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Immunohistochemical evidence for substance P-containing nerve endings in the human cortex
Brain Research, 104 (1976) 181-186
181
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Immunohistochemical evidence for substance P-containing nerve endings in the human cortex
T O M A S HOKFELT, BJORN MEYERSON, C H A R L O T T E SACHS
G{JRAN NILSSON, B E N G T P E R N O W AND
Departments of Histology and Pharmacology, Karolinska Institute, and Departments of Neurosurgery, Clinical Physiology and Neurology, Karolinska Hospital, Stockholm (Sweden) (Accepted November 19th, 1975)
In 1931, von Euler and G a d d u m 4 isolated a peptide with smooth muscle stimulating and hypotensive effects from the equine brain and intestine. Recently this peptide, called substance P (SP), has been isolated and its structure as an undecapeptide revealed by Chang and Leeman 1, and Chang, Leeman and Niall ~. Early studies by Pernow 14 indicated that, in the dog's brain, SP has a marked regional distribution and this could subsequently be confirmed in biochemicaP 9-21, and recently in radioimmunological studies 16. With antibodies to SP 12 it has recently been possible to demonstrate the cellular localization of S P - - o r SP-like immunoreactivity (SPLI) - - in the rat and cat using the indirect immunofluorescence technique
182 and the immersion in the lixative was approximately 30 min. After a total fixation time of 2-6 h and rinsing in 0.1 M phosphate buffer with 51~Josucrose added fo~-at least 24 h, 10 #m thick sections were cut on a cryostat (Dittes, Heidelberg). The sections were processed according to the indirect immunofluorescence technique oi" Coons and collaborators (see ref. 3) using antibodies to SP conjugated to bovine serum albumin prepared as described previously 1~'. The antiserum was pretreated with bovine albumin before incubation. This serum, in addition pretreated with SP (10 mg/1 ml antiserum diluted i :10), served as control serum. The sections were incubated with the SP antiserum, diluted 1:40, for 30 min at + 3 7 °C, rinsed in phosphate buffered saline (PBS), incubated with fluorescein isothiocyanate (F1TC) conjugated antibodies (commercially available from Statens Bakteriologiska Laboratorium, SBL, Stockholm, Sweden), diluted 1:4, for 30 min at + 3 7 °C, rinsed with PBS and mounted in a mixture of PBS and glycerin (1:3) and examined in a Zeiss junior fluorescence microscope. Dot- and fiberlike structures, often with a varicose appearance, were observed in most pieces of human l¥ontal and temporal cortex (Figs. 1 and 2A). Such fibers were present in all cortical layers but with a low density (Fig. 2A). Occasionally, however, small areas with a very dense network of SP-positive fibers could be observed, especially in the temporal lobe cortex (Figs. t A and B). As seen in single sections such a high density could be observed in some cases in layer I1 but was in other cases present in all cortical layers. Furthermore, the high density of SP-positive fibers was not seen in all pieces from one and the same patient. Even within one and the same section (size about 10 mm < 5 mm) the density varied markedly so that one area measuring approximately 2 m m in width contained a dense network whereas in the adjacent cortex only single fibers were seen. No predominant direction of the course of the SP-positive fibers could be seen. No SP-positive cell bodies could be observed in the areas studied. No fluorescent fibers were observed after incubation with control serum (Fig. 2B). The present histochemical findings confirm and extend earlier biochemical results of the presence of SP in the human cortex x4,19-2~. Our results demonstrate that SP is present in presumable nerve endings with an uneven distribution. Thus, the density of the SP-positive fibers varies considerably between different brain regions and also within one and the same tissue piece representing an area of 10 m m ~" 5 ram. The highest concentrations have so far been observed in the temporal cortex. It is interesting to note that a similar 'patchy' innervation has recently been observed for dopamine nerve endings in the entorhinal cortex of the rat 8. It should be emphasized, however, that it cannot be excluded that the marked regional variations and the lack of SP-positive fibers in several cortical pieces are due to technical artifacts. The tumor might have induced changes in the innervation pattern and the time-lapse between occlusion of the blood supply to the resected area and the immersion of the tissue into the fixative may vary. In parallel experiments on consecutive sections with antibodies to tyrosine hydroxytase and dopaminefl-hydroxylase, only single fluorescent fibers were observed (H6kfelt, Goldstein, Park, Meyerson and Sachs, unpublished results) and no 'islands' of catecholamine fibers could be observed.
