Organization of impulse flows and selective neuronal death in the hyperstriatum of chick embryos and chicks

Studies have been made on changes in the number of neurones per volume unit of the tissue of hyperstriatum tissue in chick embryos from the 18th day of incubation to the 3rd day after hatching. Significant interhemispheric asymmetry in migration rate and cell death was revealed in the dorsal hyperstriatum from the 19th day of embryogenesis, which may be due to asymmetry in the intensity of visual afferentation which depends on the stationary position of the embryo in the egg. In the activity of neuronal populations within the dorsal hyperstriatum at a stage which immediately precedes mass death of cells (up to 40%), a structure of impulse volley was found which is characterised by the onset of impulse discharges following each other in a form of short series. Sometimes it is possible to reveal correlation between the duration of dominating interdischarge intervals and the extremes in the recovery of excitability of neuronal pools, which indirectly indicates putative reverberative origin of impulse cyclic phenomenon.     

The organization of pulse trains in the dorsal hyperstriatum of hens in relation to the afferent input in ontogeny]

Analysis of background multicellular activity of neuronal populations in the dorsal hyperstriatum of chick embryos and baby-chicks, incubated either in darkness or under periodic illumination, revealed an impulse volley structure which is characterized by onset of discharges which follow each other in a form of small series at close intervals. These series originally may be observed in the background activity of the left dorsal hyperstriatum in "illuminated" chick embryos at the 19th day of incubation, and only at the 1st day after hatching--in the right dorsal hyperstriatum of embryos, "illuminated" although they are present in both left and right dorsal hyperstriatum in embryos incubated in darkness, the difference being presumably due to asymmetric input of visual afferentation to this structure. Series of impulse volleys with repeating intervals are considered as a reflection of the activity of local microsystems of neurons which exhibit cyclic structure and which are capable to maintain stable impulse activity in its intrinsic system of connections, which is one of the elements of a mechanism of synaptic stabilization and formation of organized neuronal complexes.     

The dynamics of the activity parameters of the neuronal populations in the dorsal hyperstriatum of hens depending on the afferent input in ontogeny]

Studies have been made on the properties of neuronal populations of the dorsal hyperstriatum of the forebrain in hens beginning from the 18th day incubation up to the 7th day after hatching, which were incubated and kept either in darkness or under periodic illumination. Among the investigated parameters of multicellular activity, mean frequency of the discharge activity was found to be most susceptible to the level of external light afferentation in ontogenesis. The discharge activity was more intense in the left hemisphere of chick embryos and chicks from illuminated group, which may be associated with asymmetrical visual afferentation resulting from embryonic position within the egg, and its modulating effect on synchronization processes in the adjacent neurones of the given structure.     

Antibodies against mouse nerve growth factor interfere in vivo with the development of avian sensory and sympathetic neurones

The monoclonal antibody 27/21 directed against mouse nerve growth factor (NGF) interferes in vivo with the survival of sensory dorsal root ganglion (DRG) neurones during the development of the quail embryo: the number of DRG neurones at embryonic day 11 (E11) was reduced by about 30% in embryos treated with the antibody between E3 and E11. Neurone numbers in the nodose ganglion were not affected. The effect of NGF antibodies on sympathetic neurones was assessed by determining the levels of the adrenergic marker enzyme tyrosine hydroxylase. Both total tyrosine hydroxylase activity and protein levels in sympathetic chains were reduced by about 30% in embryos treated with 27/21 antibody but not in embryos treated with a control antibody. The 27/21 antibody cross-reacts with chick NGF-like activity as shown in vitro by the ability of the antibody to partially block the survival activity of chick-embryo-fibroblast-conditioned medium for E9 chick DRG neurones.     

The assessment of the strength of the interaction between neurons of the cat motor cortex in a conditioned reflex to time]

The work was conducted on cats with recording multineuronal activity of the motor cortex at the elaboration of conditioned reflex to time. Strength of interaction was estimated between the adjacent and remote neurones in the limits of 0.5 m. The dynamics of strengths of interneuronal interaction in most cases did not correlate with the dynamics of impulses frequency of the studied neurones. Changes of strengths of interaction between mutually remote neurones at CRT were met more frequently than between the adjacent ones, what may serve as one more evidence of the hypothesis on more strict structure of connections within microsystems and greater plasticity of connections between microsystems.     

