mM) in a glass homogenizer with a Teflon pestle (Kontes) for normal presence was thought to be limited to the gastrointes- 30 sec at 600 rpm. Subsequent centrifugations, collection, and tinal tract, but the discovery of high levels of VIP immuno- designation of fractions were essentially as described by reactivity in neural cell lines and in mammalian brain (3-5) has Whittaker (14) and DeRobertis (15), with the following mod- established its presence in the nervous system as well. VIP thus ification. The nuclear fraction (P1) was washed once and re- joins a number of other polypeptides-including , centrifuged at 1000 X g for 10 min. The combined supernatants . neurotensin, and the enkaphalins-that have been were centrifigued at 12,000 X g for 20 min; the pellet was re- shown to occur both in gut and in brain (6-11). suspended once in the original volume of 0.32 M sucrose and A possible functional role for VIP in the central nervous recentrifuged as above. The new pellet, P2, was subfractionated system is suggested by its selective localization in brain, being by centrifugation in sucrose gradients. at highest concentration in cerebral cortex and hypothalamus For centrifugation on discontinuous density gradients, the and virtually absent in cerebellum (3). The recent demonstra- P2 pellet was resuspended in half the original volume of 0.32 tion, by immunofluorescence, of VIP in cell bodies and nerve M sucrose, and 1 ml of this suspension was layered on a dis- terminals in cortex and in hypothalamic and other nuclei (12) continuous density gradient consisting of equal amounts of 0.8 supports this possibility. M and 1.2 M sucrose and centrifuged at 51,000 X g for 2 hr We have investigated the subcellular distribution of VIP in (rotor SW39, Beckman model L ultracentrifuge). Six fractions selected areas of rat brain, on the basis that nerve-cell constit- were collected by aspiration: (i) 0.5-0.6 ml of 0.32 M sucrose; uents are localized in compartments that correspond to their (Hi) 1 ml, at the interface with 0.8 M sucrose, containing white function and that the subcellular localization of the peptide fluffy material (myelin); (iii) 1 ml of 0.8 M sucrose; (iv) 1 ml, could thus provide an indication of its function. VIP was con- at the interface between 0.8 M and 1.2 M sucrose, showing two centrated in synaptosomal (nerve ending) preparations, which distinct bands-a light, faint one on top and a heavy one un- were also enriched in dopamine and norepinephrine (NE). The derneath (no attempt was made to separate these two bands, peptide also could be released from synaptosomes by depolar- which contained the bulk of the synaptosomes); (v) 1-1.2 ml, ization with high [K+], and the release was Ca2+-dependent. containing the remainder of the 1.2 M sucrose; and (v) pellet, These findings provide evidence for a physiologic role for VIP containing mitochondria. in synaptic function, possibly as a or modu- Data are expressed for three final fractions: 1, comprising lator. fractions (i) and (ii) (lighter than 0.8 M sucrose); 2, combining (iii-v) (between 0.8 and 1.2 M); and 3, fraction (vi) (heavier MATERIALS AND METHODS than 1.2 M). VIP for iodination and for standard solutions in the radioim- Continuous sucrose gradients were formed in SW39 tubes munoassay was the natural porcine peptide (2), prepared in the with a standard gradient apparatus, by mixing equal amounts laboratory of V. Mutt, Karolinska Institute, Stockholm. 125I- of 1.6 M and 0.8 M sucrose. The resuspended P2 fraction (1 ml = 10 mg of protein) was layered on top of the gradient, which The costs of publication of this article were defrayed in part by the was then centrifuged at 51,000 X g for 120 min. By puncturing payment of page charges from funds made available to support the research which is the subject of the article. This article must therefore Abbreviations: VIP, vasoactive intestinal polypeptide; NE, norepi- be hereby marked "advertisement" in accordance with 18 U. S. C. nephrine; SDH, succinate dehydrogenase; LDH, lactate dehydroge- §1734 solely to indicate this fact. nase. 3424 Downloaded by guest on September 26, 2021 Cell Biology: Giachetti et al. Proc. Natl. Acad. Sci. USA 74 (1977) 3425

