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Spontaneous activity in the cardioaccelerator nerves of the crayfish, Astacus pallipes lereboullet
Comp. Biochem. Physiol., 1970, Vol. 33, pp. 859 to 869. Pergamon Press. Printed in Great Britain
SPONTANEOUS ACTIVITY IN THE CARDIOACCELERATOR NERVES OF THE CRAYFISH,
ASTACUS PALLIPES LEREBOULLET E. W. T A Y L O R Department of Zoology and Comparative Physiology, University of Birmingham
(Received 25 August 1969) Abstract--1. Patterned discharges have been recorded from the cardioaccelerator nerves of the prepared crayfish. The patterns vary markedly with temperature. 2. They originate from neurons in the suboesophageal ganglion, continue after partial isolation of the C.N.S. in the thorax, but breakdown when the accelerator nerve is sectioned. 3. The bursts of impulses appear to coincide with slow changes in the position and frequency of beating of the heart, indicating some form of tonic control of heart movements from the C.N.S.
INTRODUCTION THE BEATING of the crustacean heart is initiated by an intrinsic pool of neurons forming the neurogenic pacemaker which exhibits spontaneous, integrated bursts of activity driving the heart muscle (Maynard, 1960). In the Decapoda the frequency of heart-beat is controlled by inhibitory and acceleratory nerves from the central nervous system (Wiersma & Novitski, 1942; Maynard, 1953). Maynard (1960) considered that the cardioregulator nerves in the Crustacea are probably involved only in brief reflex acceleration and inhibition of the heart and regarded their effects as being relatively transitory. Wiersma & Novitski (1942) noted no consistent effects upon heart-rate following the sectioning of the cardioregulator nerves in the crayfish. Maynard (1961) has, however, recorded a resting discharge in the dorsal nerves to the heart of the lobster Hornarus and stated that these nerves may normally show tonic activity. He described the activity in the accelerator fibres as being uneven and erratic in preparations showing a low-frequency discharge and rhythmic and continuous in preparations discharging at a high frequency. As part of a wider study of respiratory and cardiovascular control in the Crustacea the spontaneous activity in the cardioregulator nerves of the crayfish, Astacus pallipes Lereboullet, has been monitored. The recordings from all four cardioregulator nerves consisted of high-frequency discharges in several different units. The activity in the cardioaccelerator nerves included bursts of impulses 859
860
E . W . TAYLOR
with several interesting properties. This paper is a description of the bursting activity recorded from the cardioacceleratornerves, and a discussion of its possible functional significance.
METHODS Male and female crayfish with a mean body-length of around 10 cm were prepared for experiments in a similar way to that described by Wiersma & Novitsld (1942). During experiments the thoracic cavity was bathed with a physiological saline the composition of which was based on information provided by Lockwood (1961). This contained 10.27 g NaCI, 0"38 g KCI, 0.45 g MgCI~, 2.5 g CaC12 and 0"4 g NaHCOa made up to 1 I. with glass distilled water. In contrast to the majority of the Decapoda the crayfish possesses only one pair of cardioaccelerator nerves (Wiersma & Novitski, 1942). These were identified on the floor of the thoracic cavity from the description by Keim (1915) and exposed for recordings by cutting and turning back the overlying sheets of flexor muscles. Their cardioaccelerator function was initially checked by stimulating them via two silver electrodes and noting an increase in heart-rate. Spontaneous activity in the prepared nerve trunks was recorded en passant by lifting the nerve on a pair of fine silver hooks into a layer of previously cooled and aerated liquid paraffin. Activity was also recorded from the proximal end of the cut nerve by taking it up into a glass suction electrode filled with saline. T h e nerve impulses were routinely recorded on tape, displayed on a cathode ray oscilloscope and during recordings monitored on an audio-amplifier. In some instances a small square of carapace was removed to expose part of the dorsal surface of the heart covered by the hypodermis. Heart movements were then recorded as changes in capacitance between the dorsal wall of the heart and a small silver plate clamped immediately above it. In this way the frequency and amplitude of heart movements were recorded on the oscilloscope via a capacitance bridge and D.C. amplifier without mechanically loading the heart. T h e temperature range during the course of all these experiments was 5-20°C. Each experimental temperature was, however, maintained fairly constant by means of saline of the appropriate temperature, and where necessary, the use of ice-blocks to cool the platform on which the preparation was mounted. T h e temperature range during experiments was always noted, latterly by means of a thermocouple inserted into the preparation. The origin of the cardioaccelerator nerves in the suboesophageal ganglion was traced through serial sections of the C.N.S. dissected from the thorax, fixed and lightly stained in osmic acid and viewed with a phase-contrast microscope.
RESULTS Activity in the intact cardioaccelerator nerve The nerve impulses recorded with hook electrodes from the unbroken cardioa c c e l e r a t o r n e r v e s w e r e a t t r i b u t a b l e , on t h e basis o f s p i k e size, to at least five s e p a r a t e u n i t s (see Fig. 1). S m a l l units, p r o b a b l y m o r e t h a n one, d i s c h a r g e d i r r e g u l a r l y a n d c o n t i n u o u s l y in m a n y p r e p a r a t i o n s a n d in w i d e l y s p a c e d r a p i d b u r s t s o f spikes in s o m e p r e p a r a t i o n s at h i g h e r t e m p e r a t u r e s . T h e r e w e r e at least t w o m e d i u m - s i z e d units, one firing i r r e g u l a r l y , t h e o t h e r in v e r y r e g u l a r p a t t e r n e d bursts. L a r g e units w h e n active e i t h e r fired m o r e o r less r e g u l a r l y a n d c o n t i n u o u s l y
FIG. 3.
Spontaneous
discharges
in crayfish accelerator temperatures
nerl.es between
recorder! from j and 2O’C.
four
different
preparations
at a range
of
FIG. 5. Short bursts of impulses recorded from a cardioaccelerator nerve. The trace was triggered by the first impulse in each burst and filmed with the camera running vertically.
b
FIG. 7.
.
Spontaneous discharges (h) after cutting
recorded from an accelerator nerve it distal to the recnrding position.
. 1
j
,,
,.
,,
,.,
(a) before
and
,
.
FIG. 9. Recordings of heart movements and activity in the right cardioaccelerator nerve of a prepared crayfish. An upward deflection of the heart trace denotes upward movement of the heart indicating slow changes in the relative position of the heart as well as systolic contractions which cause the dorsal wall of the heart to bulge.
