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The distribution of Factor S in the cockroach, Periplaneta americana, and its role in stress paralysis
J. Insect Physiol., 1969, Vol. 15, pp. 963 to 975. Pergamon Press. Printed in Great Britain
THE
DISTRIBUTION OF FACTOR S IN THE COCKROACH, PERIPLANETA AiWERICANA, AND ITS ROLE IN STRESS PARALYSIS* B. J. COOK,
M. DE LA CUESTA,
and J. G. POMONIS
Metabolism and Radiation Research Laboratory, Entomology Research Division, Agriculture Research Service, United States Department of Agriculture, Fargo, North Dakota 58102 (Received 23 December 1968) Abstract-A quantitative bioassay was used to determine that the nerve cord and head of the cockroach, Penplaneta americana (L.), contained the highest concentrations of Factor S ; the legs and body contained lesser amounts. A twofold increase in the titre of Factor S was observed by spectrofluorometry and by bioassay in insects subjected to 4 hr of mechanical stress. compared with the amounts in unstimulated insects. Houseflies, Musca dumestica (L.), injected with 30 ,ug of the active substance were immediately parrdysed: 60 per cent were unable to right themselves within 2 hr; the other 40 per cent showed a marked impairment in motor co-ordination. A substance that caused much the same biological response as Factor S was found in perfusates from electrically stimulated nerve cords of cockroaches. Exposure of the last abdominal ganglion of the cockroach to 8 pg of Factor S caused facilitation of the post-synaptic spike potential followed by after-discharges lasting 30 to 80 msec,; 12 pg of the active residue partially blocked synaptic conduction. A parallel was drawn between the synaptic responses with increasing concentrations ,of Factor S and those occurring at progressive stages in stress paralysis. Factor S showed several properties typical of neurohormones, and an involvement in the mode of action of DDT is suggested. INTRODUCTION
INSECTS subjected to excessive stress are known to release pharmacologically active agents (STERNBURG, 1963) and large amounts of these agents cause autointoxication that often results in paralysis and death. However, the chemical nature of the majority of these substances is not known though one is clearly associated with the neuroendocrine complex. DAVEY (1963) showed that cockroaches, .Per@aneta amwicuna (L.), subjected to enforced activity released a cardiac accelerator from the corpora cardiaca. More recently, KATER (1968) demonstrated that this proteinaceous heat-stable substance was released from the same organ after electrical stimulation of the brain. The effect of this substance on the general behaviour of insects remains obscure. * Mention of a proprietary product does not necessarily imply endorsement product by the United States Department of Agriculture. 963
of this
B. J. COOK,M. DE LA CTJESTA, ANDJ. G. POMONIS
964
Another pharmacologically active substance in insects was reported by BEA~MENT (1958) and STEFWWJRG et aZ. (1959). Both papers described a paralytic agent that appears in the blood of cockroaches prostrated by either electrical stimulation or by exposure to DDT; they concluded that it was released from the central nervous system. A relationship between it and Factor S, a neuromuscular excitatory substance recently obtained from insects (COOK, 1967), seemed possible. MATERIALS Extraction
AND lMETHODS
procedure
Although the general method for the extraction of Factor S was described by COOK (1967), we have modified certain details in the procedure to ensure greater consistency in the yield of the active agent. After the tissues were ground in 3 vol. of 8% trichloroacetic acid, the homogenates were allowed to stand for 2 hr at 4°C before centrifugation at 10,000 g; the supernatant obtained from the centrifugation was treated with ether (1 part ether to 2 parts supernatant). This mixture was shaken vigorously in a separator-y funnel for 5 min, the two phases were allowed to separate, and the water fraction was retained. The procedure was repeated at least three times, and if the water fraction still retained some characteristics of an emulsion, it was extracted further with ether to remove the excess lipid. Since Factor S is specifically adsorbed on aluminium hydroxide suspended in solution, the precipitate was retained and dissolved in 1 N sulphuric acid. Only a sufficient amount of acid was added to bring the pH to 3. Addition beyond this, point, even if the precipitate was not completely dissolved, had a detrimental effect on the active principle. Chrmatographic
pw$cation
of Factor S
Large extracts of whole insects were subjected to cation exchange chromatography on Amberlite IRC 50 by the method of BERGSTROM and HANSSON(1950) ; Amberlite CG-50, 100/200 mesh, was used for extracts of small portions of insect tissue. The dimensions of the column bed were 35 x 6 mm. Extract residues suspended in 1 ml or less of buffer were introduced onto the column_ The column containing the tissue extract was eluted with 25 ml of 0.2% sodium chloride and then by 25 ml of 1 N HCl. Factor S was found in the N HCI fraction from the column; this fraction was therefore taken to dryness, and the residue was dissolved in O-2 ml of ethanol. Factor S appeared as a pink spot on paper chromatograms sprayed with potassium ferricyanide. The R, region corresponding to the pink spot in the phenol-HCl system was eluted from the remainder of the chromatogram and subjected to rechromatography on paper in butanol-acetic acid-water (4 : 1 : 5) prior to bioassay. Small tissue extracts, however, were chromatographed on cellulose thin layer in a butanol-ethanol-1 N acetic acid (3 : 1 : 1) system. The neuroactive area was located on the plates as a yellow-to-brown spot with naphthoquinone-4-sulphonate.
FACTORS
IN THE COCKROACH,
PERIPLANETA
AMERICANA
965
However, this reaction was not specific for Factor S, so several regions were routinely scraped from the plates of each sample. The fractions of interest were then eluted in several ml of 95% ethanol, taken to dryness, and resuspended in 200 ~1 of physiological saline solution (TWAROG and ROEDER,1957).
Physiological preparations Isolated ventral nerve cord. Factor S showed two very characteristic effects on the spontaneous activity of the isolated nerve cord of the cockroach: a two- to threefold increase in arrhythmic activity, and recurrent trains from O-5 to 1 set in duration. These trains were frequently initiated by large bursts of high-frequency pulses lasting from 100 to 200 msec (COOK, 1967). The thoracic portion of the nerve cord from the adult American cockroach was routinely used for quantitative bioassay. The preparation and recording procedures have already been described (COOK, 1967). For studying the release of Factor S from the central nervous system, two nerve cords containing both abdominal and thoracic portions were excised from male cockroaches and placed in 80 ~1 of saline solution. One electrode from a laboratory stimulator was attached to the connective between two of the abdominal ganglia. The indifferent electrode was submerged in the saline bath. Recording electrodes were attached between the first, second, and third thoracic ganglia of the preparation. Spontaneous activity was monitored for 5 or 10 mm before stimulation. The preparation was then stimulated continuously for 30 to 45 min at a pulse strength of 1.2 V, a duration of 0.1 msec and a frequency of 100 cycles/set. Stimulation was interrupted occasionally to .obserKe spontaneous activity. During the first few minutes the cord responded to each stimulus, but as time progressed the general level of activity gradually declined, and after 30 min it was greatly depressed. At that time the saline solution was usually withdrawn and saved for bioassay or chemical analysis. Synaptic conduction in the last abdominal ganglion Post-synaptic responses were recorded from an electrode pair on one of the connectives near the fifth abdominal ganglion. The cereal nerves on the same side were stimulated by a square pulse of 0.1 msec duration and at an intensity of O-6 to 2 V and a frequency of O*fi/sec. The preparation was frequently moistened with several ~1 of physiological saline solution, and samples containing Factor S were applied in ~1 quantities to the dorsal surface of the ganglion.
Mechanical stimulation of cockroaches Adult cockroaches of both sexes were tumbled in 1 gal jars 6 in. wide. These jars were made to rotate on their own axes at 90 rev/min by the powered rollers on which they rested. After 4 hr, 30 to 40 per cent of the insects were paralysed. They were unable to right themselves once they were placed on their backs, and when they were placed upright and subjected to acute cereal stimulation, their legs moved in a slow, unto-ordinated fashion or not at all.
B.J.
966
COOK, M.DE
LA CUESTA,AND J; G.POMONIS RESULTS
Distribution of Factor S in the tissues of the American cockroach Until the substance or substances responsible for the neuroexcitatory activity can be chemically identified, we have defined its action in terms of activity units: one unit is that amount of tissue (pg/pl) required to either induce a perceptible rise in spontaneous activity (10 to 20 per cent) or elicit bursting trains on the isolated nerve cord during a 10 min period. Since increasing concentrations of Factor S brought more immediate responses from the nerve cord, an arbitrary scale was defined in units from 1 to 5 (Table 1). If an extract induced an immediate rise in spontaneous activity of 50 per cent or more with recurrent bursting trains, a designation of 5 activity units was given. TABLE I-DISTRIBUTION OF FACTOR S IN THE TISSUESOFTHE ~OCKXOACH, P. americana, DETERMINED BY QUANTITATIVEBIOASSAY
Tissue from 175 insects Nerve cord Head Body: Legs
Relative response of isolated nerve cord to dilutions of the various tissue extracts (expressed in activity units) t
Wet wt. of tissue before extraction (g)
Zone of neuroactivity* (Rf)
0.78 6.5 70.0 26.2
0.17-0.31 O-0.21 0.20-0.27 O-0.15
1 : 200 dilution (mg-equiv./ 20 CLl) 5 4 3 3
0.35 2.92 3140 llG30
1 : 1000 dilution
1 : 2000 dilution
4 3 2 1
4 3 1 0
* After chromatography on cellulose thin layer with butanol-ethanol-I N acetic acid (3 : 1 : 1). t Definition of activity units: 5= immediate rise in activity 50 per cent or more with recurrent bursting trains; 4= rise in activity to 50 per cent within 4 min with bursting trains; 3= rise in activity to 25 per cent within 6 min with bursting trains; 2= rise in activity to 25 per cent within 8 min with occasional bursting trains; 1 =either a slight rise in activity or occasional bursting trains during 1Omin period; 0 = no response over 1Omin period. $ Bodies minus nerve cord.
