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Distribution of vasoactive intestinal polypeptide in intact, stria terminalis transected and cerebral cortex isolated rats
Brain Research, 213 (1981) 455-459 © Elsevier/North-Holland Biomedical Press
455
Distribution of vasoactive intestinal polypeptide in intact, stria terminalis transected and cerebral cortex isolated rats
MIKLOS PALKOVITS*, JACQUELINE BESSON and WILLIAM ROTSZTEJN 1st Department of Anatomy, Semmelweis University Medical School, Budapest, 1-1-1450 (Hungary) and (J.B.) Unitd de Recherche de Diabdtologie et d'Etudes Radioimmunologiques des Hormones Protdiques, U. 55 de l'lnserm, H6pital St. Antoine, 75012 Paris, and (I,V.R.) U. 159 de Neuroendocrinologie de l'Inserm, 75014 Paris (France)
(Accepted January 15th, 1981) Key words: vasoactive intestinal polypeptide -- stria terminalis -- cerebral cortex
Recent radioimmuno-2,5,6,s,15,16 and immunocytochemicalS, 13,17 studies have clarified the cerebral localization of vasoactive intestinal polypeptide (VIP). VIP proved to be present in almost every major anatomical unit of the brain. Its distribution is uneven with the highest concentration in the cerebral cortex where it is found in perikarya (mainly in layers II-III) fibres and nerve terminalsS,18,17. The amygdala, suprachiasmatic nucleus and midbrain central gray matter were also shown to contain VIP immunopositive cellss,17. VIP was determined in several limbic, extrapyramidal and brain stem areas. To decide whether cortical VIP is extrinsic, VIP was measured one week after the undercutting of the cortex. The stria terminalis is known to contain VIP-fibres8,13A4,17. The transection of this pathway was aimed at elucidating the origin of abundant VIP-fibres found in the amygdala and some hypothalamic regions. CFY-strain male rats of 200 4- 10 g b. wt. were kept under standard conditions (24 4- 1 °C, 7 0 ~ humidity, 12-12 h light-dark periods, normal food, tap water ad libitum). Intact and operated animals were killed by decapitation at 08.00-09.00 h. Brains were frozen on dry-ice and cut in the coronal plane in a --10 °C cryostat. Section thickness was 300/~m. Using the micropunch technique 10 samples were taken from 8 brain areas (listed in Tables). Each sample was homogenized either in 120/~10.1 N HC1 or in 120/~1 0.1 N perchloric acid. From 5 #1 of the homogenates, protein was determined 9. VIP-concentration was measured by a specific radioimmunoassay 1. The VIP antiserum was raised in rabbits against natural porcine VIP coupled to bovine serum albumin. It exhibited no cross-reactivity with substance P, neurotensin, enkephalins, endorphins, LH-RH, somatostatin and glucagon. * To whom all correspondence and requests for reprints should be addressed at: Lab. Clin. Sci. NIMH, Bd. 10, Rm. 2D-47, 9000, Rockville Pike, Bethesda, Md. 20205, U.S.A.
456 Isolation of the cerebral cortex: the frontal cortex was undercut with a 2 mm wide glass knife cut with a diamond from a cover-glass. The knife was introduced into the skull at the level of the bregma, 5 mm from the midline, approximately in the horizontal plane (Fig. 1, cut 2). Transection of the stria terminalis: a 1.5 mm wide glass knife was introduced vertically, at the level of the bregma, 1.3 mm from the midline (Fig. 1, cut 1). The animals were decapitated on the 8th postoperative day. Transections were controlled under microscopic section and brain samples were taken out as detailed above. VIP-concentrations after isolation of the frontal cortex: there was no change in VIP concentration in the isolated frontal cortex, nor did the VIP-levels of ipsi- and
~.~~,7:.~i7!~,7n/.',t
J ~lJ I "
~-;
--A ,-"'7) sI _,/
6300
l
• " it
~
i
Fig. 1, Coronal sections of the rat forebrain with knife cuts, 1 : vertical cut to transect the stria
terminalis. 2: isolation of the frontal cortex with a horizontal cut. Drawings after KSnig and Klippel 7, Distances rostral to the interauricular line are given in/~m. Abbreviations: A, amygdala; Ah, anterior hypothalamic nucleus; B, bed nucleus of the stria terminalis (NIST); C, corpus callosum; CI, internal capsule; Co, optic chiasm; CP, caudate putamen; F, fimbria hippocampi; G, globus pallidus; M, medial forebrain bundle; Po, medial preoptic nucleus; S, stria terminalis; "k', suprachiasmatic nucleus.