SPONTANEOUS ACTIVITY I N CARDIOACCELERATOR NERVEB OF CRAYFISH
861
or in long irregular bursts, the latter particularly in preparations at low temperatures. In some preparations a large unit fired only during each of the bursts recorded from one of the medium-sized units, suggesting that some form of nervous recruitment was involved, (see Fig. 7). The occurrence of each of these units and their rates and patterns of firing varied between preparations and showed particularly complicated variations related to the experimental temperature. The effects of temperature variation on this activity will be dealt with in a later publication; the present description is concerned primarily with the activity in the medium-sized bursting unit. Activity within the bursts recorded from this unit typically started at a high frequency and fell off continuously to the end of the burst. Often the final slowing was stepwise in form and sometimes it oscillated between high and low frequencies (see Fig. 2). The frequency of the bursts, the number of spikes per burst and the
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'Q 7
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FIc. 2. The changing frequency of the impulses within the bursts of activity recorded from accelerator nerves. In both long and short bursts the impulse frequency is high at first and drops continuously, with some oscillations, to the end of the burst. overall frequency of the bursting unit varied widely between preparations. The bursts recorded from each preparation were, however, remarkably regular both in frequency and spike number (see Fig. 3 and Table 1). The various patterns of activity recorded from different preparations are related to the experimental temperature. The long infrequent bursts, containing many action potentials, were recorded from preparations at approximately 5°C, the more highly frequent short bursts came from preparations at approximately 20°C. The overall discharge frequency was highest at low temperatures.
862
E . W . TAYLOR
E x a m i n a t i o n of t h e v a r i o u s d i s c h a r g e p a t t e r n s r e c o r d e d f r o m different p r e p a r a tions revealed a precise relationship between burst-interval and the number of t i m e s t h e u n i t fired w i t h i n e a c h b u r s t . T h i s is illustrated, u s i n g r e c o r d i n g s f r o m 13 different a n i m a l s , in Fig. 4. T h e n u m b e r of spikes w i t h i n e a c h b u r s t r o s e l o g a r i t h m i c a l l y as t h e t i m e - i n t e r v a l b e t w e e n successive b u r s t s i n c r e a s e d . T h i s r e l a t i o n s h i p h o l d s for t h e d i s c h a r g e p a t t e r n s r e c o r d e d f r o m single p r e p a r a t i o n s . T h e r e c o r d e d
Ordinafes
•
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•
.~
° O
•
•
~n
..~
f0
g ff
o
I
L
Time interval between bursts,
3o sec
FIG. 4. T h e mean number of impulses per burst plotted logarithmically against mean time-interval between bursts for recordings of spontaneous activity in the accelerator nerves of 13 prepared crayfish.
TABLE
1--MEASUREMENTS
O F T H E D I S C H A R G E P A T T E R N S RECORDED F R O M T H E ACCELERATOR NERVES OF NINE CRAYFISH
Number of bursts filmed
Mean number of impulses per burst + S.D.
glean time interval between bursts +_S.D. (sec)
Overall discharge frequency of bursting unit (impulses/min)
24 21 13 12 8 14 6 16 12
2"75 + 0"28 4"68 +__0-28 7"4 __+0"96 14"25 + 3-41 14"71 + 1"98 24"07 + 3"29 32.83 + 8"08 47"7 + 11.97 126"4 + 8"94
4"13 __+1"98 4"8 __+1-15 6'35 + 3-11 13'54 __+3"14 14'89 + 1"25 15"31 + 1 "45 20.92 + 4"39 24"46 + 9"05 32"82 + 2.23
40 58"5 61"7 63 59.3 94"3 94"1 117 231
SPONTANEOUS
ACTIVITY
IN CARDIOACCELERATOR
NERVES
OF CRAYFISH
863
variations in spike number between individual bursts in any series, reflected in the standard deviations around the means in Table 1, are directly reflected in the time-intervals between adjacent bursts (see Fig. 5). This is illustrated graphically in Fig. 6. This direct relationship between the interburst period and the number of spikes in a burst in en passant recordings implies some form of negative feedback in the system, possibly from the target organ, similar to the reciprocity between the neurons of the crustacean cardiac ganglion described by Maynard (1960). The open-loop situation will be described below. I0
• o
E
•
O0
•
•
O0
5 IO
Z
I 5 Time intervol
to od]acen| burst,
I iO sec
FIG. 6. The relationship between the time-intervals separating consecutive bursts of activity in the accelerator nerve and the number of impulses per burst. The points are taken from a continuous recording of activity in a single preparation.
Progressive isolation of the nervous system in the thorax When preparing the crayfish for experiments the legs and mouthparts were cut elose to their bases and the stomach, bearing the stomatogastrie ganglion, was removed. Thus much of the peripheral nervous system in the thorax was removed prior to any recordings being taken. Regular bursting activity was obtained from the cardioaccelerator nerve after cutting the abdominal cord in the first abdominal segment, and both circumoesophageal commisures. These cuts abolished the possible influenees of centres "higher" and "lower" in the C.N.S. on the activity recorded from the eardioaccelerator nerves. The presence of these centres was inferred from electrical stimulation of the C.N.S. in the crayfish by Wiersma & Novitski (1942). The regular bursting activity would therefore, appear to originate from the central nervous system in the thorax. It continued after the other three cardioregulator nerves (i.e. the other accelerator and the two inhibitors) were cut, which appears to rule out any overriding influence of interaction between the four
864
E.W. TAYLOR
regulator nerves on the recorded activity, despite the cross-connexions in the C.N.S. demonstrated by Wiersma & Novitski (1942). Recordings from the proximal ends of cut accelerator nerves taken into suction electrodes contained the same identifiable units as the en passant recordings from hook electrodes. The overall level of activity was, however, higher. Occasionally bursts of activity were recorded from a medium-sized unit. These were, however, less regular and of a lower frequency, related to the number of spikes within each burst, than the recordings from the intact nerve. In some instances, following the recording of a series of regular bursts from an intact nerve on hook electrodes, it was cut distal to the recording position. The bursts typicaUy became less regular and were sometimes replaced by continuous, irregular firing in all units (see Fig. 7). The overall discharge frequency recorded in the open-loop situation was higher and the bursting activity less regular than that observed in en passant recordings. These changes suggest that sectioning the nerve removes some form of clamp (perhaps negative feedback from the target organ) from the spontaneously active neurons in the C.N.S. Consequently their discharge frequency rises and the clear patterning of the discharges breaks down. Hughes & Wiersma (1960) noted a similar loss of rhythmic bursts from neurons in the crayfish abdominal ganglia following isolation of the nerve cord from sensory inflow. The origins of the accelerator nerves in the central nervous system
