Avian spinal cord chimeras. Further studies on the neurological syndrome affecting the chimeras after birth

These experiments bring new information concerning the immunological status after birth of quail → chick spinal cord chimeras. Such birds have been produced using recipient flocks of chickens different from those in our previous experiments. The breakdown of tolerance after hatching has been recorded and found to vary with the origin of the embryos. Chickens of broiler JA 657 strain and of a white leghorn strain raised in Japan started to exhibit signs of neural graft rejection later in life than the white leghorn chickens from a French breeder used in our previous studies. As previously described, in two animals, long-term tolerance was observed only for allogeneic chick → chick neural tube grafts. In one chimera the neurological syndrome resulting from rejection was reversible, and no signs of immune attack of the grafted central nervous tissue could be detected at sacrifice. This and other observations reported in this article strongly support the contention that the host immune response to foreign neural tissues starts in peripheral nerves and ganglia where no blood-brain barrier exists rather than in the spinal cord. A humoral response of the host against non-polymorphic quail antigens present on fibroblasts was observed in all birds at the time of rejection.     

Development of the glycogen body of the Japanese quail, Coturnix japonica: II. Observations of electron microscopy

Transmission electron microscopic observations of the relationships of the cells of the glycogen body and those of nervous tissue in the lumbosacral spinal cord show that one day after hatching, glycogen cells at the lateral margins of the glycogen body lie in close association with elements of the neuropil in the adjacent spinal cord. Glycogen cells and their processes appear to extend into the neuropil, where they contact other glycogen cells, blood vessels, neurons, and neuroglia. Junctional complexes and synapses occur among glycogen cells, astrocytes, and oligodendrocytes. Other indications of specialized activities were surmised by the presence of annulate lamellae in continuity with extensive arrays of smooth endoplasmic reticulum in several glycogen cells. These observations enhance our earlier views that cells of the avian glycogen body are metabolically active in the synthesis and degradation of glycogen for neuronal support and myelination in the central nervous system.     

Postnatal development of a demyelinating disease in avian spinal cord chimeras

Xenogeneic spinal cord chimeras were constructed by grafting fragments of quail neural primordium into chick embryos at 2 days of incubation. Hatched birds displayed normal motor behavior for about 5 to 7 weeks, whereupon they developed a neurological syndrome; in the grafted spinal cord the pathological signs of the disease were very similar to those of the active plaques of multiple sclerosis and of the lesions of experimental allergic encephalomyelitis and neuritis, including Ia expression by brain capillary endothelia, rupture of the blood-brain barrier, leukocytic infiltration in the nervous tissue, and demyelination. In the animals at the most advanced stage of the disease an autoimmune attack occurred on the host's nervous system with the same histopathological signs.     

A monoclonal antibody recognizing a common antigen on neurons and fibroblasts in chicken and quail

A monoclonal antibody, FiN1, obtained by immunization of a mouse with homogenates of embryonic quail nodose ganglia, was found to react with a surface antigenic determinant, both in quail and chick, present on practically all neurons of the spinal cord and of the peripheral nervous system and on a subpopulation of fibroblasts. An ontogenetic study performed on tissue sections, cell suspensions and cultures showed that FiN1 defines a differentiation marker which appears relatively late in development, during the second half of embryonic life, and persists after hatching. The onset and evolution of its expression during development varies in a tissue-specific manner.     

Changes in metabolite concentration in the plasma, liver and muscle of feed restricted Japanese quail exposed to cold.

