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Functional significance of neurosecretory cells in the last abdominal ganglion of the lobster, Homarus vulgaris L
GENERAL
AND
COMPARATIVE
Functional
ENDOCRINOLOGk
Significance
Abdominal
Ganglion
B. SCHREINER,
ij,
(1969)
%@-‘t6~
of Neurosecretory of the Lobster,
H. STAALAND,
Zodogical
Laboratory,
dND
University
Received
May
Cells Homarws
A. SEMB oj
Oslo,
in t vu/g
JOIIANSSO~
Norway
16, 1969
Extirpation of the last (sixth) abdominal ganglion of the lobster (Nomar~s vulgaris L.) had no effects on the amount of chloride in the hemolymph, or on the amounts of ninhydrin-positive substances in muscles. About three months after the operation, the percentage of muscle tissue water was significantly higher in the experimental animals and remained so nine months later. It is suggested that neurosecretory cells in the last abdominal ganglion are the site of production of a hormone which is necessary for normal tissue growth after eedysis.
In addit’ion to several types of neurosecretory cells previously described from different crustaceans, the last abdominal ganglion of t’he lobster (No~~arus vulgaris L.) contains a specific type of neurosecretory cell which is very rarely observed in the other abdominal ganglia, and which we referred to as the “droplet cell” (Johansson and Schreiner, 1965). The secretory product of this cell type has specific tinctorial affinities, but it has not been possible on the basis of histological criteria to correlate the secretory a&ivity of these cells with the season of the year, or with the size or sex of the individual lobster. By extirpation of t#he last abdominal ganglion we hoped to obtain some information about the functional significance of the neurosecretory activity of the last abdominal ganglion. These reisults are reported here. MATERIAL
AND
winter season it, stayed at about 6-11°C whereas in the summer and early autumn it rose to about 1618°C. The lobsters were fed with crabs, prawns. clams, and herrings ad lib&m. Due to lack of space some individuals had to be kept together in large tanks,, but after seven months all individuals were kept separately m smaller tanks. When kept in the large ta.nks, the claws were tied, but despite this six individuals were lost, mostly due to cannibalism after moulting. When the animals were kept separately the claws were untied. The 44 surviving individuals were sacrificed at irregular interva!s, viz. 21, 42. 96, and 384 days after the operation. The fifth thoracic, the fifth and sixth (i.e., the last,) abdominal ganglia, and the samples of gonads were fixed in Bouin’s solution. The material was embedded in paraffin, sectioned at 7 p, and stained with the hematoxylin-eosin or aldehyde-fuchsin methods. Due to loss of sections, some series of sections of ganglia were incomplete and had to be discarded in the later analysis of neurosecretory activity. Pieces of abdominal muscle were carefully dried between two pieces of filter paper, the wet weight recorded, and thereafter the tissue was homogenized in distilled water for 16 minutes in a PotterElvehjem glass homogenizer and centrifuged” Protein-free extracts of the homogenates were obtained according to the method of Kalman and Lombrozo (1961)) and total free ninhydrm-positive substances (NPS) were determined according to the ninhydrin procedure of Moore and Stein (194%. Samples of hemolymph were centrifuged for 5
METHODS
The material used for this study consisted of 50 lobsters (25 8 8 and 25 0 p ) obtained from commercial sources in Bergen, Norway. The average body length was from 22-23 cm. The last abdominal ganglion was extirpated from 33 individuals by a ventral approach, whereas 17 individuals were sham-operated and served as controls. The operations were performed in May, and all individuals survived the operation. The animals were kept in running sea water. The temperature of the water was not controlled, but during the 399
400
SCHREINER,
STAALAND,
mm in a Beckman-Spinco microfuge at 15,000 rpm; to prevent clotting, the supernatant fluid was afterwards transferred to new tubes. Chloride was determined by titrating directly in the supernatant fluid according to the method of &hales and Schdes (1941). The water content of the abdominal muscle was determined as the difference in weight after the tissue had ,been carefully dried between two’ pieces of filter paper, and after drying at 105°C for about 24 hr. RESULTS
At the time of the operation, all of the 27 last (sixth) abdominal ganglia which were excised contained neurosecretory cells of the droplet type. Similarly, when the last abdominal ganglia of the controls were examined at later sacrifice, droplet cells were found in 15 out of a total of 16 individuals. In all cases this cell type was present in large numbers in the last ganglia. The droplet cells were nearly always in the droplet phase of the possible secretory cycle. As found previously (Johansson and Schreiner, 1965), this cell type was almost completely absent from the fifth thoracic ganglion and the fifth abdominal ganglion. It could not be identified in any of these ganglia in the control animals, and it was found in only three (18, 2 ? ? ) of the 28 experimental individuals. The droplet cell type was in these three cases represented by only one or two single cells. It is interesting to note that of the three individuals, two contained the droplet cell in both the thoracic and the fifth abdominal ganglia. The other nerve cells with a possible secretory activity all appeared normal in both the experimental and the control animals. No sex differences were observed in the neurosecretory activity. After recovery from the operation and resumption of feeding, no difference was observed in the behavior of experimental vs control animals. Growth, moulting, and sexual development were especially observed in those individuals which were kept for a period of more than one year. The experimental animals did not seem to differ from the control ones with regard to the rate of moulting, the fixation of the moulting season, or the in-
____ -. EXPE . --0 CONT YK , -: r -=Y;>. /’ 56 ;‘ci /’ --0’ /,0’~ AND
JOHANSSON
560
580
560
540
540
520
520
50011
I 50
20
POST-OPERATIVE
100 AGE
I 200
I 400
I 5oo
IN DAYS
FIG. 1. The amount of chloride in the hemolymph (MmCl-/I).