457 TABLE I VIP concentrations after surgical isolation of the cerebral (frontal) cortex (ng/mg protein 4- S.E.M.) Number of animals in parentheses. Regions
Sham-operated
Frontal cortex Nucleus accumbens NIST Central amygdala Suprachiasmatic nucleus
4.56 4- 0.61 (7) 4.28 ± 1.35 (5) 2.13 4- 0.63 (7) 1.31 4- 0.24 (7) 10.01 4- 1.85 (7)
Isolated cortex lpsilateral
Contralateral
3.94 i 0.76 (6) 4.22 4- 1.40 (6) 2.72 4- 0.64 (5) 4.57 4- 1.24" (6) 9.19 4- 1.85 (6)
4.22 4- 0.39 (6) 3.56 4- 1.40 (6) 3.65 4- 1.19 (5) 2.99 4- 0.98 (6) 8.53 ± 1.79 (6)
* P < 0.02. contralateral accumbens, suprachiasmatic nuclei a n d N I S T differ. I n contrast, a significant rise was detected in the ipsilateral central amygdaloid nucleus (Table I). VIP concentrations after transection of the stria terminalis: a significant fall resulted by unilateral transection in the suprachiasmatic a n d lateral amygdaloid nuclei in both sides. The ipsilateral decrease was more significant. A n ipsilateral decrease of V I P - c o n t e n t was f o u n d also in the N I S T b u t it was significant only in one o f the parallel experiments. I n other territories studied n o change was encountered (Table
I0. I n the rat, V I P - i m m u n o r e a c t i v e perikarya, as well as fibres a n d terminals, were s h o w n to be located in cortical layers II a n d llIa,la, 17. There are likely to be intrinsic TABLE II Concentrations of vasoactive intestinal peptide ( ng/mg protein) in certain brain areas 7 days after surgical transection of the stria terminalis Results are mean 4- S.E.M. Number of animals in parentheses. Sham-operated
Transection of the stria terminalis Ipsilateral
Contralateral
1st. Experiment Central amygdaloid nucleus 2.63 4- 0.64 (5) Suprachiasmatic nucleus 9.77 4- 0.54 (4) Bed nucleus of the stria terminalis 2.88 4- 0.42 (6)
1.62 4- 0.44 (6) 3.68 ± 0.48 (4)**
2.98 4- 0.72 (6)
1.04 4- 0.31 (6)**
2.65 4- 0.76 (4)
2nd Experiment Frontal cortex 4.56 4- 0.61 (7) NIST 2.13 4- 0.63 (7) Central amygdaloid nucleus 1.31 + 0.24 (7) Lateral amygdaloid nucleus 2.41 4- 0.25 (7) Suprachiasmatic nucleus 10.01 4- 1.85 (7) Median eminence 1.52 4- 0.30 (7) Ventromedial nucleus 1.05 4- 0.24 (7)
4.28 4- 0.23 1.69 4- 0.45 1.82 4- 0.38 1.06 4- 0.32 3.59 4- 0.69 0.99 4- 0.15 1.28 4- 0.15
4.17 4- 0.52 (7) 3.39 4- 1.03 (6) 2.57 4- 0.99 (7) 1.60 4- 0.18 (6)* 5.38 :t: 0.73 (7)* 0.99 4- 0.15 (7) 0.78 4- 0.13 (7)
* P < 0.05;