The cardioinhibitor and cardioaccelerator nerves of the crayfish are respectively the first and second superior nerves from the suboesophageal ganglion (Keim, 1915). The accelerator nerves leave the ganglion above the point of insertion of the ventral motor nerves supplying the second maxillipeds and have been described as segmental nerves from the ganglion supplying these appendages (Pilgrim & Wiersma, unpublished). Serial sections of the accelerator nerve and suboesophageal ganglion revealed that the nerve contains six large axons, two approximately 20/~m and four approximately 10/~m in diameter. In addition there are several smaller axons below 5/~m in diameter. Its structure does, therefore, reflect the recorded nervous activity described in the previous sections. From its point of insertion on the suboesophageal ganglion the nerve passes anteriorly, as a discrete nerve trunk, inside the ganglion for approximately 200/zm. It then begins to break up into individual axons and turns towards a large group of neurons lying ventrally in the periphery of the ganglion on each side, between the points of insertion of the ventral nerves to the first and second maxillipeds, and around 400/zm, anterior to the point of insertion of the nerve. Presumably, therefore, the bursting activity recorded in the accelerator fibres originates from one of the neurons in this group. Sectioning the nerve proximal to the recording position abolished the recorded activity, though occasionally a small unit continued to fire. The accelerator may, therefore, in common with the
S P O N T A N E O U S A C T I V I T Y I N C A R D I O A C C E L E R A T O R NERVES OF C R A Y F I S H
865
majority of decapodan segmental nerves, be mixed sensory and motor with the motor activity originating from a group of neurons in the suboesophageal ganglion. Simultaneous recordings of heart movements and activity in the accelerator nerves In the Crustacea only one of the axons from each cardioregulator nerve enters the heart ganglion and effects pacemaker activity directly (Alexandrowicz, 1932; Maynard, 1960). The other axons in the cardioaccelerator nerve end in the neurosecretory pericardial gland, run to dorsal muscles or run to the suspensory ligaments which support the heart in the pericardium (Maynard, 1953). It is probable therefore, that only a small part of the activity recorded from the cardioaccelerator nerve will have any direct effect upon the activity of the heart. Simultaneous recordings of heart movements and activity in an intact cardioaccelerator nerve were scrutinized for signs of related phenomena. Towards the end of experiments on the prepared crayfish, particularly when nerve lesions had been carried out, the heart often began to beat irregularly or to stop beating for long periods. On several occasions return of heart-beat was heralded by a prolonged burst of activity from a medinm-sized fibre in the accelerator nerve (see Fig. 8). This agrees with the observation by Maynard (1953) that stimulation of the accelerator nerves restored regular beating to a previously irregular or inactive heart and redistributed activity in the neurons of the heart ganglion into regular bursts.
I
to
,
1
I
I
I
I
I
I
I
=,
E 0
32 Time,
$ec
FIO. 8. The changing frequency of impulses during a prolonged burst of activity recorded from a previously silent accelerator nerve plotted against time and related to the onset and continuation of heart-beat.
The frequency of heart-beats in the prepared crayfish was often very variable, alternately accelerating and slowing down. This variation was associated with slow changes in the recorded position of the heart which resembled the slow rise and fall of the baseline of the crayfish E.C.G., attributed to heart "tonus fluctuations" (Ashby & Larimer, 1964). Bursts of activity in the cardioaecelerator nerve often coincided with the onset of a slow rise in the recorded position of the heart, relative to the silver
866
E.W. TAYLOR
plate, and with an associated increased frequency of heart beat (see Fig. 9). There appears, therefore, to be some relationship between the bursts of activity in the accelerator nerve and movements of the heart. It should be stressed that these bursts of activity do not bear a one-to-one relationship to heart-beat. The burst frequencies recorded from different preparations varied between approximately 2 to 20 per min whilst heart-rate was between 8 and 60 per min (i.e. in any single preparation burst frequency was approximately one-quarter of heart-rate).
DISCUSSION The rhythmic activity recorded from the accelerator nerves is of interest in a consideration of the role of peripheral sensory inflow in establishing patterns of discharge from neurons in the C.N.S. In this preparation rhythmicity was conditional upon the accelerator nerve being intact. This implies that feedback from the target organ was triggering or clamping central nervous activity, and that sensory inflow was along the nerve itself. When the nerve was sectioned nervous activity continued but was less precisely patterned. The important role of sensory inflow in the control of rhythmic movements elicited by local co-ordinating mechanisms in the nerve cord was demonstrated by Hughes & Wiersma (1960). They recorded rhythmic bursts of impulses, synchronous with swimmeret movements, from the first segmental nerve roots of the isolated abdomen which persisted until the cord was completely deafferentated, when the discharges became intermittent. The bursts of impulses recorded in the accelerator nerve are presumably driving a system which undergoes rhythmic cycles of activity analagous to the metachronism of the swimmerets. A relationship between the spontaneous activity in the accelerator nerves and heart-rate was described above. The possibility of tonic control of heart movements from the C.N.S., analogous to the vagal tone on the vertebrate heart, was discussed by Maynard (1960). He has described a few observations of spontaneous discharges in the cardioaccelerator nerves of Homarus (Maynard, 1961) but believes that central nervous control is probably limited to brief reflex acceleration or inhibition. Bearing in mind that in the present series of experiments the crayfish was eviscerated and had its blood system replaced by saline, the very high levels of spontaneous activity recorded in the cardioregular nerves may nevertheless indicate that some form of tonic control over the heart-rate is present in the intact animal. The bursts of impulses in the accelerator nerve often coincided with an increase in heart-rate plus a slowly rising wave on the baseline indicating that the heart was slowly bulging upwards. Ashby& Latimer (1964) noted that in recordings of the crayfish E.C.G. cardioacceleration induced by high CO 2 was accompanied by "an increased tonus shown by the elevated baseline". Florey (1960) studied the effects on heart movements of stimulating the cardioregulator nerves. His
SPONTANEOUS ACTIVITY IN CARDIOACCELERATOR NERVES OF CRAYFISH
867
