Neurophysiological changes associated with paralysis arising from body stress in the cockroach, Periplaneta americana

J.Insect Plrysiol., 1974,VoI.20,pp.21 to 40.PergamonPress. Printed in Great Britain

NEUROPHYSIOLOGICAL CHANGES ASSOCIATED WITH PARALYSIS ARISING FROM BODY STRESS IN THE COCKROACH, PERIPLANETA AMERICANA BENJAMIN

J. COOK*

and GERALD

G. HOLT

Metabolism and Radiation Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Fargo, North Dakota 85102, U.S.A. (Received 22 May 1973)

Abstract-Two hours after physical stress, Periplaneta americana could be separated into three behavioural categories: normal to hyperactive; torpid with ataxia; and paralysed. At 2 hr, 68 per cent were either torpid or paralysed, at 20 hr, 83 per cent were paralysed. Weight loss was a distinct physiological symptom of stress paralysis : The calculated mean loss was 9.5 mg/hr for torpid insects and 12.4 mg/hr for paralysed cockroaches, losses were four to six times larger than those occurring in starved cockroaches. However, the haemolymph osmolarities of the three categories showed no appreciable differences. Only starved and paralysed cockroaches showed a noticeable reduction in muscle fibre membrane potentials of the flexor tibia-a mean value below 40 mV for starved insects and a mean value below 50 mV for paralysed insects. Both of these categories consistently showed a lower amplitude for junctional potentials, but paralysed cockroaches showed a much higher incidence of complete failure to neural stimulation. Most muscle fibres of completely paralysed insects lost their sensitivity to direct extracellular stimulation while the loss in sensitivity was less evident in starved cockroaches. Axonal conduction on the crural nerve was not changed by stress, and the spontaneous efferent activity of completely paralysed insects was similar to the pattern of activity for normal cockroaches. Stress seemed to alter the volume and content of the intermyofibral spaces of muscles. INTRODUCTION BEAMENT (1958) noticed that Periplaneta americana (L.) subjected to prolonged mechanical immobilization often became paralysed in a matter of hours or days following release. He surmised that the enforced immobility produced a state of continuous hyperactivity in the confined insects. He tested this supposition by subjecting cockroaches to 4 to 12 hr of enforced activity. The incidence of paralysis following such stress was greater than that produced by immobilizing the insect. In addition, when blood from these paralysed cockroaches was injected into normal insects, the symptoms of paralysis often developed.

* Present address: Western Cotton Research Laboratory, A.R.S., U.S.D.A., East Broadway Road, Phoenix, Arizona 85040, U.S.A. 21

4135

22

BENJAMINJ. COOK ANDGERALDG. HOLT

EWING (1967) f ound a similar type of paralysis occurring in submissive male cockroaches, Nuuphoeta cinerea (Oliv.), that had been exposed to an aggressive encounter with another male. SANFORD (1971) observed a similar phenomenon occurring in the field cricket, Gyrilus intuger, after social stress. HESLOP and RAY (1959) found that regardless of whether cockroaches were subjected to physical or chemical (DDT intoxication) stress, the symptoms leading to stress paralysis were nearly identical ; even a difference in the chemical did not produce any variation in the sequence of events that led to paralysis (COLHOUN, 1960). Thus, a variety of stimuli of sufficient strength and duration can trigger a stress syndrome in cockroaches that will culminate in paralysis and death. The process of autointoxication seems to arise from the excessive release of pharmacologically active substances from the central nervous system (STERNBERG et al., 1959 ; COOK et al., 1969) and the neuroendocrine complex (DAVEY, 1963). We therefore made an attempt to define in some detail the neurophysiological changes that accompany the onset of paralysis in the cockroaches in the hopes of establishing the primary physiological lesion. MATERIALS

AND METHODS*

The cockroaches (P_ americana) used in this study were taken from stock colonies maintained at a temperature of 27°C and a r.h. of 40 per cent and fed on dry dog food; water was provided ad lib. Mechanical stimulation Adult cockroaches of both sexes (30 individuals randomly assorted) were tumbled in a 1 1. jar with a 15 cm diameter. All insects were subjected to stress from 8 a.m. to 12 noon to insure a uniform rhythm of exposure. The addition of several small rubber stoppers (size 00) prevented the cockroaches from clinging to the sides of the jar. The jar was rotated about its own axis at 90 rev/min by powered rollers. After 4 hr, the insects were removed to another container and held until they could be separated into behavioural categories. Starvation Similar cockroaches were placed in individual containers and deprived of food and water for 7 days before experiments were begun. However, in several instances, insects starved in excess of 10 days were utilized to get some impression of the more terminal aspects of physiological change resulting from starvation and dehydration. Huemolymph osmolurity The osmolarity of stressed cockroach haemolymph was determined from vapour pressure measurements made on a Hewlett Packard osmometer (Model 302). * The mention constitute

of a company

an endorsement

name or proprietary

by the U.S.

