Histochemistry
Histochemistry59, 249-257 (1979)
9 by Springer-Verlag 1979
Distribution of Gastrin and CCK-like Peptides in Rat Brain An ImmunocytochemicalStudy I. Lor6n, J. Alumets, R. Hfikanson, and F. Sundler Departments of Histologyand Pharmacology,Universityof Lurid Biskopsgatan 5, S-22362Lund, Sweden
Summary. The distribution of gastrin and CCK-like peptides in the rat brain was studied by immunocytochemistry using an antiserum reacting equally well with both groups of peptides. Immunoreactive nerve cell bodies were detected in all cortical areas, in the hippocampus where they were particularly numerous, in the mesencephalic central gray and in the medulla oblongata. After colchicine treatment immunoreactive material appeared also in cell bodies of the magnocellular hypothalamic system. Immunoreactive nerve fibers were widely distributed in the brain. Particularly dense accumulations were seen in the hippocampus near the ventral surface of the brain, in the caudate nucleus, in the interpeduncular nucleus, the parabrachial nucleus, the dorsal part of the medulla oblongata and in the dorsal horn of the spinal cord. In the hypothalamus immunoreactive nerve fibers were observed in all nuclei, being most frequent in the ventromedial, dorsal and lateral hypothalamic nuclei. A rich supply of nerve fibers was seen in the outer zone of the median eminence and in the neurohypophysis. From previous immunochemical analysis it appears that the peptide demonstrated in most parts of the brain is identical with CCK-8. In the neurosecretory cell bodies of the hypothalamus, the median eminence and the neurohypophysis, however, the immunoreactive material is probably identical with gastrin.
Introduction "Gastrin-like" material in brain extracts was first described by Vanderhaegen et al. (1975). Subsequent studies (Dockray et al., 1976, 1978; Muller et al., 1977; Robberecht et al., 1978; Rehfeld, 1978a, b) have indicated that the bulk of gastrin-like material is distinct from gastrin and identical with, or closely related to CCK-octapeptide (CCK-8), the COOH-terminal eight amino acid sequence of CCK. The fact that CCK-8 shares the COOH-terminal five amino acids with gastrin may explain why it was initially described as "gastrin-like". Re-
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cently Rehfeld (1978 a) was able to d e m o n s t r a t e significant a m o u n t s of gastrin-17 a n d gastrin-34 in extracts of the porcine hypophysis a n d h y p o t h a l a m u s ; in the r e m a i n d e r o f the b r a i n n o n e was found, I n r a b b i t b r a i n cortex Straus et al. (1977) described n u m e r o u s nerve cell bodies, b u t n o nerve terminals, reacting with antisera against the C O O H - t e r m i n a l tetragastrin fragment. We n o w wish to report o n the i m m u n o c y t o c h e m i c a l d i s t r i b u t i o n of gastrin a n d C C K - l i k e material in the rat brain. The results o b t a i n e d differ from those of Straus et al. (1977) in that we observed n o t only i m m u n o r e a c t i v e nerve cell bodies b u t also n u m e r o u s nerve fibers in several b r a i n regions.
Materials and Methods Adult female Sprague-Dawleyrats were perfused via the ascending aorta with 300 ml of an ice-cold 4% formaldehyde solution (paraformaldehyde dissolved in 0.1 M phosphate buffer). Various brain regions were post-fixed for 24 h in the same type of fixative as that used for the perfusion. The specimens were rinsed for at least 48 h in Tyrode buffer with 5% sucrose added. Postfixation and rinsing were carried out at 5~ C. The specimens were frozen in dry ice, and frontal sections were cut in a cryostat at - 15~ C. The sections were then processed for the immunocytochemical demonstration of gastrin-like and CCK-like peptides. The antiserum used (code no. 4562; kindly supplied by Professor J.F. Rehfeld, Arhus, Denmark) was raised in rabbits against synthetic human gastrin I, 2-17, covalently bound to albumin (Rehfeld et al., 1972). In the immunocytochemical procedure this antiserum demonstrates gastrin and CCK with about equal potency (Alumets et al., 1979). The working dilutions were 1:320 for immunofluorescence and 1:25,000 for immunoperoxidase (PAP) staining (for details on these procedures, see Alumets et al., 1979). Inactivated antiserum (excess amounts of gastrin-17 or CCK-8 were added) was used to provide control sections. Immunostaining was regarded as specific only if it failed to appear in the control sections. In one series of experiments, colchicine (8 gg/gl) was injected intraventricularly (2 gl to each of 3 rats) and the animals were killed 48 h later. Tissue specimens were processed as above; sections were cut in the region of the hypothalamus and the mesencephalon.