traces reveal that acceleration was accompanied by a slow rise and inhibition by a drop in baseline. Similar recordings were obtained from the present preparation when the cardioregulator nerves were stimulated. Any attempt to establish a functional role in the control of heart-beat for the bursting activity recorded from the accelerator nerves must include a consideration of these slow waves, the nature of which is still not clear. It is possible that the bursting unit is the accelerator fibre proper, i.e. the axon which enters the cardiac ganglion via the dorsal nerve. Stimulation of the accelerator fibres increases the rate of firing of the cardiac ganglion by eliciting, on the membrane of the follower cells, depolarizing synaptic potentials which exhibit facilitation and temporal summation (Terzuolo & Bullock, 1958). Bursts of activity in these fibres could, therefore, depolarize the follower cells and so speed up or initiate heart-beats. A control system of this kind would tend to lead to the oscillations in heart-rate which have often been described in recordings of crustacean E.C.G.'s (Maynard, 1960). It is equally possible that the bursting unit supplies the pericardial glands and causes the release of the hormone which has been shown to have a cardioacceleratory effect (Alexandrowicz & Carlisle, 1953). Cooke (1964) reported the release of excitatory material from the pericardial organ of Libinia emarginata following electrical stimulation of the cardioaccelerator nerves. The stimulus threshold for action potentials in the gland corresponded to the threshold for secretion and the excitatory effect of the fluid was proportional to the number of stimuli given. The crayfish preparation used in this investigation is not at all suited to a study of hormonal effects as its interrupted blood supply will prevent proper distribution of the hormone. Nevertheless the observed relationship of bursting activity to heart-rate could conceivably be due to the production of a hormone from the pericardial gland as it would be released into the pericardial cavity close to the heart. Neither of these possible explanations account for the slow waves associated with cardioacceleration. Slow changes in heart position and electrical activity could be due to some form of tonus fluctuation in the myocardium causing the heart to "bunch up". Another purely conjectural explanation can, however, be offered for the slow waves. The crustacean heart is a force suction pump. Systole is brought about by contraction of the myocardium against resistance in the circulation and the pull of the elastic suspensory ligaments; diastole is due to relaxation of the myocardium and distension of the heart by the pull of the ligaments. In this system stroke volume is related to the duration of diastole which, because it is a much slower process than systole is also the rate limiting step. Control of diastolic filling is, therefore, of prime importance in the regulation of heart action. This control is exerted via the tension developed in the suspensory ligaments. This is in turn controlled by the alary muscles, contraction of which increases pericardial volume, drawing blood into the pericardium, and alters the tension exerted through the suspensory ligaments on the myocardium. It is possible that the observed slow changes in heart position result from changes in tension in the suspensory ligaments. Mangold (reported in
868
E . W . TAYLOR
Maynard, 1960) observed that the rate of beat of the decapod heart was increased when the heart was stretched. The observed increase in heart-rate associated with the slow waves may be a response to stretch imposed via the suspensoryligaments. Control of tension in the suspensory ligaments could, therefore, constitute a system controlling both stroke volume and frequency of heart-beats. A]exandrowicz (1932) reported that the alary muscles are supplied with motor fibres from four segmental nerves in the thorax (the thoracic nerves II to V of Keim, 1915). The activity recorded in the accelerator nerve is, therefore, unlikely to supply the alary muscles directly. Fibres from the accelerator nerves do, however, anastomose on the suspensory ligaments and may therefore exert direct tonic influences on tension. Afferent activity in the accelerator nerves may relate to the tension developed in the suspensory ligaments. It is tempting to postulate the existence of stretch receptors in the pericardial cavity sending fibres into the accelerator nerves. Alternatively there may be sensoryinflowfrom the myocardiumor cardiac ganglion, possibly from the dendritic arborizations on the neurons of the ganglion which are thought to be stretch receptors supplying single-neuron reflex-arcs (Maynard, 1960), via the small fibres located in the dorsal nerves to the heart by Alexandrowicz (1932). T h e n a t u r e of the slow waves obtained in recordings of the crayfish E . C . G . and heart m o v e m e n t s , the relationship to heart-beat of the s p o n t a n e o u s patterned discharges r e c o r d e d f r o m the cardioaccelerator nerves and the effects of environmental variation on this activity are all subjects w o r t h y of f u r t h e r study.
REFERENCES ALEXANDROWlCZJ. S. (1932) The innervation of the heart of the Crustacea--I. Decapoda. Q. 31. microsc. Sci. 75, 181-249. ALmCANDROWICZJ. S. & CARLISLED. B. (1953) Some experiments on the function of the pericardial organs of Crustacea. 07. mar. biol. Ass. U.K. 32, 175-192. ASHBY E. A. & LAmMERJ. L. (1964) Cardiac responses of the crayfish, Procambarus simulans, to external and internal COs stress. Physiol. Zo6l. 37, 21-32. COOKE I. M. (1964) Electrical activity and release of neurosecretory material in crab pericardial organs. Comp. Biochem. Physiol. 13, 353-366. FLOm~Y E. (1960) Studies on the nervous regulation of the heart beat in decapod crustacea. 07. gen. Physiol. 43, 1061-1081. HUGHES G. M. & WIERSMA C. A. G. (1960) The co-ordination of swimmeret movements in the crayfish, Procambarus clarkii (Girard). 07. exp. Biol. 37, 657-670. KEIM W. (1915) Das Nervensystem yon Astacus fluviatilis (Potamobius astacus L.) Ein Beitrag Zur Mophologie der Dekapoden. Z. wiss. Zool. 113, 485-545. LOCKWOODA. P. M. (1961) "Ringer" solutions and some notes on the physiological basis of their ionic composition. Comp. Biochem. Physiol. 2, 241-289. MAYNARD D. M. (1953) Activity in a crustacean ganglion--I. Cardio-inhibition and acceleration in Panulious argus. Biol. Bull. 104, 156-170. MAYNARDD. M. (1960) Circulation and heart function. In The Physiology of the Crustacea (Edited by WATERMXNT. H.), Vol. I, pp. 161-226. Academic Press, New York.
SPONTANEOUS ACTIVITY I N CARDIOACCELERATOR NERVES OF CRAYFISH
869
MAYNARD D. M. (1961) Cardiac inhibition in decapod Crustacea. In Nervous Inhibitions (Edited by FLoaxY E.), pp. la, a. 178. Pergamon Press, London. TERZt:OLOC. A. & BULLOCKT. H. (1958) Acceleration and inhibition in crustacean ganglion ceils. Arch. ital. Biol. 96, 117-134. WIEaSMA C. A. G. & Novlxsxt E. (1942) The mechanism of the nervous regulation of the crayfish heart. J. exp. Biol. 19, 255-265. Key Word Index--Cardioaccelerator nerves; crayfish; Astacus pallipes; patterns of action potentials; heart control of Astacus.
