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Ultrastructural changes in the crayfish sinus gland following electrical stimulation
QENERAL
AND
COMPARATIVE
ENDOCRINOLOGY
Ultrastructural
10,
Changes following
ANN Department
376382
in the
Electrical
HEFFINGTON of Anatomy.
(1968)
The
Received
Sinus
Gland
Stimulation’
BUNT
University at Dallas,
Crayfish
AND
EBERT
of Texas Soufhujestern Texas 75B5
October
A. ASHBY Medical
School
14, 1967
Ultrastructural examination of the sinus glands of the crayfish, Procambarus clarkii, following in viva and in vitro electrical stimulation reveals significantly fewer granules within the neurosecretory axon terminals than in similarly treated but unstimulated controls. Granule depletion appears to be nonspecific with respect to the different terminal types. Most effective depletion of granules is achieved with stimuli of 0.b2.O/second, while higher frequencies are progressively less effective. Further evidence concerning the mechanism of granule release is also presented.
The crustacean sinus gland, located adjacent, to the eyestalk ganglia, contains numerous neurosecretory (NS) axon terminals derived from cell bodies lying more proximally within the eyestalk X-organ, the brain, and possibly the thoracic ganglion (Bliss et al., 1954). A multiplicity of different hormones are contained within the gland, as evidenced by physiological and biochemical (Kleinholz, 1966)) histological (Potter, 1958)) and ultrastructural studies (Bunt and Ashby, 1967). It is generally accepted that the hormones present in the axon terminals are contained within membrane-enclosed NS granules and are released by appropriate firing of the axons (Bern and Yagi, 1964). In resemblance to ordinary neurons, some NS cells have been found to possess the ability to conduct electrical impulses. Speidel (1919)) who first, described the glandular appearance of the elasmobranch Dahlgren cells, noted that electrical or pilocarpine stimulation of the spinal cord resulted in an increase in the number of stainable cytoplasmic granules. In contrast,
electrically induced depletion of stainable NS material has been reported in the corpus cardiacum of the cockroach, Blaberus craniifer (Hodgson and Geldiay, 1959) and the locust, Schistoceru gregatia (Highnam, 1961), in the central nervous system of the oyster, Crassostrea virginica (Nagabhushanam, 1964), and in the urohypophysis of the teleost, Tilapia mossambica (Fridberg et al., 1966). Ultrastructural studies on the pericardial organ of the isopod, Squilla (Knowles, 1963)) and the teleost urohypophysis (Fridberg et al., 1966) lend further support to physiological and histological evidence of hormone release from NS axons following electrical stimulation. In view of these reports, this study was undertaken to determine morphological changes at the ultrastructural level which might result from in vivo and in vitro electrical stimulation of the sinus gland of the crayfish, Procambarus clarkii. In addition, for the interpretation of compound action potentials recorded from the axon trunks, it was of further interest to determine the stimulus frequency associated with maximum granule depletion.
‘This work was supported in part by N.I.H. grant 06623-02 from N.I.N.D.B. 376
SINUS
MATERIALS
AND
GLAND
ELECTRICAL
copy as described previously (Bunt and Ashby, 1967). In Vitro Experiments: The right and left sinus glands with adhering trunks were first dissected free in saline and then drawn up into separate glass-pipette electrodes along with small droplets of saline. The stimulus was applied to the right gland while the Ieft gland served &9 an unstimulated control. The method was adapted from the technique of Easton (1964) for recording from garpike olfactory nerve (cf. Table 1 for variables). Both sinus glands were then processed and examined by electron microscopy. Data from counts of NS axon terminals were analvzed according to the z-test for the significance of difference between proportions (Duncan, 1952).
