NATIONAL RESEARCH COUNCIL OF CANADA CONSEIL NATIONAL DE RECHERCHES DU CANADA

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TECHNICAL TRANSLATION 1814 TRADUCTION TECHNIQUE

Peculiarities of reflex responses in chick embryos

(Ob osobennostyakh reflektornykh reaktsii u kurinykh embrionov)

T.P. Blinkova

Fiziologicheskii Zhurnal SSSR im, I.M. Sechenova, 48 (11): 1415-1420, 1962

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 KlA OS2

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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

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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

+38°C. 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)

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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. The relationship of heart rate and age is non­

monotonic. 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

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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%): to sound stimuli: 6- 8; to vibration: 7 - 30; to the electric

current: 8 ~ 14; and to ammonia: 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 uncon­

ditioned 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

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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. Not infre­

quently, it was violent. 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-

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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 con­

traction. 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

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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 develop­

ment, 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 embryo­

genesis. 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.

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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.

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4. Promptov, A.N. Zhurn. Obshch. Biol., 9 (2): 145, 1948.

5. Barry, A, Journ. Exp, Zool,, 85: 157, 1940,

6. Clark, E.L. and Clark, E.R. Journ, Exp. Zool,, 17: 373, 1914,

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

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

9. Kuo, Z.V. Journ. Exp. Zool,, 61: 395, 1932.

10. Preyer, W, Specielle Physiol, des Embryo, Leipzig, 1885.

11. Ray, W.S. Child Develop., 3: 175, 1932.

12. Romanoff, A.L. Anot. Rec., 89: 313, 1944.

13. Sontag, L.W. and Wallace, R.F. Am. Journ. Dis, Child,, 48, 1934.

14. Spelt, D.K. Psycho!. Bull., 35 (5): 712, 1938.

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350

300

250

\ \ ..... -..... -.J \ ..... --f50 \.--' ......

100 L..,...---1---'----'---....l....--._____. _ __.__

6 8 tO tt f* 16 t8 ZO

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

' · .. '

t I I f 4 I I f f I I I . I I I 4 I I l I C I C 4 f I t I « • ' p· ~ ~ ~ ~·- ...... -,, . ,.:. ..... : .. · -· ~· - . . - ·~

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

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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

•