Results
Distribution of tmmunoreactive Nerve Celt Bodies I m m u n o r e a c t i v e nerve cell bodies were seen in neocortex, paleocortex, mesocortex a n d archiocortex as well as in claustrum. The cell bodies were rather few in the neo- a n d mesocortex a n d occurred scattered in layers I I - V I (Fig. 1 a). They were seen to give off processes some of which could be followed for quite some distance. Occasionally single nerve cell bodies were seen in the piriform a n d e n t o r h i n a l cortex; they were fairly frequent in the cortical a m y g d a loid nuclei. I m m u n o r e a c t i v e cell bodies were most frequent in the h i p p o c a m p u s where they could be observed in the strata oriens, pyramidale, l a c u n o s u m moleculare, a n d r a d i a t u m of C A 1 - C A 3 (Fig. 1 d). Here, they were more intensely i m m u n o r e a c t i v e t h a n in other areas. Most of these cell bodies h a d several processes, some of which c o u l d be followed for rather long distances before they were seen to divide into smaller branches. I n the dentate gyrus i m m u n o r e a c tive cell bodies were seen in the hilar region a n d a l o n g the i n n e r surface of the g r a n u l e cell layer (Fig. 1 b). F r o m some of these cell bodies fibers could
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Fig. 1. Nerve cell bodies displaying CCK-like immunoreactivity in a layers II-III of the parietal cortex (x 300), b the hilar region of the dentate gyrus close to the granule cell layer (x 350), e nucleus gracilis ( x 300) and d stratum radiatum of CA3 in hippocampus ( • 320)
be followed along the inner surface of the granule cell layer which was sometimes penetrated by smaller branches from the fibres. The subiculum contained cell bodies in about the same frequency as the other hippocampal areas. Occasionally immunoreactive cell bodies were observed in the molecular layer of the dentate gyrus. A group of cell bodies was found in the ventral part of the mesencephalic central gray; some of these cell bodies occurred close to the ventricular lining (Fig. 2). Cell bodies were also seen in medulla oblongata where they were located in nucleus gracilis (Fig. 1 c). The distribution of immunoreactive cell bodies in hypothalamus is described on page 256.
Distribution of Immunoreactive Nerve Fibers Diencephalon. In epithalamus the lateral habenular nuclei contained some immunoreactive nerve fibers. In thalamus such nerve fibers were seen to run in the midline and in nuclei close to the midline, while a rather dense network of immunoreactive nerves was seen in the lateral geniculate body and the ventral thalamic nucleus. In hypothalamus immunoreactive nerve fibers seemed to be present in all nuclei; the most dense accumulation of nerves was in the ventromedial (Fig. 3 a), dorsal and lateral hypothalamic nuclei (Fig. 3 b). The ventromedial nucleus contained a network of delicate nerves while the lateral nucleus had more coarse nerves. The immunoreactive nerve fibers in the lateral and most ventral part of the preoptic area mostly had a coarse appearance while the majority of nerves in the other preoptic areas were more delicate. All other hypothalamic nuclei (anterior, posterior, periventricular, and paraventricular
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Fig. 2. Immunoreactive cell bodies in the ventral part of the mesencephalic central gray (PAP technique) ( x 100)
Fig. 3. Immunoreactive nerve fibres in hypothalamus, a A network of delicate nerve fibres in the ventromedial nucleus ( x 350), b a network of more coarse nerve fibres in the lateral nucleus ( x 375)
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hypothalamic nuclei, the ventral and dorsal premamillary nuclei as well as the mamillary complex) contained both coarse and delicate nerve fibers in a low to moderate number. On the whole, delicate nerves were more numerous than coarse ones. A dense strand of mixed coarse and delicate nerves was seen to run close to the ventral surface. Another strand of nerves was observed in the dorsal part of hypothalamus between the paraventricular and the lateral hypothalamic nuclei.