METHODS
Male and female P. chrkii (l&cm carapace length) were maintained in the laboratory in pond water and fed commercial rabbit food. In Vivo Experiments: Each crayfish was secured on a Plexiglas animal carrier with rubber bands. Both eyestalks were immobilized with dental wax and the exoskeleton of each was slit on either side, along the dorsal proximal margin of the retina and across the base of the eyestalk. The loosened exoskeleton and underlying hypodermis TABLE VARIABLES
FOR in
F’dro
1 EXPERIMENT
No. of animals
Pulse (msrc)
Frequency (per second)
2 2
6.0
0.5 0.5 2.0 40
3 3 3
80
3 3
4
2 2 2
5.0 5.0 5.0 5.0 5.0
Dmation (minutes)
vY+Y
5
RESULTS
10
In Viva Electrical Stimulation. The electrically stimulated sinus glands differ from the control glands in their content of numerous scattered axon terminals which are either empty or greatly depleted of NS
10
3
100
10 10 10
granules. No preferential depIetion of terminal types was observed following electrical stimulation; consequently, data from all granule types were pooled for statistical treatme& The percentages of such depleted terminals are presented in Table 2. In some of the emptied terminals (Fig. l), a few typical NS granules are surrounded by numerous electron-lucent vesicles. In Fig. 1, an extruded granule core lies between the axolemma and the connective tissue sheath. In other terminals (Fig. 2), NS granules
were lifted away to reveal the sinus glands. Both glands were kept moistened with physiological saline (Van Harreveld, 1936) during the period of exposure. Fine silver electrodes were placed beneath both sinus glands on the medial side of the optic peduncle, at the junction of the optic lamina. The stimulus was then applied to the right sinus gland. The following variables were employed: (1) two animals, pulse 0.3 msec, frequency 30/second, voltage 1.5V, and duration 60 minutes; (2) two animals, pulse 3.0 msec, voltage 5.OV, and duration 20 minutes. Both the right and left sinus glands were then dissected out and prepared for electron microsTABLE DEPLETION
I n viva:
In vitro:
OF
NS AXON
TERMINALS
Duration (minutes)
Frequency (per second)
20
Control
30
30
60 60
Control
10
Control
5
30
10
0.5 0.5 2.0 40
10
80
10 10
10
377
STIMULATIOh‘
100
2
FOLLOWING
ELECTRICAL
‘% Depleted
terminals
11.2% (n (n 11.9% (n (n 27.5% 10.2% (n 53.3% (n 48.4% (n
= = = = = = =
21.3%
52.1% 34.1% 20.4yo 18.2%
z
P
71) 369) 126)
1.96
<0.05
620)
3.69
4.19
<0.0006 <0.0006 <0.0006 <0.0006 <0.02 <0.06
137) 92)
116) (n = 123)
(n = 129) (n = 147) (n =
STIMULATION
132)
6.37 5.61 4.72 2.38 1.89
(20 minutes). The terminal FI G. 1. In viva stimulation It vesicles. An ebuded NS granule core lies within ieci,ive tissue sheath. X25,000.
contains a pocket
fes- KS granules plus uumerous elect1 ‘onof the plasma membrane, adjacent t’o the
SINUS
GLAND
ELECTRICAL
with cores of low electron density and rather crenated limiting membranes lie among the electron-lucent vesicles and scattered membranous profiles. This configuration is observed in the preterminal portions of the axons as well as in the terminals adjoining the blood sinus (Fig. 3). In Vitro Electrical Stimulutio~n. All electrically stimulated glands differ from the control glands in their increased percentages of depleted terminals, as presented in Table 2. From these data, it can be seen that the greatest depletion of granules is observed at. lower frequencies of stimulation, with less depletion at the higher. The ultrastructure of the terminals themselves closely!. resembles that observed in the in viva experiments, in that scattered terminals a.re well filled with granules, while others exhibit few NS granules plus numerous electron-lucent vesicles. Some of these vesicles (Fig. 4) are approximately the same diameters as the NS granules, while others (Figs. 5 and 6) resemble the smaller so-called “synaptic vesicles” which are usually grouped just beneath the plasma membrane bordering the blood sinus. Thickenings of the plasma membrane adjacent to groups of these vesicles are observed in some of t’he actively secreting terminals (Fig. 6). Many of the NS granules (Figs. 6 and 7) exhibit a clear halo between the granule core and limiting membrane, an appearance observed less frequently in the in vivo experimental and the in vitro control sinus glands. Frequently these haloed KS granules seem to be associated with t,he presence of dense granule cores between the axolemma and connective tissue sheath (Fig. 7).
Occasional terminals contain a few NS granules of low electron density surrounded by seemingly broken membranes, as noted 2. In rice st,imulation (60 minutes). The scattered vesicles and membranous profiles.
previously in the in vivo experiments very rarely in control sinus glands.
