Acta physiol. scand. 1978. 104. 499-501 From the Department of Histology and Pharmacology, Karolinska Institutet, Stockholm. the Department of Medical Pharmacology, Uppsala University, Uppsala, Sweden, the Department of Medical Biochemistry, Aarhus University, Aarhus, Denmark, the Department of Anatomy University of Minnesota, Minnesota, and the Department of Internal Medicine and Pharmacology, University of Texas, Dallas, Texas, U.S.A.
Peptide neurons in the vagus, splanchnic and sciatic nerves" BY
JAN M. LUNDBERG, TOMAS HOKFELT,GORANNILSSON, LARSTERENIUS, JENSREHFELD,ROBERTELDE,SAM1 SAlD
Peripheral nerves in general contain both motor and sensory fibres. The classical transmitter substances in the motor nerves are either acetylcholine (Loewi, 1921) or noradrenaline (Euler 1956), although a non-cholinergic, non-adrenergic component also has been described (see Burnstock 1972). In addition peptides have been observed e.g. in extracts of the vagus nerve, including substance P (SP) (Euler 1963), vasoactive intestinal polypeptide (VIP) (Said and Rosenberg 1976), gastrin (GAS) (Uvnas-Wallensten e t a / . 1977) and somatostatin (SOM) (Uvnas-Wallensten et a/. 1978). In the present communication 3 different types of peripheral nerves were studied by the indirect immunofluorescence technique using antisera to several peptides. With this approach it was attempted to define further the cellular localization of such peptides. 10 male albino rats (b.wt. 150-200 g) and 10 male guinea pigs (b.wt. 300400 g) were subjected t o the following treatments according to the techniques described by Dahlstrom (1971). The vagus nerve was ligated at the cervical level, the greater splanchnic nerve at the subdiaphragmatic level and the sciatic nerve one c m below the foramen infrapiriforme. In 5 other animals of each kind colchicine (20%) or vinblastine (1 yo) were applied locally on the nodose ganglion and on the first sacral spinal ganglion. These drugs and the ligation technique were used to increase the levels of the peptides, since in untreated animals the cell bodies and axons often contain too low amounts to be visualized with immunocytochemistry. 24 h after both types of treatment the animals were perfused with icecold 4 % paraformaldehyde and the nerves and ganglia were processed for the indirect immunofluorescence technique (see Coons 1958). Briefly, cryostat sections were incubated with a n antiserum raised to one of the peptides, SP, SOM, VIP, GAS and methionine-enkephalin (met-ENK) followed by a second incubation with fluorescein-isothiocyanate conjugated antibodies and examined in a Zeiss fluorescence microscope. The different antisera pretreated with an excess of the respective peptide served as control sera. For further details on experimental procedures including characteristics of antisera, see Lundberg et a/. (1979). It should, however, be pointed out that the GAS antiserum also cross-reacts with cholecystokinin (CCK).
* Part of these results were communicated at the 6th ETP winterschool in Zuoz, Switzerland, January 1978. 499
500
JAN M. LUNDBERG ET AL.
Fig. 1 A-C. Immunofluorescence micrographs of sections of the vagus nerve (A) and sciatic nerve (B) and the nodose ganglion (C) of the rat 24 h after ligation (A, B) or colchicine application (C) incubated with antiserum to SP (A, C) and met-ENK (B). Many SP (A) and some met-ENK (B) positive fibres are seen above the ligation and some met-ENK positive ones below (B). Small and large (arrows in C ) SP positive cells are seen in the nodose ganglion. Arrow heads in A and B indicate site of ligation. Bars indicate 50pm.
SP-, SOM-, VIP-, GAS- and met-ENK immunoreactive material were found in all three nerves studied as shown by their accumulation central (and in the sciatic nerve, partly peripheral) to the ligations, indicating a rapid axonal transport (Fig. 1 A, B). In Table I, subjective estimations have been made to schematically indicate the number of peptide containing nerves above a ligation in the guinea pig. In the rat similar distribution patterns were observed although the number of nerves appeared to be slightly lower. In the nodose ganglion high numbers of small and some large SP immunoreactive cell bodies were observed (Fig. 1 C). Moderate numbers of VIP immunoreactive cells but only few SOM and GAS positive cells could be seen, virtually all being of the small type. No met-ENK immunoreactive cells could be demonstrated in the ganglion. In the first sacral spinal ganglion numerous GAS positive neurons, partly of the large type were seen. In addition SP, SOM and VIP but no ENK immunoreactive small neurons were observed. The present results indicate that in the vagus nerve complex as well as in the sciatic nerve SP-, SOM-, (CCK-)like peptides, at least partly, are present in sensory neurons, whereas a TABLE I. Subjective estimation of the number of peptide containing axons in the vagus, splanchnic and sciatic nerve of the guinea pig. The estimates are based on analysis of accumulations of immuno= very high, + = high, = medium, reactive material above a ligation. = low and + = single. LI =Like imunoreactivity. For abbreviations of peptides see text.