Table 1. Distribution of VIP immunoreactivty in subeelhlalr 80- fractions of rat brain

VIP content, ng/g* Cru Hypothalamus Striatum Cerebral 0 Fraction (4) (4) cortex (3) + 60- 0 Homogenate 12.10 + 0.70 17.60 + 4.50 26.40 : 4.60 P1 2.60 + 0.34 3.05 + 0.55 5.51 d 0.31 S2 5.50 1 0.75 4.80 1 0.55 13.64 ± 2.44 40- P2 9.54 I 0.61 13.60 b 2.90 19.76 1 1.94 0 * Values are means + SEM. Numbers in parentheses are the numbers of determinations. L' 20- the tubes at the bottom and counting drops, 18 fractions of approximately 0.3 ml each were collected. Because this volume F-L 4' was too small to permit assays in duplicate, fractions from two gradients were usually combined and the volume of each 0 _ ..r _ fraction was brought up to 1.0 ml with distilled water. Pi S2 P2 When hypothalamic or cortical tissues were labeled in vitro FIG. 1. Subcellular distribution of immunoreactive VIP, protein, with DL-[3H]NE, blocks of tissue were dissected with a razor SDH, and NE in rat hypothalamus. Distribution is expressed as blade. Each block, weighing 25-30 mg, was incubated in 2 ml percentage of total recovered content or activity in the subcellular of Krebs-Henseleit buffer containing [3H]NE (0.2 MAM) for 20 fractions PI, S2, and P2. Values are means + SEM from four experi- min at 370 in an atmosphere at 95% 02/5% CO2. At the end of ments. o, VIP; a, protein; X, SDH; al, NE. the incubation, blocks were rinsed in Krebs-Henseleit buffer, VIP was measured by a sensitive radioimmunoassay as de- weighed and homogenized in 0.32 M sucrose, and fractionated scribed (20), with the following modifications: a high-titer as outlined above. (>1:150,000), specific antiserum was produced by immuni- Release Experiments. Experiments testing the release of the zation with VIP-bovine serum albumin conjugate, and 125I- peptide were carried out on P2 pellets prepared from cerebral labeled VIP was prepared by the lactoperoxidase method. The cortex. The pellets were resuspended in Krebs solution to obtain lower limit of sensitivity of the assay was 20 pg. The assay a protein concentration of 4 mg/ml (11.25 ml of Krebs solution showed negligible crossreactivity with these peptides: secretin, per g of original tissue). One-half milliliter of the P2 suspension glucagon, -pancreozymin, porcine pancreatic was mixed in a centrifuge tube with 0.5 ml of each of four polypeptide, substance P, somatostatin, a-endorphin [fl-lipo- variants of Krebs solution: (i) normal (5.9 mM) [K+], absent tropin-(61-76)], neurotensin, Leu-enkephalin, Met-enkephalin,, Ca2+; (i) high (55 mM) [K+], normal (1.3 mM) [Ca2+]; (iii) high , and II. [K+], absent Ca2+; and (iv) normal [K+], normal [Ca2+]. The Samples for electron microscopic examination of synapto- mixtures were incubated at 30° for 30 min, following which somal fractions were prepared as described (21). they were centrifuged at 40,000 X g for 10 min. The superna- Statistical analysis was performed by Student's t test, using tant solution was pipetted off and assayed for VIP immuno- paired or unpaired comparisons. P values less than 0.05 were reactivity. considered significant. Analytical Determinations. Protein