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Neural properties of the protocerebral neurosecretory cells of the adult cockroach Periplaneta americana
J. InsectPhysiol.,1968,Vol. 14,pp. 1785to 1792. Pergamma Press. Printed in Great Britain
NEURAL PROPERTIES OF THE PROTOCEREBRAL NEUROSECRETORY CELLS OF THE ADULT COCKROACH PERIPLANETA
J. L. GOSBEE,
AMERICANA*
J. V. MILLIGAN,
and B. N.
SMALLMAN
Departments of Biology and Physiology, Queen’s University, Kingston, Canada
Abstract-Electrical stimulation of various areas of the cockroach protocerebrum resulted in changes in the amount of neurosecretory products both in the neurosecretory cells and in the corpora cardiaca. Stimulating the neurosecretory cell area for various time intervals led to depletion of the neurosecretion from the neurosecretory system. Intracellular recordings showed the neurosecretory cells to have resting membrane potentials of 20 to 40 mV; depolarizations of 2 to 3 mV occurred spontaneously and could be modified by stimulating the corpora cardiaca. Depletion of the neurosecretory material from the corpora cardiaca was observed after depolarizing these organs for 30 min with isotonic saline solution having a potassium concentration fifty times that of normal cockroach saline. These findings support the implicit assumption that the neurosecretory cells of the brain have properties of neural cells as well as secretory cells, and that the neural and secretory activities are functionally related.
INTRODUCTION
SEVERALtheories relating to the function of insect neurosecretory systems have assumed that the secretory activity of the neurosecretory cells is controlled by electrical activity. In this laboratory we have been mainly concerned with the hypothesis proposed by VAN DERKLOOT (1955) to explain diapause in Hyalophora cecropia. VAN DJB KLOOT (1955) postulated that diapause resulted from inactivation of the neurosecretory cells as deduced from his observations on the loss of cholinesterase and electrical activity of the whole brain. However, recent studies (SCHOONHOVEN,1963; SHAPPIROet al., 1965, 1967; TYSHTCHEZNKO and MANDELSTAM,1965 ; MANSINGH and SULMAN, 1967) provide overwhelming evidence that electrical and cholinesterase activity are present in the brains of diapausing insects. All of these workers, including Van der Kloot, investigated the whole brain with no special study of the neurosecretory cells. Thus, while the experimental * This research was supported by the National Research Council of Canada, Grant Number A-2394, to B. N. S., and NRC Studentship to J. L. G. 1785
1786
J. L. GOSBEB, J. V. MILLIGAN,ANDB. N. SMALLMAN
evidence on which Van der Kloot based his hypothesis must be rejected because of the more recent investigations, one of his basic assumptions has not been tested. Van der Kloot clearly assumed that it was the cessation of electrical activity in the neurosecreto~ cells themselves which initiated diapause. Even though electrical activity and cholinesterase activity can be detected in the whole brain during diapause, the question of electrical activity in the neurosecretory cells remains central to his hypothesis. It has not yet been demonstrated for any insect neurosecretory system that neurosecretory cells exhibit electrical activity, nor that the release or synthesis of neurosecretory material is somehow controlled by electrical activity. In fish, BERNand YAGI (1965) have demonstrated electrical activity in the caudal neurosecreto~ system. Our studies were therefore designed to investigate three questions: (1) Does electrical stimulation in other parts of the brain influence the synthesis and/or release of neurosecretory products from the neurosecretory cells of the pars intercerebralis ? (2) Do the neurosecretory cells show any evidence of being capable of conducting an electrical impulse ? (3) What possible mechanism could link the conduction of an impulse with the release of the neurosecretory products from the neurohaem~ organ, the corpora cardiaca ? MATERIALS AND METHODS Experimental insects Colonies of approximately thirty Periplaneta americana were maintained on rabbit chow in 1 gal. jars at room temperature. Adults of both sexes were used. Care was taken to have adults of about the same age ( f 1 week) and sex for any one experiment. Histological methods All tissues were fixed overnight with Bouin’s fixative (PANTIN, 1962, p. 9), using 1% trichloracetic acid instead of the glacial acetic acid described by Pantin (EVEN, 1962). Th e 1ocation of the neurosecretory cells of the pars intercerebralis was determined by the in sitti staining method of DOGRAand TANDAN(1964), using their ‘Staining Procedure II’ with paraldehyde fuchsin stain. Sectioned tissues were prepared by E~EN’S (1962) paraldehyde fuchsin-staining procedure. Electrical stimulation of the protocerebrum To investigate the effect of brain neural activity on amounts of neurosecretory products in both the neurosecretory cells and the corpora cardiaca, we stimulated electrically on the periphery of the neurosecretory cell cluster. Square-wave stimuli of 5 V intensity and 5 msec duration were delivered from a Grass S4 stimulator through electrolytically tapered varnished steel insect pins at the rate of 2O/sec, using a Grass SIU 478A stimulus isolation unit. The brain and corpora cardiaca were subsequently examined histologically for changes in the amount of neurosecretory materials in comparison to sham-operated controls. In the latter the electrodes were inserted but they were not electrically stimulated.
