Download electrophysiological investigations of the heart of squill a mantis

J. Exp. Biol. (1964), 41, 689-700
With 7 text-figures
Printed in Great Britain
689
ELECTROPHYSIOLOGICAL INVESTIGATIONS OF THE
HEART OF SQUILLA MANTIS
I. THE GANGLIONIC NERVE TRUNK
BY HILARY F. BROWN
Stazione Zoologica, Naples, and University Laboratory of Physiology, Oxford
{Received 6 February 1964)
GENERAL INTRODUCTION
In most, possibly all, Crustacea the heart beat is neurogenic. Each beat is initiated
by a burst of impulses from a small group of neurones whose cell bodies lie grouped
in the ganglionic nerve trunk (g.n.t.) in the heart wall and whose axons run to the heart
muscle. Many attempts have been made by electrophysiologists to discover how these
neurones are integrated to fire rhythmic bursts (for example Maynard, 1953, 1955;
Hagiwara & Bullock, 1957; Bullock & Terzuolo, 1957). However, apart from the study
by Irisawa et al. (1962) of some aspects of heart muscle physiology in Squilla oratoria,
analysis of the complete neuromuscular system of a crustacean heart does not appear
to have been undertaken.
In the present study such an analysis has been attempted in the case of the heart of the
stomatopod, Squilla mantis. The relatively simple anatomical plan of the stomatopod
heart makes it a more favourable preparation for electrophysiological study than is the
heart of decapod Crustacea. The present paper describes the investigations made of
the ganglionic nerve trunk of Squilla mantis. A second paper (Brown, 1964 a) will be
concerned with the electrophysiological properties of the heart muscle of Squilla.
In a third paper (Brown, 19646) the mode of action of extracts of the pericardial
organs on the heart of Squilla will be considered in relation to the electrophysiology
of untreated hearts.
THE ANATOMY OF THE HEART OF SQU1J.LA MANTIS
The stomatopod heart is a long tubular structure which extends about half the total
length of the body; in Squilla mantis this amounts to about 8 cm. It shows obvious
signs of segmentation in having 13 pairs of ostial orifices and 15 pairs of lateral arteries.
Fig. 1 is a diagram of the heart of Squilla mantis, redrawn from Alexandrowicz (1934).
In his paper Alexandrowicz gave a clear and detailed account of the innervation of the
Squilla heart, of which the main points will be summarized here. The ganglionic
nerve trunk (g.n.t.) is a chain of 14-16 nerve cells which runs the length of the heart
tube outside the muscle in the mid-dorsal line. For the most part there is one nerve
cell body per heart segment, situated behind the ostial orifices. The largest cell bodies
are in the sixth to thirteenth segments and reach 180 //, in diameter, while the cell
bodies near either end of the chain are much smaller.
Alexandrowicz described two large processes arising from the cell body of each
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HILARY F. BROWN
neurone, one running forwards and one backwards in the trunk. He did not find it
possible to determine how far these processes ran in the trunk before giving off branches
to the muscle, though in some cases they seemed to branch in the third segment from
the cell body, and he found side branches of the processes of more than one neurone
in the same heart segment. He reported seeingfiveor six axons in cross-sections of the
g.n.t., except in the anterior and posterior regions of the trunk, where he found fewer.
Dorsal,
nerves
Paired
ostia
Lateral
arteries
Ganglionic
nerve
trunk
Posterior
aorta
Fig. 1. Diagram of the heart of Squilla mantis (redrawn from Alexandrowicz, 1934).
Approx. twice natural size.
Alexandrowicz also described two other types of cell processes: short dendrites which
arise from the cell bodies and the proximal regions of the longitudinal processes and
end on the muscle fibres close to the cell bodies, and thin collaterals which arise from
the other types of cell processes and ramify within the g.n.t. Three pairs of dorsal
nerves enter the g.n.t. from the central nervous system (Fig. 1).
Electrophysiological investigations of the heart of Squilla mantis
,, . . ,
691
METHODS
Material
Specimens of Squilla mantis were obtained locally at Naples and kept in running sea
water, where they would live for up to 3 weeks.
