Brain Research, 165 (1979) 79-85 © Elsevier/North-HollandBiomedicalPress
79
VASOACTIVE INTESTINAL PEPTIDE (VIP): BRAIN DISTRIBUTION, SUBCELLULAR LOCALIZATION AND EFFECT OF DEAFFERENTATION OF THE HYPOTHALAMUS IN MALE RATS
JACQUELINE BESSON, WILLIAM ROTSZTEJN, MARC LABURTHE, JACQUES EPELBAUM, ALAIN BEAUDET, CLAUDE KORDON and GABRIELROSSELIN Unit~ de Recherche de Diabdtologie et d'Etudes Radioimmunologiques des Hormones Protdiques, U.55 (Institut National de la Santd et de la Recherche M~dicale) Hdpital St. Antoine, 75012 Paris and (W. R., J. E., A. B and C. K.) Unitd de Neuroendocrinologie, I N S E R M U.159, Centre Paul Broca, 75014 Paris (France)
(Accepted July 5th, 1978)
SUMMARY We have studied the regional and subcellular distribution of vasoactive intestinal peptide (VIP) in the brain of adult male rat, using a specific radioimmunoassay. Selective deafferentation of the mediobasal hypothalamus (MBH) was also performed in order to investigate the origin of hypothalamic VIP. The highest concentrations of VIP were found in the neocortex, namely the occipital region. The brain stem, the posterior hypothalamus, and the pineal gland contained low amounts of the peptide. VIP was not detectable in the cerebellum and the neurohypophysis. After fractionation, most of the VIP was recovered from the crude mitochondrial fraction of the hypothalamus as well as the parietal cortex. However, a non-negligible portion of the activity was also found in the supernatant suggesting that the peptide is mainly located in nerve endings but also present in neuronal cell bodies and/or axons. Two weeks after complete deafferentation of the MBH, VIP concentrations of the caudal MBH (including the infundibular sulcus, the stalk, part of the ventromedial nucleus and premamillary structures) were decreased by 40 ~. In contrast, no change in VIP levels were observed in the rostral MBH, organum vasculosum of the lamina terminalis (OVLT), and cortex. This suggests that hypothalamic nerve endings containing the peptide derive from neuronal cell bodies located both outside and within the MBH.
INTRODUCTION The vasoactive intestinal peptide (VIP) isolated from porcine small intestinet7 is structurally and biologically related to secretin, glucagon and gastric inhibitory polypeptide (GIP)14, is. High concentrations of VIP have been detected by radio-
80 immunoassay throughout the gastrointestinal tract 1-3,11 in neural cell lines 19 and in the central nervous system4,S, 19. Moreover, recent immunohistochemical studies have revealed the presence of numerous VIP-positive nerve cell bodies and terminals in the brain6,11,1z. The purpose of the present study is to provide a more detailed description of VIP regional distribution and subcellular localization in the rat brain. In order to investigate the origin of hypothalamic VIP, the peptide was also assayed following selective deafferentation of the mediobasal hypothalamus (MBH). MATERIALS AND METHODS Adult male Wistar rats, weighing 210-230 g, were killed by decapitation. Olfactory bulbs, fragments of the frontoparietai and occipital cortices, striatum, hippocampus, thalamus, brain stem, cerebellum and hypothalamus were rapidly dissected on ice. The hypothalamus was further divided in anterior, median and posterior fragments. The pineal gland as well as the adeno- and neurohypophyses were also sampled. Subcellularfractionation. Thirty hypothalami or 10 fragments of parietal cortex were pooled in 0.32 M sucrose (10 ~ w/v) and homogenized with a Teflon pestle and a glass homogenizer (clearance to 0.15 mm, 10 up and down strokes) at 4 °C. The subsequent fractionation was carried out as described by Ramirez et al. 1~. The whole process was controlled by evaluation of the lactate dehydrogenase (LDH) content of the fractions, according to the method of Johnson and Whittaked °. As an additional control, the subcellular distribution of VIP was compared with that of luteinizing hormone-releasing hormone (LH-RH). Deafferentation studies. Complete deafferentations of the mediobasal hypothalamus (MBH) were performed by surgical isolation, using an Hal~tsz knife (1.5 mm radius) under stereotaxic control 9. The animals were killed by decapitation two weeks after