** P < 0.01.
(7) (5) (7) (6)** (6)** (7) (7)
458 neurons because the undercutting of the cortex did not affect VIP-levels (Table 1). A similar observation has been already mentioned by Emson and Lindvall 4. Indeed, VIP-positive cells were of the bipolar, interneuron type 13,17 and no VIP-positive fibres were found either in the internal capsule or in the pyramidal tracP 7. In the central amygdaloid nucleus the undercutting of the cortex brought about an ipsilateral rise in VIP-concentration. The reason for this is unclear as no direct neural link is known to exist between the two territories. It should be noted, however, that with immunocytochemistry V1P-fibres were demonstrated to run from the central amygdaloid nucleus along the internal capsule 17. These may reach the frontal cortex. The high amount of VIP in the amygdala, NIST and suprachiasmatic nucleus, together with previous immunocytochemical data8,1a,~4, ~7, showing an important long-projecting VIP tract relating these structures (stria terminalis), suggests that one of the essential role of VIP in the brain is linked with this pathway. The transection of the stria terminalis or lesioning of the amygdala results in a decrease of VIP-immunoreactivity in the ipsilateral stria terminalis, NIST, preoptic and anterior hypothalamic areas13,14. In our experiments unilateral stria terminalis transection decreased VIP levels in the bilateral suprachiasmatic nucleus and in the ipsilateral NIST. These reductions may be due either to the interruption of VIP innervating these nuclei by the stria terminalis and/or to the impairment of the neural input required for local VIP synthesis. Cell bodies from amygdaloid nuclei gave fibres which formed a pathway projecting to the NIST, suprachiasmatic, ventromedial nuclei and preoptic area (cf. ref. 11). Some hypothalamic fibres are decussating in the anterior commissure a. Presumably these comprise VIP-fibres running to the suprachiasmatic nucleus, as indicated by the decrease of VIP-concentration in the suprachiasmatic nucleus after stria terminalis transection. Examination of VIP-content in various amygdaloid nuclei after ST transection shows a decrease both in the ipsi- and the contralateral side of the lateral amygdaloid nucleus. This observation can be related to the observation by Quick et al. 12, showing that this operation decreased VIP-induced cyclic AMP accumulation in the amygdala. It is assumed that the stria terminalis contains not only amygdalofugal but also amygdalopetal VIP fibres or essential input to the amygdaloid VIP-synthesis.
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 Besson, J., Rotsztejn, W., Laburthe, M., Epelbaum, J., Beaudet, A., Kordon, C. and Rosselin, G., Vasoactive intestinal peptide (VIP): brain distribution, subcellular localization and effect of deafferentation of the hypothalamus in male rats, Brain Research, 165 (1979) 79-85. 3 De Olmos, J. S. and Ingram, W. R., The projection of the stria terminalis in the rat brain. An experimental study, J. cornp. Neurol., 146 (1972) 303-334. 4 Emson, P. C. and Lindvall, O., Distribution of putative neurotransmitters in the neocortex, Neuroscience, 4 (•979) 1-30. 5 Fahrenkrug, J. and Schaffalitzkyde Muckadell, O. B., Distribution of vasoactive intestinal polypeptide (VIP) in the porcine central nervous system, J. Neurochem., 31 (1978) 1445-1451.