Isolation and perfusion of the heart
The animals were decapitated and their claws were removed. The heart was exposed
by cutting away the carapace and removing the dorsal muscle blocks. The posterior
aorta was grasped with forceps and the heart was cut out from behind forwards. Some
of the underlying gonad tissue was removed as well and was trimmed away later. The
heart was quickly transferred to the perfusion dish and the posterior aorta was tied on to
the inflow cannula. The valve at the base of the posterior aorta was opened with the
cannula tip so that the fluid would flow into the heart by this route, counter to the
natural direction of flow.
Table 1. Composition of the perfusion fluid used for isolated Squilla hearts
NaCl
KC1
CaCl,
MgCla
NaHCO,
Urea
520 m-equiv./l.
13 m-equiv./l.
28 m-equiv./l.
49 m-equiv./l.
topH7
1-04 g./l.
The perfusion fluid was made up according to the formula given by Welsh (1939)
for crustacean hearts (Table 1). A little glucose was added. During the summer the
perfusion fluid was cooled to o° C. before it was placed in the reservoir bottle, insulated with glass wool (a in Fig. 2). It then passed through an oxygenating chamber
(d) and by the time it reached the perfusion dish (/) its temperature was about 170 C ,
which was still considerably cooler than the summer room-temperature in Naples.
Perfusion dishes were of glass or Perspex, with two holes in the lateral walls into
which were set the glass inflow cannula and an outflow tube leading to a filter pump,
by means of which the fluid was kept at a constant level, about half a centimetre above
the heart. The perfusion dish was mounted on the stage of a dissecting microscope,
with illumination from below. Micromanipulators were used for placing the
electrodes.
After a few minutes of perfusion the isolated heart would start to beat regularly.
It was always left for some time to adjust to perfusion conditions before recording was
started. Some hearts would continue to beat for up to 6 hr. when isolated and perfused, while others, under seemingly identical conditions, died far sooner. These
differences in vigour could not be correlated with the apparent condition of the animal.
Often animals which seemed very active had weak hearts while vigorous hearts might
come from weak animals.
Recording from the ganglionic nerve trunk
Only extracellular records were taken from the g.n.t. Conventional d.c. amplifiers
were used and the resulting signal was displayed on a Cossor oscilloscope. The elec-
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HILARY F. BROWN
trodes used were thin hooks of chlorided silver wire. The g.n.t. of the Squilla heart is
too fine and too closely attached to the muscle to be separated from it, so with fine
scissors two longitudinal cuts were made in the muscle wall on either side of the g.n.t.
for the distance of a few heart segments. As Alexandrowicz (1931) first observed in the
heart of Ligia oceanica, cutting the heart muscle has no effect on the co-ordination
of the crustacean heart beat as long as the g.n.t. is intact. The length of g.n.t. and
muscle thus freed was hooked over the silver wire electrode. The electrode was
mounted on the micro-manipulator arm (j) in Fig. 2. By moving the electrode up and
down, the g.n.t. was lifted out of the fluid for recording and replaced at other times.
A second chlorided silver electrode was placed in the perfusion dish and earthed.
Fig. 2. Diagram of the apparatus used for electrical recording from the heart of Squilla
mantis, a, Thermally insulated reservoir bottle; b, cooled perfusion fluid; c, fluid level indicator; d, aeration chamber; e, screw clip on rubber inflow lead; /, perfusion chamber; g,
heart; h, movable stage of microscope; i, outflow to filter pump;/, microelectrode or chlorided
silver hook electrode; k, micromanipulator arm; I, n, wires leading to cathode follower and
amplifier; m, microscope; o, mirror.
RESULTS
Extracellular records from the ganglionic nerve trunk
The average rate of beating of 10 isolated perfused hearts, soon after perfusion had
been started, was 17 beats/min. External electrodes, placed as described above at any
point on the g.n.t. recorded at each of these beats a certain number (N) of nerve
impulses (a burst). N might be any number between 1 and 12 but was more or less
constant for any one heart under given conditions (Fig. 3). A diagram of a sequence
of three externally recorded g.n.t. bursts is given in Fig. 4 to show the precise limits of
the durations and intervals which are referred to in discussing these records.