surgery. The MBH was dissected out and divided into a rostral part, containing the median eminence and most of the arcuate nucleus, and a caudal part, including the infundibular sulcus, the proximal stump of the stalk, part of the ventromedial nucleus and premammillary structures 16. The parietal cortex as well as a medial prechiasmatic fragment containing the organum vasculosum of the lamina terminalis (OVLT) 2° were also taken. The effectiveness of all deafferentations was verified by macroscopic examination and by measurement of L H - R H levels in the isolated islands 2°. Only animals showing a 75 ~ decrease in the L H - R H content of the islands were included in the results. VIP extraction and radioimmunoassay. The tissues were first boiled for 5 rain in deionized water in order to destroy the proteolytic enzymes 13. VIP was then extracted by sonication in 0.5 M acetic acid and lyophilized. In some experiments VIP was directly extracted with 0.1 N hydrochloric acid, so that other neuropeptides and particularly L H - R H could be simultaneously detected. VIP immunoreactivity was measured by a specific radioimmunoassay as previously described (detection sensitivity 60 pg/ml, intra-assay variation coefficient < 0.09, cross-reaction with secretin, glucagon, GIP, and the synthetic fragments 1-6, 14-28, 18-28 ofVIP < 0.3 ~)1. It was
81 verified that neither sucrose nor 0.1 N HCI interfered in the radioimmunoassay system, and that identical results for VIP content were obtained after either acetic or hydrochloric acid extraction of the samples. RESULTS Regional distribution. VIP is present in almost all rat brain structures assayed (Table I). The highest concentrations are found in the neocortex, namely the occipital region. VIP concentration are low in the brain stem, the posterior hypothalamus, the pineal gland, and not detectable in the cerebellum and the neurohypophysis. Subcellular localization. The subcellular distribution of VIP is similar in the cortex and the hypothalamus (Table II). After the first differential centrifugation, VIP is mainly recovered from the low speed supernatant S1. Only small amounts ( < 13 %) are found in the pellet P1, which contains nuclei and large membrane debris. After the second centrifugation, most of the VIP present in the supernatant S1 is recovered from the crude mitochondrial fraction (Pz), which contains myelin, synaptosomes, mitochondria, and small membrane debris. Subfractionation of the pellet (P2) on a density gradient shows that almost all endogenous VIP migrates with band B, which contains most of the synaptosomes. About one-fifth of the original amount of VIP remains in the S~ supernatant, most of which is recovered from the Ss fraction. L H - R H is mainly recovered from synaptosomal fraction (P2) and only 5 % from the microsomal supernatant ($2). After further fractionation of $2 the totality of L H - R H is recovered from the microsomal pellet (Ps). Deafferentation experiments. Complete deafferentation of the mediobasal hypothalamus (MBH) induces a 40 % decrease in VIP concentration of the caudal M B H
TABLE I Regional distribution of immunoreactive VIP in the rat brain
Values are reported as mean 4- S.E.M. of n determinations. Numbers in parentheses are the number of structures pooled for one determination, n.d. = not detectable. Structure
n
Wet weight (mg)
VIP (pg/mg)
Occipital cortex Parietal cortex Frontal cortex Anterior hypothalamus (3) Median hypothalamus (3) Posterior hyptothalamus (3) Hippocampus Striatum Olfactory bulbs (4) Thalamus Brain stem Cerebellum (4) Adenohypophysis (6) Neurohypophysis (10) Pineal gland (8)
5 5 5 3 3 3 6 6 3 5 6 3 3 2 1
108 -4- 14 160 4- 9 36 4- 4 19.2 4- 0.3 26.3 ± 0.6 13.3 4- 0.7 104 4- 7 72 4- 3 76 =t=3 36 4- 4 219 4- 8 219 4- 4 ~ 10 ~, 1 ~ 1
302 4- 16 265 4- 15 169 4- 20 60 4- 6 39 4- 5 20 4- 1 69 4- 4 58 4- 9 58 4- 8 34 4- 3 24 4- 1 n.d. 42 4- 3 n.d. 16
II
120"**
51,000
P~ A B C
P3 S~
262 34 215 47 6 15 127 n.d. 119 28
100 13 82 18 2 6 49 -45 11
%
0.94 0.11 1.12 0.69 0.02 0.72 0.44 n.d, 0.33 0.11
Units**
LDH
100 12 119 73 2 77 47 -35 12
%*
82 7 69 12 2 5 38 1.7 41 5.6
Tissue (rig~g)
VIP
Hypothalamus
100 9 84 15 2 6 46 2 50 7
%*
1.3 0.08 0.95 0,42 0.07 0.45 0.34 n.d. 0.22 n.d.