Development of gamma-glutamyl transpeptidase activity in primary cultures of neurones and glial cells derived from the cerebral hemispheres of chick embryos

The development of gamma-glutamyl transpeptidase (GGT) activity in neurones and glial cells was studied in primary cell cultures derived from the cerebral hemispheres of chick embryos. GGT activity was found in both basic types of nervous tissue cells. It was always higher in glial cell cultures, where it was up to 2.3-fold the values in neurone-enriched cultures. If the culture medium contained foetal calf serum, the GGT activity of both types of nerve cells was higher than in the presence of inactivated calf serum. Comparison with the in vivo situation showed that the level of GGT activity in nerve cell cultures was significantly lower. Between the seventh day of embryogenesis and the third day of postnatal development of the nerve cells, there were marked differences between the GGT activity of cells maintained under in vitro conditions and cells of the same age in brain tissue homogenate. GGT activity in cerebral hemisphere homogenates from a 17-day-old embryos amounted to 4-fold the activity in a primary glial cell culture and to 16-fold the value in a neurone-enriched culture from hemispheres at the same stage of development.     

Redox status of the oviduct and CDC2 activity in 2-cell stage embryos in heat-stressed mice

Mammalian preimplantation embryos are vulnerable to heat stress. However, the mechanisms by which maternal heat stress compromises embryonic development are unclear. We hypothesized that the loss of developmental competence in maternally heat-stressed embryos results from enhanced oxidative stress in the oviducts. In experiment 1, oviducts and zygotes were collected from mice that were heat-stressed at 35 degrees C and 60% relative humidity for 12 h on the day of pregnancy as well as from control mice. The zygotes were cultured for 84 h to assess their development, and the H(2)O(2) level, glutathione concentration, and free radical scavenging activity (FRSA) were measured in the oviduct. In experiment 2, zygotes were cultured for 22 h to reach the late G(2) phase in the 2-cell stage, and Cdc2 activity was assessed using immunoblotting. A high percentage (87.6%) of control embryos developed to morulae or blastocysts, whereas the majority (67.4%) of the heat-stressed group arrested at the 2-cell stage. Although heat stress did not alter the FRSA or glutathione concentration in the oviducts, the H(2)O(2) level (P < 0.01) and its ratio to the FRSA (P < 0.05) significantly increased in the heat-stressed group. The Cdc2 activation at the 2-cell stage, as shown by the ratio of the dephosphorylated form to the phosphorylated form, was evident in control embryos but absent in heat-stressed embryos, and the level was similar to that in embryos blocked at the 2-cell stage (positive control). These results indicate that maternal heat stress enhances oxidative stress in the oviducts and that loss of developmental competence in maternally heat-stressed embryos correlates with a defect in Cdc2 activity at the 2-cell stage.     

Sex differences in the regulation of embryonic brain aromatase

Oestrogen formed from androgen by aromatization plays a critical role in the sexual differentiation of the male brain and behaviour. A question which has still to be answered is what regulates the gender-specific changes in aromatase activity forming oestrogen during sensitive periods of brain growth. Using a primary cell culture technique and sexed embryos, we have shown that in the fetal mouse brain, oestrogen formation in the male is neuronal rather than glial and aromatase activity is regionally localized, being higher in the hypothalamus than in the cortex. The aromatase activity measured from cells in culture has the same enzyme binding affinity (apparent K_m 40 nM) as intact brain samples. Neurones developing in the embryonic male brain (embryonic day (ED) 15) contain higher aromatase activity (V_max, 895 fmol/h/mg protein) than the female (V_max, 604). Although a sex difference exists at early stages of embryonic development (ED 13), the embryonic aromatase system is regulated by steroids later in fetal development. The developing aromatase-containing neuroblasts probably form processes which connect to other aromatase neurones. Immunoreactive staining with an aromatase polyclonal antibody identifies an increase in numbers of aromatase-immunoreactive hypothalamic neuronal cell bodies following testosterone treatment. Testosterone treatment also causes both stimulation of neurite growth and branching as well as functional maturation of aromatase neurones. In particular, there is an increase in aromatase activity per neurone as well as a dramatic increase in the number of neurones expressing the enzyme. Both the functional and morphological changes depend on androgen receptor stimulation for several days in vitro. This conclusion is supported by colocalization studies which reveal a high number of fetal hypothalamic aromatase neurones co-expressing androgen receptor. We conclude that testosterone influences the growth of male hypothalamic neurones containing aromatase at a sensitive period of brain development. Endogenous steroid inhibitors of aromatase, probably formed within the neuroglia, also play a role in the control of oestrogen production. An endogenous 5-reduced metabolite of testosterone, 5-androstanedione, is almost as potent in inhibiting neuronal hypothalamic aromatase activity (K_i = 23 nM) as the synthetic non-steroidal inhibitors such as the imidazole, fadrozole, and the triazoles, arimidex and letrozole. It is clear that the oestrogen-forming capacity of the male hypothalamus has the special characteristics and plasticity of regulation which could affect brain differentiation at specific steroid-sensitive stages in ontogeny.     