The nerve impulses recorded externally from one point on the g.n.t. were simple
Electrophysiological investigations of the heart of Squilla mantis
693
triphasic spikes and were almost always exactly the same height throughout a sequence of several beats. (Any such sequence is termed a 'run'.) This suggests that
repeated activity of the same structure was being recorded. On one occasion, a glass
microelectrode was used to explore a region of a g.n.t. containing a cell body. An
external record of the impulse burst, exactly similar to that recorded with hook
electrodes, was obtained, but only when the electrode tip was close up against the cell
body itself. This suggests that activity of the cell body rather than of cell processes
was responsible for the recorded signals. Indirect support for this conclusion, too
detailed to be given here, comes also from recordings from the ganglionic nerve
trunks of decapod hearts.
w.
rmn
•
rmt-1
1 sec.
MmV.
Fig. 3. Typical extracellular records from the ganglionic nerve trunks of spontaneously
beating Squilla hearts, (a), (b), (c) recorded from the same heart at different times, (d) From
the g.n.t. of a second heart. Spikes re-touched.
Burst duration
lm
PU'Se
intervals
4+fBurst interval
-
Fig. 4. Diagram of an extracellular record from the g.n.t. during a sequence of three heart
beats, to illustrate the terms used. This is a' run' of three' bursts'.
When two hook electrodes were placed at different points of the g.n.t., the second
one recorded at a given beat the same number of impulses as did the first. Typically,
the impulses in this second record were spaced at the same intervals as those recorded
at the first electrode, but the whole record at the second site was slightly displaced in
time (Fig. 5 (a)). It appears likely that all the nerve cells in the g.n.t. fire in succession
N times per beat. In most of the nine hearts from which records were taken simultaneously from two regions of the g.n.t., each nerve impulse was invariably recorded
at the same one of the two electrodes before being recorded at the other (as in
Fig. 5(a)). In four of the nine hearts this was at the anterior recording site; in two of
the nine hearts the impulses all appeared first at the posterior recording site. In the
45
Exp. Biol. 41, 4
694
HILARY F. BROWN
remaining three hearts, though most of the impulses were first recorded at one and the
same site, the last impulse or last few impulses of some or all bursts were first recorded
at the other site (Fig. 5 (b)). In this last group the delay between the electrodes was
the same for all the impulses (though reversible in sign) suggesting that the impulses
are initiated in the anterior or posterior regions of the chain, beyond the two recording
sites. In a typical case the recording sites were at the levels of cell 7 and cell 11. This
conclusion is supported by the results of the cutting and ligaturing experiments
described below.
1mV.[
100 msec.
Fig. 5. Simultaneous extracellular records from two points on the ganglionic nerve trunk of
Squilla mantis hearts. Records from two hearts, (a) and (6). The dotted lines are drawn in to
emphasize the time differences at the two recording points. In (6) the last 5 spikes reversed
their direction of travel. In the top record of (b) the large, slow component is from the muscle.
Tracings of original records.
The site of burst initiation
The perfused heart was cut transversely or ligatured at different points in an
attempt to locate the region responsible for initiating the rhythm. Some portions of
the g.n.t. so isolated would continue to initiate beats in the muscle isolated with them.
When the heart was divided in two at about the level of cells 7 and 8, one half would
continue to beat at the previous rate while the other half either beat more slowly or
was completely quiescent. In portions which were initially quiescent sporadic beats
would usually start up after a few minutes and beating would later become more
regular and faster.
The anterior and posterior portions of a few hearts were further subdivided by
several transverse cuts or ligatures. The cells of the g.n.t. were afterwards stained with
methylene blue to verify the positions of the cuts. Only some of the portions continued beating immediately after isolation and these (denoted by the cells they contained) are listed in Table 2. They beat considerably faster than the whole heart had
done.
The middle region of the heart (that containing cells 6-10) usually remained
quiescent when isolated by transverse cuts but the amount of spontaneity varied in
Electrophysiological investigations of the heart of Squilla mantis
695
different hearts. Sometimes even single neurones of this region which had been
separated from their neighbours would, some time after isolation, set up slow beating
of the muscle isolated with them. In two cases (f in Table 2) several portions of the
heart were left beating immediately after cutting.