and
DISCUSSI0-h
The increased depletion of NS granules within sinus gland terminals following electrical stimulation supports reports that NS cells possess the ability to conduct electrical impulses and that these impulses cause release of the hormone secretory products both in vivo (Harris, 1947, 1960; Cross and Harris, 1952; Bissett et aE., 1963; Hayward and Smith, 1964)) and also in vitro (Cooke, 1964; Douglas and Poisner, 1964; Haller et al., 1965; Sachs et al., 1967). The ultrastructural changes after the in z&o and in vitro electrical stimulation of the sinus gland axons closely resemble those report,ed in the teleost urohypophysis (Fridberg et al., 1966) and isopod pericardial organ (Knowles, 1963)) following similar electrical stimulation in viva. In agreement with both studies, here the decrease in numbers and electron density of the NS granules is accompanied by the appearance of both large and small electron lucent vesicles within the axon terminals. In contrast to the report by Knowles (1963)) however, large vesicles surrounding smaller vesicles have been observed only rarely. The finding of dense NS granule cores between the axon plasma membrane and blood sinus basement membrane in actively secreting terminals lends further support to the hypothesis (Bunt and Ashby, 1967) that the mechanism of secretion in the crayfish sinus gland closely resembles that reported in the corpus cardiacum of insects (Smith and Smith, 1964; Normann, 1965) and in the rat, anterior pituitary (Lever and Peterson, 1960; Farquhar, 1961) and exocrine pancreas (Palade, 1959). The increased numbers of “synaptic vesicles” in actively secreting terminals are evidently
contains only four moderately dense NS granules, of small vesicles borders a thickened region of the plasma membrane beneath the connective t,issuesheath. X25,000. FIG. 3. In viuo stimulation (20 minutes). A preterminal axon, surrounded by processes of a supporting cell, contains no NS granules of high electron density. Smudgy granules and vesicles of low electron density are grouped around the periphery and in the center of the axon. X25,000. FIG.
plus
terminal A group
379
STIMULATIOK
FIG. 4. In vitro vesicles,
x25,000.
whose
stimulation (J/second frequency). moderately dense somewhat flocculant
h few typical SS cont,ents surround
granules denser,
lie among eccentrically
numerous placed
large cores.
SINUS
GLAKD
ELECTRICAL
related to the release mechanism, but the significance of these vesicles and the thickenings of the adjacent axolemma, which closely resemble those reported in NS axons of the corpus cardiacum of Calliphora (Johnson, 1966)) remains uncertain. The significance of t,he greater release of secretory granules from the sinus glands following lower frequencies of in vitro electrical stimulation is also not readily apparent. The results are consistent, however, with the report of greater release of cardioexcitatory hormone from the pericardial organs of the spider crab, Libinia, following lower frequencies of electrical stimulation than after higher frequencies with the same voltage and duration of stimulation (Cooke, 1964). Since it is not feasible at this time to obtain intracellular records of electrical activity from these axons, the physiological firing rate can only be estimated. The results presented here suggest t,hat, based on maximum granule depletion from the terminals, the range may be under a.O/second. The failure to demonstrate increased depletion at the higher frequencies may indicate an induced hyperpolarization which limits the response frequency or merely an inability of the fibers to respond to each pulse in the stimulus train above a certain rate. The somewhat greater granule depletion achieved in the in vitro preparation than in the in viva experiments of comparable frequencies of stimulation may reflect t,he interruption of axoplasmic flow in the former, limiting effective replenishment of the terminals from the preterminal regions. REFERENCES BERN, H. A., ASD MAGI, K. (1964). Electrophysiology of nemosecretory systems. In “Proceedings of the Srcond International Congress of Endocrinology.” pp. 5’77-583. BISSETT, G. IV., HILTON, S. M., AND POISNER, 9. FIG. 5. In NS granules, FIG. 6. In collections of FIG. 7. In ules. Granule Few”synaptic
STIMULATION
381
M. (1963). Parallel assays of vasopressin and osytocin in blood on localized electrical stimulation of the hypothalamus. J. Physiol. 169, 40~. BLISS, D. E., DURAND, J. B., AND WELSH, J. H. (1954). Neurosecretory systems in decapod Crustacea. Z. Zellforsch. Mikroskop. Anat. Abt. Hktochem. 39, 520436. BUST, A. H., AND ASHBY, E. A. (1967). Ultrastructure of the sinus gland of the crayfish, Procambarus clarkii. Gen. Comp. Endocrinol. 9, 334-342. COOKE, I. M. (1964). Electrical activity and release of neurosecretory material in crab pericardial organs. Comp. Biochem. Physiol. 13, 353-366. CROSS, B. A., AND HARRIS, G. W. (1952). The role of the neurohypophysis in the milk-ejection reflex. J. Endocrinol. 8, 148-161. DouGL.~~, W. W. AND POISNER, A. M. (1964). Stimulus-secretion coupling in a neurosecretory organ and the role of calcium in the release of vasopressin from the neurohypophysis. J. Physiol. 172, l-18. DUNCAN, A. C. (1952). “Quality Control and Industrial Statistics.” Irwin Press, Chicago, Illinois. EASTON, D. M. (1965). Impulses at the artifactual nerve end. Cold Spring Harbor Symp. Quant. Biol. 00, 1525. FARQUHAR, M. G. (1961). Origin and fate of secretory granules in cells of the anterior pituitary gland. Trans. N. k’. Acad. Sci. 23, 346351. FRIDBERG. G., IWISAKI, S., YAGI, K., BERN, H. A., RILSOX, D. M., AND NISHIOK.~, R. S. (1966). Relation of impulse conduction to electrically induced release of neurosecretory material from the urophysis of the teleost fish, Tilapia mossambica. J. Exptl. Zool. 161, 137-150. H.~LLER, E. W., S.~CHS, H., SPERELAKIS, N., AND SH.~RE, L. (1965). Release of vasopressin from isolated guinea pig posterior pituitaries. Am. J. Physiol. 209, 79-83. HARRIS, G. W. (1947). The innervation and actions of the neurohypophysis; an investigation using the method of remote-control stimulation. Phil. Trans. Roy. Sot. London Ser. B. 232, 385441. HARRIS, G. W. (1960). Central control of pituitary secretion. In “Nemophysiology” (J. Field, H.
