Autonomic Drug Effects on the Heart Rate of

Early Rat Embryos

MAURICE ROBKIN,2 THOMAS H. SHEPARD 3 AND DAVID BAUM 3,4
(WITH TECHNICAL ASSISTANCE OF GLENDA BASS 3)
Department of Nuclear Engineering, B F - 1 0 , 2 and Department of Pediatrics,3
University of W a s h i n g t o n , Seattle, W a s h i n g t o n 981 95

ABSTRACT We examined the effect of cardioactive drugs on the heart rate

of intact rat embryos at day 11 (25 somites) of gestation, the stage of develop-
ment prior to cardiac innervation. Tested were the autonomic drugs methoxa-
mine and isoproterenol, the P-adrenergic receptor blocker propranolol, and the
phosphodiesterase inhibitor theophylline. Our observations indicated that
(1) methoxamine had no effect; (2) isoproterenol and theophylline caused in-
creases in the heart rate; and (3) propranolol inhibited the effect of isoproterenol
but did not affect the response to theophylline. We concluded that our results
are consistent with (1) Ahlquist's ('48) original concept of a and @ adrenergic
receptors and the observation that the heart has no a receptors, (2) with the
role of cyclic-AMP in the model of the adrenergic receptor proposed by Suther-
land ('68),and (3) with the observations that the embryonic heart at this stage
of development is not yet innervated and so is without the vagal reflex arc
(Hogg, '57; Gomez, '58).

Drugs affecting cardiac function are
widely used. In many instances they are
administered to pregnant women, and if
they cross the placenta, the embryo itself
may be affected.
In this laboratory, in research on the
effects of drugs on the development of
critical stages of embryogenesis, we have
used pregnant rats in days lOY2-12V2 of
gestation as an experimental model with
particular concentration on day 11. At the
latter age, rat embryos have about 24-26
somites and correspond approximately to
a gestational age in humans of about
23-26 days (Shepard, '69).
The fully developed normal mammalian
heart is controlled in part by the ziuto-
nomic nervous system so that observed
responses to an administered drug may
be due not only to direct effects on the
heart cells but also to indirect effects of
stimulation or depression of neural re-
flexes. In the case of embryos the heart
is functioning and pumping blood, but
has not yet achieved innervation or its
adult form. (Hogg, '57; Gomez, '58). With-
out innervation the reflexive responses to
cardiotropic drugs will be absent, leaving
only those responses that are the result
of direct action on the heart.
To our knowledge the only published
study of these direct responses of the
hearts of rat embryos at the gestational
age of our model is Hall's ('57). He studied
the effects of acetylcholine and epineph-
rine on isolated intact hearts and heart
fragments from rat embryos of day 10V2
to 12 of gestation. It is not known whether
or not a flow restriction due to a-adren-
ergic-mediated peripheral vasoconstriction
will cause a direct pressure change at the
heart which will in turn affect the heart
rate. The assumption that, in absence of
cardiac innervation and the vagal reflex,
the responses that Hall observed might
also apply to the heart of the intact em-
bryo must therefore be made with some
caution.
In his study of intact rat embryos
Adolph ('65) administered catecholamines
at day 16 or later. By day 16 the rat heart
has attained its adult form and the auto-
nomic nerves have grown into the rnyo-
cardium (Hogg, '57; Gomez, '58). He also
did not completely separate the embryos
from the maternal circulation. The changes
Received May 30, '72. Accepted July 31, '73.
1 This work was supported i n part by NIH grants,
HD00180,HD00836, and HD06775.
4 Present address: Department of Pediatrics, Stan-
ford University, Stanford, California.