Department

product

of Agriculture.

in this paper

does

not

NEUROPHYSIOLOGICAL

CHANGES FROM BODY STRESS IN THE COCKROACH

23

Physiological preparations The flexor tibialis muscles in the metathoracic Nerve muscle preparation. segment of the cockroach afforded us a rapid means of determining neuromuscular changes with a minimum of dissection. The insects were secured dorsal side down in soft ‘dental wax while the right metathoracic leg was drawn out and across a shallow basin made of the same wax and filled with saline solution. The femur of this l.eg was then fastened across the basin with wax. After the cuticle of the posterior surface of the femur was carefully removed, the exposed flexor muscle was allowed to equilibrate for 15 to 20 min in a saline solution of the following composition: 9.32 g NaCl, 0.77 g KCl, 0.50 g CaCl,, O-18 g NaHCO,, 0.01 g NaHaPO, to 1 1. of distilled water (BECHT et al., 1960). A small silver-silver chloride ground wire was placed in the bath, and a recording glass micropipette filled with 3 M KC1 was then positioned just above the muscle fibres with a micromanipulator. Fibre impalements were accomplished by a light tap on the preparation base plate. Intracellular potentials were amplified with an electrometer as previously described (COOK et al., 1971) and recorded from a Tektronix 564B oscilloscope with a trace-recording camera. The flexor muscle was stimulated indirectly from a pair of platinum electrodes under the fifth metathoracic nerve. A square pulse of O-1 msec duration was used. Motor nerve preparation. Spontaneous motor nerve traffic was monitored by placing a pair of fine platinum electrodes underneath the fifth nerve between the metathoracic ganglion and the severed end of the nerve in the coxa. Axonal conduction was determined by taking up the distal cut portion of the crural nerve in a suction electrode in the region of the femur and stimulating the same nerve through a pair of platinum electrodes under the proximal cut end. Both stimulated and spontaneous nerve impulses were amplified by a Grass PSI1 through a high-impedance probe and monitored on an oscilloscope. Muscle ultrastructure For this study the retractor unguis muscle was used because it could be quickly removesd from the leg with a minimum of tissue damage. We assumed that the ultrastructural changes in the retractor unguis would be similar to those occurring in the flexor tibia. The procedures for tissue preparation and observation under the electron microscope were similar to those previously described by HOLMAN and COOK (1972). RESULTS

Behavioural changes in response to stress During the exposure to the mechanical stress of tumbling, the cockroaches gradually became exhausted, and the initial attempts to maintain equilibrium by running with the moving surface of the jar eventually gave way to a passive acceptance of the churning that resulted from motion of the jar and its contents. Immediately after stress, most of the insects remained quiet for 30 to 4.5 min,

24

BENJAMIN

J. COOK AND GERALD G. HOLT

that is, they showed little spontaneous movement and no response to tactile stimulation. However, after this recovery period, as many as 50 per cent displayed normal behaviour. The period of exhaustion meant that we might easily misinterpret behaviour if we made our judgements too soon, so no attempt was made to separate the cockroaches into behavioural categories for 2 hr. After this time, behaviour was classified as follows: (1) Normal to hyperactive. Insects in the first category responded to a slight vibration of the cage and tactile stimulation in a co-ordinated manner. They also showed some evidence of spontaneous activity. Certain members of this group occasionally displayed hyperactive behaviour, that is, a slight vibration of the cage or tactile stimulation induced wild running behaviour that lasted for several minutes. However, most cockroaches in the category remained still and occasionally assumed the subordinate posture described by EWING (1967 j-limbs tucked beneath the body and the head under the shield of the pronotum. (2) Torpid with ataxia. The insects in the second category were generally insensitive to slight vibrations and tactile stimulation. However, a sharp pinch of the cerci would provoke slow, often unto-ordinated, movements of the legs with some forward progression. When such insects were placed on their backs, they could still right themselves though with difficulty. (3) Paralysed. Although the paralysed insects occasionally showed movements and tremors of the legs, they were incapable of forward progression, even after sharp cereal stimulation, The righting reflex was also missing. Fig. 1 shows the percentage of 300 insects that showed particular physiological symptoms at certain times after the 4 hr exposure to physical stress. Within 2 hr, 68 per cent of the cockroaches were either torpid with ataxia (32 per cent) or paralysed (36 per cent), Within 20 hr, 83 per cent were paralysed. The rapid onset of paralysis is emphasized by the fact that only 10 per cent were classified as torpid at 20 hr. Thus, a cockroach subjected to physical stress tended to progress inevitably to paralysis and death despite apparently normal behaviour shortly after stress. Also, the stressed cockroaches preferred to remain still and showed little inclination to eat or drink. The lids of the cages containing the stressed insects showed excessive condensation suggesting a loss of spiracular control and/or accelerated respiration, In addition, the paralysed cockroaches were particularly susceptible to bacterial infection: tissue lysis and putrefaction were often evident within 12 hr after the onset of paralysis. Weight loss during and following stress The progressive development of dehydration, another characteristic physiological symptom induced by mechanical stress, could be quantitated simply by weighing individual insects. Also, since HESLOP and RAY (1959) had suggested that desiccation might be responsible for the neuromuscular disorders observed in stress-paralysed insects, we subjected a group of cockroaches to starvation in an effort to establish whether the loss of food and water were indeed a causative agent of paralysis. Fig. 2 shows the results. The mean weight loss for starved