Units**
LDH
100 6 73 32 5 35 26 -25 --
%*
23t 54 157 11 11 n.d. 83
Tissue (ng/g)
LH-RH
100 23 68 5 5 -36
%*
* Per cent of the values measured in the homogenate, ** L D H activity is expressed as pmole N A D H ~ / h / m g wet weight. *** Sucrose gradient. A: 0.32-0.8 M sucrose interface containing myelin; B: 0.8-1,2 M sucrose interface containing synaptosomes; C: mitochondrial a n d synaptosomal pellet, n.d. =~ not detectable.
20 60
17,000 100,000
Homogena~
3
1000
P1 S1 S~
(min)
(g)
Tissue (rig~g)
VIP
Time
Speed
Fraction
Cortex
Centrifugation
Subcellular localization of immunoreactive VIP in cerebral cortex and hypothalamus of male rat
TABLE
I'0
83 TABLE III Effect of total deafferentation of the mediobasal hypothalamus (MBH) on VIP concentrations o f the rostral and caudal MBH, the 0 VLT and the cortex
Values are given as mean 4- S.E.M. with number of animals in parentheses. Structure
Rostral MBH Caudal MBH OVLT Cortex
Control
Complete deafferentation
V1P/Protein (ng/mg)
LH-RH/Protein (rig~rag)
V1P/Protein (ng/mg)
LH-RH/Protein (ng/mg)
3.9 3.1 3.1 4.9
18.0 4- 1.4 (12) 8.7 + 1.4 (12) 0.4 4- 0.1 (12) --
3.3 1.8 2.9 5.2
4.6 -4- 0.5 (6)* 2.1 4- 0.8 (6)* 0.5 4- 0.1 (6) --
-4- 0.3 (12) 4- 0.4 (12) 4- 0.3 (12) -4- 0.9 (6)
-4- 0.5 (6) i 0.3 (6)* 4- 0.5 (6) 4- 0.6 (6)
* P < 0.01. The protein content of the tissue samples from operated animals were not significantly different from that of controls. (Table III). In contrast, there is no significant reduction of VIP levels in the rostral MBH, O V L T and cortex. L H - R H concentration in the OVLT is not modified by complete deafferentation of the MBH. DISCUSSION The present study shows that immunoreactive VIP is present in most rat brain structures, as previously reported for different species3,8,19. VIP concentrations reported here are consistent with those found by Emson et al. a in the hypothalamus. However, they are significantly higher than those found by Giachetti et al. in the cortex, the hypothalamus and the striatum 8. The reasons of this discrepancy are not known, but they could be due to differences in the experimental procedures and/or specificity of the antibodies used in both studies. The highest concentrations of VIP found in the cerebral cortex confirm the results of other radioimmunoassays performed in dog 19 and rat brain s, as well as immunohistochemical observations in rat 6. VIP concentrations of the anterior hypothalamus seem to be higher than those found in its posterior part; however, such a difference is not reported by Emson et al. 4. VIP subcellular localization in the hypothalamus and parietal cortex is similar. In both structures most of the VIP is recovered from the crude mitochondrial fraction. The good correlation between VIP concentrations and L D H activity in band A, B and C after gradient centrifugation confirms that VIP present in P2 is mainly contained in nerve endings 21. This conclusion is in good agreement with previous observations made by Giachetti et al. s on the rat striatum and Emson et al. 4 on the hypothalamus. In addition, a non-negligible fraction of the activity is also found in the supernatant (Sz). This contrasts with the distribution of other neuropeptides, such as L H - R H 7 and somatostatinS; only traces of these peptides were found in the supernatant Sz, as confirmed here for L H - R H . The presence of VIP in the supernatant S~ cannot be explained by contamination due to disrupted synaptosomes, since further centrifugation of $2 shows that the VIP content of S~ is