183
Fig. I. lmmunofluorescence micrographs of the temporal cortex after incubation with SP antiserum. A dense network of SP-positive fibers is observed as seen in a low (A) and high (B) power magnification. Arrows point to structures with an unspecific fluorescence. Surface of the brain to the right in A. Magnifications × 160 (A) and ×400 (B).
184
Fig. 2. lmmunofluorescence micrographs o1' the temporal lobe alter incubation with SP antiscrmn (A) or control serum (SP antiserum pretreated with SP) (B). An area with only few fibers (arrow~) is shown in A. B represents a semiconsecutive section to 1B. Note the almost total lack of fluorescent fibers. Magnification 400.
185 A l t h o u g h the i m m u n o f l u o r e s c e n c e o b s e r v e d in this study is specific for SP in the sense t h a t it is a b o l i s h e d after p r e t r e a t m e n t o f the SP a n t i s e r u m with SP, it c a n n o t be e x c l u d e d t h a t cross-reactivity occurs between o u r a n t i b o d i e s a n d an SP-like peptide(s). In fact, in the h u m a n b r a i n Zetler 2°,21 has d e m o n s t r a t e d the existence o f several active p e p t i d e s in c r u d e SP p r e p a r a t i o n s . In the rat extensive n e t w o r k s o f SP-positive fibers have been observed m a i n l y in the b r a i n stem a n d spinal c o r d a n d to a smaller extent in certain limbic cortical areas, whereas only very few fibers are seen in the n e o c o r t e x v. Thus, species differences m a y exist with r e g a r d to d i s t r i b u t i o n a n d / o r c r o s s - r e a c t i n g peptides. The origin o f the cortical SP-positive fibers is at p r e s e n t unclear. In the r a t we have o b s e r v e d SP-positive cell bodies in the i n t e r p e d u n c u l a r nucleus and the medial h a b e n u l a e , as well as in spinal ganglia 7 ( a n d u n p u b l i s h e d observations). T h e f u n c t i o n a l significance o f the present results is unclear. Based on neurop h y s i o l o g i c a l studies SP has been a t t r i b u t e d a role as an e x c i t a t o r y t r a n s m i t t e r or m o d u l a t o r in various b r a i n areasS, 1°,15 as well as in p a r t o f the p r i m a r y sensory n e u r o n s 9. The presence o f SP o r S P L I in p r o b a b l e nerve t e r m i n a l s in some cortical areas o f the h u m a n b r a i n seem to be in c o n f o r m i t y with this hypothesis. These studies were s u p p o r t e d by grants f r o m the Swedish M e d i c a l R e s e a r c h C o u n c i l (04X-2887, 04X-3521, 04X-2886), M a g n u s Bervalls Stiftelse, H a r a l d och G r e t a Jeanssons Stiftelse a n d K n u t och Alice W a l l e n b e r g s Stifte!se. T h e skilful technical assistance o f Miss A. Nyg~trds, Miss G. Thulin a n d Miss E. Tirdn is gratefully a c k n o w l e d g e d .
