Calcitonin Gene-Related Peptide in Human Celiac/Superior Mesenteric and Inferior Mesenteric Ganglia Immunohistochemical Localization and Coexistence with Substance Pa M. DEL FIACCO, A. FLORIS, M. L. LAI, R. MONTISCI, AND M. QUARTU Department of Cytomorphology University of Cagliari 09124 Cagliari, Italy
Prevertebral sympathetic ganglia of laboratory animals contain large amounts of calcitonin gene-related peptide (CGRPI-like immunoreactivity (CGRP-I), localized to nerve fibers and terminals,’-*which form synaptic contacts with the principal ganglionic cells,3 and to small intensity fluorescent (SIF) c e h 2In this report we present evidence for the presence of CGRP in the human celiac/superior mesenteric (CSM) and inferior mesenteric (IM) ganglia, and compare its distribution with that, previously shown,4 of substance P (SP). The indirect immunofluorescence technique was applied to tissue sections obtained from autopsic specimens of CSM and surgical specimens of IM. Either single immunostainings for CGRP alone and SP alone on consecutive tissue sections or double immunostainings for both CGRP and SP were carried out. A rabbit anti-CGRP serum (Peninsula RAS-6009-N) and a rat anti-SP monoclonal antibody5 were used as primary antibodies. CGRP-I marked a large amount of nerve fibers and terminal-like structures (FIG.1). No labeled cell bodies were observed. Nerve bundles within the ganglia contained variable amounts of CGRP-like immunoreactive (CGRP-IR) fibers. Isolated IR filaments were seen leaving the bundles and branching among the cell clusters. They eventually formed dense terminal-like plexuses encircling many principal ganglionic cells. Such plexuses showed a patchy distribution within the CSM ganglion, so that the CGRP innervation appeared to be restricted to a subpopulation of postganglionic neurons. Double immunostaining showed that the two peptides are often colocafized (FIG.2 ) . However, fibers containing exclusively CGRP-I as well as fibers containing only SP-I could be identified. Moreover, particularly in the CSM ganglion, SP-IR innervation appeared more widely diffused over the cell clusters. Our results suggest a role for CGRP in regulating the postganglionic activity of prevertebral ganglia in man. Data obtained from experimental studies on laboratory animals show that most of the CGRP-IR innervation of the prevertebral sympathetic ganglia is of sensory origin;2a proportion of the CGRP-IR elements originates from the intestinal intramural neurons;* moreover, a preganglionic origin for the CGRP-I may be considered,6 although in situ hybridization data speak against it.’
This work was funded by grants from the Italian MURST and CNR. 473
FIGURE 1. Tissue sections of an autopsic specimen of CSM ganglion complex from a 53year-old female subject (A, B) and of a surgical specimen of IM ganglion from a 50-year-old male subject (C). CGRP-I is revealed with a tetramethyl-rhodarnine isothiocyanate-conjugated secondary anti-rabbit serum (Jackson). The imrnunoreaction labels bundles of nerve fibers, most of which show thick varicosities (A), and plexiforrn beaded filaments around principal ganglionic cells (B, C). The fluorescence over the cell bodies is nonspecifically due to lipofuscin accumulation. Magnification: 210 x .
DEL FIACCO ef at.: CGRP IN MESENTERIC GANGLlA
475
FIGURE 2. Double immunostaining for CGRP (A), revealed with a tetramethyl-rhodamine isothiocyanate-conjugated secondary anti-rabbit serum (Jackson), and for SP (B), revealed with a dichlorotriazinylamino-fluorescein-conjugatedanti-rat serum (Jackson) on the same tissue section of an autopsic specimen of CSM ganglionic complex from a 40-year-old male subject. The two peptides are colocalized in most neuronal processes showing thick varicosities. However, elements containing CGRP-I alone or SP-I alone are also detectable. Moreover, a delicate network of thin SP-IR fibers can frequently be observed over areas, occupied by cell clusters, that appear almost totally devoid of CCRP-I. One of these areas is visible close to the left superior corner of the micrographs. Magnification: 2 1 0 ~ .
476
ANNALS NEW YORK ACADEMY OF SCIENCES
Fibers showing colocalization of peptides are likely to be of a sensory nature.' However, as primary sensory neurons containing CGRP alone or SP alone are present in dorsal root ganglia,' nerve fibers immunoreactive to either CGRP or SP may also participate in these circuits. This neuronal circuitry would allow for short-loop autonomic reflexes in which central transmission is not required. In addition, CGRP-containing elements in the prevertebral ganglia may represent the afferent limb of the peripheral intestino-intestinal reflex arc.* REFERENCES 1.
2. 3.
4. 5. 6.
7.
LEE, Y., K. T A K A M IS. , GIRGIS, C. J. HILLYARD, I. M A C ~ N T Y RP.E C. , EMSON& M. TOHYAMA. 1985. Neuroscience 15: 122771237, LINDH, B., T. HOKFELT& L . G . E L F V I N 1988. . Neuroscience 26: 1037-1071. LEE, Y., N . HAYASHI, C. J . HILLYARD, S. 1. GIRGIS,I. MACINTYRE, P. C. EMSON& M. TOHYAMA. 1987. Brain Res. 407: 149-151. LEVANTI, M. C., R. MONTISCI, M. D. ROSA,P. SEDDA & M. DEL FIACCO.1988. Basic Appl. Histochem. 32: 145-152. ~ C. MILSTEIN.1979. Proc. Natl. Acad. Sci. USA 76: CUELLO,A. C., G. G A L F R& 3532-3536. YAMAMOTO, K., E . SENBA, T. MATSUNACA & M. TOHYAMA. 1989. Brain Res. 487: 158- 164. R ~ T H E L YM., I , C. B. METZ & P. K. L U N D .1989. Neuroscience 29: 225-239.