SPONTANEOUS ACTIVITY IN THE CARDIOACCELERATOR NERVES OF THE CRAYFISH,
ASTACUS PALLIPES LEREBOULLET E. W. T A Y L O R Department of Zoology and Comparative Physiology, University of Birmingham
(Received 25 August 1969) Abstract--1. Patterned discharges have been recorded from the cardioaccelerator nerves of the prepared crayfish. The patterns vary markedly with temperature. 2. They originate from neurons in the suboesophageal ganglion, continue after partial isolation of the C.N.S. in the thorax, but breakdown when the accelerator nerve is sectioned. 3. The bursts of impulses appear to coincide with slow changes in the position and frequency of beating of the heart, indicating some form of tonic control of heart movements from the C.N.S.
INTRODUCTION THE BEATING of the crustacean heart is initiated by an intrinsic pool of neurons forming the neurogenic pacemaker which exhibits spontaneous, integrated bursts of activity driving the heart muscle (Maynard, 1960). In the Decapoda the frequency of heart-beat is controlled by inhibitory and acceleratory nerves from the central nervous system (Wiersma & Novitski, 1942; Maynard, 1953). Maynard (1960) considered that the cardioregulator nerves in the Crustacea are probably involved only in brief reflex acceleration and inhibition of the heart and regarded their effects as being relatively transitory. Wiersma & Novitski (1942) noted no consistent effects upon heart-rate following the sectioning of the cardioregulator nerves in the crayfish. Maynard (1961) has, however, recorded a resting discharge in the dorsal nerves to the heart of the lobster Hornarus and stated that these nerves may normally show tonic activity. He described the activity in the accelerator fibres as being uneven and erratic in preparations showing a low-frequency discharge and rhythmic and continuous in preparations discharging at a high frequency. As part of a wider study of respiratory and cardiovascular control in the Crustacea the spontaneous activity in the cardioregulator nerves of the crayfish, Astacus pallipes Lereboullet, has been monitored. The recordings from all four cardioregulator nerves consisted of high-frequency discharges in several different units. The activity in the cardioaccelerator nerves included bursts of impulses 859
860
E . W . TAYLOR
with several interesting properties. This paper is a description of the bursting activity recorded from the cardioacceleratornerves, and a discussion of its possible functional significance.
METHODS Male and female crayfish with a mean body-length of around 10 cm were prepared for experiments in a similar way to that described by Wiersma & Novitsld (1942). During experiments the thoracic cavity was bathed with a physiological saline the composition of which was based on information provided by Lockwood (1961). This contained 10.27 g NaCI, 0"38 g KCI, 0.45 g MgCI~, 2.5 g CaC12 and 0"4 g NaHCOa made up to 1 I. with glass distilled water. In contrast to the majority of the Decapoda the crayfish possesses only one pair of cardioaccelerator nerves (Wiersma & Novitski, 1942). These were identified on the floor of the thoracic cavity from the description by Keim (1915) and exposed for recordings by cutting and turning back the overlying sheets of flexor muscles. Their cardioaccelerator function was initially checked by stimulating them via two silver electrodes and noting an increase in heart-rate. Spontaneous activity in the prepared nerve trunks was recorded en passant by lifting the nerve on a pair of fine silver hooks into a layer of previously cooled and aerated liquid paraffin. Activity was also recorded from the proximal end of the cut nerve by taking it up into a glass suction electrode filled with saline. T h e nerve impulses were routinely recorded on tape, displayed on a cathode ray oscilloscope and during recordings monitored on an audio-amplifier. In some instances a small square of carapace was removed to expose part of the dorsal surface of the heart covered by the hypodermis. Heart movements were then recorded as changes in capacitance between the dorsal wall of the heart and a small silver plate clamped immediately above it. In this way the frequency and amplitude of heart movements were recorded on the oscilloscope via a capacitance bridge and D.C. amplifier without mechanically loading the heart. T h e temperature range during the course of all these experiments was 5-20°C. Each experimental temperature was, however, maintained fairly constant by means of saline of the appropriate temperature, and where necessary, the use of ice-blocks to cool the platform on which the preparation was mounted. T h e temperature range during experiments was always noted, latterly by means of a thermocouple inserted into the preparation. The origin of the cardioaccelerator nerves in the suboesophageal ganglion was traced through serial sections of the C.N.S. dissected from the thorax, fixed and lightly stained in osmic acid and viewed with a phase-contrast microscope.
RESULTS Activity in the intact cardioaccelerator nerve The nerve impulses recorded with hook electrodes from the unbroken cardioa c c e l e r a t o r n e r v e s w e r e a t t r i b u t a b l e , on t h e basis o f s p i k e size, to at least five s e p a r a t e u n i t s (see Fig. 1). S m a l l units, p r o b a b l y m o r e t h a n one, d i s c h a r g e d i r r e g u l a r l y a n d c o n t i n u o u s l y in m a n y p r e p a r a t i o n s a n d in w i d e l y s p a c e d r a p i d b u r s t s o f spikes in s o m e p r e p a r a t i o n s at h i g h e r t e m p e r a t u r e s . T h e r e w e r e at least t w o m e d i u m - s i z e d units, one firing i r r e g u l a r l y , t h e o t h e r in v e r y r e g u l a r p a t t e r n e d bursts. L a r g e units w h e n active e i t h e r fired m o r e o r less r e g u l a r l y a n d c o n t i n u o u s l y
FIG. 3.
Spontaneous
discharges
in crayfish accelerator temperatures
nerl.es between
recorder! from j and 2O’C.
four
different
preparations
at a range
of
FIG. 5. Short bursts of impulses recorded from a cardioaccelerator nerve. The trace was triggered by the first impulse in each burst and filmed with the camera running vertically.
b
FIG. 7.
.
Spontaneous discharges (h) after cutting
recorded from an accelerator nerve it distal to the recnrding position.
. 1
j
,,
,.
,,
,.,
(a) before
and
,
.
FIG. 9. Recordings of heart movements and activity in the right cardioaccelerator nerve of a prepared crayfish. An upward deflection of the heart trace denotes upward movement of the heart indicating slow changes in the relative position of the heart as well as systolic contractions which cause the dorsal wall of the heart to bulge.