9: 3 5 4 4 .
TERATOLOGY. 35
36 MAURICE ROBKIN, THOMAS H. SHEPARD A N D DAVID BAUM

he observed in embryonic heart rate when flex effect on the heart. To test this rea-
the catecholamines were administered to soning, methoxamine (Vasoxyl, Burroughs
the pregnant female did not agree with Wellcome), an a-adrenergic stimulator
those observed when the drugs were given (Goodman and Gilman, '70) was intro-
to the embryo, and he thus raised ques- duced in various concentrations. In a
tions concerning the penetration of the final test of peripheral vasconstriction
drug through the placenta and secondary (yolk-sac vessels) an extremely large dose
effects due to alteration of uterine blood of methoxamine was administered which
flow leading to embryonic hypoxia. Al- resulted in death of the embryos.
though our experiments deal with fewer According to Sutherlands model, the
compartments we are still faced with the function of the adrenergic preceptor and
uncertainty of whether or not the drug of cyclic-AMP (CAMP) are independent.
reaches the embryonic heart in an un- If this is the case, for doses resulting in
changed state. submaximal total responses, the responses
Insofar as comparisons between the re- to the p-receptor-stimulator isoproterenol,
sponses of either the hearts of intact or- and to increased c-AMP due to the phos-
ganisms or the excised hearts and heart phodiesterase-inhibitor theophylline
fragments can be made, our results may should be additive in any order of admin-
be compared with Hall's. That is, in our istration. Blocking the p-adrenergic re-
experiments, as in Hall's, the embryonic ceptor with propranolol should have no
hearts were completely separated from effect on the response to increased levels
the pregnant female and any secondary of C-AMP.
effects. Further, Hall also examined the The enzyme phosphodiesterase facili-
response to epinephrine of hearts from tates the regulation of cellular concen-
day 101/2-11?42 embryos. The positive trations of c-AMP by catalyzing its metab-
chronotropic response that he observed is olism. The level of c-AMP may be increased
consistent with a similar response to iso- by administration of theophylline, an in-
proterenol that we observed. hibitor of phosphodiesterase.
In the case of intact embryos we are
METHODS AND MATERIALS
not aware of any published study of the
response of the heart to cardiotropic drugs The embryos were explanted according
during the gestational period with which to the method of New and Stein ('64) and
we have concerned ourselves. It would installed in a life support system with
be very difficult to apply the methods of circulating medium (denoted by the term
previous workers to the hearts of intact PLASMOM). The ambient environment of
embryos as small as these. In our study the embryos is controlled and we have
we have made use of a new in vitro cul- demonstrated good growth and develop-
ture technique for early rat embryos (Rob- ment of the embryos for culture periods
kin et al., '72) to examine the effects of up to 24 h in this system (Robkin et al.,
methoxamine, isoproterenol, propranolol, '72). For some of the experiments (see
and theophylline. below), New's ('67) circulator was used.
The model we have hypothesized is Using the PLASMOM system we are able
based on: (1) Ahlquist's ('48) concept of to observe and record the heartbeat of
a and p receptors; (2) the generally ac- embryos by a technic of transillumination
cepted point of view that there are no (Y (Robkin et al., '72). The recording is con-
receptors in the heart (Nickerson and tinuous and permits a detailed and exact
Chan, '61; Moran and Perkins, '61; Moran analysis of the embryonic heart-rate re-
et al., '62) and (3) Sutherland et al.'s sponses to various agents. We estimate
('68) model of adenyl cyclase and second that the heart rate may be determined
messenger, adenosine 3',5'-monophosphate with better than 1% accuracy.
(cyclic-AMP). The initial filling of the PLASMOM re-
If the embryonic heart is not innervated quires about 6 ml of serum, which is im-
there can be no vagal reflex stimulated mediately circulated and warmed. The
via peripheral a-adrenergic receptors, and embryos are simultaneously explanted
administration of an a-adrenergic stimula- and attached to rafts in Hanks' balanced
tor in subtoxic doses should have no re- salt solution (BSS) at room temperature.