84 mainly recovered from the supernatant Sa. Recovery of V1P from the supernatant thus suggests that the peptide is also present in neuronal cell bodies and/or axons, a conclusion which is in keeping with recent immunocytochemical data 6 11. The significant decrease (40~o) in VIP concentration of the caudal MBH following complete deafferentation of the hypothalamus suggests that a relatively high p r o p o r t i o n of the peptide is present in nerve endings originating from neuronal cell bodies located outside the island. Nevertheless, the largest fraction of V I P is unaffected by the lesions. This intrinsic V I P might be present in nerve terminals whose cell bodies are located within the island; such VIP-containing cell bodies have been observed in the ventromedial hypothalamus of the mouse tl. This interpretation would also be in keeping with our subcellular fractionation data which show a substantial recovery of V I P in the supernatant $3. Presence of the neuropeptide in non-neuronal cells is unlikely but cannot be formally excluded. At any rate, the unequal distribution of V I P in various structures of the central nervous system and its presence within nerve endings is compatible with the hypothesis that the peptide m a y exhibit neurotransmitter functions a,4,8,19. ACKNOWLEDGEMENTS We thank D. Hui Bon H o a and E. Pattou for their skillful technical assistance and C. Brunet for the careful preparation of the manuscript. This work was supported by the D616gation G6n6rale 5. la Recherche Scientifique (77-7-0504).
REFERENCES 1 Besson, J., Laburthe, M., Bataille, D., Dupont, C, and Rosselin, G., Vasoactive intestinal peptide (VIP): tissue distribution in the rat as measured by radioimmunoassay and by radioreceptor assay, Acta Endocr. (Kbh.), 87 (1978) 799-810. 2 Bloom, S. R., Bryant, M. G. and Polak, J. M., Distribution of gut hormones, Gut, 16 (1975) 821. 3 Bryant, M. G., Bloom, S. R., Polak, J. M., Albuquerque, R. H., Modlin, I. and Pearse, A. G. E., Possible dual role for vasoactive intestinal peptide as gastrointestinal hormone and neurotransmitter substance, Lancet, I (1976) 991-993. 4 Emson, P. C., Fabrenkrug, J., Schaffalitsky de Muckadell, O. B., Jessel, T. M. and Iversen, L. L., Vasoactive intestinal polypeptide (VIP): vesicular localization and potassium-evoked release from rat hypothalamus, Brain Research, 143 (1978) 174-178. 5 Epelbaum, J., Brazeau, P., Tsang, D., Brawer, J. and Martin, J. B., Subcellular distribution of radioimmunoassayable somatostatin in rat brain, Brain Research, 126 (1977) 309-323. 6 Fuxe, K., H/Jkfelt, T., Said, S. I. and Mutt, V., Vasoactive intestinal peptide and the nervous system: immunohistochemical evidence for localization in central and peripheral neurons, particularly intracortical neurons of the cerebral cortex, Neurosci. Lett., 5 (1977) 241-246. 7 Gautron, J. P., Pattou, E. and Kordon, C., New data on the subcellular distribution of LH-RH in various structures of the rat hypothalamus, Molec. cell. Endocr., 8 (1977) 81-92. 8 Giachetti, A., Said, S. I., Reynolds, R. and Koniges, F. C., Vasoactive intestinal polypeptide in brain: localization in and release from isolated nerve terminals, Proc. nat. Acad. Sci. (Wash.), 74 (1977) 3424-3428. 9 Hal~isz, B. and Pupp, L., Hormone secretion of the anterior pituitary gland after physical interruption of all nervous pathways to the hypophysiotropic area, Endocrinology, 77 (1965) 553-562. 10 Johnson, N. K. and Whittaker, V. P., Lactate dehydrogenase as a cytoplasmic marker in brain, Biochem. J., 88 (1963) 404--409,