was determined ac- cording to Lowry et al. (16). The sucrose concentration was RESULTS estimated by measuring the refractive index in a Goldberg refractometer. Succinate dehydrogenase [succinate:(acceptor) VIP in subcellular fractions oxidoreductase, EC 1.3.99.1] (SDH) activity was measured as The peptide levels were particularly high in homogenates from described by Pennington (17), 1 unit being equivalent to a cerebral cortex-(26.4 ng/g of tissue), being twice as high as in change in A of 1.0 per 15 min. Lactate dehydrogenase (L-lac- hypothalamus and greater than in striatum (Table 1). Differ- tate:NAD+ oxidoreductase, EC 1.1.1.27) (LDH) was measured ential centrifugation of these homogenates yielded the classical spectrophotometrically (18) as follows. Triton X-100 was added subcellular fractions (14)-i.e., cell nuclei and debris (Pi); mi- to each sample to a final concentration of 0.05%, to release the crosomes (S2); and crude synaptosomes, also containing mito- occluded activity. Aliquots of the sample were then added to chondria (P2). The P2 fractions characteristically contained the a reaction mixture consisting of 1 mM Na pyruvate, 0.1 mM bulk of the proteins, the mitochondrial enzyme SDH, and the NADH, and 0.05 M Na phosphate buffer (pH 7.4) to a volume neurotransmitter NE (measured in hypothalamic preparations) of 2 ml; the oxidation of NADH was followed with a recording or dopamine (measured in striatal preparations) (Fig. 1). When spectrophotometer (Gilson) for 5 min, and the enzyme activity examined with the electron microscope after fixation with in the sample was calculated from the change in A at 340 nm; glutaraldehyde, the P2 pellets showed several synaptosomal 1 unit of enzyme equals 1 umol of NADH oxidized per min. NE profiles, some containing synaptic vesicles and vacuoles (Fig. and dopamine were determined spectrophotometrically after 2). extraction with HCI04 (0.4 M final concentration), purification In each of the three brain areas examined, the concentration with alumina, and oxidation according to Chang (19). of VIP was highest in the P2 fraction (P < 0.01) (Table 1, Fig. When [3H]NE was used to label noradrenergic neurones, the 1). The distribution of VIP, relative to its total recovered con- 3H present in the fractions was determined directly in an aliquot tent, was similarly highest in the crude synaptosomal fractions. (50 Ml) of sucrose by adding 10 ml of Aquasol (New England Thus, the percentages of VIP in P2 fractions were 54.4 for hy- Nuclear) and assaying for radioactivity with a liquid scintilla- pothalamus, 61.8 for striatum, and 50.9 for cerebral cortex tion spectrometer. Earlier experiments had shown that under (Table 2). The relative specific activity of VIP (i.e., its per- our experimental conditions most of the radioactivity (>85%) centage content relative to that of protein) was also highest in represented unchanged [3H]NE. the P2 fraction in each instance. Downloaded by guest on September 26, 2021 3426 Cell Biology: Giachetti et al. Proc. Nati. Acad. Sci. USA 74 (1977)

Table 2. Distribution of relative specific activity (RSA)* of VIP in subcellular fractions of rat brain Hypothalamus Striatum Cerebral cortex (4) (4) (3) %of %of %of Fraction total activity RSA total activity RSA total activity RSA

P1 14.8 ± 1.6 0.75 ± 0.08 14.2 4 1.2 0.72 - 0.06 14.4 + 1.3 0.93 4f 0.17 S2 30.8 ± 2.4 0.96 ± 0.07 24.1 4 4.4 0.78 ± 0.17 34.7 4 2.4 0.77 + 0.09 P2 54.4 ± 2.0 1.14 k 0.05 61.8 ± 4.2 1.30 1 0.14 50.9 i 1.1 1.34 : 0.07 * Relative specific activity (RSA) is percentage VIP content relative to that of protein. Values are means + SEM. Numbers in parentheses are the numbers of determinations.

