Neural III.
Control
of Circulation
in
Neurotransmitters
GUNTER LIEBESWAR, EARL MAYER1
JAMES
E. GOLDMAN,
New York University School of Medicine Public Health Research Institute of the
and City
Department
of New York,
DURING THE LAST 20 yr invertebrate preparations have proved useful for relating the properties of single identified nerve cells to behavior (16, 19). Most studies have examined how the electrophysiological properties of individual neurons and their functional or morphological patterns of interconnection contribute to the behavior of an organism. Identified cells have also been used to examine basic biochemical properties of neurons, including transmitter synthesis and utilization (24, 29, 30, 37). Relativelv little work has been carried out on relating transmitter biochemistry either to cellular properties or to behavior. Yet studies of the cellular basis of behavior are likely to be most effective if the biochemical aspects of cellular function are also taken into consideration. It therefore seemed important to attempt to speci f y significant segments of the neural circuit controlling a behavioral svstem in terms of the transmitter biochemistry of its individual cells. Six identified motoneurons of the abdominal ganglion of Aplysia californica are part of a larger network controlling circulation. The properties of these cells, plus some of the command interneurons that control have been described their firing patterns, previously (21, 28). The heart excitor cell causes an acceleration of the myo~&IE genie heart beat that may outlast the firing of the cell bv more than 1 min. The heart inhibitors, LD,,rl and LDn12, have shorter lasting inhibitory effects on the strength and frequency of heart beat. LBvC1, LBVCS, and are vasomotor cells which cause artewvx3 rial constriction. In this paper we identify the neurotransmitters used by these six Received
for
publication
December 2’7, 1974.
JOHN
KOESTER, of Neurobiology New York
AND
City
and Behavior, lOOI
motoneurons: the three vasoconstrictors and two heart inhibitors are cholinergic, and the heart excitor is serotonergic. Preliminary reports of these data have appeared elsewhere (20, 26). MATERIALS
Animals
AND
and
their
METHODS
prepal-ation
Aplysia californica, weighing 70-140 g, were supplied by the Pacific Bio-Marine Supply CO., Venice, Calif., and kept in well-aerated aquaria of artificial seawater (ASW, made from Instant Ocean, Aquarium Systems, Inc., Wickliffe, Ohio) at 15OC. The semi-intact preparation allowing simultaneous recording of blood pressure and heart rate and intracellular recording from cardiovascular motoneurons has been described previously (28). A nimals were pinned to the wax floor of a small aquarium, the internal organs exposed by a longitudinal slit through the foot, the digestive tract removed, and a second slit in the body wall was then made to expose the gill (Fig. IA). The heart was perfused with ASW supplemented with 0.1% glucose and 3 PM choline through a cannula placed in the efferent vein of the gill. Blood pressure was recorded from the proximal cut end of the gastroesophageal artery. The abdominal ganglion, with its major nerves intact, was mounted on a substage and its connective tissue capsule removed by microdissection. In some pharmacological experiments the ganglion was removed at the start of the experiment to eliminate centrally w mediated fluctuations in heart rate (21). Another preparation was used in some experiments for recording contractions of the aortic constrictor musculature (Fig. 1B). Here the abdominal aorta was perfused backward at constant pressure. The heart was removed through a small slit in the Pericardium to insure free flow of ASW through ;he aorta when the vascular muscle was relaxed.
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768
LIEBESWAR,
Anterior
Plexiglas
Aorta
i;/ -
GOLDMAN,
!\
Substage
Abdominal
Cannula Vein
Aorta
to Efferent of GIII
FIG. 1. Experimental preparations. A: experimental preparation for simultaneous recording of blood pressure and intracellular recording from cardiovascular motoneurons (see ref 28). B: a modification for recording contractions of the aortic sphincter musculature. The heart is removed through a small slit in the pericardium (dotted line), and the abdominal aorta is perfused backward from a constant-pressure reservoir. Contraction of the aorta impeded flow from the reservoir and caused an increase of pressure in the cannula.
When the muscle contracted, an increase in pressure was recorded in the perfusion cannula. Pharmacological
methods
Neurotransmitters and blocking drugs were dissolved in ASW immediately before the experiment. They were applied to the effector organs through the cannula to the efferent vein of the gill (Fig. 1A) or through the cannula to the abdominal aorta (Fig. lB), In some experiments small pulses of putative transmitters dissolved in ASW were injected into the perfusion tubing. This was a convenient method for imitating the action of firing a motoneuron.
KOESTER,
AND
MAYER1
In pilot studies, the putative transmitter was tested on the heart or on the abdominal aorta in geometrically progressing concentrations before and after addition of a competitive blocking drug, and the parallel shift in the doseresponse curve was measured (8). For the experiments reported in this paper blocking drugs were used in concentrations for which 8 > A/A, > 4, i.e., the concentration (A) of the agonist which had a given effect in the presence of the antagonist was 4-8 times greater than the concentration (A,) of the agonist, which had the same effect in the absence of the antagonist. The following drugs were used: acetylcholine chloride (Sigma Chemical Co., St. Louis), arecoline hydrochloride (Sigma), atropine sulfate (Schwarz/Mann, Orangeburg, N.Y.), benzoquinonium chloride (gift of Sterling-Winthrop Research Institute, Rensselaer, N.Y.), D-2-bromolysergic acid diethylamide = BOL-148 (Sandoz, Basle), cinanserin hydrochloride (gift of E. R. Squibb & Sons, Princeton, N.J.), dopamine hydrochloride (Sigma), epinephrine USP (Parke, Davis & Co., Detroit, Mich.), y-aminon-butyric acid (Sigma), L-glutamic acid (Sigma), hexamethonium bromide (Sigma), levarterenol bitartrate (Winthrop Laboratories, New York), u-lysergic acid diethylamide = LSD (Sandoz, Basle), methysergide bimaleate (gift of Sandoz Pharmaceuticals, Hanover, N. J-), serotonin creatinine sulfate (Schwarz/Mann), tetraethylammonium chloride (Eastman Kodak, Rochester, N.Y.), d-tubocurarine chloride (Schwarz/Mann). BOL-148 and LSD were kindly provided by the Center for Studies of Narcotic and Drug Abuse, National Institute of Mental Health, Rockville, Md. Biochemical
methods
We used pressure injections to introduce [3H]choline (15.8 Ci/mmol, Amersham-Searle) or L-[3H] tryptophan (5.6 Ci/mmol, New England Nuclear Corp., Boston, Mass.) into the cell bodies of cardiovascular motoneurons. The details of the preparation of radioactive compounds for injections and the technique of pressure injection have been described previously (4). Cardiovascular motoneurons were first impaled with conventional microelectrodes and* identified by their motor effects (28). The nerve cells were then reimpaled with double-barrel micropipettes, suitable for both pressure injection and intracellular recording. The average volume of motoneuron cell bodies was estimated from their diameters as about 3 nl, and injection volumes were estimated to be less than 0.5 nl. While being inoften hyperpolarized tranjected, neurons INJECTIONS.