SPONTANEOUS ACTIVITY I N CARDIOACCELERATOR NERVEB OF CRAYFISH
861
or in long irregular bursts, the latter particularly in preparations at low temperatures. In some preparations a large unit fired only during each of the bursts recorded from one of the medium-sized units, suggesting that some form of nervous recruitment was involved, (see Fig. 7). The occurrence of each of these units and their rates and patterns of firing varied between preparations and showed particularly complicated variations related to the experimental temperature. The effects of temperature variation on this activity will be dealt with in a later publication; the present description is concerned primarily with the activity in the medium-sized bursting unit. Activity within the bursts recorded from this unit typically started at a high frequency and fell off continuously to the end of the burst. Often the final slowing was stepwise in form and sometimes it oscillated between high and low frequencies (see Fig. 2). The frequency of the bursts, the number of spikes per burst and the
15
o
5
o
'Q 7
.~
\
~o
.E_ 4
sec
"i:5
3
6
$e¢
4
b
sec
FIc. 2. The changing frequency of the impulses within the bursts of activity recorded from accelerator nerves. In both long and short bursts the impulse frequency is high at first and drops continuously, with some oscillations, to the end of the burst. overall frequency of the bursting unit varied widely between preparations. The bursts recorded from each preparation were, however, remarkably regular both in frequency and spike number (see Fig. 3 and Table 1). The various patterns of activity recorded from different preparations are related to the experimental temperature. The long infrequent bursts, containing many action potentials, were recorded from preparations at approximately 5°C, the more highly frequent short bursts came from preparations at approximately 20°C. The overall discharge frequency was highest at low temperatures.
862
E . W . TAYLOR
E x a m i n a t i o n of t h e v a r i o u s d i s c h a r g e p a t t e r n s r e c o r d e d f r o m different p r e p a r a tions revealed a precise relationship between burst-interval and the number of t i m e s t h e u n i t fired w i t h i n e a c h b u r s t . T h i s is illustrated, u s i n g r e c o r d i n g s f r o m 13 different a n i m a l s , in Fig. 4. T h e n u m b e r of spikes w i t h i n e a c h b u r s t r o s e l o g a r i t h m i c a l l y as t h e t i m e - i n t e r v a l b e t w e e n successive b u r s t s i n c r e a s e d . T h i s r e l a t i o n s h i p h o l d s for t h e d i s c h a r g e p a t t e r n s r e c o r d e d f r o m single p r e p a r a t i o n s . T h e r e c o r d e d
Ordinafes
•
IO0
•
.~
° O
•
•
~n
..~
f0
g ff
o
I
L
Time interval between bursts,
3o sec
FIG. 4. T h e mean number of impulses per burst plotted logarithmically against mean time-interval between bursts for recordings of spontaneous activity in the accelerator nerves of 13 prepared crayfish.
TABLE
1--MEASUREMENTS
O F T H E D I S C H A R G E P A T T E R N S RECORDED F R O M T H E ACCELERATOR NERVES OF NINE CRAYFISH
Number of bursts filmed
Mean number of impulses per burst + S.D.
glean time interval between bursts +_S.D. (sec)
Overall discharge frequency of bursting unit (impulses/min)
24 21 13 12 8 14 6 16 12
2"75 + 0"28 4"68 +__0-28 7"4 __+0"96 14"25 + 3-41 14"71 + 1"98 24"07 + 3"29 32.83 + 8"08 47"7 + 11.97 126"4 + 8"94
4"13 __+1"98 4"8 __+1-15 6'35 + 3-11 13'54 __+3"14 14'89 + 1"25 15"31 + 1 "45 20.92 + 4"39 24"46 + 9"05 32"82 + 2.23
40 58"5 61"7 63 59.3 94"3 94"1 117 231
SPONTANEOUS
ACTIVITY
IN CARDIOACCELERATOR
NERVES
OF CRAYFISH
863
variations in spike number between individual bursts in any series, reflected in the standard deviations around the means in Table 1, are directly reflected in the time-intervals between adjacent bursts (see Fig. 5). This is illustrated graphically in Fig. 6. This direct relationship between the interburst period and the number of spikes in a burst in en passant recordings implies some form of negative feedback in the system, possibly from the target organ, similar to the reciprocity between the neurons of the crustacean cardiac ganglion described by Maynard (1960). The open-loop situation will be described below. I0
• o
E
•
O0
•
•
O0
5 IO
Z
I 5 Time intervol
to od]acen| burst,
I iO sec
FIG. 6. The relationship between the time-intervals separating consecutive bursts of activity in the accelerator nerve and the number of impulses per burst. The points are taken from a continuous recording of activity in a single preparation.
Progressive isolation of the nervous system in the thorax When preparing the crayfish for experiments the legs and mouthparts were cut elose to their bases and the stomach, bearing the stomatogastrie ganglion, was removed. Thus much of the peripheral nervous system in the thorax was removed prior to any recordings being taken. Regular bursting activity was obtained from the cardioaccelerator nerve after cutting the abdominal cord in the first abdominal segment, and both circumoesophageal commisures. These cuts abolished the possible influenees of centres "higher" and "lower" in the C.N.S. on the activity recorded from the eardioaccelerator nerves. The presence of these centres was inferred from electrical stimulation of the C.N.S. in the crayfish by Wiersma & Novitski (1942). The regular bursting activity would therefore, appear to originate from the central nervous system in the thorax. It continued after the other three cardioregulator nerves (i.e. the other accelerator and the two inhibitors) were cut, which appears to rule out any overriding influence of interaction between the four
864
E.W. TAYLOR
regulator nerves on the recorded activity, despite the cross-connexions in the C.N.S. demonstrated by Wiersma & Novitski (1942). Recordings from the proximal ends of cut accelerator nerves taken into suction electrodes contained the same identifiable units as the en passant recordings from hook electrodes. The overall level of activity was, however, higher. Occasionally bursts of activity were recorded from a medium-sized unit. These were, however, less regular and of a lower frequency, related to the number of spikes within each burst, than the recordings from the intact nerve. In some instances, following the recording of a series of regular bursts from an intact nerve on hook electrodes, it was cut distal to the recording position. The bursts typicaUy became less regular and were sometimes replaced by continuous, irregular firing in all units (see Fig. 7). The overall discharge frequency recorded in the open-loop situation was higher and the bursting activity less regular than that observed in en passant recordings. These changes suggest that sectioning the nerve removes some form of clamp (perhaps negative feedback from the target organ) from the spontaneously active neurons in the C.N.S. Consequently their discharge frequency rises and the clear patterning of the discharges breaks down. Hughes & Wiersma (1960) noted a similar loss of rhythmic bursts from neurons in the crayfish abdominal ganglia following isolation of the nerve cord from sensory inflow. The origins of the accelerator nerves in the central nervous system