1 CHANG, M. M., AND LEEMAN, S. E., Isolation of a sialogogic peptide from bovine hypothalamic tissue and its characterization as substance P., J. biol. Chem., 245 (1970) 4784-4790. 2 CHANG, M. M., LEEMAN, S. E., AND NIALE, H. D., Amino-acid sequence of substance P, Nature New Biol., 232 (1971) 86-87. 3 COONS, A.H., Fluorescent antibody methods. In J. F. DANIELLI (Ed.), General Cytochemical Methods, Academic Press, New York, 1958, pp. 399422. 4 EULER, U. S. VON, AND GADDUM, J. H., An unidentified depressor substance in certain tissue extracts, J. Physiol. (Lond,), 72 (1931) 74-87. 5 HENRY, J. L., KRNJEVI(~, K., AND MORRIS, M. E., Excitatory action of substance P on the spinal cord, Fed. Proe., 33 (1974) 548. 6 HOKFELT,T., KELLERTH,J.-O., NILSSON,G., AND PERNOW, B., Experimental immunohistochemical studies on the localization and distribution of substance P in cat primary sensory neurons, Brain Research, 100 (1975) 235-252. 7 HOKFELT,T., KELLERTH,J.-O., NILSSON,G., AND PERNOW,B., Morphological support for a trans-
mitter or modulator role of substance P: immunohistochemical localization in the central nervous system and in primary sensory neurons, Science, 190 (1975) 889 890. 8 HOKFELT, T., LJUNGDAHL,/~., FUXE, K., AND JOHANSSON,O., Dopamine nerve terminals in the rat limbic cortex: aspects of the dopamine hypothesis of schizophrenia, Science, 184 (1974) 177
179. 9 KONISHI,S., AND OTSUKA, M., The effects of substance P and other peptides on spinal neurons of the frog, Brain Research, 65 (1974) 297~410. 10 KRNJEVl(:, K., AND MORRIS, M.E., An excitatory action of substance P on cuneate neurons, Canad. J. physiol. Pharmacol., 52 (1974) 736-744. 11 NILSSON,G., HOKFELT,T., AND PERNOW, B., Distribution of substance P-like immunoreactivity
186
12 13 14 15 16 17
18 19
20 21
in the rat central nervous system as revealed by inmlunohistochemistry, Med. I~io/, I[]., 52 (t974) 424-427. NILSSON, G., PERNOW, B., FISHER, G. H., AND FOLKERS, K., Presence of substance P-like immtmoreactivity in plasma l¥orn man and clog, Acta physiol, sc,and., 94 (1975) 542 544. PEASE, D. C., Buffered formaldehyde as a killing agent and primary fixative t'or electron microscopy, Anat. Rec., 142 (1962) 342. PERNOW, B., Studies on substance P. Purification, occurrence and biological actions, Acta phy,siol. scand., 29, Suppl. 105 (1953) 1 90. PH~LUS, J. W., AND LIMACHER, J. J., Substance P excitation of cerebral cortica! Betz cells, Brain Research, 69 (1974) 158-163. POWELL, D., LEEMAN, S. F,., TREGEAR, G. W., NIALL, H. D., AND POTTS, J. T. JR, Radioimmunoassay for substance P, Nature New Biol., 241 (1973) 252-254. TAKAHASH~, T., KONISH~, S., POWELL, D., LEEMAN, S. E., AND OTSUKA, M., Identification of ihe motoneuron-depolarizing peptide in bovine dorsal roots as hypothalamic substance P, Brain Research, 73 (1974) 59 69. TAKAHASHJ, T., AND OTSUKA, M., Regional distribution of substance P in the spinal cord and nerve roots of the cat and the effect of dorsal root section, Brabt Research, 87 (19"75) 1 I I. ZETLER, G., Substanz P, ein Polypeptid aus Darm und Gehirn mit depressiven, hyperalgetischen und Morphin-antagonistischen Wirkungen auf das Zentralnervensystem, Nauto,n-Schmiedebergs Arch. exp. Path. PharmJk., 228 (1956) 513-538. ZETLER, G., Zwei neue pharmakologisch aktive Polypeptide in einem Substanz P-haltigen Hirnextrakt, Naunyn-Schmie:tebergs, Arch. exp. Path. Pharmak., 242 (1961) 330-352. ZETLER, G., Distribution of petidergic neurons in mammalian brain. In W. BARGMANN AND B. SCHARRER (Eds.), Aspects O/Neuroendocrit:ology, Springer, Berlin, 1970, pp. 287-295.
