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Lobster heart: Electrophysiology of single cells including effects of the regulator nerves
Camp. Biochem. PhysioL, 1971, Vol. 39A, pp, 643 to 648. Pergamon Press. Printed in Great Britain
LOBSTER HEART: ELECTROPHYSIOLOGY OF SINGLE CELLS INCLUDING EFFECTS OF THE REGULATOR NERVES MARK HALLETT* The Biological Laboratories,
Harvard University,
Cambridge,
Massachusetts
02138
Abstract-l. The electrical event underlying contraction of lobster heart muscle ceils is the excitatory junctional potential (ejp) and the beat of the heart fotlows a series of ejp’s. 2. The ejp’s are capable of facilitation and summation and the muscle cells are polyneuronally innervated. 3. The cardiac regulator nerves have no demonstrable direct effect on the electrical activity of the muscle cells. 4. Gamma-amino butyric acid (G-ABA) has no clear influence on the muscle cells and direct inhibition of these cells may be lacking. INTRODUCTION THE RHYTHMIC activity
of the heart of the lobster (Homarus ame&anus) is controlled by a nine-cell ganglion which lies in the interior of the heart. This ganglion is essentially autonomous ; it has its own pacemaker system and produces periodic bursts of action potentials in its efferent nerves. The ganglion has been a model system for the study of the interaction of nerve cells (Horridge, 1965). The heart muscle has been known for many years to be striated, but until recently its physiological properties were not investigated. It appears now that the electrophysiological behavior of the heart muscle is quite similar to that of crustacean peripheral striated muscle. The action potentials that originate in the ganglion produce in the muscle fibers a series of excitatory junctional potentials (ejp’s) which is the electrical event leading to muscle contraction (Anderson & Cooke, 1969, 1970; Van der Kloot, 1970). There is no electrotonic connection between laterally adjacent muscle fibres (Anderson & Cooke, 1970) and the muscle fibres seem to be polyneuronally innervated. The cardiac regulator nerves which originate from the central nervous system were described for the lobster by Alexandrowicz (1932). There are a pair of inhibitory nerves (one on each side) and two pair of accelerator nerves. These nerves run in separate paths distant from the heart and then join in common nerves on each side of the heart before innervating it. They have synaptic endings on the ganglionic nerve cells and the effects have been studied in detail (Terzuolo * Present address: Laboratory Bethesda, Maryland 20014.
of Neurobiology, 643
National
Institute
of Mental Health,
644
MARK HALLETT
&
Bullock, 1958). Anatomical studies by Alexandrowicz and, in particular, by Maynard on Punulirus (Maynard, 1961) suggest that the regulator nerves (at least the inhibitor) have processes which go out to the muscle through the elIerent branches of the ganghon. The physiological effects in the muscle cells of stimulation of these nerves have not been studied in the lobster or, in fact, in any of its relatives. If effects could be demonstrated, they would verify the anatomicai studies. MATERIALS
AND METHODS
The heart was isolated for ele~troph~iological study Ieaving it connected only to a small portion of the dorsal carapace. The heart was pinned to a pamffin~filled dish and covered by and perfused through an ostium with cold (1619°C) lobster perfusion fluid (Cole, 1941). Glass micropipettes were utilized for intracellular recording and were filled with 3 M KCl. The technique of Woodbury & Brady (1956) of a dangling microelectrode was used to record from moving heart muscle. Electric recording was through a Bioelectric Neutralized Input Capacity Amplifier and displayed on a Tektronix SO2 Dual Beam Oscilloscope. Heart tension transduced through a Grass FT 03B strain gauge attached to three anterior arteries of the heart was displayed on the second beam. Intracellular recording from a single cell could be maintained for over one-half hour. The inhibitor and accelerator nerves were found by dissection through the tissues on the carapace about 3-4 cm cephalad to the heart. They were stimulated with suction electrodes. RESULTS
The intracellular electrical activity of muscle cells during normal rhythmic was activity is illustrated in Fig. 1. For fifty cells the average resting potential -50 mV, average maximum depolarization was 23 mV and average burst duration was 280 msec. The potential variation appears to be a series of numerous Figure la shows the electrical ejp’s. Never was spike-like activity observed.