Lack of effect of 2.45-GHz microwave radiation on the development of preimplantation embryos of mice

The development of preimplantation embryos after exposure to microwave radiation was studied. Female CD-1 mice were induced to superovulate, mated, and exposed to 2.45-GHz microwave or sham radiation for 3 h at power densities of 9 mW/cm² and 19 mW/cm² on either day 2 or 3 of pregnancy (plug day was considered day 1). Another group of mice was exposed to heat stress by placing the dams in an environmental room at an ambient temperature of 38 °C and relative humidity at 62% for 3 h on day 2 of pregnancy. All groups were euthanized on day 4 of pregnancy and embryos were recovered by flushing excised uterine horns. Embryos were examined for abnormalities and classified by the developmental stages. They were then treated with hypotonic solution and dissociated for counting blastomeres. Heat stress caused stunted development of embryos, but no remarkable effect of microwave radiation could be found on the development of preimplantation embryos.     

Effect of the sex-linked dwarf gene on thyrotrophic and somatotrophic axes in the chick embryo

Plasma concentrations of thyroxine (T4), triiodothyronine (T3), reversed triiodothyronine (rT3), and insulin-like growth factors I and II (IGF-I, IGF-II) together with peripheral 5'-monodeiodination activity were measured in both normal and sex-linked dwarf embryos between day 14 of incubation and day 1 posthatch. Plasma T4 levels increased gradually during embryonic development while T3 concentrations remained low until day 20, when a sharp increase was observed. rT3 levels also increased from day 14 and dropped on day 20 when T3 levels started to increase. 5'-monodeiodination activity was high on day 14 of incubation, decreased thereafter, and showed an increase at the time of air sac penetration together with increased T3 levels. At this stage, differences between normal and dwarf embryos were observed; the latter had lower nonsignificant 5'-Monodeiodination activity and lower (P less than 0.01) plasma T3 levels. Plasma IGF-II levels were high during the whole embryonic period studied. Dwarf embryos had lower (P less than 0.05) IGF-II levels at the time of hatching. IGF-I levels were high on days 14 and 16, declined afterwards, and started to increase again around hatching. With the exception of T3 and IGF-II levels, introduction of the dwarf gene did not cause major changes in the hormonal parameters studied. This may explain the identical body weight at hatching.     

Cross-correlation as a method for discriminating impulses on multineuronal recordings

Impulses series, singled out from multineuronal activity by means of amplitude discriminator, were analyzed by creating crosscorrelation histograms. The authors consider that in studying of net brain activity with crosscorrelation method of analysis "window" discrimination of impulses series in multineuronal record is a quite correct method and may be used along with discrimination of spikes by form. It is shown that the size of the "window", used for singling out of separate impulses series, is not a determining parameter in search of temporal dependence in the activity of the investigated cells, as final discrimination of the impulses of dependently acting neuronal pairs occurs at the level of the crosscorrelation method itself. The step of the analysis under the use of crosscorrelation method is of decisive significance for revealing of direct interconnections between neurones in the cases when these connections are masked on histograms with wide peaks reflecting the influences of common sources, on the recorded cells.     

An analysis of interneuronal connections in the auditory cortex of awake cats]

A statistical analysis has been made of the interaction of the auditory cortex units in alert cats with chronically implanted electrodes. Three neurones with an amplitude ratio of 4:2:1 were singled out from the multineuronal activity. The dependence between the firing of two neurones was determined by the cross interval histograms. The relationships between 78 pairs of units were studied in 26 three units microsystems. About a third of the studied pairs functioned independently. The number of pairs with one-way and two-way connections was about equal (26 and 30 respectively). The neurones which generated spikes of high and medium amplitude, had the largest number of two-way connections. One-way connections were equally represented in all the three neurones, though with regard to direction they depended on the amplitude characteristics of the spikes. In neurones with large and medium spikes, output connections predominated, while in neurones with small spikes input connections predominated considerably. The connection could be of inhibitory, excitatory or mixed type. The inhibitory type of connections was the most frequent occurrence (57 out of 86). At prolonged recording (6 to 16 min) of spike activity, most of the functional connections persisted.     