Telencephalon. A small aggregation of immunoreactive nerve fibers was seen in tuberculum olfactorium. Within the limbic lobe nerves could be demonstrated in the septal area, nucleus accumbens, hippocampus and in the amygdaloid, entorhinal and cingulate cortex. In the septum a moderate number of immunoreactive nerve fibers was located in the ventral part close to the lateral ventricle. The ventral and medial part of the nucleus accumbens contained a narrow strand of nerves. Immunoreactive nerve fibers could be seen in all subdivisions of the hippocampal formation but a particularly dense accumulation was seen in the pyramidal cell layer and in areas that reached the ventral surface of the brain. The lowest number of nerves was seen in the molecular layer of the dentate gyrus as well as in the strata of alveus and oriens. All nuclei in the amygdaloid complex contained immunoreactive nerves; they were most numerous in the medial, central and cortical nuclei. The entorhinal cortex as well as the piriform cortex contained a moderate number of nerves. In all other cortical areas, a small number of immunoreactive nerve fibers could be seen in the different layers. In the pathways of the limbic system nerves were located in the medial forebrain bundle area and in stria terminalis. The caudate nucleus was densely innervated by delicate nerves, especially its ventral, medial and dorsomedial part. Mesencephalon. In mesencephalon a dense system of coarse nerves was found in the dorsal and lateral parts of the interpeduncular nucleus. A rather dense supply was seen also in the nucleus of the medial geniculate body as well as in the superior and inferior collicle. In the central gray only a few immunoreacrive nerves were seen. Ports, Medulla Oblongata and Spinal Cord. In pons and medulla oblongata immunoreactive nerve fibers were seen in several areas. A rich supply was observed in the parabrachial nucleus (Fig. 4 a), and in the dorsal part of medulla oblongata, i.e. nucleus gracilis, nucleus cuneatus, area postrema, and in the nucleus of the solitary tract (as well as in the solitary tract). A horizontal bundle with a moderate number of nerve fibers was seen running in the ventral part of the reticular formation (Fig. 4b) ; other parts of the reticular formation contained scattered fibers. A moderate to low number of nerve fibers was seen in several other areas of pons and medulla oblongata such as raphe dorsalis and magnus, the medial longitudinal fascicle, the substantia gelatinosa of the trigeminal nerve, the nucleus of the mesencephalic tract, the medial and lateral lemniscus, the cochlear nucleus, the cerebellar peduncles, the nucleus of the spinal tract of the trigeminal nerve, the cuneate fascicle, the vagal and hypoglossal nuclei.
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Fig. 4. Immunoreactive nerve fibres in a the parabrachial nucleus (up is medial and right is dorsal in picture), b the ventral part (ventral is to the left) of the reticular formation ( x 4 2 0 ) and e lamina I and II of the spinal cord ( x 310)
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Fig. 5. Immunoreactive nerve cell bodies and nerve fibres (PAP stained) in a the magnocellular part of the paraventricular nucleus ( x 125)- viii indicates the third ventricle (note non-specific staining of the ependymal layer) and b the circular nucleus ( x 125), c band of immunoreactive nerves in the exter~a] zone of the median eminence (arrows) ( x 1O0), d immunofluorescent nerve fibres in the posterior Iobe of the hypophysis ( x 300); PI indicates the pars intermedia
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In the spinal cord immunoreactive nerve fibers were seen in the lamina I and II (Fig. 4c) as well as along the medial part of the dorsal horn. In other areas of the gray matter no or only very few nerves were observed.
The Magnocellular Hypothalamic System and the Hypophysis Only after intraventricular injection of colchicine were immunoreactive cell bodies seen in the paraventricular nucleus (Fig. 5 a), (in the magnocellular part and along the third ventricle), and in nucleus circularis (Fig. 5 b). Immunoreactive nerve fibers formed a dense band in the outer zone of the median eminence (Fig. 5c); they were less numerous in the deeper zone. The neurohypophysis was richly supplied with nerves (Fig. 5 d), while the adenohypophysis contained only few nerves.