Despite this variability it seems that the cells with the greatest intrinsic rhythmicity
and the highest spontaneous firing rate are those near the ends of the g.n.t. This is in
keeping with the finding that within an intact g.n.t. the impulses appear to be initiated
in the anterior or posterior portions of the chain.
Table 2. Portions of Squilla hearts which continued beating when isolated
The numbers refer to the contained cells.
No. of
Posterior halves
hearts
Cell 12
Cell 13
•Cells 14 and 15
•Cells 13, 14 and 15
Cells 11
\
12
• 13, 14 and 15/
Anterior halves
Cell 1
•Cells 1, 2 and 3
Cells 5 and 6
Cells
1
1
1
2
if
I
1
1
• Portion not further subdivided.
+ More than one portion of the heart left beating immediately after cutting.
The number of impulses in the g.n.t. burst
The number of impulses in the burst fired by the g.n.t. was related to the interval
between the bursts. In any one heart the number was fairly constant over a short
time (several minutes) but over a longer period the heart slowed and the number of
impulses per burst increased. Fig. 6 illustrates this relationship for a single heart. The
recordings were taken over a period of 1^ hr. The correlation coefficient for the points
in Fig. 6 is 0-98, which is highly significant.
Fig. 7 shows the relationship between the number of impulses per g.n.t. burst and
the burst interval for many hearts plotted on the same graph. Each reading gives the
average number of impulses per burst and the average burst interval for a run of 4-5
beats of a spontaneously beating heart. From some hearts more than one set of observations is included, taken at intervals of \ hr. or more. Fourteen readings from ten
hearts are included in the graph. The records analysed included not only extracellular
records from the g.n.t., where impulse number and burst interval can be seen directly,
but also intracellular records from the heart muscle. In these latter, as is shown by
recording simultaneously from the g.n.t. and from the muscle (see Brown, 1964a),
the number and spacing of the peaks of the junction potential recorded in the muscle
at a heart beat correspond exactly to those of the nerve impulses in the g.n.t. burst
initiating them.
One point in Fig. 7 differs widely from the others. It was taken from a heart
45-2
696
HILARY F. BROWN
that was beating very slowly at the time. If this point is included, then the correlation
coefficient for the readings is 0-39 and P > o-i. If it is omitted, the correlation
coefficient for the remaining points is 0-90, which is highly significant (P < o*oi). Its
exclusion seems justified, for the rate of beating had become very slow, and the heart
was probably in poor condition. Two previous readings from the same heart both fall
near the other points.
10
r 6
o
o
I
0
2
4
6
Average number of impulses per burst
8
Fig. 6. The average number of impulses per burst plotted against burst interval in a spontaneously beating Squilla heart as the rate of beating dropped over a period of i j hr. Each
point on the graph represents the average values for a run of three to five bursts.
There is a little evidence that the composition of the g.n.t. burst may be affected by
proprioceptive impulses from the dendritic endings of the neurones on the muscle
fibres. On one occasion when the muscle was being stimulated directly with long square
pulses of current (1-5 sec. duration and up to 50/tA. intensity) it was observed that
the g.n.t. fired every time the stimulus was given, though with a delay of 50 msec.
This long delay indicates that the g.n.t. was not directly excited by the electrical
stimulus (as when the stimulating electrode was placed directly on the g.n.t.) but that
burst firing was being regulated by impulses from the proprioceptive endings, presumably stimulated mechanically by the muscle movement.
Electrophysiological investigations of the heart of Squilla mantis
697
The pattern of impulses within the g.n.t. burst
The intervals between the impulses in a g.n.t. burst averaged 45 msec, but they were
not all the same length. Within one run recorded from a heart the spacing of impulses
within the bursts remained remarkably constant. When, as was usually the case, two
bursts of a run were both composed of the same number of impulses, the comparable
intervals (first, second, third, etc.) within the two bursts were of almost exactly equal
•s 6
o
I
o
o
o
o
o
o
o
o
.0
2
4
6
Average number of Impulses per burst
Fig. 7. The relation between the number of impulses per burst (abscissa) and the burst interval
in seconds (ordinate) for ten spontaneously beating hearts of Squilla mantis. Each point represents average values for a run of three to five bursts.
length. In all the runs recorded from any one heart the pattern of impulses within the
bursts remained similar although the number of impulses per burst varied from run to
run. Thus in the bursts shown in Fig. 3 (a) thefirstinterval between impulses was longer
than the second, while the third and subsequent intervals lengthened again, and the
sixth and last was noticeably the longest. Fig. 3 (b) and (c) show two other runs recorded
from the same heart. The relative lengths of the intervals within the bursts followed
the same pattern (second interval shorter than the first, then interval length progressively increasing) although in each run the number of impulses per burst differed.