The cardioinhibitor and cardioaccelerator nerves of the crayfish are respectively the first and second superior nerves from the suboesophageal ganglion (Keim, 1915). The accelerator nerves leave the ganglion above the point of insertion of the ventral motor nerves supplying the second maxillipeds and have been described as segmental nerves from the ganglion supplying these appendages (Pilgrim & Wiersma, unpublished). Serial sections of the accelerator nerve and suboesophageal ganglion revealed that the nerve contains six large axons, two approximately 20/~m and four approximately 10/~m in diameter. In addition there are several smaller axons below 5/~m in diameter. Its structure does, therefore, reflect the recorded nervous activity described in the previous sections. From its point of insertion on the suboesophageal ganglion the nerve passes anteriorly, as a discrete nerve trunk, inside the ganglion for approximately 200/zm. It then begins to break up into individual axons and turns towards a large group of neurons lying ventrally in the periphery of the ganglion on each side, between the points of insertion of the ventral nerves to the first and second maxillipeds, and around 400/zm, anterior to the point of insertion of the nerve. Presumably, therefore, the bursting activity recorded in the accelerator fibres originates from one of the neurons in this group. Sectioning the nerve proximal to the recording position abolished the recorded activity, though occasionally a small unit continued to fire. The accelerator may, therefore, in common with the
S P O N T A N E O U S A C T I V I T Y I N C A R D I O A C C E L E R A T O R NERVES OF C R A Y F I S H
865
majority of decapodan segmental nerves, be mixed sensory and motor with the motor activity originating from a group of neurons in the suboesophageal ganglion. Simultaneous recordings of heart movements and activity in the accelerator nerves In the Crustacea only one of the axons from each cardioregulator nerve enters the heart ganglion and effects pacemaker activity directly (Alexandrowicz, 1932; Maynard, 1960). The other axons in the cardioaccelerator nerve end in the neurosecretory pericardial gland, run to dorsal muscles or run to the suspensory ligaments which support the heart in the pericardium (Maynard, 1953). It is probable therefore, that only a small part of the activity recorded from the cardioaccelerator nerve will have any direct effect upon the activity of the heart. Simultaneous recordings of heart movements and activity in an intact cardioaccelerator nerve were scrutinized for signs of related phenomena. Towards the end of experiments on the prepared crayfish, particularly when nerve lesions had been carried out, the heart often began to beat irregularly or to stop beating for long periods. On several occasions return of heart-beat was heralded by a prolonged burst of activity from a medinm-sized fibre in the accelerator nerve (see Fig. 8). This agrees with the observation by Maynard (1953) that stimulation of the accelerator nerves restored regular beating to a previously irregular or inactive heart and redistributed activity in the neurons of the heart ganglion into regular bursts.
I
to
,
1
I
I
I
I
I
I
I
=,
E 0
32 Time,
$ec
FIO. 8. The changing frequency of impulses during a prolonged burst of activity recorded from a previously silent accelerator nerve plotted against time and related to the onset and continuation of heart-beat.
The frequency of heart-beats in the prepared crayfish was often very variable, alternately accelerating and slowing down. This variation was associated with slow changes in the recorded position of the heart which resembled the slow rise and fall of the baseline of the crayfish E.C.G., attributed to heart "tonus fluctuations" (Ashby & Larimer, 1964). Bursts of activity in the cardioaecelerator nerve often coincided with the onset of a slow rise in the recorded position of the heart, relative to the silver
866
E.W. TAYLOR
plate, and with an associated increased frequency of heart beat (see Fig. 9). There appears, therefore, to be some relationship between the bursts of activity in the accelerator nerve and movements of the heart. It should be stressed that these bursts of activity do not bear a one-to-one relationship to heart-beat. The burst frequencies recorded from different preparations varied between approximately 2 to 20 per min whilst heart-rate was between 8 and 60 per min (i.e. in any single preparation burst frequency was approximately one-quarter of heart-rate).
DISCUSSION The rhythmic activity recorded from the accelerator nerves is of interest in a consideration of the role of peripheral sensory inflow in establishing patterns of discharge from neurons in the C.N.S. In this preparation rhythmicity was conditional upon the accelerator nerve being intact. This implies that feedback from the target organ was triggering or clamping central nervous activity, and that sensory inflow was along the nerve itself. When the nerve was sectioned nervous activity continued but was less precisely patterned. The important role of sensory inflow in the control of rhythmic movements elicited by local co-ordinating mechanisms in the nerve cord was demonstrated by Hughes & Wiersma (1960). They recorded rhythmic bursts of impulses, synchronous with swimmeret movements, from the first segmental nerve roots of the isolated abdomen which persisted until the cord was completely deafferentated, when the discharges became intermittent. The bursts of impulses recorded in the accelerator nerve are presumably driving a system which undergoes rhythmic cycles of activity analagous to the metachronism of the swimmerets. A relationship between the spontaneous activity in the accelerator nerves and heart-rate was described above. The possibility of tonic control of heart movements from the C.N.S., analogous to the vagal tone on the vertebrate heart, was discussed by Maynard (1960). He has described a few observations of spontaneous discharges in the cardioaccelerator nerves of Homarus (Maynard, 1961) but believes that central nervous control is probably limited to brief reflex acceleration or inhibition. Bearing in mind that in the present series of experiments the crayfish was eviscerated and had its blood system replaced by saline, the very high levels of spontaneous activity recorded in the cardioregular nerves may nevertheless indicate that some form of tonic control over the heart-rate is present in the intact animal. The bursts of impulses in the accelerator nerve often coincided with an increase in heart-rate plus a slowly rising wave on the baseline indicating that the heart was slowly bulging upwards. Ashby& Latimer (1964) noted that in recordings of the crayfish E.C.G. cardioacceleration induced by high CO 2 was accompanied by "an increased tonus shown by the elevated baseline". Florey (1960) studied the effects on heart movements of stimulating the cardioregulator nerves. His
SPONTANEOUS ACTIVITY IN CARDIOACCELERATOR NERVES OF CRAYFISH
867
