Post-hatching Day Research Articles

Sexually dimorphic expression of the genes encoding ribosomal proteins L17 and L37 in the song control nuclei of juvenile zebra finches

Studies evaluating the role of steroid hormones in sexual differentiation of the zebra finch song system have produced complicated and at times paradoxical results, and indicate that additional factors may be critical. Therefore, in a previous study we initiated a screen for differential gene expression in the telencephalon of developing male and female zebra finches. The use of cDNA microarrays and real-time quantitative PCR revealed increased expression of the genes encoding ribosomal proteins L17 and L37 (RPL17 and RPL37) in the male forebrain as a whole. Preliminary in situ hybridization data then indicated enhanced expression of both these genes in song control regions. Two experiments in the present study quantified the mRNA expression. The first utilized 25-day-old male and female zebra finches. The second compared a separate set of juveniles to adults of both sexes to both re-confirm enhanced expression in juvenile males and to determine whether it is limited to developing animals. In Experiment 1, males exhibited increased expression of both RPL17 and RPL37 compared to females in Area X, the robust nucleus of the arcopallium (RA), and the ventral ventricular zone (VVZ), which may provide neurons to Area X. Experiment 2 replicated the sexually dimorphic expression of these genes at post-hatching day 25, and documented that the sex differences are eliminated or greatly reduced in adults. The results are consistent with the idea that these ribosomal proteins may influence sexual differentiation of Area X and RA, potentially regulating the genesis and/or survival of neurons.

Activities of 3β-HSD and aromatase in slices of developing and adult zebra finch brain

Sex steroids influence the development and function of the songbird brain. Developmentally, the neural circuitry underlying song undergoes masculine differentiation under the influence of estradiol. In adults, estradiol stimulates song behavior and the seasonal growth of song control circuits. There is good reason to believe that these neuroactive estrogens are synthesized in the brain. At all ages, estrogens could act at the lateral ventricle, during migration, or where song nuclei exist or will form. We investigated the activity of two critical steroidogenic enzymes, 3β-hydroxysteroid dehydrogenase/isomerase (3β-HSD) and aromatase, using a slice culture system. Sagittal brain slices were collected from juvenile (posthatch day 20) and adult zebra finches containing either the lateral ventricle, where neurons are born, or the telencephalic song nuclei HVC and RA. The slices were incubated with 3H-dehydroepiandrosterone or 3H-androstenedione. Activity was determined by isolating certain products of 3β-HSD (5α-androstanedione, 5β-androstanedione, estrone, and estradiol) and aromatase (estrone and estradiol). Activities of both 3β-HSD and aromatase were detected in all slices and were confirmed using specific enzyme inhibitors. We found no significant difference in activity between adult males and females in either region for either enzyme. Juvenile female slices containing the lateral ventricle, however, showed greater levels of 3β-HSD activity than did similar slices from age-matched males. Determination of the activity of these critical steroidogenic enzymes in slice culture has implications for the role of neurosteroids in brain development.

Dynamic changes of apoptosis and expression of Bcl-2 family members in the posthatch hippocampus of Bengalese finches

The hippocampus of songbirds plays an important role in spatial memory, and probably in song learning. Although prolonged neuronal generation and apoptosis are thought to be closely correlated with memory function, natural changes of the number of neurons and in apoptosis in the hippocampus of songbirds have not been fully investigated during development and in the adult. In the current study, we examined developmental changes in the volume and the number of neurons and apoptotic cells in the hippocampus of songbirds ( Lonchura striata) from posthatch day (P5) to adulthood. Apoptotic cells were determined by Nissl staining and immunohistochemistry for cleaved caspase-3, a key apoptotic caspase executioner. The expression levels of Bcl-2 family member mRNA and protein, including Bcl-2, Bcl-x L and Bax, were also investigated. Our results indicated that: (1) the hippocampus volume significantly increased from P5 to P60, although the number of neurons remained stable in all studied stages; (2) the number of apoptotic cells was highest at P45, based either on the Nissl staining or on the immunohistochemistry for caspase-3; (3) Bcl-2 mRNA expression was high from P5 to adulthood, while Bax mRNA declined abruptly from P5 to adulthood, and Bcl-x mRNA was high after P45. Bcl-2 protein was only detected at P5 and P15, while detection of Bcl-x L and Bax proteins paralleled levels of mRNA expression. Our study provides detailed changes of apoptosis in the posthatch songbird hippocampus, suggesting an important role for caspase-3 and Bcl-2 family members in hippocampus apoptosis.