PROTOCRRRRRAL NRUROSRCRRT ORY CRLLS OF COCKROACH
1787
Intracellular recordings Glass micropipettes manufactured by methods ADRIAN(1956) were inserted by a micromanipulator
similar to those described by into the neurosecretory cells. All electrodes were filled with 3 M KC1 by boiling under reduced pressure for several minutes. Electrodes with resistances of less than 10 MQ and tip potentials greater than 10 mV were discarded. The KC1 made contact with Ag-AgCl electrodes through an Agar-Ringer gel (ADRIAN,1956). The Ag-AgCl electrodes were connected to a neutralized input capacity amplifier (Bioelectric Instruments NF 1). From this amplifier the signal was fed into a Tektronix 3A3 amplifier to be displayed on a Tektronix RM565 cathode-ray oscilloscope. Photographs of the tracings were obtained with a Grass Kymograph camera from a ‘ slave’ oscilloscope. Fine steel electrodes were used to stimulate the corpora cardiaca while intracellular recordings were being made from the neurosecretory cell. The stimuli were generated by a Grass S4 stimulator and delivered to the electrodes through a Grass SIU 478A isolation unit. Depolarization studies
The corpora cardiaca were bathed for 30 min either in normal cockroach Ringer’s solution (ROEDER,1953), or in an isotonic solution which had fifty times the normal concentration of potassium; the sodium concentration was decreased to maintain isotonicity. The concentration of CaCl, and glucose remained constant at 1.81 mM and 22.2 mM respectively. The effect of these solutions on the amount of neurosecretory material in the corpora cardiaca was determined histologically. RESULTS In situ staining revealed about 100 cells closely grouped along the midline of
the pars intercerebralis. Individual cells measured about 20 ~1in dia. In the living cockroach they appeared bluish-white under the proper lighting conditions. The close grouping and superficial location of these large cells greatly facilitated accurate placement of the electrodes both for stimulation and recording. Evidence that electrical stimulation of the brain affects the distribution of neurosecretory material
To test whether electrical stimulation affected the distribution of neurosecretory material, twenty adult females were used in four test groups of five cockroaches each. The control group consisted of two sham-operated animals for each of the four test groups, and two unoperated controls. The cockroaches were stimulated on the periphery of the neurosecretory cell area for 0.5, 1, 5, or 15 min. The sham-operated animals had the electrodes inserted for the same periods of time. Histological examination revealed that all sham-operated animals had approximately the same amount of neurosecretory material in the neurosecretory cells and in the corpora cardiaca as did the unoperated cockroaches. In the experimental
1788
J.
L. GOSBEE, J. V. MILLIGAN, AND B. N. %ALLMAN
animals various amounts of neurosecretory materials were present. Fig. 1 shows typical sections of the pars intercerebralis neurosecretory cells from shamoperated and experimental cockroaches. Cockroaches stimulated for 0.5 min had more neurosecretory material than the control group while those stimulated for 1 min had approximately the same amount. Cockroaches stimulated for 5 min or for 15 min had less neurosecretory material than the control group. The corpora cardiaca showed responses parallel to those described for the neurosecretory cells; the cockroaches stimulated for O-5 or 1 min had more neurosecretory material here than the control group while those stimulated for 5 or 15 min had less. Evidence that newosecretory cells show electrical activity Intracellular recordings were obtained from neurosecretory cells of several insects. Table 1 shows the mean resting membrane potentials of neurosecretory cells of eighteen insects. The large standard deviations indicate considerable variation among the cells of each insect, while the range of 17 to 47 mV represents a large variation of the means between insects. TABLE ~--RESTING MEMBRANEPOTENTIALSMEASURED FROM NEUROSECRETORY CELLSOF THE COCKROACH PARSIN’IZRCEREBRALIS
Electrode resistance Sex F F F F F F F F F F F M M M M M M M
(MQ) 20 10 10 10 1.5 18 10 10 20 25 25 10 14 10 10 12 10 26
No. of cells measured
Mean resting membrane potential +l SD. (mV)
of electrical activity
5 10
23 + 8.6 27f 11.6 27+ 8.2 35 + 8.9 41 f 17.9 44 f 14.5 18f 4.5 19f 4.0 23 + 8.9 28 f 9.9 47 * 15*0 33 f 14.2 17f 7.0 21 f 11.3 21 f 6.5 21 + 5.5 21 + 4.3 33k11.7
-
11 6 30 11 13 8 9 16 15 25 5 11 8 14 9 17
Detection
While measuring the membrane resting potentials, ‘ spontaneous’ electrical activity, in the form of 2 to 3 mV depolarizations lasting 5 to 12 msec, was sometimes observed. Table 2 shows the amplitude and duration of these depolarizations
FIG. 1. Neurosecretory cells of the cockroach pars intercerebralis stained with paraldehyde fuchsin following sham operation (S), or stimulated for 0"5, 1, 5, or 15 rain.
FIG. 2. Oscilloscope tracings of potentials recorded intracellularly from cockroach neurosecretory cells before (A) and during (B) stimulation. Calibration pulses (C) are of 2 mV amplitude and 10 msec duration.