Discussion
This study illustrates that antibodies reacting with the COOH-terminal end of CCK and gastrin can be used to demonstrate nerve fibers and cell bodies with a wide distribution in the rat brain. Under the conditions of immunocytochemistry the antiserum reacts equally well with gastrin-17 and CCK-8. From immunochemical studies it appears that gastrin-17 occurs in only very small amounts in brain and that the little there is occurs in the hypothalamus (Rehfeld, 1978 a). CCK-like peptides on the other hand are found in very large amounts in brain extracts. Immunochemical analysis has revealed that the predominant form of CCK in such extracts is CCK-8 (Rehfeld, 1978a, b). Hence, it appears likely (although by no means certain) that the immunoreactive material demonstrated by us in nerve fibers and nerve cell bodies of the central nervous system represents CCK-8. Other CCK-like components, if they exist, may of course contribute. The immunoreactive material in the nerves of the hypophysis may represent gastrin-17 since in the porcine hypophysis Rehfeld (1978b) failed to find CCK while demonstrating fairly large amounts of gastrin-17. The presence of a gastrin-like peptide (probably identical with CCK-8) in fibers and cell bodies of the rat brain has been described by H6kfelt et al. (1978). The immunoreactive material was located in cell bodies in cortical areas including hippocampus while nerve fibers were observed in the hypothalamus and the amygdala. On the whole, our observations on the distribution of the CCK-like peptides in the brain agree with the findings of H6kfelt et al. (1978). In addition, however, we find immunoreactive cell bodies and nerve fibers in several other areas. Besides the hippocampal and cortical cell bodies also described by H6kfelt et al. (1978) cell bodies occur in hypothalamus, mesencephalon and medulla oblongata. Immunoreactive nerve fibers are present in most regions of the brain. The projections for the various groups of immunoreactive cell bodies are not known. Several major pathways such as the medial forebrain bundle area and stria terminalis within the limbic system contain immunoreactive nerve fibers. In the brain stem immunoreactive nerve fibers are located in pathways such as the medial longitudinal fascicle and the medial and lateral lemniscus.
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Following colchicine treatment immunoreactive material appears in cell bodies of the magnocellular part of the paraventricular nuclei. Since these nuclei form part of the neurosecretory system which supplies nerves to the hypophysis it is of interest that the immunoreactive material in the nerves of the hypophysis probably represents gastrin-17 and gastrin-34 rather than CCK-8 (see Rehfeld, 1978). It is not inconceivable that the nerves storing the gastrins originate in the magnocellular neurosecretory system of the hypothalamus. Acknowledgements. Grant support from the Swedish Medical Research Council (04X-4499). References Alumets, J., Hfikanson, R., Schlegel, W., Sj61und, K., Sundler, F.: Distribution and ontogeny of CCK cells and their relation to gastrin cells. Cell Tissue Res. to be published (1979) Dockray, G.J. : Immunochemical evidence of cholecystokinin-like peptides in brain. Nature 264, 568-570 (1976) Dockray, G.J., Gregory, R.A., Hutchison, J.B., Harris, J.I., Runswick, M.J.: Isolation, structure and biologicalactivity oftwo cholecystokinin octapeptides from sheep brain. Nature 274, 711 713 (1978) H6kfelt, T., Elde, R., Fuxe, K., Johansson, O., Ljungdahl, •., Goldstein, M., Luft, R., Efendic, S., Nilsson, G., Terenius, L., Ganten, D., Jeffcoate, S.L., Rehfeld, J., Said, S., Perez de la Mora, M., Possani, L., Tapia, R., Teran, L., Palacios, R. : Aminergic and peptidergic pathways in the nervous system with special reference to the hypothalamus. In: The Hypothalamus. S. Reichlin, R.J. Baldessarini and J.B. Martin (eds.), pp. 69-135. New York: Raven Press 1978 Muller, J.E., Straus, E., Yalow, R.S. : Cholecystokinin and its COOH-terminal octapeptide in the pig brain. Proc. Natl. Acad. Sci. USA 74, 3035 3037 (1977) Rehfeld, J.F. : Localisation of gastrins to neuro- and adenohypophysis. Nature 271, 771 773 (1978 a) Rehfeld, J.F.: Immunochemical studies on cholecystokinin. II. Distribution and molecular heterogeneity in the central nervous system and small intestines of man and hog. J. Biol. Chem. 253, 4022 4030 (1978b) Rehfeld, J.F., Stadil, F., Rubin, B.: Production and evaluation of antibodies for the radioimmunoassay of gastrin. Scand. J. Clin. Lab. Invest. 30, 221~32 (1972) Robberecht, P., Deschodt-Lanckman, M., Vanderhaegen, J.J. : Demonstration of biological activity of brain gastrin-like peptidic material in the human: Its relationship with the COOH-terminal octapeptide of cholecystokinin. Proc. Natl. Acad. Sci. USA 75, 524-528 (1978) Straus, E., Muller, J.E., Choi, H.-S., Paronetto, F., Yalow, R.: Immunohistochemical localization in rabbit brain of a peptide resembling COOH-terminal octapeptide of cholecystokinin. Proc. Natl. Acad. Sci. USA 74, 3033 3034 (1977) Vanderhaegen, J.J., Signeau, J.C., Gepts, W.: New peptide in the vertebrate CNS reacting with antigastrin antibodies. Nature 257, 604-605 (1975)