 DRUG EFFECTS ON HEART RATE OF RAT EMBRYOS
Immediately after explantation the em-
bryos in their chamber are inserted into
the circulator. The chamber contains
about 2 ml of Hank's BSS and the result-
ing mixture is allowed to return to the
desired temperature. The temperature
within the embryo chamber is maintained
at 38.5 I+ 0.3" C. Table 1 shows the elec-
trolyte composition of Hanks' BSS and
human serum.
As soon as the temperature has stabil-
ized, the embryos are examined with a
binocular dissecting microscope to deter-
mine whether a vigorous heartbeat and
good yolk-sac circulation are present. Oc-
casionally we observed circulatory stasis
in the yolk sac which we considered a
warning of impending embryonic dis-
tress and excluded the preparation from
analysis.
A low power laser (University Labora-
tories, Model 200) illuminates the embryo
and a lens is used to focus an image of
the heart onto the sensitive face of a pho-
tomultiplier tube. The flicker caused by
the beating heart is detected and recorded
on a strip-chart recorder (Brush Instru-
ment Co., Model Mark 220). Since there
are two or three embryos explanted in
the chamber, we can usually find at least
one where the silhouette of the beating
heart can be detected by the phototube.
If all three embryos are suitable they
can be used in an experiment by rapidly
adjusting the position of the chamber so
as alternately to focus the image of the
heart of each embryo in succession upon
the photomultiplier tube. A baseline heart
rate is determined at the start of each
experiment.
With the circulating volume and flow
rates used, a mixing time of about 1 min
is estimated (approximately 2 complete
circuits). The hearts begin to respond
1-2 min after injection of the drug, which
TABLE 1
Electrolyte composition of culture solutions
Hank's Serum Medium
Ion BSS (nominal) (approximate)
meqll meqll meqll
Na+ 142 140 140
K+ 5.8 4.9 5.1
Ca++ 1.3 5.5 4.7
Mg+ 0.9 2.1 1.8
c1- 146 102 112
37
presumably is a measure of the combined
effect of mixing time in the circulating
medium and diffusion time into the yolk-
sac circulation. The injection is made on
the upstream side of the heater. We have
observed no resulting temperature change
in the circulating medium at the location
of the embryos as determined by a rapidly
responding needle-mounted thermistor
probe in the embryo chamber (Yellow
Spring Instrument Co., Model 507). Al-
though the heart rate has been shown to
be a sensitive function of ambient tem-
perature (Robkin et al., '72) we have
shown by analysis of the strip-chart re-
cording after administration of up to 0.4
ml of Hanks medium without drugs that
no significant changes in the heart rate
occurs. Changes less than twice the ac-
curacy of the measurement (2-4 beats/
min) were considered insignificant since
spontaneous variations of the heart rate
of this magnitude were often observed
while taking baseline recordings. Any
larger response to an injection can be
ascribed to the drugs. While the recorder
is running, the injection i s made and a
continuing record obtained.
The record was analyzed unless the
heartbeat became too irregular to inter-
pret or the rate dropped below 100 beats/
min which was taken as evidence of irrep-
arable injury to the embryo. If analysis
of the record showed continuing decrease
of the heart rate or other anomalous be-
havior the embryo was considered abnor-
mal and the experiment was discarded.
In some cases the embryo did not respond
to the drug, but in the absence of other
anomalous behavior the experiment was
included in the results.
The embryos were examined for stage
of development and somite number shortly
after the termination of the experiments,
which usually lasted 2-3 h. The day-11
embryos conformed to our previous defini-
tions (Shepard et al., '70) which at the
start of the observations coincide with
crown-rump length of 3 mm and a somite
count of 25. An increase in somite num-
ber over those of littermate controls oc-
curred during the experiment, indicating
continuing development of the embryos.
Four protocols were followed to analyze
the model. In each, before any drugs were
administered, the heartbeat of the em-
38 MAURICE ROBKIN, THOMAS H. SHEPARD AND DAVID BAUM
bryos was recorded for up to 20 min to
ascertain that they were stable. In a given