crease in body length at each moult. Nor did removal of the last abdominal ganglion prevent sexual maturation in males, or the occurrence of external ova in females. Dissection of the animals indicated that at the time of sacrifice the rectum of the experimental animals was somewhat dilated compared with that of the control individuals. Measurements of the rectal diameter of individuals examined at 96 days of postoperative age showed this difference to be highly significant, whereas the difference was not significant in the animals examined at 384 days post-operative age. The results of the determination of chloride in the hemolymph are given in Fig. 1, the NPS in muscles in Fig. 2, the percentage of muscle tissue water in Fig. 3. The removal of the last abdominal ganglion had no significant effect on the contents of chloride in the hemolymph, or on the contents of NPS in muscles. The fluctuation observed
360 -
I
-
l
---
0 CONTROL
EXPERIMENTAL
I 20
i
I 50
POST-OPERATIVE
I 100 AGE
i 200
-380
I 400
I
IN DAYS
FIG. 2. The amount of NPS in muscles (mMNP/S kg tissue water).
NEUROSECRETPON
IN
HO??‘LarU.S
401
probably an effect due to the disruption of the innervation of the rectal muscles in the operated animals and needs no more comment. Of more interest are the results which must be attributed to a possible endocrine activity of the last abdominai ganglion (Fig. 3). During ecdysis a transient hydration takes place in crustaceans, and the subsequent replacement of the absorbed water by I-d--I 20 i 50 100 200’ T&G proteins may be regarded as t’he true growth POST-OPERATIVE AGE IN DAYS in these animals (Lowndes and Panikkar, 194143; Passano, 1960). At 42 days postFIG. 3. The percentage of muscle tissue water operative age, no difference was found in the (g water/100 g wet tissue). Vertical lines indicate content of muscle Cssue water of the two standard error. groups of lobsters, whereas at 96 days postin these values in both the experimental and operative age the water content of the conthe control individuals during the experi- trol animals was significantly lower than mental period are probably due to changes that of the experimental animals. Almost in the salinity of the sea water (Florkin, all observed moults took place between 1961-62). these two dates. The possibi1it.y therefore In contrast to these negative findings, the exists that, the absence of the last abdomi-, removal of the last ganglion did result in a nal ganglion prevented the normal replacesignificant increase of the water content of ment of water by proteins which should the muscles of the animals (Fig. 3). Where- have taken place during this time interval. as no effects were obvious in the samples The fact that the situation was the same t.aken 21 and 42 days after the operation, more than nine months later (i.e., at 384 the water content had increased signifi- days post-operative age) indicates that the cantly at 96 days after the operation, and process of tissue growth was. probably it stayed at this level about a year later stopped, not only delayed. If these assumpwhen the last animals were sacrificed. tions are correct, the Endings may suggest that the last abdominal ganglion is the site DISCUSSION of production of a growt.h hormone which is As menConed above, neurosecretory cells necessary for normal tissue growth in i’le of the droplet type may occassionally be lobster. The fact that this ganglion ,is found in other ganglia of the ventral cord characterized by the presence of large of the lobster. Because of this, the presence numbers of a specific type of neurosecretory of one or two droplet cells in the fifth ab- cells suggeststhat these neurosecretory cells dominal ganglion of three of the experi- may be the source of this hormone. mental animals Ln the present study is no indication of a compensatory activity of We are indebted to Professor 6. Roll&en for the neurosecretory cells of this ganglion. Nor were any signs of a regeneration of the working facilities and permission to keep OUT ventral nerve cord observed. Thus, with the lobsters at the Institute of Marine Research, Directorate of Fisheries, Bergen, to candreal. limitations based on the fact that only the Gundersen and Mr. E. Bakken for all help in fifth thoracic and the fifth abdominal gang- taking care of the animals, to Mrs. I. Szetren for lia were examined for activity, the results doing the histological work, and to ca.nd.mag. obtained must be attributed to the loss of J. Fyhn for assistance in some of the chemical the last abdominal ganglion. determinations. This work was suppori;ed by a The dilatation of the rectum which was grant from the “Fridthjof Nansen’s Fund for the observed in the experimental animals is Advancement of Science and the Humanities.”
