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Immunohistochemical localization of substance P and ACTH-like activity in the central nervous system of the earthworm Lumbricus terrestris L.
Acta histochem. 66, 262 - 268 (1980)
2fl(l Department of Anatomy, Histology and Embryology Semmelweis University Medical School Budapest, Hungary
Immunohistochemical localization of substance P and ACTH-like activity in the central nervous system of the earthworm Lumbricus terrestris L. By BELA AROS, TrBOR WENGER, BELA VIGH and INGEBORG VIGH-TEICHMANN With 3 figures (Received October 31, 1979)
Summary The peroxidase-antiperoxidase (PAP) method was used for the immunohistological demonstra· tion of substance P and ACTH in the cerebral and subesophageal ganglia of the earthworm, Lumbricus terrestris L. With rabbit antibody to substance P a positive immunoreaction was found in nerve cells smaller than the type A neurons of the cerebral ganglion. Their perikarya and nerve processes as well as their terminal enlargements in the synaptic zone were immunoreactive. ACTH-like activity was visible in a larger perikaryon type of the A neurons. Their nerve processes did not show any reaction. However, a part of the nerve terminals of the synaptic zone and a few neurons of type B of the cerebral ganglion, further some cells of the subesophageal ganglion reacted positively.
Introduction Neurosecretory cells were described in the centra.l nervous system of the earthworm by SCHARRER (1937) and studied by many authors (OTREMBA 1961; ARos and VIGH 1961; AROS et al. 1965; HERLANT-MEEWIS 1966; ZIMlllRMANN 1971; SCHOUMACKER and VAN DAMME 1971; a. 0.). In addition to the neurosecretory peptidergic type A cells monoamine producing neurons were also demonstrated (lit. see AROS et al. [1977]; DE VRIES-SCHOUMACKER [1977]). Histochemically, the GOMORI-positive type A neurons of the cerebral ganglion were found to contain a protein rich in sulphydryl (SH), disulfid (SS) and carboxyl (COOH) groups (TEICHMANN et al. 1966). The GOMoRI-positive substance of a part of the A cells (AI type) contains numerous free SH groups and a few SS groups. It is digestible with pepsin but not with trypsin. The neurosecretory material of the other A neurons (A2 type) is characterized by a rich content of disulfid groups and a lesser amollnt of sulphydryl groups, further by the presence of tyrosine and tryptophan. The A2 neurons do not stain with aldehyde fuchsin after pepsin and trypsin digestion, respectively (TEICHMANN et al. 1966). TORK et al. (1966) found that 35 8_ labelled cystcinc and methionine were incorporated into both the neurosecretory A neurons and the large, motoric GOMoRI-negative B cells as well as into the synaptic zones of thc cerebral and subesophageal ganglia.
Immunohistochemical localization of substance P
263
The introduction of immunocytochemical methods revealed new data about the chemical composition of materials elaborated in the neurons of the central nervous system of the earthworm. Recent works showed that immunoreactive neurophysin and vasopressin could not be detected in the aldehyde fuchsin-positive neurosecretory cells (REMY and DUBOIS 1979), but substances like pancreatic polypeptide or vasoactive intestinal peptide were present (SFNDLER et al. 1977). The opiate-like materials p-endorphin and leu-enkephalin were demonstrated in neurons of the cerebral ganglion (ALUMETS et al. 1979) while
Materials and methods The animals (Lumbricus terrestris L.) collected in January and February, were fixed in BOUINHOLLAND solution for 24 h. Following fixation, the tissue was dehydrated in graded alcohols, cleared in toluene and embedded in paraplast. Serial 7 pm sections of the 6 most anterior segments of the animal~ were mounted on glass slides. For immunocytochemical localization of ACTH and substance P the peroxidase-anti-peroxidase method was U>led (STERNBERGER et a!. 1970; STERNBERGER 1974). Rabbit antibodies to 1-32ACTHl) and to synthetic substance ]>2) were employed to detect respective tissue antigens. The final dilution of the primary antisera wa~; Anti-ACTH 1; 4,000, anti-substance PI; 3,000. The sections were incubated with the antiserum in a hydratdd chamber at 4 °C for 48 h. Peroxidase labelled sheep anti-rabbit-y-globulin (PAP from Dako Ltd., Danmark) was used as the secondary antiserum (dilution 1; 50, incubation time 10 and 30 min, respectively). Incubated sections were deveiope(l for peroxi(lase using 3,:~' -diaminobenzidine tetra-HCl (Sigma) and H 2 0 2. The specificity of the staining was controlled by incubating either with normal rabbit serum 01' with specific antiscrum absorbed with respective antigen. Further, simple peroxidase reaction was performed to dntect endogeneou~ enzyme aetivity. III a(ldition, the central nerVOUR ~ystem of the animals was investigated by routine histological mnthod~ as w<,ll as by special tecll1lique~ like rapid GOLGI impregnation, zinc-iodide-Os0 4 method (J ABOI'ERO 1964) and cllromn a.lum hematoxylin staining (BARGMA;\;N 1949).