run each drug administration was fol-
lowed by a period of recording the heart
rates until they had stopped changing
and the heart had reached a new stable
rate.
The effective response threshold doses
in our preparations were established so
that by using only slightly larger dosages
we might avoid approaching any maximal
responses when drugs were used in com-
bination. The threshold concentrations in
the circulator were approximately 0.2 pg/
ml isoproterenol hydrochloride (Isuprel,
Winthrop) and 25 pglml theophylline
(Crystalline, Mallinkrodt). In our experi-
ments we used approximately 0.5 pglml
isoproterenol and 50 pg/ml theophylline.
For each experiment isoproterenol and
propranolol were freshly diluted from the
ampoul and theophylline was freshly dis-
solved dry powder.
The amounts added to the circulating
medium were 4 pg of isoproterenol hydro-
chloride, 5 pg of propranolol hydrochloride
(Inderal, Ayerst) (equimolar with the iso-
proterenol), and 425 pg of theophylline.
The volume of solution added at each ad-
ministration of drugs was 0.1 ml. The
volume of circulating medium in most of
these preparations is about 8 ml, so that
we are able to achieve reasonable titers
of introduced materials with relatively
small amounts. The medium used was
human serum, collected in the laboratory
and stored frozen for short periods of time.
Shepard and Tanimura (‘70, private com-
munication) found no significant differ-
ences in the results of culturing rat em-
bryos with rat serum or fresh human
serum.
In the first protocol the order of admin-
istration of the drugs was isoproterenol,
propranolol, and theophylline. In the sec-
ond, it was propranolol, isoproterenol, and
theophylline. In the third, it was theo-
phylline, isoproterenol, and propranolol.
The amounts of drug administered in
the methoxamine experiments were 4, 8,
20, 100, 1000, and 40,000 pg injected into
approximately 10 ml of circulating medium
in each case. New’s (’67) circulator was
used in the first three of these (4, 8, 20 pg)
and the embryos were followed with the
dissecting microscope. The PLASMOM
was used for all the other cases.
Table 2 gives an outline of the three
protocols followed with the cardiotropic
drugs. The time intervals indicated be-
tween injections are the smallest, average,
and largest values required to reach a
steady state heart rate for a few minutes
after the changes in heart rate for the
various embryos that made up an experi-
mental group.
The assumption was made that in the
dosage range we worked with, the response
was proportional to the concentration of
drug in the circulating medium. The data
were analyzed on the basis of percentage
change of heart rate per unit concentra-
tion of circulating drugs. Composite plots
were made by normalizing the data to the
percentage changes corresponding to
0.5 pglml isoproterenol, 0.6 pg/ml pro-
pranolol, and 50 pglml theophylline. All
of the experiments used approximately
these concentrations so that the normali-
zation allowed all of the runs in a given
protocol to be plotted on a single graph
with a common basis for comparison.
RESULTS
Figures 1-3 show the composite results
for the three protocols involving the p-
adrenergic receptor. A summary of these
results is shown in table 3. Comparison of
the responses to isoproterenol and theo-
phylline from protocol to protocol was
made based on Student’s t test (two-tailed).
As can be seen in table 2 and figure 2,
even though isoproterenol broke through
the propranolol p-block in some cases, the
response was very reduced. We also see
that the hearts responded to theophylline
even after the p-receptor was blocked by
propranolol (table 3, figs. 2, 5).
Figures 4-6 show a typical response of
a day-11 embryo to each protocol. These
curves illustrate how closely the response
of the heart can be analyzed when the
embryo is monitored continuously during
a run. The heart rates shown are as
recorded.
Methoxamine has not been reported to
have an effect other than to stimulate the
adrenergic a-receptors (Goodman and Gil-
man, ’70, p. 510). The usual physiological
dose of methoxamine is approximately
7 mg for an adult human. This dose uni-
formly distributed in a 70-kg man’s serum
would give a concentration of 2 pglml.
With concentrations of methoxamine up
DRUG EFFECTS ON HEART RATE OF RAT EMBRYOS 39

