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Title/Titre:

1814

Peculiarities of reflex responses in chick embryos

(Ob osobennostyakh reflektornykh reaktsii u kurinykh
embrionov)

Author/Auteur:

T.P. Blinkova

Reference/Reference:

Fiziologicheskii Zhurnal SSSR im, I.M. Sechenova,

48 (11): 1415-1420, 1962

Translator/Traducteur:

Mrs. G. Smirnoff

UNIVERSITY Of Wr\H:RlOU
ENGINEERING ~ SCllNC

LIBRARY

IECHNICAL REP.ORT COllEC1\0N

Canada Institute for

Scientific and Technical
Information

Institut canadien de
!'information scientifique et
technique

Ottawa, Canada
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PECULIARITIES OF REFLEX RESPONSES IN CHICK EMBRYOS

An organism adapts itself to the external environment through changes in

its unconditioned and conditioned reflex reactions in response to changing
conditions of existence.

,,

In evolutionary physiology, it is important to

elucidate the pathways and mechanisms of evolution of these adaptive reactions.

Of particular interest is the study of their ontogenetic formation during
the period of embryonic development,
The authors who discovered the formation of temporary connections in
chick embryos (Gos, 1933; Hunt, 1949) and in the human fetus (Ray, 1932; Sontag
and Wallace, 1934; Spelt, 1938) during the period of embryogenesis merely
recorded the fact without attempting to analyze the mechanism of these reflex
connections.

Our attempt to develop a temporary connection during the period

of embryogenesis of hens had been successful (Blinkova, 1960).

Consequently,

further investigations were directed to the study of characteristics of a

temporary connection in embryos.

The correlation of morphological, biochemical

and electrophysiological data on the maturation of different levels of the C.N.S.,

coupled with the study of functional characteristics of temporary connections in
embryos, will probably enable us in the future to understand the mechanism of
formation of an elementary embryonal temporary connection as a basis of
conditioned reflex activity of adult animals.

Methods

Works described in the literature, with rare exceptions, had been carried
out under conditions of acute experimentation (Preyer, 1885; E,L. Clark and
E.R. Clark, 1914; Kuo, 1932; Volokhov, 1951; Bogdanov, 1960 and others), as the
problems faced by the authors required wide access to the embryo.

However, the

- 3 -

method of acute experimentation has some negative aspects.

The membrane

dissection alone, not to mention the rougher interference by the experimenter,

disrupts the normal course of physiological functions.
In a previous work (Blinkova, 1960), a temporary connection had been
developed during the period of embryogenesis, under conditions of chronic
experimentation.

The presence of a reflex had been determined from visual

observations and cinematographic recording of general motor reactions.

In the present work, which was also carried out under conditions of
chronic experimentation, motor reactions and the ECG (electrocardiogram) of
chick embryos were recorded throughout the entire period of development of the
temporary connection, starting from its first manifestation.
By using the method of chronic insertion of electrodes we were able to
follow, under optimum conditions of embryonic development, the evolution of the
heartbeat frequency from the first days of embryogenesis up to the time of
hatching and during the first days of postembryonic life.

The ECG and motor

responses were recorded through copper electrodes inserted in the poles of the
egg.

To avoid trauma, the electrodes were inserted undPr the shell at a depth

of 3 - 5 mm, without their touching the embryo directly.

The embryo was placed

in a specially screened chamber where the temperature was constantly kept at

+38C.

The biopotentials were amplified through an AC.;..coupled amplifier and

recorded w1th an ink-pen recorder.

The ECG and motor responses of chicks were

recorded under conditions of unrestricted behaviour in a heated chamber to

avoid their cooling.
Experiments in the study of unconditioned reflex reactions were carried
out on 62, and conditioned reflex reactions on 35 chick embryos,
The temporary connection was developed by the mota-defensive method during
the last week of embryonic development, starting from the 14th day.

A tone of

2,000 Hz * , 80 db, imitating to a certain extent the squeaking of a chick, served

as a conditioned stimulus.

An electric current of approximately 0.3 rna (3.5 v)

was applied as an unconditioned reinforcement.

The stimulating current was fed

through the same electrodes that were used for recording the ECG and motor
responses of embryos.

The action of an individual sound stimulus lasted

In the original - 2,000 sec. (Translator)

- 4 -

3 - 5 sec.