Table 1 summarizes the distribution of Factor S determined by quantitative bioassay and represents one of three experiments. In undiluted samples, several chromatographic fractions from each tissue showed neuroactivity, but as the fractions were diluted, only a single area eventually displayed biological activity. When isolated nerve cords were perfused with 20 ~1 of a 1 to 2000 dilution of fraction Rt O-17 to O-31 from the extracted nerve cords, a 25 per cent increase in arrhythmic activity coupled with bursting trains were evident within 4 min after application. This 20 ~1 sample was equivalent to O-01 of a nerve cord or 35 pg of nerve tissue, wet weight. The same dilution from extracts of the cockroach body caused a response from the nerve cord within 10 min; the 20 ~1 aliquot in this instance contained the equivalent of 3.2 mg wet weight. Extracts from the cockroach leg showed no neuroactivity with sample equivalents of 180 pg of tissue.
FACTOR S IN THE COCKROACH, PERIPLANETA
AMERICANA
967
A comparison of these results clearly indicates that the nerve cord and the head are at least a hundred-fold more potent than the body and leg regions. If we take into account the activity units designated for each body region at the 1 to 2000 level, the nerve cord could be as much as four hundred-fold more potent than the body. Although the majority of Factor S is contained in the central nervous system, this fact alone does not establish it as a transmitter. Yet it is an essential corollary to any further considerations regarding the neurotrophic properties of this compound or compounds. Relation
of titre of Factor S to stress paralysis
Since the American cockroach suffers a severe neuromuscular disorder after exposure to sustained mechanical stress (BEAMENT, 1958), the titre of Factor S might account for the onset of paraIysis observed by both BEAMENT(1958) and STERNBURG et aZ. (1959). Therefore, 650 normal cockroaches (590 g) were extracted and the extract was purified to determine the amount of Factor S that was present. A second group of equal number and approximate weight (560 g) were mechanically stimulated by tumbling in jars for 4 hr; 50 to 60 per cent of this group were either hyperactive or paralysed. The tumbled insects were then extracted and the extract was purified. A quantitative comparison was made between the two groups by employing the fluorescent complex that Factor S forms with potassium ferricyanide (COOK, 1967). One-fifth of the extract (95 g of tissue) from both treated and untreated groups was spotted on Whatman No. 1 filter paper, chromatographed in phenol-O*1 N HCI (1 : 1) and sprayed with potassium ferricyanide. Factor S, appearing as a pink spot in each sample, was eluted in 95% ethanol. A reference blank was obtained by eluting the region between the two samples that had the same R, and approximate area. An emission spectrum of the eluates was obtained with an Aminco Bowman spectrofluorometer. A peak emission was obtained for the complex at 350 rn,u (Fig. 1). A twofold increase in the titre of Factor S was observed in the stimulated cockroaches over that in the unstimulated insects. The blank showed essentially no fluorescence in the same region of the spectrum. Quantitative bioassay with the isolated nerve cord on extracts from the two groups of insects confirmed the spectrofluorometric results. Physiological saline solutions containing 9.5 g of tissue/O.5 ml of saline showed 1 activity unit for the untreated groups, ‘while the same amount of tissue/saline from the stimulated cockroaches showed 3 activity units. Response of hozlsefies to injections of Factor S Since houseflies contain Factor S (COOK, 1967), they were injected with various concentrations of the extract known to contain the neuroactive substance. The extract residue was suspended in housefly saline solution (BREBBIA and
968
B. J. COOK,M.
DE LA
CUESTA, ANDJ. G. POMONIS
LUDWIG, 1962), and each insect was injected with 1 ,ul or less in the region of the pronotum.
IO-
200
I 250
300 350 WAVELENGTH
400
450
500
FIG. 1. Comparison of fluorescent emission spectra of potassium ferricyanide derivative of Factor S from equal weights of normal vs. stimulated cockroaches. (A) Cockroaches stimulated 4 hr, (B) unstimulated cockroaches, (C) blank filter paper and reagent excitation 300 rnp. Before treatment, insects were subjected to cold anaesthesia, but they usually revived prior to injection. Controls injected with eluates from blank chromatograms showed no abnormal behaviour. The two dashed lines in Fig. 2 trace the time course of recovery for houseflies injected with 1.5pg of Factor S. The curve of open circles represents the number of houseflies that were able to right themselves and move about to some extent. The second curve (closed circles) traces the course of total recovery after the injection of 1s ,ug of Factor S. For the first hour after injection,
TIME
IN HR
FIG. 2. The time course of response of houseflies to injections with various amounts of Factor S. Curves A (15 pg), C (30 pg), E (60 pg) show percentage of insects able to turn over. Curves B (15 pg) and D (30 pg) show percentage of insects with no ataxia.
FACTOR S IX THE COCKROACX,
PERTPLAXETA
A&IERI&Ah-A
969
95 to 100 per cent of the treated flies were either totally paralysed or unable to co-ordinate their movements. The curve traced by the solid line and open triangles represents the response of houseflies treated with 30 pg of Factor S. At this concentration, 83 per cent were still completely paralysed 1 hr after injection; after 2 hr, 60 per cent were paralysed, and the remaining 40 per cent showed abnormal behaviour. The solid and open squares in Fig. 2 represent the response of houseflies treated with 60 pg of Factor S. At this concentration, 90 per cent of the individuals were paralysed after 24 hr, and the remaining 10 per cent could right themselves but still showed abnormal behavioural characteristics. Immediately after each injection, the flies generally displayed a flaccid type of paralysis. When they were able to move and right themselves, they showed a marked ataxia, occasional tremors, and excessive salivation. As the flies approached normal behaviour, occasional clonic convulsions were evident in some individuals. In contrast to the results with houseflies, cockroaches, injected with several hundred pg of Factor S showed only transitory indications of ataxia and no knockdown. This anomaly might be explained by the fact that the cockroach is more readily able to metabolize Factor S than the housefly. Also, an active enzyme system for the degradation of Sternburg’s neuroactive substance seems to be well documented (EATON and STERNBURG, 1967). This is certainly supported by the observation that although 30 to 40 per cent of the cockroaches subjected to stress were paralysed after 4 hr, some recovered quite rapidly from the effects of paralysis and appeared normal within 30 to 45 min. The release of pwoactive
substance from the isolated nerve cord
Electrical stimulation of the isolated nerve cord of the cockroach clearly caused the release of a neuroactive agent in 8 out of 10 experiments. The typical response of the isolated nerve cord to the application of perfusates from stimulated nerve cords is shown in Fig. 3. The neuroactive material or materials in the perfusate increased the level of arrhythmic activity within several minutes after application and often caused recurrent bursting trains. The character of this response has a definite resemblance to that found with tissue extracts of Factor S. The identity of the material found in perfusates with Factor S was further substantiated by experiments on synaptic conduction in the last abdominal ganglion. In some preparations when several ~1 of the perfusate were placed on the dorsal surface of the terminal ganglion, a definite and persistent after-discharge was produced. In other experiments, an increase in the threshold for the post-synaptic response was observed. It is almost a certainty that more than one active substance is being released from the nerve cord during electrical stimulation, but our present methods of spot test analysis are not sufficiently sensitive to detect the material of interest in the perfusates. Nevertheless, it is encouraging to note that neuroactive materials can be released into perfusates from stimulated nerve cords, and it lends support to the possibility that Factor S is indeed released from the central nervous system, as indicated by the in vivo experiments on stimulated cockroaches.