to approximately 100 pglml of medium,

we were unable to observe an effect on
the heart rate of the day-11 embryos
tested. Visual examination of the yolk-sac
vessels with a dissecting microscope with
a comparator eyepiece did not indicate
any change in vessel diameter even with
a dose of 4000 pglml, which killed the
embryos.
In protocol 1 , isoproterenol was the first
drug administered. The resulting response
of the heart rate may be taken as repre-
sentative for isoproterenol administered
by itself. In protocol 3, theophylline was
the first drug administered and the re-
sponse in this case may be taken as rep-
resentative for theophylline. These changes
were taken as the reference responses for
the purpose of the discussion below.
In protocol 2 , a p-adrenergic blockade
was first established with propranolol, and
z m p - tested by next administering isoproterenol.
In the 14 embryos (day 11 of gestation)
examined, the heart was unresponsive in
8 of them and accelerated in 6 of them by
an amount that was greatly reduced from
the reference value. The average response
was a factor of 10 smaller than the refer-
ence response, indicating that the p-re-
ceptor was blocked. Following the isopro-
terenol, theophylline was administered.
The resulting acceleration of the heart
was not significantly different from the
reference increase seen in protocol 3,
which implies that the theophylline re-
sponse was not affected by the blockage
of the p receptor. On the other hand,
when theophylline followed isoproterenol
(protocol l), the resulting increase in the
heart rate was smaller than the reference
theophylline increase with marginal sig-
nificance ( P = 0.055). Similarly, when iso-
proterenol followed theophylline (protocol
3), the resulting increase due to the p
simulation was again smaller than the
reference, with marginal significance
( P = 0.055).
DISCUSSION
According to the model proposed by
Ahlquist ('48) the adrenergic receptors
acted upon by sympathomimetic drugs
can be classified according to whether
their stimulation results in excitation
(a-adrenergic receptors) or inhibition (p-
adrenergic receptors) of the cells on which
they are located. One prominent exception
40 MAURICE ROBKIN, THOMAS H. SHEPARD AND DAVID BAUM

35 Theophylline

30

PI

-
25
P
-.
$ 20
5
9,
h
15
2
\
c
10
P
9,

F
5

-
124 6 8 0 2 4 7
I
12
Time (min)
I I
20
-
112 4 6 8

Fig. 1 Summary of responses of day-11 embryo group subjected to protocol 1. All re-
sponses have been normalized (see text). Zero time mark represents actual time of adminis-
tration of each drug.

