- Email: [email protected]
An electron microscopic study on enkephalin-like immunoreactive nerve fibers in the celiac ganglion of guinea pigs
142
Brain Research, 252 (1982) 142-145 Elsevier Biomedical Press
An electron microscopic study on enkephalin-like immunoreactive nerve fibers in the celiac ganglion of guinea pigs HISATAKE KONDO and RYOGO YUI Department of Anatomy, Niigata University School of Medicine, Niigata 951, (Japan) (Accepted July 27th, 1982) Key words: enkephalin nerve - - fine structure - - celiac ganglion - - guinea pig
Enkephalin-like immunoreactive nerve fibers in the celiac ganglion of guinea pigs were characterized by a high population of large granular vesicles mixed with small clear vesicles. The immunoreactive material is confined to the large granular vesicles. The immunoreactive nerve fibers formed many axo-dendritic as well as axo-somatic synapses and also formed a few synapses with presumed preganglionic axons containing numerous vesicles. The immunoreactive fibers were regarded as presynaptic at these synapses. These findings suggest that enkephalin might play a role as a neurotransmitter or neuromodulator in the ganglionic transmission of this prevertebral ganglion. On the basis of light microscopic immunohistochemical studies, positive immunoreactivity with antisera to several biologically active peptides has been revealed in autonomic ganglial,2,3,4,L Fine structural features and synaptic relations of neuronal elements showing the immunoreactivity to these peptides are crucial to consider the functional significance of the peptides in the ganglionic transmission. We have attempted the immuno-electron microscopic studies on the celiac ganglion of guinea pigs and have so far reported the ultrastructure of substance P- and VIP-like immunoreactive nerve fibers and their synaptic relations~,% The present study, as the third of a series of this attempt, was undertaken to clarify the morphological characteristics of enkephalin-like immunoreactive nerve fiber s and to examine the possibility that enkephalin might be a neurotransmitter or neuromodulator in this sympathetic ganglion. Young albino guinea pigs of either sex of body weight 200-250 g were used in the present study. The animals were anesthetized (Nembutal, 35 mg/kg) and perfused with 4 ~ paraformaldehyde in phosphate buffer, pH 7.4, via the ascending aorta for 15 min. Celiac ganglia were dissected out and immersed in the same fixative for an additional 2 h at 4 °C. Sections, 20/~m thick, were cut using a Vibratome 0006-8993/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
(Oxford Instrument). The sections were incubated for 20 h at 4 °C with rabbit anti-methionine (met-) enkephalin antiserum (UCB-Bioproducts S. A., Belgium 1672/002) at the ddution of 1:600. The sites of antigen-antibody reaction were visualized with the peroxidase-antiperoxidase (PAP) method according to Sternberger 10. As the control serum, the anti-enkephalin antiserum was immunoabsorbed with synthetic met-enkephalin (10/zg/ml, 1:600 diluted antiserum). After completion of the PAP procedure, the sections were postfixed with 1 ~ OSO4, dehydrated and embedded in Epon 812. Thin sections were examined with brief staining by uranyl acetate. A total of 90 immunoreactive nerve fibers were identified in electron microscopy (Figs. 1, 2 and 3). They were 0.3-1.3/~m in diameter and enclosed by Schwann cell and ganglionic satellite cell sheaths. The immunoreactive fibers contained a high population of large granular vesicles, 90 nm in diameter as well as small clear vesicles, 45 nm in diameter. The large granular vesicles had a distinct halo between the core and the limiting membrane of the vesicles, and immunoreactive materials were rather precisely localized on the core, resulting in the conspicuous increase in density of the core. The electron density of axoplasmic matrix was in general slightly high as
143
Fig. 1. Enkephalin-like immunoreactive nerve fibers ('k') in synaptic contact (arrows) with an unlabeled postganglionic dendrite (D). Note the presence of abundant large granular vesicles showing conspicuous increase in density of the core. x 20,250. Fig. 2. Axo-somatic synapses (arrows) between an immunoreactive fiber ('k') and a principal neuron soma (S) and between a nonimmunoreactive fiber (n) and the neuron soma × 30,800. Fig. 3. Axo-axonic synapses (thick arrows) between an immunoreactive fiber ('k') and a non-reactive fiber (n) containing small clear vesicles predominantly (a) and between an immunoreactive fiber (~t') and a non-reactive fiber (n) containing large granular vesicles predominantly (b). Note the difference in density of the core of large granular vesicles in between the immunoreactive fiber and the non-reactive fiber. A thin arrow indicates another synapse on a dendritic or somatic spine. × 30,800.