970
B. J. COOK,M.
DE LA
CTJESTA, ANDJ. G. POMONIS
Effects of Factor S on synaptic conduction Although the endogenous activity of neurons in the isolated nerve cord of the cockroach provides an extremely sensitive preparation for the bioassay of neuroactive agents, it is difficult to ascribe any specific function to a substance on the basis of its application to such a preparation since sensory, motor, and inemuncial elements are present. The synapses of the last abdominal ganglion provide a preparation with greater functional significance. This ganglion contains a key synapse in the well-known evasion reflex arc in the American cockroach. A study of the effects of Factor S on such a preparation might give some indication of the function of this substance in the central nervous system. Fig. 4 shows one of the ten experiments with various concentrations of Factor S. The normal post-synaptic response in the terminal ganglion is shown in Fig. 4(A) and (B). I mmediately after applying 8 pg of Factor S to the dorsal surface of the ganglion, a rise in spontaneous central activity together with recurrent trains were often observed. After 1 mm, the giant fibre spike potential showed a marked depression followed by a distinct after-discharge (Fig. 4C). These after-discharges consisted of a short series of spikes following the arrival of a single maximal pre-synaptic volley. Normally, after-discharges are produced only after a period of synaptic inactivity; once the synapse has adapted, a single spike response occurs to each limital stimulus of the cereal nerve (PTJMPHREY and RAWDON-SMITH, 1937). Not only were the after-discharges of the post-synaptic response persistent in treated ganglion, but they and the giant fibre spike showed facilitation (Fig. 4D and E). Exposure of ganglion to 12 s of active residue caused conduction failure, and an increase in stimulus intensity (1 to 2 V) was required to reinstate the postsynaptic response. Ganglions perfused with 1 ,~l of saline solution containing O-4 pg of Factor S showed a persistent after-discharge with the giant fibre response.
Synaptic conduction in stress-paralysed insects HESLOP and RAY (1959) observed recurrent trains of impulses passing along the abdominal nerve cord in stress-paralysed cockroaches, and these trains were associated with synaptic facilitation in the last abdominal ganglion. Moreover, after thorough washings of the body cavities of paralysed insects, the trains ceased entirely in several cases, and facilitation could no longer be detected in the ganglion. These observations suggested a possible involvement of central synapses in the neuromuscular disorders apparent in stressed insects. In an effort to clarify these observations and correlate the changes in synaptic conduction during the course of paralysis with the effects of various concentrations of Factor S, a number of cockroaches were subjected to mechanical stress as described earlier. After 4 hr of tumbling in the jars, the cockroaches could be placed in various categories according to the behaviour that they displayed (Table 2).
A
B C
!) t:
F G
FIG. 3. T h e effect of perfusates from stimulated nerve cords on spontaneous activity of the isolated nerve cord of the cockroach. (A) N o r m a l activit3, ; (B) 3 min after application of perfusate from stimulated nerve cords ; (C) 5 re.in ; (D) 7 rain ; (E) 9 rain; (F) 11 m i n ; (G) 13 rain; (H) reversible block at 15 rain elapsed time: time mark 50 msec.
A b---4
B C
b----4 °
•
mr
D
ilr,i
' '
EJ FIG. 4. Post-synaptic response of terminal ganglion in P. americana to a perfusion of 2/,1 (8/zg) of Factor S. (A) Several normal spike potentials, time mark 2 reset; (B) spike potential 1 rain after perfusion of 2/zl of saline; (C) spike potential and after-discharge 1 min after perfusion with Factor S, time mark 5 msec; (D) same preparation 7 rain after application, time mark 10 msec; (E) same preparation 7 min after application, time mark 3 msec.
]
I
I
o~ ,-"
....
~
II,
X
r
•
rn
0
©
FACTOR S IN THE COCKROACH, P.ERIPi.ANZTA
TABLE
~-RESPONSES
OF CENTRAL NERVOUS SYSTEM OF
DURING
Condition of insect. Normal to hyperactive Torpid with ataxia Paralysed
971
AMERICANA
P. americana
THE COURSE OF STRESS PARALYSIS
NO. examined
Endogenous activity of abdominal nerve cord (No. showing trains)
Post-synaptic responses of the last abdominal ganglion No. showing afterdischarge
No. showing increased threshold
No. showing conduction failure
17
5
4
3
0
17 25
8 1
6 9
11 16
2 10
1. Normal to hyperactive. Many insects in this category appeared more or less normal but some were hyperactive and easily upset by the slightest stimulus. 2. Torpid with ataxia. These insects could move and right themselves with diEculty and showed an inability to co-ordinate. 3. Paralysed. These insects occasionally showed tremors in the legs but were unable to right themselves and would not move, even after severe stimulation of the cerca. ‘These categories seem to suggest the behavioural pattern sequence that occurs during the time course of paralysis induction. The majority of the insects (60 to 70 per cent) subjected to 4 hr of tumbling A neurophysiological examination of were designated as normal to hyperactive. these cockroaches revealed recurrent trains and after-discharge in 5 out of 17 individuals. Torpid cockroaches often showed an after-discharge in the postsynaptic response to a single cereal stimulus. This was evident even at the threshold of response, and it was a persistent phenomenon. Insects in this same category also showed an elevated threshold for post-synaptic response that often required an increasing voltage intensity (3-10 V) to establish the response. Generally, the stimulus threshold for synaptic conduction in normal cockroaches was between 0.8 and 2.5 V at 0.1 msec duration and O-5 cycles/set. Torpid cockroaches often showed a certain synaptic instability, indicated by an intermittent response to single pre-synaptic volleys (2 to 3 in 5 volleys). This instability was also evident in hyperactive cockroaches. A blockade in synaptic conduction (no response to 15 V) was generally found in paralysed insects. If there was a synaptic response, it was considerably above normal threshold levels. Although conduction failure did occur in paralysed insects, the axons of the giant fibres could still conduct an impulse, and endogenous activity was still evident on connectives from the abdominal nerve cord of many individuals (Fig. 5). HESLOP and RAY (1959) suggested that the neuromuscular disturbance in stressed insects may be a consequence of a reduction in haemolymph volume which, in turn, diminishes the transport of metabolites. It is quite true that
972
B.
J. COOK,M.
DE
LA CUESTA,ANDJ. G. POMONIS
insects placed under stress for 6 to 8 hr are subject to severe desiccation, but this may simply be a consequence of a functional failure of the spiracular muscles resulting from paralysis. Nevertheless, the majority of insects that we observed after tumbling 4 hr showed little or no evidence of desiccation. Thus, it is unlikely that the paralysis that we observed arose from an excessive concentration of ions and metabolites in haemolymph. In fact, stress paralysis appears to be the direct consequence of a failure in synaptic conduction (Fig. 5), and when one compares the post-synaptic responses in this section with those of the preceding one, it is evident that there is a definite correlation between increasing concentrations of Factor S and the inductive stages of paralysis.
DISCUSSION
BEAMENT (1958) was the first to note that cockroaches, P. americana, are affected by a form of paralysis during or after long periods of mechanical immobilization. Parabiotic and blood injection experiments suggested that paralysis was caused by a blood-borne agent. Beament further noted that the integrity of the nervous system was essential if paralysis were to occur, for when the nerves leading to the leg muscles were severed prior to the application of physical stress, the incidence of paralysis decreased markedly. At first glance, the conditions employed by Beament to induce paralysis seemed far removed from those normally encountered by cockroaches. Nevertheless, EWING (1967), while studying fighting behaviour between male cockroaches (Naupheta niterea), noted that subordinates frequently became sluggish and stiff. Their righting reflex disappeared and a state of semi-paralysis affected the abdomen and limbs. He further observed that insects, once entering upon this state, did not seem to recover and they soon died. HESLOP and RAY (1959) theorized that any stimulus with sufficient strength could trigger a stress syndrome culminating in paralysis. In fact, they observed that the pattern of oxygen consumption and the sequence of tremors, convulsions, and eventual paralysis were similar in cockroaches, regardless of whether chemical (DDT) or physical stimulation (mechanical immobilization) were employed. Such intensive stimulation of the central nervous system undoubtedly overwhelms the delicate integrating mechanisms of the central synapses, resulting in the release of neuropharmacologically active agents into the haemolymph. This view is certainly supported by the following: (1) the increased titre of Factor S in stress-paralysed insects, (2) the obvious concentration of Factor S in the central nervous system, and (3) the presence of Factor S-like substances in perfusates from electrically stimulated nerve cords. STERNBURG et al. (1959) f ound a substance in the cockroach that appeared to have some relationship to the neuromuscular dysfunction described by BEAMENT (1958). This agent, found in the blood of DDT-treated cockroaches, induced hyperexcitability in the isolated nerve cord of the cockroach, and when injected into DDT-resistant houseflies, the symptoms of intoxication soon developed.