The impulse pattern of the bursts from another heart (Fig. 3 (d)) was not the same,
for the first interval within the bursts was not consistently longer than the second, but
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HILARY F. BROWN
the bursts from this heart did show the same progressive lengthening of the later
intervals. Measurements of the impulse intervals in bursts recorded from the g.n.t.'s
of thirteen hearts showed that the only completely consistent feature of their pattern
was that the last interval was always as long as, or longer than, the penultimate interval
and was usually markedly longer.
DISCUSSION
The impulses recorded externally from any one point of the g.n.t. appear to represent repeated firings of the same structure, probably a cell body. This is borne out by
the records taken simultaneously from two points of a g.n.t., which showed a constant delay between the two recording electrodes for all impulses, indicating that all
the impulses travel along the same ' firing channel' within a g.n.t. and not in several
parallel pathways. The simplest hypothesis is that the cells fire in order, one after
another from the initiating cell forward or backward, each cell directly stimulating the
one next to it to fire. This is hard to reconcile with Alexandrowicz's histological
observations that the bipolar processes of the g.n.t. neurones run forwards and backwards within the g.n.t. for some segments before branching and that cross-sections of
the g.n.t. showed five or six axons. However, there must be some branching nearer
the cell body than this, for, in those heart segments which beat when isolated from the
rest of the heart by two transverse cuts, some of the axons to the muscle must have left
the g.n.t. within the segment containing the cell body.
On the basis of his histological findings, Alexandrowicz suggested that excitation
might pass from one g.n.t. neurone to the next by way of the muscle. Thus the firing
of one neuron would cause contraction of the muscle fibres it innervated, which in
turn would excite the dendrites of another neurone and so on. However, it can be
assumed that the speed at which the nerve impulses travel along the processes in the
g.n.t. is not faster than the conduction velocities found in general for crustacean peripheral nerves (between 1 and 5 m./sec). Since the average value for conduction velocity
along the g.n.t. found in the present study was 1-5 m./sec., it seems unlikelythat there are
more breaks in the 'firingchannel' than simple synapses between neurones. Furthermore, careful removal of nearly all the muscle on either side of the g.n.t. for a distance
of several segments did not disturb the firing of the nerve cells as long as the g.n.t. was
unharmed, suggesting that the muscle is not a normal link in the excitation pathway.
Irisawa & Irisawa (1957) cut the g.n.t. of the heart of Squilla oratoria transversely
in front of the 13th cell and then further forward segment by segment. In the seven
cases they tested, they found that the segment containing cell 13 always beat after
isolation at the same rate as had the whole heart and that the rate of beating of segments further forward decreased progressively. Cell 11 was in three cases out of seven
quiescent after isolation, cell 10 was quiescent in six cases out of seven. They suggested that the cell of the 13th segment is the pacemaker and governs the rate of the
heart beat. But in the present investigation of Squilla mantis, although spontaneity
was similarly found to be greatest in the cells towards either end of the g.n.t. chain,
simultaneous recording from two points on the g.n.t. showed that in intact hearts,
beating spontaneously, the bursts were initiated as frequently in the anterior as in the
posterior region of the g.n.t. and that initiation could shift from one of these regions to
the other in the same g.n.t.
Electrophysiological investigations of the heart of Squilla mantis
699
Maynard (1955) considered that every neurone in the lobster g.n.t. might be capable
of spontaneous activity but that in a normal burst only the first to fire did so spontaneously and this drove the others. Similarly, in Squilla mantis, while most cells of
the g.n.t. show some spontaneity in that even the quiescent cells in the middle of the
chain would often start up slow firing some time after isolation, those which fire most
readily when isolated are those towards the ends of the chain (i.e. in those regions
where burst firing is initiated in the intact heart). The cell with greatest spontaneity
following isolation may be one of a number of cells towards the front or back of the
chain, which again suggests that no one cell in the chain of sixteen is invariably 'the
pacemaker'.
