Development of spontaneous motility in chick embryos. Participation of the central cholinergic mechanism

Development of the effectivensess of the systemic administration of oxotremorin (200 micrograms/kg egg weight) on spontaneous motility in chick embryos was studied from the 11th to the 19th day of incubation. 1) Oxotremorin activated spontaneous motility from the 15th day of incubation; subsequently, its effect increased with the embryo's age. 2) Glycine (100 mg/kg e.w.) blocked the activating effect of oxotremorin, again in a direct correlation to the embryo's age. 3) The spontaneous motility of spinal embryos was activated significantly less, thereby demonstrating the importance of the supraspinal component of the effect of oxotremorin. 4) The results are evaluated primarily as evidence of the role of the cholinergic mechanism (Everett 1964) in embryonal development of supraspinal control of the spontaneous activity of spinal motoneurones.     

Development of spontaneous motility in chick embryos. The spinal and supraspinal component of the inhibitory effect of glycine and GABA.

Development of the effect of glycine and gamma-aminobutyric acid [GABA] on spontaneous motility was studied in 11- to 19-day-old chick embryos under normal conditions and after acute and chronic decapitation. Chronic decapitation was performed on the 2nd day of incubation. Glycine (100 mg/kg egg weight) and GABA (103 mg/kg egg weight) (applied onto the shell membrane) demonstrably inhibited spontaneous motility only from the 15th day of incubation, the inhibitory effect increasing with the embryo's age. When administered together in half doses, glycine and GABA completely inhibited spontaneous motility for the first time in 19-day-old embryos. Neither amino acid influenced depression of motility immediately after decapitation, but 24 and 48 hours after, in 17- and 19-day-old embryos, they had a paradoxical effect, i.e. they transiently activated motor activity and even caused motor paroxysms. After chronic decapitation, both glycine and GABA again had a mild, protracted inhibitory effect. A comparison of spontaneous motility in normal and chronically decapitated embryos showed that the role of supraspinal factors in spinal motor output increases significantly with development of the chick embryo from the 15th day of incubation and that inhibition of these supraspinal factors plays the decisive role in the effect of glycine and GABA.     

The acute and the chronic effects of imipramine on the spontaneous motility in chick embryos

The effect of imipramine on the spontaneous motility and development of chick embryos was studied from the 4th to the 19th day of incubation. On acute administration (a single dose of 12.5 of 25 mg/kg egg weight), imipramine already induced significant depression of spontaneous motility in 11-day embryos--an effect which increased significantly after the 15th day of incubation. The similar effect of imipramine in spinal embryos testifies to its direct action on the spinal cord and draws attention to certain details of the role of supraspinal structures of the CNS in the acute effect of imipramine. The chronic administration of imipramine showed that it had an almost 100% lethal effect from 4th to the 7th day of incubation. Between the 8th and the 10th day it caused longlasting depression of spontaneous motility. When it was administered between the 11th and 16th day of incubation, no significant effect on the development of spontaneous motor activity was found in chick embryos.     

Development of spontaneous motility in chick embryos. Interaction of reserpine and apomorphine

The effect of apomorphine (1 mg/kg egg weight) on a base of the pre-administration of reserpine (2.5 mg/kg e.w.) 30 min or 2 or 4 hours previously was studied in chick embryos from the 13th to 19th day of incubation. Only the depressant effect of apomorphine was manifested in 13-day embryos. In 15-day embryos summation of the depressant effect of both drugs was recorded for the first time. In 17-day and especially 19-day embryos apomorphine raised the frequency of spontaneous movements high above the level of reserpine-induced depression of motor activity. This activating effect of apomorphine depended upon the supraspinal parts of the CNS, as it disappeared in 17-day embryos after decentralization of the spinal cord (particularly after chronic decapitation). We consider these findings to be further experimental evidence of the participation of catecholaminergic (in particular dopaminergic) central systems in supraspinal control of embryonic motor activity.     