Quail fed ad libitum and 50% ad libitum were cold exposed for several weeks, during time control quail remained at 21 degrees C. The concentration of plasma glucose, FFA, and uric acid, tissue glycogen and carcass fat content was measured at the end of the cold exposure period. Quail fed ad libitum showed no significant change in the levels of plasma and tissue metabolites, or the carcass fat content, following cold exposure. The feed consumption by the cold exposed quail increased, and the mean body weight showed little variation from that of the controls. Feed restricted quail which were cold exposed lost significantly more weight, and had a lower ranked fat content than their controls. Whereas feed restriction caused a lowering of the liver glycogen concentration in both treatment groups, muscle glycogen levels were higher than in quail fed ad libitum. However, cold exposure was not accompanied by a change in muscle and liver glycogen levels in feed restricted quail. Feed restricted quail at 21 degrees C were hypoglycaemic and hyperlipaemic compared to quail fed ad libitum, but cold exposed feed restricted quail had a much higher plasma glucose concentration than the controls. The ranked carcass fat content was inversely related to plasma FFA level in both control and cold exposed feed restricted quail. It is suggested that both a glycolytic and lipid mobilizing response to cold is obtained in quail whose body reserves are not spared from catabolism by adequate dietary nutrient absorption, and the possibility of gluconeogenesis from precursors produced by proteolysis is indicated.     

The existence of a glycogenic body in the spinal cord of a cyprinodontiform teleost,Jenynsia lineata (Jenyns 1842)

Summary A distinct spinal cord structure is described in the teleostean fish J. lineata wich occupies the medial dorsal aspect of the cord, and extends in depth from the dorsal surface to the tectum of the central canal. This structure is characterized by the presence of large quantities of glycogen; this feature makes it similar in certain aspects to the glycogenic body of birds. The name of glycogenic body has been proposed for this structure.     

Development of spinal cord bioelectric activity in spinal chick embryos and its behavioral implications

Embryonic behavior of the chick is the product of spontaneous multiunit burst discharges within the ventral spinal cord. The present study describes the ontogeny of spinal cord burst discharges in embryos which were deprived of brain input by removing several neural tube segments of 2-day embryos at cervical or mid-thoracic levels. Characteristics of bioelectric activity present in both intact and chronically transected cords are: (a) the appearance of spike discharges; (b) the organization of unit discharges into synchronized multiunit bursts; (c) the establishment of intracord synchronization of burst discharges over wide expanses of cord tissue; (d) an increase in burst duration and complexity at 7 days due to the appearance of the burst afterdischarge; (e) an increase in the amount of burst activity from 6 to 13 days followed by a decline until hatching at 21 days; (f) a shift from periodic to irregular patterns of burst activity at 13 days; and (g) the existence of the cord burst discharge as a correlate of embryonic movement. Several differences were found between burst activity from chronic spinal and intact embryos: (a) cervical spinal embryos were significantly less active than controls from 15 through 19 days; and (b) long sequences of unusual repetitive burst afterdischarges appeared in chronic spinal embryos by 13 days. The results indicate that the transected embryonic spinal cord is remarkably autogenous in function, although patterns of activity unique to the transected cord appear and increase in prominence during later stages of incubation.     

The blood-brain barrier of the chick glycogen body (corpus gelatinosum) and its functional implications

Among recent vertebrates only birds possess a glycogen body (corpus gelatinosum), located in the rhomboidal sinus of the lumbosacral region of the spinal cord and separated from the neural tissue proper. Because of the specific topographical situation of this circumventricular organ, the structure of its vascular system is of special interest with respect to the still unsolved functional problems. The existence of a blood-brain barrier is demonstrated by the exclusion of intravascularly injected tracer (horseradish peroxidase), and immunocytochemical demonstration of glucose transporter-1 as a functional marker and of neurothelin, occludin and ZO-1 as structural markers. Alkaline phosphatase and gamma-glutamyltransferase activities, two enzyme reactions frequently used for demonstration of an established blood-brain barrier in vitro, were localized histochemically on the plasmalemma of glycogen body cells and were absent from the endothelium. In addition, local enlargements of the intercellular space were observed by transmission and scanning electron microscopy. In accordance with the concept of a third circulation the cerebrospinal fluid may be the vehicle for distributing substances originating in the glycogen body to the CNS, while the vascular endothelium maintains the internal milieu by virtue of its dynamic barrier functions.     