The sum of VIP levels in the three subcellular fractions Release of VIP from synaptosomes consistently exceeded the peptide content in the original ho- mogenate (Table 1). Raising the K+ concentration in the incubation medium from 5.9 to 55 mM in the presence of Ca2+ evoked an increase in VIP Further purification of synaptosomal fractions concentration in the medium over a SO-min period, from 91 ± On subfractionation of the crude synaptosomal fractions by 8 to 173 h 18 pg/ml (15 determinations, P < 0.0005). Omission centrifugation in discontinuous sucrose gradients (Table 3), VIP of Ca2+ from the medium abolished the apparent release of the immunoreactivity was recovered mainly in the purified sy- peptide from cortical synaptosomes under the same conditions naptosomes which were also richest in dopamine and in pro- (six determinations, p < 0.0025). The absence of Ca2+ itself was teins; the purified mitochondrial pellet contained most of the associated with a fall in VIP concentration from 91 4 8 to 79 SDH activity. :19 pg/ml, but the difference was not significant. With continuous sucrose-gradient centrifugation (Fig. 3), mitochondria (SDH) sedimented in a band of heavy sucrose DISCUSSION (fractions 3-6), distinct from the band (fractions 6-10) con- The abundance of VIP-like immunoreactivity in the brain areas taining the bulk of LDH activity, an enzyme considered to be examined, especially in cerebral cortex, confirms earlier studies a synaptosomal marker (18). The distribution of VIP (fractions on the distribution of this peptide in dog brain (3) and is in 6-12) followed closely that of LDH. The highest specific ac- agreement with immunohistochemical observation in rats (12). tivity of VIP (1.81) occurred in fraction 11, whereas that of In this respect, the regional distribution of VIP differs from that LDH (1.71) was in fraction 9. In the experiment shown in Fig. of most other brain peptides-e.g., substance P and neuro- 3, the hypothalamic preparation had been incubated in vitro with [3H]NE before the isolation of synaptosomes. The bulk of the labeled neurotransmitter was found in the same band of sucrose (fractions 6-12) containing the major portions of LDH and VIP. The peak specific activity of [3HJNE (1.84) was in fraction 10. Due to slight variations in the collection of fractions, the results of multiple experiments could not be combined, but six experiments with striatal and hypothalamic tissues gave essentially the same distribution as shown. We also verified the integrity of the synaptosomal preparation by showing that the [3H]NE uptake was via an active process: incubation on ice was a followed by little incorporation of radioactivity in the major 0. synaptosomal peak. crJ Q

2 4 6 8 10 12 14 16 18 Fraction number FIG. 3. Fractionation of hypothalamic synaptosomes on con- FIG. 2. Electron micrograph of synaptosomal preparation from tinuous sucrose gradients. Distributions ofVIP (0), protein (o), SDH rat hypothalamus, showing synaptosomal profiles including mito- (A), LDH (0), and [3HJNE (-) are expressed as percentages of their chondria and small vesicles ofvariable size. One synaptic cleft is seen total activity or content per fraction. Crude synaptosomes, corre- near the center. Section was fixed in glutaraldehyde and osmium te- sponding to 0.1 g of original tissues, were centrifuged at 51,000 X g troxide and stained on the grid with uranyl acetate and lead citrate for 120 min at 4°. Gradient density decreases from left (1.6 M) to (18). (X20,000.) right. Downloaded by guest on September 26, 2021 Cell Biology: Giachetti et al. Proc. Nati. Acad. Sci. USA 74 (1977) 3427

Table 3. Distribution of VIP, dopamine, and SDHhin subcellular fractions of rat striatum Recovered activity, % of total VIP Dopamine SDH Protein Fractions (8) (2) (3) (4) 1. Lighter than 0.8 M (myelin) 18.5 ± 1.1 0 1.2 4 1.2 24.6 A 1.6 2. Between 0.8 and 1.2 M (synaptosomes) 51.2 : 1.9 69.0 10.3 ± 2.5 39.5 + 2.8 3. Denser than 1.2 M (mitochondria) 30.4 + 2.3 31.0 88.4 1 3.4 35.9 + 1.9 Values are means + SEM. Numbers in parentheses are the numbers of determinations.