Results With rab hit antibody to I-Jubstance P we got a strong reaction without considerable background in the cerebral ganglion. Here, only small immunoreactive cell bodies were seen lying in the cortical region of the ganglion (Fig. la). The perikarya were pear-shaped and mainly unipolar (Fig. 1 b, c). Also their nerve processes gave a fltrong reaction. We could follow these nerve processes to the neuropil, a synaptic I) The anti-ACTH was kindly provided by Dr. L. KARTESZI (MTA, KOKI, Hungary). 2) We are indebted t·o 17'
])1'.
B. KRRDET,HlfE
(CNR~,
France) for the gift of anti-substance P.
264
B. Auos et ul.
Fig. 1 a to c. Immunohistochemical localization of substance P in the cerebral ganglion. X 120; b) The processes of th£> neurOll~ (arrow8) run to the synaptic zone (8) of the ganglion. X 280; c) The strongly reacting pcrikarya are smaller than A-type cells (A). In the synaptic zone (8) varicose thickenings of the nerve fibers_ X 280.
a) Small ne uronal perikarya (P) in the dorsal superficial part of the ganglion.
Immunohistochemical localization of substance P
,
265
a
Fig. 2a to c. ~ e urons of the corebral ganglion demonstrated with impregnation techniques. a) Dark A ·type neurons with zinc-iodide-osmium method. Arrow8 the axons of the cells, B type B neurons, 8 syraptic zone. X 450; b) Rapid GOLGI method dcm on strates some large neuronal perikarya (P) and the synaptic zone (8) of the ganglion . X 120; c) Small perikarya (P) and t he ir processes running to the synaptic zonc (8). x 120.
zone, situated below the cellular layer (Fig. la, b, c). There were periodic thickenings and termjnal enlargements on these fibers on the level of the synaptic zone. Some of the small neurons exhibited only a medium dense reaction. All immunoreactive neuronal perikarya were similar to the G01VIORI-positive type A cells demonstrable by chrome alum hematoxylin staining after previous oxidation with potassium permanganate sulfuric acid . They also resembled those A cells seen in zinc-iodide-Os0 4 preparations and in slides stained with the rapid GOLGI method (Fig. 2a, h). However, it has to be stressed that the immunoreactive cells appeared to be somewhat smaller in size than the A neurons. Similar small neurons were de-
266
B.
AIWS
et al.
Fig. 3a to d. ACTH-l ikc activit,y ill t he ee t'ob m l an rl s ubesop hagea l ga ng lia . a) View of react ing p erik a rya (arrow8 ) in the p eripltc ral, d orsal p art of t he cerebra l ganglion. X 120; b) Par t of t h e cerebral gan gli on . Most of the p ositive cell s a re A -typ e perik a ry a (A) . X 280 ; c) Positiv e type IJ cells ( cm 'ow ) be low th e layer of type A p ori ka rya. x 2 80; d) A fow p ositiyc neurons ( arJ'ows) in the subcf;ophagpal gang lion. X 280.