NEUROPHYSIOLOGICAL

CHANGES

FROM

BODY

STRESS

IN

THE

COCKROACH

25

60 50 40 30 ij;

Paralyzed

20.1 IO I 2

IO

Time,

20

hr

FIG. 1. The percentage of 300 cockroaches (thirty insects per group; 10 replicates) showing specific behavioural patterns at various times after physical stress. Paralysed and Torpid: Regression equation: y = 66 + 1.3x. Number of obser -

vatj.ons: 30. Paralysed: Regression equation: y = 28 + 2.7~. Number of observations : 30.

insects after 7 days exceeded that for paralysed insects at 20 hr by 62 mg. However, the starved insects displayed normal behaviour, and paralysis did not develop, even after 14 days of starvation, though these insects occasionally showed symptoms of ataxia and sluggishness 1 to 2 hr before death. In addition, the weight loss in the ,physically stressed cockroaches progressed four to six times faster than in the starved insects: the calculated mean loss was 2.3 mg/hr in starved cockroaches compared with 9.5 mg/hr for torpid and 12.4 mg/hr for paralysed cockroaches. The rate of loss was even greater during stress. Individual weight losses before and after stress can be estimated from the group totals shown in Table 1. The initial Iaverage individual weight for three replicates of 30 cockroaches each was 813 mg, and the mean individual weight immediately after stress was 73.5 mg which represents an average loss of 19.5 mg/cockroach per hr. The weight losses in the stressed insects certainly reflected an active process of dehydration and a corresponding reduction in haemolymph volume. In fact, an estimate of the percentage of weight loss for all behavioural classes can be

BENJAMINJ. COOKANDGERALDG. HOLT

26

Paralyzed

‘t-_2

IO

hr

20

hr

day

FIG. 2. Individual weight losses for torpid, paralysed, and starved cockroaches at various times after stress. Torpid: Regression equation: y = 741- 9.3x. Number of observations: 75. Paralysed: Regression equation: y = 698 - 9%. Number of observations: 120. Starved: Number of observations: 64. Average dry weight = 215 mg; SD. = f 59 mg. TABLE l-Loss

IN WEIGHTOF COCKROACHES BEFOREANDAFTER4 hr OF PHYSICALSTRESS Group weights (g)*

Initial 22-9 26.2 24.1

Immediately after stress

2 hr after stress

21.0 23.6 21-7

20.1 21.7 20.4

* Thirty cockroaches per group; three replicates.

calculated from the data in Table 1. For example, the difference between the weight before treatment and at 2 hr after treatment was 123 mgjcockroach, a 15.2 per cent loss. EDNEY (1968) calculated that when Periplaneta larvae lost 12 per cent of their wet weight, the haemolymph osmolarity would increase from 410 to 607 mosmoles (if we assume that there was no ion-regulating mechanism in the haemolymph), but WALL (1970) failed to record such an increase and suggested that excess solutes are removed from the haemolymph during desiccation. Our measurements of haemolymph osmolarity in normal cockroaches and in the two behavioural classes of physically stressed insects (Fig. 3) agree with the observations of WALL. Mean osmolarity showed a gradual increasing slope from normal to

NEUROPHYSIOLOGICAL

CHANGES

FROM

BODY

STRESS

paralysed insects, but the increase fell well within (1970) Sor cockroaches after 7 days of dehydration.

IN

THE

the range

T

27

COCKROACH

reported

by WALL

I

I

FIG.