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APLYSIA
CARDIOVASCULAR
sien tly. Less frequently, a depolarization was observed which initiated a burst of spikes. When the depolarization outlasted the end of the injection by longer than 5 min the cell was arbitrarily considered damaged and discarded. INCUBATIONS
AFTER INJECTION. After an injection of [“HIcholine the whole preparation was incubated for 1 h at 2PC in ASW containing 0.1 y0 glucose and 50 ‘FM choline. After an injection of L-[3H] tryptophan, the aquarium with the preparation was perfused continuously at 21% with ASW supplemented with glucose, amino acids, and vitamins (4).
Analysis
of injected
radioactiuity
OF [~H~ACETYLCHOLINE. One hour after injection, whole ganglia were homogenized at O°C with acetone-formic acid 85:15 (v/v)* Choline compounds were separated by high-voltage paper electrophoresis at pH 4.7 (11) .
DETERMINATION
OF [~HISEROTONIN. Six hours after injection, single cell bodies were dissected and homogenized at O°Cl in 0.2 M perchloric acid containing 0.2 mg of bovine serum albumin and 50 nmol of serotonin creatinine sulfate; soluble radioactivity was analyzed by ionexchange chromatography, high-voltage paper electrophoresis, and descending paper chromatography (4). In experiments using L-[3H] tryptoof tryptophan into the incorporation phan, protein was also measured (38).
DETERMINATION
RESULTS
Transmitter
of heart-excitor
neuron
RBffE
We found that accelerated the heart. Threshold concentration ranged from 5 X 10-10 to 1 X 10-g M (Fig. 2). The main action of serotonin was to increase heart rate, but it also affected blood pressure. Sometimes, especially at concentrations slightly above threshold, serotonin produced an initial increase in blood pressure; with higher concentrations, it commonly decreased pulse pressure. The increase in heart. rate typically outlasted the perfusion with serotonin (Fig. 2). We also tested acetylcholine, dopamine, epinephrine, norepinephrine, y-amino-n-butyric acid, and L-glutamic acid in concentrations up to lo-SM. Only dopamine shared a positive chronotropic effect with serotonin, but the PHARMACOLOGICAL
serotonin
in
STUDIES.
low
concentrations
NEUROTRANSMITTERS
769
concentrations necessary to achieve a cornparable effect- were 45 IL 12 (n = 3) times larger than those of serotonin. We next compared the effects of firing the neuron with the effects of injecting =klE a pulse of serotonin. Firing the motoneuron by passing depolarizing current through the microelectrode for 10 s and injecting 0.10 ml of 2.5 X 10-T M serotonin increased heart rate in similar ways (Fig. 3). Biphasic changes in blood pressure were observed: an initial increase, followed by a decrease. A biphasic effect of RBHE on blood pressure has been previously described by Mayeri et al. (28), who suggested that the increase in blood pressure might be caused by a direct positive inotropic effect of the RB,, neurotransmitter on heart muscle. They attributed the decrease in pressure either to a partial refractoriness of the heart muscle at- high rates of beating or to a decrease in diastolic filling time. To obtain further evidence that serotonin is the transmitter released by RB,,, we tested the effects of several serotonin-blocking agents on both the injection of serotonin into the perfusion cannula and the firing of RB,,. D-lysergic acid die thylamide (10-S to lo=--” M), D-2-bromolysergic acid diethylamide (10 -6 to 1O-5 M), and methysergide (10 -6 to 2 X 10-E M) were tested. These have been reported to block the effect of serotonin on other molluscan hearts (36, 44, 46, 47), although they have agonist activity as well (44, 46). A great variability action was in the intensity of the blocking observed for each of the drugs. When first perfused through the heart the blocking agents themselves increased heart rate; later, heart rate often slowed and became irregular. Thus, it was difficult to determine precisely the efficacy of these drugs as blocking agents. More convincing blockage was obtained with cinanserin hydrochloride (2 X lO--5 to agent 4 X 10-E M>, a serotonin-blocking first described by Krapcho et al. (23). The agonist activity of cinanserin on the heart of Aplysia was small. Cinanserin blocked both the effect of firing RBHE and of a pulse injection of serotonin (Fig. 4). Washing with ASW partially restored the effect of firing RBNE and the effect of injecting serotonin.
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LIEBESWAR,
770
GOLDMAN,
KOESTER,
AND
MAYER1
Beats
/min
Blood Pressure
1IOmm
5 x lO-‘O M
H2°
1
25
25
3 20 I5
2x10-’
M 25
3 20
15
4 x10-’ 5-HT
M
I min
FIG. 2. Effect of serotonin on blood pressure and heart rate. Experimental Fig. 1A except that the abdominal ganglion had been removed. The denervated the efferent vein of the gill alternately with ASW and with ASW containing 4 X IO-9 M). A bar below the record of blood pressure indicates perfusion with STUDIES. Since serotonin synthesis from tryptophan can be used to identify serotonergic cells (4), we examined whether KB,, was able to synthesize this transmitter. We injected [SH] tryptophan directly into the cell body of RB,, and found that the neuron synthesized significant amounts of [3H]serotonin (Table 1). Conversion to serotonin and incorporation into protein were the same as those previously found in other neurons of the RB cluster (4) .l BIOCHEMICAL
1 Frazier et al. (‘7) originally described the RB cluster of the abdominal ganglion as a group of 15-20 cells with common electrophysiological and morphological characteristics. RB,, is the only neuron of this cluster whose function has yet been identified.
arrangement as shown in heart was perfused from serotonin (5 X lo-10 to serotonin.