Results of Experiments

Our task consisted in determining the periods of time during which the
circuit was temporarily connected in the C.N.S. of the chick embryo and in
studying its characteristics,

Prior to this, we carried out a series of

experiments to study the unconditioned motor and heart reflexes to various

stimuli, inasmuch as these reflexes form the basis of all temporary connections.
At first we followed the evolution of heart-contraction frequency, starting
from the 6th day of embryonic development up to the moment of hatching.

Heart-

contraction frequency throughout the entire embryogenesis is quite variable

and fluctuates, as a rule, between 200 and 280 beats per

minute.

When recorded

over an extended period of time, its fluctuations can reach, on the average,
30% of the initial background.
monotonic.

The relationship of heart rate and age is non-

A change in the heartbeat frequency towards acceleration begins on

the 19th day of embryonic development and continues after hatching, including, as
shown by our observations, the first two days of postembryonic life.

According

to data of A.N. Promptov (1948) and O.V. Bogdanov (1960), this acceleration
continues during the following days (Figure 1).
The unconditioned motor and heart reactions to sound stimuli, vibration,
ammonia, and the electric current were observed from the 6th day of embryonic
development.

When studying the embryo's response reactions to ammonia, the

latter was let through the chamber and rapidly replaced by air at room temperature.
Two openings were drilled for this purpose in the air chamber, at an angle that
prevented the stream of air-and-ammonia mixture and of pure air, when fed into
the chamber, from pressing the embryo,
The motor reactions to all these stimuli were expressed quite distinctly
and exhibited throughout all the days of embryonic development.

Whereas the

motor reaction to sound stimuli and ammonia was extinguished rapidly when evoked

- 5 -

by vibration and an electric current, it was extinguished with great difficulty.

On the 19th - 20th day of embryogenesis, reflex motor responses became somewhat
depressed, which corroborates A.A. Volokbov's findings (1951).
Reflex heart responses to these same stimuli were recorded throughout all
the days of embryogenesis, but only in 67%, rather than in all of experimental
embryos.

The response reaction waspredominantly that of acceleration,

The

change in heart-contraction frequency in relation to the initial background was

(in%):
current:

to sound stimuli:
8

6- 8; to vibration:

14; and to ammonia:

7 - 30;

to the electric

10- 42 (Figure 2).

The latent period of response reaction to ammonia was 4 - 5 sec, and to

other stimuli 0.5 - 1.5 sec.

Not infrequently, after vibration was applied, the

heart-contraction frequency decelerated for about 30 sec to 1 min.

In the course of work, it was found that while individual stimuli evoked
a moderate acceleration of heart activity, their combined effect gradually
lowered the background frequency of heartbeats.

As can be seen in Figure 1, 2,

the curve, each point of which represents an arithmetical mean of background

indices of heartbeat frequency before the application of one or another stimulus,
is situated much below the curve of evolution of heartbeat frequency (Figure 1, 1).
Figure 1 illustrates the deceleration of heartbeat frequency under the combined
effect of the applied stimuli.

Starting from the 19th day of development, the

acceleration of heartbeats, which occurs in the norm, was even more pronounced
under the combined effect of stimuli,
We proceeded to develop the temporary connection after the motor reaction
to the sound stimulus hatl been extinguished.

When developing the temporary

connection during the first two days, in one group of embryos conditioned and
unconditioned stimuli were applied simultaneously, while in the second group
the delay of the conditioned stimulus was gradually increased to 5 sec.

It

was later revealed that the coincidence in time of the conditioned and unconditioned stimuli did not accelerate the development of the temporary connection.
Its first manifestation was noted at the 7th- 9th combination, i.e., on the
1st - 2nd day of development (Figure 3).

The percentage of positive responses

increased from experiment to experiment, reaching a maximum on the 4th day of

- 6 -

experimentation

lin most cases this was the 17th day of embryonic development),

after which it showed a certain decrease (Figure 4).

Inasmuch as there is constant spontaneous motor activity in chick embryos,
it was sometimes difficult to differentiate the manifestation of a temporary
connection from a chance coincidence of spontaneous motor activity with the
moment of application of a conditioned stimulus (dubious instances were not
taken into account).

Differential inhibition was not developed as the temporary

connection was not sufficiently stable.