3. Osmolalities of haemolymph for unstressed, torpid, and paralysed cockroaches 2 hr after stress.

Neurophysiological changes associated with stress paralysis Alth.ough the experiments of BEAMENT (1958) left little doubt that a chemical agent capable of causing paralysis is present in the blood of paralysed cockroaches, the precise details of the neurophysiological changes occurring during the development of paralysis were left undetermined. The following experiments were made in an attempt to provide these details. Insects for the study were selected 2 hr after stress from the four physiological categories mentioned. At this time, some insects showed signs of dehydration. The change in spontaneous motor nerve discharge, axonal conduction, and the flexor muscle twitch response to direct extracellular stimulation in stressed insects j.s shown in Table 2. Each such response was consecutively determined on individual insects within the symptomatic groupings listed. Only two instances of conduct.ion failure were observed in paralysed insects and just a single instance of spontaneous efferent discharge failure in the same category. However, the failure of the twitch response was quite evident in paralysed cockroaches. No detectable change occurred in the character of axonal conduction on the crural nerve after stress. As Fig. 4(C) and (F) show, both fast and slow axon responses were observed in completely paralysed and untreated cockroaches at Also, the spontaneous efferent activity (Fig. 41) similar voltage thresholds. of a completely paralysed insect was similar to that of normal cockroaches. Nevertheless, occasional recurrent trains were detected in torpid and paralysed insects though no attempt was made to quantitate such events.

15 15 15 15 15

Normal Torpid

Starved 7-l 1 days Paralysed (with leg movement) Paralysed (no movement)

Physiological condition of insects

No. of insects tested

15 15 14

15 1.5

No. with activity

Spontaneous efferent activity in fifth nerve

15 14 14

15 15

No. responding

Axonal conduction in fifth nerve

13 10 2

No. responding ~ 15 15

Response of flexor muscle to direct extracellular stimulation

TABLE 2-VARIOUS NEUROPHYSIOLOGICAL CHANGES ASSOCIATED WITH STRESS PARALYSIS

F +I

c!

$

8

% u

F E

2 ’

29

FIG. 4. Effect of physical stress on the response of the flexor tibiae muscle and the fifth metathoracic nerve of the cockroach. (A) An intracellularly recorded fast response from a normal muscle fibre. (B) An intracellularly recorded response of a normal muscle fibre to direct extracellular stimulation. (C) Response of a normal fifth metathoracic nerve to stimulation showing both fast and slow conducting fibres. (D) An intracellular record of an attenuated fast response from a muscle fibre of a paralysed cockroach. (E) An intracellularly recorded response of a muscle fibre (paralysed cockroach) that has lost some of its responsiveness to (F) Fast and slow conducting fibres in the direct extracellular stimulation. fifth metathoracic nerve of a paralysed insect. (G) Small spontaneous excitatory postsynaptic potentials (E, potentials) recorded from a muscle fibre in a torpid cockroach. (H) A record from a muscle fibre (paralysed insect) that has lost its re(I) Spontaneous motor impulses sponsiveness to direct extracellular stimulation. recorded from the severed end of the crural nerve in a paralysed insect. Calibration: 20 mV and 5 msec per division (A), 10 mV and 2 msec (B), 5 msec (C), 20 I~V and 10 msec (D), 20 mV and 5 msec (E), 5 msec (F), 1 mV and 50 msec (G), 20 mV and 5 msec (H), and 50 msec (I).

31

FIG. 7(A) A neuromuscular junction in the retractor unguis muscle of a normal cockroach. Ax, Axon; M, mitochondrion; SV, presynaptic vesicles; P, postsynaptic pillar, intermyofibril space (arrow); scale mark, 1 pm. (B) Cross-section of an axon (Ax) in a cleft of the rectractor unguis muscle of a starved and dehydrated cockroach (7 days). M, Mitochondrion, condensed intermyofibril space (arrow); scale mark, 1 pm. (C) Several neuromuscular junctions in the retractor unguis muscle of a completely paralysed cockroach. Ax, Axon; P, postsynaptic pillar, distended intermyofibril space (arrow); scale mark, 1 pm. (D) A neuromuscular junction from a paralysed cockroach showing a high density of presynaptic vesicles in the nerve terminal. Scale mark, 0.5 pm.