+++++
++
N. vagus N. splanchnicus major N. ischiadicus
+++
+++
SP-LI
SOM-LI
VIP-LI
GAS-(CCK-) met-ENK-LI LI
+++++ +++ +++
+ + +
+++
+ + +++
+
++
++ +++ +
PEPTIDE NEURONS IN PERIPHERAL NERVES
50 I
met-ENK-like peptide may be contained in axons of motor fibres. The nature of the fibres containing the peptides in the splanchnic nerves is at present under investigation. Similar distribution patterns have also been observed in immunohistochemical studies on these three types of nerves in the cat (Lundberg, Uvnas-Wallensten, Hokfelt et al., in preparation). Thus, in addition to the previously identified SP and SOM immunoreactive cells in sensory ganglia (Hokfelt et al. 1976) two further subpopulations have been identified containing a GAS-(CCK-)like and a VIP-like peptide, respectively. Finally, ENK-like material is present in peripheral nerves with a probable central origin. The localization of the peripheral innervation areas and of putative opioid receptors of such ENK positive fibers projecting e.g. in the sciatic nerve remains to be elucidated. This study was supported by grants from the Swedish Medical Research Council (04X-2887, 12X-5189, 25X-5065, 04X-3521), Magnus Bergvalls Stiftelse, Harald Jeanssons Stiftelse and Harald and Greta Jeanssons Stiftelse. The skilful technical assistance of Miss Gun Norell is gratefully acknowledged.
References BURNSTOCK, G., Purinergic nerves. Pharmacol. R ~ L )1972. . 24. 509-581. A. H., Fluorescent Antibody Methods. In: General Cytochemical Methods. Ed.: J . F. DANlELLI. Academic Press, New York 1958. 399422. DAHLSTROM, A., Effects of vinblastine and colchicine o n monoamine containing neurons of the rat with special regard to the axoplasrnic transport of arnine granules. Acta neuropath. (Berl.). 1971. Suppl. V. 226-237. EULER,U. S. von, Noradrenaline. Thomas. Springfield, Ill. 1956. EULER, U. S. von, Substance P in subcellular particles in peripheral nerves. Ann. N . Y . Acad. Sci. 1963. 104. 4 4 9 4 6 1 . HOKFELT,T., R. E m f , 0. JOHANSSON, R . LUFT, G. NILSSONand A. ARIMURA, Immunohistochemical evidence for separate populations of somatostatin-containing and substance P-containing primary afferent neurons. Neurosciencr 1976. I . 13 1-1 36. LOEWI,O., Uber humorale Ubertragbarkeit der Herznervenwirkung. Pfliig. Arch. ges. Physiol. 1921. 189. 239. LUNDBERG, J. M., T. HOKFELT,G. NILSSON,L. TERENlUS, J. REHFELD, R. P. ELDE,M . GOLDSTEIN and S. SAID,Peripheral peptide and catecholamine containing neurons in the vagus, splanchnic, hypogastric and pelvic nerves. Occurrence and distribution of substance P-, somatostatin-, VIP-, gastrin (CCK)and enkephalin-like immunoreactivity and of catecholamine synthesizing enzymes in truncs and ganglia. Neuroscience Submitted. SAID,S. and R. ROSENBERG, Vasoactive intestinal polypeptide: Abundant immunoreactivjty in neural cell lines and normal nervous tissue. Science 1976. 192. 907-908. UvNAs-WALLfNsTm, K., S. EFENDIC and R. LUFT,Occurrence of somatostatin-like immunoreactivity in the vagal nerves. Acta physiol. scanrl. 1978. 102. 248-250. UVNAS-WALLENSTEN, K., J. F. REHFELD and B. UVNAS,Heptadecapeptide gastrin in the vagal nerve. Proc. nut. Acnd. Sci. (Wash.) 1977. 74. 5707-5710.