459 6 Giachetti, A., Said, S. I., Reynolds, R. C. and Koniges, F. C., Vasoactive intestinal polypeptide in brain: localization and release from isolated nerve terminals, Proc. nat. ,4cad. Sci. (Wash.), 74 (1977) 3424-3428. 7 K6nig, J. F. R. and Klippel, R. A., The Rat Brain: ,4 Stereotaxic ,4tlas of the Forebrain andLower Parts of the Brain Stem: Williams and Wilkins, Baltimore, 1963. 8 Lor6n, I., Emson, P. C., Fahrenkrug, J., Bj6rklund, A., Alumets, J., Hhkanson, R. and Sundler, F., Distribution of vasoactive intestinal polypeptide in the rat and mouse brain, Neuroscience, 4 (1979) 1953-1976. 9 Lowry, O. H., Rosebrough, N. Y., Farr, A. L. and Randall, R. J., Protein measurement with the Foline phenol reagent, J. biol. Chem., 193 (1951) 265-275. 10 Palkovits, M., Isolated removal of hypothalamic or other brain nuclei of the rat, Brain Research, 59 (1973) 449-450. 11 Palkovits, M. and Z~lborszky, L., Neural connections of the hypothalamus. In P. J. Morgane and J. Panksepp (Eds.), ,4natomy of the Hypothalamus, Vol. 1, Marcel Dekker, New York, 1980, pp. 379-509. 12 Quik, M., Emson, P. C., Fahrenkrug, J. and Iversen, L. L., Effect of kainic acid injections and other brain lesions on vasoactive intestinal peptide (VIP)-stimulated formation of cAMP in rat brains, Naunyn-Schmiedeberg's Arch. Pharmacol., 306 (1979) 281-286. 13 Roberts, G. W., Woodhams, P. L., Bryant, M. Crow, T. J., Bloom, S. R. and Polak, J. M., VIP in rat brain: evidence for a major pathway linking the amygdala and hypothalamus via the stria terminalis, Histochemistry, 65 (1980) 103-119. 14 Roberts, G. W., Woodhams, P. L., Crow, T. J. and Polak, J. M., Loss of immunoreactive VIP in the bed nucleus following lesions of the stria terminalis, Brain Research, 195 (1980) 471-475. 15 Said, S. I. and Roseberg, R. N., Vasoactive intestinal polypeptide: abundant immunoreactivity in neural cell lines and normal nervous tissue, Science, 192 (1976) 907-908. 16 Samson, W. K., Said, S. I. and McCann, S. M., Radioimmunologic localization of vasoactive intestinal polypeptide in hypothalamic and extrahypothalamic sites in the rat brain, Neurosci. Lett., 12 (1979) 265-269. 17 Sims, K. B., Hoffman, D. L., Said, S. I. and Zimmerman, E. A., Vasoactive intestinal peptide (VIP) in mouse and rat brain: an immunocytochemical study, Brain Research, 186 (1980) 165-184.
455
Distribution of vasoactive intestinal polypeptide in intact, stria terminalis transected and cerebral cortex isolated rats
MIKLOS PALKOVITS*, JACQUELINE BESSON and WILLIAM ROTSZTEJN 1st Department of Anatomy, Semmelweis University Medical School, Budapest, 1-1-1450 (Hungary) and (J.B.) Unitd de Recherche de Diabdtologie et d'Etudes Radioimmunologiques des Hormones Protdiques, U. 55 de l'lnserm, H6pital St. Antoine, 75012 Paris, and (I,V.R.) U. 159 de Neuroendocrinologie de l'Inserm, 75014 Paris (France)
(Accepted January 15th, 1981) Key words: vasoactive intestinal polypeptide -- stria terminalis -- cerebral cortex
Recent radioimmuno-2,5,6,s,15,16 and immunocytochemicalS, 13,17 studies have clarified the cerebral localization of vasoactive intestinal polypeptide (VIP). VIP proved to be present in almost every major anatomical unit of the brain. Its distribution is uneven with the highest concentration in the cerebral cortex where it is found in perikarya (mainly in layers II-III) fibres and nerve terminalsS,18,17. The amygdala, suprachiasmatic nucleus and midbrain central gray matter were also shown to contain VIP immunopositive cellss,17. VIP was determined in several limbic, extrapyramidal and brain stem areas. To decide whether cortical VIP is extrinsic, VIP was measured one week after the undercutting of the cortex. The stria terminalis is known to contain VIP-fibres8,13A4,17. The transection of this pathway was aimed at elucidating the origin of abundant VIP-fibres found in the amygdala and some hypothalamic regions. CFY-strain male rats of 200 4- 10 g b. wt. were kept under standard conditions (24 4- 1 °C, 7 0 ~ humidity, 12-12 h light-dark periods, normal food, tap water ad libitum). Intact and operated animals were killed by decapitation at 08.00-09.00 h. Brains were frozen on dry-ice and cut in the coronal plane in a --10 °C cryostat. Section thickness was 300/~m. Using the micropunch technique 10 samples were taken from 8 brain areas (listed in Tables). Each sample was homogenized either in 120/~10.1 N HC1 or in 120/~1 0.1 N perchloric acid. From 5 #1 of the homogenates, protein was determined 9. VIP-concentration was measured by a specific radioimmunoassay 1. The VIP antiserum was raised in rabbits against natural porcine VIP coupled to bovine serum albumin. It exhibited no cross-reactivity with substance P, neurotensin, enkephalins, endorphins, LH-RH, somatostatin and glucagon. * To whom all correspondence and requests for reprints should be addressed at: Lab. Clin. Sci. NIMH, Bd. 10, Rm. 2D-47, 9000, Rockville Pike, Bethesda, Md. 20205, U.S.A.