Received October 30, 1978
Histochemistry59, 249-257 (1979)
9 by Springer-Verlag 1979
Distribution of Gastrin and CCK-like Peptides in Rat Brain An ImmunocytochemicalStudy I. Lor6n, J. Alumets, R. Hfikanson, and F. Sundler Departments of Histologyand Pharmacology,Universityof Lurid Biskopsgatan 5, S-22362Lund, Sweden
Summary. The distribution of gastrin and CCK-like peptides in the rat brain was studied by immunocytochemistry using an antiserum reacting equally well with both groups of peptides. Immunoreactive nerve cell bodies were detected in all cortical areas, in the hippocampus where they were particularly numerous, in the mesencephalic central gray and in the medulla oblongata. After colchicine treatment immunoreactive material appeared also in cell bodies of the magnocellular hypothalamic system. Immunoreactive nerve fibers were widely distributed in the brain. Particularly dense accumulations were seen in the hippocampus near the ventral surface of the brain, in the caudate nucleus, in the interpeduncular nucleus, the parabrachial nucleus, the dorsal part of the medulla oblongata and in the dorsal horn of the spinal cord. In the hypothalamus immunoreactive nerve fibers were observed in all nuclei, being most frequent in the ventromedial, dorsal and lateral hypothalamic nuclei. A rich supply of nerve fibers was seen in the outer zone of the median eminence and in the neurohypophysis. From previous immunochemical analysis it appears that the peptide demonstrated in most parts of the brain is identical with CCK-8. In the neurosecretory cell bodies of the hypothalamus, the median eminence and the neurohypophysis, however, the immunoreactive material is probably identical with gastrin.
Introduction "Gastrin-like" material in brain extracts was first described by Vanderhaegen et al. (1975). Subsequent studies (Dockray et al., 1976, 1978; Muller et al., 1977; Robberecht et al., 1978; Rehfeld, 1978a, b) have indicated that the bulk of gastrin-like material is distinct from gastrin and identical with, or closely related to CCK-octapeptide (CCK-8), the COOH-terminal eight amino acid sequence of CCK. The fact that CCK-8 shares the COOH-terminal five amino acids with gastrin may explain why it was initially described as "gastrin-like". Re-
0301-5564/79/0059/0249/$01.80
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cently Rehfeld (1978 a) was able to d e m o n s t r a t e significant a m o u n t s of gastrin-17 a n d gastrin-34 in extracts of the porcine hypophysis a n d h y p o t h a l a m u s ; in the r e m a i n d e r o f the b r a i n n o n e was found, I n r a b b i t b r a i n cortex Straus et al. (1977) described n u m e r o u s nerve cell bodies, b u t n o nerve terminals, reacting with antisera against the C O O H - t e r m i n a l tetragastrin fragment. We n o w wish to report o n the i m m u n o c y t o c h e m i c a l d i s t r i b u t i o n of gastrin a n d C C K - l i k e material in the rat brain. The results o b t a i n e d differ from those of Straus et al. (1977) in that we observed n o t only i m m u n o r e a c t i v e nerve cell bodies b u t also n u m e r o u s nerve fibers in several b r a i n regions.
Materials and Methods Adult female Sprague-Dawleyrats were perfused via the ascending aorta with 300 ml of an ice-cold 4% formaldehyde solution (paraformaldehyde dissolved in 0.1 M phosphate buffer). Various brain regions were post-fixed for 24 h in the same type of fixative as that used for the perfusion. The specimens were rinsed for at least 48 h in Tyrode buffer with 5% sucrose added. Postfixation and rinsing were carried out at 5~ C. The specimens were frozen in dry ice, and frontal sections were cut in a cryostat at - 15~ C. The sections were then processed for the immunocytochemical demonstration of gastrin-like and CCK-like peptides. The antiserum used (code no. 4562; kindly supplied by Professor J.F. Rehfeld, Arhus, Denmark) was raised in rabbits against synthetic human gastrin I, 2-17, covalently bound to albumin (Rehfeld et al., 1972). In the immunocytochemical procedure this antiserum demonstrates gastrin and CCK with about equal potency (Alumets et al., 1979). The working dilutions were 1:320 for immunofluorescence and 1:25,000 for immunoperoxidase (PAP) staining (for details on these procedures, see Alumets et al., 1979). Inactivated antiserum (excess amounts of gastrin-17 or CCK-8 were added) was used to provide control sections. Immunostaining was regarded as specific only if it failed to appear in the control sections. In one series of experiments, colchicine (8 gg/gl) was injected intraventricularly (2 gl to each of 3 rats) and the animals were killed 48 h later. Tissue specimens were processed as above; sections were cut in the region of the hypothalamus and the mesencephalon.