The craniocaudal extent of the glycogen body in the domestic chicken

All birds have a glial enclosed, glycogen-containing structure in the lumbosacral region of their spinal cords. Recently, a dorsal, central, glycogen-rich area surrounding the central canal in the brachial spinal cord was described in domestic chickens. In order to topographically delineate and histochemically describe this structure, fresh, frozen serial sections of chicken brains and spinal cords were processed for glycogen content, phosphorylase, succinic dehydrogenase and cholinesterase activities. The glycogen-rich area surrounding the central canal in the lumbosacral region is found at all levels of the spinal cord and lower medulla. In the upper medulla, it is located in the midline floor immediately ventral to the ependyma. It persists in this position until the level of the oculomotor complex where it ends. Phosphorylase positive regions closely parallel the glycogen distribution. No succinic dehydrogenase or cholinesterase activities are found in these areas.     

Post-hatching thyroid development and body growth in precocial vs altricial birds

Thyroid growth, thyroid function and body growth are markedly different in developing precocial Japanese quail and altricial Ring doves despite comparability in incubation period, hatchling size, adult body weight and adult serum thyroid hormone concentrations. In quail thyroid activity is high during the perinatal period, declines shortly after hatching, then gradually attains adult function. In contrast, in doves, there is no perinatal peak of thyroid activity. Thyroid function is low at hatching and increases steadily during the first week. Serum hormone concentrations vary around a mean similar to that of adults during the remainder of the nestling and fledgling periods.     

Body mass regulation during incubation in female common eiders Somateria mollissima

We investigated changes in incubation behaviour induced by body fuel depletion in incubating female common eiders, which, in contrast to pelagic seabirds, fast despite being close to marine food sources. In the Svalbard Archipelago, electronic scales were placed under eider nests and the incubation of six birds was prolonged by using wax-filled eggs. Based on changes in the rate of body mass loss in normally incubating females and in ten captive birds that did not incubate, body reserves neared depletion on average four days after hatching. During prolonged incubation, females took more frequent and longer recesses. Nest attentiveness consequently decreased, but was still high. In contrast to recesses during normal incubation, during which body mass of the birds decreased, mass remained constant during the recesses of prolonged incubation. The body stores of female eiders seemed to enable them to complete incubation with a limited safety margin. A further drop in body mass is avoided when a critical body mass is reached, because birds then start feeding enough to maintain mass while continuing incubation. Presumably, a similar mechanism will enable eiders to continue incubation when body reserves are prematurely depleted before hatching.     

A brachial glycogen body in the spinal cord of the domestic chicken.

When cervical segments 14 to 15 of the chicken spinal cord are cut transversely and studied by routine histological and histochemical methods, an onion-shaped region, filled with thread-like fibers, is seen to surround the ependymal cells of the central canal and to be bounded laterally by the neural elements of the spinal gray matter. This area is negative for succinic dehydrogenase, beta-hydroxybutyrate dehydrogenase and cholinesterase activity, but very strongly periodic acid-Schiff positive. Diastase controls show the positive material to be glycogen. Parasagittal sections through this cervical region and into the upper thoracic cord, show the glycogen-rich region to extend longitudinally throughout the region. Because of its location and histochemical characterization, which are similar to that of the ventral portion of the glycogen body, the term brachial glycogen body is proposed for this structure.     

Activation of the serotonergic system in chick spinal cord during hatching.