The distribution of expression of doublecortin (DCX) mRNA and protein in the zebra finch brain

Using in situ hybridization, we measured the distribution of expression of doublecortin (DCX), a microtubule-associated protein, in zebra finch adult and nestling (P9–11) brains. In adult brain, DCX mRNA was detected mainly in the mesopallium (M), medial striatum (MSt), septum, Area X, diencephalon, telencephalic subventricular zone (SVZ), and Purkinje cells in the cerebellum. The expression at posthatch day 9 (P9) was heavy in almost the entire telencephalon and showed heavier expression in SVZ and song regions such as the high vocal center (HVC) and the robust nucleus of arcopallium (RA). Outside of the telencephalon at P9, we found distinct label in nucleus ovoidalis (OV), nucleus spiriformis lateralis (SpL), and nucleus subpretectalis (SP) in the midbrain, almost the entire diencephalon including nucleus dorsomedialis posterior thalami (DMP), stratum griseum et fibrosum superficiale (SGF) in optic tectum, and Purkinje cells in cerebellum. Most of the heavily labeled areas by in situ hybridization overlapped with immunohistochemical staining for DCX, indicating that DCX mRNA is probably translated into protein in those regions. No sex difference was found in DCX expression at P9 or in the adult except that Area X was labeled only in the adult male. The intensity of expression in the adult was significantly lower than that at P9, which suggests a particular role for DCX in early song bird brain development. If DCX is predominantly expressed in migrating neurons, as suggested from studies in mammals, the present results offer no evidence for a sex difference in neuronal migration.

Morphometry of otoliths in chicken macula lagena

The macula lagena located at the apical end of the cochlea in birds is characterized by the presence of numerous otoliths with unclear sensory functions. These otoliths are reported to be similar to those in the vestibular system but their detailed features in morphology are unknown. In the present study, we examined the number, size and shape of otoliths from the macula lagena in Chinese domestic chickens ( Gallus Ling Nan) with a scanning electron microscope for morphometry. For chickens aged 10–15 post-hatch days, the otoliths in each macula lagena were counted to be 16,055 ± 4038 (mean ± S.D., n = 4). The average length and width were 12.98 ± 3.70 μm and 5.10 ± 1.48 μm ( n = 526 otoliths), respectively. The ratio of length to width for the otolith was 2.58 ± 0.39 ( n = 526 otoliths) and remained relatively constant despite their variations in physical size. Almost all the otoliths were in regular shape and appeared like isolated cylinders with smooth facets at each end, but a few of them (0.025% of 64,221 otoliths screened) were found to be in odd shapes, such as T-shape and cross-shape. The results suggest that otoliths in the macula lagena and those in the vestibular system of bird's inner ear have similar physical properties and may play a similar role in sensing gravitational and acceleration signals.

Post-hatching growth and development of the pectoral and pelvic limbs in the black noddy, Anous minutus

The black tern ( Anous minutus) uses a semi-precocial growth strategy. Terrestrial locomotor capacity occurs soon after hatching, but pectoral limb development is delayed and flight is not possible until about post-hatching day 50. A growth series (hatchlings to fledglings) was used to explore how limb musculoskeletal development varied with body mass. In the pelvic limb, bone lengths scaled isometrically or with negative allometry. Gastrocnemius muscle mass and the failure load and stiffness of the tibiotarsus scaled isometrically. In the pectoral limb, pectoralis and supracoracoideus muscle masses increased with strong positive allometry that was mirrored by increases in wing bone strength and stiffness. Bending strength ( σ ult) and modulus ( E) remained fairly constant throughout development to fledging for all limb bones. The moment of inertia ( I) scaled with negative allometry for the tibiotarsus and with strong positive allometry in the wing bones. Differences in σ ult and E of the tibiotarsus between pre-fledged chicks and adults was due, primarily, to increases in bone density rather than increases in the moment of inertia of the skeletal elements, whereas σ ult of wing bones was a function of increases in both bone density and I. Early development of functional pelvic limbs in tree-nesting birds is relatively unusual, and presumably reflects a familial trait that does not appear to compromise breeding success in this species.