tensin-which are found in low concentrations in the cortex and 1. Said, S. I. & Mutt, V. (1970) "Polypeptide with broad biological resembles that of -like or cholecystokinin-like immu- activity: Isolation from ," Science 169, 1217- noreactivity (22, 23). 1218. Our experiments show that isolated nerve terminals (sy- 2. Said, S. I. & Mutt, V. (1972) "Isolation from porcine intestinal wall naptosomes) from cerebral cortex, hypothalamus, and striatum of a vasoactive octacosapeptide related to secretin and to gluca- of rat brain are enriched in VIP. This enrichment was evident gon." Eur. J. Biochem. 28, 199-204. 3. Said, S. I. & Rosenberg, R. (1976) "Vasoactive intestinal poly- in the crude synaptosomal fractions (P2 pellets) and became peptide: Abundant immunoreactivity in neural cell lines and more pronounced with their progressive purification by cen- normal nervous tissues," Science 192, 907-908. trifugation on discontinuous or continuous sucrose gradients. 4. Bryant, M. G., Bloom, S. R., Polak, J. M., Albuquerque, R. H., In these fractions, the highest concentrations (and relative Modlin, I. & Pearse, A. G. E. (1976) "Possible dual role for va- specific activity) of the peptide coincided closely with those of soactive intestinal peptide as gastrointestinal and neu- NE and of LDH, a synaptosomal enzyme. The increase in total rotransmitter substance," Lancet i, 991-993. VIP immunoreactivity after fractionation of the homogenates 5. Larsson, L.-I., Fahrenkrug, J., Schaffalitzky de Muckadell, O., is at present unexplained, though a similar finding was reported Sundler, F., HAkanson, R. & Rehfeld, J. F. (1976) "Localization for brain neurotensin (24). of vasoactive intestinal polypeptide (VIP) to central and pe- The finding that VIP is concentrated in ripheral neurons," Proc. Natl. Acad. Sci. USA 73,3197-3200. synaptosomal 6. Hokfelt, T., Efendic, S., Hellerstrom, C., Johansson, O., Luft, R. preparations agrees with immunocytochemical localization of & Arimura, A. (1975) "Cellular localization of somatostatin in the peptide, done with the same antibody, in nerve terminals endocrine-like cells and neurons of the rat with special references of the neocortex, neostriatum, central amygdaloid nucleus, and to the A1-cells of the pancreatic islets and to the hypothalamus," suprachiasmatic, medial preoptic, and anterior hypothalamic Acta Endocrinol. 80 (Suppl. 200):5-41. nuclei (12). 7. Leeman, S. E. & Mroz, E. A. (1974) "Substance P," Life Sci. 15, The demonstrated K+-evoked release of VIP from synapto- 2033-2044. somes and the prevention of this release with omission of Ca2+ 8. Carraway, R. & Leeman, S. E. (1973) "The isolation of a new suggest that the peptide may be released from nerve terminals hypotensive peptide, neurotensin, from bovine hypothalami," by physiologic-e.g., depolarizing-stimuli. Similar observa- J. Biol. Chem. 248,6854-6861. 9. Orci, L., Baetens, O., Rufener, C., Brown, M., Vale, W. & Guil- tions have been made for NE (25) and more recently for sub- lemin,.R. (1976) "Evidence for immunoreactive neurotensin in stance P (26). dog intestinal mucosa," Life Sci. 19, 559-562. Our findings that isolated nerve terminals are enriched in 10. Hughes,. J., Smith, T. W., Kosterlitz, H. W., Fothergill, L. A., VIP and that they may release it with increased K+ concen- Morgan, B. A. & Morris, H. R. (1975) "Identification of two re- tration in the presence of Ca2+ lend support to the possibility lated pentapetides from the brain with potent opiate agonist that VIP may have a physiologic role in synaptic function, either activity," Nature 258, 577-579. as a transmitter or as a modulator of the actions of other trans- 11. Pearse, A. G. E. (1976) "Peptides in brain and intestine," Nature mitters (27). The selective distribution of the peptide in cerebral 262,92-94. cortex, especially in layers II-IV of the neocortex, within as- 12. Fuxe, K., Hokfelt, T., Said, S. I. & Mutt, V. (1977) "Vasoactive sociation neurons (12), suggests a special in intestinal polypeptide and the nervous system: Immunohisto- role integrative chemical evidence