FACTOR S IN THE COCKROACH,
PERIPLANETA
AMERICANA
973
The active principle was soluble in water, methanol, and ethanol but not in nonpolar solvents. Although the chemical structure of the substance was not determined, it was chemically, chromatographically, and biologically distinct from acetylcholine, epinephrine, norepinephrine, histamine, y-aminobutyric acid, and 5hydroxytryptamine. In further studies, HAT;VKINS and STERN~URG(1964) reported the occurrence of their DDT toxin in the blood of treated crayfish. This factor from the crayfish stimulated the isolated nerve cord of cockroaches as well as the crayfish and was chromatographically and chemically similar to the agent isolated from DDTtreated cockroaches. It was tentatively concluded from spot-test analyses that the neuroactive substance was an aromatic amine with a possible ester group. Several of the chemical properties of the DDT-toxin showed a similarity to Factor S (COOK, 1967): (1) both substances give a pink colour with p-nitroanaline and a yellow colour with p-dimethylaminobenzaldehyde, and (2) the solubilities and susceptibility to oxidation of Factor S agree with those described by HAWKINS and STERNBURG(1964). It is also interesting to compare the pharmacological effects of Factor S on synaptic conduction in the sixth abdominal ganglion with those reported by EATON and STERNBURG(1967) for DDT-treated cockroaches. They found that the cereal-giant fibre synapse became increasingly unstable as intoxication progressed, and when the insects reached the final stages of prostration, the synapse was completely blocked. They also showed that the DDT toxin occasionally induced after-discharges in the post-synaptic response of the metathoracic ganglion. When isolated metathoracic segments were thoroughly profused with saline and then treated with the DDT toxin, a complete block of the post-synaptic response was obtained. Synaptic conduction in the terminal ganglion was partially blocked with 10 to 12 pg of Factor S, and O-4 to 4 pg of residue was sufficient to produce an after-discharge (Fig. 4). The successive stages leading to stress paralysis in tumbled cockroaches showed a progressive increase in threshold and finally a blockade of the post-synaptic response in the last abdominal ganglion (Table 2). Thus, a close similarity, if not identity, is suggested between Factor S and the DDT neurotoxin. The initial site of action for DDT is clearly on the sensory neurons. ROEDER and WEIANT (1946, 1948), using isolated nerve preparations, showed that extremely low concentrations of DDT initiated trains of impulses in the sensory neurons, and the isolated central nervous system was affected only at relatively high concentrations. A molecular mechanism for interaction of DDT with neural components was proposed by MATSUMURAand O’BRIEN (1966). They found that the addition of~DDT to nerve cord homogenates caused a shift in the U.V. spectrum and gave rise to a new fluorescent emission band, suggesting the formation of a chargetransfer complex with certain neural proteins. Perhaps this charge-transfer complex of DDT with components in the sensory fibres of the insect initiates the recurrent trains of impulses described by ROEDER and WEIANT (1946). Since intense sensory stimulation does increase the level of Factor S in tumbled
974
B.J.
COOK,M.DE
LA
CUESTA,AND J. G.POMONIS
cockroaches (Fig. l), it is reasonable to assume that DDT might do the same thing, Indeed, if this proves to be the case, Factor S could explain the ataxia and eventual paralysis produced in insects treated with DDT (Figs. 1 and 2). After-discharge is a phenomenon associated with synaptic transmission (BOISTEL, 1968), and it serves as an indicator of the level of excitability within the synapse of the last abdominal ganglion of the cockroach. A number of pharmacological agents alter the threshold of synaptic excitability, producing a persistent after-discharge in this insect; certain anti-cholinesterases are among the most potent, namely DFP. ROEDERet al. (1947) found that within several minutes after application of lo+ M DFP, a single presynaptic stimulation caused an afterdischarge that lasted between 15 and 20 sec. HODGSON and WRIGHT (1963) noted that both epinephrine and norepinephrine produced after-discharges in the terminal ganglion, but their duration in no way compared with that found with DFP. The action of Factor S resembles that of the catecholamines. Exposure of the terminal ganglion to O-4 pg of residue caused after-discharges to appear within 1 to 3 min. When preparations were treated with 4 pg of residue, facilitation of the spike potential was evident together with after-discharges lasting from 30 to 80 msec. The loss of motor co-ordination in injected houseflies and stressed cockroaches suggests that Factor S has a direct effect on motor neuron pools or an indirect one via the intemeurons of the central nervous system. Undoubtedly, the central control of efferent activity is accomplished by either modulation in the frequency of impulses or changes in the number of impulses within a series (USHERWOOD, 1967). Both elements constitute the pulsed code which transmits information from one neuron to another. It is interesting to note that Factor S does appear to induce recurrent bursting trains in the central nervous system and on motor axons leading from the metathoracic ganglion (COOK, 1967). Perhaps this substance (or substances) functions as a modulator of neurons that controls various neuron pools by changing the threshold for excitation or in some manner modulates the frequency of impulses. FLOREY (1967) defined modulator substances as ‘ . . . any compound of cellular and nonsynaptic origin that affects the excitability of nerve cells and represents a normal link in the regulatory mechanisms that govern the performance of the nervous system. Such modulator substances can effect the responsiveness of nerve cells to transsynaptic actions of presynaptic neurons and they can alter the tendency to spontaneous activity’. If Factor S does prove to be a modulator, it might arise from active neurons or glial cells. In spite of the obvious effects of Factor S on the central nervous system, one must still not overlook the possibility that it acts upon the peripheral myoneural junction. VAN DER KLOOT (1960) observed that Factor S had an excitatory effect on the nerve-muscle junction in the crayfish. Support for this contention in insects was given by COOK (1967). In either event, the main site of action for Factor S (at the moment) seems to be on synaptic junctions, and it demonstrates some of the properties of a neurohormone: (1) concentration in the central nervous system, (2) release from the central nervous system, and (3) alters the excitability of post-synaptic response that could be attributed to either a transmitter or a modulator substance.
FACTORS IN THECOCKROACH, PERIPLANETA
AMERICANA
975
Acknowle&ements-We are grateful for the able technical assistance of Mrs. PATRICIA KRAMERand Miss CYNTHIAHOPP. REFERENCES BF.AMENT J. W. L. (1958) A paralysing agent in the blood of cockroaches. J. Insect Physiol. 2, 199-214. BERGSTROM S. and HANSONNG. (1950) The use of Arnberhte IRC 50 for the purification of adrenaline and histamine. Acta physiol. stand. 22, 87-92. BOISTBLJ. (1968) The synaptic transmission and related phenomena in insects. Adv. Insect Physiol. 5, l-64. BREBBIAD. R. and LUDWIG D. (1962) Saline solutions for maintaining the isolated heart of the house Ay, Musca domestica L. Ann. ent. Sot. Am. 55, 131-135. COOK B. J, (1967) An investigation of Factor S, a neuromuscular excitatory substance from insects and crustacea. Biol. Bull., Woods Hole 133, 526-538. DAVEY K. G. (1963) The release by enforced activity of the cardiac accelerator from the corpus cardiacum of Periplaneta awicana. J. Insect Physiol. 9, 375-381. EATON J. L. and STERNBURG J. G. (1967) Temperature effects on nerve activity in DDTtreated American co&roaches. r. econ. Ent. 60, 1358-1364. EWING L. S. (1967) Fighting and death from stress in a cockroach. Science, N.Y. 155, 1035-1036. FLOREYE. (1967) Neurotransmitters and modulators in the animal kingdom. Fed. PYOC. 26, 1164-1178. HAWKINS W. B. and STERNSURG J. (1964) Some chemical characteristics of a DDT-induced neuroactive substance from cockroaches and crayf%h. J. econ. Ent. 57, 241-247. HESLOP J. P. and RAY J. W. (1959) The reaction of the cockroach Periplaneta americana L. to bodily stress and DDT. g. Insect Physiol. 3, 395401. HODGSONE. S. and WRIGHT A. M. (1963) Action of epinephrine and related compounds upon the insect nervous system. Gen. camp. Endocr. 3, 519-525. KATER S. B. (1968) Cardioaccelerator release in Periplaneta americana (L.). Science, N.Y. 160, 765-766. NIATSUMURAF. and O’BRIEN R. D. (1966) Interactions of DDT with components of American cockroach nerve. Agric. Fd Chem. 14, 39-43. PUMPHREY R. J. and RAW~ON-SMITH A. F. (1937) Transmission of nervous impulses through the last abdominal ganglion of the cockroach. Proc. R. Sot. (B) 122,106-118. ROI~DERK. D., KENNEDYN. K., and SAMSONE. A. (1947) Synaptic conduction to giant fibers of the cockroach and the action of anti-cholinesterases. 3. NeurophysioZ. 10, l-10. ROEDEFX K. D. and WEIANT E. A. (1946) The site of action of DDT in the cockroach. Science, N.Y. 103, 304-306. ROEDEFC K. D. and WEIANTE. A, (1948) The effect of DDT on sensory and motor structures in the cockroach leg. r. cell. camp. Physiol. 30, 147-172. STERNS-G J. (1963) Autointoxication and some stress phenomena. A. Rev. Ent. 8, 19-38. STERNBURG J., CHANG S. C., and KEARNs C. W. (1959) The release of a neuroactive agent by the American cockroach after exposure to DDT or electrical stimulation. J. eccm. Ent. 52, 1070-1076. TWAROG B. M. and ROEDERK. D. (1957) Pharmacological observations on the desheathed last abdominal ganglion of the cockroach. Ann. ent. Sot. Am. 50,231-237. USHERWOODP. N. R. (1967) Insect neuromuscular mechanisms. Am. Zool. 7, 553-582. VAN DER’KLOOT W. G. (1960) Factor S-a substance which excites crustacean muscle. J. Neurochem. 5, 245-252.