Flc. 3. Cross-sections of cockroach corpora cardiaca following bathing in normal Ringer's solution (A) and following bathing in high K + Ringer's solution (B). The neurosecretory material has been darkly stained by paraldehyde fuchsin.
PROTOCEREBRAL
NEUROSECRBTORY
CELLS
OF
1789
COCKROACH
measured in six cells of five insects. Stimulation of the corpora cardiaca modified the frequency and amplitude of the depolarizations in one animal. Fig. 2 shows that before stimulation the frequency was less than 1 per 100 msec trace while after stimulation it was about 2 per 100 msec trace. In addition, the potentials occurring after stimulation were significantly smaller in amplitude than before stimulation (P < 0405). The first depolarization is tightly locked to the stimulus having a latency of 11.0 f 5.3 msec. The second has a latency of 44.5 + 8-5 msec after the beginning of the first potential. TABLE 2-AMPLITUDE AND DURATIONOF POTENTIALS RRCORDED IN NEUROSECRRTORY CELLS OF THE
Cell no.
No. of potentials measured
PROTOCBREBRIJM
Mean amplitude +l S.D. (mv)
Mean duration +1 S.D. (msec)
Stim.
16-5
6
1.3 f 0.21
7.7 * 1.03
+
7-3 7-3 7-3
16 4 8
2.7 k 0.33 1.7 + 0.65 4.5 * 1.0
4.9 f 0.68 4.8 + 0.96 8.3 f 0.87
+
9-2
2
21-1 21-1
6 26
1.5 + 0.64 1.2 f 0.28
6.8 f 1.72 6.6 k 1.47
21-6 21-6 21-6 21-6 21-6 21-6 21-6
23 9 9 6 27 25 304
4.7 f 1.6 3.0 f 0.28 2.5 ?I0.47 2.4 + 0.90 2.0 + 0.67 1.8 + 0.80 2.0 f 0.74
9.8 + 1.51 9.8 + 1.17 ll*Ort 1.00 12.6 + 2.25 7.9 ;t 1.33 9.3 +_3.34 8.9 + 1.5
59
2.3 zk0.58 1.3 + 0.39 1.5 + 0.47
12.0 + 1.99 10.8 + 1.63 10.7 f 1.62
23-l * 23-l * 23-l*
SW 59:
1.3
-
Spont .
+ + + + + + + + + + + + + + +
Potentials were measured without stimulation (spont.) or with stimulation (stim.) of the corpora cardiaca. * Some of these potentials are shown in Fig. 2. t First potential occurring after stimulus artifact. $ Second potential occurring after stimulus artifact.
Evidence that depolarization of the corpora cardiaca aflects the release of neurosecretory materials Depolarization of the axons is a characteristic of neural activity but its transient nature makes the study of release of neurosecretory materials during depolarization
1790
J. L. GOSBEE,J. V. MILLIGAN,ANDB. N. SMALLMAN
difficult. For this reason we attempted a more prolonged depolarization by bathing the corpora cardiaca in situ in a high-potassium Ringer’s; the KC1 of the normal Ringer’s was increased from 2.68 mM KC1 to 134 mM while the concentration of NaCl was decreased from 154 mM to 22.7 mM. Fig. 3 shows that the corpora cardiaca bathed in the high [K+] had much less neurosecretory material than those bathed in the normal Ringer’s. The neurosecretory cells of both groups had approximately the same amounts of neurosecretory materials. The same results were obtained in four replications of this experiment.
DISCUSSION These results show that prolonged electrical stimulation of the brain of Periplaneta americana depletes the amount of neurosecretory material in the neurosecretory cells of the pars intercerebralis and in the corpora cardiaca. The observed depletion may result from increased release of neurosecretory material, or decreased synthesis of the material, or some combination of both. Our methods do not permit us to decide which of these alternatives is correct. These results are similar to those observed on the corpora cardiaca of Blaberus cranizyer by HODGSON and GELDIAY (1959), on the goldfish preoptic neurosecretory cells by JASINSKI et al. (1966), and on the caudal neurohaemal organ of the teleost fish, Tilapia, by FRIDBERGet al. (1966). The microelectrode studies were directed towards obtaining some basic information on the electrical characteristics of insect neurosecretory cells. The demonstrations of resting membrane potentials of 20 to 40 mV and of depolarizations of 2 to 3 mV lasting for 5 to 12 msec suggest that electrical activity does occur in the neurosecretory cells of the pars intercerebralis. An interesting characteristic of these cells is the long duration of the depolarization. The range of 5 to 12 msec agrees well with the duration of potentials observed in other neurosecretory systems, e.g. YAGI et al. (1963) reported 8 msec duration from the leech: MORITA et al. (1961) and ISHIBA.SHI(1962) recorded 8 to 10 msec duration from the caudal neurosecretory system of the eel; BENNETTand Fox (1962) obtained 5 to 10 msec duration from skates and flukes. If the depolarizations we measured resulted from conducted potentials, then they are much smaller in amplitude than one would expect. This low amplitude may be a reflection of the unipolar characteristic of the insect neurosecretory cell. ROEDER(1963) h as suggested that the conducted potential of an insect unipolar neuron may not invade the cell body. Since we were recording intracellularly from the cell body, the small depolarizations observed may be a reflection of electrical activity occurring somewhere on the axon close to the cell body. The fact that stimulation of the corpora cardiaca could modify these depolarizations, both in frequency and amplitude, suggests a neural connexion between the neurosecretory cell and the corpora cardiaca. We cannot be certain of the pathway involved but it may be the neurosecretory axon itself.