vitro stimulation (0.5/second frequency, B-minute duration). The center terminal contains few plus numerous small “synaptic vesicles.” X25,000. vitro stimulation (a/second frequency). The axon terminal contains haloed NS granules plus “synaptic vesicles” grouped beneath thickenings in the plasmalemma. X25,000. vitro stimulation (2/second frequency). The terminal contains decreased numbers of NS grancores lacking limiting membranes lie between the axolemma and connective tissue sheath. vesi&s” are present here. X25,000.
382
BUNT
AND
W. Magoun, and V. E. Hall, eds.), ed. 2, pp. 1001-1038. Waverly Press, Baltimore. H.~Yw.~RD, J. N., AND SMITH, W. K. (1964). Antidiuretic response to electrical stimulation in brain stem of the monkey. Am. J. Physiol. 206, 15-20. HIGHNAM, K. C. (1961). Induced changes in the a.mounts of material in the neurosecretory system of the desert locust. Nature 191, 199200. HODGSON, E. S., AND GELDIAY, S., (1959). Experimentally induced release of neuroseeretory material from roach Corpora cardiaca. Biol. Bull. 117, 275-283. JOHNSON, B. (1966). Ultrastructure of probable sitrs of release of neurosecretory materials in an insect, Calliphoru stygia Fabr. (Diptera). Gen. Comp. Endocrinol. 6, 99-108. KLEINHOLZ, L. H. (1966). Separation and purification of crustacean eyestalk hormones. Am. Zool. 6, 161-167. KNOWLES, F. G. W. (1963). Techniques in the study of neurosecretion. Z/L “Techniques in Endocrine Research” (P. Eckstein and F. G. W. Knowles, eds.), pp. 5765. Academic Press, New York. LEVER, J. D., AND PETERSON, R. (1960). Cellular identities in the pars distalis of the rat pituitary. Trans. N. Y. Acad. Sci. 22, 504-508. NAGABHUSHANAM, R. (1964). Neurosecretory changes in the nervous system of the oyster,
ASHBY
C’rasaostrea pcrimental 14.
virgiraica, conditions.
induced Indinn
by various (‘sJ. Eqtl. Biol. 2,
T. C. (1965). The neurosecretory system of the adult Calliphora erythrocephala. I. The fine structure of the corpus cardiacum with some observations on adjacent organs. Z. Zellforsch. Mikroskop. Anat. Abt. Histochem. 67, 461601. P.~L.~DE, G. E. (1959). Functional changes in the structure of cell components. In “Subcellular Particles” (T. Hayashi, ed.), pp. 64-83. Am. Physiol. Sot.. Washington, D. C. POTTER, D. D. (1958). Observations on the neurosecretory system of portunid crabs. In “Second International Symposium on Neurosecretion” (W. Bargmann, B. Hanstrom, E. Scharrer, B. Scharrer, eds.), pp. 113-118. Springer, Berlin. SACHS, H., SHARE, L., OSINCHAK, J., AND CARPI, A. (1967). Capacity of the neurohypophysis to release vasopressin. Endocrinology 81, 755-770. SMITH, U., AND SMITH, D. S. (1966). Observations on the serretory processes in the corpus cardiscum of the stick insect, CarausilLs morosus. J. Cell Sci. 1, 59-66. SPEIDEL, C. C. (1919). Gland-cells of internal secretion in thr spinal cord of the skates. Carnegie Inst. Wash. 13, 1-31. VAN HARREVELD, A. (1936). A physiological solution for fresh water crustaceans. Proc. Sot. Erptl. Biol. Med. 34, 428-432. NORMANN,
AND
COMPARATIVE
ENDOCRINOLOGY
Ultrastructural
10,
Changes following
ANN Department
376382
in the
Electrical
HEFFINGTON of Anatomy.
