J. Inwcr Physiol. Vol. 39, No. 8, pp. 701-707. 1993 Printed in Great Britain. All rights reserved

0022-1910/93 $6.00 + 0.00 Copyright 0 1993 Pergamon Press Ltd

Brain Factors Control the Activity of Prothoracic Gland Nerves in the Cockroach Periplaneta americana KLAUS RICHTER*

Received 3 December 1992

Neurotropic factors of the brain with stimulating effects on the discharging pattern of the prothoracic gland nerve in the cockroach Periplaneta americana were investigated. Homogenates of brains from 13- to 15- and from 2Oday-old last-instar larvae as well as of corpora cardiaca from 15- to M-day-old larvae stimulated spike activity of the prothoracic gland nerve in in situ and in vitro experiments. The neurotropic effect was dose dependent in a range of 0.75 to three brain equivalents and was mainly due to an increase of the small type of action potentials. The increase of spike activity in preparations without the suboesophageal ganglion differed from those with this ganglion by a factor of 103. Therefore, the effect of brain homogenates on spike activity in the prothoracic gland nerve is possibly dependent on the control of the prothoracic ganglion by the suboesophageal ganglion. The neurotropic brain factors were inactivated by pronase treatment and methanotic extraction. Geltiltration on Sephadex G 100 resulted in two active fractions ranging near the upper and the lower exclusion limit.

Periplaneta americana Brain factors Prothoracic gland innervation Moult regulation

INTRODUCTION

Studies on moult regulation in insects have mainly concentrated on the neurohormonal regulation of the prothoracic glands by the prothoracicotropic hormones from neurosecretory cells of the brain (Gilbert, 1989; Koolman, 1990). However, anatomical evidence of pro- thoracic gland innervation from the suboesophageal, prothoracic and mesothoracic ganglia in Blattodea, Coleoptera, Diptera and Lepidoptera (Herman, 1967; Richter, 1986) as well as some physiological indications of nervous influences on prothoracic gland activity (Mala and Sehnal, 1978; Richter, 1983, 1985; Okajima et al., 1989; Budd et al., 1992) suggest that the control of the moulting glands, at least in these insects, is considerably more complex.

The anatomy of Peripianeta offers some advantages for experiments on prothoracic gland innervation (Richter, 1985, 1993). The prothoracic gland of Periplaneta americana is innervated by neurones of the prothoracic ganglion, via the prothoracic gland nerve (Birkenbeil and Agricola, 1980). In this nerve two types of efferent action potentials are differentiated in the pattern of discharges (Richter, 1983, 1993). Under exper- imental conditions, one type is characterized by an amplitude of 225 PV and a duration of 1.2 ms. The other

*SLhsische Akademie der Wissenschaften zu Leipzig, D-6900 Jena, PF 322, Germany.

type of spike potential has an amplitude of 30 JJV and a duration of 2.3 ms. The electrophysiological features of these two types correspond to those characteristic of motor (the large type) and neurosecretory axons (the small type) as are known in insects (Cook and Milligan, 1972; Orchard and Finlayson, 1976; Zaretsky and Loher, 1983) as well as in vertebrates (Poulain et al., 1977). The small and slow type of action potentials is known also from prothoracic gland nerves of Mamestra brussicae (Okajima and Watanabe, 1989). It was inter- preted as the nervous activity of bundles of neuro- secretory axons in the prothoracic gland nerve (Birkenbeil, 1991; Richter, 1985, 1993).

In Periplunetu the spike activity of the prothoracic gland nerve could be shown in a functional correlation to secretory activities of the prothoracic gland (Richter, 1993). The first peak of ecdysteroids produced by the prothoracic gland between days 18 and 23 of the last larval instar is accompanied by an important increase in the small potential type in the prothoracic gland nerve. The large increase in ecdysteroid titre at the end of the moulting period, on the other hand, is not correlated with the small type of action potentials. The large type of spike potential does not show any significant corre- lations with the secretory activity of the prothoracic gland.

Except for the fact that the function of the first ecdysone peak in cockroaches is unknown as yet, the gland is obviously dependent on nervous connections to

701

702 KLAUS RICHTER

produce the first ecdysteroid peak, as shown by exper- iments with transections of the prothoracic gland nerve in vivo (Richter, 1985).

The spike activities of the neurones of the prothoracic gland nerve in Periplaneta are inhibited by a GABA- ergic mechanism, connected to the suboesophageal ganglion (Richter, 1983, 1986, 1989, 1993). GABA causes an inhibition of ecdysone secretion in pro- thoracic glands as well as an inhibition of discharges of small action potentials in the prothoracic gland nerve. During the first peak of ecdysteroid production by the prothoracic gland this inhibition seems to be lost, resulting in an increase in the small potentials (Richter, 1993).