PROTOCEREBRAL NEUROSECRETORY CELLSOF COCKROACH
1791
The depletion of neurosecretory material from the corpora cardiaca, when they were bathed in a Ringer’s solution containing high concentrations of potassium, suggests that depolarization is the mechanism by which a conducted potential could effect the release of neuros~reto~ materials. When the potential invades the axon endings it would depolarize them, and this could lead to the release of the neurosecretory materials. The long duration of the potentials might facilitate the release by keeping the membrane depolarized sufficiently long for the release to occur. In conclusion, the findings reported here support the assumption by VAN DER KLOOT (1955), and many others, that the secretory activity of the neurosecretory cells is controlled by nervous activity in other brain cells and in the neurosecreto~ cells themselves. The neurosecreto~ cells may be the centre for transduction of information from a nervous code into a chemical code; that is, from conducted potentials to hormones. The problem of how such transduction occurs seems central to understanding the mechanisms controlling development and diapause in insects. REFERENCES ADRIANR. H. (1956) The effect of internal and external potassium concentration on the membrane potential of frog muscle. J. PhysioE. 133, 631-658. BENNETT M. and Fox S. (1962) Electrophysiology of caudal neurosecretory cells in the skate and fluke. Gen. camp. Endocr. 2, 77-95. BERN H. A. and YAGI K. (1965) Electrophysiology of neurosecretory systems. Proc. 2nd int. Congr. Endocr. pp. 577-583. Excerpta Medica Foundation, New York. DOGRAG. S. and TANDENB. K. (1964) Adaptation of certain histological techniques for in situ demonstration of the neuroendocrine system of insects and other animals. Quart. J. micr. Sci. 105,455-466. EVEN A. B. (1962) An improved aldehyde fuchsin staining technique for neurosecretory products in insects. Trans. Am. micr. Sot. 81, 94-96. FRIDBERGG., IWASAKIS., YAGI K., BERN H., WILSON D., and NISHIOKAR. (1966) Relation of impulse conduction to electrically induced release of neurosecretory materials from the urophysis of the teleost fish, Xilapia mossambica. J. exp. Zool. 161, 137-149. HODGSONE. and GELDIAY S. (1959) Experimentally induced release of neurosecretory materials from roach corpora cordiaca. Biol. Bull., Woods Hole 117, 275-283. ISHIBASHIT. (1962) Electrical activity of the caudal neurosecretory cells in the eel ~~uiZZa japonica with special reference to synaptic transmission. Gen. corn& Endocr. 2,415-424. JASINSKIA., GORBMANA., and HARA “I’. (1966) Rate of movement and redistribution of stainable neurosecretory granules in hypothalamic neurons. Science, IV. Y. 154,776-778. MANSINGHA. and SMALLMANB. N. (1967) The cholinergic system in insect diapause. J. Insect Physiol. 13, 4-47-467. MORITAH., ISHIBASHIT., and YAMASHITA S. (1961) Synaptic transmission in neurosecretory cells. Nature, Land. 191, 183. PANTINC. F. (1962) Notes on ~icroscopjcaZ Tech~i~s~or Zoologists. Cambridge University Press, Cambridge. ROEDERK. D. (1953) Insect Physiology. John Wiley, New York. ROEDERK. D. (1963) Nerwe Cells and Insect Behavior. Harvard University Press, Cambridge, Mass. SCHOONHOVEN L. M. (1963) Spontaneous electrical activity in the brains of diapausing insects. Science, N.Y. 141, 173-174.
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SHAPPIROD. G., EICHENBAUMD., and LOCKE B. (1965) Cholinesterase in the brain of the cecropia silkmoth in relation to the control of neurosecretion and diapause. Am. Zool. 5, 698699. SHAPPIROD. G., EICHENBAUMD., and LOCKE B. (1967) Chohnesterase in the brain of the cecropia silkmoth during metamorphosis and pupal diapause. Biol. Bull., Woods Hole 132, 108-125. TYSHTCHENKO V. and MANDEUTAMJ. (1965) A study of spontaneous electrical activity and localization of cholinesterase in the nerve ganglia of Antheraea pe~yni G&r. at different stages of metamorphosis and in pupal diapause. J. Znsect Physiol. 11, 1233-1239. VAN DERKLOOT W. G. (1955) The control of neurosecretion and diapause by physiological changes in the brain of the cecropia silkworm. Biol. Bull., Woods Hole 109, 276-294. YAW K., BERN H., and HAGADORN I. (1963) Action potentials of neurosecretory neurons in the leech Thercnnyson rude. Gen. camp. Endocr. 3,490-495.