456 Isolation of the cerebral cortex: the frontal cortex was undercut with a 2 mm wide glass knife cut with a diamond from a cover-glass. The knife was introduced into the skull at the level of the bregma, 5 mm from the midline, approximately in the horizontal plane (Fig. 1, cut 2). Transection of the stria terminalis: a 1.5 mm wide glass knife was introduced vertically, at the level of the bregma, 1.3 mm from the midline (Fig. 1, cut 1). The animals were decapitated on the 8th postoperative day. Transections were controlled under microscopic section and brain samples were taken out as detailed above. VIP-concentrations after isolation of the frontal cortex: there was no change in VIP concentration in the isolated frontal cortex, nor did the VIP-levels of ipsi- and
~.~~,7:.~i7!~,7n/.',t
J ~lJ I "
~-;
--A ,-"'7) sI _,/
6300
l
• " it
~
i
Fig. 1, Coronal sections of the rat forebrain with knife cuts, 1 : vertical cut to transect the stria
terminalis. 2: isolation of the frontal cortex with a horizontal cut. Drawings after KSnig and Klippel 7, Distances rostral to the interauricular line are given in/~m. Abbreviations: A, amygdala; Ah, anterior hypothalamic nucleus; B, bed nucleus of the stria terminalis (NIST); C, corpus callosum; CI, internal capsule; Co, optic chiasm; CP, caudate putamen; F, fimbria hippocampi; G, globus pallidus; M, medial forebrain bundle; Po, medial preoptic nucleus; S, stria terminalis; "k', suprachiasmatic nucleus.
457 TABLE I VIP concentrations after surgical isolation of the cerebral (frontal) cortex (ng/mg protein 4- S.E.M.) Number of animals in parentheses. Regions
Sham-operated
Frontal cortex Nucleus accumbens NIST Central amygdala Suprachiasmatic nucleus
4.56 4- 0.61 (7) 4.28 ± 1.35 (5) 2.13 4- 0.63 (7) 1.31 4- 0.24 (7) 10.01 4- 1.85 (7)
Isolated cortex lpsilateral
Contralateral
3.94 i 0.76 (6) 4.22 4- 1.40 (6) 2.72 4- 0.64 (5) 4.57 4- 1.24" (6) 9.19 4- 1.85 (6)
4.22 4- 0.39 (6) 3.56 4- 1.40 (6) 3.65 4- 1.19 (5) 2.99 4- 0.98 (6) 8.53 ± 1.79 (6)
* P < 0.02. contralateral accumbens, suprachiasmatic nuclei a n d N I S T differ. I n contrast, a significant rise was detected in the ipsilateral central amygdaloid nucleus (Table I). VIP concentrations after transection of the stria terminalis: a significant fall resulted by unilateral transection in the suprachiasmatic a n d lateral amygdaloid nuclei in both sides. The ipsilateral decrease was more significant. A n ipsilateral decrease of V I P - c o n t e n t was f o u n d also in the N I S T b u t it was significant only in one o f the parallel experiments. I n other territories studied n o change was encountered (Table
I0. I n the rat, V I P - i m m u n o r e a c t i v e perikarya, as well as fibres a n d terminals, were s h o w n to be located in cortical layers II a n d llIa,la, 17. There are likely to be intrinsic TABLE II Concentrations of vasoactive intestinal peptide ( ng/mg protein) in certain brain areas 7 days after surgical transection of the stria terminalis Results are mean 4- S.E.M. Number of animals in parentheses. Sham-operated
Transection of the stria terminalis Ipsilateral
Contralateral
1st. Experiment Central amygdaloid nucleus 2.63 4- 0.64 (5) Suprachiasmatic nucleus 9.77 4- 0.54 (4) Bed nucleus of the stria terminalis 2.88 4- 0.42 (6)