402
SCHREINER,
STAALAND,
REFERENCES M. (1961-62). Regulation anisomotique extracellulaire, regulation isomotique intracellulaire et euryhalinite. Ann. Xoc. Roy. 2001. Belg. 92, X3-186. JOHANSSON, A. S., AND SOHREINER, B. (1965). Neurosecreto’ry cells in the ventral ganglia of the lobster, Homarus vulgaris L. Gen. Comp. Endocrinol. 5, 55%567. KALMAN, S. M., AND LOMBROZO, M. E. (1961). The effect of estradiol on the free amino acids of the rat uterus. J. Pharmacol. Exptl. Therap. 131, FLORKIN,
265-269. LOWNDES,
A. G.,
AND
PANIKKAR,
N. K. (1941-43).
AND
JOHANSSON
9 note on the changes in water content of the lobster (Homarus vuEgaris M.-EDW.) during moult. J. Marin. Biol. Assoc. 25, 111-112. MOORE, S., AND STEIN, W. H. (1948). Photometric ninhydrin method for use in the chromatography of amino acids. J. Biol. Chem. 176, 367-388. PASSANO, L. M. (1966). Molting and its control. In “The Physiology of Crustacea” (T. H. Waterman> ed.) Vol. 1, pp. 473-536. Academic Press, New York. SCHALES, O., AND &HALES, S. (1941). A simple and accurate method for determination of chloride in biological fluids. J. Biol. Chem. 140, 879-884.
AND
COMPARATIVE
Functional
ENDOCRINOLOGk
Significance
Abdominal
Ganglion
B. SCHREINER,
ij,
(1969)
%@-‘t6~
of Neurosecretory of the Lobster,
H. STAALAND,
Zodogical
Laboratory,
dND
University
Received
May
Cells Homarws
A. SEMB oj
Oslo,
in t vu/g
JOIIANSSO~
Norway
16, 1969
Extirpation of the last (sixth) abdominal ganglion of the lobster (Nomar~s vulgaris L.) had no effects on the amount of chloride in the hemolymph, or on the amounts of ninhydrin-positive substances in muscles. About three months after the operation, the percentage of muscle tissue water was significantly higher in the experimental animals and remained so nine months later. It is suggested that neurosecretory cells in the last abdominal ganglion are the site of production of a hormone which is necessary for normal tissue growth after eedysis.