85 11 Larsson, L. I., Fahrenkrug, J., Schaffalitsky de Muckadell, O. B., Sundler, F., Hakanson, R. and Rehfeld, J. F., Localization of vasoactive intestinal polypeptide (VIP) to central and peripheral neurons, Proc. nat. Acad. Sci. (Wash.), 73 (1976) 3197-3200. 12 Larsson, L. I., Edvinson, L., Fahrenkrug, J., Hakanson, R., Owman, Ch., Schaffalitzky de Muckadell, O. B. and Sundler, F., Immunohistochemicallocalization of a vasodilatory polypeptide (VIP) in cerebrovascular nerves, Brain Research, (1976) 400-404. 13 Mutt, V., Preparation of highly purified secretin, Ark. Kemi., 15 (1959) 69-74. 14 Mutt, V. and Said, S. I., Structure of the porcine vasoactive intestinal octacosapeptide, the aminoacid sequence. Use of kallikrein in its determination, Europ. J. Biochem., 42 (1974) 581-589. 15 Ramirez, V. D., Gautron, J. P., Epelbaum, J., Pattou, E., Zamora, A. and Kordon, C., Distribution of LH-RH in subcellular fractions of mediobasal hypothalamus, Molec. cell. Endocr., 3 (1975) 330-350. 16 Rotsztejn, W. H., Charli, J. L., Pattou, E. and Kordon, C., Stimulation by dopamine of luteinizing hormone-releasing hormone (LH-RH) release from the mediobasal hypothalamus in male rats, Endocrinology, 101 (1977) 1475-1483. 17 Said, S. I. and Mutt, V., Polypeptide with broad biological activity: isolation from small intestine, Science, 169 (1970) 1217-1218. 18 Said, S. I. and Mutt, V., Isolation from porcine intestinal wall of a vasoactive octacosapeptide related to secretin and to glucagon, Europ. J. Biochem., 28 (1972) 199-204. 19 Said, S. I. and Rosenberg, R. N., Vasoactive intestinal polypeptide: abundant immunoreactivity in neural cell lines and normal nervous tissue, Science, 192 (1976) 907-908. 20 Weiner, R. I., Pattou, E., Kerdelhue, B. and Kordon, C., Differential effects of hypothalamic deafferentation upon luteinizinghormone-releasing hormone in the median eminence and organum vasculosum of the lamina terminalis, Endocrinology, 97 (1975) 1597-1600. 21 Whittaker, V. P., Michaelson, I. A. and Kirkland, R. S. A., The separation of synaptic vesicles from nerve endings particles (synaptosomes), Biochem. J., 90 (1964) 293-303.
79
VASOACTIVE INTESTINAL PEPTIDE (VIP): BRAIN DISTRIBUTION, SUBCELLULAR LOCALIZATION AND EFFECT OF DEAFFERENTATION OF THE HYPOTHALAMUS IN MALE RATS
JACQUELINE BESSON, WILLIAM ROTSZTEJN, MARC LABURTHE, JACQUES EPELBAUM, ALAIN BEAUDET, CLAUDE KORDON and GABRIELROSSELIN Unit~ de Recherche de Diabdtologie et d'Etudes Radioimmunologiques des Hormones Protdiques, U.55 (Institut National de la Santd et de la Recherche M~dicale) Hdpital St. Antoine, 75012 Paris and (W. R., J. E., A. B and C. K.) Unitd de Neuroendocrinologie, I N S E R M U.159, Centre Paul Broca, 75014 Paris (France)
(Accepted July 5th, 1978)
SUMMARY We have studied the regional and subcellular distribution of vasoactive intestinal peptide (VIP) in the brain of adult male rat, using a specific radioimmunoassay. Selective deafferentation of the mediobasal hypothalamus (MBH) was also performed in order to investigate the origin of hypothalamic VIP. The highest concentrations of VIP were found in the neocortex, namely the occipital region. The brain stem, the posterior hypothalamus, and the pineal gland contained low amounts of the peptide. VIP was not detectable in the cerebellum and the neurohypophysis. After fractionation, most of the VIP was recovered from the crude mitochondrial fraction of the hypothalamus as well as the parietal cortex. However, a non-negligible portion of the activity was also found in the supernatant suggesting that the peptide is mainly located in nerve endings but also present in neuronal cell bodies and/or axons. Two weeks after complete deafferentation of the MBH, VIP concentrations of the caudal MBH (including the infundibular sulcus, the stalk, part of the ventromedial nucleus and premamillary structures) were decreased by 40 ~. In contrast, no change in VIP levels were observed in the rostral MBH, organum vasculosum of the lamina terminalis (OVLT), and cortex. This suggests that hypothalamic nerve endings containing the peptide derive from neuronal cell bodies located both outside and within the MBH.