tected in rapid GOLCn impregnations as well (Fig. 2c ). Th e perikMya of the large B neurons did not react with t he a ntibody to substance P. Tn the subesophageal ganglion , immllnoreactive cells coulrl not he demonstrated. The ne uropil appeared to he negative. "L'sing ra bbit antibo(lies to 1- 32ACTH we observed a posit iv e illllllunoreaction fin, t of all in type A cell bodies. The~e illlllllmoreact ive perika r ya were of larger size thi1n those which reacted wit.h substance P antibodies (Fig . ~ a h, b). A part of the type A neurons were negative. It is noteworth that the nerve processes seldom react ed with ACTH antibodies. Most of the type B neurons were also negative, hut
Immunohistochemical localization of substance P
267
some of them showed medium or strong activity (Fig. 3a, b, c). There were some immunoreactive neurons in the sub esophageal ganglion as well. These perikarya lay ventrolaterally in the rostral part of the ganglion (Fig. 3c). In the control sections stained either after incubation with normal rabbit serum or with primary antiserum saturated with respective antigen, we failed to demonstrate any specific immunoreactive sites in the ganglia investigated. Further, no endogeneous peroxidase activity was visible in the nervous tissue.
Discussion SUNDER ot al. (1977) were the fir~t authors to report on neurons containing immunoreactive materials - a pancreatic polypeptide or a vasoactive intestinal peptide - in the earthworm. Recently, RiMY and DUBOIS (1979) found positive immunoreaction when using a-endorphin antiserum in ct'rcbral (~ supraesophageal) and subesophageal ganglia of Dendrobaena 8ubrubicunda (Lumbricidae). a negative result was obtained with f3-endorphin, neurophysin and vasopressin antisera, further with anti-a· and anti-f3.MSH. The a-endorphin-like material was detected in 4 neurons of the subesophageal ganglion. 2 of the cells were conical in shape and located in the rostral region, the other pair of pear.shaped perikarya lay close to the origin of the last nerve of the ganglion. More recently, _-1.LF)IETS et al. (1979) reported on the presence of leu.enkephalin and f3-endorphin-like materiah in Lumbricus ternstris. In the cerebral ganglion many perikarya of different sizes and numerous nerve fibers were found to display intense leu-enkephalin immunoreaction. These positive fibers extended into the circumpharyngeal connectives, too. Only a few immunoreactive cell boclie8 and fibers wnre observed in the subesophageal and in the abdominal ganglia. The f3-endorphin reactive cells seemed to be larger in size and much fewer in number, but tlleir distribution was fonnrl to be similar to that of the enkephalin reactive cells. The results of our present immunocytochemical work indicate that the polypeptides substance P and ACTH, or closely related peptides, exist in neurons of thc cerebral and subesophageal ganglia of the earthworm. ACTH-immunoreactive material was found in a part of both the Go:>IORI-positivo A and tho GO~IOIU-negative B type neurons of the cerebral ganglion and in some cells of the subesophageal ganglion. Thc significance of the presence of the ACTH-like activity in the perikarya is unknown. Substance P seems to be located in a small typc of neuronal cell bodies being some\rhat smaller than the G03IORI-positive A type cells of the cerebral ganglion. This and the demonstration of Himilarly shaped, small cells in GOLGI impregnations give some ground to speculate that the substance P neurons may represent a separate cell type. Nerve processes and terminals in the synaptic zone also gave a positive immunoreaction with substance P antibodies. Tnt he synaptic zone, we described various types of synapses, some of them werc snppos('d to br' peptidergic in naturc (Anos et a1. 1977). Presumably, a part of these peptidergic synapsAs belongs to the fibers which react with anti-substance P. This suggests that substance P may playa role in peptidergic synaptic transmission in invertebrate nervous tissue. Further electron microscopic immunocytochemical studies are needed to identify the immunoreactive neurons, and their terminals in the synaptic zone and to distinguish them from other types doscribed earlier.
Literature ALUMETS, J., HAKANSON, R., SUNDLER, F., and THORELL, J., Neuronal localization of immunoreactive enkephalin and tJ-endorphin in the earthworm. Nature 279, 805-806 (1979). AROS, B., and VIGE, B .. Xeul'Osecretory act.ivity of the central and peripheral nervous system in the earthworm. Acta bioI. Acad. Sci. hung. 12, 169-186 (1961).