FIG. 1. Top traces represent intracellular recordings from heart muscle cells. Bottom traces show tension of the heart. In part (a) three recordings were taken from the same cell whose resting potential was - 35 mV. Part (b) shows recordings from three different cells from different places in the same heart; the resting potentials were - 35 mV, -40 mV and - 43 mV respectiveIy.
LOBSTER
HEART:
ELECTROPHYSIOLOGY
OF SINGLE
CELLS
645
activity from the same cell for three different beats and Fig. lb shows a single record from three different cells obtained at short time intervals. While the pattern was roughly similar each time in a single cell, different cells showed different patterns-some simple and some more complex in their pattern of ejp’s. Comparing the potential variations in a single cell from beat to beat, it was fairly clear that the initial part of the pattern was more constant than the terminal part. Tension followed the electrical activity with a delay of approximately 50 msec. The degree of depolarization might change from time to time or in different situations. Heart contraction seemed stronger with greater depolarization. Also, in those rare cases when there were two phases in the electrical activity, the tension traces were notched. Tension, therefore, seems to be related to the magnitude of the potential change. To learn more about the physiological properties of the muscle cells it was possible to cut away the ganglion cells and stimulate, by a suction electrode, a branch of the ganglion running to the muscle. (When the ganglion was partially cut, its rhythmicity was destroyed, but individual neurons fired randomly and in the muscle cells simple ejp’s of various sizes could be recorded. An example of these ejp’s is shown in Fig. 2a.) When the ganglion was completely cut away
b and c 2 set
FIG. 2. Record (a) was taken from a preparation where the ganglion is partially cut and shows two different types of ejp’s (resting potential - 50 mv). Record (b) shows facilitation of ejp’s produced by stimulation of nerve from the ganglion to the heart at a rate of 2/set (resting potential -48 mv). Part (c), from the ssrne cell, shows summation as well as facilitation with a stimulating rate of 6/set. from the heart, no areas of muscle were active and the muscle cells were essentially electrically silent. Using graded voltage pulses for stimulation of the nerve bundle, no ejp’s could be produced in some muscle fibers while ejp’s of one to four different sizes could be produced in others. By using the lowest voltage that produced an ejp, one could presumably study the properties of a single nervemuscle junction. Figure 2b illustrates facilitation of ejp’s evoked by repetitive stimulation at this voltage. Stimulation of the nerve at a higher rate. would produce summation as well as facilitation. As illustrated in Fig. 2c about 4 ejp’s
MARK HAILETT
646
summate to a progressively higher level after which an additional ejp prolongs a plateau level. The problem of the effect of the regulator nerves on the heart muscle cells was also investigated. With sufficient stimulation of the inhibitor nerve the heart arrests in diastole, the ganglion ceases its rhythmic burst-like activity (Maynard, 1961) and several ganglion cells show hyperpolarizing potentials (Terzuolo &
(a1 3
Inhibition
2omvL
(b)
Acceleration FIG. 3. Top traces represent records of intracellular potential of heart muscle cells and bottom traces show heart tension. (a) and (b) are from the same cell (whose resting potential was -40 mV) and show the effect of stimulation of the cardiac inhibitor nerve at ten times/set and the cardiac accelerator nerve at thirty times/set respectively.