181
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Immunohistochemical evidence for substance P-containing nerve endings in the human cortex
T O M A S HOKFELT, BJORN MEYERSON, C H A R L O T T E SACHS
G{JRAN NILSSON, B E N G T P E R N O W AND
Departments of Histology and Pharmacology, Karolinska Institute, and Departments of Neurosurgery, Clinical Physiology and Neurology, Karolinska Hospital, Stockholm (Sweden) (Accepted November 19th, 1975)
In 1931, von Euler and G a d d u m 4 isolated a peptide with smooth muscle stimulating and hypotensive effects from the equine brain and intestine. Recently this peptide, called substance P (SP), has been isolated and its structure as an undecapeptide revealed by Chang and Leeman 1, and Chang, Leeman and Niall ~. Early studies by Pernow 14 indicated that, in the dog's brain, SP has a marked regional distribution and this could subsequently be confirmed in biochemicaP 9-21, and recently in radioimmunological studies 16. With antibodies to SP 12 it has recently been possible to demonstrate the cellular localization of S P - - o r SP-like immunoreactivity (SPLI) - - in the rat and cat using the indirect immunofluorescence technique
182 and the immersion in the lixative was approximately 30 min. After a total fixation time of 2-6 h and rinsing in 0.1 M phosphate buffer with 51~Josucrose added fo~-at least 24 h, 10 #m thick sections were cut on a cryostat (Dittes, Heidelberg). The sections were processed according to the indirect immunofluorescence technique oi" Coons and collaborators (see ref. 3) using antibodies to SP conjugated to bovine serum albumin prepared as described previously 1~'. The antiserum was pretreated with bovine albumin before incubation. This serum, in addition pretreated with SP (10 mg/1 ml antiserum diluted i :10), served as control serum. The sections were incubated with the SP antiserum, diluted 1:40, for 30 min at + 3 7 °C, rinsed in phosphate buffered saline (PBS), incubated with fluorescein isothiocyanate (F1TC) conjugated antibodies (commercially available from Statens Bakteriologiska Laboratorium, SBL, Stockholm, Sweden), diluted 1:4, for 30 min at + 3 7 °C, rinsed with PBS and mounted in a mixture of PBS and glycerin (1:3) and examined in a Zeiss junior fluorescence microscope. Dot- and fiberlike structures, often with a varicose appearance, were observed in most pieces of human l¥ontal and temporal cortex (Figs. 1 and 2A). Such fibers were present in all cortical layers but with a low density (Fig. 2A). Occasionally, however, small areas with a very dense network of SP-positive fibers could be observed, especially in the temporal lobe cortex (Figs. t A and B). As seen in single sections such a high density could be observed in some cases in layer I1 but was in other cases present in all cortical layers. Furthermore, the high density of SP-positive fibers was not seen in all pieces from one and the same patient. Even within one and the same section (size about 10 mm < 5 mm) the density varied markedly so that one area measuring approximately 2 m m in width contained a dense network whereas in the adjacent cortex only single fibers were seen. No predominant direction of the course of the SP-positive fibers could be seen. No SP-positive cell bodies could be observed in the areas studied. No fluorescent fibers were observed after incubation with control serum (Fig. 2B). The present histochemical findings confirm and extend earlier biochemical results of the presence of SP in the human cortex x4,19-2~. Our results demonstrate that SP is present in presumable nerve endings with an uneven distribution. Thus, the density of the SP-positive fibers varies considerably between different brain regions and also within one and the same tissue piece representing an area of 10 m m ~" 5 ram. The highest concentrations have so far been observed in the temporal cortex. It is interesting to note that a similar 'patchy' innervation has recently been observed for dopamine nerve endings in the entorhinal cortex of the rat 8. It should be emphasized, however, that it cannot be excluded that the marked regional variations and the lack of SP-positive fibers in several cortical pieces are due to technical artifacts. The tumor might have induced changes in the innervation pattern and the time-lapse between occlusion of the blood supply to the resected area and the immersion of the tissue into the fixative may vary. In parallel experiments on consecutive sections with antibodies to tyrosine hydroxytase and dopaminefl-hydroxylase, only single fluorescent fibers were observed (H6kfelt, Goldstein, Park, Meyerson and Sachs, unpublished results) and no 'islands' of catecholamine fibers could be observed.