62
THE
DISTRIBUTION OF FACTOR S IN THE COCKROACH, PERIPLANETA AiWERICANA, AND ITS ROLE IN STRESS PARALYSIS* B. J. COOK,
M. DE LA CUESTA,
and J. G. POMONIS
Metabolism and Radiation Research Laboratory, Entomology Research Division, Agriculture Research Service, United States Department of Agriculture, Fargo, North Dakota 58102 (Received 23 December 1968) Abstract-A quantitative bioassay was used to determine that the nerve cord and head of the cockroach, Penplaneta americana (L.), contained the highest concentrations of Factor S ; the legs and body contained lesser amounts. A twofold increase in the titre of Factor S was observed by spectrofluorometry and by bioassay in insects subjected to 4 hr of mechanical stress. compared with the amounts in unstimulated insects. Houseflies, Musca dumestica (L.), injected with 30 ,ug of the active substance were immediately parrdysed: 60 per cent were unable to right themselves within 2 hr; the other 40 per cent showed a marked impairment in motor co-ordination. A substance that caused much the same biological response as Factor S was found in perfusates from electrically stimulated nerve cords of cockroaches. Exposure of the last abdominal ganglion of the cockroach to 8 pg of Factor S caused facilitation of the post-synaptic spike potential followed by after-discharges lasting 30 to 80 msec,; 12 pg of the active residue partially blocked synaptic conduction. A parallel was drawn between the synaptic responses with increasing concentrations ,of Factor S and those occurring at progressive stages in stress paralysis. Factor S showed several properties typical of neurohormones, and an involvement in the mode of action of DDT is suggested. INTRODUCTION
INSECTS subjected to excessive stress are known to release pharmacologically active agents (STERNBURG, 1963) and large amounts of these agents cause autointoxication that often results in paralysis and death. However, the chemical nature of the majority of these substances is not known though one is clearly associated with the neuroendocrine complex. DAVEY (1963) showed that cockroaches, .Per@aneta amwicuna (L.), subjected to enforced activity released a cardiac accelerator from the corpora cardiaca. More recently, KATER (1968) demonstrated that this proteinaceous heat-stable substance was released from the same organ after electrical stimulation of the brain. The effect of this substance on the general behaviour of insects remains obscure. * Mention of a proprietary product does not necessarily imply endorsement product by the United States Department of Agriculture. 963
of this
B. J. COOK,M. DE LA CTJESTA, ANDJ. G. POMONIS
964
Another pharmacologically active substance in insects was reported by BEA~MENT (1958) and STEFWWJRG et aZ. (1959). Both papers described a paralytic agent that appears in the blood of cockroaches prostrated by either electrical stimulation or by exposure to DDT; they concluded that it was released from the central nervous system. A relationship between it and Factor S, a neuromuscular excitatory substance recently obtained from insects (COOK, 1967), seemed possible. MATERIALS Extraction
AND lMETHODS
procedure
Although the general method for the extraction of Factor S was described by COOK (1967), we have modified certain details in the procedure to ensure greater consistency in the yield of the active agent. After the tissues were ground in 3 vol. of 8% trichloroacetic acid, the homogenates were allowed to stand for 2 hr at 4°C before centrifugation at 10,000 g; the supernatant obtained from the centrifugation was treated with ether (1 part ether to 2 parts supernatant). This mixture was shaken vigorously in a separator-y funnel for 5 min, the two phases were allowed to separate, and the water fraction was retained. The procedure was repeated at least three times, and if the water fraction still retained some characteristics of an emulsion, it was extracted further with ether to remove the excess lipid. Since Factor S is specifically adsorbed on aluminium hydroxide suspended in solution, the precipitate was retained and dissolved in 1 N sulphuric acid. Only a sufficient amount of acid was added to bring the pH to 3. Addition beyond this, point, even if the precipitate was not completely dissolved, had a detrimental effect on the active principle. Chrmatographic
pw$cation
of Factor S
Large extracts of whole insects were subjected to cation exchange chromatography on Amberlite IRC 50 by the method of BERGSTROM and HANSSON(1950) ; Amberlite CG-50, 100/200 mesh, was used for extracts of small portions of insect tissue. The dimensions of the column bed were 35 x 6 mm. Extract residues suspended in 1 ml or less of buffer were introduced onto the column_ The column containing the tissue extract was eluted with 25 ml of 0.2% sodium chloride and then by 25 ml of 1 N HCl. Factor S was found in the N HCI fraction from the column; this fraction was therefore taken to dryness, and the residue was dissolved in O-2 ml of ethanol. Factor S appeared as a pink spot on paper chromatograms sprayed with potassium ferricyanide. The R, region corresponding to the pink spot in the phenol-HCl system was eluted from the remainder of the chromatogram and subjected to rechromatography on paper in butanol-acetic acid-water (4 : 1 : 5) prior to bioassay. Small tissue extracts, however, were chromatographed on cellulose thin layer in a butanol-ethanol-1 N acetic acid (3 : 1 : 1) system. The neuroactive area was located on the plates as a yellow-to-brown spot with naphthoquinone-4-sulphonate.
FACTORS
IN THE COCKROACH,
PERIPLANETA
AMERICANA
965
However, this reaction was not specific for Factor S, so several regions were routinely scraped from the plates of each sample. The fractions of interest were then eluted in several ml of 95% ethanol, taken to dryness, and resuspended in 200 ~1 of physiological saline solution (TWAROG and ROEDER,1957).
Physiological preparations Isolated ventral nerve cord. Factor S showed two very characteristic effects on the spontaneous activity of the isolated nerve cord of the cockroach: a two- to threefold increase in arrhythmic activity, and recurrent trains from O-5 to 1 set in duration. These trains were frequently initiated by large bursts of high-frequency pulses lasting from 100 to 200 msec (COOK, 1967). The thoracic portion of the nerve cord from the adult American cockroach was routinely used for quantitative bioassay. The preparation and recording procedures have already been described (COOK, 1967). For studying the release of Factor S from the central nervous system, two nerve cords containing both abdominal and thoracic portions were excised from male cockroaches and placed in 80 ~1 of saline solution. One electrode from a laboratory stimulator was attached to the connective between two of the abdominal ganglia. The indifferent electrode was submerged in the saline bath. Recording electrodes were attached between the first, second, and third thoracic ganglia of the preparation. Spontaneous activity was monitored for 5 or 10 mm before stimulation. The preparation was then stimulated continuously for 30 to 45 min at a pulse strength of 1.2 V, a duration of 0.1 msec and a frequency of 100 cycles/set. Stimulation was interrupted occasionally to .obserKe spontaneous activity. During the first few minutes the cord responded to each stimulus, but as time progressed the general level of activity gradually declined, and after 30 min it was greatly depressed. At that time the saline solution was usually withdrawn and saved for bioassay or chemical analysis. Synaptic conduction in the last abdominal ganglion Post-synaptic responses were recorded from an electrode pair on one of the connectives near the fifth abdominal ganglion. The cereal nerves on the same side were stimulated by a square pulse of 0.1 msec duration and at an intensity of O-6 to 2 V and a frequency of O*fi/sec. The preparation was frequently moistened with several ~1 of physiological saline solution, and samples containing Factor S were applied in ~1 quantities to the dorsal surface of the ganglion.
Mechanical stimulation of cockroaches Adult cockroaches of both sexes were tumbled in 1 gal jars 6 in. wide. These jars were made to rotate on their own axes at 90 rev/min by the powered rollers on which they rested. After 4 hr, 30 to 40 per cent of the insects were paralysed. They were unable to right themselves once they were placed on their backs, and when they were placed upright and subjected to acute cereal stimulation, their legs moved in a slow, unto-ordinated fashion or not at all.
B.J.
966
COOK, M.DE
LA CUESTA,AND J; G.POMONIS RESULTS
Distribution of Factor S in the tissues of the American cockroach Until the substance or substances responsible for the neuroexcitatory activity can be chemically identified, we have defined its action in terms of activity units: one unit is that amount of tissue (pg/pl) required to either induce a perceptible rise in spontaneous activity (10 to 20 per cent) or elicit bursting trains on the isolated nerve cord during a 10 min period. Since increasing concentrations of Factor S brought more immediate responses from the nerve cord, an arbitrary scale was defined in units from 1 to 5 (Table 1). If an extract induced an immediate rise in spontaneous activity of 50 per cent or more with recurrent bursting trains, a designation of 5 activity units was given. TABLE I-DISTRIBUTION OF FACTOR S IN THE TISSUESOFTHE ~OCKXOACH, P. americana, DETERMINED BY QUANTITATIVEBIOASSAY
Tissue from 175 insects Nerve cord Head Body: Legs
Relative response of isolated nerve cord to dilutions of the various tissue extracts (expressed in activity units) t
Wet wt. of tissue before extraction (g)
Zone of neuroactivity* (Rf)
0.78 6.5 70.0 26.2
0.17-0.31 O-0.21 0.20-0.27 O-0.15
1 : 200 dilution (mg-equiv./ 20 CLl) 5 4 3 3
0.35 2.92 3140 llG30
1 : 1000 dilution
1 : 2000 dilution
4 3 2 1
4 3 1 0
* After chromatography on cellulose thin layer with butanol-ethanol-I N acetic acid (3 : 1 : 1). t Definition of activity units: 5= immediate rise in activity 50 per cent or more with recurrent bursting trains; 4= rise in activity to 50 per cent within 4 min with bursting trains; 3= rise in activity to 25 per cent within 6 min with bursting trains; 2= rise in activity to 25 per cent within 8 min with occasional bursting trains; 1 =either a slight rise in activity or occasional bursting trains during 1Omin period; 0 = no response over 1Omin period. $ Bodies minus nerve cord.