Bullock, 1968). Figure 3a shows an example of the effect of inhibitor nerve stimulation in the cardiac cell. There was a cessation of electrical activity, but no clear influence on the cell itself was detected other than would be expected from the indirect effect from lack of input from the ganglion. For example, no i~ibitory junctional potentials were observed. Occasionally there was an escape beat and the magnitude of the potential change was not reduced; thus, there was no evidence for an increased muscle membrane conductance caused by the inhibitor nerve stimulation. Following inhibitor nerve stimulation there was post-inhibitor rebound with depolarization of the cell and tension of the whole heart of greater magnitude. Stimulation of the accelerator nerve causes an increased frequency of bursts from the ganglion and an increased number of action potentials per burst (Maynard, 1961). In th e muscle cell there was an increased number of ejp’s in each burst with concomitant increased amount of depolarization. This is shown in Fig. 3b. As with inhibitor nerve stimulation no direct effect was observed on the muscfe ceff (for example, ejp’s correlated with nerve stimulation). Gamma-aminobutyric acid (GABA) is thought to be the transmitter substance at inhibitory nerve-muscle junctions in crustacea (Otsuka et al., 1966). Studies with GABA were undertaken to see if cardiac muscle responds to this drug in a way similar to peripheral muscle. Unfortunately GABA mimics the effects of inhibitor
LOBSTER
HEART:
ELE~ROP~SIOLOGY
OF SINGLE
CELLS
647
nerve stimulation on the ganglion (Cooke, 1962) and thus its effect on the heart muscle can be indirect. Injection of 1 ml of 10-s M GABA into an ostium of the heart, where it can directly contact the ganglion, did indeed mimic inhibitory nerve stimulation and the muscle cell’s potential remained at the resting level. On the other hand, external application with a medicine dropper of a similar amount of GABA near the tip of the microelectrode had no visible effect on the electrical activity of the cell. This is similar to the case of L~~~~~heart where GABA had no effect on ejp’s produced by stimulation of an efferent nerve from the ganglion (Abbott et al., 1969). External application of other drugs, however, can influence the muscle. Glutamate has been proposed to be the transmitter in excitatory crustacean nervemuscle junctions (Takeuchi and Takeuchi, 1964), and, as such, is a likely candidate for the cardiac nerve-muscle junctions. Applications of several ml of 10m3 M glutamate on the external surface of the heart caused the muscle cell membrane to depolarize by several mV and decreased the magnitude of the potential change in the cell’s rhythmic activity. Tonus of the whole heart was increased, but each contraction was decreased in magnitude. This change is also precisely what was found for the L~~~~~ heart (Abbott et al., 1969). DISCUSSION
In general the physiolo~cal properties of lobster heart muscle cells, found here, are similar to those of crustacean peripheral muscle. The electrical events underlying contraction are ejp’s and these are capable of facilitation and, as shown for the first time here, summation. In normal activity contraction of the heart is preceded by a series of ejp’s. There is evidence for pol~euronal innervation: different ejp’s could be produced by variable stimulating voltage in the nervemuscle preparation and different sized ejp’s could be seen in the heart with the partially destroyed ganglion. As in the periphery not every nerve fiber innervates all muscle cells, and since the cells are electricaily separate, this is a sufficient explanation for the different appearance of the potential variation from cell to cell in the normally beating heart. Basically, these findings confirm those of the previous studies (Anderson & Cooke, 1969, 1970; Van der Kloot, 1970). No evidence could be found here for direct innervation of the heart muscle by the cardiac regulator nerves. All effects of stimulating the regulator nerves could be explained by indirect effects through the ganglion. The anatomical work could not be verified physiologically. In fact since external application of GABA had no obvious electrical effect, there does not seem to be specialization of the muscle cell surface to respond to GABA and direct inhibitory innervation is unlikely. This lack of inhibition would be a major difference in physiological properties between cardiac and peripheral muscle. The property seems to be shared with LimEus heart muscle. The effects of glutamate are in direct contrast to those of GABA and are compatible with the idea that glutamate is the excitatory transmitter for the heart-muscle junctions. 22