Compared with other biogenic amines, octopamine shows the most distinct positive neurotropic effect on the small potentials in the prothoracic gland nerve. As a provisional conclusion, octopamine may be a stimulator in contrast to GABA (as an inhibitor) of the prothoracic gland and the prothoracic gland nerve of P. americana (Richter, 1993).

In Periplaneta, the small axons of the prothoracic gland nerve contain neurosecretory peptide granules (Birkenbeil, 1991), whose content has not been identified as yet. These neuropeptides are supposed to participate in the nervous regulation of the prothoracic gland in cockroaches. It is the aim of this investigation to look for further factors, especially peptide factors, of the central nervous system which are implicated in nervous regulation of the prothoracic gland.

MATERIALS AND METHODS

Animals

Prothoracic glands from laboratory-bred P. americana last-instar larvae were dissected as described previously (Richter, 1983). To make the determination of their stage as precise as possible, the animals were selected when freshly moulted from mass culture and held in groups in piacryl containers with perforated lids (15 x 25 x 10 cm) under constant conditions (28°C 5060% r.h., constant light-dark cycle (12/12 h), water and food (standard rat pellets, pulverized) ad libitum, with folded paper as hiding facilities). The mean length of the moulting interval under these conditions was 30.5 + 0.6 days (n = 366).

To record extracellular nervous activity in situ the thoracic region of larvae hxed by needles in a wax dish was dissected to expose the prothoracic gland branch of nerve 4 rlb from the prothoracic ganglion (Richter and Gersch, 1983). For recordings in vitro the gland and the prothoracic ganglion, connected by the prothoracic gland nerve, were kept in small glass dishes with 1 ml Ringer’s solution according to Yamasaki and Narahashi (1959). For maintaining the nervous activity under in vitro conditions, Ringer’s solution must be aerated with oxygen by a small capillary tube. The temperature in all recordings was 20-22°C.

Bioassay

The efferent activity of the prothoracic gland nerve was obtained extracellularily by recordings with suction electrodes (Fig. 1) as described previously (Richter, 1983; Richter and Gersch, 1983). After adapting the prep- aration to the experimental conditions for 5 min nervous activity was recorded as a control of each preparation. Thereafter the homogenates or fractions in question were applied to the perfusion solution (in situ) or to the bath volume (in vitro). Nervous activity was recorded in 5 min intervals following addition of brain homogenates up to 30 min.

Preparation of crude extract

Whole brains as well as separated corpora cardiaca with the corpora allata attached were collected by dissection and stored at -23°C. Crude brain and cor- pora cardiaca extracts were prepared by homogenizing in an ice cooled Potter homogenizer with water. The homogenates were centrifuged at 4°C with 10,000 g until a clear supernatant was obtained. After precipitation of lipids at -23°C overnight the homogenates were cen- trifuged again, the supematant freeze-dried and then re-dissolved and diluted for administration to prep- arations in adequate volumes of Ringer’s solution.

Pronase treatment was carried out with 5 PUK-units (SERVA) to 50 brain equivalents and 30 min incubation at 40°C.

Gel jiltration

Columns (1.3 x 75 cm) with Sephadex G 100 gel were eluted with 0.2 M acidic acid (9 ml/h). The column was

Oxygen

FIGURE I. System for recording nervous activity from the prothor- acic gland nerve in oitro PG, Prothoracic gland; SC, connectives between prothoracic ganglion and suboesophageal ganglion; I, pro- thoracic ganglion; N4, N5, 4th and 5th segmental nerves; R, Ringer’s

solution.

ACTIVITY OF PROTHORACIC GLAND NERVES IN I’. AMERICANA 703

equilibrated by separating a mixture of arginine (mol. wt 174) and blue dextran (average mol. wt 2,000,000).

Crude brain homogenates dissolved in the eluant were applied as samples of 500 ~1 to the column. Fractions of 2.5 ml were collected. Fractions were dried in a rotation evaporator (bath temperature 35°C) and redissolved in Ringer’s solution for testing.

RESULTS

Efect of brain homogenates in situ

There are two characteristic transient peaks in spike activity of the prothoracic gland nerve during the last larval instar of P. americana (Richter, 1993). The first peak appears between days 12 and 15 and the second one between days 20 and 22. The two peaks are characterized by a large increase of discharges of the small type of action potentials. Larvae of days 9 or 10 were chosen as preparations for recording neurotropic effects to avoid interference of effects of brain homogenates with the natural variations in spike pattern typical for later periods of the last-instar of Periplaneta. In this period the spike activity of the prothoracic gland nerve is at a low level.