The nature of the spontaneity of the neurones and of their integration for coordinated burst firing needs more investigation. Although the muscle does not seem
to be a link in the conduction path of impulses along the g.n.t., it remains possible
that proprioceptive impulses from the dendritic endings of the g.n.t. cells on the
muscle fibres can affect the pattern of impulses in the burst, as was apparently happening in the case reported above (see Results).
Hagiwara & Bullock (1957) took intracellular recordings from the g.n.t. neurones of
the lobster. They found that repetitive presynaptic stimulation results in synaptic
potentials whose amplitude bears an inverse relation to frequency. From the larger
synaptic potentials (at lower rates) more spikes arise than from the smaller ones. The
spikes are initiated at the base of the axon and do not invade the soma of the neurone
and 'wipe out' the depolarization there. Thus the synaptic potential can persist and
determine the number of spikes in the burst. Two findings suggest that in Squilla
the process which determines the composition of the burst is of a similar nature.
First, there is the correlation between increase in the number of impulses per burst
and increasing burst interval and, secondly, the lengthening of the impulse intervals
towards the end of the bursts, which implies an exponential decline of the process
initiating the impulses. However, the nerve impulses recorded from the g.n.t. of
Squilla appear to be firings of the nerve cell bodies which cannot therefore be carrying
graded synaptic potentials determining burst composition.
It is possible that the region of graded potentials and impulse initiation is in some
other part of the g.n.t. neurone in Squilla, say at the base of the dendrites, so that the
soma as well as the axon here carries all-or-none impulses. Alternatively, the small
cells of the front and back regions of the g.n.t. where the bursts are normally initiated
may not carry all-or-none impulses in the same way as do the other cells. Recordings
were not obtained from these cells, which are smaller and less accessible than the
central neurones of the g.n.t. chain.
SUMMARY
1. With an external hook electrode placed upon the ganglionic nerve trunk of the
isolated heart of Squilla mantis a burst of a small number (3-12) of nerve impulses was
recorded at each heart beat.
2. The number of impulses per burst showed a direct correlation with interval
between bursts.
3. The only consistent feature of impulse pattern within the bursts was a lengthening of the intervals between impulses towards the ends of the bursts.
700
HILARY F . BROWN
4. Electrodes at two points on the ganglionic nerve trunk each recorded the same
number of impulses at a burst. The delay between the two recording points was the
same for all impulses, and usually all the impulses were, in a given heart, recorded
travelling in the same direction, though this could be either forwards or backwards
along the chain.
5. It is suggested that each cell in the chain of 16 fires in succession the same
number of times during a burst and that the impulses travel along the same ' firing
channel' within the ganglionic nerve trunk.
6. Cells near the two ends of the chain showed the greatest spontaneity when
isolated by transverse cuts or ligatures. Coupled with the records obtained from two
points, this suggests that the bursts are initiated in the front or back regions of the
chain, but not invariably by the same one of the 16 cells.
This work formed part of a D.Phil thesis for Oxford University. I should like to
thank my supervisor Miss R. J. Banister for her help, the Director and staff of the
Stazione Zoologica, Naples, for laboratory facilities, and the D.S.I.R. for the postgraduate studentship which supported me.
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Note added in proof
Watanabe and Takeda (1963) have recorded intracellularly from the neurones of the
ganglionic nerve trunk of the heart of Squilla oratoria. Their results suggest that firing
of the cell body of each neurone is not essential for conduction along the chain, for
parallel axons, functionally linked to one another and to the soma axon by sideconnections, carry propagated impulses past the somata before these have discharged.
They conclude that transmission across the side-connections takes place with a high
safety factor, and probably electrically so that the whole g.n.t. acts as a single unit.
WATANABE, A. & TAKEDA, K. (1963). The spread of excitation among neurons in the heart ganglion
of the Stomatopod, Squilla oratoria. J. Gen. Physiol. 46, 773—801.