Transmitter of LD,, heart-inhibitor neurons and LBv, vasoconstrictor neurons STUDIES. Pharmacology neurons. Acetylcholine decreased heart rate, with a threshold between 2.5 X lo-lo and 5 X lo-lo M (Fig. 5). Perfusion with low concentrations of acetylcholine also decreased pulse pressure and induced irregularities of the heart beat. None of the other transmitter candidates, tested in concentrations below 1O-5 M, decreased heart rate. A pulse injection of acetylcholine (0.25 ml of a 6.25 X 10-S M solution in ASW) mimicked the decrease in heart rate and pulse pressure induced by firing a heart-inhibitor neuron (Fig. 6). Tetraethylammonium chloride (1 - 2 X PHARMACOLOGICAL
of heart-inhibitor
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APLYSIA
CARDIOVASCULAR
771
NEUROTRANSMITTERS
HEART RATE
-I IO BLOOD PRESSURE
IOmm I H2°
I
25 mV
0.10 ml 2.5 K 10-7M 5-HT
39 AP
1 min
Blood pressure and heart rate as affected by firing the heart-excitor neuron RB,,, (39 action (AP)) and by pulse of serotonin (0.10 ml of a 2.5 X 10-T M solution in ASW). Spike activity in by direct depolarizing current. The cell was hyperpolarized before and after the deRBEIE was produced polarization. Serotonin was injected into the cannula perfusing the heart via the efferent vein of the gill. FIG.
3.
potentials
1O-4 M) and benzoquinonium chloride (1 - 2 X 10-S M) effectively blocked bot.h the effect of firing an LD,, neuron and the effect of a pulse injection of acetylcholine. These two drugs were chosen because they had previously been found to be effective antagonists of acetylcholine in the heart of Venus mercenaria (27, 45). Benzoquinonium blocked the negative chronotropic effects of the transmitter of and of injected acetylcholine more ~&II effectively than it blocked their negative inotropic effects (Fig. 7). Reiter (34) has made qualitatively similar observations in a study of the effects of acetylcholine and benzoquinonium in the heart of Aplysia Zimacina. The effect of benzoquinonium on the inhibition caused both by firing LDm and injecting acetylcholine was partially restored after washing with ASW (Fig. 7). Atropine, curare, and hexamethonium, apTABLE
Cell
1.
Serotonin
synthesis
No. of Determinations
plied in concentrations up to 10-d M, did not block the effect of acetylcholine. Arecoline, a cholinomimetic which increases potassium conductance of Aplysia pleural ganglia neurons (17, lS), was found to be an effective inhibitor of the heart (Fig. 8). Pharmacology of vasoconstrictor neurons. The transmitters previously tested on the heart were assayed for their effects on the aortic sphincter musculature in concentrations up to 10 -4 M. Only acetylcholine was found to cause constriction of the sphincter. Its threshold lay between 5 X IO-7 and 1 X 10-G M (Fig. 9), approximately three orders of magnitude above that of its threshold for the heart. A pulse injection of acetylcholine (0.10 ml of a 2.5 X 10-S M solution in ASW) mimicked the response to firing a vasoconstrictor neuron (Fig. 10).
in RB neurons
Total Radioactivity in Cell Body, pmol
RB13E
5
0.8-6.0
RB
7
0.3-3.4
Conversion [3H]Serotonin
to
y0 of Total
7.8+ 8.2 2
Incorporation into Protein radioactivity
1.8
10.6
1.3
10.4
t 2.3 + - 2.7
Values are means & standard error. RB cell values from Eisenstadt et al. (4), but including two additional RB cells. Note that the value 3H eluted together with serotonin has been replaced by the value for identified [aH]serotonin. Under our conditions of scintillation counting, 1 pmol = 2,200 cpm.
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772
LIEBESWAR,
GOLDMAN,
KOESTEK,
AND
MAYER1
15 3
Beats /rn~n
/min
Beats 35 30 25 20 15
IO
IO 5
BLOOD PRESSURE
I
PRESSURE
I
2.5 x IO-“M
20mm
BLOOD
-
IO m m “20
H2O
22AP
35 30 25 20 15 IO
I
20 mm
IxIO-~M
-
H2O
++.---I
50 mV
I min
FIG. 4. Cinanserin blockade of the effect of firing the heart-excitor neuron RI),, and the effect of injecting serotonin. Records in the left column were taken before, those in the middle column during, and those in the right column after the perfusion with cinanserin hydrochloride (4 X 10-G was fired by direct stimulation of the w RB,, cell. Small bars below the blood pressure record in the lower half of the figure indicate pulse of serotonin (0.25 ml of a 2.5 X 10-7 M solution). In these records RB,, was kept hyperpolarized between firing periods. These records are all from one experiment.
The blocking agent hexamethonium chloride (1 X lO-4 M) antagonized equally the contraction of the abdominal aorta caused by firing the vasoconstrictor neuron and the contraction caused by a pulse injection of acetylcholine (Fig. 11). Washing with ASW reversed the effect. Curare (1 X 1O-4 M) also antagonized the action of acetylcholine in the abdominal aorta, although in the same molar concentration it was less effective than hexamethonium. STUDIES. Ace tylcholine synthesis from choline can be used to identify cholinergic neurons (4). To provide further evidence- that acetylcholine is the transmitter of the heart inhibitor and the vasoconstrictor neurons, we determined whether these neurons were able to synthesize BIOCHEMICAL
2x
IO-“M ACh
I min
FIG. 5. Effect of acetylcholine on blood pressure and heart rate. The denervated heart was perfused from the efferent vein of the gill alternately with ASW and with ASW containing acetylcholine (2.5 X 10-10 to 2 X 10-g M). A bar below the record of blood pressure indicates perfusion with acetylcholine.
Beats /min 20 HEART RATE
1 15
r4.e
IO 5
20mm I H2O
h LDHI
--i
L--
I
mV
-.-
0.25ml 156 AP
30
6.25
I min
x 10-8M
ACh
FIG. 6.
Blood pressure and heart rate as affected by firing a heart-inhibitor neuron LD,, (156 action potentials) and by a pulse injection of acetylcholine (0.25 ml of a 6.25 X 10-S M solution in seawater). Spike activity in LD,,, was produced by direct depolarizing current. The cell was hyperpolarized before and after the depolarization. Acetylcholine was injected into the cannula perfusing the heart via the efferent vein of the gill.