c o m p a r e d to t h a t o f u n l a b e l e d n e u r o n a l profiles. F i f t y - t w o o f 90 labeled fibers identified in the present study f o r m e d a direct a p p o s i t i o n to unlabeled n e u r o n a l profiles w i t h o u t intervening Schwann cell sheaths. T h e y included 34 d e n d r i t e s (Fig. 1) a n d 13 s o m a o f p r i n c i p a l ganglion cells (Fig. 2) a n d 5 vesicle-containing n e u r o n a l profiles (Fig. 3).
The dendrites were characterized b y neurofilaments, neurotubules, m i t o c h o n d r i a , ribosomes, b u t few vesicles. The u n l a b e l e d vesicle-containing n e u r o n a l profiles were characterized b y n u m e r o u s vesicles consisting o f small clear spherical vesicles, 45 n m in diameter, a n d large g r a n u l a r ones, 90 n m in diameter, b u t n o r i b o s o m e s were f o u n d in a n y o f them.
144 These profiles are presumed to be preganglionic axons ~. Of those vesicle-containing neuronal profiles, 3 contained small clear vesicles predominantly and the other 2 contained large granular vesicles more numerously. At all contact sites an accumulation of vesicles, mainly small clear ones, associated with electron dense material was seen along the apposed plasma membrane of the labeled fibers. The immunoreactive granular vesicles were frequently seen displaced from the contact sites. A cytoplasmic density was slightly present on the inside of the apposed plasma membrane of unlabeled dendrites and soma. However, no cytoplasmic density with accumulations of vesicles was seen underneath the apposed membranes of unlabeled vesicle-containing neuronal profiles. After incubation with the control serum, no neuronal elements showing the immunoreactivity were observed either in light or electron microscopy. The present study revealed that a high population of large granular vesicles mixed with small clear vesicles is the characteristic feature of the enkephalin-like immunoreactive nerve fibers, in which the immunoreactivity is clearly confined to the large granular vesicles. It also revealed that the immunoreactive fibers form many axo-dendritic as well as axo-somatic and a few axo-axonic contacts. It is suggested on the basis of morphological criteria for chemical synapses 8, that all these contacts are synaptic sites, whereby the immunoreactive fibers are presynaptic. This ultrastructural feature of the immunoreactive fibers is similar to that of VIP-like immunoreactive nerve fibers in the same ganglion 6. However, there exists a marked difference in the proportions of 3 types of synapses formed by these two fibers. Among synapses formed by VIP fibers,
the axo-dendritic type is predominant in number, followed by the axo-axonic one and the axo-somatic type is uncommon. On the other hand, the axosomatic type is rather numerous and the axo-axonic one is rare among those formed by enkephalin fibers although the axo-dendritic type are most numerous among them. This difference indicates that there exist at least two subpopulations in nerve fibers containing abundant large granular vesicles in terms of peptides included. In physiological studies so far published 7, enkephalinergic presynaptie inhibition on the substance P-mediated slow EPSP has been demonstrated in the inferior mesenteric ganglion of guinea pigs. Some of the unlabeled vesicle-containing neuronal profiles making synaptic contact with the immunoreactive fiber in the present material, show ultrastructural features similar to those of the substance P-like immunoreactive nerve fibers reported earlier 5 in terms of the composition of vesicles. Thus, it is likely that some of the axo-axonic synapses formed by the immunoreactive fibers might be the site of enkephalinergic presynaptie inhibition on the substance P-mediated postganglionic response. However, the much more frequent occurrence of the axo-dendritic and axo-somatic synapses formed by the immunoreactive fibers implies that enkephalin may exert its main direct influence on postganglionic principal neurons through these synaptic pathways.