NEUROPHYSIOLOGICAL

CHANGES

FROM

BODY

33

STRESS IN THE COCKROACH

Intracellular recordings were restricted to the surface fibres in the central portion of the flexor tibia muscle. So we would have reasonable assurance of obtaining a ‘fast’ (twitch) rather than a ‘slow’ (tonic) response. When the fast axon was stimulated in the fifth metathoracic nerve, an electrical response of fairly consistent amplitude was recorded from the muscle fibre. Such an event took the form of a large postsynaptic potential that evoked a variable response from the muscle fibre itself. This latter response was occasionally indicated by a slight inflexion in the rising phase of the potential. A typical fast response from the flexor tibia muscle of a normal cockroach is shown in Fig. 4A. Generally, slow responses occurred near the points of muscle insertion on the integument, and they showed a noticeable facilitation during repetitive excitation. The data in Fig. 5 represent the results of intracellular measurements from 1.5 different muscle preparations in each of the behavioural categories, with 10 to 20 fibres being sampled from each muscle preparation. Normal resting

roaches

Torpid

potentials

100

resting

Paralyzed

roaches potentials

.

resting

roaches potentials

Starved

roaches

resting

potentials

I-

75

50

25 ;I:,

49 59 69 79

9 1929394959

L&L l.dL 293949S6979

2939495969 79

9 19293949596979

91929394959

Lb!_ 19 293949

59

9 19293949

mV

FIG. 5. Histograms of intracellular potentials recorded from the flexor tibiae muscles of 15 insects in each of the designated categories. The number beneath each column represents the maximum millivolt reading for a 9 mV range; e.g., 29 represents the range from 20 to 29 mV.

The histogram for resting potentials in the normal cockroach agrees with a reported range of 40 to 70 mV for resting potentials in the skeletal muscles of the coxa of the same insect (BECHT et al., 1960). Those for torpid cockroaches showed a slight shift to lower values that became more evident in paralysed insects in which the mean resting potential was below 50 mV. However, the most dramatic

34

BENJAMINJ. COOKAND GERALDG. HOLT

depolarizing shift occurred in starved cockroaches: the mean resting potential fell well below 40 mV. This progressive drop in the resting membrane potential undoubtedly reflects the active process of dehydration occurring in both stressed and starved insects. Thus, one might anticipate a corresponding drop in the amplitude of junctional potentials. Such a drop was quite evident in starved cockroaches, but the muscle fibres of paralysed insects with their comparatively higher resting potentials showed even lower mean junctional potential values. Most fibres either failed to respond to neural stimulation or showed a marked attenuation in amplitude (Fig. 4D), but a few fibres in paralysed cockroaches had junctional potentials of normal amplitude. Without electrical stimulation, preparations of the flexor tibia muscle often showed a variety of small depolarizing postsynaptic potentials. Most of these spontaneous potentials originate from motor neurons in the metathoracic ganglion since they disappeared when the fifth nerve was cut. We had hoped to study the effects of stress on the frequency of minature end plate potentials (MEPPs), but when the crural nerve was severed, the frequency of MEPPs was drastically reduced, and no quantitative study was possible. PIEK and MANTEL (1970) reported a similar experience in their studies of the thoracic muscles of a number of insects. Nevertheless, we did observe MEPPs on several occasions in muscle fibres of both normal and torpid insects after the fifth nerve had been cut. Thus, MEPPs are associated with the quantal release of transmitter substances in insects, as suggested by USHERWOOD (1963a). Two types of spontaneous depolarizing postsynaptic potentials (SPSPs) were observed in the cockroach flexor tibia muscle that conformed in character to the types E, and E, potentials described by PIEK and MANTEL (1970). The amplitudes for E, potentials ranged from 20 to 50 mV but these potentials were seen only occasionally. The smaller E, potentials (0.5-14 mV) occurred frequently (Fig. 4G). No spontaneous hyperpolarizing potentials were observed. The pattern of spontaneous junctional activity in the flexor tibia muscle of the cockroach seemed to parallel the observations of HOYLE (1966) and PIEK and MANTEL (1970) concerning skeletal muscle in other insects. Usually the activity pattern for both normal and stressed cockroaches alternated between long periods of fairly regular discharge with intermittent bursts and periods of silence. However, as the physiological symptoms of paralysis developed, the firing frequency of the E, potentials at active recording sites seemed to increase; it reached a peak in insects that showed leg movement but no forward progression (Table 3). This circumstance seems to be supported by the close similarity in the average frequency for E, potentials at active sites in both normal and starved cockroaches despite the wide differences in their physiological states. Also we occasionally obtained evidence of recurrent trains on records from both torpid and paralysed (with leg movement) insects (Fig. 6). Once the cockroaches became completely paralysed, the frequency of E, dropped abruptly from 7*9/set in partially paralysed (with leg movement) insects to 1*6/set in completely paralysed insects. A corresponding drop occurred in the frequency of active recording sites. Thus,

3-C~A1icm

IN SPONTANEOUS

Normal Torpid Paralysed (movement, no progression) Paralysed (no movement) Starved (7 days)