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APLYSIA
CARDIOVASCULAR
Beats
773
NEUROTRANSMITTERS Beats/min
/min 25
%L “REA”T”E’
-I
20
-----JUPi
RATE
-0 .-*s*.\e~t-
20 I5
--
15
@-?@I
1
I HEART
IO
‘, BLOOD PRESSURE
20mm
I
“20 5x10 I 1,
A LDHl
‘IryJ
w 107 AP
wd
5
BLOOD PRESSURE
IOmm I
-0 OM I
m b
129AP
“20
30mV 20 I5
dL
IO 126AP 5
-7
1x10
’ M 1
20 I5
0.25ml 1.25 x10-‘M ACh
20mm I
IO
“20
5
2x10-‘M
-
1
Benzoquinonium blockade of the effect of firing a heart-inhibitor neuron LD,,, and the effect of injecting acetyhholine. Records in the left column were taken before, those in the middle column during, and those in the right column after the perfusion with benzoquinonium chloride (2 X 10-5 M). LD,, was fired by direct stimulation of the cell. Note that the number of action potentials is higher in the record under benzoquinonium than in the record under control conditions. Phar. macological studies in the heart-inhibitor neurons were made difficult by the fact that repeated firing of these neurons often soon led to a weakening oi their motor effect. Small bars below the blood pressure record in the lower half of the figure indicate. a pulse injection of acetylcholine (0.25 ml of a 1.25 X 10-T M solution). FIG.
7.
[“HlacetyIcholine from [“HIcholine injected into their cell bodies. Both the heart-inhibitor neurons, LD,,, and the vasoconstrictor neurons, LB,,, converted choline into acetylcholine to an extent similar to that of cholinergic neurons R2 and LlO (Table Z)? Thus, both the pharmacological and the biochemical data argue strongly for the cholinergic nature of the heart inhibitor and the vasocons trictor neurons. DISCUSSI6N
We have provided both and biochemical evidence
pharmacological that the heart
2 Cells R2 and LlO are presumed to be cholinergic on the basis of their high endogenous levels of ace tylcholine (29) and choline acetyltransferase (11). Additional supporting evidence from pharmacological and synaptic release experiments is available for LlO (15, 22j.
20 15
IO 5
4 x10-‘M ARECOLINE HYDROCHLORIDE
I min
FIG. 8. Effect of arecoline on blood pressure and heart rate (compare with Fig. 5). A bar below the record of blood pressure indicates perfusion with ASW containing arecoline hydrochloride (5 X 10-S M to 4 X 10-7 M).
excitor neuron (RB,,,) is serotonergic, and that the two LDu, heart inhibitor neurons and the three LB,c vasoconstrictor neurons are cholinergic (Fig. 12). Serotonin
rnediates
excitation
to heart
Three lines of evidence indicate that serotonin is the neurotransmitter used by RB,, to mediate excitation to the heart. First, we found that serotonin applied directly to the heart mimics the motor effect produced by firing RB,,. The sensitivity of the heart to serotonin is high and about 40 times greater than that for dopamine, the only other neurotransmitter tested which causes heart acceleration. A high sensitivity to serotonin has also been reported for the hearts of a variety of mollusks, including several species of Aplysia (5, 14, 47). Second, cinanserin blocks both the effects of serotonin and of on the heart. Third, RBIIE spike activity significant quantities RBEIE can synthesize or serotonin from tryptophan. The amount
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LIEBESWAR,
774 4
4
~xIO-~M
I
r
IxIO-~M
I
2 ~16~
M
-
GOLDMAN,
KOESTER,
I
r
20cm “20
4xl0M ACh
--J I min
FIG. 9.
Effect of acetylcholine on the vasoconstrictor musculature of the abdominal aorta. Experimental arrangement as shown in Fig. 1B. The abdominal ganglion was removed and the denervated aorta perfused backward from the constantpressure reservoir with seawater containing acetylcholine (5 X lo- 7 to 4 X 10-S M). The vertical arrows indicate the start and the end of the perfusion with acetylcholine. There is a delay with which acetylcholine reaches and leaves the aorta after switching between the solutions.
synthesized is of the same order of magnitude as that synthesized by other RB cells and by the giant metacerebral cells of Aplysia (4). There is both pharmacological (9, 10, 32) and biochemical (43) evidence that the two symmetrical giant metacerebral
AND
MAYER1
cells of Afdysia, located in the cerebral ganglion, are serotonergic. These data from the injection experiments are important since transmitter synthesis shows cellular specificity. Eisenstadt of et al. (4) found that cholinergic neurons Aplysia did not synthesize serotonin from injected tryptophan; conversely, the serotonergic RB cells did not synthesize acetylcholine from injected choline. Another characteristic apparently specific to serotonergic neurons is the ability to transport serotonin along their axons (12). We have preliminary evidence that RBuE transports newly synthesized serotonin into its axon in the pericardial nerve. The conclusion that RBuE uses serotonin as a transmitter is further strengthened by evidence that the heart of Aplysia receives sero tonergic innervation. First, Taxi and Gautron (40) have demonstrated the presence of serotonergic nerve terminals in the heart using histochemical and autoradiographic techniques. Second, Chase et al. (3) and Carpenter et al. (2) have demonstrated serotonin in moderately high concentrations in the auricle and ventricle of Aplysia heart. Carpenter et al. (2) also found a serotonin-uptake mechanism in the heart, which appears to be an active process. Third, we have found that the isolated Aplysia heart, containing the terminal regions of the motoneurons, can synthesize
5cm H20
BLOOD PRESSURE
50 mV
WC -
I I-k--
Lf-IOZAP
FIG. 10. Effect on vascular muscle neuron and by a pulse injection of in blood pressure brought about by Fig. 1A. Since the heart is far more aorta, an injection of acetylcholine Fig. 1B. The effect of firing LBvC perfusing the abdominal aorta. The
30 set
99AP
0.1 ml 2.5 x Kf5M ACh
30 set
of the abdominal aorta produced by firing an LB,, vasoconstrictor acetylcholine (0.10 ml of a 2.5 X 10-S solution in ASW). A: increase firing LB,, recorded in the experimental arrangement as shown in sensitive to acetylcholine than the vasoconstrictor musculature in the cannot mimic this effect. B: experimental arrangement as shown in was mimicked by a pulse injection of acetylcholine into the cannula heart had been removed.
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AZ’LYSZA
TABLE
Acetylcholine
2.