268
B. AROS et aI., Immunohistochemical localization of substance P
AROS, B., VIGH, B., and TEICHMANx. 1., The structure of the neurosecretory system of the earthworm. In: LENHoHsEK Memory Symp. BioI. Acad. Sci. hung. 5, 3()3-316 (1965). - and VIGH-TEICHMANN, 1., Intra- and extraganglionic nerve endings formed by neurosecretory cells of tho cerebral ganglion of the earthworm (Lumbricus terrestris L.). Cell Tiss. Res. 180, 537 - 553 (1977). BAHGilIANN, W., tber ,\io J1curosekretorisehe Verknupfung von Hypothalamus un,l Neurohypophyse. Z. ZeIIforseh. 34,610-6:34 (1949). DE VRIES-SCHOU!\IACKER, H., Fluoroseence awl ultrastructural localization of aminergic neurons in the nerve cord of Eiswiafoetida (Annelida-Oligochaeta). CeIl Tiss. Res. 185, 351- 360 (1977). HERLANT-MEEW1S, R., Les cellule>; neurosecrotriccs de la chaine nervouso d'Eisenia foetida. Z. ZellfoI'sch. 69, :1l9-325 (1966). JABOlSERO, V., Ubor dio Brauchbarkeit dcr Osmiumtetroxyrl-Zinkjodid-Methode zur Analyse del' vegotativen Periphcrie. Acta neuroveg. (Wien) 26, 184-210 (1964). OTREIIlBA, P., Beobachtungen an ncurosekretorischen Zellen des Regenwurmes (Lumbricus spec.). Z. ZeIIforsch. 54,421- 436 (1961). REMY, CH., awl DUBOIS, M. P., Localisation par immunofluorescence de peptides analogues it I'exendorphine dans les ganglion infra-oesophagiens tlu lombricide Dendrobaena subrubicunda EISEN. Experientia 35, 137 -1:38 (1979). SCHARRER, B., Ubor sekretorisoh tiitige Nervenzellen bei wirbellosen Tieren. Naturwissenschaften 25,131-138 (1937). SCHOUMACKER, H., an,l VAN DAMME, N., MAILLET's Os04-ZnI2 fixative and alcian blue staining in the study of neurosecretion; invertebrates. Stain Techno!. 46, 2:33-237 (1971). STEUNBERGER, L. A., Immunocytochemistry. Englewood Cliffs: Prentice Hall 1974. HAHDY, P. H., .Jr., CUC[TLIS, J. J., and MEYER, H. G., The unlabeled antibody enzymo method of immunohistochemistry: preparation an,l properties of soluble antigen-antibody complex (horseradish peroxidase-antiperoxidase) and its use in i,lentification of Hpirochetes. J. Ristochern. Cytochem. 18,315-:333 (1970). SUNDLER, F., HAKANSON, H., ALUMETR, J., and WALLES, B., Neuronal localizat,ion of pancreatic polypeptide (PP) and vasoaetive intestinal peptide (VIP) immunoreactivity in the earthworm (Lumbricus terrestri8). Brain Ro,.;. Bull. 2, 61-65 (1977). TEICHMANN, 1., AHOS, B., and VWH, B., Histochemical studies on GOMORI-positive substances. III. Examination of tho earthworm's neuroseoretory system (Lumbricu8 herculeu8, Eisenirt foetida). Acta bioI. Acad. Sci. hung. 17, :129-357 (1966). TORK, 1., Auos, B., KISS, .1., and VIGH, B., Morphological examination of the metabolism in the secretory cells of the nervous system. J. Autol'adiographic stucly of tho ineorporation of S35 labelled cYHteine and methionino into the neurosecretory system of the earthworm (Eisenirt foetirla, J.dtmbricu8 herculeu8). Acta bioI. Acad. Sci. hung. 17,185-198 (1966). ZIMMERMANN, P., Die zentralnervDse Kontroll" tI"r Dehydration bei Lumbricu8 ten'estris L. Z. Zellforsch. 112,551-571 (1971). Address of the authors: Prof. Dr. BELA AROS, Dr. TIBOR WENGER, Dozenten Dr. BELA VIGH and Dr. INGEBORG VIGH-TEICHMANN, 2nd Department of Anatomy Histology, and Embryology, SOTE, TuzoltO utca 58, R - 1094 Budapest.