COONS,
'
Peptide neurons in the vagus, splanchnic and sciatic nerves" BY
JAN M. LUNDBERG, TOMAS HOKFELT,GORANNILSSON, LARSTERENIUS, JENSREHFELD,ROBERTELDE,SAM1 SAlD
Peripheral nerves in general contain both motor and sensory fibres. The classical transmitter substances in the motor nerves are either acetylcholine (Loewi, 1921) or noradrenaline (Euler 1956), although a non-cholinergic, non-adrenergic component also has been described (see Burnstock 1972). In addition peptides have been observed e.g. in extracts of the vagus nerve, including substance P (SP) (Euler 1963), vasoactive intestinal polypeptide (VIP) (Said and Rosenberg 1976), gastrin (GAS) (Uvnas-Wallensten e t a / . 1977) and somatostatin (SOM) (Uvnas-Wallensten et a/. 1978). In the present communication 3 different types of peripheral nerves were studied by the indirect immunofluorescence technique using antisera to several peptides. With this approach it was attempted to define further the cellular localization of such peptides. 10 male albino rats (b.wt. 150-200 g) and 10 male guinea pigs (b.wt. 300400 g) were subjected t o the following treatments according to the techniques described by Dahlstrom (1971). The vagus nerve was ligated at the cervical level, the greater splanchnic nerve at the subdiaphragmatic level and the sciatic nerve one c m below the foramen infrapiriforme. In 5 other animals of each kind colchicine (20%) or vinblastine (1 yo) were applied locally on the nodose ganglion and on the first sacral spinal ganglion. These drugs and the ligation technique were used to increase the levels of the peptides, since in untreated animals the cell bodies and axons often contain too low amounts to be visualized with immunocytochemistry. 24 h after both types of treatment the animals were perfused with icecold 4 % paraformaldehyde and the nerves and ganglia were processed for the indirect immunofluorescence technique (see Coons 1958). Briefly, cryostat sections were incubated with a n antiserum raised to one of the peptides, SP, SOM, VIP, GAS and methionine-enkephalin (met-ENK) followed by a second incubation with fluorescein-isothiocyanate conjugated antibodies and examined in a Zeiss fluorescence microscope. The different antisera pretreated with an excess of the respective peptide served as control sera. For further details on experimental procedures including characteristics of antisera, see Lundberg et a/. (1979). It should, however, be pointed out that the GAS antiserum also cross-reacts with cholecystokinin (CCK).
* Part of these results were communicated at the 6th ETP winterschool in Zuoz, Switzerland, January 1978. 499
500
JAN M. LUNDBERG ET AL.
Fig. 1 A-C. Immunofluorescence micrographs of sections of the vagus nerve (A) and sciatic nerve (B) and the nodose ganglion (C) of the rat 24 h after ligation (A, B) or colchicine application (C) incubated with antiserum to SP (A, C) and met-ENK (B). Many SP (A) and some met-ENK (B) positive fibres are seen above the ligation and some met-ENK positive ones below (B). Small and large (arrows in C ) SP positive cells are seen in the nodose ganglion. Arrow heads in A and B indicate site of ligation. Bars indicate 50pm.
SP-, SOM-, VIP-, GAS- and met-ENK immunoreactive material were found in all three nerves studied as shown by their accumulation central (and in the sciatic nerve, partly peripheral) to the ligations, indicating a rapid axonal transport (Fig. 1 A, B). In Table I, subjective estimations have been made to schematically indicate the number of peptide containing nerves above a ligation in the guinea pig. In the rat similar distribution patterns were observed although the number of nerves appeared to be slightly lower. In the nodose ganglion high numbers of small and some large SP immunoreactive cell bodies were observed (Fig. 1 C). Moderate numbers of VIP immunoreactive cells but only few SOM and GAS positive cells could be seen, virtually all being of the small type. No met-ENK immunoreactive cells could be demonstrated in the ganglion. In the first sacral spinal ganglion numerous GAS positive neurons, partly of the large type were seen. In addition SP, SOM and VIP but no ENK immunoreactive small neurons were observed. The present results indicate that in the vagus nerve complex as well as in the sciatic nerve SP-, SOM-, (CCK-)like peptides, at least partly, are present in sensory neurons, whereas a TABLE I. Subjective estimation of the number of peptide containing axons in the vagus, splanchnic and sciatic nerve of the guinea pig. The estimates are based on analysis of accumulations of immuno= very high, + = high, = medium, reactive material above a ligation. = low and + = single. LI =Like imunoreactivity. For abbreviations of peptides see text.