In addit’ion to several types of neurosecretory cells previously described from different crustaceans, the last abdominal ganglion of t’he lobster (No~~arus vulgaris L.) contains a specific type of neurosecretory cell which is very rarely observed in the other abdominal ganglia, and which we referred to as the “droplet cell” (Johansson and Schreiner, 1965). The secretory product of this cell type has specific tinctorial affinities, but it has not been possible on the basis of histological criteria to correlate the secretory a&ivity of these cells with the season of the year, or with the size or sex of the individual lobster. By extirpation of t#he last abdominal ganglion we hoped to obtain some information about the functional significance of the neurosecretory activity of the last abdominal ganglion. These reisults are reported here. MATERIAL
AND
winter season it, stayed at about 6-11°C whereas in the summer and early autumn it rose to about 1618°C. The lobsters were fed with crabs, prawns. clams, and herrings ad lib&m. Due to lack of space some individuals had to be kept together in large tanks,, but after seven months all individuals were kept separately m smaller tanks. When kept in the large ta.nks, the claws were tied, but despite this six individuals were lost, mostly due to cannibalism after moulting. When the animals were kept separately the claws were untied. The 44 surviving individuals were sacrificed at irregular interva!s, viz. 21, 42. 96, and 384 days after the operation. The fifth thoracic, the fifth and sixth (i.e., the last,) abdominal ganglia, and the samples of gonads were fixed in Bouin’s solution. The material was embedded in paraffin, sectioned at 7 p, and stained with the hematoxylin-eosin or aldehyde-fuchsin methods. Due to loss of sections, some series of sections of ganglia were incomplete and had to be discarded in the later analysis of neurosecretory activity. Pieces of abdominal muscle were carefully dried between two pieces of filter paper, the wet weight recorded, and thereafter the tissue was homogenized in distilled water for 16 minutes in a PotterElvehjem glass homogenizer and centrifuged” Protein-free extracts of the homogenates were obtained according to the method of Kalman and Lombrozo (1961)) and total free ninhydrm-positive substances (NPS) were determined according to the ninhydrin procedure of Moore and Stein (194%. Samples of hemolymph were centrifuged for 5
METHODS
The material used for this study consisted of 50 lobsters (25 8 8 and 25 0 p ) obtained from commercial sources in Bergen, Norway. The average body length was from 22-23 cm. The last abdominal ganglion was extirpated from 33 individuals by a ventral approach, whereas 17 individuals were sham-operated and served as controls. The operations were performed in May, and all individuals survived the operation. The animals were kept in running sea water. The temperature of the water was not controlled, but during the 399
400
SCHREINER,
STAALAND,
mm in a Beckman-Spinco microfuge at 15,000 rpm; to prevent clotting, the supernatant fluid was afterwards transferred to new tubes. Chloride was determined by titrating directly in the supernatant fluid according to the method of &hales and Schdes (1941). The water content of the abdominal muscle was determined as the difference in weight after the tissue had ,been carefully dried between two’ pieces of filter paper, and after drying at 105°C for about 24 hr. RESULTS
At the time of the operation, all of the 27 last (sixth) abdominal ganglia which were excised contained neurosecretory cells of the droplet type. Similarly, when the last abdominal ganglia of the controls were examined at later sacrifice, droplet cells were found in 15 out of a total of 16 individuals. In all cases this cell type was present in large numbers in the last ganglia. The droplet cells were nearly always in the droplet phase of the possible secretory cycle. As found previously (Johansson and Schreiner, 1965), this cell type was almost completely absent from the fifth thoracic ganglion and the fifth abdominal ganglion. It could not be identified in any of these ganglia in the control animals, and it was found in only three (18, 2 ? ? ) of the 28 experimental individuals. The droplet cell type was in these three cases represented by only one or two single cells. It is interesting to note that of the three individuals, two contained the droplet cell in both the thoracic and the fifth abdominal ganglia. The other nerve cells with a possible secretory activity all appeared normal in both the experimental and the control animals. No sex differences were observed in the neurosecretory activity. After recovery from the operation and resumption of feeding, no difference was observed in the behavior of experimental vs control animals. Growth, moulting, and sexual development were especially observed in those individuals which were kept for a period of more than one year. The experimental animals did not seem to differ from the control ones with regard to the rate of moulting, the fixation of the moulting season, or the in-
____ -. EXPE . --0 CONT YK , -: r -=Y;>. /’ 56 ;‘ci /’ --0’ /,0’~ AND
JOHANSSON
560
580
560
540
540
520
520
50011
I 50
20
POST-OPERATIVE
100 AGE
I 200
I 400
I 5oo
IN DAYS
FIG. 1. The amount of chloride in the hemolymph (MmCl-/I).
crease in body length at each moult. Nor did removal of the last abdominal ganglion prevent sexual maturation in males, or the occurrence of external ova in females. Dissection of the animals indicated that at the time of sacrifice the rectum of the experimental animals was somewhat dilated compared with that of the control individuals. Measurements of the rectal diameter of individuals examined at 96 days of postoperative age showed this difference to be highly significant, whereas the difference was not significant in the animals examined at 384 days post-operative age. The results of the determination of chloride in the hemolymph are given in Fig. 1, the NPS in muscles in Fig. 2, the percentage of muscle tissue water in Fig. 3. The removal of the last abdominal ganglion had no significant effect on the contents of chloride in the hemolymph, or on the contents of NPS in muscles. The fluctuation observed
360 -
I
-
l
---
0 CONTROL
EXPERIMENTAL
I 20
i
I 50
POST-OPERATIVE
I 100 AGE
i 200
-380
I 400
I
IN DAYS
FIG. 2. The amount of NPS in muscles (mMNP/S kg tissue water).