NEURAL PROPERTIES OF THE PROTOCEREBRAL NEUROSECRETORY CELLS OF THE ADULT COCKROACH PERIPLANETA
J. L. GOSBEE,
AMERICANA*
J. V. MILLIGAN,
and B. N.
SMALLMAN
Departments of Biology and Physiology, Queen’s University, Kingston, Canada
Abstract-Electrical stimulation of various areas of the cockroach protocerebrum resulted in changes in the amount of neurosecretory products both in the neurosecretory cells and in the corpora cardiaca. Stimulating the neurosecretory cell area for various time intervals led to depletion of the neurosecretion from the neurosecretory system. Intracellular recordings showed the neurosecretory cells to have resting membrane potentials of 20 to 40 mV; depolarizations of 2 to 3 mV occurred spontaneously and could be modified by stimulating the corpora cardiaca. Depletion of the neurosecretory material from the corpora cardiaca was observed after depolarizing these organs for 30 min with isotonic saline solution having a potassium concentration fifty times that of normal cockroach saline. These findings support the implicit assumption that the neurosecretory cells of the brain have properties of neural cells as well as secretory cells, and that the neural and secretory activities are functionally related.
INTRODUCTION
SEVERALtheories relating to the function of insect neurosecretory systems have assumed that the secretory activity of the neurosecretory cells is controlled by electrical activity. In this laboratory we have been mainly concerned with the hypothesis proposed by VAN DERKLOOT (1955) to explain diapause in Hyalophora cecropia. VAN DJB KLOOT (1955) postulated that diapause resulted from inactivation of the neurosecretory cells as deduced from his observations on the loss of cholinesterase and electrical activity of the whole brain. However, recent studies (SCHOONHOVEN,1963; SHAPPIROet al., 1965, 1967; TYSHTCHEZNKO and MANDELSTAM,1965 ; MANSINGH and SULMAN, 1967) provide overwhelming evidence that electrical and cholinesterase activity are present in the brains of diapausing insects. All of these workers, including Van der Kloot, investigated the whole brain with no special study of the neurosecretory cells. Thus, while the experimental * This research was supported by the National Research Council of Canada, Grant Number A-2394, to B. N. S., and NRC Studentship to J. L. G. 1785
1786
J. L. GOSBEB, J. V. MILLIGAN,ANDB. N. SMALLMAN
evidence on which Van der Kloot based his hypothesis must be rejected because of the more recent investigations, one of his basic assumptions has not been tested. Van der Kloot clearly assumed that it was the cessation of electrical activity in the neurosecreto~ cells themselves which initiated diapause. Even though electrical activity and cholinesterase activity can be detected in the whole brain during diapause, the question of electrical activity in the neurosecretory cells remains central to his hypothesis. It has not yet been demonstrated for any insect neurosecretory system that neurosecretory cells exhibit electrical activity, nor that the release or synthesis of neurosecretory material is somehow controlled by electrical activity. In fish, BERNand YAGI (1965) have demonstrated electrical activity in the caudal neurosecreto~ system. Our studies were therefore designed to investigate three questions: (1) Does electrical stimulation in other parts of the brain influence the synthesis and/or release of neurosecretory products from the neurosecretory cells of the pars intercerebralis ? (2) Do the neurosecretory cells show any evidence of being capable of conducting an electrical impulse ? (3) What possible mechanism could link the conduction of an impulse with the release of the neurosecretory products from the neurohaem~ organ, the corpora cardiaca ? MATERIALS AND METHODS Experimental insects Colonies of approximately thirty Periplaneta americana were maintained on rabbit chow in 1 gal. jars at room temperature. Adults of both sexes were used. Care was taken to have adults of about the same age ( f 1 week) and sex for any one experiment. Histological methods All tissues were fixed overnight with Bouin’s fixative (PANTIN, 1962, p. 9), using 1% trichloracetic acid instead of the glacial acetic acid described by Pantin (EVEN, 1962). Th e 1ocation of the neurosecretory cells of the pars intercerebralis was determined by the in sitti staining method of DOGRAand TANDAN(1964), using their ‘Staining Procedure II’ with paraldehyde fuchsin stain. Sectioned tissues were prepared by E~EN’S (1962) paraldehyde fuchsin-staining procedure. Electrical stimulation of the protocerebrum To investigate the effect of brain neural activity on amounts of neurosecretory products in both the neurosecretory cells and the corpora cardiaca, we stimulated electrically on the periphery of the neurosecretory cell cluster. Square-wave stimuli of 5 V intensity and 5 msec duration were delivered from a Grass S4 stimulator through electrolytically tapered varnished steel insect pins at the rate of 2O/sec, using a Grass SIU 478A stimulus isolation unit. The brain and corpora cardiaca were subsequently examined histologically for changes in the amount of neurosecretory materials in comparison to sham-operated controls. In the latter the electrodes were inserted but they were not electrically stimulated.