Results
Distribution of tmmunoreactive Nerve Celt Bodies I m m u n o r e a c t i v e nerve cell bodies were seen in neocortex, paleocortex, mesocortex a n d archiocortex as well as in claustrum. The cell bodies were rather few in the neo- a n d mesocortex a n d occurred scattered in layers I I - V I (Fig. 1 a). They were seen to give off processes some of which could be followed for quite some distance. Occasionally single nerve cell bodies were seen in the piriform a n d e n t o r h i n a l cortex; they were fairly frequent in the cortical a m y g d a loid nuclei. I m m u n o r e a c t i v e cell bodies were most frequent in the h i p p o c a m p u s where they could be observed in the strata oriens, pyramidale, l a c u n o s u m moleculare, a n d r a d i a t u m of C A 1 - C A 3 (Fig. 1 d). Here, they were more intensely i m m u n o r e a c t i v e t h a n in other areas. Most of these cell bodies h a d several processes, some of which c o u l d be followed for rather long distances before they were seen to divide into smaller branches. I n the dentate gyrus i m m u n o r e a c tive cell bodies were seen in the hilar region a n d a l o n g the i n n e r surface of the g r a n u l e cell layer (Fig. 1 b). F r o m some of these cell bodies fibers could
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Fig. 1. Nerve cell bodies displaying CCK-like immunoreactivity in a layers II-III of the parietal cortex (x 300), b the hilar region of the dentate gyrus close to the granule cell layer (x 350), e nucleus gracilis ( x 300) and d stratum radiatum of CA3 in hippocampus ( • 320)
be followed along the inner surface of the granule cell layer which was sometimes penetrated by smaller branches from the fibres. The subiculum contained cell bodies in about the same frequency as the other hippocampal areas. Occasionally immunoreactive cell bodies were observed in the molecular layer of the dentate gyrus. A group of cell bodies was found in the ventral part of the mesencephalic central gray; some of these cell bodies occurred close to the ventricular lining (Fig. 2). Cell bodies were also seen in medulla oblongata where they were located in nucleus gracilis (Fig. 1 c). The distribution of immunoreactive cell bodies in hypothalamus is described on page 256.
Distribution of Immunoreactive Nerve Fibers Diencephalon. In epithalamus the lateral habenular nuclei contained some immunoreactive nerve fibers. In thalamus such nerve fibers were seen to run in the midline and in nuclei close to the midline, while a rather dense network of immunoreactive nerves was seen in the lateral geniculate body and the ventral thalamic nucleus. In hypothalamus immunoreactive nerve fibers seemed to be present in all nuclei; the most dense accumulation of nerves was in the ventromedial (Fig. 3 a), dorsal and lateral hypothalamic nuclei (Fig. 3 b). The ventromedial nucleus contained a network of delicate nerves while the lateral nucleus had more coarse nerves. The immunoreactive nerve fibers in the lateral and most ventral part of the preoptic area mostly had a coarse appearance while the majority of nerves in the other preoptic areas were more delicate. All other hypothalamic nuclei (anterior, posterior, periventricular, and paraventricular
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Fig. 2. Immunoreactive cell bodies in the ventral part of the mesencephalic central gray (PAP technique) ( x 100)
Fig. 3. Immunoreactive nerve fibres in hypothalamus, a A network of delicate nerve fibres in the ventromedial nucleus ( x 350), b a network of more coarse nerve fibres in the lateral nucleus ( x 375)
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hypothalamic nuclei, the ventral and dorsal premamillary nuclei as well as the mamillary complex) contained both coarse and delicate nerve fibers in a low to moderate number. On the whole, delicate nerves were more numerous than coarse ones. A dense strand of mixed coarse and delicate nerves was seen to run close to the ventral surface. Another strand of nerves was observed in the dorsal part of hypothalamus between the paraventricular and the lateral hypothalamic nuclei.