CARDIOVASCULAR
synthesis
NEUROTRANSMITTERS
in LD
and LB
No. of Determinations
Cell
LDHI
sero tonin from exogenously supplied phan (unpublished observations).
values
tryp to-
Acetylcholine mediates inhibition to heart and excitation to arteries Three types of evidence support the conclusion that the LD,, heart inhibitors and the LB,, vasoconstrictor cells are cholinergic. First, acetylcholine was the only transmitter tested which mimics the effects of these motor cells on heart and aorta at low concentrations. This confirms several previous reports on the high sensitivity of the Aplysia heart to the inhibitory effects of
-A
/
I
1 5QmV
~
I
Control
of Circulation
in
Neurotransmitters
GUNTER LIEBESWAR, EARL MAYER1
JAMES
E. GOLDMAN,
New York University School of Medicine Public Health Research Institute of the
and City
Department
of New York,
DURING THE LAST 20 yr invertebrate preparations have proved useful for relating the properties of single identified nerve cells to behavior (16, 19). Most studies have examined how the electrophysiological properties of individual neurons and their functional or morphological patterns of interconnection contribute to the behavior of an organism. Identified cells have also been used to examine basic biochemical properties of neurons, including transmitter synthesis and utilization (24, 29, 30, 37). Relativelv little work has been carried out on relating transmitter biochemistry either to cellular properties or to behavior. Yet studies of the cellular basis of behavior are likely to be most effective if the biochemical aspects of cellular function are also taken into consideration. It therefore seemed important to attempt to speci f y significant segments of the neural circuit controlling a behavioral svstem in terms of the transmitter biochemistry of its individual cells. Six identified motoneurons of the abdominal ganglion of Aplysia californica are part of a larger network controlling circulation. The properties of these cells, plus some of the command interneurons that control have been described their firing patterns, previously (21, 28). The heart excitor cell causes an acceleration of the myo~&IE genie heart beat that may outlast the firing of the cell bv more than 1 min. The heart inhibitors, LD,,rl and LDn12, have shorter lasting inhibitory effects on the strength and frequency of heart beat. LBvC1, LBVCS, and are vasomotor cells which cause artewvx3 rial constriction. In this paper we identify the neurotransmitters used by these six Received
for
publication
December 2’7, 1974.
JOHN
KOESTER, of Neurobiology New York
AND
City
and Behavior, lOOI
motoneurons: the three vasoconstrictors and two heart inhibitors are cholinergic, and the heart excitor is serotonergic. Preliminary reports of these data have appeared elsewhere (20, 26). MATERIALS
Animals
AND
and
their
METHODS
prepal-ation
Aplysia californica, weighing 70-140 g, were supplied by the Pacific Bio-Marine Supply CO., Venice, Calif., and kept in well-aerated aquaria of artificial seawater (ASW, made from Instant Ocean, Aquarium Systems, Inc., Wickliffe, Ohio) at 15OC. The semi-intact preparation allowing simultaneous recording of blood pressure and heart rate and intracellular recording from cardiovascular motoneurons has been described previously (28). A nimals were pinned to the wax floor of a small aquarium, the internal organs exposed by a longitudinal slit through the foot, the digestive tract removed, and a second slit in the body wall was then made to expose the gill (Fig. IA). The heart was perfused with ASW supplemented with 0.1% glucose and 3 PM choline through a cannula placed in the efferent vein of the gill. Blood pressure was recorded from the proximal cut end of the gastroesophageal artery. The abdominal ganglion, with its major nerves intact, was mounted on a substage and its connective tissue capsule removed by microdissection. In some pharmacological experiments the ganglion was removed at the start of the experiment to eliminate centrally w mediated fluctuations in heart rate (21). Another preparation was used in some experiments for recording contractions of the aortic constrictor musculature (Fig. 1B). Here the abdominal aorta was perfused backward at constant pressure. The heart was removed through a small slit in the Pericardium to insure free flow of ASW through ;he aorta when the vascular muscle was relaxed.
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768
LIEBESWAR,
Anterior
Plexiglas
Aorta
i;/ -
GOLDMAN,
!\
Substage
Abdominal
Cannula Vein
Aorta
to Efferent of GIII
FIG. 1. Experimental preparations. A: experimental preparation for simultaneous recording of blood pressure and intracellular recording from cardiovascular motoneurons (see ref 28). B: a modification for recording contractions of the aortic sphincter musculature. The heart is removed through a small slit in the pericardium (dotted line), and the abdominal aorta is perfused backward from a constant-pressure reservoir. Contraction of the aorta impeded flow from the reservoir and caused an increase of pressure in the cannula.
When the muscle contracted, an increase in pressure was recorded in the perfusion cannula. Pharmacological
methods
Neurotransmitters and blocking drugs were dissolved in ASW immediately before the experiment. They were applied to the effector organs through the cannula to the efferent vein of the gill (Fig. 1A) or through the cannula to the abdominal aorta (Fig. lB), In some experiments small pulses of putative transmitters dissolved in ASW were injected into the perfusion tubing. This was a convenient method for imitating the action of firing a motoneuron.
KOESTER,
AND
MAYER1
In pilot studies, the putative transmitter was tested on the heart or on the abdominal aorta in geometrically progressing concentrations before and after addition of a competitive blocking drug, and the parallel shift in the doseresponse curve was measured (8). For the experiments reported in this paper blocking drugs were used in concentrations for which 8 > A/A, > 4, i.e., the concentration (A) of the agonist which had a given effect in the presence of the antagonist was 4-8 times greater than the concentration (A,) of the agonist, which had the same effect in the absence of the antagonist. The following drugs were used: acetylcholine chloride (Sigma Chemical Co., St. Louis), arecoline hydrochloride (Sigma), atropine sulfate (Schwarz/Mann, Orangeburg, N.Y.), benzoquinonium chloride (gift of Sterling-Winthrop Research Institute, Rensselaer, N.Y.), D-2-bromolysergic acid diethylamide = BOL-148 (Sandoz, Basle), cinanserin hydrochloride (gift of E. R. Squibb & Sons, Princeton, N.J.), dopamine hydrochloride (Sigma), epinephrine USP (Parke, Davis & Co., Detroit, Mich.), y-aminon-butyric acid (Sigma), L-glutamic acid (Sigma), hexamethonium bromide (Sigma), levarterenol bitartrate (Winthrop Laboratories, New York), u-lysergic acid diethylamide = LSD (Sandoz, Basle), methysergide bimaleate (gift of Sandoz Pharmaceuticals, Hanover, N. J-), serotonin creatinine sulfate (Schwarz/Mann), tetraethylammonium chloride (Eastman Kodak, Rochester, N.Y.), d-tubocurarine chloride (Schwarz/Mann). BOL-148 and LSD were kindly provided by the Center for Studies of Narcotic and Drug Abuse, National Institute of Mental Health, Rockville, Md. Biochemical
methods
We used pressure injections to introduce [3H]choline (15.8 Ci/mmol, Amersham-Searle) or L-[3H] tryptophan (5.6 Ci/mmol, New England Nuclear Corp., Boston, Mass.) into the cell bodies of cardiovascular motoneurons. The details of the preparation of radioactive compounds for injections and the technique of pressure injection have been described previously (4). Cardiovascular motoneurons were first impaled with conventional microelectrodes and* identified by their motor effects (28). The nerve cells were then reimpaled with double-barrel micropipettes, suitable for both pressure injection and intracellular recording. The average volume of motoneuron cell bodies was estimated from their diameters as about 3 nl, and injection volumes were estimated to be less than 0.5 nl. While being inoften hyperpolarized tranjected, neurons INJECTIONS.