The objectives of the present study were to determine the levels of serotonin (5-HT), its major catabolic metabolite, 5-hydroxyindoleacetic acid (5-HIAA), and norepinephrine (NE) in chick spinal cord before, during, and after hatching and also to determine if changes in the levels of these chemicals are directly related to the hatching behavior. The levels of 5-HT, 5-HIAA, and NE were measured by high performance liquid chromatography with electrochemical detection in whole spinal cords of 20-day-old "pre-hatching" embryos, 21-day-old "normal hatching" embryos, 0-day-old "post-hatching" chicks, and 0-day-old "glass egg hatching" chicks. NE was measured but no significant differences were found in NE levels among experimental groups. The concentration of 5-HT was elevated in chick embryo spinal cords during normal hatching compared to pre-hatching embryos and post-hatching chicks. The concentration of 5-HIAA was elevated during and after normal hatching compared to pre-hatching embryos. However, neither 5-HT nor 5-HIAA levels were found to be elevated in chick spinal cords during glass egg hatching compared to pre-hatching embryos or post-hatching chicks. Therefore, there appears to be an activation of the serotonergic system in chick spinal cord related to the specific event of hatching but this activation is not directly related to the movements common to both hatching and glass egg hatching.     

Pathfinding during spinal tract formation in the chick-quail chimera analysed by species-specific monoclonal antibodies.

In order to analyse the spinal tract formation at early stages of development in avian embryos, chick-quail spinal cord chimeras were prepared and species-specific monoclonal antibodies (MAb) were developed. MAbs CN, QN and CQN uniquely stained chick, quail, and both chick and quail nervous tissues, respectively. All three antibodies appeared to bind to the same membrane molecule, but to different epitopes. Cord reversal revealed the features of axonal growth of both cord interneurons and dorsal root ganglion cells. Quail cord interneurons grew along an originally ventral marginal layer in the quail cord transplanted in a reversed position, then turned toward the ventral side at the boundary between the graft and the host, and grew along the host chick ventral marginal layer. Central axons of dorsal root ganglia were restricted to the ventrolateral region of the cord which originally formed the dorsal funiculus. These results suggest that cord interneurons and dorsal root ganglion cells actively select to grow along specific regions of the cord and that spinal tract formation appears to be determined by cord cells, and not by sclerotome cells.     

2',3'-Cyclic nucleotide 3'-phosphohydrolase in the developing chick brain and spinal cord

Developmental changes of the 2',3'-cyclic nucleotide 3'-phosphohydrolase activity in the chick brain and spinal cord are reported. The greater part of increase in enzyme activity occurred between 18 days of incubation and 3 days after hatching in the whole brain, and between 18 and 21 days of incubation in the spinal cord. These periods are those of active myelination in the chick brain and spinal cord, respectively. The possibility was emphasized that 2',3'-cyclic nucleotide 3'-phosphohydrolase can be used as a marker for the myelin sheaths in the developing central nervous system. Comparisons were also made among the developmental changes in the forebrain, midbrain, brain stem, cerebellum, and spinal cord.     

Vulnerability of myelin development of the chick to the herbicide 2,4-dichlorophenoxyacetic butyl ester

Fertilized hens' eggs were treated externally with 2,4-Dichlorophenoxyacetic butyl ester (2,4-D b.e.) (3.1 mg/egg) immediately before starting incubation, and after different times of incubation (5, 10 and 15 days). Controls were treated externally with ether. Hatchability studies demonstrated that fetotoxic effects of 2,4-D b. e. were similar on the 0, 5 and 10 incubation day, but the 15 Day Group improved the hatching percentage. One day after hatching, chicks were decapitated, and CNS tissue was dissected. Myelin markers, as cerebrosides and CNP, were determined in cerebrum, cerebellum, brain stem and spinal cord of the four groups. They were reduced in cerebrum and brain stem of the 0, 5 and 10 Day Groups, but in the 15 Day Group they were in normal levels. Cerebellum presented normal myelin marker contents in each group studied, while spinal cord only presented decreased marker contents in the 5 Day Group. UDP galactose-ceramide galactosyl transferase (EC 2.4.1.45) activity was reduced in whole brain of chicks born from eggs treated preincubation. The results show the importance of time drug application and suggest that the vulnerable period in CNS development includes proliferation and development of myelin forming cells. Among CNS regions, cerebrum and brain stem seem to be the most vulnerable to the toxic action of 2,4-D b.e. in the chick.     