648
MARK HA~LLETT REFERENCES
ABBOTTB. C., LANG F. & PARNASI. (1969) Physiological properties of the heart and cardiac ganglion of Limulur polyphemus. Conzp. B&hem. Physiol. 28, 149-l 58. ALEZUNDROWXCZ J. (1932) The innervation of the heart of crustacea. I. Decapoda. Quart. J. Microscop. Sci. 75, 181-249. ANDERSON M. & COOKEI. M. (1969) Neummuscular transmission in the heart of the lobster Homaru~ americanus. Experimentia Supp~~~~rn 15, 166-168. ANDERSONM. & COOKEI. M. (1970) Electrophysiology of the heart of the lobster Homa~us americanus. J. gen. Physiol. 55, 137. COLE W. (1941) A perfusing solution for the lobster (Homarus) heart and the effects of its constituent ions on the heart. J. gen. Physiol. 25, l-6. COOKE I. M. (1962) The neurohumoral regulation of the crustacean heart. Ph.D. thesis, Harvard University. HORRIDGEG. A. (1965) In Structure and Function in the Nervous Systems of Invertebrates (Edited by BULLOCKT. H. & HORRIDGEG, A.), pp. 990-995. W. H. Freeman & Co., San Francisco. MAYNARDD. (1961) Cardiac inhibition in Decapod crustacea. In Nervous Inhibition (Edited by FLO~Y I?.), pp. 144-178. Pergamon Press, Oxford. OTSUKAM., IWRSONL. L., HALL 2. W. & KRAVITZE. A. (1966) Release of gamma-aminobutyric acid from inhibitory nerves of lobster. PYOC.natn. Acad. Ski. U.S.A. 56, lllO111s. TAKEUCHIA. & TAKEUCHIN. (1964) The effect on crayfish muscle of iontophoretically applied glutamate. 3. Physiol., Lond. 170, 296-317. TERZUOLOC. & BULLOCKT. (1958) Acceleration and inhibition in crustacean ganglion cetls. Arch. it&. Biol. %, 117-134. VAN DERKLOOT W. (1970) The electrophysiology of muscle fibers in the hearts of decapod crustaceans. _Y. exp. 2001. 174, 367-380. WOODBURYJ, & BRADYA. (1956) Intraceilular recording from moving tissues with a flexibly mounted ul~a~croelec~ode. Science 123, 100. Key Word Index-Lobster heart; cardiac ganglion; crustacean muscle; excitatory junctiona1 potential; cardiac regulator nerves; gamma-aminobutyric acid; glutamate.
LOBSTER HEART: ELECTROPHYSIOLOGY OF SINGLE CELLS INCLUDING EFFECTS OF THE REGULATOR NERVES MARK HALLETT* The Biological Laboratories,
Harvard University,
Cambridge,
Massachusetts
02138
Abstract-l. The electrical event underlying contraction of lobster heart muscle ceils is the excitatory junctional potential (ejp) and the beat of the heart fotlows a series of ejp’s. 2. The ejp’s are capable of facilitation and summation and the muscle cells are polyneuronally innervated. 3. The cardiac regulator nerves have no demonstrable direct effect on the electrical activity of the muscle cells. 4. Gamma-amino butyric acid (G-ABA) has no clear influence on the muscle cells and direct inhibition of these cells may be lacking. INTRODUCTION THE RHYTHMIC activity
of the heart of the lobster (Homarus ame&anus) is controlled by a nine-cell ganglion which lies in the interior of the heart. This ganglion is essentially autonomous ; it has its own pacemaker system and produces periodic bursts of action potentials in its efferent nerves. The ganglion has been a model system for the study of the interaction of nerve cells (Horridge, 1965). The heart muscle has been known for many years to be striated, but until recently its physiological properties were not investigated. It appears now that the electrophysiological behavior of the heart muscle is quite similar to that of crustacean peripheral striated muscle. The action potentials that originate in the ganglion produce in the muscle fibers a series of excitatory junctional potentials (ejp’s) which is the electrical event leading to muscle contraction (Anderson & Cooke, 1969, 1970; Van der Kloot, 1970). There is no electrotonic connection between laterally adjacent muscle fibres (Anderson & Cooke, 1970) and the muscle fibres seem to be polyneuronally innervated. The cardiac regulator nerves which originate from the central nervous system were described for the lobster by Alexandrowicz (1932). There are a pair of inhibitory nerves (one on each side) and two pair of accelerator nerves. These nerves run in separate paths distant from the heart and then join in common nerves on each side of the heart before innervating it. They have synaptic endings on the ganglionic nerve cells and the effects have been studied in detail (Terzuolo * Present address: Laboratory Bethesda, Maryland 20014.