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APLYSIA
CARDIOVASCULAR
sien tly. Less frequently, a depolarization was observed which initiated a burst of spikes. When the depolarization outlasted the end of the injection by longer than 5 min the cell was arbitrarily considered damaged and discarded. INCUBATIONS
AFTER INJECTION. After an injection of [“HIcholine the whole preparation was incubated for 1 h at 2PC in ASW containing 0.1 y0 glucose and 50 ‘FM choline. After an injection of L-[3H] tryptophan, the aquarium with the preparation was perfused continuously at 21% with ASW supplemented with glucose, amino acids, and vitamins (4).
Analysis
of injected
radioactiuity
OF [~H~ACETYLCHOLINE. One hour after injection, whole ganglia were homogenized at O°C with acetone-formic acid 85:15 (v/v)* Choline compounds were separated by high-voltage paper electrophoresis at pH 4.7 (11) .
DETERMINATION
OF [~HISEROTONIN. Six hours after injection, single cell bodies were dissected and homogenized at O°Cl in 0.2 M perchloric acid containing 0.2 mg of bovine serum albumin and 50 nmol of serotonin creatinine sulfate; soluble radioactivity was analyzed by ionexchange chromatography, high-voltage paper electrophoresis, and descending paper chromatography (4). In experiments using L-[3H] tryptoof tryptophan into the incorporation phan, protein was also measured (38).
DETERMINATION
RESULTS
Transmitter
of heart-excitor
neuron
RBffE
We found that accelerated the heart. Threshold concentration ranged from 5 X 10-10 to 1 X 10-g M (Fig. 2). The main action of serotonin was to increase heart rate, but it also affected blood pressure. Sometimes, especially at concentrations slightly above threshold, serotonin produced an initial increase in blood pressure; with higher concentrations, it commonly decreased pulse pressure. The increase in heart. rate typically outlasted the perfusion with serotonin (Fig. 2). We also tested acetylcholine, dopamine, epinephrine, norepinephrine, y-amino-n-butyric acid, and L-glutamic acid in concentrations up to lo-SM. Only dopamine shared a positive chronotropic effect with serotonin, but the PHARMACOLOGICAL
serotonin
in
STUDIES.
low
concentrations
NEUROTRANSMITTERS
769
concentrations necessary to achieve a cornparable effect- were 45 IL 12 (n = 3) times larger than those of serotonin. We next compared the effects of firing the neuron with the effects of injecting =klE a pulse of serotonin. Firing the motoneuron by passing depolarizing current through the microelectrode for 10 s and injecting 0.10 ml of 2.5 X 10-T M serotonin increased heart rate in similar ways (Fig. 3). Biphasic changes in blood pressure were observed: an initial increase, followed by a decrease. A biphasic effect of RBHE on blood pressure has been previously described by Mayeri et al. (28), who suggested that the increase in blood pressure might be caused by a direct positive inotropic effect of the RB,, neurotransmitter on heart muscle. They attributed the decrease in pressure either to a partial refractoriness of the heart muscle at- high rates of beating or to a decrease in diastolic filling time. To obtain further evidence that serotonin is the transmitter released by RB,,, we tested the effects of several serotonin-blocking agents on both the injection of serotonin into the perfusion cannula and the firing of RB,,. D-lysergic acid die thylamide (10-S to lo=--” M), D-2-bromolysergic acid diethylamide (10 -6 to 1O-5 M), and methysergide (10 -6 to 2 X 10-E M) were tested. These have been reported to block the effect of serotonin on other molluscan hearts (36, 44, 46, 47), although they have agonist activity as well (44, 46). A great variability action was in the intensity of the blocking observed for each of the drugs. When first perfused through the heart the blocking agents themselves increased heart rate; later, heart rate often slowed and became irregular. Thus, it was difficult to determine precisely the efficacy of these drugs as blocking agents. More convincing blockage was obtained with cinanserin hydrochloride (2 X lO--5 to agent 4 X 10-E M>, a serotonin-blocking first described by Krapcho et al. (23). The agonist activity of cinanserin on the heart of Aplysia was small. Cinanserin blocked both the effect of firing RBHE and of a pulse injection of serotonin (Fig. 4). Washing with ASW partially restored the effect of firing RBNE and the effect of injecting serotonin.
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LIEBESWAR,
770
GOLDMAN,
KOESTER,
AND
MAYER1
Beats
/min
Blood Pressure
1IOmm
5 x lO-‘O M
H2°
1
25
25
3 20 I5
2x10-’
M 25
3 20
15
4 x10-’ 5-HT
M
I min
FIG. 2. Effect of serotonin on blood pressure and heart rate. Experimental Fig. 1A except that the abdominal ganglion had been removed. The denervated the efferent vein of the gill alternately with ASW and with ASW containing 4 X IO-9 M). A bar below the record of blood pressure indicates perfusion with STUDIES. Since serotonin synthesis from tryptophan can be used to identify serotonergic cells (4), we examined whether KB,, was able to synthesize this transmitter. We injected [SH] tryptophan directly into the cell body of RB,, and found that the neuron synthesized significant amounts of [3H]serotonin (Table 1). Conversion to serotonin and incorporation into protein were the same as those previously found in other neurons of the RB cluster (4) .l BIOCHEMICAL
1 Frazier et al. (‘7) originally described the RB cluster of the abdominal ganglion as a group of 15-20 cells with common electrophysiological and morphological characteristics. RB,, is the only neuron of this cluster whose function has yet been identified.
arrangement as shown in heart was perfused from serotonin (5 X lo-10 to serotonin.