Sensitivity of embryonic spontaneous motility to the GABA agonists baclofen and muscimol

The development of the sensitivity of spontaneous motor activity to the GABA agonists baclofen (10 mg/kg egg weight, systemic administration) and muscimol (0.8 mg/kg e.w., systemic administration) was tested in 11-day to 19-day-old chick embryos. 1) Baclofen already significantly depressed the frequency of spontaneous movements in 11-day embryos; its effect attained the maximum (85% depression of spontaneous motility) in 13-day embryos. After the 15th day of incubation, it reduced spontaneous motor activity by 50-60%. In spinal embryos, baclofen had the same, but a quantitatively more pronounced effect, demonstrated from its direct action on the spinal cord uninfluenced by supraspinal modulation, which began to be manifested after the 15th day of incubation. 2) Muscimol did not begin to inhibit spontaneous motility significantly until the 13th day of incubation. Subsequently, the latent period of its effect shortened, its duration lengthened and, lastly, its quantitative result also increased. 3) A comparison of the effect of GABA (Sedlácek 1978), muscimol and baclofen in 17-day chick embryos showed that the depressive effect increased in the sequence baclofen less than GABA less than muscimol, but that GABA took effect faster than the others. The results testify that the maturation of the individual elements of the GABA-ergic central inhibition system is a complex process.     

Bicuculline activation of embryonic spontaneous motility

The activating effect of bicuculline on spontaneous central motor output activity was studied in chick embryos from the 11th to the 19th day of incubation by means of spontaneous motility. When applied onto the embryonic membranes, bicuculline [1 mg X kg-1 egg weight] significantly activated embryonic motility from the 15th day of incubation. In 15-day embryos it increased spontaneous motility 2.5-fold and in 17- and 19-day embryos 3.5-fold. The role of supraspinal factors in the activating effect of bicuculline increased with development. In 15-day embryos it accounted for 56.7% and in 17-day embryos for already 84.6% of the total effect of bicuculline. Antagonism was manifested between bicuculline and the inhibitory amino acids glycine and GABA; in the case of GABA it was quantitatively more pronounced. The results of this study of development of the activating effect of bicuculline and its antagonism with gamma-aminobutyric acid are evaluated from the aspect of the connecting-up and development of central GABA-ergic components in the regulation of embryonic motility.     

Development of the differential effect of oxazepam on spontaneous and activated motility in chick embryos

Development of the acute depressant effect of oxazepam (in a dose of 10 mg/kg egg weight) on spontaneous and activated motor activity was studied in chick embryos (incubation age 11-19 days). The depressant effect of oxazepam was demonstrated for the first time in 13-day-old embryos. From the 15th day of incubation it led to prolonged depression of embryonic motility. From the 15th day the depressant effect of oxazepam depended on the connections of the central motor output with the prosencephalon. In mesencephalic, rhombencephalic and spinal preparations the effect of oxazepam was insignificant. In 13- and 15-day-old embryos oxazepam blocked the activating effect of strychnine and pentylenetetrazol, but not the picrotoxin-induced activation. The picrotoxin activation of embryonic motility was incompletely blocked for the first time in 17-day-old and completely only in 19-day-old embryos. The results are discussed with reference to heterochronia of development of the various components in the complex GABA receptor.     

An attempt to activate the spontaneous motility in chick embryos with tetanus toxin

The effect of tetanus toxin on spontaneous motor activity was studied in chick embryos between the 11th and 19th day of incubation. The toxin--dose 20 mg/kg egg weight ( = 2.86 X 10(3) mouse MLD) in 25 microliters isotonic NaCl solution--was injected into the tibial or the wing muscles. Tetanus toxin induced demonstrable activation of embryonic motility from the 15th day of incubation onwards. Activation attained 155-200% of resting activity. The activating effect was manifested for the first time by motor paroxysms in 17-day and particularly 19-day embryos. Tetanus toxin activation was effectively depressed by glycine (100 mg/kg e.w.) and GABA (100 mg/kg e.w.), the former having a stronger effect than the latter. The effect of tetanus toxin on spinal embryos was relatively more pronounced, while the depressant effect of the inhibitory amino acids (especially glycine) was weaker. The results are evaluated as further evidence that central inhibitory mechanisms are connected up in regulation of the spontaneous motor output activity of chick embryos on about the 15th day of incubation.     

Opioid elements in development of the spontaneous motility of chick embryos

The effects of the acute and chronic administration of a pure opioid antagonist--naltrexone--was studied in chick embryos from the 4th to the 19th day of incubation. In acute administration, naltrexone (40 mg/kg egg weight) induced paroxysmal activation of spontaneous motility in both normal and spinal embryos from the 13th-15th day of incubation. Activation attained 3- to 4-fold the resting activity of chick embryos of the same ages. The chronic administration of naltrexone (7.46 +/- 1.18 mg/kg e.w. per 24 h) from the 4th to the 16th day of incubation was not manifested either in the embryos' somatic development or in the weight of the brain hemispheres, but it depressed the development of spontaneous motility to 26.1-75.8% of the activity of the control embryos. This developmental effect was not demonstrably correlated either to the length of time for which naltrexone was administered, or to when, in the course of incubation, it was administered to the chick embryos. The results are evaluated as evidence of the participation of opioid elements in the development and effectuation of central motor input functions in the early stages of ontogenetic development.     