INTRODUCTION The vasoactive intestinal peptide (VIP) isolated from porcine small intestinet7 is structurally and biologically related to secretin, glucagon and gastric inhibitory polypeptide (GIP)14, is. High concentrations of VIP have been detected by radio-
80 immunoassay throughout the gastrointestinal tract 1-3,11 in neural cell lines 19 and in the central nervous system4,S, 19. Moreover, recent immunohistochemical studies have revealed the presence of numerous VIP-positive nerve cell bodies and terminals in the brain6,11,1z. The purpose of the present study is to provide a more detailed description of VIP regional distribution and subcellular localization in the rat brain. In order to investigate the origin of hypothalamic VIP, the peptide was also assayed following selective deafferentation of the mediobasal hypothalamus (MBH). MATERIALS AND METHODS Adult male Wistar rats, weighing 210-230 g, were killed by decapitation. Olfactory bulbs, fragments of the frontoparietai and occipital cortices, striatum, hippocampus, thalamus, brain stem, cerebellum and hypothalamus were rapidly dissected on ice. The hypothalamus was further divided in anterior, median and posterior fragments. The pineal gland as well as the adeno- and neurohypophyses were also sampled. Subcellularfractionation. Thirty hypothalami or 10 fragments of parietal cortex were pooled in 0.32 M sucrose (10 ~ w/v) and homogenized with a Teflon pestle and a glass homogenizer (clearance to 0.15 mm, 10 up and down strokes) at 4 °C. The subsequent fractionation was carried out as described by Ramirez et al. 1~. The whole process was controlled by evaluation of the lactate dehydrogenase (LDH) content of the fractions, according to the method of Johnson and Whittaked °. As an additional control, the subcellular distribution of VIP was compared with that of luteinizing hormone-releasing hormone (LH-RH). Deafferentation studies. Complete deafferentations of the mediobasal hypothalamus (MBH) were performed by surgical isolation, using an Hal~tsz knife (1.5 mm radius) under stereotaxic control 9. The animals were killed by decapitation two weeks after surgery. The MBH was dissected out and divided into a rostral part, containing the median eminence and most of the arcuate nucleus, and a caudal part, including the infundibular sulcus, the proximal stump of the stalk, part of the ventromedial nucleus and premammillary structures 16. The parietal cortex as well as a medial prechiasmatic fragment containing the organum vasculosum of the lamina terminalis (OVLT) 2° were also taken. The effectiveness of all deafferentations was verified by macroscopic examination and by measurement of L H - R H levels in the isolated islands 2°. Only animals showing a 75 ~ decrease in the L H - R H content of the islands were included in the results. VIP extraction and radioimmunoassay. The tissues were first boiled for 5 rain in deionized water in order to destroy the proteolytic enzymes 13. VIP was then extracted by sonication in 0.5 M acetic acid and lyophilized. In some experiments VIP was directly extracted with 0.1 N hydrochloric acid, so that other neuropeptides and particularly L H - R H could be simultaneously detected. VIP immunoreactivity was measured by a specific radioimmunoassay as previously described (detection sensitivity 60 pg/ml, intra-assay variation coefficient < 0.09, cross-reaction with secretin, glucagon, GIP, and the synthetic fragments 1-6, 14-28, 18-28 ofVIP < 0.3 ~)1. It was