PROTOCRRRRRAL NRUROSRCRRT ORY CRLLS OF COCKROACH
1787
Intracellular recordings Glass micropipettes manufactured by methods ADRIAN(1956) were inserted by a micromanipulator
similar to those described by into the neurosecretory cells. All electrodes were filled with 3 M KC1 by boiling under reduced pressure for several minutes. Electrodes with resistances of less than 10 MQ and tip potentials greater than 10 mV were discarded. The KC1 made contact with Ag-AgCl electrodes through an Agar-Ringer gel (ADRIAN,1956). The Ag-AgCl electrodes were connected to a neutralized input capacity amplifier (Bioelectric Instruments NF 1). From this amplifier the signal was fed into a Tektronix 3A3 amplifier to be displayed on a Tektronix RM565 cathode-ray oscilloscope. Photographs of the tracings were obtained with a Grass Kymograph camera from a ‘ slave’ oscilloscope. Fine steel electrodes were used to stimulate the corpora cardiaca while intracellular recordings were being made from the neurosecretory cell. The stimuli were generated by a Grass S4 stimulator and delivered to the electrodes through a Grass SIU 478A isolation unit. Depolarization studies
The corpora cardiaca were bathed for 30 min either in normal cockroach Ringer’s solution (ROEDER,1953), or in an isotonic solution which had fifty times the normal concentration of potassium; the sodium concentration was decreased to maintain isotonicity. The concentration of CaCl, and glucose remained constant at 1.81 mM and 22.2 mM respectively. The effect of these solutions on the amount of neurosecretory material in the corpora cardiaca was determined histologically. RESULTS In situ staining revealed about 100 cells closely grouped along the midline of
the pars intercerebralis. Individual cells measured about 20 ~1in dia. In the living cockroach they appeared bluish-white under the proper lighting conditions. The close grouping and superficial location of these large cells greatly facilitated accurate placement of the electrodes both for stimulation and recording. Evidence that electrical stimulation of the brain affects the distribution of neurosecretory material
To test whether electrical stimulation affected the distribution of neurosecretory material, twenty adult females were used in four test groups of five cockroaches each. The control group consisted of two sham-operated animals for each of the four test groups, and two unoperated controls. The cockroaches were stimulated on the periphery of the neurosecretory cell area for 0.5, 1, 5, or 15 min. The sham-operated animals had the electrodes inserted for the same periods of time. Histological examination revealed that all sham-operated animals had approximately the same amount of neurosecretory material in the neurosecretory cells and in the corpora cardiaca as did the unoperated cockroaches. In the experimental
1788
J.
L. GOSBEE, J. V. MILLIGAN, AND B. N. %ALLMAN
animals various amounts of neurosecretory materials were present. Fig. 1 shows typical sections of the pars intercerebralis neurosecretory cells from shamoperated and experimental cockroaches. Cockroaches stimulated for 0.5 min had more neurosecretory material than the control group while those stimulated for 1 min had approximately the same amount. Cockroaches stimulated for 5 min or for 15 min had less neurosecretory material than the control group. The corpora cardiaca showed responses parallel to those described for the neurosecretory cells; the cockroaches stimulated for O-5 or 1 min had more neurosecretory material here than the control group while those stimulated for 5 or 15 min had less. Evidence that newosecretory cells show electrical activity Intracellular recordings were obtained from neurosecretory cells of several insects. Table 1 shows the mean resting membrane potentials of neurosecretory cells of eighteen insects. The large standard deviations indicate considerable variation among the cells of each insect, while the range of 17 to 47 mV represents a large variation of the means between insects. TABLE ~--RESTING MEMBRANEPOTENTIALSMEASURED FROM NEUROSECRETORY CELLSOF THE COCKROACH PARSIN’IZRCEREBRALIS
Electrode resistance Sex F F F F F F F F F F F M M M M M M M
(MQ) 20 10 10 10 1.5 18 10 10 20 25 25 10 14 10 10 12 10 26
No. of cells measured
Mean resting membrane potential +l SD. (mV)
of electrical activity
5 10
23 + 8.6 27f 11.6 27+ 8.2 35 + 8.9 41 f 17.9 44 f 14.5 18f 4.5 19f 4.0 23 + 8.9 28 f 9.9 47 * 15*0 33 f 14.2 17f 7.0 21 f 11.3 21 f 6.5 21 + 5.5 21 + 4.3 33k11.7
-
11 6 30 11 13 8 9 16 15 25 5 11 8 14 9 17
Detection
While measuring the membrane resting potentials, ‘ spontaneous’ electrical activity, in the form of 2 to 3 mV depolarizations lasting 5 to 12 msec, was sometimes observed. Table 2 shows the amplitude and duration of these depolarizations
FIG. 1. Neurosecretory cells of the cockroach pars intercerebralis stained with paraldehyde fuchsin following sham operation (S), or stimulated for 0"5, 1, 5, or 15 rain.
FIG. 2. Oscilloscope tracings of potentials recorded intracellularly from cockroach neurosecretory cells before (A) and during (B) stimulation. Calibration pulses (C) are of 2 mV amplitude and 10 msec duration.