183
Fig. I. lmmunofluorescence micrographs of the temporal cortex after incubation with SP antiserum. A dense network of SP-positive fibers is observed as seen in a low (A) and high (B) power magnification. Arrows point to structures with an unspecific fluorescence. Surface of the brain to the right in A. Magnifications × 160 (A) and ×400 (B).
184
Fig. 2. lmmunofluorescence micrographs o1' the temporal lobe alter incubation with SP antiscrmn (A) or control serum (SP antiserum pretreated with SP) (B). An area with only few fibers (arrow~) is shown in A. B represents a semiconsecutive section to 1B. Note the almost total lack of fluorescent fibers. Magnification 400.
185 A l t h o u g h the i m m u n o f l u o r e s c e n c e o b s e r v e d in this study is specific for SP in the sense t h a t it is a b o l i s h e d after p r e t r e a t m e n t o f the SP a n t i s e r u m with SP, it c a n n o t be e x c l u d e d t h a t cross-reactivity occurs between o u r a n t i b o d i e s a n d an SP-like peptide(s). In fact, in the h u m a n b r a i n Zetler 2°,21 has d e m o n s t r a t e d the existence o f several active p e p t i d e s in c r u d e SP p r e p a r a t i o n s . In the rat extensive n e t w o r k s o f SP-positive fibers have been observed m a i n l y in the b r a i n stem a n d spinal c o r d a n d to a smaller extent in certain limbic cortical areas, whereas only very few fibers are seen in the n e o c o r t e x v. Thus, species differences m a y exist with r e g a r d to d i s t r i b u t i o n a n d / o r c r o s s - r e a c t i n g peptides. The origin o f the cortical SP-positive fibers is at p r e s e n t unclear. In the r a t we have o b s e r v e d SP-positive cell bodies in the i n t e r p e d u n c u l a r nucleus and the medial h a b e n u l a e , as well as in spinal ganglia 7 ( a n d u n p u b l i s h e d observations). T h e f u n c t i o n a l significance o f the present results is unclear. Based on neurop h y s i o l o g i c a l studies SP has been a t t r i b u t e d a role as an e x c i t a t o r y t r a n s m i t t e r or m o d u l a t o r in various b r a i n areasS, 1°,15 as well as in p a r t o f the p r i m a r y sensory n e u r o n s 9. The presence o f SP o r S P L I in p r o b a b l e nerve t e r m i n a l s in some cortical areas o f the h u m a n b r a i n seem to be in c o n f o r m i t y with this hypothesis. These studies were s u p p o r t e d by grants f r o m the Swedish M e d i c a l R e s e a r c h C o u n c i l (04X-2887, 04X-3521, 04X-2886), M a g n u s Bervalls Stiftelse, H a r a l d och G r e t a Jeanssons Stiftelse a n d K n u t och Alice W a l l e n b e r g s Stifte!se. T h e skilful technical assistance o f Miss A. Nyg~trds, Miss G. Thulin a n d Miss E. Tirdn is gratefully a c k n o w l e d g e d .