(1968)
The
Received
Sinus
Gland
Stimulation’
BUNT
University at Dallas,
Crayfish
AND
EBERT
of Texas Soufhujestern Texas 75B5
October
A. ASHBY Medical
School
14, 1967
Ultrastructural examination of the sinus glands of the crayfish, Procambarus clarkii, following in viva and in vitro electrical stimulation reveals significantly fewer granules within the neurosecretory axon terminals than in similarly treated but unstimulated controls. Granule depletion appears to be nonspecific with respect to the different terminal types. Most effective depletion of granules is achieved with stimuli of 0.b2.O/second, while higher frequencies are progressively less effective. Further evidence concerning the mechanism of granule release is also presented.
The crustacean sinus gland, located adjacent, to the eyestalk ganglia, contains numerous neurosecretory (NS) axon terminals derived from cell bodies lying more proximally within the eyestalk X-organ, the brain, and possibly the thoracic ganglion (Bliss et al., 1954). A multiplicity of different hormones are contained within the gland, as evidenced by physiological and biochemical (Kleinholz, 1966)) histological (Potter, 1958)) and ultrastructural studies (Bunt and Ashby, 1967). It is generally accepted that the hormones present in the axon terminals are contained within membrane-enclosed NS granules and are released by appropriate firing of the axons (Bern and Yagi, 1964). In resemblance to ordinary neurons, some NS cells have been found to possess the ability to conduct electrical impulses. Speidel (1919)) who first, described the glandular appearance of the elasmobranch Dahlgren cells, noted that electrical or pilocarpine stimulation of the spinal cord resulted in an increase in the number of stainable cytoplasmic granules. In contrast,
electrically induced depletion of stainable NS material has been reported in the corpus cardiacum of the cockroach, Blaberus craniifer (Hodgson and Geldiay, 1959) and the locust, Schistoceru gregatia (Highnam, 1961), in the central nervous system of the oyster, Crassostrea virginica (Nagabhushanam, 1964), and in the urohypophysis of the teleost, Tilapia mossambica (Fridberg et al., 1966). Ultrastructural studies on the pericardial organ of the isopod, Squilla (Knowles, 1963)) and the teleost urohypophysis (Fridberg et al., 1966) lend further support to physiological and histological evidence of hormone release from NS axons following electrical stimulation. In view of these reports, this study was undertaken to determine morphological changes at the ultrastructural level which might result from in vivo and in vitro electrical stimulation of the sinus gland of the crayfish, Procambarus clarkii. In addition, for the interpretation of compound action potentials recorded from the axon trunks, it was of further interest to determine the stimulus frequency associated with maximum granule depletion.
‘This work was supported in part by N.I.H. grant 06623-02 from N.I.N.D.B. 376
SINUS
MATERIALS
AND
GLAND
ELECTRICAL
copy as described previously (Bunt and Ashby, 1967). In Vitro Experiments: The right and left sinus glands with adhering trunks were first dissected free in saline and then drawn up into separate glass-pipette electrodes along with small droplets of saline. The stimulus was applied to the right gland while the Ieft gland served &9 an unstimulated control. The method was adapted from the technique of Easton (1964) for recording from garpike olfactory nerve (cf. Table 1 for variables). Both sinus glands were then processed and examined by electron microscopy. Data from counts of NS axon terminals were analvzed according to the z-test for the significance of difference between proportions (Duncan, 1952).