to this classification was the cardiac ex-
citatory receptors which seem to be only
P me.
According to this concept, a-adrenergic-
stimulating agents affect the heart only
by the stimulation of a reflex originating
from a peripheral receptor. Thus, in the
mature animal, methoxamine, an a-adren-
ergic stimulator, causes peripheral vaso-
constriction and increased blood pressure
leading to the bradycardiogenic vagal
refiex arising in the carotid baroreceptor.
In the absence of cardiac innervation
this model would predict the absence of
any cardiac response to an a-adrenergic-
stimulating drug. Since our studies were
performed on embryos, at a dosage of up
to 1000 times the adult human physiolog-
ical dose, the lack of a response is prob-
ably consistent with an unresponsive or
absent peripheral a receptor, an unre-
sponsive peripheral vascular bed, or an
incomplete nervous reflex arc between
the baroreceptor and cells of the heart.
Our results cannot directly distinguish
between these. However, since no effect
on yolk-sac vessels was observed, the pe-
ripheral vessel a-receptors may not be
functioning, if, in fact, yolk-sac vessels
have such receptors.
In the model proposed by Sutherland
et al. ('68) the adrenergic p receptor is
adenyl cyclase which catalyzes the forma-
tion of cyclic-AMP within the cells of the
receptor organ which in turn acts as sec-
ond messenger leading by a sequence of
enzyme amplification steps to enhance-
ment of the rate of synthesis of the energy
source for cardiac muscle. To function ef-
ficiently as a messenger the cyclic-AMP
must be destroyed rapidly to preserve the
control mechanism. With rapid destruction
the level of cyclic-AMP is highly depend-
ent upon the impulse rate of the sym-
pathetic nerves at the receptors. Inhibition
of the phosphodiesterase that metabolizes
 DRUG EFFECTS ON HEART RATE O F RAT EMBRYOS
1 Isoproterenol
Isoproterenol
4
n
12 4 6 8 12 4 6 8
Time ( m i n )
Fig. 2 Summary of responses of day-11 em-
bryo group subjected to protocol 2. Initial ad-
ministration of the p-blocker propranol is not
shown as there were no responses observed. Re-
sponses shown have been normalized (see text).
Zero time mark represents actual time of admin-
istration of each drug.
the cyclic-AMP is equivalent to an increase
of the impulse rate of the sympathetic
nerves (not yet present in the embryonic
heart), resulting in an increased heart
rate. Similarly, addition of a p-adrenergic
stimulator is also equivalent to an increase
in the impulse rate of the sympathetic
cardiac nerves.
Our results imply that the site of action
of the p-adrenergic stimulator is proximal
in the chain of events to that of c-AMP,
consistent with Sutherlands model. How-
ever, the hypothesis that the actions are
completely independent is not firmly es-
tablished. Even at the low dosages used
(2 X threshold) there was some indica-
tion of a saturation effect. That is, in
both protocols 1 and 3, the following tachy-
cardiogenic drug had a smaller effect per
unit dose. We may note that, in protocol
3, the observation of a reduced effect with
isoproterenol is not consistent with Suther-
lands third criterion that theophylline
will potentiate a p-adrenergic stimulator
(Sutherland et al., '68; Meyer, '70).
This reduced effect is not simply that
due to a limiting upper heart rate, since
it was observed with protocol 1. In that
procedure, the initial isoproterenol accel-
eration was followed by propranolol which
returned the heart rate essentially to base-
line values before the theophylline was
41
35 -
30 -
z 25-
:.
-
:
c 20-
.s
-
b - r T - r l
0 2 4 6 8 2 4 6 8
Time (min)
Fig. 3 Summary of responses of day-11 rat
embryo group subjected to protocol 3. Final ad-
ministration of the p-blocker propranolol is not
shown, but all embryos had strongly decelerated
heart rates. Responses shown have been normal-
ized (see text). Zero time mark represents actual
time of administration of each drug.
administered. The increase due to the
theophylline was considerably less than
that due to the isoproterenol. Thus it can-
not be the case that the response to theo-
phylline brought the heart rate to any
upper limit.
The results may perhaps be explained
by a depletion during the initial accelera-
tion of some other required compound
which leads to a reduced response with a
following drug. This hypothesis is consist-
ent with the observation in protocol 2 that
if the initial acceleration due to isoproter-
enol is first inhibited by the p blockade
then the theophylline response is not sig-
nificantly different from the reference
response to theophylline. Such a model
would also be consistent with the results
of protocol 1 if the recovery time for this
compound were long compared to the in-
terval between the administration of iso-
proterenol and theophylline. Unfortunate-
ly, the level of significance for rejecting
the hypothesis of additivity is sufficiently
 42 MAURICE ROBKIN, THOMAS H. SHEPARD AND DAVID BAUM

TABLE 3

Summa?y of experimental results

Protocol 1 Protocol 2 Protocol 3

A B C D E F
Isoproterenol Theophylline Isoproterenol Theophylline Theophylline Isoproterenol