1 CHANG, M. M., AND LEEMAN, S. E., Isolation of a sialogogic peptide from bovine hypothalamic tissue and its characterization as substance P., J. biol. Chem., 245 (1970) 4784-4790. 2 CHANG, M. M., LEEMAN, S. E., AND NIALE, H. D., Amino-acid sequence of substance P, Nature New Biol., 232 (1971) 86-87. 3 COONS, A.H., Fluorescent antibody methods. In J. F. DANIELLI (Ed.), General Cytochemical Methods, Academic Press, New York, 1958, pp. 399422. 4 EULER, U. S. VON, AND GADDUM, J. H., An unidentified depressor substance in certain tissue extracts, J. Physiol. (Lond,), 72 (1931) 74-87. 5 HENRY, J. L., KRNJEVI(~, K., AND MORRIS, M. E., Excitatory action of substance P on the spinal cord, Fed. Proe., 33 (1974) 548. 6 HOKFELT,T., KELLERTH,J.-O., NILSSON,G., AND PERNOW, B., Experimental immunohistochemical studies on the localization and distribution of substance P in cat primary sensory neurons, Brain Research, 100 (1975) 235-252. 7 HOKFELT,T., KELLERTH,J.-O., NILSSON,G., AND PERNOW,B., Morphological support for a trans-
mitter or modulator role of substance P: immunohistochemical localization in the central nervous system and in primary sensory neurons, Science, 190 (1975) 889 890. 8 HOKFELT, T., LJUNGDAHL,/~., FUXE, K., AND JOHANSSON,O., Dopamine nerve terminals in the rat limbic cortex: aspects of the dopamine hypothesis of schizophrenia, Science, 184 (1974) 177
179. 9 KONISHI,S., AND OTSUKA, M., The effects of substance P and other peptides on spinal neurons of the frog, Brain Research, 65 (1974) 297~410. 10 KRNJEVl(:, K., AND MORRIS, M.E., An excitatory action of substance P on cuneate neurons, Canad. J. physiol. Pharmacol., 52 (1974) 736-744. 11 NILSSON,G., HOKFELT,T., AND PERNOW, B., Distribution of substance P-like immunoreactivity
186
12 13 14 15 16 17
18 19
20 21
in the rat central nervous system as revealed by inmlunohistochemistry, Med. I~io/, I[]., 52 (t974) 424-427. NILSSON, G., PERNOW, B., FISHER, G. H., AND FOLKERS, K., Presence of substance P-like immtmoreactivity in plasma l¥orn man and clog, Acta physiol, sc,and., 94 (1975) 542 544. PEASE, D. C., Buffered formaldehyde as a killing agent and primary fixative t'or electron microscopy, Anat. Rec., 142 (1962) 342. PERNOW, B., Studies on substance P. Purification, occurrence and biological actions, Acta phy,siol. scand., 29, Suppl. 105 (1953) 1 90. PH~LUS, J. W., AND LIMACHER, J. J., Substance P excitation of cerebral cortica! Betz cells, Brain Research, 69 (1974) 158-163. POWELL, D., LEEMAN, S. F,., TREGEAR, G. W., NIALL, H. D., AND POTTS, J. T. JR, Radioimmunoassay for substance P, Nature New Biol., 241 (1973) 252-254. TAKAHASH~, T., KONISH~, S., POWELL, D., LEEMAN, S. E., AND OTSUKA, M., Identification of ihe motoneuron-depolarizing peptide in bovine dorsal roots as hypothalamic substance P, Brain Research, 73 (1974) 59 69. TAKAHASHJ, T., AND OTSUKA, M., Regional distribution of substance P in the spinal cord and nerve roots of the cat and the effect of dorsal root section, Brabt Research, 87 (19"75) 1 I I. ZETLER, G., Substanz P, ein Polypeptid aus Darm und Gehirn mit depressiven, hyperalgetischen und Morphin-antagonistischen Wirkungen auf das Zentralnervensystem, Nauto,n-Schmiedebergs Arch. exp. Path. PharmJk., 228 (1956) 513-538. ZETLER, G., Zwei neue pharmakologisch aktive Polypeptide in einem Substanz P-haltigen Hirnextrakt, Naunyn-Schmie:tebergs, Arch. exp. Path. Pharmak., 242 (1961) 330-352. ZETLER, G., Distribution of petidergic neurons in mammalian brain. In W. BARGMANN AND B. SCHARRER (Eds.), Aspects O/Neuroendocrit:ology, Springer, Berlin, 1970, pp. 287-295.