Condition of insects

TABLE

18 15 IO 10 12

No. of insects tested

EXCITATORY

63 75 49 46 70

Total number of penetrations

POSTSYNAPTIC

3.5 24 26 4 18

DEVELOPMENT

192 232 131 17 164

Total scan times for active sites (set)

~~~~~~~~~~

Percentage active sites

POTENTIALS

1073 1557 1047 28 826

Total No. (Ez) potentials

OFSTRESS

5.6 6.7 7.9 I.6 5-o

Average potentials/ set

PARALYSIS

36

BENJAMINJ. COOKANDGERALDG. HOLT

shown in Table 3, 35 per cent of the penetrations in normal untreated cockroaches had Es potentials, but only 4 per cent of the muscle fibres of completely paralysed insects showed activity.

FIG. 6. Small spontaneous excitatory postsynaptic potentials (E, potentials) recorded from a fibre of the flexor tibiae muscle of a paralysed (with leg movement) cockroach. Calibration: 500 PV and 1 sec.

When muscle fibres of unstressed cockroaches were stimulated directly, either a graded or a neurally evoked response was obtained, depending on the position of the stimulating and recording electrodes in relation to the geometry of innervation. Generally, if the two electrodes were within 0.5 mm of each other a graded response could be obtained (see such a response recorded with an intracellular electrode, Fig. 4B). However, as stress paralysis developed, the muscle fibres gradually lost their graded responsiveness to direct stimulation, as shown in Fig. (4E) and Table 4. Finally, in completely paralysed insects, most muscle TABLE

~-INTRACELLULAR FXTRACELLULAR

Physiological condition of insects Normal Starved 7-11 days Paralysed (with leg movement) Paralysed (no

RESPONSE

OF

STIMULATION

No. of insects tested

THE

FLEXOR

MUSCLE

DURINGDEVELOPMENT

OF

P. americana

OFSTRFSS

TO DIRECT

PARALYSIS

Total number of penetrations

No. of graded responses

5 6 6

26 26 24

21 7 9

5 8 7

0 11 8

5

20

1

1

18

No. of neurally evoked responses

No. of no responses

fibres failed to respond to stimulation (Fig. 4H and Table 4), and no twitch response was observed (Table 2). Starved insects did not loose muscle excitability as judged by the observed twitch response (Table 2). Nevertheless, intracellular records showed a larger percentage of failure in starved insects, probably a consequence of desiccation in superficial fibres.

NEUROPHYSIOLOGICAL CHANGES FROM BODY STRESS IN THE COCKROACH

37

Changes ;in muscle ultrastructure A myoneural junction in the retractor unguis muscle of a normal cockroach is shown in Fig. 7(A). There is a large cluster of synaptic vesicles in the nerve terminal opposite a postsynaptic pillar. The axon is ensheathed in glial elements and the intermyofibril spaces are filled with a granular substrate. Fig. 7(B) shows an axon passing between myofibrils in the retractor unguis muscle of a cockroach starved for 7 days. In this sample there was both an apparent reduction in the intermyofibril spacing and a marked increase in the granular substrate. Even th’e mitochondria show signs of disintegration. Fig. 7(C) shows several myoneural junctions from a completely paralysed cockroach. Such specimens occasiorrally revealed a noticeable swelling in the intermyofibril spacing and a webbing of the granular substrate. Also some nerve-muscle junctions in paralysed insects had a high density of synaptic vesicles near the presynaptic membrane (Fig. 7D) or in the axoplasm, similar to the situation described by REES and USHERWOOD (1972) in degenerating motor nerve terminals. How,ever, it should be emphasized that the ultrastructural changes just described were not consistently found in cockroaches from a given behavioural category. Samples from both paralysed and starved insects often appeared normal. Nevertheless, the changes observed seemed to be characteristic for a behavioural class. Starved cockroaches never showed swelling in the intermyofibril spacing nor a high density of synaptic vesicles in nerve terminals, and paralysed insects failed to show any reduction in the intermyofibril spacing or increase in the density of the granular substrate. We have not included the normal to hyperactive category of stressed insects in the tables and graphs because this group showed no detectable difference from the normal. DISCUSSION

The notion that the process of dehydration alone causes neuromuscular dysfunction in stress-paralysed insects can be dismissed for a number of reasons: (1) Starved and dehydrated cockroaches did not develop the symptoms of paralysis in 7 days. Further, if insects were allowed food and water after this time, they quickly :returned to a normal physiological state. However, cockroaches subjected to physical stress seldom reverted to a normal condition. (2) Haemolymph ionregulating mechanisms seem to function in a normal manner in both paralysed and starved cockroaches. Thus, excessive accumulation of ions along the outer surfaces of muscle fibres and nerves is not likely until either the blood volume reaches a critical level or the normal tissue ion storage sites exceed their limits. (3) The mean resting potentials of paralysed insects were 10 to 1.5 mV higher than those of starved insects. Nevertheless, the junctional potentials of paralysed insects showed a disproportionately lower value than those of starved insects. (4) As dehydration progressed even the obvious reduction in blood volume with its implied restrictions on circulation was somewhat alleviated by the gradual appearance of a membranous air-filled sac in the abdominal cavity.