+++++
++
N. vagus N. splanchnicus major N. ischiadicus
+++
+++
SP-LI
SOM-LI
VIP-LI
GAS-(CCK-) met-ENK-LI LI
+++++ +++ +++
+ + +
+++
+ + +++
+
++
++ +++ +
PEPTIDE NEURONS IN PERIPHERAL NERVES
50 I
met-ENK-like peptide may be contained in axons of motor fibres. The nature of the fibres containing the peptides in the splanchnic nerves is at present under investigation. Similar distribution patterns have also been observed in immunohistochemical studies on these three types of nerves in the cat (Lundberg, Uvnas-Wallensten, Hokfelt et al., in preparation). Thus, in addition to the previously identified SP and SOM immunoreactive cells in sensory ganglia (Hokfelt et al. 1976) two further subpopulations have been identified containing a GAS-(CCK-)like and a VIP-like peptide, respectively. Finally, ENK-like material is present in peripheral nerves with a probable central origin. The localization of the peripheral innervation areas and of putative opioid receptors of such ENK positive fibers projecting e.g. in the sciatic nerve remains to be elucidated. This study was supported by grants from the Swedish Medical Research Council (04X-2887, 12X-5189, 25X-5065, 04X-3521), Magnus Bergvalls Stiftelse, Harald Jeanssons Stiftelse and Harald and Greta Jeanssons Stiftelse. The skilful technical assistance of Miss Gun Norell is gratefully acknowledged.
References BURNSTOCK, G., Purinergic nerves. Pharmacol. R ~ L )1972. . 24. 509-581. A. H., Fluorescent Antibody Methods. In: General Cytochemical Methods. Ed.: J . F. DANlELLI. Academic Press, New York 1958. 399422. DAHLSTROM, A., Effects of vinblastine and colchicine o n monoamine containing neurons of the rat with special regard to the axoplasrnic transport of arnine granules. Acta neuropath. (Berl.). 1971. Suppl. V. 226-237. EULER,U. S. von, Noradrenaline. Thomas. Springfield, Ill. 1956. EULER, U. S. von, Substance P in subcellular particles in peripheral nerves. Ann. N . Y . Acad. Sci. 1963. 104. 4 4 9 4 6 1 . HOKFELT,T., R. E m f , 0. JOHANSSON, R . LUFT, G. NILSSONand A. ARIMURA, Immunohistochemical evidence for separate populations of somatostatin-containing and substance P-containing primary afferent neurons. Neurosciencr 1976. I . 13 1-1 36. LOEWI,O., Uber humorale Ubertragbarkeit der Herznervenwirkung. Pfliig. Arch. ges. Physiol. 1921. 189. 239. LUNDBERG, J. M., T. HOKFELT,G. NILSSON,L. TERENlUS, J. REHFELD, R. P. ELDE,M . GOLDSTEIN and S. SAID,Peripheral peptide and catecholamine containing neurons in the vagus, splanchnic, hypogastric and pelvic nerves. Occurrence and distribution of substance P-, somatostatin-, VIP-, gastrin (CCK)and enkephalin-like immunoreactivity and of catecholamine synthesizing enzymes in truncs and ganglia. Neuroscience Submitted. SAID,S. and R. ROSENBERG, Vasoactive intestinal polypeptide: Abundant immunoreactivjty in neural cell lines and normal nervous tissue. Science 1976. 192. 907-908. UvNAs-WALLfNsTm, K., S. EFENDIC and R. LUFT,Occurrence of somatostatin-like immunoreactivity in the vagal nerves. Acta physiol. scanrl. 1978. 102. 248-250. UVNAS-WALLENSTEN, K., J. F. REHFELD and B. UVNAS,Heptadecapeptide gastrin in the vagal nerve. Proc. nut. Acnd. Sci. (Wash.) 1977. 74. 5707-5710.
COONS,
'