81 verified that neither sucrose nor 0.1 N HCI interfered in the radioimmunoassay system, and that identical results for VIP content were obtained after either acetic or hydrochloric acid extraction of the samples. RESULTS Regional distribution. VIP is present in almost all rat brain structures assayed (Table I). The highest concentrations are found in the neocortex, namely the occipital region. VIP concentration are low in the brain stem, the posterior hypothalamus, the pineal gland, and not detectable in the cerebellum and the neurohypophysis. Subcellular localization. The subcellular distribution of VIP is similar in the cortex and the hypothalamus (Table II). After the first differential centrifugation, VIP is mainly recovered from the low speed supernatant S1. Only small amounts ( < 13 %) are found in the pellet P1, which contains nuclei and large membrane debris. After the second centrifugation, most of the VIP present in the supernatant S1 is recovered from the crude mitochondrial fraction (Pz), which contains myelin, synaptosomes, mitochondria, and small membrane debris. Subfractionation of the pellet (P2) on a density gradient shows that almost all endogenous VIP migrates with band B, which contains most of the synaptosomes. About one-fifth of the original amount of VIP remains in the S~ supernatant, most of which is recovered from the Ss fraction. L H - R H is mainly recovered from synaptosomal fraction (P2) and only 5 % from the microsomal supernatant ($2). After further fractionation of $2 the totality of L H - R H is recovered from the microsomal pellet (Ps). Deafferentation experiments. Complete deafferentation of the mediobasal hypothalamus (MBH) induces a 40 % decrease in VIP concentration of the caudal M B H
TABLE I Regional distribution of immunoreactive VIP in the rat brain
Values are reported as mean 4- S.E.M. of n determinations. Numbers in parentheses are the number of structures pooled for one determination, n.d. = not detectable. Structure
n
Wet weight (mg)
VIP (pg/mg)
Occipital cortex Parietal cortex Frontal cortex Anterior hypothalamus (3) Median hypothalamus (3) Posterior hyptothalamus (3) Hippocampus Striatum Olfactory bulbs (4) Thalamus Brain stem Cerebellum (4) Adenohypophysis (6) Neurohypophysis (10) Pineal gland (8)
5 5 5 3 3 3 6 6 3 5 6 3 3 2 1
108 -4- 14 160 4- 9 36 4- 4 19.2 4- 0.3 26.3 ± 0.6 13.3 4- 0.7 104 4- 7 72 4- 3 76 =t=3 36 4- 4 219 4- 8 219 4- 4 ~ 10 ~, 1 ~ 1
302 4- 16 265 4- 15 169 4- 20 60 4- 6 39 4- 5 20 4- 1 69 4- 4 58 4- 9 58 4- 8 34 4- 3 24 4- 1 n.d. 42 4- 3 n.d. 16
II
120"**
51,000
P~ A B C
P3 S~
262 34 215 47 6 15 127 n.d. 119 28
100 13 82 18 2 6 49 -45 11
%
0.94 0.11 1.12 0.69 0.02 0.72 0.44 n.d, 0.33 0.11
Units**
LDH
100 12 119 73 2 77 47 -35 12
%*
82 7 69 12 2 5 38 1.7 41 5.6
Tissue (rig~g)
VIP
Hypothalamus
100 9 84 15 2 6 46 2 50 7
%*
1.3 0.08 0.95 0,42 0.07 0.45 0.34 n.d. 0.22 n.d.
Units**
LDH
100 6 73 32 5 35 26 -25 --
%*
23t 54 157 11 11 n.d. 83
Tissue (ng/g)
LH-RH
100 23 68 5 5 -36
%*
* Per cent of the values measured in the homogenate, ** L D H activity is expressed as pmole N A D H ~ / h / m g wet weight. *** Sucrose gradient. A: 0.32-0.8 M sucrose interface containing myelin; B: 0.8-1,2 M sucrose interface containing synaptosomes; C: mitochondrial a n d synaptosomal pellet, n.d. =~ not detectable.