38

BENJAMINJ. COOKANDGERALDG. HOLT

Although cockroaches seemed to show some adaption to environmental stress, the stamina of an individual insect can be remarkably reduced in a subsequent encounter with stress. This fact was particularly well illustrated by an incident that occurred in our studies. A certain portion of the colony of cockroaches was exposed to 6 hr of cold because a heating failure in midwinter reduced the holding room temperature to - 3°C. Although 80 to 90 per cent of the adult cockroaches survived this exposure and seemed quite normal, the ability to withstand physical stress was markedly reduced. After 2 hr of tumbling, more than 60 per cent of the insects were either torpid or paralysed. This loss in vigour persisted in the colony for almost 2 months. Certainly a better understanding of such environmental stressors and the relationship to the vigour of insect populations could be of considerable importance in planning an effective insect control programme. Unfortunately, the relevance of our findings to such natural stressors as cold shock and overcrowding is not immediately apparent. As emphasized by HOYLE (1957), the evidence of direct external stimulation of normal insect muscle must be viewed with caution since no insect muscle has yet been shown to produce a propagated spike. Also the distributed nerve endings in muscle seem to have a lower threshold than the muscle fibres themselves to extracellular stimulation. Such nerve-mediated muscle responses show no change in latency or amplitude unless another axon is recruited as the stimulus intensity is increased. Nevertheless, we found it possible to excite the flexor tibia muscle directly by placing a pair of stimulating electrodes on the surface of the muscle and an intracellular recording electrode close by. The electrical response of the fibres under the cathodal electrode was graded according to the intensity of the stimulus (Fig. 4B). These graded depolarizations and the progressive shortening of the interval between stimulus and response support our assumption of a direct response. USHERWOOD (1963b) reported a similar response from denervated muscle fibres of the locust, Schistocerca gregaria. Moreover, CERF et al. (1959) found that when insect muscle fibres were subjected to brief intracellular depolarizing pulses, a graded response was obtained with a vanishing interval between the stimulus and response as the intensity of the stimulus was increased. Finally, the response that we observed seemed to be a local rather than a propagated event, for it was not recorded when the intracellular electrode was more than O-5 mm away from the cathode. The seemingly unimpaired generation and conduction of efferent impulses on the crural nerve of paralysed insects together with the complete failure or pronounced attenuation of evoked excitatory postsynaptic potentials (EPSP) seem to imply that transmission failure occurs at the myoneural junction. Moreover, the nearly simultaneous loss of both evoked and spontaneous EPSPs suggest that this failure has resulted from an excessive release of transmitter substance from the presynaptic nerve terminals followed by a desensitization of the postsynaptic sites. However, the progressive loss of direct responsiveness of muscle to externally applied current in paralysed cockroaches confound such a straightforward explanation. In fact, our ultrastructural studies indicate that changes

NEUROPHYSIOLOGICAL CHANGES FROM

BODY STRESS IN THE COCKROACH

39

can occur in both the volume and content of the intermyofibrial spaces. Such changes might reflect or cause altered ionic fluxes which in turn could introduce drastic changes in the electrical properties of muscle fibre membranes. Thus, the actual cause of paralysis remains obscure because it was not possible to determine whether synaptic failure preceded the loss in the responsiveness of muscle fibres, and it is entirely possible that both events proceed more or less simultaneously. Perhaps a careful pharmacological analysis of some of the substances released during stress (BEAMENT, 1958; STERNBURG et al., 1959; DAVEY, 1963; COOK et al., 1969; CASIDA and MADDRELL, 1971) may eventually explain the temporal sequencing of