% Heartrate
change ( t SE)
per PcLglml 47r5 0.10 * 0.01 4.7+ 1.8 0.16rt0.02 0.15 & 0.02 37k2
Number of
embryos 11 11 14 14 11 10
P Reference B vs E Avs C DvsE Reference F vs A
0.055 < 0.001 N.S. 0.055
Values are based on Student’s t statistic for the differences between the isoproterenol response i n protocol 1 and the othe
protocols, and between the theophylline response in protocol 3 and the other protocols, with the hypothesis that all response
are the same. Isoproterenol is the first drug administered in protocol 1 and theophylline is the first drug administered i r
protocol 3. The positive value of the isoproterenol response in protocol 2 is the result of the drug breaking through the pro
pranolol blockade with small accelerations in 6 of the 14 cases. The heart-rate changes shown are computed per unit con
centration of circulating drug SO that the data making up the average value in a given column can be combined.

210
I
.F
:B 200

90.

80. 0
0
.54pgm/ml
I soproterenol
0
170. 0 +o 0 56 r g m /ml 0 . 0 0
0 4 Theophylline 0 .
0 . 0
.67p g m /m I 5 0
0 .
+ @

0
Propronolol 0 . 0
160. I I I I I i
10 20 30 40 50 60
Time (min)
Fig. 4 Detail of response of a day-11 rat embryo to protocol 1. Concentrations shown are
for this particular case.

ambiguous that any attempt at model
making must be taken as highly specu-
lative.
In summary, our results are consistent
with the concept that there are only p-
adrenergic receptors in the fetal rat heart
and their stimulation leads to an increase
in the transmitter, adenyl cyclase, and in
“second messenger,” cyclic-AMP (Suther-
land et al., ’68), thence to an increase in
the energy source and tachycardia. By-
passing the p receptor by directly affect-
 DRUG EFFECTS ON HEART RATE OF RAT EMBRYOS 43

\ '"1 0
0
.
0 .

Propranolol Isoproterenol Theophylline

I50 I I I I i 1

2201
210

0
0
0

0
0

.63pgm/ml
* 0

0 .
Propronolol
O O . O O O O O

0 0 0 0 0 0.

eo
0
*
.51 p g r n / r n l
Isoprolerenol

- 4
55 p q m / rnl
Theophylline
160 1 I 1 I I I I
0 10 20 30 40 50 60
Time (min)
Fig. 6 Detail of response of a day-11 rat embryo to protocol 3. Concentrations shown are
for this particular case.

ing the level of cyclic-AMP with theophyl- LITERATURE CITED

line also leads to tachycardia. Our findings
are also in keeping with observations that
the rat heart is not innervated On
day 11 of gestation and O d Y drugs that
directly affect the heart wfl be effective.
Adolph, E. F. 1965 Capacities for regulation of
heart rate in fetal, infant and adult rats. Am. J.
Physiol., 209: 1095-1105.
Ahlquist, R. P. 1948 A study of adrenotropic
receptors. Am. J. Physiol., 153: 586-600.
Gomez, H. 1958 The development of the in-
44 MAURICE ROBKIN, THOMAS H. SHEPARD AND DAVID BAUM
nervation of the heart i n the rat embryo. Anat.
Rec., 130: 53-71.
Goodman, L. S., and A. Gilman 1970 The Phar-
macological Basis of Therapeutics. 4th Ed. Mac-
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Govier, W. C., N. C. Mosal, P. Whittington and
A. H. Broom 1966 Myocardial alpha and
beta adrenergic receptors as demonstrated by
atrial functional refractory-period changes. J.
Pharmacol. Exp. Ther., 154: 255-263.
Hall, E. K. 1957 Acetylcholine and epinephrine
effects on the embryonic rat heart. J. Cell. Comp.
Physiol., 49: 187-200.
Hogg, I. E. 1957 Developing innervation of the
heart in the albino rat. Anat. Rec., 127: 470
(abst.).
Meyer, S. E. 1970 Adenylcyclase a s a compo-
nent of the adrenergic receptor. CIBA Sympo-
sium on Molecular Properties of Drug Receptors.
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