Telencephalon. A small aggregation of immunoreactive nerve fibers was seen in tuberculum olfactorium. Within the limbic lobe nerves could be demonstrated in the septal area, nucleus accumbens, hippocampus and in the amygdaloid, entorhinal and cingulate cortex. In the septum a moderate number of immunoreactive nerve fibers was located in the ventral part close to the lateral ventricle. The ventral and medial part of the nucleus accumbens contained a narrow strand of nerves. Immunoreactive nerve fibers could be seen in all subdivisions of the hippocampal formation but a particularly dense accumulation was seen in the pyramidal cell layer and in areas that reached the ventral surface of the brain. The lowest number of nerves was seen in the molecular layer of the dentate gyrus as well as in the strata of alveus and oriens. All nuclei in the amygdaloid complex contained immunoreactive nerves; they were most numerous in the medial, central and cortical nuclei. The entorhinal cortex as well as the piriform cortex contained a moderate number of nerves. In all other cortical areas, a small number of immunoreactive nerve fibers could be seen in the different layers. In the pathways of the limbic system nerves were located in the medial forebrain bundle area and in stria terminalis. The caudate nucleus was densely innervated by delicate nerves, especially its ventral, medial and dorsomedial part. Mesencephalon. In mesencephalon a dense system of coarse nerves was found in the dorsal and lateral parts of the interpeduncular nucleus. A rather dense supply was seen also in the nucleus of the medial geniculate body as well as in the superior and inferior collicle. In the central gray only a few immunoreacrive nerves were seen. Ports, Medulla Oblongata and Spinal Cord. In pons and medulla oblongata immunoreactive nerve fibers were seen in several areas. A rich supply was observed in the parabrachial nucleus (Fig. 4 a), and in the dorsal part of medulla oblongata, i.e. nucleus gracilis, nucleus cuneatus, area postrema, and in the nucleus of the solitary tract (as well as in the solitary tract). A horizontal bundle with a moderate number of nerve fibers was seen running in the ventral part of the reticular formation (Fig. 4b) ; other parts of the reticular formation contained scattered fibers. A moderate to low number of nerve fibers was seen in several other areas of pons and medulla oblongata such as raphe dorsalis and magnus, the medial longitudinal fascicle, the substantia gelatinosa of the trigeminal nerve, the nucleus of the mesencephalic tract, the medial and lateral lemniscus, the cochlear nucleus, the cerebellar peduncles, the nucleus of the spinal tract of the trigeminal nerve, the cuneate fascicle, the vagal and hypoglossal nuclei.
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Fig. 4. Immunoreactive nerve fibres in a the parabrachial nucleus (up is medial and right is dorsal in picture), b the ventral part (ventral is to the left) of the reticular formation ( x 4 2 0 ) and e lamina I and II of the spinal cord ( x 310)
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Fig. 5. Immunoreactive nerve cell bodies and nerve fibres (PAP stained) in a the magnocellular part of the paraventricular nucleus ( x 125)- viii indicates the third ventricle (note non-specific staining of the ependymal layer) and b the circular nucleus ( x 125), c band of immunoreactive nerves in the exter~a] zone of the median eminence (arrows) ( x 1O0), d immunofluorescent nerve fibres in the posterior Iobe of the hypophysis ( x 300); PI indicates the pars intermedia
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In the spinal cord immunoreactive nerve fibers were seen in the lamina I and II (Fig. 4c) as well as along the medial part of the dorsal horn. In other areas of the gray matter no or only very few nerves were observed.
The Magnocellular Hypothalamic System and the Hypophysis Only after intraventricular injection of colchicine were immunoreactive cell bodies seen in the paraventricular nucleus (Fig. 5 a), (in the magnocellular part and along the third ventricle), and in nucleus circularis (Fig. 5 b). Immunoreactive nerve fibers formed a dense band in the outer zone of the median eminence (Fig. 5c); they were less numerous in the deeper zone. The neurohypophysis was richly supplied with nerves (Fig. 5 d), while the adenohypophysis contained only few nerves.