1.62 4- 0.44 (6) 3.68 ± 0.48 (4)**
2.98 4- 0.72 (6)
1.04 4- 0.31 (6)**
2.65 4- 0.76 (4)
2nd Experiment Frontal cortex 4.56 4- 0.61 (7) NIST 2.13 4- 0.63 (7) Central amygdaloid nucleus 1.31 + 0.24 (7) Lateral amygdaloid nucleus 2.41 4- 0.25 (7) Suprachiasmatic nucleus 10.01 4- 1.85 (7) Median eminence 1.52 4- 0.30 (7) Ventromedial nucleus 1.05 4- 0.24 (7)
4.28 4- 0.23 1.69 4- 0.45 1.82 4- 0.38 1.06 4- 0.32 3.59 4- 0.69 0.99 4- 0.15 1.28 4- 0.15
4.17 4- 0.52 (7) 3.39 4- 1.03 (6) 2.57 4- 0.99 (7) 1.60 4- 0.18 (6)* 5.38 :t: 0.73 (7)* 0.99 4- 0.15 (7) 0.78 4- 0.13 (7)
* P < 0.05;
** P < 0.01.
(7) (5) (7) (6)** (6)** (7) (7)
458 neurons because the undercutting of the cortex did not affect VIP-levels (Table 1). A similar observation has been already mentioned by Emson and Lindvall 4. Indeed, VIP-positive cells were of the bipolar, interneuron type 13,17 and no VIP-positive fibres were found either in the internal capsule or in the pyramidal tracP 7. In the central amygdaloid nucleus the undercutting of the cortex brought about an ipsilateral rise in VIP-concentration. The reason for this is unclear as no direct neural link is known to exist between the two territories. It should be noted, however, that with immunocytochemistry V1P-fibres were demonstrated to run from the central amygdaloid nucleus along the internal capsule 17. These may reach the frontal cortex. The high amount of VIP in the amygdala, NIST and suprachiasmatic nucleus, together with previous immunocytochemical data8,1a,~4, ~7, showing an important long-projecting VIP tract relating these structures (stria terminalis), suggests that one of the essential role of VIP in the brain is linked with this pathway. The transection of the stria terminalis or lesioning of the amygdala results in a decrease of VIP-immunoreactivity in the ipsilateral stria terminalis, NIST, preoptic and anterior hypothalamic areas13,14. In our experiments unilateral stria terminalis transection decreased VIP levels in the bilateral suprachiasmatic nucleus and in the ipsilateral NIST. These reductions may be due either to the interruption of VIP innervating these nuclei by the stria terminalis and/or to the impairment of the neural input required for local VIP synthesis. Cell bodies from amygdaloid nuclei gave fibres which formed a pathway projecting to the NIST, suprachiasmatic, ventromedial nuclei and preoptic area (cf. ref. 11). Some hypothalamic fibres are decussating in the anterior commissure a. Presumably these comprise VIP-fibres running to the suprachiasmatic nucleus, as indicated by the decrease of VIP-concentration in the suprachiasmatic nucleus after stria terminalis transection. Examination of VIP-content in various amygdaloid nuclei after ST transection shows a decrease both in the ipsi- and the contralateral side of the lateral amygdaloid nucleus. This observation can be related to the observation by Quick et al. 12, showing that this operation decreased VIP-induced cyclic AMP accumulation in the amygdala. It is assumed that the stria terminalis contains not only amygdalofugal but also amygdalopetal VIP fibres or essential input to the amygdaloid VIP-synthesis.