of Neurobiology, 643
National
Institute
of Mental Health,
644
MARK HALLETT
&
Bullock, 1958). Anatomical studies by Alexandrowicz and, in particular, by Maynard on Punulirus (Maynard, 1961) suggest that the regulator nerves (at least the inhibitor) have processes which go out to the muscle through the elIerent branches of the ganghon. The physiological effects in the muscle cells of stimulation of these nerves have not been studied in the lobster or, in fact, in any of its relatives. If effects could be demonstrated, they would verify the anatomicai studies. MATERIALS
AND METHODS
The heart was isolated for ele~troph~iological study Ieaving it connected only to a small portion of the dorsal carapace. The heart was pinned to a pamffin~filled dish and covered by and perfused through an ostium with cold (1619°C) lobster perfusion fluid (Cole, 1941). Glass micropipettes were utilized for intracellular recording and were filled with 3 M KCl. The technique of Woodbury & Brady (1956) of a dangling microelectrode was used to record from moving heart muscle. Electric recording was through a Bioelectric Neutralized Input Capacity Amplifier and displayed on a Tektronix SO2 Dual Beam Oscilloscope. Heart tension transduced through a Grass FT 03B strain gauge attached to three anterior arteries of the heart was displayed on the second beam. Intracellular recording from a single cell could be maintained for over one-half hour. The inhibitor and accelerator nerves were found by dissection through the tissues on the carapace about 3-4 cm cephalad to the heart. They were stimulated with suction electrodes. RESULTS
The intracellular electrical activity of muscle cells during normal rhythmic was activity is illustrated in Fig. 1. For fifty cells the average resting potential -50 mV, average maximum depolarization was 23 mV and average burst duration was 280 msec. The potential variation appears to be a series of numerous Figure la shows the electrical ejp’s. Never was spike-like activity observed.
FIG. 1. Top traces represent intracellular recordings from heart muscle cells. Bottom traces show tension of the heart. In part (a) three recordings were taken from the same cell whose resting potential was - 35 mV. Part (b) shows recordings from three different cells from different places in the same heart; the resting potentials were - 35 mV, -40 mV and - 43 mV respectiveIy.
LOBSTER
HEART:
ELECTROPHYSIOLOGY
OF SINGLE
CELLS
645
activity from the same cell for three different beats and Fig. lb shows a single record from three different cells obtained at short time intervals. While the pattern was roughly similar each time in a single cell, different cells showed different patterns-some simple and some more complex in their pattern of ejp’s. Comparing the potential variations in a single cell from beat to beat, it was fairly clear that the initial part of the pattern was more constant than the terminal part. Tension followed the electrical activity with a delay of approximately 50 msec. The degree of depolarization might change from time to time or in different situations. Heart contraction seemed stronger with greater depolarization. Also, in those rare cases when there were two phases in the electrical activity, the tension traces were notched. Tension, therefore, seems to be related to the magnitude of the potential change. To learn more about the physiological properties of the muscle cells it was possible to cut away the ganglion cells and stimulate, by a suction electrode, a branch of the ganglion running to the muscle. (When the ganglion was partially cut, its rhythmicity was destroyed, but individual neurons fired randomly and in the muscle cells simple ejp’s of various sizes could be recorded. An example of these ejp’s is shown in Fig. 2a.) When the ganglion was completely cut away
b and c 2 set