Table 1 summarizes the distribution of Factor S determined by quantitative bioassay and represents one of three experiments. In undiluted samples, several chromatographic fractions from each tissue showed neuroactivity, but as the fractions were diluted, only a single area eventually displayed biological activity. When isolated nerve cords were perfused with 20 ~1 of a 1 to 2000 dilution of fraction Rt O-17 to O-31 from the extracted nerve cords, a 25 per cent increase in arrhythmic activity coupled with bursting trains were evident within 4 min after application. This 20 ~1 sample was equivalent to O-01 of a nerve cord or 35 pg of nerve tissue, wet weight. The same dilution from extracts of the cockroach body caused a response from the nerve cord within 10 min; the 20 ~1 aliquot in this instance contained the equivalent of 3.2 mg wet weight. Extracts from the cockroach leg showed no neuroactivity with sample equivalents of 180 pg of tissue.
FACTOR S IN THE COCKROACH, PERIPLANETA
AMERICANA
967
A comparison of these results clearly indicates that the nerve cord and the head are at least a hundred-fold more potent than the body and leg regions. If we take into account the activity units designated for each body region at the 1 to 2000 level, the nerve cord could be as much as four hundred-fold more potent than the body. Although the majority of Factor S is contained in the central nervous system, this fact alone does not establish it as a transmitter. Yet it is an essential corollary to any further considerations regarding the neurotrophic properties of this compound or compounds. Relation
of titre of Factor S to stress paralysis
Since the American cockroach suffers a severe neuromuscular disorder after exposure to sustained mechanical stress (BEAMENT, 1958), the titre of Factor S might account for the onset of paraIysis observed by both BEAMENT(1958) and STERNBURG et aZ. (1959). Therefore, 650 normal cockroaches (590 g) were extracted and the extract was purified to determine the amount of Factor S that was present. A second group of equal number and approximate weight (560 g) were mechanically stimulated by tumbling in jars for 4 hr; 50 to 60 per cent of this group were either hyperactive or paralysed. The tumbled insects were then extracted and the extract was purified. A quantitative comparison was made between the two groups by employing the fluorescent complex that Factor S forms with potassium ferricyanide (COOK, 1967). One-fifth of the extract (95 g of tissue) from both treated and untreated groups was spotted on Whatman No. 1 filter paper, chromatographed in phenol-O*1 N HCI (1 : 1) and sprayed with potassium ferricyanide. Factor S, appearing as a pink spot in each sample, was eluted in 95% ethanol. A reference blank was obtained by eluting the region between the two samples that had the same R, and approximate area. An emission spectrum of the eluates was obtained with an Aminco Bowman spectrofluorometer. A peak emission was obtained for the complex at 350 rn,u (Fig. 1). A twofold increase in the titre of Factor S was observed in the stimulated cockroaches over that in the unstimulated insects. The blank showed essentially no fluorescence in the same region of the spectrum. Quantitative bioassay with the isolated nerve cord on extracts from the two groups of insects confirmed the spectrofluorometric results. Physiological saline solutions containing 9.5 g of tissue/O.5 ml of saline showed 1 activity unit for the untreated groups, ‘while the same amount of tissue/saline from the stimulated cockroaches showed 3 activity units. Response of hozlsefies to injections of Factor S Since houseflies contain Factor S (COOK, 1967), they were injected with various concentrations of the extract known to contain the neuroactive substance. The extract residue was suspended in housefly saline solution (BREBBIA and
968
B. J. COOK,M.
DE LA
CUESTA, ANDJ. G. POMONIS
LUDWIG, 1962), and each insect was injected with 1 ,ul or less in the region of the pronotum.
IO-
200
I 250
300 350 WAVELENGTH
400
450
500
FIG. 1. Comparison of fluorescent emission spectra of potassium ferricyanide derivative of Factor S from equal weights of normal vs. stimulated cockroaches. (A) Cockroaches stimulated 4 hr, (B) unstimulated cockroaches, (C) blank filter paper and reagent excitation 300 rnp. Before treatment, insects were subjected to cold anaesthesia, but they usually revived prior to injection. Controls injected with eluates from blank chromatograms showed no abnormal behaviour. The two dashed lines in Fig. 2 trace the time course of recovery for houseflies injected with 1.5pg of Factor S. The curve of open circles represents the number of houseflies that were able to right themselves and move about to some extent. The second curve (closed circles) traces the course of total recovery after the injection of 1s ,ug of Factor S. For the first hour after injection,
TIME
IN HR
FIG. 2. The time course of response of houseflies to injections with various amounts of Factor S. Curves A (15 pg), C (30 pg), E (60 pg) show percentage of insects able to turn over. Curves B (15 pg) and D (30 pg) show percentage of insects with no ataxia.
FACTOR S IX THE COCKROACX,
PERTPLAXETA
A&IERI&Ah-A
969
95 to 100 per cent of the treated flies were either totally paralysed or unable to co-ordinate their movements. The curve traced by the solid line and open triangles represents the response of houseflies treated with 30 pg of Factor S. At this concentration, 83 per cent were still completely paralysed 1 hr after injection; after 2 hr, 60 per cent were paralysed, and the remaining 40 per cent showed abnormal behaviour. The solid and open squares in Fig. 2 represent the response of houseflies treated with 60 pg of Factor S. At this concentration, 90 per cent of the individuals were paralysed after 24 hr, and the remaining 10 per cent could right themselves but still showed abnormal behavioural characteristics. Immediately after each injection, the flies generally displayed a flaccid type of paralysis. When they were able to move and right themselves, they showed a marked ataxia, occasional tremors, and excessive salivation. As the flies approached normal behaviour, occasional clonic convulsions were evident in some individuals. In contrast to the results with houseflies, cockroaches, injected with several hundred pg of Factor S showed only transitory indications of ataxia and no knockdown. This anomaly might be explained by the fact that the cockroach is more readily able to metabolize Factor S than the housefly. Also, an active enzyme system for the degradation of Sternburg’s neuroactive substance seems to be well documented (EATON and STERNBURG, 1967). This is certainly supported by the observation that although 30 to 40 per cent of the cockroaches subjected to stress were paralysed after 4 hr, some recovered quite rapidly from the effects of paralysis and appeared normal within 30 to 45 min. The release of pwoactive
substance from the isolated nerve cord
Electrical stimulation of the isolated nerve cord of the cockroach clearly caused the release of a neuroactive agent in 8 out of 10 experiments. The typical response of the isolated nerve cord to the application of perfusates from stimulated nerve cords is shown in Fig. 3. The neuroactive material or materials in the perfusate increased the level of arrhythmic activity within several minutes after application and often caused recurrent bursting trains. The character of this response has a definite resemblance to that found with tissue extracts of Factor S. The identity of the material found in perfusates with Factor S was further substantiated by experiments on synaptic conduction in the last abdominal ganglion. In some preparations when several ~1 of the perfusate were placed on the dorsal surface of the terminal ganglion, a definite and persistent after-discharge was produced. In other experiments, an increase in the threshold for the post-synaptic response was observed. It is almost a certainty that more than one active substance is being released from the nerve cord during electrical stimulation, but our present methods of spot test analysis are not sufficiently sensitive to detect the material of interest in the perfusates. Nevertheless, it is encouraging to note that neuroactive materials can be released into perfusates from stimulated nerve cords, and it lends support to the possibility that Factor S is indeed released from the central nervous system, as indicated by the in vivo experiments on stimulated cockroaches.