METHODS
Male and female P. chrkii (l&cm carapace length) were maintained in the laboratory in pond water and fed commercial rabbit food. In Vivo Experiments: Each crayfish was secured on a Plexiglas animal carrier with rubber bands. Both eyestalks were immobilized with dental wax and the exoskeleton of each was slit on either side, along the dorsal proximal margin of the retina and across the base of the eyestalk. The loosened exoskeleton and underlying hypodermis TABLE VARIABLES
FOR in
F’dro
1 EXPERIMENT
No. of animals
Pulse (msrc)
Frequency (per second)
2 2
6.0
0.5 0.5 2.0 40
3 3 3
80
3 3
4
2 2 2
5.0 5.0 5.0 5.0 5.0
Dmation (minutes)
vY+Y
5
RESULTS
10
In Viva Electrical Stimulation. The electrically stimulated sinus glands differ from the control glands in their content of numerous scattered axon terminals which are either empty or greatly depleted of NS
10
3
100
10 10 10
granules. No preferential depIetion of terminal types was observed following electrical stimulation; consequently, data from all granule types were pooled for statistical treatme& The percentages of such depleted terminals are presented in Table 2. In some of the emptied terminals (Fig. l), a few typical NS granules are surrounded by numerous electron-lucent vesicles. In Fig. 1, an extruded granule core lies between the axolemma and the connective tissue sheath. In other terminals (Fig. 2), NS granules
were lifted away to reveal the sinus glands. Both glands were kept moistened with physiological saline (Van Harreveld, 1936) during the period of exposure. Fine silver electrodes were placed beneath both sinus glands on the medial side of the optic peduncle, at the junction of the optic lamina. The stimulus was then applied to the right sinus gland. The following variables were employed: (1) two animals, pulse 0.3 msec, frequency 30/second, voltage 1.5V, and duration 60 minutes; (2) two animals, pulse 3.0 msec, voltage 5.OV, and duration 20 minutes. Both the right and left sinus glands were then dissected out and prepared for electron microsTABLE DEPLETION
I n viva:
In vitro:
OF
NS AXON
TERMINALS
Duration (minutes)
Frequency (per second)
20
Control
30
30
60 60
Control
10
Control
5
30
10
0.5 0.5 2.0 40
10
80
10 10
10
377
STIMULATIOh‘
100
2
FOLLOWING
ELECTRICAL
‘% Depleted
terminals
11.2% (n (n 11.9% (n (n 27.5% 10.2% (n 53.3% (n 48.4% (n
= = = = = = =
21.3%
52.1% 34.1% 20.4yo 18.2%
z
P
71) 369) 126)
1.96
<0.05
620)
3.69
4.19
<0.0006 <0.0006 <0.0006 <0.0006 <0.02 <0.06
137) 92)
116) (n = 123)
(n = 129) (n = 147) (n =
STIMULATION
132)
6.37 5.61 4.72 2.38 1.89
(20 minutes). The terminal FI G. 1. In viva stimulation It vesicles. An ebuded NS granule core lies within ieci,ive tissue sheath. X25,000.
contains a pocket
fes- KS granules plus uumerous elect1 ‘onof the plasma membrane, adjacent t’o the
SINUS
GLAND
ELECTRICAL
with cores of low electron density and rather crenated limiting membranes lie among the electron-lucent vesicles and scattered membranous profiles. This configuration is observed in the preterminal portions of the axons as well as in the terminals adjoining the blood sinus (Fig. 3). In Vitro Electrical Stimulutio~n. All electrically stimulated glands differ from the control glands in their increased percentages of depleted terminals, as presented in Table 2. From these data, it can be seen that the greatest depletion of granules is observed at. lower frequencies of stimulation, with less depletion at the higher. The ultrastructure of the terminals themselves closely!. resembles that observed in the in viva experiments, in that scattered terminals a.re well filled with granules, while others exhibit few NS granules plus numerous electron-lucent vesicles. Some of these vesicles (Fig. 4) are approximately the same diameters as the NS granules, while others (Figs. 5 and 6) resemble the smaller so-called “synaptic vesicles” which are usually grouped just beneath the plasma membrane bordering the blood sinus. Thickenings of the plasma membrane adjacent to groups of these vesicles are observed in some of t’he actively secreting terminals (Fig. 6). Many of the NS granules (Figs. 6 and 7) exhibit a clear halo between the granule core and limiting membrane, an appearance observed less frequently in the in vivo experimental and the in vitro control sinus glands. Frequently these haloed KS granules seem to be associated with t,he presence of dense granule cores between the axolemma and connective tissue sheath (Fig. 7).
Occasional terminals contain a few NS granules of low electron density surrounded by seemingly broken membranes, as noted 2. In rice st,imulation (60 minutes). The scattered vesicles and membranous profiles.
previously in the in vivo experiments very rarely in control sinus glands.