2fl(l Department of Anatomy, Histology and Embryology Semmelweis University Medical School Budapest, Hungary
Immunohistochemical localization of substance P and ACTH-like activity in the central nervous system of the earthworm Lumbricus terrestris L. By BELA AROS, TrBOR WENGER, BELA VIGH and INGEBORG VIGH-TEICHMANN With 3 figures (Received October 31, 1979)
Summary The peroxidase-antiperoxidase (PAP) method was used for the immunohistological demonstra· tion of substance P and ACTH in the cerebral and subesophageal ganglia of the earthworm, Lumbricus terrestris L. With rabbit antibody to substance P a positive immunoreaction was found in nerve cells smaller than the type A neurons of the cerebral ganglion. Their perikarya and nerve processes as well as their terminal enlargements in the synaptic zone were immunoreactive. ACTH-like activity was visible in a larger perikaryon type of the A neurons. Their nerve processes did not show any reaction. However, a part of the nerve terminals of the synaptic zone and a few neurons of type B of the cerebral ganglion, further some cells of the subesophageal ganglion reacted positively.
Introduction Neurosecretory cells were described in the centra.l nervous system of the earthworm by SCHARRER (1937) and studied by many authors (OTREMBA 1961; ARos and VIGH 1961; AROS et al. 1965; HERLANT-MEEWIS 1966; ZIMlllRMANN 1971; SCHOUMACKER and VAN DAMME 1971; a. 0.). In addition to the neurosecretory peptidergic type A cells monoamine producing neurons were also demonstrated (lit. see AROS et al. [1977]; DE VRIES-SCHOUMACKER [1977]). Histochemically, the GOMORI-positive type A neurons of the cerebral ganglion were found to contain a protein rich in sulphydryl (SH), disulfid (SS) and carboxyl (COOH) groups (TEICHMANN et al. 1966). The GOMoRI-positive substance of a part of the A cells (AI type) contains numerous free SH groups and a few SS groups. It is digestible with pepsin but not with trypsin. The neurosecretory material of the other A neurons (A2 type) is characterized by a rich content of disulfid groups and a lesser amollnt of sulphydryl groups, further by the presence of tyrosine and tryptophan. The A2 neurons do not stain with aldehyde fuchsin after pepsin and trypsin digestion, respectively (TEICHMANN et al. 1966). TORK et al. (1966) found that 35 8_ labelled cystcinc and methionine were incorporated into both the neurosecretory A neurons and the large, motoric GOMoRI-negative B cells as well as into the synaptic zones of thc cerebral and subesophageal ganglia.
Immunohistochemical localization of substance P
263
The introduction of immunocytochemical methods revealed new data about the chemical composition of materials elaborated in the neurons of the central nervous system of the earthworm. Recent works showed that immunoreactive neurophysin and vasopressin could not be detected in the aldehyde fuchsin-positive neurosecretory cells (REMY and DUBOIS 1979), but substances like pancreatic polypeptide or vasoactive intestinal peptide were present (SFNDLER et al. 1977). The opiate-like materials p-endorphin and leu-enkephalin were demonstrated in neurons of the cerebral ganglion (ALUMETS et al. 1979) while
Materials and methods The animals (Lumbricus terrestris L.) collected in January and February, were fixed in BOUINHOLLAND solution for 24 h. Following fixation, the tissue was dehydrated in graded alcohols, cleared in toluene and embedded in paraplast. Serial 7 pm sections of the 6 most anterior segments of the animal~ were mounted on glass slides. For immunocytochemical localization of ACTH and substance P the peroxidase-anti-peroxidase method was U>led (STERNBERGER et a!. 1970; STERNBERGER 1974). Rabbit antibodies to 1-32ACTHl) and to synthetic substance ]>2) were employed to detect respective tissue antigens. The final dilution of the primary antisera wa~; Anti-ACTH 1; 4,000, anti-substance PI; 3,000. The sections were incubated with the antiserum in a hydratdd chamber at 4 °C for 48 h. Peroxidase labelled sheep anti-rabbit-y-globulin (PAP from Dako Ltd., Danmark) was used as the secondary antiserum (dilution 1; 50, incubation time 10 and 30 min, respectively). Incubated sections were deveiope(l for peroxi(lase using 3,:~' -diaminobenzidine tetra-HCl (Sigma) and H 2 0 2. The specificity of the staining was controlled by incubating either with normal rabbit serum 01' with specific antiscrum absorbed with respective antigen. Further, simple peroxidase reaction was performed to dntect endogeneou~ enzyme aetivity. III a(ldition, the central nerVOUR ~ystem of the animals was investigated by routine histological mnthod~ as w<,ll as by special tecll1lique~ like rapid GOLGI impregnation, zinc-iodide-Os0 4 method (J ABOI'ERO 1964) and cllromn a.lum hematoxylin staining (BARGMA;\;N 1949).