1 H6kfelt, T., Elfvin, L.-G., Elde, R., Schultzberg, M., Goldstein, M. and Luft, R., Occurrence of somatostatln-like immunoreactivity in some principal sympathetic noradrenergic neurons. Proc. nat. Acad. Sci. U.S.A., 74 (1977) 3587-3591. 2 H6kfelt, T., Elfvin, L.-G., Schultzberg, M., Fuxe, K., Said, S. I., Mutt, V. and Goldstein, M., Immunohistochemical evidence of vasoactive intestinal polypeptidecontaining neurons and nerve fibers in sympathetic ganglia, Neuroscience, 2 (1977) 885-896. 3 H6kfelt, T., Elfvin, L.-G., Schultzberg, M., Goldstein, M. and Nilsson, G., On the occurrence of substance
P-containing fibers in sympathetic ganglia: immunohistochemical evidence, Brain Research, 132 (1977) 2941. 4 Kondo, H. and Yui, R., Enkephalin-like immunoreactivity in the SIF cells of sympathetic ganglia of frogs, Biomed. Res., 2 (1981) 338-340. 5 Kondo, I-I. and Yui, R., An electron microscopic study on substance P-like immunoreactive nerve fibers in the celiac ganglion of guinea pigs, Brain Research, 222 (1981) 134-137. 6 Kondo, H. and Yui, R., An electron microscopic study on VIP-like immunoreactive nerve fibers in the celiac
The authors wish to thank Dr. Tsuneo Fujita for his encouragement during this work and Miss Hiroko Kato for her expert secretarial help. This work was supported by Grant 56440019 from the Ministry of Education, Science and Culture of Japan to H.K.
145 ganglion of guinea pigs, Brain Research, 237 (1982) 227-231. 7 Konishi, S., Tsunoo, A., Yanaihara, N. and Otsuka, M., Peptidergic excitatory and inhibitory synapses in mammalian sympathetic ganglia: roles of substance P and enkephalin, Biomed. Res., 1 (1980) 528-536. 8 Pappas, G. D. and Waxman, S. G., Synaptic fine structure - - morphological correlates of chemical and electronic transmission. In G. D. Pappas and D. P. Purpura (Eds.), Structure and Function of Synapses, Raven Press,
New York, (1972) pp. 1-43. 9 Schultzberg, M., H~Skfelt, T., Ternius, L., Elfvin, L.-G., Lundberg, J. M., Brandt, J., Elde, R. and Goldstein, M., Enkephalin immunoreactive nerve terminals and cell bodies in sympathetic ganglia of the guinea pig and rat, Neuroscience, 4 (1979) 249-270. 10 Sternberger, L. A., Immunocytochemistry, 2nd edn., John Wiley and Sons Inc., New York, 1979, pp. 104169.
Brain Research, 252 (1982) 142-145 Elsevier Biomedical Press
An electron microscopic study on enkephalin-like immunoreactive nerve fibers in the celiac ganglion of guinea pigs HISATAKE KONDO and RYOGO YUI Department of Anatomy, Niigata University School of Medicine, Niigata 951, (Japan) (Accepted July 27th, 1982) Key words: enkephalin nerve - - fine structure - - celiac ganglion - - guinea pig
Enkephalin-like immunoreactive nerve fibers in the celiac ganglion of guinea pigs were characterized by a high population of large granular vesicles mixed with small clear vesicles. The immunoreactive material is confined to the large granular vesicles. The immunoreactive nerve fibers formed many axo-dendritic as well as axo-somatic synapses and also formed a few synapses with presumed preganglionic axons containing numerous vesicles. The immunoreactive fibers were regarded as presynaptic at these synapses. These findings suggest that enkephalin might play a role as a neurotransmitter or neuromodulator in the ganglionic transmission of this prevertebral ganglion. On the basis of light microscopic immunohistochemical studies, positive immunoreactivity with antisera to several biologically active peptides has been revealed in autonomic ganglial,2,3,4,L Fine structural features and synaptic relations of neuronal elements showing the immunoreactivity to these peptides are crucial to consider the functional significance of the peptides in the ganglionic transmission. We have attempted the immuno-electron microscopic studies on the celiac ganglion of guinea pigs and have so far reported the ultrastructure of substance P- and VIP-like immunoreactive