We therefore confined ourselves to

extinguishing it acutely on the 17th day of development of the chick embryo,

when the percentage of positive responses in experiments is usually highest.
As a rule, the motor reaction was extinguished after 4 - 5 nonreinforcements
of the sound stimulus.

Inasmuch as a temporary connection is inconstant in its

manifestation, we considered the extinction complete when no motor reaction was

observed after 7 - 8 separate sound applications.
In order to ascertain that we were indeed witnessing a temporary connection
rather than motor reactions arising from increased reflex excitability, control
experiments were set up.

In these experiments, as many sound stimuli and

stimuli by the electric current were applied to the control chick embryo as to
the experimental embryo, without, however, making them coincide in time.

No

motor reaction to the sound stimulus was observed in such cases.

The presence and stability of the temporary connection were also checked
during the first hours of postembryonic life of the experimental animals.

The

motor reaction to sound which had been applied as a conditioned stimulus during
the period of embryogenesis was extinguished.

Its first applications evoked in

chicks a defensive motor reaction, accompanied by a vocal reaction.

quently, it was violent.

Not infre-

In this case, it was also extinguished after 3 - 5

nonreinforcements of the sound stimulus.

At the same time, the motor reaction

to sound in the control chicks was either absent or resembled an orientational

reflex.
Although the development of vegetative temporary connections was not the
main purpose of our investigations, we also recorded the vegetative components,
in addition to recording general movements.

We registered the change in heart-

..
- 7 -

beat frequency towards deceleration in 67% of experimental embryos.

This

deceleration was characterized by inconstancy, and usually represented 6 - 8%

of the initial background.

These changes were most pronounced on the 17th -

19th day of development.

Thus, temporary connections in chick embryos are characterized by the
following features:

relative rapidity

~f

development, inconstancy of

manifestation, and ease of extinction during both the period of embryogenesis

and the early postembryonic period.

Discussion of Results

What were we able to perceive that was new by using the method of chronic
insertion of electrodes?

The . nature of the curve obtained by us during the

evolution of heart-contraction frequency up to the 19th day of embryonic

development does not differ in principle from the data of Romanoff (1944).
Barry (1940), and others, except that it indicates a higher frequency of contraction.

The literature contains information (Promptov, 1948; Bogdanov, 1960)

to the effect that the heartbeat rate in chicks gradually increases during the
first days after hatching.

The authors attribute this to the increased tonus

of sympathetic nerves as a result of the simulating effect of factors of the

external environment.

We observed this gradual increase in the rate of heart

contractions starting from the 19th day of embryonic life.

In all probability,

the unavoidable loss of blood caused by the dissection of embryonic membranes

disrupts the fine regulation of physiological functions, and explains why the
investigators did not observe the phenomenon noted by us,
Unconditioned reflex shifts of the heart were observed from the 6th day
of embryonic development.

It is interesting to note that individual stimuli

evoked an increase in heartbeat frequency, but after every stimulation a new,

lower level of frequency was established,

This pattern may indicate that during

the earlier period of embryogenesis, the nervous centres of the embryo that are

:w;-

- 8 -

responsible for the unconditioned reflex regulation of heart activity can

respond to various external stimuli only by brief excitation, after which their
adaptive capacity becomes rapidly exhausted.

Such limited capacity is

characteristic of every function undergoing a process of evolution.

During the

later stages of embryogenesis, shortly before hatching, when the nervous centres
mature and become readied for regulating the heart functions of the hatched
chick in response to external influences, the latter stimulate their activity,
evoking an adequate acceleratory reaction.
Observations show that unconditioned reflex motor reactions are evoked by
all stimuli and throughout all the days of development.

During the last days

of embryogenesis, the motor activity becomes depressed, which corroborates A.A.

Volokhov's data (1951).

Thus, while motor reactions during the last days of

embryogenesis become somewhat depressed, the heartbeat rate during the same days
increases under the influence of the developing tonus of sympathetic nerves.
This may indicate the relative independence and nonsimultaneity of formation of
functions in the various divisions of the C.N.S.
When forming a temporary connection during the period of embryonic development, we first witness the appearance of conditioned motor reactions, and then
the vegetative (heart) shifts as components of this temporary connection.
Experiments in the evolution of unconditioned reflex heart reactions show that
only towards the end of embryogenesis is there an adequate reaction in response
to external stimuli.

The acceleration of heartbeats on the background of the

conditioned motor reaction during the last days of embryonic development thus
becomes understandable.