The effect of β-bungarotoxin, or geniculate ganglion lesion on taste bud development in the chick embryo

Chick taste bud (gemmal) primordia normally appear on embryonic day (E) 16 and incipient immature, spherical-shaped buds at E17. In ovo injection of beta-bungarotoxin at E12 resulted in a complete absence of taste buds in lower beak and palatal epithelium at developmental ages E17 and E21. However, putative gemmal primordia (solitary clear cells; small, cell groupings) remained, lying adjacent to salivary gland duct openings as seen in normal chick gemmal development. Oral epithelium was immunonegative to neural cell adhesion molecule (NCAM) suggesting gemmal primordia are nerve-independent. Some NCAM immunoreactivity was evident in autonomic ganglion-like cells and nerve fibers in connective tissue. After unilateral geniculate ganglion/otocyst excision on E2.5, at developmental ages E18 and posthatching day 1, approximately 12% of surviving ipsilateral geniculate ganglion cells sustained approximately 54% of the unoperated gemmal counts. After E18, proportional stages of differentiation in surviving developing buds probably reflect their degree of innervation, as well as rate of differentiation. Irrespective of the degree of geniculate ganglion damage, the proportion of surviving buds can be sustained at the same differentiated bud stage as on the unoperated side, or may differentiate to a later bud stage, consistent with the thesis that bud maturation, maintenance, and survival are nerve-dependent.

Early post-hatching sex differences in cell proliferation and survival in the quail telencephalic ventricular zone and intermediate medial mesopallium

Previous studies indicated that avian telencephalic areas related to learned behavior, such as song perception and production, are sexually dimorphic. Our study focused on the eventual occurrence of dimorphism in the intermediate medial mesopallium, an area associated with learning in non-singing birds. During early post-hatching life (days 1 and 5) cell proliferation and survival of newborn cells were studied by means of 5-bromo-2-deoxy-uridine immunocytochemistry. Programmed cell death (apoptosis) was investigated at post-hatching day 10. The ventricular zone, intermediate medial part of mesopallium and lateral septal area was analyzed using stereological methods for cell counts. Short-term experiments revealed significantly higher numbers of newborn cells in male ventricular zone of mesopallium compared to female at post-hatching day 1. Long-term survival until post-hatching day 20 showed significantly higher numbers of labeled cells in the male compared to female intermediate medial part of mesopallium, which is the final destination of migrating cells born in the overlying ventricular zone. The vast majority of these early post-hatching newborn cells residing in the intermediate medial part of mesopallium expressed a neuronal phenotype. In addition to neurogenesis, higher numbers of apoptotic figures were found in the male intermediate medial part of mesopallium at post-hatching day 10, suggesting that cell death plays a role in the control of telencephalic regional cell density in males. Our findings indicate that sex-specific mechanisms possibly stimulate increased cell genesis and survival, as well as the counteracting event of increased apoptotic cell death that characterized the male intermediate medial part of mesopallium.