970
B. J. COOK,M.
DE LA
CTJESTA, ANDJ. G. POMONIS
Effects of Factor S on synaptic conduction Although the endogenous activity of neurons in the isolated nerve cord of the cockroach provides an extremely sensitive preparation for the bioassay of neuroactive agents, it is difficult to ascribe any specific function to a substance on the basis of its application to such a preparation since sensory, motor, and inemuncial elements are present. The synapses of the last abdominal ganglion provide a preparation with greater functional significance. This ganglion contains a key synapse in the well-known evasion reflex arc in the American cockroach. A study of the effects of Factor S on such a preparation might give some indication of the function of this substance in the central nervous system. Fig. 4 shows one of the ten experiments with various concentrations of Factor S. The normal post-synaptic response in the terminal ganglion is shown in Fig. 4(A) and (B). I mmediately after applying 8 pg of Factor S to the dorsal surface of the ganglion, a rise in spontaneous central activity together with recurrent trains were often observed. After 1 mm, the giant fibre spike potential showed a marked depression followed by a distinct after-discharge (Fig. 4C). These after-discharges consisted of a short series of spikes following the arrival of a single maximal pre-synaptic volley. Normally, after-discharges are produced only after a period of synaptic inactivity; once the synapse has adapted, a single spike response occurs to each limital stimulus of the cereal nerve (PTJMPHREY and RAWDON-SMITH, 1937). Not only were the after-discharges of the post-synaptic response persistent in treated ganglion, but they and the giant fibre spike showed facilitation (Fig. 4D and E). Exposure of ganglion to 12 s of active residue caused conduction failure, and an increase in stimulus intensity (1 to 2 V) was required to reinstate the postsynaptic response. Ganglions perfused with 1 ,~l of saline solution containing O-4 pg of Factor S showed a persistent after-discharge with the giant fibre response.
Synaptic conduction in stress-paralysed insects HESLOP and RAY (1959) observed recurrent trains of impulses passing along the abdominal nerve cord in stress-paralysed cockroaches, and these trains were associated with synaptic facilitation in the last abdominal ganglion. Moreover, after thorough washings of the body cavities of paralysed insects, the trains ceased entirely in several cases, and facilitation could no longer be detected in the ganglion. These observations suggested a possible involvement of central synapses in the neuromuscular disorders apparent in stressed insects. In an effort to clarify these observations and correlate the changes in synaptic conduction during the course of paralysis with the effects of various concentrations of Factor S, a number of cockroaches were subjected to mechanical stress as described earlier. After 4 hr of tumbling in the jars, the cockroaches could be placed in various categories according to the behaviour that they displayed (Table 2).
A
B C
!) t:
F G
FIG. 3. T h e effect of perfusates from stimulated nerve cords on spontaneous activity of the isolated nerve cord of the cockroach. (A) N o r m a l activit3, ; (B) 3 min after application of perfusate from stimulated nerve cords ; (C) 5 re.in ; (D) 7 rain ; (E) 9 rain; (F) 11 m i n ; (G) 13 rain; (H) reversible block at 15 rain elapsed time: time mark 50 msec.
A b---4
B C
b----4 °
•
mr
D
ilr,i
' '
EJ FIG. 4. Post-synaptic response of terminal ganglion in P. americana to a perfusion of 2/,1 (8/zg) of Factor S. (A) Several normal spike potentials, time mark 2 reset; (B) spike potential 1 rain after perfusion of 2/zl of saline; (C) spike potential and after-discharge 1 min after perfusion with Factor S, time mark 5 msec; (D) same preparation 7 rain after application, time mark 10 msec; (E) same preparation 7 min after application, time mark 3 msec.
]
I
I
o~ ,-"
....
~
II,
X
r
•
rn
0
©
FACTOR S IN THE COCKROACH, P.ERIPi.ANZTA
TABLE
~-RESPONSES
OF CENTRAL NERVOUS SYSTEM OF
DURING
Condition of insect. Normal to hyperactive Torpid with ataxia Paralysed
971
AMERICANA
P. americana
THE COURSE OF STRESS PARALYSIS
NO. examined
Endogenous activity of abdominal nerve cord (No. showing trains)
Post-synaptic responses of the last abdominal ganglion No. showing afterdischarge
No. showing increased threshold
No. showing conduction failure
17
5
4
3
0
17 25
8 1
6 9
11 16
2 10
1. Normal to hyperactive. Many insects in this category appeared more or less normal but some were hyperactive and easily upset by the slightest stimulus. 2. Torpid with ataxia. These insects could move and right themselves with diEculty and showed an inability to co-ordinate. 3. Paralysed. These insects occasionally showed tremors in the legs but were unable to right themselves and would not move, even after severe stimulation of the cerca. ‘These categories seem to suggest the behavioural pattern sequence that occurs during the time course of paralysis induction. The majority of the insects (60 to 70 per cent) subjected to 4 hr of tumbling A neurophysiological examination of were designated as normal to hyperactive. these cockroaches revealed recurrent trains and after-discharge in 5 out of 17 individuals. Torpid cockroaches often showed an after-discharge in the postsynaptic response to a single cereal stimulus. This was evident even at the threshold of response, and it was a persistent phenomenon. Insects in this same category also showed an elevated threshold for post-synaptic response that often required an increasing voltage intensity (3-10 V) to establish the response. Generally, the stimulus threshold for synaptic conduction in normal cockroaches was between 0.8 and 2.5 V at 0.1 msec duration and O-5 cycles/set. Torpid cockroaches often showed a certain synaptic instability, indicated by an intermittent response to single pre-synaptic volleys (2 to 3 in 5 volleys). This instability was also evident in hyperactive cockroaches. A blockade in synaptic conduction (no response to 15 V) was generally found in paralysed insects. If there was a synaptic response, it was considerably above normal threshold levels. Although conduction failure did occur in paralysed insects, the axons of the giant fibres could still conduct an impulse, and endogenous activity was still evident on connectives from the abdominal nerve cord of many individuals (Fig. 5). HESLOP and RAY (1959) suggested that the neuromuscular disturbance in stressed insects may be a consequence of a reduction in haemolymph volume which, in turn, diminishes the transport of metabolites. It is quite true that
972
B.
J. COOK,M.
DE
LA CUESTA,ANDJ. G. POMONIS
insects placed under stress for 6 to 8 hr are subject to severe desiccation, but this may simply be a consequence of a functional failure of the spiracular muscles resulting from paralysis. Nevertheless, the majority of insects that we observed after tumbling 4 hr showed little or no evidence of desiccation. Thus, it is unlikely that the paralysis that we observed arose from an excessive concentration of ions and metabolites in haemolymph. In fact, stress paralysis appears to be the direct consequence of a failure in synaptic conduction (Fig. 5), and when one compares the post-synaptic responses in this section with those of the preceding one, it is evident that there is a definite correlation between increasing concentrations of Factor S and the inductive stages of paralysis.
DISCUSSION
BEAMENT (1958) was the first to note that cockroaches, P. americana, are affected by a form of paralysis during or after long periods of mechanical immobilization. Parabiotic and blood injection experiments suggested that paralysis was caused by a blood-borne agent. Beament further noted that the integrity of the nervous system was essential if paralysis were to occur, for when the nerves leading to the leg muscles were severed prior to the application of physical stress, the incidence of paralysis decreased markedly. At first glance, the conditions employed by Beament to induce paralysis seemed far removed from those normally encountered by cockroaches. Nevertheless, EWING (1967), while studying fighting behaviour between male cockroaches (Naupheta niterea), noted that subordinates frequently became sluggish and stiff. Their righting reflex disappeared and a state of semi-paralysis affected the abdomen and limbs. He further observed that insects, once entering upon this state, did not seem to recover and they soon died. HESLOP and RAY (1959) theorized that any stimulus with sufficient strength could trigger a stress syndrome culminating in paralysis. In fact, they observed that the pattern of oxygen consumption and the sequence of tremors, convulsions, and eventual paralysis were similar in cockroaches, regardless of whether chemical (DDT) or physical stimulation (mechanical immobilization) were employed. Such intensive stimulation of the central nervous system undoubtedly overwhelms the delicate integrating mechanisms of the central synapses, resulting in the release of neuropharmacologically active agents into the haemolymph. This view is certainly supported by the following: (1) the increased titre of Factor S in stress-paralysed insects, (2) the obvious concentration of Factor S in the central nervous system, and (3) the presence of Factor S-like substances in perfusates from electrically stimulated nerve cords. STERNBURG et al. (1959) f ound a substance in the cockroach that appeared to have some relationship to the neuromuscular dysfunction described by BEAMENT (1958). This agent, found in the blood of DDT-treated cockroaches, induced hyperexcitability in the isolated nerve cord of the cockroach, and when injected into DDT-resistant houseflies, the symptoms of intoxication soon developed.