As a first experimental series, effects of brain hom- ogenates on the prothoracic gland nerve in situ were measured. The homogenates were prepared from brains and corpora cardiaca at different times of the last larval instar. The effect of homogenates was dependent on the age of the donors of brains or corpora cardiaca and consisted of increases of efferent spike activity in the prothoracic gland nerve. The strongest effect was ob- served with brain homogenates from larvae between days 13 and 15 and of day 20. Prior to day 13 or later

to day 20 no substantial effect of brain homogenates was observed. Fifteen min after application of a homogenate corresponding to three brain equivalents, the increase of spike activity was about 100% (Fig. 2). In comparison, application of homogenates of corpora cardiaca also resulted in an increase of prothoracic gland nerve ac- tivity. The main effect was induced by corpora cardiaca from days 15 and 16. The increase induced by a hom- ogenate corresponding to three pair corpora cardiaca equivalents was comparable to that of active brain homogenates. Homogenates of corpora cardiaca pre- pared from larvae of other days of the moulting interval, did not show any neurotropic effect.

Homogenate of muscle tissue, as a control, applied to the same type of preparation did not show any effect comparable to brain or corpora cardiaca homogenates. A change in spike frequency of 3 + 27% in comparison to untreated preparations only was detected.

Eflect of brain homogenates in vitro

The spike activity of the prothoracic gland nerve measured under in situ conditions was sometimes disturbed by respiration and other body movements. Therefore, an isolated preparation (last-instar larvae of days 9 or lo), consisting of the prothoracic ganglion together with the prothoracic gland, both connected by the prothoracic gland nerve was used under in vitro conditions. The course of spike activity of such a preparation is shown in Fig. 3.

During the first 5 min of measuring a small and transient peak of activity was to observe as an unspecific postpreparatory excitation effect. Thereafter, during the next 25 min the activity decreased slowly to a steady level of about 1 imp&e/s. Administration of a homogenate of three brain equivalents from larvae of days 14/15 of

I I I I I I - 5 10 15 20 25 30 b.e.

Time (days)

FIGURE 2. Change of nervous activity (%) in the prothoracic gland nerve from larvae of the day 9-10, 15 min after administration of homogenates, containing three brain equivalents (solid bar) or three pair corpora cardiaca equivalents (open bar) of different age in the last larval instar (time, days) in in siru preparations. For comparison the course of spike activity (-_O--) in the prothoracic gland nerve (Imp./s) during the whole moulting interval is shown (after Rich;er, 1993). Each column

is the mean (+ SEM) of IO-15 measurements.

704 KLAUS RICHTER

16

2

T

I I I t I 5 10 15 20 25 30

Time (min)

FIGURE 3. Effect of brain homogenate (three brain equivalents) from days 14/15 of the last larval instar on the nervous activity of the prothoracic gland nerve under in vitro conditions. The preparation consisted of the prothoracic ganglion only. -O--, Untreated control; -@-, administration of brain homogenate, 5 min before start of recording. Each point is the mean (f SEM) of 15 measurements;

l significance (P = 5%).

I I I f I I 5 10 1s 20 25 30

Time (min)

FIGURE 4. Effect of brain homogenate (three brain equivalents) from days 14/15 of the last larval instar on nervous activity of the prothor- acic gland nerve under in vitro conditions. The preparation consisted of the prothoracic ganglion connected with the suboesophageal ganglion. Thick lines, whole, undifferentiated nervous activity; thin lines, effect differentiated for the small action potentials only. -_O-, Untreated control; -a--, administration of brain homogenate, 5 min before start of recording. Each point is the mean (&SEM) of 10-15

measurements.

the moulting interval resulted in a doubling of spike activity within 2 min. This increase remained constant during the following 30 min. A slight increase was noticed and became statistically significant 5 min after administration (Fig. 3).

The effect of brain homogenates on spike activity in the prothoracic gland nerve depended on the connection of the prothoracic ganglion to the suboesophageal ganglion. In preparations in which the functional con- nection between these two ganglia was intact, the spike activity in untreated controls remained on a low level, as a result of the inhibitory effect of the suboesophageal ganglion on the spike activity of the neurones in the prothoracic ganglion (Richter, 1985). Administration of three brain equivalents to this kind of preparation resulted in an increase of spike activity only during the first 15 min of the experimental interval. Later, the effect was suppressed by about 50% (Fig. 4). The inhibiting effect of the suboesophageal ganglion could be shown by comparison of the spike activity after administration of homogenate to preparations with and without connec- tion to the suboesophageal ganglion, respectively. The increase of spike activity in preparations without sub- oesophageal ganglion differed from those with this ganglion by a factor of lo3 (Fig. 5).