events that lead to paralysis. In conclusion, it is interesting to compare the changes that occurred at the peripheral myoneural junction during the development of stress parafysis with those that occurred in the terminal ganglion in an earlier study (COOK et al., 1969). In the previous study, at least 40 per cent of the insects in the torpid with ataxia ca.tegory showed some impairment of the transmission process at the cereal nerve to giant fibre synapse in the terminal ganglion, and nearly 50 per cent of the cockroaches in this class showed evidence of recurrent training along the connectives between the fifth and sixth abdominal ganglia. This recurrent training and the increase in the frequency of firing seemed to occur in the metathoracic ganglion of torpid and paralysed cockroaches, as judged from the data in Table 2 and Fi,g. 6. However, both the resting potentials and the fast junctional potentials in the flexor tibia muscle of torpid cockroaches appeared almost normal (Fig. 5). Thus, the neural instability and synaptic dysfunction associated with stress paralysis, first seem to occur in the central nervous system and only show up in the peripheral synapses during the terminal stages of paralysis. Acknc~wledgements-We would like to thank Mr. HUGH SPENCERof the Department of Physiology, University of Manitoba, for reading and criticizing the manuscript. We are indebted to Mr. JOHN REINECKEof this laboratory for the electron microscopy, and we also wish to express our appreciation to Mrs. JANE DICICCO, Mr. DAVIDOWENS, and Miss CYNTHIAHOPP for their competent technical assistance. REFERENCES J. W. L. (1958) A paralysing agent in the blood of cockroaches. J. Insect Physiol. 2, 199-214. BECHT G., HOYLE G., and USHERWOOD P. N. R. (1960) Neuromuscular transmission in the coxal muscles of the cockroach. J. Insect Physiol. 4, 191-201. CASIDA J, E. and MADDRRLL H. P. (1970) Diuretic hormone release on poisoning Rho&us with insecticide chemicals. Pest. Biochem. Physiol. 1, 71-83. CERF J. A., GRUNDFEST H., HOYLE G., and MCCANN F. V. (1959) The mechanism of dual responsiveness in muscle fibres of the grasshopper Romalea microptwa. J. gen. Physiol. 43, 377-395. COLHOUN E. H. (1960) Approaches to mechanisms of insecticidal action. J. agvic. Fd Chem. 8 25;!-257. Cook B. J., DE LA CUESTAM., and POMONISJ. G. (1969) The distribution of Factor S in the cockroach, Periptaneta americana, and its role in stress paralysis. J. Insect Physiol. 15, 963-975. BEAMENT

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BENJAMIN J. COOK ANDGERALD G. HOLT

COOK B. J., LONG J. B., and OWENS D. (1971) An inexpensive solid state amplifier for

measuring bioelectric potentials. Camp. Biochem. Physiol. 4OA, 385-390. DAVEY K. G. (1963) The release by enforced activity of the cardiac accelerator from the

corpus cardiacurn of Periplaneta americana. J. Insect Physiol. 9, 375-381. EDNEY E. B. (1968) The effect of water loss on the haemolymph of Arenivaga sp. and Periplaneta americana. Comp. Biochem. Physiol. 25, 149-158. EWING L. S. (1967) Fighting and death from stress in a cockroach. Science, New York 155, 1035-1036. HESLOP J. P. and RAY J. W. (1959) The reaction of the cockroach Periplaneta americana L. to bodily stress and DDT. r. Insect Physiol. 3, 395-401. HOLMANG. M. and COOK B. J. (1972) Isolation, partial purification and characterization of a peptide which stimulates the hindgut of the cockroach, Leucophaea maderae (Fabr.). Biol. Bull., Woods Hole 142, 446-460. HOYLE G. (1957) Nervous control of insect muscles. In Recent Advances in Invertebrate Physiology (Ed. by SCHEERB. T.). University of Oregon Press. HOYLE G. (1966) Functioning of the inhibitory conditioning axon innervating insect muscles. J. exp. Biol. 44, 429-453. PIEK T. and MANTELP. (1970) A study of different types of action potentials and miniature potentials in insect muscles. Comp. Biochem. Physiol. 34, 935-951. REJZS D. and USHERWOOD P. N. R. (1972) Fine structure of normal and degenerating motor axons and nerve-muscle synapses in the locust, Schistocerca gregaria. Comp. Biochem. Physiol. 43A, 83-101. SANFORDF. (1971) Antagonistic behavior in dominant subordinate relationships in a possible social stress response in the field cricket, Gyrilus intager. Ph.D. Thesis, University of Oklahoma. STERNBURG J., CHANGS. C., and KEARNSC. W. (1959) The release of a neuroactive agent by the American cockroach after exposure to DDT or electrical stimulation. 3. econ. ht. 52, 1070-1076. USHERWOOD P. N. R. (1963a) Spontaneous miniature potentials from insect muscle fibres. J. Physiol. 169, 149-160. USHERWOODP. N. R. (1963b) Response of insect muscle to denervation-II. Changes in neuromuscular transmission. J. Insect Physiol. 9, 81 l-825. WALL B. J. (1970) Effects of dehydration and rehydration on Periplaneta americana. J. Insect Physiol. 16, 1027-1042.