Development of spontaneous motility in chick embryos. Interaction of strychnine, glycine and GABA.

The interaction of strychnine (1 mg/kg egg weight), glycine (100 mg/kg egg weight) and GABA (103 mg/kg egg weight) on spontaneous motor activity recorded by the method of Kovach (1970) in intact eggs was studied in chick embryos from the 11th to 21st day of incubation. In 11- and 13-day embryos, neither of the amino acids influenced strychnine activation of spontaneous motility. From the 15th incubation day, strychnine activation was distinctly affected by both amino acids, but the maximum effect was observed on the 19th day. Glycine had a stronger inhibitory effect, since it prevented strychnine convulsions from developing, whereas GABA only modified them. It can be concluded from the results that glycine-sensitive and GABA-sensitive mechanisms of embryonal spontaneous motility do not begin to take effect in chick embryos until the 15th day of incubation.     

Development of spontaneous motility in chick embryos. Further components of aminergic mechanisms

The participation of central monoaminergic systems in the regulation of spontaneous motility in developing chick embryos was tested by systemic administration of several drugs, which affect the different sites of central aminergic systems. Amphetamine (2 mg.kg-1 egg weight) evoked an age-dependent depression of spontaneous motility, which first occurred in a significant fashion on day 13 of incubation. Two thirds of this depression depended upon supraspinal influences. The effect of p-chlorophenylalanine (100 mg.kg-1 e.w.) consisted from day 15 of incubation in a short-lasting depression followed by partial recovery of resting motility. alpha-methyldopa (100 mg.kg-1 e.w.) depressed the spontaneous motility from day 13 of incubation, without any signs of recovery within the first hour after drug administration. The most pronounced depressive effect was evoked by melatonin (25 mg.kg-1 e.w.) even in 11-day-old embryos. Along with the increased depth of depression the recovery of motility declined until full cessation of motility was achieved in 17-day-old embryos. The results were interpreted as constituting further evidence for the involvement of central aminergic systems in the development of supraspinal control over spontaneous motor activity generated by the CNS.     

Sensitivity of the generator of spontaneous motility in chick embryos to the acute and chronic administration of MPTP.

The acute and chronic effect of 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP) on spontaneous motor activity and its development was studied in chick embryos. 1. From the 13th day of incubation, the acute effect of MPTP (30 mg/kg e.w., up to 60 min after administration) consisted in significant depression of spontaneous motility. From the 17th day, the effect of MPTP in supraspinal compartments of the CNS also began to participate in this depression. 2. The subacute effect of MPTP (up to 24 h after a single dose) was lethal for 11-day-old embryos. Conversely, in older embryos resting motility partly recovered, with signs of an inverse correlation to the embryo's age. The final effect, however, consisted in absolute failure of the hatching process 3. The chronic effect of MPTP (3.57 mg/kg e.w./24 h, from the 4th to the 16th day of incubation) led to a developmental reduction of spontaneous motor activity, chiefly from the 8th to 12th day of incubation. 4. The interaction of nialamide (25 mg/kg e.w.), a blocker of monoaminooxidase produced disparate results with the effect of MPTP in young and old embryos.     

Development of the cerebrospinal fluid in chick embryos. Physicochemical properties.

The development of the physicochemical properties of the cerebrospinal fluid (CSF) was studied in chick embryos from the 9th day of incubation up to hatching. Some of these properties were compared with the corresponding blood or blood plasma properties. During the second half of incubation the CSF pressure rose from 13.2 plus or minus 0.18 mm H2O in 9-day-old embryos to 80.7 plus or minus 0.48 mm H2O just prior to hatching. The critical stages of this development were the 13th to 15th and the 19th to 21st day of incubation. In 13- and 15-day-old embryos, CSF pressure fell sharply after the intracerebral injection of ouabain, but in 19-day embryos it was unaffected. Except for the 15th and 19th incubation day, the CSF pH was always lower than the plasma pH. From the 11th day of incubation up to hatching, the CSF pH fell from 7.36 plus or minus 0.002 to 7.2 plus or minus 0.005. On the 11th and 13th day, specific CSF resistance was higher than plasma resistance, whereas from the 17th incubation day it was significantly lower than the plasma value. During the second half of incubation, specific CSF resistance fell from 1.059 times 10(6) to 0.824 times 10(6) omega mm.m(-1). A difference between the D.C. potential of the venous blood and the CSF appeared for the first time in 15-day-old embryos, the CSF being negative in relation to the blood. By the end of the incubation period this potential difference rose to 10.82 times 0.07 mv.     