and
DISCUSSI0-h
The increased depletion of NS granules within sinus gland terminals following electrical stimulation supports reports that NS cells possess the ability to conduct electrical impulses and that these impulses cause release of the hormone secretory products both in vivo (Harris, 1947, 1960; Cross and Harris, 1952; Bissett et aE., 1963; Hayward and Smith, 1964)) and also in vitro (Cooke, 1964; Douglas and Poisner, 1964; Haller et al., 1965; Sachs et al., 1967). The ultrastructural changes after the in z&o and in vitro electrical stimulation of the sinus gland axons closely resemble those report,ed in the teleost urohypophysis (Fridberg et al., 1966) and isopod pericardial organ (Knowles, 1963)) following similar electrical stimulation in viva. In agreement with both studies, here the decrease in numbers and electron density of the NS granules is accompanied by the appearance of both large and small electron lucent vesicles within the axon terminals. In contrast to the report by Knowles (1963)) however, large vesicles surrounding smaller vesicles have been observed only rarely. The finding of dense NS granule cores between the axon plasma membrane and blood sinus basement membrane in actively secreting terminals lends further support to the hypothesis (Bunt and Ashby, 1967) that the mechanism of secretion in the crayfish sinus gland closely resembles that reported in the corpus cardiacum of insects (Smith and Smith, 1964; Normann, 1965) and in the rat, anterior pituitary (Lever and Peterson, 1960; Farquhar, 1961) and exocrine pancreas (Palade, 1959). The increased numbers of “synaptic vesicles” in actively secreting terminals are evidently
contains only four moderately dense NS granules, of small vesicles borders a thickened region of the plasma membrane beneath the connective t,issuesheath. X25,000. FIG. 3. In viuo stimulation (20 minutes). A preterminal axon, surrounded by processes of a supporting cell, contains no NS granules of high electron density. Smudgy granules and vesicles of low electron density are grouped around the periphery and in the center of the axon. X25,000. FIG.
plus
terminal A group
379
STIMULATIOK
FIG. 4. In vitro vesicles,
x25,000.
whose
stimulation (J/second frequency). moderately dense somewhat flocculant
h few typical SS cont,ents surround
granules denser,
lie among eccentrically
numerous placed
large cores.
SINUS
GLAKD
ELECTRICAL
related to the release mechanism, but the significance of these vesicles and the thickenings of the adjacent axolemma, which closely resemble those reported in NS axons of the corpus cardiacum of Calliphora (Johnson, 1966)) remains uncertain. The significance of t,he greater release of secretory granules from the sinus glands following lower frequencies of in vitro electrical stimulation is also not readily apparent. The results are consistent, however, with the report of greater release of cardioexcitatory hormone from the pericardial organs of the spider crab, Libinia, following lower frequencies of electrical stimulation than after higher frequencies with the same voltage and duration of stimulation (Cooke, 1964). Since it is not feasible at this time to obtain intracellular records of electrical activity from these axons, the physiological firing rate can only be estimated. The results presented here suggest t,hat, based on maximum granule depletion from the terminals, the range may be under a.O/second. The failure to demonstrate increased depletion at the higher frequencies may indicate an induced hyperpolarization which limits the response frequency or merely an inability of the fibers to respond to each pulse in the stimulus train above a certain rate. The somewhat greater granule depletion achieved in the in vitro preparation than in the in viva experiments of comparable frequencies of stimulation may reflect t,he interruption of axoplasmic flow in the former, limiting effective replenishment of the terminals from the preterminal regions. REFERENCES BERN, H. A., ASD MAGI, K. (1964). Electrophysiology of nemosecretory systems. In “Proceedings of the Srcond International Congress of Endocrinology.” pp. 5’77-583. BISSETT, G. IV., HILTON, S. M., AND POISNER, 9. FIG. 5. In NS granules, FIG. 6. In collections of FIG. 7. In ules. Granule Few”synaptic
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M. (1963). Parallel assays of vasopressin and osytocin in blood on localized electrical stimulation of the hypothalamus. J. Physiol. 169, 40~. BLISS, D. E., DURAND, J. B., AND WELSH, J. H. (1954). Neurosecretory systems in decapod Crustacea. Z. Zellforsch. Mikroskop. Anat. Abt. Hktochem. 39, 520436. BUST, A. H., AND ASHBY, E. A. (1967). Ultrastructure of the sinus gland of the crayfish, Procambarus clarkii. Gen. Comp. Endocrinol. 9, 334-342. COOKE, I. M. (1964). Electrical activity and release of neurosecretory material in crab pericardial organs. Comp. Biochem. Physiol. 13, 353-366. CROSS, B. A., AND HARRIS, G. W. (1952). The role of the neurohypophysis in the milk-ejection reflex. J. Endocrinol. 8, 148-161. DouGL.~~, W. W. AND POISNER, A. M. (1964). Stimulus-secretion coupling in a neurosecretory organ and the role of calcium in the release of vasopressin from the neurohypophysis. J. Physiol. 172, l-18. DUNCAN, A. C. (1952). “Quality Control and Industrial Statistics.” Irwin Press, Chicago, Illinois. EASTON, D. M. (1965). Impulses at the artifactual nerve end. Cold Spring Harbor Symp. Quant. Biol. 00, 1525. FARQUHAR, M. G. (1961). Origin and fate of secretory granules in cells of the anterior pituitary gland. Trans. N. k’. Acad. Sci. 23, 346351. FRIDBERG. G., IWISAKI, S., YAGI, K., BERN, H. A., RILSOX, D. M., AND NISHIOK.~, R. S. (1966). Relation of impulse conduction to electrically induced release of neurosecretory material from the urophysis of the teleost fish, Tilapia mossambica. J. Exptl. Zool. 161, 137-150. H.~LLER, E. W., S.~CHS, H., SPERELAKIS, N., AND SH.~RE, L. (1965). Release of vasopressin from isolated guinea pig posterior pituitaries. Am. J. Physiol. 209, 79-83. HARRIS, G. W. (1947). The innervation and actions of the neurohypophysis; an investigation using the method of remote-control stimulation. Phil. Trans. Roy. Sot. London Ser. B. 232, 385441. HARRIS, G. W. (1960). Central control of pituitary secretion. In “Nemophysiology” (J. Field, H.