Results With rab hit antibody to I-Jubstance P we got a strong reaction without considerable background in the cerebral ganglion. Here, only small immunoreactive cell bodies were seen lying in the cortical region of the ganglion (Fig. la). The perikarya were pear-shaped and mainly unipolar (Fig. 1 b, c). Also their nerve processes gave a fltrong reaction. We could follow these nerve processes to the neuropil, a synaptic I) The anti-ACTH was kindly provided by Dr. L. KARTESZI (MTA, KOKI, Hungary). 2) We are indebted t·o 17'
])1'.
B. KRRDET,HlfE
(CNR~,
France) for the gift of anti-substance P.
264
B. Auos et ul.
Fig. 1 a to c. Immunohistochemical localization of substance P in the cerebral ganglion. X 120; b) The processes of th£> neurOll~ (arrow8) run to the synaptic zone (8) of the ganglion. X 280; c) The strongly reacting pcrikarya are smaller than A-type cells (A). In the synaptic zone (8) varicose thickenings of the nerve fibers_ X 280.
a) Small ne uronal perikarya (P) in the dorsal superficial part of the ganglion.
Immunohistochemical localization of substance P
,
265
a
Fig. 2a to c. ~ e urons of the corebral ganglion demonstrated with impregnation techniques. a) Dark A ·type neurons with zinc-iodide-osmium method. Arrow8 the axons of the cells, B type B neurons, 8 syraptic zone. X 450; b) Rapid GOLGI method dcm on strates some large neuronal perikarya (P) and the synaptic zone (8) of the ganglion . X 120; c) Small perikarya (P) and t he ir processes running to the synaptic zonc (8). x 120.
zone, situated below the cellular layer (Fig. la, b, c). There were periodic thickenings and termjnal enlargements on these fibers on the level of the synaptic zone. Some of the small neurons exhibited only a medium dense reaction. All immunoreactive neuronal perikarya were similar to the G01VIORI-positive type A cells demonstrable by chrome alum hematoxylin staining after previous oxidation with potassium permanganate sulfuric acid . They also resembled those A cells seen in zinc-iodide-Os0 4 preparations and in slides stained with the rapid GOLGI method (Fig. 2a, h). However, it has to be stressed that the immunoreactive cells appeared to be somewhat smaller in size than the A neurons. Similar small neurons were de-
266
B.
AIWS
et al.
Fig. 3a to d. ACTH-l ikc activit,y ill t he ee t'ob m l an rl s ubesop hagea l ga ng lia . a) View of react ing p erik a rya (arrow8 ) in the p eripltc ral, d orsal p art of t he cerebra l ganglion. X 120; b) Par t of t h e cerebral gan gli on . Most of the p ositive cell s a re A -typ e perik a ry a (A) . X 280 ; c) Positiv e type IJ cells ( cm 'ow ) be low th e layer of type A p ori ka rya. x 2 80; d) A fow p ositiyc neurons ( arJ'ows) in the subcf;ophagpal gang lion. X 280.