Interaction of beta-carboline with chick embryo spontaneous motility.

The effect of beta-carboline (beta-CCE) on spontaneous motility and its development was studied in chick embryos between the 11th and 19th day of incubation. 1. Acutely administered beta-CCE (7.5 mg/kg e.w.) already induced significant activation of motility in 11-day-old embryos. From the 17th day of incubation activation acquired a paroxysmal character. 2. In spinal embryos (decapitated on the second day of incubation) there was no such activating effect, demonstrating that it is associated with supraspinal components of the CNS. 3. In chronic administration from the fourth day of incubation (1.55 +/- 0.24 mg/kg e.w./24h), beta-CCE led to reduced development of spontaneous motility. The effect was concentrated in the period between the fourth and eighth day of incubation. The chronic administration of beta-CCE augmented the activating effect of metrazol and weakened GABA-inhibition of spontaneous motility. 4. On the basis of their findings, the authors express the hypothesis that the benzodiazepine beta-CCE-sensitive component of the complex GABA receptor evidently already functions from the beginning of the second half of incubation of chick embryos.     

Brachially innervated ectopic hindlimbs in the chick embryo. II. The role of supraspinal input in the loss of limb motility

Brachially innervated grafted hindlimbs display a progressive loss of motility as development proceeds. However, the virtually immobile grafted hindlimbs of E20 embryos exhibited strong, synchronous contractions of gastrocnemius and tibialis muscles upon intraperitoneal injection of strychnine nitrate (20 μg). This result indicated that the marked behavioral deficit was not due to an inability of the motoneurons that innervate the immobile grafted hindlimbs to initiate and propagate action potentials, but was probably the result of an effective loss of motoneuron excitation. To examine the hypothesis that interaction with the supraspinal nervous system is involved in the reduction of grafted hindlimb activity, the normal forelimb and grafted hindlimb movements of chronic spinal embryos were examined. The normal forelimbs of chronic spinal embryos exhibited the same number of movements as normal embryos at all stages examined. Thus the deficit in grafted hindlimb motility is not comparable to the behavior of the normal forelimb in chronic spinal embryos and is, therefore, unlikely to be due to a lack of excitation from the supraspinal nervous system. The possibility of an inhibitory influence via supraspinal projections was examined in chronic spinal embryos that had brachially innervated grafted hindlimbs. After E12, the grafted hindlimbs of chronic spinal embryos displayed significantly fewer movements than the normal forelimbs of chronic spinal embryos but significantly more movements than the brachial hindlimb of embryos with intact spinal cords. By E18, however, both spinal and nonspinal brachial hindlimbs were equally dysfunctional. Thus prevention of supraspinal communication transiently reduces but does not prevent the emergence of motor dysfunction in the brachially innervated hindlimbs, which appears to be due to motoneurons not receiving sufficient net excitation, from spinal circuits, to propagate action potentials to the muscles. © 1993 John Wiley & Sons, Inc.     

Are spontaneous activity and supraspinal control co-responsible for functional development of the generator of embryonic motility?

The effect of chronic administration of phenobarbital (from the 4th to the 16th day of incubation) combined with chronic spinal decentralization (decapitation at stage 11-13) on the development of spontaneous motor activity (recorded on the 17th day of incubation) was studied in 4 to 17-day-old chick embryos. 1. Combination of the two experimental treatments led to summation of their negative effect on development; spontaneous motor activity fell to 15.2% of the control value (to 26.9% after the isolated administration of phenobarbital and to 30.5% after isolated decapitation). 2. Metrazol activation of motor activity (100 mg/kg egg weight) after combination of the two factors was relatively no different from the results after their isolated use. 3. The acute administration of GABA (100 mg/kg e.w.) likewise induced relatively the same depression of spontaneous motility as after isolated chronic decapitation and the isolated chronic administration of phenobarbital. The results confirm the hypothesis of the significance of spontaneous activity of the spinal motoneurones for their survival and their actual functional development during the embryonic period.     