FIG. 2. Record (a) was taken from a preparation where the ganglion is partially cut and shows two different types of ejp’s (resting potential - 50 mv). Record (b) shows facilitation of ejp’s produced by stimulation of nerve from the ganglion to the heart at a rate of 2/set (resting potential -48 mv). Part (c), from the ssrne cell, shows summation as well as facilitation with a stimulating rate of 6/set. from the heart, no areas of muscle were active and the muscle cells were essentially electrically silent. Using graded voltage pulses for stimulation of the nerve bundle, no ejp’s could be produced in some muscle fibers while ejp’s of one to four different sizes could be produced in others. By using the lowest voltage that produced an ejp, one could presumably study the properties of a single nervemuscle junction. Figure 2b illustrates facilitation of ejp’s evoked by repetitive stimulation at this voltage. Stimulation of the nerve at a higher rate. would produce summation as well as facilitation. As illustrated in Fig. 2c about 4 ejp’s
MARK HAILETT
646
summate to a progressively higher level after which an additional ejp prolongs a plateau level. The problem of the effect of the regulator nerves on the heart muscle cells was also investigated. With sufficient stimulation of the inhibitor nerve the heart arrests in diastole, the ganglion ceases its rhythmic burst-like activity (Maynard, 1961) and several ganglion cells show hyperpolarizing potentials (Terzuolo &
(a1 3
Inhibition
2omvL
(b)
Acceleration FIG. 3. Top traces represent records of intracellular potential of heart muscle cells and bottom traces show heart tension. (a) and (b) are from the same cell (whose resting potential was -40 mV) and show the effect of stimulation of the cardiac inhibitor nerve at ten times/set and the cardiac accelerator nerve at thirty times/set respectively.
Bullock, 1968). Figure 3a shows an example of the effect of inhibitor nerve stimulation in the cardiac cell. There was a cessation of electrical activity, but no clear influence on the cell itself was detected other than would be expected from the indirect effect from lack of input from the ganglion. For example, no i~ibitory junctional potentials were observed. Occasionally there was an escape beat and the magnitude of the potential change was not reduced; thus, there was no evidence for an increased muscle membrane conductance caused by the inhibitor nerve stimulation. Following inhibitor nerve stimulation there was post-inhibitor rebound with depolarization of the cell and tension of the whole heart of greater magnitude. Stimulation of the accelerator nerve causes an increased frequency of bursts from the ganglion and an increased number of action potentials per burst (Maynard, 1961). In th e muscle cell there was an increased number of ejp’s in each burst with concomitant increased amount of depolarization. This is shown in Fig. 3b. As with inhibitor nerve stimulation no direct effect was observed on the muscfe ceff (for example, ejp’s correlated with nerve stimulation). Gamma-aminobutyric acid (GABA) is thought to be the transmitter substance at inhibitory nerve-muscle junctions in crustacea (Otsuka et al., 1966). Studies with GABA were undertaken to see if cardiac muscle responds to this drug in a way similar to peripheral muscle. Unfortunately GABA mimics the effects of inhibitor
LOBSTER
HEART:
ELE~ROP~SIOLOGY
OF SINGLE
CELLS
647
nerve stimulation on the ganglion (Cooke, 1962) and thus its effect on the heart muscle can be indirect. Injection of 1 ml of 10-s M GABA into an ostium of the heart, where it can directly contact the ganglion, did indeed mimic inhibitory nerve stimulation and the muscle cell’s potential remained at the resting level. On the other hand, external application with a medicine dropper of a similar amount of GABA near the tip of the microelectrode had no visible effect on the electrical activity of the cell. This is similar to the case of L~~~~~heart where GABA had no effect on ejp’s produced by stimulation of an efferent nerve from the ganglion (Abbott et al., 1969). External application of other drugs, however, can influence the muscle. Glutamate has been proposed to be the transmitter in excitatory crustacean nervemuscle junctions (Takeuchi and Takeuchi, 1964), and, as such, is a likely candidate for the cardiac nerve-muscle junctions. Applications of several ml of 10m3 M glutamate on the external surface of the heart caused the muscle cell membrane to depolarize by several mV and decreased the magnitude of the potential change in the cell’s rhythmic activity. Tonus of the whole heart was increased, but each contraction was decreased in magnitude. This change is also precisely what was found for the L~~~~~ heart (Abbott et al., 1969). DISCUSSION