for localization in central and peripheral activity. The significance of the prevalence of VIP in hy- neurons, particularly intracortical neurons of the cerebral cortex," pothalamic and striatal nerve terminals is undetermined. Be- Neurosci. Lett., in press. cause VIP is a potent vasodilator, is located in cerebrovascular 13. Glowinsky, J. & Iversen, L. L. (1966) "Regional studies of cate- nerves (28), and augments cerebral blood flow in intact animals cholamines in the rat brain-I. The disposition of 3H-nor- (unpublished observations), the peptide may serve as a mediator epinephrine, 3H-dopamine and 3H-DOPA in various regions of of cerebral . The possibility that VIP, like other the brain," J. Neurochem. 13, 655-669. brain peptides, may also influence neuronal function without 14. Whittaker, V. P. (1959) "The isolation and characterization of affecting synaptic transmission per se remains to be acetylcholine-containing particles from brain," Biochem. J. 72, (29, 30) 694-706. investigated. 15. DeRobertis, E. (1975) "Synaptic receptors," in Isolation and Molecular Biology (Marcel Dekker, Inc., New York). 16. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. We thank Dr. Rene Frenkel, Department of Biochemistry, for help (1951) "Protein measurement with the folin phenol reagent," J. with the LDH assay and Mrs. Shirley M. Harvey, Mrs. J.inda Dick, and Biol. Chem. 193,265-275. Mr. Wallace T. Ford, Jr., for excellent technical assistance. This work 17. Pennington, R. J. (1961) "Biochemistry of dystrophic muscle. was supported in part by Grant HL-19300 and Center Award HL- Mitochondrial succinate-tetrazolium reductase and adenosine 14187 from the National Institutes of Health, and by a grant from the triphosphatase," Biochem. J. 80, 649-654. American Cancer Society. 18. Johnson, M. K. & Whittaker, V. P. (1963) "Lactate dehydrogenase Downloaded by guest on September 26, 2021 Boy: Giamuaet-et al Proc. NaUi#.Ac.ScUA 74:1#7

as a cytoplasmic marker in brain," Biochemi. J. 88,404-409. 25. Blaustein, M. P., Johnson, E. M., Jr. & Needleman, P. (1972) 19. Chang, C. C. (1964) "A sensitive method for spectrophotofluo- "Calcium-dependent norepinephrine release from presynaptic rometric assay of ," Int. J. Neuropharmacol. 3, nerve endings in vitro," Proc. Natl. Acad. Sci. USA 89, 2237- 643-649. 2240. 20. Said, S. I. & Faloona, G. R. (1975) "Elevated plasma and tissue 26. Schenker, C., Mroz E. A. & Leeman, S. E. (1976) "Release of levels of vasoactive intestinal polypeptide in the watery diarrhea substance P from isolated nerve endings," Nature 264, 790- syndrome due to pancreatic, bronchogenic and other tumors," 792. N. Engl. J. Med. 293,155-160. 27. Giachetti, A., Rosenberg, R. N. & Said, S. I. (1976) "Vasoactive 21. Warberg, J., Eskay, R. L., Barnea, A., Reynolds, R. C. & Porter, intestinal polypeptide in brain synaptosomes," Lancet ii, J. C. (1977) "Release of releasing hormone 741-742. and thyrotropin releasing hormone from a synaptosome-enriched 28. Larsson, L.-I., Edvinsson, L., Fahrenkrug, J., Hakanson, R., fraction of hypothalamic homogenates," Endocrinology 100, Owman, Ch., Schaffalitzky de Muckadell, 0. & Sundler, F. (1976) 814-825. "Immunohistochemical localization of a vasodilatory polypeptide 22. Vanderhaeghen, J. J., Signeau, J. C. & Gepts, W. (1975) "New (VIP) in cerebrovascular nerves," Brain Res. 113,400-404. peptide in the vertebrate CNS reacting with antigastrin anti- 29. Nicoll, R. A. (1975) "Peptide receptors in CNS," in Handbook bodies," Nature 257, 604-605. of Psychopharmacology, eds. Iversen, L. L., Iversen, S. D. & 23. Dockray, G. J. (1976) "Immunochemical evidence of cholecys- Snyder, S. H. (Plenum Press, New York), Vol. 4, pp. 229-263. tokinin-like peptide in brain," Nature 264,568-570. 30. Barker, J. L. (1977) "Physiological roles of peptides in the nervous 24. Uhl, G. R. & Snyder, S. H. (1976) "Regional and subcellular dis- system," in Peptides in Neurobiology, ed. Gainer, H. (Plenum tributions of brain neurotensin," Life Sci. 19, 1827-1832. Press, New York), pp. 295-43. Downloaded by guest on September 26, 2021