Development of spontaneous motility in chick embryos. Sensitivity to aminergic transmitters.

The consequences of systemic administration of aminergic transmitters (n-adrenaline 16 microgram/kg egg weight; serotonin 2.5 and 5 mg/kg e.w.; dopamine 2.5 and 5 mg/kg e.w.) for the spontaneous motility and heart rate of 11- to 19-day chick embryos were studied intack eggs. The following results were characteristic for all three transmitters: a) when administered to 11- and 13-day embryos their effect was non-significant; the first signs of activity did not appear until the 15th day of incubation. The effect on 17- and 19-day embryos was stronger. b) After the 15th day of incubation, all these transmitters had a predominantly inhibitory effect on spontaneous motility; in 17- and 19-day embryos this acquired a periodic character. c) The changes in spontaneous motility did not correlate significantly in any way with the relatively small heart rate changes. It is concluded from the results that aminergic mechanisms begin to participate in regulation of the spontaneous motility of chick embryos from the 15th day of incubation, and not before.     

Changes in the development of spontaneous motility in chick embryos after the chronic administration of GABA

From the 4th to the 16th day of incubation, GABA was administered continuously to chick embryos in a mean dose of 9.04 +/- 0.98 mg/kg e.w./24 h. On the 17th day of incubation, spontaneous motility was evaluated from the frequency of spontaneous movements as resting motility and motility after the acute administration of GABA (100 mg/kg e.w.), bicuculline (1 mg/kg e.w.) and oxazepam (10 mg/kg e.w.). 1) The chronic administration of GABA reduced the spontaneous motor activity of the experimental embryos to 38.4-47.8% of the control value. To obtain this effect it was sufficient to administer GABA between the 4th and the 8th day of incubation. 2) The inhibitory effect of the acute administration of GABA in the experimental embryos was only half its effect in the controls. Conversely, the relative size of bicuculline activation of motility was distinctly greater in the experimental embryos, which were also significantly more sensitive to oxazepam. The results show that GABA has a dual effect during embryogenesis--a) an early effect between the 4th and 8th day of incubation causing a chronic debt in the development of spontaneous motor activity, and b) an inhibitory effect as a central transmitter, which begins to be manifested in embryonic spontaneous motility of chick embryos on about the 15th day of incubation.     

Development of spontaneous motility in chick embryos. Normal development and the activating effect of strychnine and picrotoxin.

The longitudinal development of spontaneous motility in chick embryos was studied by Kovach's method (Kovach 1970) from the 10th day of incubation up to hatching, in completely intact eggs. From the 10th to 12th day of incubation, very low amplitude movements of a burst character predominated in spontaneous motility. From the 13th day, both low and high amplitude movements could be distinguished. From the 18th day, high amplitude movements alternating with intervals of motor inactivity preponderated. This discontinuous motility, which was most pronounced on the 20th day of incubation, changed to periodic strong hatching movements. Reduction of spontaneous motility after the 17th day of incubation was not confirmed. Strychnine already activated spontaneous motility in 11-day embryos, but typical convulsions did not appear until the 15th incubation day. With picrotoxin, motility was likewise stimulated in 11-day embryos and paroxysmal activation did not occur until the 15th incubation day. In older embryos, convulsions were gradually succeeded by a continuous increase in spontaneous motility. The effect of picrotoxin had a much longer latent period than the effect of strychnine.     

Embryonic spontaneous motility after acute and chronic administration of kainate

Changes in spontaneous motility after the acute and chronic administration of kainate were studied in 11- to 19-day-old chick embryos. 1. After acute administration, kainate (20 mg/kg e.w.) already depressed motility in 11-day-old embryos. From the 17th day it induced explosive activation of embryonic motility, but never in chronic spinal preparations. 2. The chronic administration of kainate (2.56 +/- 0.62 mg/kg e.w./24 h) reduced embryonic motor activity. The effect already developed after administering kainate from the 4th to the 8th day of incubation. Prolonged administration made no important difference to the results. Chronic administration was followed by histopathological changes in the nervous tissue. These were mainly of an oedematous type and affected the glia and the brain capillaries, whereas pyknotic changes were found in the large neurones. 3. The results showed that the CNS is already sensitive to the neurotoxic effect of kainate from the early stages of embryogenesis and that the picture of the reaction of the embryonic CNS is closely correlated to the degree of maturation.