In general the physiolo~cal properties of lobster heart muscle cells, found here, are similar to those of crustacean peripheral muscle. The electrical events underlying contraction are ejp’s and these are capable of facilitation and, as shown for the first time here, summation. In normal activity contraction of the heart is preceded by a series of ejp’s. There is evidence for pol~euronal innervation: different ejp’s could be produced by variable stimulating voltage in the nervemuscle preparation and different sized ejp’s could be seen in the heart with the partially destroyed ganglion. As in the periphery not every nerve fiber innervates all muscle cells, and since the cells are electricaily separate, this is a sufficient explanation for the different appearance of the potential variation from cell to cell in the normally beating heart. Basically, these findings confirm those of the previous studies (Anderson & Cooke, 1969, 1970; Van der Kloot, 1970). No evidence could be found here for direct innervation of the heart muscle by the cardiac regulator nerves. All effects of stimulating the regulator nerves could be explained by indirect effects through the ganglion. The anatomical work could not be verified physiologically. In fact since external application of GABA had no obvious electrical effect, there does not seem to be specialization of the muscle cell surface to respond to GABA and direct inhibitory innervation is unlikely. This lack of inhibition would be a major difference in physiological properties between cardiac and peripheral muscle. The property seems to be shared with LimEus heart muscle. The effects of glutamate are in direct contrast to those of GABA and are compatible with the idea that glutamate is the excitatory transmitter for the heart-muscle junctions. 22
648
MARK HA~LLETT REFERENCES
ABBOTTB. C., LANG F. & PARNASI. (1969) Physiological properties of the heart and cardiac ganglion of Limulur polyphemus. Conzp. B&hem. Physiol. 28, 149-l 58. ALEZUNDROWXCZ J. (1932) The innervation of the heart of crustacea. I. Decapoda. Quart. J. Microscop. Sci. 75, 181-249. ANDERSON M. & COOKEI. M. (1969) Neummuscular transmission in the heart of the lobster Homaru~ americanus. Experimentia Supp~~~~rn 15, 166-168. ANDERSONM. & COOKEI. M. (1970) Electrophysiology of the heart of the lobster Homa~us americanus. J. gen. Physiol. 55, 137. COLE W. (1941) A perfusing solution for the lobster (Homarus) heart and the effects of its constituent ions on the heart. J. gen. Physiol. 25, l-6. COOKE I. M. (1962) The neurohumoral regulation of the crustacean heart. Ph.D. thesis, Harvard University. HORRIDGEG. A. (1965) In Structure and Function in the Nervous Systems of Invertebrates (Edited by BULLOCKT. H. & HORRIDGEG, A.), pp. 990-995. W. H. Freeman & Co., San Francisco. MAYNARDD. (1961) Cardiac inhibition in Decapod crustacea. In Nervous Inhibition (Edited by FLO~Y I?.), pp. 144-178. Pergamon Press, Oxford. OTSUKAM., IWRSONL. L., HALL 2. W. & KRAVITZE. A. (1966) Release of gamma-aminobutyric acid from inhibitory nerves of lobster. PYOC.natn. Acad. Ski. U.S.A. 56, lllO111s. TAKEUCHIA. & TAKEUCHIN. (1964) The effect on crayfish muscle of iontophoretically applied glutamate. 3. Physiol., Lond. 170, 296-317. TERZUOLOC. & BULLOCKT. (1958) Acceleration and inhibition in crustacean ganglion cetls. Arch. it&. Biol. %, 117-134. VAN DERKLOOT W. (1970) The electrophysiology of muscle fibers in the hearts of decapod crustaceans. _Y. exp. 2001. 174, 367-380. WOODBURYJ, & BRADYA. (1956) Intraceilular recording from moving tissues with a flexibly mounted ul~a~croelec~ode. Science 123, 100. Key Word Index-Lobster heart; cardiac ganglion; crustacean muscle; excitatory junctiona1 potential; cardiac regulator nerves; gamma-aminobutyric acid; glutamate.