nerve fibers and their synaptic relations~,% The present study, as the third of a series of this attempt, was undertaken to clarify the morphological characteristics of enkephalin-like immunoreactive nerve fiber s and to examine the possibility that enkephalin might be a neurotransmitter or neuromodulator in this sympathetic ganglion. Young albino guinea pigs of either sex of body weight 200-250 g were used in the present study. The animals were anesthetized (Nembutal, 35 mg/kg) and perfused with 4 ~ paraformaldehyde in phosphate buffer, pH 7.4, via the ascending aorta for 15 min. Celiac ganglia were dissected out and immersed in the same fixative for an additional 2 h at 4 °C. Sections, 20/~m thick, were cut using a Vibratome 0006-8993/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
(Oxford Instrument). The sections were incubated for 20 h at 4 °C with rabbit anti-methionine (met-) enkephalin antiserum (UCB-Bioproducts S. A., Belgium 1672/002) at the ddution of 1:600. The sites of antigen-antibody reaction were visualized with the peroxidase-antiperoxidase (PAP) method according to Sternberger 10. As the control serum, the anti-enkephalin antiserum was immunoabsorbed with synthetic met-enkephalin (10/zg/ml, 1:600 diluted antiserum). After completion of the PAP procedure, the sections were postfixed with 1 ~ OSO4, dehydrated and embedded in Epon 812. Thin sections were examined with brief staining by uranyl acetate. A total of 90 immunoreactive nerve fibers were identified in electron microscopy (Figs. 1, 2 and 3). They were 0.3-1.3/~m in diameter and enclosed by Schwann cell and ganglionic satellite cell sheaths. The immunoreactive fibers contained a high population of large granular vesicles, 90 nm in diameter as well as small clear vesicles, 45 nm in diameter. The large granular vesicles had a distinct halo between the core and the limiting membrane of the vesicles, and immunoreactive materials were rather precisely localized on the core, resulting in the conspicuous increase in density of the core. The electron density of axoplasmic matrix was in general slightly high as
143
Fig. 1. Enkephalin-like immunoreactive nerve fibers ('k') in synaptic contact (arrows) with an unlabeled postganglionic dendrite (D). Note the presence of abundant large granular vesicles showing conspicuous increase in density of the core. x 20,250. Fig. 2. Axo-somatic synapses (arrows) between an immunoreactive fiber ('k') and a principal neuron soma (S) and between a nonimmunoreactive fiber (n) and the neuron soma × 30,800. Fig. 3. Axo-axonic synapses (thick arrows) between an immunoreactive fiber ('k') and a non-reactive fiber (n) containing small clear vesicles predominantly (a) and between an immunoreactive fiber (~t') and a non-reactive fiber (n) containing large granular vesicles predominantly (b). Note the difference in density of the core of large granular vesicles in between the immunoreactive fiber and the non-reactive fiber. A thin arrow indicates another synapse on a dendritic or somatic spine. × 30,800.
c o m p a r e d to t h a t o f u n l a b e l e d n e u r o n a l profiles. F i f t y - t w o o f 90 labeled fibers identified in the present study f o r m e d a direct a p p o s i t i o n to unlabeled n e u r o n a l profiles w i t h o u t intervening Schwann cell sheaths. T h e y included 34 d e n d r i t e s (Fig. 1) a n d 13 s o m a o f p r i n c i p a l ganglion cells (Fig. 2) a n d 5 vesicle-containing n e u r o n a l profiles (Fig. 3).
The dendrites were characterized b y neurofilaments, neurotubules, m i t o c h o n d r i a , ribosomes, b u t few vesicles. The u n l a b e l e d vesicle-containing n e u r o n a l profiles were characterized b y n u m e r o u s vesicles consisting o f small clear spherical vesicles, 45 n m in diameter, a n d large g r a n u l a r ones, 90 n m in diameter, b u t n o r i b o s o m e s were f o u n d in a n y o f them.