The increase in spike activity after administration of brain homogenates was mainly due to an increase of the small type of action potentials. As shown in Fig. 4 course and amount of the entire nervous activity are identical with course and amount of the small type of spikes in the prothoracic gland nerve during the experimental interval.

Experiments with different concentrations of brain homogenates showed a dose dependence of the neuro- tropic effect. An extract containing the equivalent of 0.75

I I I I I I 5 10 15 20 25 30

Time (min)

FIGURE 5. Increase of nervous activity in the prothoracic gland nerve of an isolated prothoracic ganglion (-_O--) and of a ganglion in functional connection to the suboesophageal ganglion (-a-) in comparison to untreated controls (%) under in vitro conditions. Administration of brain homogenate corresponding to three brain

equivalents, 5 min before start of recording.

ACTIVITY OF PROTHORACIC GLAND NERVES IN P. AMERICANA 705

% 1000 x

.,

.? s 800

m

z 0 600

2

z 400 0

FJ 2 200 k 0

Brain equivalents

0 0.75

0 1.5

0 3

0 6

5 10 15 20 25 30

Time (min)

FIGURE 6. Dependence of the neurotropic effect in the prothoracic gland nerve on the concentration of brain homogenate. Prothoracic ganglion in vitro; homogenate administration 5 min before start of

recordings.

brain was sufficient to stimulate spike activity by 200% 20 min following administration.

The effect reached saturation with a homogenate concentration of three brain equivalents (about lOOO%, 20 min after administration). Higher concentrations did not lead to a further increase of nervous activity (Fig. 6).

Preliminary experiments to characterize the brain factor

The neurotropic activity of brain homogenates was lost after extraction with ethanol as well as by treatment with the enzyme pronase. Pronase inactivated by boiling (3 min at 1OO’C) did not destroy the neurotropic activity of the brain homogenate. Extraction of brain homogen- ates with methanol resulted also in a loss of neurotropic activity. On the other hand, heating the homogenate for

I I I I 5 10 15 20 25 30

Time (min)

FIGURE 7. Nervous activity in the prothoracic gland nerve of a separated prothoracic ganglion in oirro after administration of brain homogenates treated in different ways. -_O-, Activity of a normal, untreated preparation (control); -•--, homogenate corresponding to three brain equivalents; -_O-, ditto homogenate, methanolic extract; - x -, ditto homogenate, inactivated by boiling for 3 min; --O- -, ditto homogenate, inactivated by incubation with pronase; ---o---,

ditto homogenate, after incubation with inactivated pronase.

Blue Dextran Arginin 1.0

10 20 30 40 50 60 70

Effluent volume (ml)

FIGURE 8. Gel filtration of a brain homogenate (200 brains) on Sephadex G 100. The column was calibrated with blue dextran and arginin as molecular weight markers. Solid bar, increase of nervous

activity in the prothoracic gland nerve; --, E280nm.

2 min to 100°C was without influence on the activity of the homogenate (Fig. 7).

In a preliminary experiment, a homogenate of 100 brains including corpora cardiaca was separated by gel filtration on Sephadex G 100. The collected tissue was homogenized in ice cooled water and applied to the column as a sample of 500 ~1, purified by centrifugation.

Of all fractions neurotropic activity was detected in two peaks (Fig. 8), one near the upper exclusion limit and the other before the lower exclusion limit. When tested on the prothoracic gland nerve activity both fractions showed about the same effectiveness.

DISCUSSION

It was shown in previous investigations that ecdy- steroid production in the prothoracic gland of P. amer- icana in the last larval instar is regulated at least in two steps (Richter, 1993). One step, namely the main peak of ecdysteroid production immediately before moult is dependent on a PTTH-like hormone, which has not been characterized as yet sufficiently in Periplaneta (Richter, 1992). Another step of moulting gland regu- lation occurs in about the middle of the moulting interval. In this period, between day 12 and 15, a first ecdysteroid peak starts to appear. This first peak reaches its maximum on day 20 (Shaaya, 1978; Richter, 1985). Already on day 20 the brain produces a PTTH-like factor, which stimulates ecdysone production in the prothoracic gland in vitro (Richter, 1992). At the begin- ning of this period, between days 12 and 15, the spike activity of the prothoracic gland nerve increases by about 100% to a transient peak. This means that the first ecdysone peak of the prothoracic gland is characterized by an increase in prothoracic gland nerve activity at its beginning and a PTTH-activity of the brain in its main phase, Nevertheless, in earlier investigations the nervous

706 KLAUS RICHTER

activity of the prothoracic gland nerve was shown to be essential for normal function of the gland in this period (Srivastava et al., 1977; Richter, 1985).