Flc. 3. Cross-sections of cockroach corpora cardiaca following bathing in normal Ringer's solution (A) and following bathing in high K + Ringer's solution (B). The neurosecretory material has been darkly stained by paraldehyde fuchsin.
PROTOCEREBRAL
NEUROSECRBTORY
CELLS
OF
1789
COCKROACH
measured in six cells of five insects. Stimulation of the corpora cardiaca modified the frequency and amplitude of the depolarizations in one animal. Fig. 2 shows that before stimulation the frequency was less than 1 per 100 msec trace while after stimulation it was about 2 per 100 msec trace. In addition, the potentials occurring after stimulation were significantly smaller in amplitude than before stimulation (P < 0405). The first depolarization is tightly locked to the stimulus having a latency of 11.0 f 5.3 msec. The second has a latency of 44.5 + 8-5 msec after the beginning of the first potential. TABLE 2-AMPLITUDE AND DURATIONOF POTENTIALS RRCORDED IN NEUROSECRRTORY CELLS OF THE
Cell no.
No. of potentials measured
PROTOCBREBRIJM
Mean amplitude +l S.D. (mv)
Mean duration +1 S.D. (msec)
Stim.
16-5
6
1.3 f 0.21
7.7 * 1.03
+
7-3 7-3 7-3
16 4 8
2.7 k 0.33 1.7 + 0.65 4.5 * 1.0
4.9 f 0.68 4.8 + 0.96 8.3 f 0.87
+
9-2
2
21-1 21-1
6 26
1.5 + 0.64 1.2 f 0.28
6.8 f 1.72 6.6 k 1.47
21-6 21-6 21-6 21-6 21-6 21-6 21-6
23 9 9 6 27 25 304
4.7 f 1.6 3.0 f 0.28 2.5 ?I0.47 2.4 + 0.90 2.0 + 0.67 1.8 + 0.80 2.0 f 0.74
9.8 + 1.51 9.8 + 1.17 ll*Ort 1.00 12.6 + 2.25 7.9 ;t 1.33 9.3 +_3.34 8.9 + 1.5
59
2.3 zk0.58 1.3 + 0.39 1.5 + 0.47
12.0 + 1.99 10.8 + 1.63 10.7 f 1.62
23-l * 23-l * 23-l*
SW 59:
1.3
-
Spont .
+ + + + + + + + + + + + + + +
Potentials were measured without stimulation (spont.) or with stimulation (stim.) of the corpora cardiaca. * Some of these potentials are shown in Fig. 2. t First potential occurring after stimulus artifact. $ Second potential occurring after stimulus artifact.
Evidence that depolarization of the corpora cardiaca aflects the release of neurosecretory materials Depolarization of the axons is a characteristic of neural activity but its transient nature makes the study of release of neurosecretory materials during depolarization
1790
J. L. GOSBEE,J. V. MILLIGAN,ANDB. N. SMALLMAN
difficult. For this reason we attempted a more prolonged depolarization by bathing the corpora cardiaca in situ in a high-potassium Ringer’s; the KC1 of the normal Ringer’s was increased from 2.68 mM KC1 to 134 mM while the concentration of NaCl was decreased from 154 mM to 22.7 mM. Fig. 3 shows that the corpora cardiaca bathed in the high [K+] had much less neurosecretory material than those bathed in the normal Ringer’s. The neurosecretory cells of both groups had approximately the same amounts of neurosecretory materials. The same results were obtained in four replications of this experiment.
DISCUSSION These results show that prolonged electrical stimulation of the brain of Periplaneta americana depletes the amount of neurosecretory material in the neurosecretory cells of the pars intercerebralis and in the corpora cardiaca. The observed depletion may result from increased release of neurosecretory material, or decreased synthesis of the material, or some combination of both. Our methods do not permit us to decide which of these alternatives is correct. These results are similar to those observed on the corpora cardiaca of Blaberus cranizyer by HODGSON and GELDIAY (1959), on the goldfish preoptic neurosecretory cells by JASINSKI et al. (1966), and on the caudal neurohaemal organ of the teleost fish, Tilapia, by FRIDBERGet al. (1966). The microelectrode studies were directed towards obtaining some basic information on the electrical characteristics of insect neurosecretory cells. The demonstrations of resting membrane potentials of 20 to 40 mV and of depolarizations of 2 to 3 mV lasting for 5 to 12 msec suggest that electrical activity does occur in the neurosecretory cells of the pars intercerebralis. An interesting characteristic of these cells is the long duration of the depolarization. The range of 5 to 12 msec agrees well with the duration of potentials observed in other neurosecretory systems, e.g. YAGI et al. (1963) reported 8 msec duration from the leech: MORITA et al. (1961) and ISHIBA.SHI(1962) recorded 8 to 10 msec duration from the caudal neurosecretory system of the eel; BENNETTand Fox (1962) obtained 5 to 10 msec duration from skates and flukes. If the depolarizations we measured resulted from conducted potentials, then they are much smaller in amplitude than one would expect. This low amplitude may be a reflection of the unipolar characteristic of the insect neurosecretory cell. ROEDER(1963) h as suggested that the conducted potential of an insect unipolar neuron may not invade the cell body. Since we were recording intracellularly from the cell body, the small depolarizations observed may be a reflection of electrical activity occurring somewhere on the axon close to the cell body. The fact that stimulation of the corpora cardiaca could modify these depolarizations, both in frequency and amplitude, suggests a neural connexion between the neurosecretory cell and the corpora cardiaca. We cannot be certain of the pathway involved but it may be the neurosecretory axon itself.