Prevertebral sympathetic ganglia of laboratory animals contain large amounts of calcitonin gene-related peptide (CGRPI-like immunoreactivity (CGRP-I), localized to nerve fibers and terminals,’-*which form synaptic contacts with the principal ganglionic cells,3 and to small intensity fluorescent (SIF) c e h 2In this report we present evidence for the presence of CGRP in the human celiac/superior mesenteric (CSM) and inferior mesenteric (IM) ganglia, and compare its distribution with that, previously shown,4 of substance P (SP). The indirect immunofluorescence technique was applied to tissue sections obtained from autopsic specimens of CSM and surgical specimens of IM. Either single immunostainings for CGRP alone and SP alone on consecutive tissue sections or double immunostainings for both CGRP and SP were carried out. A rabbit anti-CGRP serum (Peninsula RAS-6009-N) and a rat anti-SP monoclonal antibody5 were used as primary antibodies. CGRP-I marked a large amount of nerve fibers and terminal-like structures (FIG.1). No labeled cell bodies were observed. Nerve bundles within the ganglia contained variable amounts of CGRP-like immunoreactive (CGRP-IR) fibers. Isolated IR filaments were seen leaving the bundles and branching among the cell clusters. They eventually formed dense terminal-like plexuses encircling many principal ganglionic cells. Such plexuses showed a patchy distribution within the CSM ganglion, so that the CGRP innervation appeared to be restricted to a subpopulation of postganglionic neurons. Double immunostaining showed that the two peptides are often colocafized (FIG.2 ) . However, fibers containing exclusively CGRP-I as well as fibers containing only SP-I could be identified. Moreover, particularly in the CSM ganglion, SP-IR innervation appeared more widely diffused over the cell clusters. Our results suggest a role for CGRP in regulating the postganglionic activity of prevertebral ganglia in man. Data obtained from experimental studies on laboratory animals show that most of the CGRP-IR innervation of the prevertebral sympathetic ganglia is of sensory origin;2a proportion of the CGRP-IR elements originates from the intestinal intramural neurons;* moreover, a preganglionic origin for the CGRP-I may be considered,6 although in situ hybridization data speak against it.’
This work was funded by grants from the Italian MURST and CNR. 473
FIGURE 1. Tissue sections of an autopsic specimen of CSM ganglion complex from a 53year-old female subject (A, B) and of a surgical specimen of IM ganglion from a 50-year-old male subject (C). CGRP-I is revealed with a tetramethyl-rhodarnine isothiocyanate-conjugated secondary anti-rabbit serum (Jackson). The imrnunoreaction labels bundles of nerve fibers, most of which show thick varicosities (A), and plexiforrn beaded filaments around principal ganglionic cells (B, C). The fluorescence over the cell bodies is nonspecifically due to lipofuscin accumulation. Magnification: 210 x .
DEL FIACCO ef at.: CGRP IN MESENTERIC GANGLlA
475
FIGURE 2. Double immunostaining for CGRP (A), revealed with a tetramethyl-rhodamine isothiocyanate-conjugated secondary anti-rabbit serum (Jackson), and for SP (B), revealed with a dichlorotriazinylamino-fluorescein-conjugatedanti-rat serum (Jackson) on the same tissue section of an autopsic specimen of CSM ganglionic complex from a 40-year-old male subject. The two peptides are colocalized in most neuronal processes showing thick varicosities. However, elements containing CGRP-I alone or SP-I alone are also detectable. Moreover, a delicate network of thin SP-IR fibers can frequently be observed over areas, occupied by cell clusters, that appear almost totally devoid of CCRP-I. One of these areas is visible close to the left superior corner of the micrographs. Magnification: 2 1 0 ~ .
476
ANNALS NEW YORK ACADEMY OF SCIENCES
Fibers showing colocalization of peptides are likely to be of a sensory nature.' However, as primary sensory neurons containing CGRP alone or SP alone are present in dorsal root ganglia,' nerve fibers immunoreactive to either CGRP or SP may also participate in these circuits. This neuronal circuitry would allow for short-loop autonomic reflexes in which central transmission is not required. In addition, CGRP-containing elements in the prevertebral ganglia may represent the afferent limb of the peripheral intestino-intestinal reflex arc.* REFERENCES 1.
2. 3.
4. 5. 6.
7.
LEE, Y., K. T A K A M IS. , GIRGIS, C. J. HILLYARD, I. M A C ~ N T Y RP.E C. , EMSON& M. TOHYAMA. 1985. Neuroscience 15: 122771237, LINDH, B., T. HOKFELT& L . G . E L F V I N 1988. . Neuroscience 26: 1037-1071. LEE, Y., N . HAYASHI, C. J . HILLYARD, S. 1. GIRGIS,I. MACINTYRE, P. C. EMSON& M. TOHYAMA. 1987. Brain Res. 407: 149-151. LEVANTI, M. C., R. MONTISCI, M. D. ROSA,P. SEDDA & M. DEL FIACCO.1988. Basic Appl. Histochem. 32: 145-152. ~ C. MILSTEIN.1979. Proc. Natl. Acad. Sci. USA 76: CUELLO,A. C., G. G A L F R& 3532-3536. YAMAMOTO, K., E . SENBA, T. MATSUNACA & M. TOHYAMA. 1989. Brain Res. 487: 158- 164. R ~ T H E L YM., I , C. B. METZ & P. K. L U N D .1989. Neuroscience 29: 225-239.