There is no information about the physiological significance of this first activity period. Unpublished observations in our laboratory suggested that some behavioural changes, like reduction of food and water intake and modifications in locomotor activity occur between days 12 and 15.

In Peripluneta, efferent nervous activity of neuro- secretory axons of the prothoracic gland nerve originates from the prothoracic ganglion and is inhibited in the first period of the moulting interval, i.e. up to days 12-15. In previous experiments, GABA could be shown to be an inhibitor of the spike activity of neurosecretory axons in the prothoracic gland nerve as well as of ecdysteroid secretion in the prothoracic gland. In Peripluneta, this GABA-ergic mechanism originates from the suboeso- phageal ganglion (Richter, 1989) as was also shown in Man&u larvae by Jungreis and Omilianowski (1980) and Gibbs (1984) and in blowfly larvae by Kauser et al. (1988). This inhibition may be assumed to cease at the beginning of the first activity period of the gland, starting about day 12 (Richter, 1992).

The neurotrophic brain factors are supposed to be candidates in disinhibiting the moulting gland for the first secretory interval by stimulating the prothoracic gland nerve activity. This is supported by the fact that the appearance of the brain factors is restricted to the few days around the beginning of this period. Further- more, the appearance of the factors is accompanied by an increase in efferent spike activity in the nervus corporis cardiaci II in this period (Richter, 1981), which is supposed to be characteristic of transportation of neurosecretory products from the brain to the corpora cardiaca and of their release.

In the corpora cardiaca homogenates the neurotropic effect on the prothoracic gland nerve appears l-2 days later than the effect of the brain homogenates. The effectiveness on the prothoracic gland nerve activity are similar in homogenates of one pair corpora cardiaca and one brain. The tissue volume of a corpus cardiacum in Periplunetu (according to the protein content) is only l/20 that of a brain (Baumann, personal communi- cation). This means that the concentration of the neuro- tropic factors in the corpora cardiaca is higher than in the brain. Because of the relative high concentration in the corpora cardiaca the factors are supposed to be produced in the brain and stored in a high amount for release in the corpora cardiaca. Beside the effects of brain homogenates of days 14/15 on prothoracic gland nerve activity were also observed with brain homogen- ates of day 20, but not with homogenates of corpora cardiaca (Fig. 2). This second neurotropic activity period of the brain appears at the beginning of the second peak of spike activity in the prothoracic gland nerve (Richter, 1993). In the present investigation this period was disre- garded. A comparison of the scarce information about implications of nervous activities in moulting gland

regulation suggests that in various insect species these relations are different.

Experiments on the role of prothoracic gland inner- vation in regulation of ecdysone production and release in the prothoracic gland beside Periplunetu are described by Okajima et al. (Okajima and Kumagai, 1989; Okajima and Watanabe, 1989; Okajima et al., 1989) on M. brussicue. In the larvae of this lepidopterous insect efferent nervous activity in prothoracic gland nerves was shown to inhibit ecdysone release. Disinhibition takes place by afferent nervous activity in the prothoracic gland nerves elicited by elevation of the trehalose level in the haemolymph. In Mumestru there is no information about the participation of the brain and the sub- oesophageal ganglion in this regulatory system. In larvae of Gulleria mellonellu, Mali and Sehnal(l978) described activating as well as inhibiting influences of the CNS on the prothoracic gland, not further detailed as yet. An inhibitory influence of the three thoracic ganglia via the brain on ecdysone release in the prothoracic glands was shown in G. mellonella by Predel(1992), whereas the role of the nerves running to the prothoracic glands remains contradictory.

Our experiments lead to the conclusion that the prothoracic gland of Periplunetu is inhibited by neuronal pathways from the suboesophageal ganglion (Richter, 1989) and disinhibited by brain factors effective on neurosecretory neurons of the prothoracic gland nerve.

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Acknowledgemenu-The author is grateful to Mrs G. Radtke and

Mrs R. Meissner for excellent technical assistance and to Professor

J. Koolman (Marburg), Professor H. Penzlin (Jena) and Dr G.-A.

Bijhm (Jena) for critical reading of the manuscript.