The insignificant stifts observed before the 17th -

19th day do not always indicate the evolution of this function.

The relative drop in the percentage of positive responses of temporary
connections after the 17th day of development can be attributed to the
depression of motor activity, which indicates the lowering of excitability of
the higher divisions of the C.N.S. in embryos during the last stage of embryogenesis.

It then becomes clear why the temporary connection in a chick embryo

prior to hatching becomes highly unstable and inconstant, while in the first hours
of postembryonic life of chicks it is characterized by great distinctness and
intensity.

- 9 -

The rapid extinction of the conditioned motor reaction during embryogenesis

and after the hatching of chicks seems paradoxical.

It can hardly be assumed

that the internal inhibitory function in the embryo is developed to any

significant degree.

It is more likely that the rapidity with which the

developed temporary connection is extinguished stems from its instability and

relatively weak excitability of the nervous centres.

Conclusions

1.

The somatic and vegetative functions in the C.N.S. of a chick embryo

are not formed simultaneously.

First to form are the unconditioned reflex

motor reactions, and then the vegetative, in particular, the heart reactions.
2.

The formation and evolution of unconditioned reflex heart reactions

is completed towards the 19th day of embryogenesis.

3.

Conditioned motor reflexes are observed from the 14th - 15th day of

embryonic development, and are characterized by inconstancy of manifestation,

rapid extinction, and depression before hatching.

References

1.

Blinkova, T.P. Ezhegodnik IEM AMN SSSR za l960g. (Yearbook of the Institute
of Exp. ~1ed., Acad. Hed. Sc. USSR for 1960). Leningrad. 1961.

2.

Bogdanov, O.V. Stanovlenie regulyatsii serdechnoi deyatel'nosti u kur i

golubei v rannem ontogeneze (Evolution of heart-activity regulation
in hens and pigeons during early ontogenesis). Diss. Leningrad.
1960.

3.

Volokhov, A.A. Zakonomernosti ontogeneza nervnoi deyatel'nosti (Ontogenetic

patterns of nervous activity). Medgiz. 1951.

- 10 -

4.

Promptov, A.N.

Zhurn. Obshch. Biol., 9 (2): 145, 1948.

5.

Barry, A,

6.

Clark, E.L. and Clark, E.R.

7.

Gos, M. Bull. Soc. Sci. Liege, (4-5): 194, 1933; (6-7): 246, 1933.
by E. Hunt, 1949.

8.

Hunt, E.

9.

Kuo, Z.V.

10.

Preyer, W,

11.

Ray, W.S.

12.

Romanoff, A.L.

13.

Sontag, L.W. and Wallace, R.F.

14.

Spelt, D.K.

Journ. Exp, Zool,, 85: 157, 1940,

Journ, Exp. Zool,, 17: 373, 1914,

Journ. Comp, a, Physiol, Psych,, 42 (2): 107, 1949.

Journ. Exp. Zool,, 61: 395, 1932.
Specielle Physiol, des Embryo, Leipzig, 1885.
Child Develop., 3: 175, 1932.
Anot. Rec., 89: 313, 1944.
Am. Journ. Dis, Child,, 48, 1934.

Psycho!. Bull., 35 (5): 712, 1938.

Cited

- 11 -

350
300
250

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Fig. 1
The evolution of heart-contraction frequency (1)
and its decleration under the combined effect of
stimuli (2)
Along the ordinate axis - frequency of heart
contractions per minute; along the abscissa axis
- developmental days during the period of embryogenesis

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....... -,,
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Fig. 2
Change in the ECG under the effect of ammonia (a) and an electric
current (b) on the 16th arid 19th days of development
In (a), arrows indicate the beginning and end of stimulation by
20% ammonia; the 'packaged peaking' in (b) indicates the moment
of stimulation by an electric current of 5 v, 30 Hz. The figures
indicate the frequency of heart contractions per 6 sec. Time
mark: 1 sec


- 12 -

Fig. 3
Development of a temporary connection in embryo No. 17
a, b - on the 14th day, B - on the 16th day of develop-
ment. The figures represent the sequential combination
numbers. Time mark: 1 sec.
70

Fig. 4
Change in the percentage of positive responses
during the development of a temporary connection
Along the ordinate axis - percentage of positive
reponses in the experiment; along the abscissa
axis - developmental days during the period of
embryogenesis