Some basic chemical components of the cerebrospinal fluid in developing chick embryos.

The development of the chloride ion, glucose and total protein concentration was investigated in the cerebrospinal fluid of 11- to 21-day-old chick embryos and compared with their development in the blood plasma. Developmental changes in the chloride concentration in the plasma and CSF were very small, but it was always higher in the CSF than in the plasma. The plasma/CSF ratio fell during development, from 0.906 in 11-day-old embryos to 0.778 at the end of incubation. The CSF glucose concentration fell up to the 19th day of incubation, but a significant increase was recorded shortly before hatching. The plasma glucose concentration rose throughout the whole of the investigated period of embryogenesis. Up to the 19th day the P/CSF ratio rose from 1.59 to 4.05 and in 21-day-old embryos fell to 2.47. The developmental increase in the plasma total protein concentration was accompanied by the reverse process in the CSF. During the second half of incubation the P/CSF ratio rose from 1.88 to 7.9 Calculation of total osmolarity from the Na+, K+, Ca2+, Cl- and glucose concentration showed permanent hyperosmolarity of the CSF compared with the plasma. The P/CSF ratio was maintained within limits of 0.94 to 0.98.     

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.     

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.     

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.     

Control of blood pressure mediated by baroreflex changes of heart rate in the chicken embryo (Gallus gallus)

Pharmacological manipulation of peripheral resistance via sodium nitroprusside and phenylephrine was used to study baroreflex function over the latter two-thirds of incubation in embryonic chickens. From day 9 to day 19 of incubation, there is a positive linear relation between heart rate and blood pressure, indicating the feedforward action of arterial pressure on heart rate. A reciprocal relationship between blood pressure and heart rate became pronounced during the last 3 days of incubation. For the purpose of the study, gain of the baroreflex was calculated as maximal gain (only those embryos that demonstrated the response) or average gain (all embryos). Maximal gain increased progressively from 13 +/- 7 beats. min(-1). kPa(-1) at 18 days to 105 +/- 83 beats. min(-1). kPa(-1) in 2-day-old hatchlings. The percentage of embryos older than 18 days with baroreflex responses increased from 33% on day 19 to 56% on day 21, indicating that baroreflex regulation begins late in incubation ( approximately 90% incubation time), and the gain of this reflex exhibits a maturation over the final 3 days of incubation.     

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 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.     

The infraslow potential oscillations in developing chick embryo brain. Correlations with neuronal activity.

The correlations between infraslow potential oscillations [ISPO] and EEG activity were studied in the brain hemispheres and optic lobes of chick embryos from the 15th to 21st day of incubation. 1. The ISPO of the optic lobes remained unchanged during and after peripheral optic stimulation -- not only in 17-day-old embryos (the prefunctional stage), but also in 19- and 21-day-old embryos, in which optic evoked activity in the optic tectum is already well developed. 2. The intracerebral administration of strychnine, GABA and sodium glutamate had no effect on the ISPO of the brain hemispheres in 15-day-old embryos. 3. The effect of these neurotropic drugs in 20-day-old embryos varied. Strychnine evoked concomitant activation of ISPO and the EEG, sodium glutamate simultaneously depressed both activities, while GABA inhibited EEG activity without affecting ISPO patterns. 4. These results supported our conclusion that neuronal activity plays a secondary role in the ISPO generation process in developing brain tissue.     

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.     

Effects of the acute and chronic administration of chlordiazepoxide on the development of spontaneous motility in chick embryos

The effects of the acute and chronic administration of chlordiazepoxide on spontaneous motility and on the reactivity of the generator of embryonic motility were studied in chick embryos from the 4th to the 19th day of incubation. 1. The acute administration of chlordiazepoxide (5 mg/kg e.w.) significantly depressed spontaneous motility from the 13th day of incubation. 2. The chronic administration of chlordiazepoxide (12.2 mg/kg e.w./24 h) from the 4th to the 8th, 12th and 16th day of incubation enhanced the reduction of the spontaneous motility of 17-day-old embryos. 3. The chronic administration of chlordiazepoxide significantly modified the activity of both activators (strychnine, metrazol, bicuculline, picrotoxin) and inhibitors (GABA, chlordiazepoxide) of the spontaneous motility of chick embryos.     

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.     

Immunoreactive insulin and insulin-like activity of the blood in ontogenesis of the hen]

Studies have been made on immunoreactive insulin (IRI) and insulin-like activity (ILA) in the blood serum of chick embryos (from the 10th day of incubation), chicks and adult hens up to 1 year old. It was shown that IRI content of embryonic blood is relatively low and remains approximately constant during incubation. During postnatal ontogenesis, the level of IRI increases, the increase being most significant at the 1st day after hatching and between the 2nd and the 5th months. With respect to IRI level, 5-month chicks are similar to adult hens. Being assayed by the method of isolated epididymal rat fat, ILA was not found in the blood serum of chick embryos. It was observed in all test samples only from the 30th day after hatching. It is suggested that at this period of postnatal life, some factors are formed in the blood which increase ILA without changes of the insulin content of the blood. After the 30th day, no evident shifts were observed in ILA, although it reached maximum in adult hens. By absolute values, ILA of the blood in chicks was several times higher than the corresponding levels of IRI.     