traces reveal that acceleration was accompanied by a slow rise and inhibition by a drop in baseline. Similar recordings were obtained from the present preparation when the cardioregulator nerves were stimulated. Any attempt to establish a functional role in the control of heart-beat for the bursting activity recorded from the accelerator nerves must include a consideration of these slow waves, the nature of which is still not clear. It is possible that the bursting unit is the accelerator fibre proper, i.e. the axon which enters the cardiac ganglion via the dorsal nerve. Stimulation of the accelerator fibres increases the rate of firing of the cardiac ganglion by eliciting, on the membrane of the follower cells, depolarizing synaptic potentials which exhibit facilitation and temporal summation (Terzuolo & Bullock, 1958). Bursts of activity in these fibres could, therefore, depolarize the follower cells and so speed up or initiate heart-beats. A control system of this kind would tend to lead to the oscillations in heart-rate which have often been described in recordings of crustacean E.C.G.'s (Maynard, 1960). It is equally possible that the bursting unit supplies the pericardial glands and causes the release of the hormone which has been shown to have a cardioacceleratory effect (Alexandrowicz & Carlisle, 1953). Cooke (1964) reported the release of excitatory material from the pericardial organ of Libinia emarginata following electrical stimulation of the cardioaccelerator nerves. The stimulus threshold for action potentials in the gland corresponded to the threshold for secretion and the excitatory effect of the fluid was proportional to the number of stimuli given. The crayfish preparation used in this investigation is not at all suited to a study of hormonal effects as its interrupted blood supply will prevent proper distribution of the hormone. Nevertheless the observed relationship of bursting activity to heart-rate could conceivably be due to the production of a hormone from the pericardial gland as it would be released into the pericardial cavity close to the heart. Neither of these possible explanations account for the slow waves associated with cardioacceleration. Slow changes in heart position and electrical activity could be due to some form of tonus fluctuation in the myocardium causing the heart to "bunch up". Another purely conjectural explanation can, however, be offered for the slow waves. The crustacean heart is a force suction pump. Systole is brought about by contraction of the myocardium against resistance in the circulation and the pull of the elastic suspensory ligaments; diastole is due to relaxation of the myocardium and distension of the heart by the pull of the ligaments. In this system stroke volume is related to the duration of diastole which, because it is a much slower process than systole is also the rate limiting step. Control of diastolic filling is, therefore, of prime importance in the regulation of heart action. This control is exerted via the tension developed in the suspensory ligaments. This is in turn controlled by the alary muscles, contraction of which increases pericardial volume, drawing blood into the pericardium, and alters the tension exerted through the suspensory ligaments on the myocardium. It is possible that the observed slow changes in heart position result from changes in tension in the suspensory ligaments. Mangold (reported in
868
E . W . TAYLOR
Maynard, 1960) observed that the rate of beat of the decapod heart was increased when the heart was stretched. The observed increase in heart-rate associated with the slow waves may be a response to stretch imposed via the suspensoryligaments. Control of tension in the suspensory ligaments could, therefore, constitute a system controlling both stroke volume and frequency of heart-beats. A]exandrowicz (1932) reported that the alary muscles are supplied with motor fibres from four segmental nerves in the thorax (the thoracic nerves II to V of Keim, 1915). The activity recorded in the accelerator nerve is, therefore, unlikely to supply the alary muscles directly. Fibres from the accelerator nerves do, however, anastomose on the suspensory ligaments and may therefore exert direct tonic influences on tension. Afferent activity in the accelerator nerves may relate to the tension developed in the suspensory ligaments. It is tempting to postulate the existence of stretch receptors in the pericardial cavity sending fibres into the accelerator nerves. Alternatively there may be sensoryinflowfrom the myocardiumor cardiac ganglion, possibly from the dendritic arborizations on the neurons of the ganglion which are thought to be stretch receptors supplying single-neuron reflex-arcs (Maynard, 1960), via the small fibres located in the dorsal nerves to the heart by Alexandrowicz (1932). T h e n a t u r e of the slow waves obtained in recordings of the crayfish E . C . G . and heart m o v e m e n t s , the relationship to heart-beat of the s p o n t a n e o u s patterned discharges r e c o r d e d f r o m the cardioaccelerator nerves and the effects of environmental variation on this activity are all subjects w o r t h y of f u r t h e r study.
REFERENCES ALEXANDROWlCZJ. S. (1932) The innervation of the heart of the Crustacea--I. Decapoda. Q. 31. microsc. Sci. 75, 181-249. ALmCANDROWICZJ. S. & CARLISLED. B. (1953) Some experiments on the function of the pericardial organs of Crustacea. 07. mar. biol. Ass. U.K. 32, 175-192. ASHBY E. A. & LAmMERJ. L. (1964) Cardiac responses of the crayfish, Procambarus simulans, to external and internal COs stress. Physiol. Zo6l. 37, 21-32. COOKE I. M. (1964) Electrical activity and release of neurosecretory material in crab pericardial organs. Comp. Biochem. Physiol. 13, 353-366. FLOm~Y E. (1960) Studies on the nervous regulation of the heart beat in decapod crustacea. 07. gen. Physiol. 43, 1061-1081. HUGHES G. M. & WIERSMA C. A. G. (1960) The co-ordination of swimmeret movements in the crayfish, Procambarus clarkii (Girard). 07. exp. Biol. 37, 657-670. KEIM W. (1915) Das Nervensystem yon Astacus fluviatilis (Potamobius astacus L.) Ein Beitrag Zur Mophologie der Dekapoden. Z. wiss. Zool. 113, 485-545. LOCKWOODA. P. M. (1961) "Ringer" solutions and some notes on the physiological basis of their ionic composition. Comp. Biochem. Physiol. 2, 241-289. MAYNARD D. M. (1953) Activity in a crustacean ganglion--I. Cardio-inhibition and acceleration in Panulious argus. Biol. Bull. 104, 156-170. MAYNARDD. M. (1960) Circulation and heart function. In The Physiology of the Crustacea (Edited by WATERMXNT. H.), Vol. I, pp. 161-226. Academic Press, New York.
SPONTANEOUS ACTIVITY I N CARDIOACCELERATOR NERVES OF CRAYFISH
869
MAYNARD D. M. (1961) Cardiac inhibition in decapod Crustacea. In Nervous Inhibitions (Edited by FLoaxY E.), pp. la, a. 178. Pergamon Press, London. TERZt:OLOC. A. & BULLOCKT. H. (1958) Acceleration and inhibition in crustacean ganglion ceils. Arch. ital. Biol. 96, 117-134. WIEaSMA C. A. G. & Novlxsxt E. (1942) The mechanism of the nervous regulation of the crayfish heart. J. exp. Biol. 19, 255-265. Key Word Index--Cardioaccelerator nerves; crayfish; Astacus pallipes; patterns of action potentials; heart control of Astacus.