Transmitter of LD,, heart-inhibitor neurons and LBv, vasoconstrictor neurons STUDIES. Pharmacology neurons. Acetylcholine decreased heart rate, with a threshold between 2.5 X lo-lo and 5 X lo-lo M (Fig. 5). Perfusion with low concentrations of acetylcholine also decreased pulse pressure and induced irregularities of the heart beat. None of the other transmitter candidates, tested in concentrations below 1O-5 M, decreased heart rate. A pulse injection of acetylcholine (0.25 ml of a 6.25 X 10-S M solution in ASW) mimicked the decrease in heart rate and pulse pressure induced by firing a heart-inhibitor neuron (Fig. 6). Tetraethylammonium chloride (1 - 2 X PHARMACOLOGICAL
of heart-inhibitor
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APLYSIA
CARDIOVASCULAR
771
NEUROTRANSMITTERS
HEART RATE
-I IO BLOOD PRESSURE
IOmm I H2°
I
25 mV
0.10 ml 2.5 K 10-7M 5-HT
39 AP
1 min
Blood pressure and heart rate as affected by firing the heart-excitor neuron RB,,, (39 action (AP)) and by pulse of serotonin (0.10 ml of a 2.5 X 10-T M solution in ASW). Spike activity in by direct depolarizing current. The cell was hyperpolarized before and after the deRBEIE was produced polarization. Serotonin was injected into the cannula perfusing the heart via the efferent vein of the gill. FIG.
3.
potentials
1O-4 M) and benzoquinonium chloride (1 - 2 X 10-S M) effectively blocked bot.h the effect of firing an LD,, neuron and the effect of a pulse injection of acetylcholine. These two drugs were chosen because they had previously been found to be effective antagonists of acetylcholine in the heart of Venus mercenaria (27, 45). Benzoquinonium blocked the negative chronotropic effects of the transmitter of and of injected acetylcholine more ~&II effectively than it blocked their negative inotropic effects (Fig. 7). Reiter (34) has made qualitatively similar observations in a study of the effects of acetylcholine and benzoquinonium in the heart of Aplysia Zimacina. The effect of benzoquinonium on the inhibition caused both by firing LDm and injecting acetylcholine was partially restored after washing with ASW (Fig. 7). Atropine, curare, and hexamethonium, apTABLE
Cell
1.
Serotonin
synthesis
No. of Determinations
plied in concentrations up to 10-d M, did not block the effect of acetylcholine. Arecoline, a cholinomimetic which increases potassium conductance of Aplysia pleural ganglia neurons (17, lS), was found to be an effective inhibitor of the heart (Fig. 8). Pharmacology of vasoconstrictor neurons. The transmitters previously tested on the heart were assayed for their effects on the aortic sphincter musculature in concentrations up to 10 -4 M. Only acetylcholine was found to cause constriction of the sphincter. Its threshold lay between 5 X IO-7 and 1 X 10-G M (Fig. 9), approximately three orders of magnitude above that of its threshold for the heart. A pulse injection of acetylcholine (0.10 ml of a 2.5 X 10-S M solution in ASW) mimicked the response to firing a vasoconstrictor neuron (Fig. 10).
in RB neurons
Total Radioactivity in Cell Body, pmol
RB13E
5
0.8-6.0
RB
7
0.3-3.4
Conversion [3H]Serotonin
to
y0 of Total
7.8+ 8.2 2
Incorporation into Protein radioactivity
1.8
10.6
1.3
10.4
t 2.3 + - 2.7
Values are means & standard error. RB cell values from Eisenstadt et al. (4), but including two additional RB cells. Note that the value 3H eluted together with serotonin has been replaced by the value for identified [aH]serotonin. Under our conditions of scintillation counting, 1 pmol = 2,200 cpm.
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772
LIEBESWAR,
GOLDMAN,
KOESTEK,
AND
MAYER1
15 3
Beats /rn~n
/min
Beats 35 30 25 20 15
IO
IO 5
BLOOD PRESSURE
I
PRESSURE
I
2.5 x IO-“M
20mm
BLOOD
-
IO m m “20
H2O
22AP
35 30 25 20 15 IO
I
20 mm
IxIO-~M
-
H2O
++.---I
50 mV
I min
FIG. 4. Cinanserin blockade of the effect of firing the heart-excitor neuron RI),, and the effect of injecting serotonin. Records in the left column were taken before, those in the middle column during, and those in the right column after the perfusion with cinanserin hydrochloride (4 X 10-G was fired by direct stimulation of the w RB,, cell. Small bars below the blood pressure record in the lower half of the figure indicate pulse of serotonin (0.25 ml of a 2.5 X 10-7 M solution). In these records RB,, was kept hyperpolarized between firing periods. These records are all from one experiment.
The blocking agent hexamethonium chloride (1 X lO-4 M) antagonized equally the contraction of the abdominal aorta caused by firing the vasoconstrictor neuron and the contraction caused by a pulse injection of acetylcholine (Fig. 11). Washing with ASW reversed the effect. Curare (1 X 1O-4 M) also antagonized the action of acetylcholine in the abdominal aorta, although in the same molar concentration it was less effective than hexamethonium. STUDIES. Ace tylcholine synthesis from choline can be used to identify cholinergic neurons (4). To provide further evidence- that acetylcholine is the transmitter of the heart inhibitor and the vasoconstrictor neurons, we determined whether these neurons were able to synthesize BIOCHEMICAL
2x
IO-“M ACh
I min
FIG. 5. Effect of acetylcholine on blood pressure and heart rate. The denervated heart was perfused from the efferent vein of the gill alternately with ASW and with ASW containing acetylcholine (2.5 X 10-10 to 2 X 10-g M). A bar below the record of blood pressure indicates perfusion with acetylcholine.
Beats /min 20 HEART RATE
1 15
r4.e
IO 5
20mm I H2O
h LDHI
--i
L--
I
mV
-.-
0.25ml 156 AP
30
6.25
I min
x 10-8M
ACh
FIG. 6.
Blood pressure and heart rate as affected by firing a heart-inhibitor neuron LD,, (156 action potentials) and by a pulse injection of acetylcholine (0.25 ml of a 6.25 X 10-S M solution in seawater). Spike activity in LD,,, was produced by direct depolarizing current. The cell was hyperpolarized before and after the depolarization. Acetylcholine was injected into the cannula perfusing the heart via the efferent vein of the gill.