Anaerobic metabolism in fowl embryos during normal incubation

Lactate concentration in blood, liver, yolk, amniotic and allantoic fluid and blood pyruvate was measured in embryos in the final week of incubation. Blood lactate was low up to day 18. The blood lactate/pyruvate ratio and liver lactate increased from day 19 until hatching. From day 14 to 19, lactate concentration in amniotic fluid remained constant, it increased 2-fold in yolk and 10-fold in allantoic fluid. There was only a 48% net accumulation of lactate in the three cavities. In conclusion, fowl embryos do not turn to anaerobic metabolism until the hatching process starts on day 19.     

Proteinase activity in Ascaris suum eggs, hatching fluid, and excretions-secretions.

Hatching fluid and the excretions and secretions (E.S.) of hatched larvae of Ascaris suum revealed proteinase activity when assayed by 2 different procedures employing collagen or casein as substrates. Both assays apparently detected similar levels of proteinase activity in hatching fluid and E.S. of hatched larvae. The Anson (casein substrate) assay worked best in 0.05 M phosphate buffer, pH 8.0. The Azocoll (collagen substrate) assay worked best in 0.05 M borate buffer at pH 8.8. Azocoll assays done at temperatures ranging from 25 to 65 C revealed maximal proteinase activity at 55 C. Analysis of hatching fluid from 18-, 21-, and 28-day-old embryos and of extracts from sonicated 0- to 28-day-old developmental stages showed that proteinase activity increased markedly 18 days after embryonation had begun. Prior to the 18th day of embryonation proteinase levels were relatively low.     

Changes in plasma testosterone levels during the peri-hatching period in the chicken.

Blood was obtained by heart puncture from 19-day-old Black Sex link chicken embryos and from Black Sex link chickens at 1.5, 6, or 24 h post-hatching. Plasma testosterone was determined by gas chromatography-mass spectrometry associated with stable isotope dilution. At 19 days the plasma of male and female chick embryos contains measurable amounts of testosterone and levels do not differ between sexes. After hatching plasma testosterone gradually declines from pre-hatch concentrations in males and females, but in all the post-hatch ages studied, plasma testosterone was significantly higher in male than in female chicks. These results indicate that in male chickens, contrary to mammals at birth, there is no surge in plasma testosterone at hatching.     

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.     

Acoustic and somatosensory evoked responses in the brain hemispheres of chick embryos.

The author explored the auditory projection in the brain hemispheres of 16- to 21-day-old chick embryos, using biaural stimulation, and the somatosensory projection, using electrical stimulation of the contralateral sciatic nerve. The first auditory evoked responses appeared on the surface of the hemisphere at the beginning of the 18th day of incubation and were localized in its mediolateral part. Up to hatching, the latent period of the surface response shortened from 76.3 msec to 28.9 msec and its amplitude augmented from 10.6 muV to 36.2 muV. If the electrode was plunged into the tissue, the evoked responses with the optimum latent period and amplitude parameters were recorded at a depth of 2-2.5 mm (latent period 20.2 msec, amplitude 40-45 muV). The maximum surface somatosensory responses were found in the medial occipital quadrant of the contralateral hemisphere. They developed from the second half of the 17th day of incubation. Up to the end of incubation the mean latent period shortened from 58.3 msec to 21.6 msec and the mean amplitude increased from 11.8 muV to 28.7 muV. What was at first a simple negative wave developed into a positive-negative complex by the end of incubation. Evoked responses at a depth of about 3.5 mm from the surface of the hemisphere had the optimum parameters (latent period 18.4 msec, amplitude 30.2 muV).     

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.     

Simulated biological effects of microgravity on phospholipid and energy metabolism of chicken embryonic brain cells studied by 31P-NMR spectroscopy

Levels of phosphomonoester (PME), phosphodiester (PDE), ATP and pH in brain cells of chicken embryos rotated for 24 h in a clinostat during the period of hatching the 13th day (E13) and 15th day (E15) embryos were investigated by using 31P-NMR spectroscopy. Significant increases in the values of PME, ATP and pH were identified after E13 rotating for 24 h. With the same treatment, differences were obtained in the phospholipid and energy metabolism of E15, but no significant levels have been reached . The calorimetric assay (malachite green method) was used for measuring the activity of total ATPase. A dramatic decrease was evident in the activity of ATPase in brain cells of rotated E13 and E15. The former is more sensitive than the latter. All the levels mentioned above could restore in 24 h after the rotation stopped, except that the level of ATP was still higher than the control.     

Simulated biological effects of microgravity on phospholipid and energy metabolism of chicken embryonic brain cells studied by ~(31)P-NMR spectroscopy

Levels of phosphomonoester (PME), phosphodiester (PDE), ATP and pH in brain cells of chicken embryos rotated for 24 h in a clinostat during the period of hatching the 13th day (E13) and 15th day (E15) embryos were investigated by using 31P-NMR spectroscopy. Significant increases in the values of PME, ATP and pH were identified after E13 rotating for 24 h. With the same treatment, differences were obtained in the phospholipid and energy metabolism of E15, but no significant levels have been reached . The calorimetric assay (malachite green method) was used for measuring the activity of total ATPase. A dramatic decrease was evident in the activity of ATPase in brain cells of rotated E13 and E15. The former is more sensitive than the latter. All the levels mentioned above could restore in 24 h after the rotation stopped, except that the level of ATP was still higher than the control.