NEUROSECRETPON
IN
HO??‘LarU.S
401
probably an effect due to the disruption of the innervation of the rectal muscles in the operated animals and needs no more comment. Of more interest are the results which must be attributed to a possible endocrine activity of the last abdominai ganglion (Fig. 3). During ecdysis a transient hydration takes place in crustaceans, and the subsequent replacement of the absorbed water by I-d--I 20 i 50 100 200’ T&G proteins may be regarded as t’he true growth POST-OPERATIVE AGE IN DAYS in these animals (Lowndes and Panikkar, 194143; Passano, 1960). At 42 days postFIG. 3. The percentage of muscle tissue water operative age, no difference was found in the (g water/100 g wet tissue). Vertical lines indicate content of muscle Cssue water of the two standard error. groups of lobsters, whereas at 96 days postin these values in both the experimental and operative age the water content of the conthe control individuals during the experi- trol animals was significantly lower than mental period are probably due to changes that of the experimental animals. Almost in the salinity of the sea water (Florkin, all observed moults took place between 1961-62). these two dates. The possibi1it.y therefore In contrast to these negative findings, the exists that, the absence of the last abdomi-, removal of the last ganglion did result in a nal ganglion prevented the normal replacesignificant increase of the water content of ment of water by proteins which should the muscles of the animals (Fig. 3). Where- have taken place during this time interval. as no effects were obvious in the samples The fact that the situation was the same t.aken 21 and 42 days after the operation, more than nine months later (i.e., at 384 the water content had increased signifi- days post-operative age) indicates that the cantly at 96 days after the operation, and process of tissue growth was. probably it stayed at this level about a year later stopped, not only delayed. If these assumpwhen the last animals were sacrificed. tions are correct, the Endings may suggest that the last abdominal ganglion is the site DISCUSSION of production of a growt.h hormone which is As menConed above, neurosecretory cells necessary for normal tissue growth in i’le of the droplet type may occassionally be lobster. The fact that this ganglion ,is found in other ganglia of the ventral cord characterized by the presence of large of the lobster. Because of this, the presence numbers of a specific type of neurosecretory of one or two droplet cells in the fifth ab- cells suggeststhat these neurosecretory cells dominal ganglion of three of the experi- may be the source of this hormone. mental animals Ln the present study is no indication of a compensatory activity of We are indebted to Professor 6. Roll&en for the neurosecretory cells of this ganglion. Nor were any signs of a regeneration of the working facilities and permission to keep OUT ventral nerve cord observed. Thus, with the lobsters at the Institute of Marine Research, Directorate of Fisheries, Bergen, to candreal. limitations based on the fact that only the Gundersen and Mr. E. Bakken for all help in fifth thoracic and the fifth abdominal gang- taking care of the animals, to Mrs. I. Szetren for lia were examined for activity, the results doing the histological work, and to ca.nd.mag. obtained must be attributed to the loss of J. Fyhn for assistance in some of the chemical the last abdominal ganglion. determinations. This work was suppori;ed by a The dilatation of the rectum which was grant from the “Fridthjof Nansen’s Fund for the observed in the experimental animals is Advancement of Science and the Humanities.”
402
SCHREINER,
STAALAND,
REFERENCES M. (1961-62). Regulation anisomotique extracellulaire, regulation isomotique intracellulaire et euryhalinite. Ann. Xoc. Roy. 2001. Belg. 92, X3-186. JOHANSSON, A. S., AND SOHREINER, B. (1965). Neurosecreto’ry cells in the ventral ganglia of the lobster, Homarus vulgaris L. Gen. Comp. Endocrinol. 5, 55%567. KALMAN, S. M., AND LOMBROZO, M. E. (1961). The effect of estradiol on the free amino acids of the rat uterus. J. Pharmacol. Exptl. Therap. 131, FLORKIN,
265-269. LOWNDES,
A. G.,
AND
PANIKKAR,
N. K. (1941-43).
AND
JOHANSSON
9 note on the changes in water content of the lobster (Homarus vuEgaris M.-EDW.) during moult. J. Marin. Biol. Assoc. 25, 111-112. MOORE, S., AND STEIN, W. H. (1948). Photometric ninhydrin method for use in the chromatography of amino acids. J. Biol. Chem. 176, 367-388. PASSANO, L. M. (1966). Molting and its control. In “The Physiology of Crustacea” (T. H. Waterman> ed.) Vol. 1, pp. 473-536. Academic Press, New York. SCHALES, O., AND &HALES, S. (1941). A simple and accurate method for determination of chloride in biological fluids. J. Biol. Chem. 140, 879-884.