Effects of 2 G hypergravity exposure on Bobwhite (Colinus virginianus) and Japanese quail (Coturnix coturnix japonica)

We compared reproductive fitness and early postnatal growth of Bobwhite (Colinus virginianus) and Japanese (Coturnix coturnix japonica) quail incubated and hatched during 2 G centrifugation. Fertilized Bobwhite and Japanese quail eggs were placed in portable incubators on the 8-ft International Space Station Test Bed (ISSTB) Centrifuge at NASA Ames Research Center. The quail eggs were incubated throughout hatching and reared until Postnatal day (P)4 at either 1.0, 1.2 or 2.0 G. Two days before hatching, candling revealed significantly greater numbers of viable Bobwhite than Japanese quail eggs at all g-loads. Bobwhite quail exhibited significantly better hatching success at all g-loads than did Japanese quail. Bobwhite hatchlings were sensitive to gravitational loading as evidenced by reduced postnatal body mass and length of 2 G hatchlings relative to 1 G control hatchlings. In contrast, mass and length of Japanese quail hatchlings were unaffected by 1.2 or 2 G exposure. Together, our findings provide evidence for superior viability and hatching success in Bobwhite quail relative to Japanese quail, coupled with greater sensitivity of postnatal body growth and development to 2 G loading. Bobwhite quail may be better suited than Japanese quail for scientific studies on space biology platforms.     

Avian spinal cord chimeras. I. Hatching ability and posthatching survival in homo- and heterospecific chimeras

Quail-chick spinal cord chimeras were constructed by grafting isotopically, at the brachial level, the neural tube of a quail embryo into a chick of the same developmental stage. The chimeras were allowed to hatch and their behavior and survival after birth were observed. We found that if white Leghorns of the rapid-feathering strain were taken as hosts, the ability of the operated embryos to hatch was higher than in the slow-feathering wild-type chickens. The important point arising from this study is that the establishment of the neuronal circuits and of the connexions of the grafted neurons to their peripheral and central targets occurs between cells of two different species in such a way that normal behavior of the chimera is ensured. These animals can stand, walk, and fly as normal chickens do. Moreover, the size reached by the fragment of quail spinal cord implanted into the chick axial structures is larger than it would have been in the donor at the same age. This results in perfectly normal morphogenesis of the vertebrae which develop from the chick somites at the level of the graft. The pigment pattern of the chick feathers colonized by quail melanoblasts of graft origin is very close to that of the quail, albeit somewhat different, probably due to the different size of the feathers in the two species. Normality of the chimeras is only transient. During the second month of their life they develop a neurological syndrome characterized first by the paralysis of the wings and later by their inability to stand. In strong contrast, spinal cord chimeras constructed between two histoincompatible chickens, remain healthy and seem to develop a complete tolerance to the graft. What seems to be the development of an immune rejection of the grafted neural tube in the quail-chick spinal cord chimeras is now under investigation.     

Culture system for embryos of blue-breasted quail from the blastoderm stage to hatching.

The blue-breasted quail (Coturnix chinensis), the smallest species in the order Galliforms, is a candidate model animal for avian developmental engineering because it is precocious and prolific. This species requires 17 days to hatch and 8 to 9 weeks to mature to an adult body weight of about 50 g, whereas the Japanese quail (Coturnix japonica) requires 16 days to hatch and 6 to 8 weeks to mature to an adult body weight of 100 to 150 g. The early embryo is the most challenging embryonic stage in terms of culture and manipulation for avian biotechnology. We have evaluated various conditions for the culture of blue-breasted quail embryos from the blastoderm stage to hatching. A hatchability rate of 26% (10/39) is among the best of the various culture conditions examined in the present study and the embryo culture system should facilitate advances in avian biotechnology.