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APLYSIA
CARDIOVASCULAR
Beats
773
NEUROTRANSMITTERS Beats/min
/min 25
%L “REA”T”E’
-I
20
-----JUPi
RATE
-0 .-*s*.\e~t-
20 I5
--
15
@-?@I
1
I HEART
IO
‘, BLOOD PRESSURE
20mm
I
“20 5x10 I 1,
A LDHl
‘IryJ
w 107 AP
wd
5
BLOOD PRESSURE
IOmm I
-0 OM I
m b
129AP
“20
30mV 20 I5
dL
IO 126AP 5
-7
1x10
’ M 1
20 I5
0.25ml 1.25 x10-‘M ACh
20mm I
IO
“20
5
2x10-‘M
-
1
Benzoquinonium blockade of the effect of firing a heart-inhibitor neuron LD,,, and the effect of injecting acetyhholine. Records in the left column were taken before, those in the middle column during, and those in the right column after the perfusion with benzoquinonium chloride (2 X 10-5 M). LD,, was fired by direct stimulation of the cell. Note that the number of action potentials is higher in the record under benzoquinonium than in the record under control conditions. Phar. macological studies in the heart-inhibitor neurons were made difficult by the fact that repeated firing of these neurons often soon led to a weakening oi their motor effect. Small bars below the blood pressure record in the lower half of the figure indicate. a pulse injection of acetylcholine (0.25 ml of a 1.25 X 10-T M solution). FIG.
7.
[“HlacetyIcholine from [“HIcholine injected into their cell bodies. Both the heart-inhibitor neurons, LD,,, and the vasoconstrictor neurons, LB,,, converted choline into acetylcholine to an extent similar to that of cholinergic neurons R2 and LlO (Table Z)? Thus, both the pharmacological and the biochemical data argue strongly for the cholinergic nature of the heart inhibitor and the vasocons trictor neurons. DISCUSSI6N
We have provided both and biochemical evidence
pharmacological that the heart
2 Cells R2 and LlO are presumed to be cholinergic on the basis of their high endogenous levels of ace tylcholine (29) and choline acetyltransferase (11). Additional supporting evidence from pharmacological and synaptic release experiments is available for LlO (15, 22j.
20 15
IO 5
4 x10-‘M ARECOLINE HYDROCHLORIDE
I min
FIG. 8. Effect of arecoline on blood pressure and heart rate (compare with Fig. 5). A bar below the record of blood pressure indicates perfusion with ASW containing arecoline hydrochloride (5 X 10-S M to 4 X 10-7 M).
excitor neuron (RB,,,) is serotonergic, and that the two LDu, heart inhibitor neurons and the three LB,c vasoconstrictor neurons are cholinergic (Fig. 12). Serotonin
rnediates
excitation
to heart
Three lines of evidence indicate that serotonin is the neurotransmitter used by RB,, to mediate excitation to the heart. First, we found that serotonin applied directly to the heart mimics the motor effect produced by firing RB,,. The sensitivity of the heart to serotonin is high and about 40 times greater than that for dopamine, the only other neurotransmitter tested which causes heart acceleration. A high sensitivity to serotonin has also been reported for the hearts of a variety of mollusks, including several species of Aplysia (5, 14, 47). Second, cinanserin blocks both the effects of serotonin and of on the heart. Third, RBIIE spike activity significant quantities RBEIE can synthesize or serotonin from tryptophan. The amount
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LIEBESWAR,
774 4
4
~xIO-~M
I
r
IxIO-~M
I
2 ~16~
M
-
GOLDMAN,
KOESTER,
I
r
20cm “20
4xl0M ACh
--J I min
FIG. 9.
Effect of acetylcholine on the vasoconstrictor musculature of the abdominal aorta. Experimental arrangement as shown in Fig. 1B. The abdominal ganglion was removed and the denervated aorta perfused backward from the constantpressure reservoir with seawater containing acetylcholine (5 X lo- 7 to 4 X 10-S M). The vertical arrows indicate the start and the end of the perfusion with acetylcholine. There is a delay with which acetylcholine reaches and leaves the aorta after switching between the solutions.
synthesized is of the same order of magnitude as that synthesized by other RB cells and by the giant metacerebral cells of Aplysia (4). There is both pharmacological (9, 10, 32) and biochemical (43) evidence that the two symmetrical giant metacerebral
AND
MAYER1
cells of Afdysia, located in the cerebral ganglion, are serotonergic. These data from the injection experiments are important since transmitter synthesis shows cellular specificity. Eisenstadt of et al. (4) found that cholinergic neurons Aplysia did not synthesize serotonin from injected tryptophan; conversely, the serotonergic RB cells did not synthesize acetylcholine from injected choline. Another characteristic apparently specific to serotonergic neurons is the ability to transport serotonin along their axons (12). We have preliminary evidence that RBuE transports newly synthesized serotonin into its axon in the pericardial nerve. The conclusion that RBuE uses serotonin as a transmitter is further strengthened by evidence that the heart of Aplysia receives sero tonergic innervation. First, Taxi and Gautron (40) have demonstrated the presence of serotonergic nerve terminals in the heart using histochemical and autoradiographic techniques. Second, Chase et al. (3) and Carpenter et al. (2) have demonstrated serotonin in moderately high concentrations in the auricle and ventricle of Aplysia heart. Carpenter et al. (2) also found a serotonin-uptake mechanism in the heart, which appears to be an active process. Third, we have found that the isolated Aplysia heart, containing the terminal regions of the motoneurons, can synthesize
5cm H20
BLOOD PRESSURE
50 mV
WC -
I I-k--
Lf-IOZAP
FIG. 10. Effect on vascular muscle neuron and by a pulse injection of in blood pressure brought about by Fig. 1A. Since the heart is far more aorta, an injection of acetylcholine Fig. 1B. The effect of firing LBvC perfusing the abdominal aorta. The
30 set
99AP
0.1 ml 2.5 x Kf5M ACh
30 set
of the abdominal aorta produced by firing an LB,, vasoconstrictor acetylcholine (0.10 ml of a 2.5 X 10-S solution in ASW). A: increase firing LB,, recorded in the experimental arrangement as shown in sensitive to acetylcholine than the vasoconstrictor musculature in the cannot mimic this effect. B: experimental arrangement as shown in was mimicked by a pulse injection of acetylcholine into the cannula heart had been removed.
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AZ’LYSZA
TABLE
Acetylcholine
2.
CARDIOVASCULAR
synthesis
NEUROTRANSMITTERS
in LD
and LB
No. of Determinations
Cell
LDHI
sero tonin from exogenously supplied phan (unpublished observations).
values
tryp to-
Acetylcholine mediates inhibition to heart and excitation to arteries Three types of evidence support the conclusion that the LD,, heart inhibitors and the LB,, vasoconstrictor cells are cholinergic. First, acetylcholine was the only transmitter tested which mimics the effects of these motor cells on heart and aorta at low concentrations. This confirms several previous reports on the high sensitivity of the Aplysia heart to the inhibitory effects of
-A
/
I
1 5QmV
~
I