PROTOCEREBRAL NEUROSECRETORY CELLSOF COCKROACH
1791
The depletion of neurosecretory material from the corpora cardiaca, when they were bathed in a Ringer’s solution containing high concentrations of potassium, suggests that depolarization is the mechanism by which a conducted potential could effect the release of neuros~reto~ materials. When the potential invades the axon endings it would depolarize them, and this could lead to the release of the neurosecretory materials. The long duration of the potentials might facilitate the release by keeping the membrane depolarized sufficiently long for the release to occur. In conclusion, the findings reported here support the assumption by VAN DER KLOOT (1955), and many others, that the secretory activity of the neurosecretory cells is controlled by nervous activity in other brain cells and in the neurosecreto~ cells themselves. The neurosecreto~ cells may be the centre for transduction of information from a nervous code into a chemical code; that is, from conducted potentials to hormones. The problem of how such transduction occurs seems central to understanding the mechanisms controlling development and diapause in insects. REFERENCES ADRIANR. H. (1956) The effect of internal and external potassium concentration on the membrane potential of frog muscle. J. PhysioE. 133, 631-658. BENNETT M. and Fox S. (1962) Electrophysiology of caudal neurosecretory cells in the skate and fluke. Gen. camp. Endocr. 2, 77-95. BERN H. A. and YAGI K. (1965) Electrophysiology of neurosecretory systems. Proc. 2nd int. Congr. Endocr. pp. 577-583. Excerpta Medica Foundation, New York. DOGRAG. S. and TANDENB. K. (1964) Adaptation of certain histological techniques for in situ demonstration of the neuroendocrine system of insects and other animals. Quart. J. micr. Sci. 105,455-466. EVEN A. B. (1962) An improved aldehyde fuchsin staining technique for neurosecretory products in insects. Trans. Am. micr. Sot. 81, 94-96. FRIDBERGG., IWASAKIS., YAGI K., BERN H., WILSON D., and NISHIOKAR. (1966) Relation of impulse conduction to electrically induced release of neurosecretory materials from the urophysis of the teleost fish, Xilapia mossambica. J. exp. Zool. 161, 137-149. HODGSONE. and GELDIAY S. (1959) Experimentally induced release of neurosecretory materials from roach corpora cordiaca. Biol. Bull., Woods Hole 117, 275-283. ISHIBASHIT. (1962) Electrical activity of the caudal neurosecretory cells in the eel ~~uiZZa japonica with special reference to synaptic transmission. Gen. corn& Endocr. 2,415-424. JASINSKIA., GORBMANA., and HARA “I’. (1966) Rate of movement and redistribution of stainable neurosecretory granules in hypothalamic neurons. Science, IV. Y. 154,776-778. MANSINGHA. and SMALLMANB. N. (1967) The cholinergic system in insect diapause. J. Insect Physiol. 13, 4-47-467. MORITAH., ISHIBASHIT., and YAMASHITA S. (1961) Synaptic transmission in neurosecretory cells. Nature, Land. 191, 183. PANTINC. F. (1962) Notes on ~icroscopjcaZ Tech~i~s~or Zoologists. Cambridge University Press, Cambridge. ROEDERK. D. (1953) Insect Physiology. John Wiley, New York. ROEDERK. D. (1963) Nerwe Cells and Insect Behavior. Harvard University Press, Cambridge, Mass. SCHOONHOVEN L. M. (1963) Spontaneous electrical activity in the brains of diapausing insects. Science, N.Y. 141, 173-174.
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SHAPPIROD. G., EICHENBAUMD., and LOCKE B. (1965) Cholinesterase in the brain of the cecropia silkmoth in relation to the control of neurosecretion and diapause. Am. Zool. 5, 698699. SHAPPIROD. G., EICHENBAUMD., and LOCKE B. (1967) Chohnesterase in the brain of the cecropia silkmoth during metamorphosis and pupal diapause. Biol. Bull., Woods Hole 132, 108-125. TYSHTCHENKO V. and MANDEUTAMJ. (1965) A study of spontaneous electrical activity and localization of cholinesterase in the nerve ganglia of Antheraea pe~yni G&r. at different stages of metamorphosis and in pupal diapause. J. Znsect Physiol. 11, 1233-1239. VAN DERKLOOT W. G. (1955) The control of neurosecretion and diapause by physiological changes in the brain of the cecropia silkworm. Biol. Bull., Woods Hole 109, 276-294. YAW K., BERN H., and HAGADORN I. (1963) Action potentials of neurosecretory neurons in the leech Thercnnyson rude. Gen. camp. Endocr. 3,490-495.