vitro stimulation (0.5/second frequency, B-minute duration). The center terminal contains few plus numerous small “synaptic vesicles.” X25,000. vitro stimulation (a/second frequency). The axon terminal contains haloed NS granules plus “synaptic vesicles” grouped beneath thickenings in the plasmalemma. X25,000. vitro stimulation (2/second frequency). The terminal contains decreased numbers of NS grancores lacking limiting membranes lie between the axolemma and connective tissue sheath. vesi&s” are present here. X25,000.
382
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AND
W. Magoun, and V. E. Hall, eds.), ed. 2, pp. 1001-1038. Waverly Press, Baltimore. H.~Yw.~RD, J. N., AND SMITH, W. K. (1964). Antidiuretic response to electrical stimulation in brain stem of the monkey. Am. J. Physiol. 206, 15-20. HIGHNAM, K. C. (1961). Induced changes in the a.mounts of material in the neurosecretory system of the desert locust. Nature 191, 199200. HODGSON, E. S., AND GELDIAY, S., (1959). Experimentally induced release of neuroseeretory material from roach Corpora cardiaca. Biol. Bull. 117, 275-283. JOHNSON, B. (1966). Ultrastructure of probable sitrs of release of neurosecretory materials in an insect, Calliphoru stygia Fabr. (Diptera). Gen. Comp. Endocrinol. 6, 99-108. KLEINHOLZ, L. H. (1966). Separation and purification of crustacean eyestalk hormones. Am. Zool. 6, 161-167. KNOWLES, F. G. W. (1963). Techniques in the study of neurosecretion. Z/L “Techniques in Endocrine Research” (P. Eckstein and F. G. W. Knowles, eds.), pp. 5765. Academic Press, New York. LEVER, J. D., AND PETERSON, R. (1960). Cellular identities in the pars distalis of the rat pituitary. Trans. N. Y. Acad. Sci. 22, 504-508. NAGABHUSHANAM, R. (1964). Neurosecretory changes in the nervous system of the oyster,
ASHBY
C’rasaostrea pcrimental 14.
virgiraica, conditions.
induced Indinn
by various (‘sJ. Eqtl. Biol. 2,
T. C. (1965). The neurosecretory system of the adult Calliphora erythrocephala. I. The fine structure of the corpus cardiacum with some observations on adjacent organs. Z. Zellforsch. Mikroskop. Anat. Abt. Histochem. 67, 461601. P.~L.~DE, G. E. (1959). Functional changes in the structure of cell components. In “Subcellular Particles” (T. Hayashi, ed.), pp. 64-83. Am. Physiol. Sot.. Washington, D. C. POTTER, D. D. (1958). Observations on the neurosecretory system of portunid crabs. In “Second International Symposium on Neurosecretion” (W. Bargmann, B. Hanstrom, E. Scharrer, B. Scharrer, eds.), pp. 113-118. Springer, Berlin. SACHS, H., SHARE, L., OSINCHAK, J., AND CARPI, A. (1967). Capacity of the neurohypophysis to release vasopressin. Endocrinology 81, 755-770. SMITH, U., AND SMITH, D. S. (1966). Observations on the serretory processes in the corpus cardiscum of the stick insect, CarausilLs morosus. J. Cell Sci. 1, 59-66. SPEIDEL, C. C. (1919). Gland-cells of internal secretion in thr spinal cord of the skates. Carnegie Inst. Wash. 13, 1-31. VAN HARREVELD, A. (1936). A physiological solution for fresh water crustaceans. Proc. Sot. Erptl. Biol. Med. 34, 428-432. NORMANN,