Discussion
This study illustrates that antibodies reacting with the COOH-terminal end of CCK and gastrin can be used to demonstrate nerve fibers and cell bodies with a wide distribution in the rat brain. Under the conditions of immunocytochemistry the antiserum reacts equally well with gastrin-17 and CCK-8. From immunochemical studies it appears that gastrin-17 occurs in only very small amounts in brain and that the little there is occurs in the hypothalamus (Rehfeld, 1978 a). CCK-like peptides on the other hand are found in very large amounts in brain extracts. Immunochemical analysis has revealed that the predominant form of CCK in such extracts is CCK-8 (Rehfeld, 1978a, b). Hence, it appears likely (although by no means certain) that the immunoreactive material demonstrated by us in nerve fibers and nerve cell bodies of the central nervous system represents CCK-8. Other CCK-like components, if they exist, may of course contribute. The immunoreactive material in the nerves of the hypophysis may represent gastrin-17 since in the porcine hypophysis Rehfeld (1978b) failed to find CCK while demonstrating fairly large amounts of gastrin-17. The presence of a gastrin-like peptide (probably identical with CCK-8) in fibers and cell bodies of the rat brain has been described by H6kfelt et al. (1978). The immunoreactive material was located in cell bodies in cortical areas including hippocampus while nerve fibers were observed in the hypothalamus and the amygdala. On the whole, our observations on the distribution of the CCK-like peptides in the brain agree with the findings of H6kfelt et al. (1978). In addition, however, we find immunoreactive cell bodies and nerve fibers in several other areas. Besides the hippocampal and cortical cell bodies also described by H6kfelt et al. (1978) cell bodies occur in hypothalamus, mesencephalon and medulla oblongata. Immunoreactive nerve fibers are present in most regions of the brain. The projections for the various groups of immunoreactive cell bodies are not known. Several major pathways such as the medial forebrain bundle area and stria terminalis within the limbic system contain immunoreactive nerve fibers. In the brain stem immunoreactive nerve fibers are located in pathways such as the medial longitudinal fascicle and the medial and lateral lemniscus.
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Following colchicine treatment immunoreactive material appears in cell bodies of the magnocellular part of the paraventricular nuclei. Since these nuclei form part of the neurosecretory system which supplies nerves to the hypophysis it is of interest that the immunoreactive material in the nerves of the hypophysis probably represents gastrin-17 and gastrin-34 rather than CCK-8 (see Rehfeld, 1978). It is not inconceivable that the nerves storing the gastrins originate in the magnocellular neurosecretory system of the hypothalamus. Acknowledgements. Grant support from the Swedish Medical Research Council (04X-4499). References Alumets, J., Hfikanson, R., Schlegel, W., Sj61und, K., Sundler, F.: Distribution and ontogeny of CCK cells and their relation to gastrin cells. Cell Tissue Res. to be published (1979) Dockray, G.J. : Immunochemical evidence of cholecystokinin-like peptides in brain. Nature 264, 568-570 (1976) Dockray, G.J., Gregory, R.A., Hutchison, J.B., Harris, J.I., Runswick, M.J.: Isolation, structure and biologicalactivity oftwo cholecystokinin octapeptides from sheep brain. Nature 274, 711 713 (1978) H6kfelt, T., Elde, R., Fuxe, K., Johansson, O., Ljungdahl, •., Goldstein, M., Luft, R., Efendic, S., Nilsson, G., Terenius, L., Ganten, D., Jeffcoate, S.L., Rehfeld, J., Said, S., Perez de la Mora, M., Possani, L., Tapia, R., Teran, L., Palacios, R. : Aminergic and peptidergic pathways in the nervous system with special reference to the hypothalamus. In: The Hypothalamus. S. Reichlin, R.J. Baldessarini and J.B. Martin (eds.), pp. 69-135. New York: Raven Press 1978 Muller, J.E., Straus, E., Yalow, R.S. : Cholecystokinin and its COOH-terminal octapeptide in the pig brain. Proc. Natl. Acad. Sci. USA 74, 3035 3037 (1977) Rehfeld, J.F. : Localisation of gastrins to neuro- and adenohypophysis. Nature 271, 771 773 (1978 a) Rehfeld, J.F.: Immunochemical studies on cholecystokinin. II. Distribution and molecular heterogeneity in the central nervous system and small intestines of man and hog. J. Biol. Chem. 253, 4022 4030 (1978b) Rehfeld, J.F., Stadil, F., Rubin, B.: Production and evaluation of antibodies for the radioimmunoassay of gastrin. Scand. J. Clin. Lab. Invest. 30, 221~32 (1972) Robberecht, P., Deschodt-Lanckman, M., Vanderhaegen, J.J. : Demonstration of biological activity of brain gastrin-like peptidic material in the human: Its relationship with the COOH-terminal octapeptide of cholecystokinin. Proc. Natl. Acad. Sci. USA 75, 524-528 (1978) Straus, E., Muller, J.E., Choi, H.-S., Paronetto, F., Yalow, R.: Immunohistochemical localization in rabbit brain of a peptide resembling COOH-terminal octapeptide of cholecystokinin. Proc. Natl. Acad. Sci. USA 74, 3033 3034 (1977) Vanderhaegen, J.J., Signeau, J.C., Gepts, W.: New peptide in the vertebrate CNS reacting with antigastrin antibodies. Nature 257, 604-605 (1975)
Received October 30, 1978