tected in rapid GOLCn impregnations as well (Fig. 2c ). Th e perikMya of the large B neurons did not react with t he a ntibody to substance P. Tn the subesophageal ganglion , immllnoreactive cells coulrl not he demonstrated. The ne uropil appeared to he negative. "L'sing ra bbit antibo(lies to 1- 32ACTH we observed a posit iv e illllllunoreaction fin, t of all in type A cell bodies. The~e illlllllmoreact ive perika r ya were of larger size thi1n those which reacted wit.h substance P antibodies (Fig . ~ a h, b). A part of the type A neurons were negative. It is noteworth that the nerve processes seldom react ed with ACTH antibodies. Most of the type B neurons were also negative, hut
Immunohistochemical localization of substance P
267
some of them showed medium or strong activity (Fig. 3a, b, c). There were some immunoreactive neurons in the sub esophageal ganglion as well. These perikarya lay ventrolaterally in the rostral part of the ganglion (Fig. 3c). In the control sections stained either after incubation with normal rabbit serum or with primary antiserum saturated with respective antigen, we failed to demonstrate any specific immunoreactive sites in the ganglia investigated. Further, no endogeneous peroxidase activity was visible in the nervous tissue.
Discussion SUNDER ot al. (1977) were the fir~t authors to report on neurons containing immunoreactive materials - a pancreatic polypeptide or a vasoactive intestinal peptide - in the earthworm. Recently, RiMY and DUBOIS (1979) found positive immunoreaction when using a-endorphin antiserum in ct'rcbral (~ supraesophageal) and subesophageal ganglia of Dendrobaena 8ubrubicunda (Lumbricidae). a negative result was obtained with f3-endorphin, neurophysin and vasopressin antisera, further with anti-a· and anti-f3.MSH. The a-endorphin-like material was detected in 4 neurons of the subesophageal ganglion. 2 of the cells were conical in shape and located in the rostral region, the other pair of pear.shaped perikarya lay close to the origin of the last nerve of the ganglion. More recently, _-1.LF)IETS et al. (1979) reported on the presence of leu.enkephalin and f3-endorphin-like materiah in Lumbricus ternstris. In the cerebral ganglion many perikarya of different sizes and numerous nerve fibers were found to display intense leu-enkephalin immunoreaction. These positive fibers extended into the circumpharyngeal connectives, too. Only a few immunoreactive cell boclie8 and fibers wnre observed in the subesophageal and in the abdominal ganglia. The f3-endorphin reactive cells seemed to be larger in size and much fewer in number, but tlleir distribution was fonnrl to be similar to that of the enkephalin reactive cells. The results of our present immunocytochemical work indicate that the polypeptides substance P and ACTH, or closely related peptides, exist in neurons of thc cerebral and subesophageal ganglia of the earthworm. ACTH-immunoreactive material was found in a part of both the Go:>IORI-positivo A and tho GO~IOIU-negative B type neurons of the cerebral ganglion and in some cells of the subesophageal ganglion. Thc significance of the presence of the ACTH-like activity in the perikarya is unknown. Substance P seems to be located in a small typc of neuronal cell bodies being some\rhat smaller than the G03IORI-positive A type cells of the cerebral ganglion. This and the demonstration of Himilarly shaped, small cells in GOLGI impregnations give some ground to speculate that the substance P neurons may represent a separate cell type. Nerve processes and terminals in the synaptic zone also gave a positive immunoreaction with substance P antibodies. Tnt he synaptic zone, we described various types of synapses, some of them werc snppos('d to br' peptidergic in naturc (Anos et a1. 1977). Presumably, a part of these peptidergic synapsAs belongs to the fibers which react with anti-substance P. This suggests that substance P may playa role in peptidergic synaptic transmission in invertebrate nervous tissue. Further electron microscopic immunocytochemical studies are needed to identify the immunoreactive neurons, and their terminals in the synaptic zone and to distinguish them from other types doscribed earlier.
Literature ALUMETS, J., HAKANSON, R., SUNDLER, F., and THORELL, J., Neuronal localization of immunoreactive enkephalin and tJ-endorphin in the earthworm. Nature 279, 805-806 (1979). AROS, B., and VIGE, B .. Xeul'Osecretory act.ivity of the central and peripheral nervous system in the earthworm. Acta bioI. Acad. Sci. hung. 12, 169-186 (1961).
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B. AROS et aI., Immunohistochemical localization of substance P
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