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 Besson, J., Rotsztejn, W., Laburthe, M., Epelbaum, J., Beaudet, A., Kordon, C. and Rosselin, G., Vasoactive intestinal peptide (VIP): brain distribution, subcellular localization and effect of deafferentation of the hypothalamus in male rats, Brain Research, 165 (1979) 79-85. 3 De Olmos, J. S. and Ingram, W. R., The projection of the stria terminalis in the rat brain. An experimental study, J. cornp. Neurol., 146 (1972) 303-334. 4 Emson, P. C. and Lindvall, O., Distribution of putative neurotransmitters in the neocortex, Neuroscience, 4 (•979) 1-30. 5 Fahrenkrug, J. and Schaffalitzkyde Muckadell, O. B., Distribution of vasoactive intestinal polypeptide (VIP) in the porcine central nervous system, J. Neurochem., 31 (1978) 1445-1451.
459 6 Giachetti, A., Said, S. I., Reynolds, R. C. and Koniges, F. C., Vasoactive intestinal polypeptide in brain: localization and release from isolated nerve terminals, Proc. nat. ,4cad. Sci. (Wash.), 74 (1977) 3424-3428. 7 K6nig, J. F. R. and Klippel, R. A., The Rat Brain: ,4 Stereotaxic ,4tlas of the Forebrain andLower Parts of the Brain Stem: Williams and Wilkins, Baltimore, 1963. 8 Lor6n, I., Emson, P. C., Fahrenkrug, J., Bj6rklund, A., Alumets, J., Hhkanson, R. and Sundler, F., Distribution of vasoactive intestinal polypeptide in the rat and mouse brain, Neuroscience, 4 (1979) 1953-1976. 9 Lowry, O. H., Rosebrough, N. Y., Farr, A. L. and Randall, R. J., Protein measurement with the Foline phenol reagent, J. biol. Chem., 193 (1951) 265-275. 10 Palkovits, M., Isolated removal of hypothalamic or other brain nuclei of the rat, Brain Research, 59 (1973) 449-450. 11 Palkovits, M. and Z~lborszky, L., Neural connections of the hypothalamus. In P. J. Morgane and J. Panksepp (Eds.), ,4natomy of the Hypothalamus, Vol. 1, Marcel Dekker, New York, 1980, pp. 379-509. 12 Quik, M., Emson, P. C., Fahrenkrug, J. and Iversen, L. L., Effect of kainic acid injections and other brain lesions on vasoactive intestinal peptide (VIP)-stimulated formation of cAMP in rat brains, Naunyn-Schmiedeberg's Arch. Pharmacol., 306 (1979) 281-286. 13 Roberts, G. W., Woodhams, P. L., Bryant, M. Crow, T. J., Bloom, S. R. and Polak, J. M., VIP in rat brain: evidence for a major pathway linking the amygdala and hypothalamus via the stria terminalis, Histochemistry, 65 (1980) 103-119. 14 Roberts, G. W., Woodhams, P. L., Crow, T. J. and Polak, J. M., Loss of immunoreactive VIP in the bed nucleus following lesions of the stria terminalis, Brain Research, 195 (1980) 471-475. 15 Said, S. I. and Roseberg, R. N., Vasoactive intestinal polypeptide: abundant immunoreactivity in neural cell lines and normal nervous tissue, Science, 192 (1976) 907-908. 16 Samson, W. K., Said, S. I. and McCann, S. M., Radioimmunologic localization of vasoactive intestinal polypeptide in hypothalamic and extrahypothalamic sites in the rat brain, Neurosci. Lett., 12 (1979) 265-269. 17 Sims, K. B., Hoffman, D. L., Said, S. I. and Zimmerman, E. A., Vasoactive intestinal peptide (VIP) in mouse and rat brain: an immunocytochemical study, Brain Research, 186 (1980) 165-184.