Variation in development of rat embryos at the presomite period.

Rat embryos at a single gestational time in the presomite period were studied for their variation in development and their fate after culture. They were explanted at 8 A.M. on day 9 of gestation from timed-pregnant Sprague-Dawley rats which were obtained by mating between 8 and 10 A.M. (plug day = day 0). In the first experiment, a total of 203 embryos from 20 litters were examined for their variation in development. Several dimensions of embryo/egg cylinder were measured and development of various embryonic/extraembryonic structures were assessed using a scoring system that we developed for the present study. Embryos were then divided into different stages of development based on their scores using the staging system that we developed previously. A large variation in developmental stage was demonstrated; the youngest embryo was at the early primitive streak stage with no signs of amniotic folds and the oldest one was at the late neural plate stage with a foregut pocket but without visible somites. No strong correlation was demonstrated between developmental stage and size of embryo/egg cylinder, nor between developmental stage and development of the proamniotic tube, ectoplacental cavity, or allantois. In the second experiment, embryos were explanted at the same time and those at different stages were cultured separately in rotating bottles and their outcomes were compared after 49 hours. The difference in mean somites number of embryos cultured from the mid primitive streak and late neural plate stages was 6.1. This difference corresponds to approximately 10 hours based on the known linear increase of somites number on day 11 of approximately 0.6 somites per hour. These results indicate a large variation in development of presomite period embryos supposedly of the same gestational age and suggest the importance of careful staging at the time of explantation if precision is needed for whole embryo culture experiments.     

Contribution of the superior atrioventricular cushion to the left ventricular infundibulum. Experimental study on the chick embryo

The role of the superior atrioventricular cushion in the normal development of the left ventricular infundibulum was experimentally studied in the chick embryo. 178 embryos at stages 19-24 of Hamburger and Hamilton were selectively labeled using gelatin-india ink; afterward embryos were reincubated until the mature heart stage, in which the final location of the labels was determined. In addition, anatomical microscopic studies were carried out on the chick embryo heart at different stages of the development. 91 embryos were obtained at the mature heart stage, 46 of which were normal. In 82,6% of these 46 embryos labels were found in the left ventricular infundibulum and were distributed in the following regions: (1) base of the free portion of the anteroseptal mitral leaflet (mitroaortic continuity); (2) the same region plus the left surface of the anterior basal portion of the ventricular septum, and (3) the left surface of the anterior basal portion of the ventricular septum. Anatomical microscopic studies showed that the superior atrioventricular cushion appears at stage 18, fusing with the inferior cushion at stage 28. Our results permit us to conclude that the superior atrioventricular cushion plays an important role in the normal development of the left ventricular infundibulum, and it contributes in the posterolateral and anteromedial wall formation.     

Multineuronal cortical activity in dogs with a defensive instrumental conditioned reflex

By methods of correlation analysis, interrelations were studied of neurones with high, mean and low amplitudes of spikes singled out by amplitude principle from background multineuronal activity of the motor and somatosensory cortical zones, recorded by chronically implanted semi-microelectrodes in dogs. In trained animals, a high positive correlation was observed of singled out neurones discharges, particularly between the neurones with low and mean amplitude of spikes. By the method of cross-interval histogram, dependent relations were revealed of the neurones with different amplitude characteristic mainly of one-sided excitatory character.     

Correlation between the amplitude of action potentials and the rate of conduction along axons of the output cells of the sensomotor cortex

Antidromic responses of neighbouring neurones in micro-areas of the sensorimotor cortex to the stimulation of fibers of the pyramidal tract as well as of the red nucleus and thalamic nuclei VPL and MGB, were studied in acute experiments on unanesthetized immobilized cats. Depending on the velocity of conduction along the axon, the neurones of all the categories were divided into fast and slow cells. When examining the two neuronal groups most differing in AP amplitude (N1 and N3), it was found that N1 neurones were mainly fast-conducting and N3 neurones-- slow-conducting. The conclusion is made that at multineuronal recording, each of the examined categories of the output neurones is characterized by positive correlation between AP amplitude and the axon conduction velocity and consequently, the size of the cell.     