144 These profiles are presumed to be preganglionic axons ~. Of those vesicle-containing neuronal profiles, 3 contained small clear vesicles predominantly and the other 2 contained large granular vesicles more numerously. At all contact sites an accumulation of vesicles, mainly small clear ones, associated with electron dense material was seen along the apposed plasma membrane of the labeled fibers. The immunoreactive granular vesicles were frequently seen displaced from the contact sites. A cytoplasmic density was slightly present on the inside of the apposed plasma membrane of unlabeled dendrites and soma. However, no cytoplasmic density with accumulations of vesicles was seen underneath the apposed membranes of unlabeled vesicle-containing neuronal profiles. After incubation with the control serum, no neuronal elements showing the immunoreactivity were observed either in light or electron microscopy. The present study revealed that a high population of large granular vesicles mixed with small clear vesicles is the characteristic feature of the enkephalin-like immunoreactive nerve fibers, in which the immunoreactivity is clearly confined to the large granular vesicles. It also revealed that the immunoreactive fibers form many axo-dendritic as well as axo-somatic and a few axo-axonic contacts. It is suggested on the basis of morphological criteria for chemical synapses 8, that all these contacts are synaptic sites, whereby the immunoreactive fibers are presynaptic. This ultrastructural feature of the immunoreactive fibers is similar to that of VIP-like immunoreactive nerve fibers in the same ganglion 6. However, there exists a marked difference in the proportions of 3 types of synapses formed by these two fibers. Among synapses formed by VIP fibers,
the axo-dendritic type is predominant in number, followed by the axo-axonic one and the axo-somatic type is uncommon. On the other hand, the axosomatic type is rather numerous and the axo-axonic one is rare among those formed by enkephalin fibers although the axo-dendritic type are most numerous among them. This difference indicates that there exist at least two subpopulations in nerve fibers containing abundant large granular vesicles in terms of peptides included. In physiological studies so far published 7, enkephalinergic presynaptie inhibition on the substance P-mediated slow EPSP has been demonstrated in the inferior mesenteric ganglion of guinea pigs. Some of the unlabeled vesicle-containing neuronal profiles making synaptic contact with the immunoreactive fiber in the present material, show ultrastructural features similar to those of the substance P-like immunoreactive nerve fibers reported earlier 5 in terms of the composition of vesicles. Thus, it is likely that some of the axo-axonic synapses formed by the immunoreactive fibers might be the site of enkephalinergic presynaptie inhibition on the substance P-mediated postganglionic response. However, the much more frequent occurrence of the axo-dendritic and axo-somatic synapses formed by the immunoreactive fibers implies that enkephalin may exert its main direct influence on postganglionic principal neurons through these synaptic pathways.
1 H6kfelt, T., Elfvin, L.-G., Elde, R., Schultzberg, M., Goldstein, M. and Luft, R., Occurrence of somatostatln-like immunoreactivity in some principal sympathetic noradrenergic neurons. Proc. nat. Acad. Sci. U.S.A., 74 (1977) 3587-3591. 2 H6kfelt, T., Elfvin, L.-G., Schultzberg, M., Fuxe, K., Said, S. I., Mutt, V. and Goldstein, M., Immunohistochemical evidence of vasoactive intestinal polypeptidecontaining neurons and nerve fibers in sympathetic ganglia, Neuroscience, 2 (1977) 885-896. 3 H6kfelt, T., Elfvin, L.-G., Schultzberg, M., Goldstein, M. and Nilsson, G., On the occurrence of substance
P-containing fibers in sympathetic ganglia: immunohistochemical evidence, Brain Research, 132 (1977) 2941. 4 Kondo, H. and Yui, R., Enkephalin-like immunoreactivity in the SIF cells of sympathetic ganglia of frogs, Biomed. Res., 2 (1981) 338-340. 5 Kondo, I-I. and Yui, R., An electron microscopic study on substance P-like immunoreactive nerve fibers in the celiac ganglion of guinea pigs, Brain Research, 222 (1981) 134-137. 6 Kondo, H. and Yui, R., An electron microscopic study on VIP-like immunoreactive nerve fibers in the celiac
The authors wish to thank Dr. Tsuneo Fujita for his encouragement during this work and Miss Hiroko Kato for her expert secretarial help. This work was supported by Grant 56440019 from the Ministry of Education, Science and Culture of Japan to H.K.
145 ganglion of guinea pigs, Brain Research, 237 (1982) 227-231. 7 Konishi, S., Tsunoo, A., Yanaihara, N. and Otsuka, M., Peptidergic excitatory and inhibitory synapses in mammalian sympathetic ganglia: roles of substance P and enkephalin, Biomed. Res., 1 (1980) 528-536. 8 Pappas, G. D. and Waxman, S. G., Synaptic fine structure - - morphological correlates of chemical and electronic transmission. In G. D. Pappas and D. P. Purpura (Eds.), Structure and Function of Synapses, Raven Press,
New York, (1972) pp. 1-43. 9 Schultzberg, M., H~Skfelt, T., Ternius, L., Elfvin, L.-G., Lundberg, J. M., Brandt, J., Elde, R. and Goldstein, M., Enkephalin immunoreactive nerve terminals and cell bodies in sympathetic ganglia of the guinea pig and rat, Neuroscience, 4 (1979) 249-270. 10 Sternberger, L. A., Immunocytochemistry, 2nd edn., John Wiley and Sons Inc., New York, 1979, pp. 104169.