20 60
17,000 100,000
Homogena~
3
1000
P1 S1 S~
(min)
(g)
Tissue (rig~g)
VIP
Time
Speed
Fraction
Cortex
Centrifugation
Subcellular localization of immunoreactive VIP in cerebral cortex and hypothalamus of male rat
TABLE
I'0
83 TABLE III Effect of total deafferentation of the mediobasal hypothalamus (MBH) on VIP concentrations o f the rostral and caudal MBH, the 0 VLT and the cortex
Values are given as mean 4- S.E.M. with number of animals in parentheses. Structure
Rostral MBH Caudal MBH OVLT Cortex
Control
Complete deafferentation
V1P/Protein (ng/mg)
LH-RH/Protein (rig~rag)
V1P/Protein (ng/mg)
LH-RH/Protein (ng/mg)
3.9 3.1 3.1 4.9
18.0 4- 1.4 (12) 8.7 + 1.4 (12) 0.4 4- 0.1 (12) --
3.3 1.8 2.9 5.2
4.6 -4- 0.5 (6)* 2.1 4- 0.8 (6)* 0.5 4- 0.1 (6) --
-4- 0.3 (12) 4- 0.4 (12) 4- 0.3 (12) -4- 0.9 (6)
-4- 0.5 (6) i 0.3 (6)* 4- 0.5 (6) 4- 0.6 (6)
* P < 0.01. The protein content of the tissue samples from operated animals were not significantly different from that of controls. (Table III). In contrast, there is no significant reduction of VIP levels in the rostral MBH, O V L T and cortex. L H - R H concentration in the OVLT is not modified by complete deafferentation of the MBH. DISCUSSION The present study shows that immunoreactive VIP is present in most rat brain structures, as previously reported for different species3,8,19. VIP concentrations reported here are consistent with those found by Emson et al. a in the hypothalamus. However, they are significantly higher than those found by Giachetti et al. in the cortex, the hypothalamus and the striatum 8. The reasons of this discrepancy are not known, but they could be due to differences in the experimental procedures and/or specificity of the antibodies used in both studies. The highest concentrations of VIP found in the cerebral cortex confirm the results of other radioimmunoassays performed in dog 19 and rat brain s, as well as immunohistochemical observations in rat 6. VIP concentrations of the anterior hypothalamus seem to be higher than those found in its posterior part; however, such a difference is not reported by Emson et al. 4. VIP subcellular localization in the hypothalamus and parietal cortex is similar. In both structures most of the VIP is recovered from the crude mitochondrial fraction. The good correlation between VIP concentrations and L D H activity in band A, B and C after gradient centrifugation confirms that VIP present in P2 is mainly contained in nerve endings 21. This conclusion is in good agreement with previous observations made by Giachetti et al. s on the rat striatum and Emson et al. 4 on the hypothalamus. In addition, a non-negligible fraction of the activity is also found in the supernatant (Sz). This contrasts with the distribution of other neuropeptides, such as L H - R H 7 and somatostatinS; only traces of these peptides were found in the supernatant Sz, as confirmed here for L H - R H . The presence of VIP in the supernatant S~ cannot be explained by contamination due to disrupted synaptosomes, since further centrifugation of $2 shows that the VIP content of S~ is
84 mainly recovered from the supernatant Sa. Recovery of V1P from the supernatant thus suggests that the peptide is also present in neuronal cell bodies and/or axons, a conclusion which is in keeping with recent immunocytochemical data 6 11. The significant decrease (40~o) in VIP concentration of the caudal MBH following complete deafferentation of the hypothalamus suggests that a relatively high p r o p o r t i o n of the peptide is present in nerve endings originating from neuronal cell bodies located outside the island. Nevertheless, the largest fraction of V I P is unaffected by the lesions. This intrinsic V I P might be present in nerve terminals whose cell bodies are located within the island; such VIP-containing cell bodies have been observed in the ventromedial hypothalamus of the mouse tl. This interpretation would also be in keeping with our subcellular fractionation data which show a substantial recovery of V I P in the supernatant $3. Presence of the neuropeptide in non-neuronal cells is unlikely but cannot be formally excluded. At any rate, the unequal distribution of V I P in various structures of the central nervous system and its presence within nerve endings is compatible with the hypothesis that the peptide m a y exhibit neurotransmitter functions a,4,8,19. ACKNOWLEDGEMENTS We thank D. Hui Bon H o a and E. Pattou for their skillful technical assistance and C. Brunet for the careful preparation of the manuscript. This work was supported by the D616gation G6n6rale 5. la Recherche Scientifique (77-7-0504).
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