Development of VIP in the peripheral nervous system of avian embryo

The distribution of the VIP containing structures was studied in the gut and in the paravertebral sympathetic ganglia of the quail and chick embryos by immunocytochemistry. In the gut, development of peptidergic nerves followed a craniocaudal gradient. Immunoreactive fibres were first visible in the oesophagus at day 9 in the quail and day 10 in the chick, at 12 days they extended over the whole length of the gut. Cell bodies were localized at day 9 in the foregut and observed in the mid- and hind-gut just before hatching. Transplantations on the chorioallantoic membrane of fragments of various parts of the digestive tract clearly demonstrated that VIP nerve cell bodies belonged to the intrinsic innervation of the gut. Besides the gut, sympathetic paravertebral ganglia contained cells with VIP immunoreactivity detected at day 9 and 10 in quail and chick respectively. In order to find out whether VIP containing neurons differentiated normally in chick embryos in which quail neural crest cells had been implanted at an early stage of development we looked for the appearance of peptidergic neurones in the following situations: when the quail neural primordium had been grafted orthotopically and isochronically into chick host (1) at the adrenomedullary (somites 18-24) and (2) at the vagal (somites 1-7) levels of the neural axis. In all conditions VIP immunoreactivity was observed in quail cells located either in the sympathetic paravertebral ganglia of the trunk at the level of the graft or in the enteric ganglia according to the graft was made at the adrenomedullary and vagal levels respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transforming growth factor alpha (TGFalpha) modulates the effect of genomic imprinting and prolongs the development of parthenogenetic murine embryos]

The effect of transforming growth factor alpha (TGF alpha) on the development of diploid parthenogenetic mouse embryos (CBA x C57BL/6)F1 was studied. The embryos were in vitro treated with the TGF alpha at the stage of morula. Upon reaching the blastocyst stage, each embryo was implanted into uterus of a pseudopregnant female. At a dose of 5 ng/ml, the TGF alpha was found to improve development of parthenogenetic embryos before implantation, increase significantly the number of developing blastocysts, and promote embryo implantation into uterus. After treatment with TGF alpha at a dose of 10 ng/ml, 4% of parthenogenetic embryos reached the stage of 30-45 somites and had forelimb and hindlimb buds; the embryo size from vertex to sacrum was 2.0 to 3.8 mm. A well-developed placenta was observed in 6% of TGF alpha-treated parthenogenetic embryos that reached the somite stages. In the parthenogenetic embryos with the most prominent development (42-45 somites) treated with 10 ng/ml of TGF alpha, the placental diameter was 4.0 to 4.2 mm on day 12 of gestation, which is close to the placental size of the normal (fertilized) 11-day-old mouse embryos. Our results suggest that endogenous TGF alpha can modulate the effects of genomic imprinting significantly improving formation of trophoblast derivatives and promoting longer postimplantation development of parthenogenetic embryos.     

Axon routing at the optic chiasm after enzymatic removal of chondroitin sulfate in mouse embryos

The effects of removing chondroitin sulfate from chondroitin sulfate proteoglycan molecules on guidance of retinal ganglion cell axons at the optic chiasm were investigated in a brain slice preparation of mouse embryos of embryonic day 13 to 15. Slices were grown for 5 hours and growth of dye-labeled axons was traced through the chiasm. After continuous enzymatic digestion of the chondroitin sulfate proteoglycans with chondroitinase ABC, which removes the glycosaminoglycan chains, navigation of retinal axons was disrupted. At embryonic day 13, before the uncrossed projection forms in normal development, many axons deviated from their normal course, crossing the midline at aberrant positions and invading the ventral diencephalon. In slices from embryonic day 14 embryos, axons that would normally form the uncrossed projection at this stage failed to turn into the ipsilateral optic tract. In embryonic day 15 slices, enzyme treatment caused a reduction of the uncrossed projection that develops at this stage. Growth cones in enzyme-treated slices showed a significant increase in the size both before and after they crossed the midline. This indicates that responses of retinal axons to guidance signals at the chiasm have changed after removal of the chondroitin sulfate epitope. We concluded that the chondroitin sulfate moieties of the proteoglycans are involved in patterning the early phase of axonal growth across the midline and at a later stage controlling the axon divergence at the chiasm.