FACTOR S IN THE COCKROACH,
PERIPLANETA
AMERICANA
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The active principle was soluble in water, methanol, and ethanol but not in nonpolar solvents. Although the chemical structure of the substance was not determined, it was chemically, chromatographically, and biologically distinct from acetylcholine, epinephrine, norepinephrine, histamine, y-aminobutyric acid, and 5hydroxytryptamine. In further studies, HAT;VKINS and STERN~URG(1964) reported the occurrence of their DDT toxin in the blood of treated crayfish. This factor from the crayfish stimulated the isolated nerve cord of cockroaches as well as the crayfish and was chromatographically and chemically similar to the agent isolated from DDTtreated cockroaches. It was tentatively concluded from spot-test analyses that the neuroactive substance was an aromatic amine with a possible ester group. Several of the chemical properties of the DDT-toxin showed a similarity to Factor S (COOK, 1967): (1) both substances give a pink colour with p-nitroanaline and a yellow colour with p-dimethylaminobenzaldehyde, and (2) the solubilities and susceptibility to oxidation of Factor S agree with those described by HAWKINS and STERNBURG(1964). It is also interesting to compare the pharmacological effects of Factor S on synaptic conduction in the sixth abdominal ganglion with those reported by EATON and STERNBURG(1967) for DDT-treated cockroaches. They found that the cereal-giant fibre synapse became increasingly unstable as intoxication progressed, and when the insects reached the final stages of prostration, the synapse was completely blocked. They also showed that the DDT toxin occasionally induced after-discharges in the post-synaptic response of the metathoracic ganglion. When isolated metathoracic segments were thoroughly profused with saline and then treated with the DDT toxin, a complete block of the post-synaptic response was obtained. Synaptic conduction in the terminal ganglion was partially blocked with 10 to 12 pg of Factor S, and O-4 to 4 pg of residue was sufficient to produce an after-discharge (Fig. 4). The successive stages leading to stress paralysis in tumbled cockroaches showed a progressive increase in threshold and finally a blockade of the post-synaptic response in the last abdominal ganglion (Table 2). Thus, a close similarity, if not identity, is suggested between Factor S and the DDT neurotoxin. The initial site of action for DDT is clearly on the sensory neurons. ROEDER and WEIANT (1946, 1948), using isolated nerve preparations, showed that extremely low concentrations of DDT initiated trains of impulses in the sensory neurons, and the isolated central nervous system was affected only at relatively high concentrations. A molecular mechanism for interaction of DDT with neural components was proposed by MATSUMURAand O’BRIEN (1966). They found that the addition of~DDT to nerve cord homogenates caused a shift in the U.V. spectrum and gave rise to a new fluorescent emission band, suggesting the formation of a chargetransfer complex with certain neural proteins. Perhaps this charge-transfer complex of DDT with components in the sensory fibres of the insect initiates the recurrent trains of impulses described by ROEDER and WEIANT (1946). Since intense sensory stimulation does increase the level of Factor S in tumbled
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B.J.
COOK,M.DE
LA
CUESTA,AND J. G.POMONIS
cockroaches (Fig. l), it is reasonable to assume that DDT might do the same thing, Indeed, if this proves to be the case, Factor S could explain the ataxia and eventual paralysis produced in insects treated with DDT (Figs. 1 and 2). After-discharge is a phenomenon associated with synaptic transmission (BOISTEL, 1968), and it serves as an indicator of the level of excitability within the synapse of the last abdominal ganglion of the cockroach. A number of pharmacological agents alter the threshold of synaptic excitability, producing a persistent after-discharge in this insect; certain anti-cholinesterases are among the most potent, namely DFP. ROEDERet al. (1947) found that within several minutes after application of lo+ M DFP, a single presynaptic stimulation caused an afterdischarge that lasted between 15 and 20 sec. HODGSON and WRIGHT (1963) noted that both epinephrine and norepinephrine produced after-discharges in the terminal ganglion, but their duration in no way compared with that found with DFP. The action of Factor S resembles that of the catecholamines. Exposure of the terminal ganglion to O-4 pg of residue caused after-discharges to appear within 1 to 3 min. When preparations were treated with 4 pg of residue, facilitation of the spike potential was evident together with after-discharges lasting from 30 to 80 msec. The loss of motor co-ordination in injected houseflies and stressed cockroaches suggests that Factor S has a direct effect on motor neuron pools or an indirect one via the intemeurons of the central nervous system. Undoubtedly, the central control of efferent activity is accomplished by either modulation in the frequency of impulses or changes in the number of impulses within a series (USHERWOOD, 1967). Both elements constitute the pulsed code which transmits information from one neuron to another. It is interesting to note that Factor S does appear to induce recurrent bursting trains in the central nervous system and on motor axons leading from the metathoracic ganglion (COOK, 1967). Perhaps this substance (or substances) functions as a modulator of neurons that controls various neuron pools by changing the threshold for excitation or in some manner modulates the frequency of impulses. FLOREY (1967) defined modulator substances as ‘ . . . any compound of cellular and nonsynaptic origin that affects the excitability of nerve cells and represents a normal link in the regulatory mechanisms that govern the performance of the nervous system. Such modulator substances can effect the responsiveness of nerve cells to transsynaptic actions of presynaptic neurons and they can alter the tendency to spontaneous activity’. If Factor S does prove to be a modulator, it might arise from active neurons or glial cells. In spite of the obvious effects of Factor S on the central nervous system, one must still not overlook the possibility that it acts upon the peripheral myoneural junction. VAN DER KLOOT (1960) observed that Factor S had an excitatory effect on the nerve-muscle junction in the crayfish. Support for this contention in insects was given by COOK (1967). In either event, the main site of action for Factor S (at the moment) seems to be on synaptic junctions, and it demonstrates some of the properties of a neurohormone: (1) concentration in the central nervous system, (2) release from the central nervous system, and (3) alters the excitability of post-synaptic response that could be attributed to either a transmitter or a modulator substance.
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Acknowle&ements-We are grateful for the able technical assistance of Mrs. PATRICIA KRAMERand Miss CYNTHIAHOPP. REFERENCES BF.AMENT J. W. L. (1958) A paralysing agent in the blood of cockroaches. J. Insect Physiol. 2, 199-214. BERGSTROM S. and HANSONNG. (1950) The use of Arnberhte IRC 50 for the purification of adrenaline and histamine. Acta physiol. stand. 22, 87-92. BOISTBLJ. (1968) The synaptic transmission and related phenomena in insects. Adv. Insect Physiol. 5, l-64. BREBBIAD. R. and LUDWIG D. (1962) Saline solutions for maintaining the isolated heart of the house Ay, Musca domestica L. Ann. ent. Sot. Am. 55, 131-135. COOK B. J, (1967) An investigation of Factor S, a neuromuscular excitatory substance from insects and crustacea. Biol. Bull., Woods Hole 133, 526-538. DAVEY K. G. (1963) The release by enforced activity of the cardiac accelerator from the corpus cardiacum of Periplaneta awicana. J. Insect Physiol. 9, 375-381. EATON J. L. and STERNBURG J. G. (1967) Temperature effects on nerve activity in DDTtreated American co&roaches. r. econ. Ent. 60, 1358-1364. EWING L. S. (1967) Fighting and death from stress in a cockroach. Science, N.Y. 155, 1035-1036. FLOREYE. (1967) Neurotransmitters and modulators in the animal kingdom. Fed. PYOC. 26, 1164-1178. HAWKINS W. B. and STERNSURG J. (1964) Some chemical characteristics of a DDT-induced neuroactive substance from cockroaches and crayf%h. J. econ. Ent. 57, 241-247. HESLOP J. P. and RAY J. W. (1959) The reaction of the cockroach Periplaneta americana L. to bodily stress and DDT. g. Insect Physiol. 3, 395401. HODGSONE. S. and WRIGHT A. M. (1963) Action of epinephrine and related compounds upon the insect nervous system. Gen. camp. Endocr. 3, 519-525. KATER S. B. (1968) Cardioaccelerator release in Periplaneta americana (L.). Science, N.Y. 160, 765-766. NIATSUMURAF. and O’BRIEN R. D. (1966) Interactions of DDT with components of American cockroach nerve. Agric. Fd Chem. 14, 39-43. PUMPHREY R. J. and RAW~ON-SMITH A. F. (1937) Transmission of nervous impulses through the last abdominal ganglion of the cockroach. Proc. R. Sot. (B) 122,106-118. ROI~DERK. D., KENNEDYN. K., and SAMSONE. A. (1947) Synaptic conduction to giant fibers of the cockroach and the action of anti-cholinesterases. 3. NeurophysioZ. 10, l-10. ROEDEFX K. D. and WEIANT E. A. (1946) The site of action of DDT in the cockroach. Science, N.Y. 103, 304-306. ROEDEFC K. D. and WEIANTE. A, (1948) The effect of DDT on sensory and motor structures in the cockroach leg. r. cell. camp. Physiol. 30, 147-172. STERNS-G J. (1963) Autointoxication and some stress phenomena. A. Rev. Ent. 8, 19-38. STERNBURG J., CHANG S. C., and KEARNs C. W. (1959) The release of a neuroactive agent by the American cockroach after exposure to DDT or electrical stimulation. J. eccm. Ent. 52, 1070-1076. TWAROG B. M. and ROEDERK. D. (1957) Pharmacological observations on the desheathed last abdominal ganglion of the cockroach. Ann. ent. Sot. Am. 50,231-237. USHERWOODP. N. R. (1967) Insect neuromuscular mechanisms. Am. Zool. 7, 553-582. VAN DER’KLOOT W. G. (1960) Factor S-a substance which excites crustacean muscle. J. Neurochem. 5, 245-252.
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