Pathology of goose haemorrhagic polyomavirus infection in goose embryos

Goose embryos were infected with goose haemorrhagic polyomavirus (GHPV) onto the chorioallantoic membrane (CAM) in order to examine the effect of GHPV on the embryos and to obtain data on whether embryos could develop into infected, virus-shedding goslings, as well as to present an accurate biological method for virus titration. The reported method of infection could offer a possibility to express the virus titre as the median embryo infective dose (EID50). As a special pathological feature of the disease, extensive cerebral haemorrhages were observed, which protruded the skullcap in many cases. Some embryos infected with 101.25 or 100.25 EID50/0.2 ml were able to hatch; however, they were in poor physical condition and died by post-hatching day 4 showing haemorrhagic nephritis and enteritis of geese. Virus shedding was revealed by polymerase chain reaction. The ability of some of the infected goose embryos to hatch may indicate the potency of GHPV to spread vertically, although this needs further study for confirmation. Pathologie du polyomavirus hémorragique de l'oie (GHPV) pour les embryons d'oie Des embryons d'oie ont été infectés par le polyomavirus hémorragique de l'oie (GHPV) au niveau de la membrane chorio-allantoïdienne (CAM) dans le but (i) d'examiner l'effet du GHPV sur les embryons et (ii) d'obtenir des données sur le développement éventuel de l'embryon dans les conditions d'infection, la diffusion du virus par les oisons, ainsi que (iii) de présenter une méthode biologique précise pour le titrage du virus. La méthode d'infection qui est présentée permet d'exprimer le titre du virus en DIE50. Une caractéristique de la pathologie de cette maladie est l'importance des hémorragies cérébrales observées qui, dans de nombreux cas, rendent proéminente la calotte crânienne. Quelques embryons infectés avec 101.25 DIE50/0.2 ml ou 100.25 DIE50/0.2 ml ont pu éclore; toutefois, ils étaient dans de mauvaises conditions physiques et sont morts 4 jours après l'éclosion présentant une néphrite hémorragique entérite de l'oie (HNEG). La diffusion du virus a été mise en évidence par PCR. Le fait que quelques embryons d'oie infectés éclosent permet d'indiquer que le virus peut se transmettre verticalement, mais des études complémentaires sont nécessaires pour le confirmer. Pathologie der Infektion mit hämorrhagischen Gänsepolyomavirus in Gänseembryonen Es wurden Gänseembryonen mit dem hämorrhagischen Gänsepolyomavirus (GHPV) mittels Inokulation auf die Chorioallantoismembran infiziert, um (i) den Effekt von GHPV auf Embryonen zu untersuchen, (ii) Erkenntnisse darüber zu gewinnen, ob sich infizierte Embryonen zu Virus ausscheidenden Gänseküken entwickeln können und (iii) eine exakte biologische Methode für die Virustitration präsentieren zu können. Die beschriebene Methode könnte eine Möglichkeit darstellen, den Virustiter als EID50 zu bestimmen. Als eine spezifische pathologische Veränderung der Erkrankung wurden extensive zerebrale Hämorrhagien, die sich in vielen Fällen bis unter die Schädeldecke ausstreckten, beobachtet. Einige Embryonen, die mit 101,25 oder 100,25 EID50/0,2 ml infiziert worden waren, konnten schlüpfen, aber waren in schlechtem Allgemeinzustand und starben 4 Tage nach dem Schlupf, wobei sie das Krankheitsbild der hämorrhagische Nephritis und Enteritis der Gänse (HNEG) zeigten. Die Virusausscheidung wurde mittels PCR nachgewiesen. Die Schlupffähigkeit einiger infizierter Embryonen weist auf eine mögliche vertikale Ausbreitung von GHPV hin, obwohl weitere Untersuchungen nötig sind, um dies zu bestätigen. Patología causada por la infección por Poliomavirus hemorrágico del ganso (GHPV) en embriones de ganso Se infectaron embriones de ganso con Poliomavirus hemorrágico del ganso (GHPV) en la membrana corioalantoidea (CAM) con la finalidad de (i) examinar el efecto de GHPV en los embriones y (ii) obtener datos sobre la posibilidad de que los embriones infectados puedan nacer y dar lugar a gansos que excretan virus, así como también (iii) presentar un método biológico preciso para titular el virus. El método descrito puede ofrecer la posibilidad de expresar el título vírico como EID50. Se observaron, como lesión especial de la enfermedad, hemorragias cerebrales extensas, que afectaban el cráneo en múltiples casos. Algunos embriones infectados con 101.25 EID50/0.2 ml o 100.25 EID50/0.2 ml fueron capaces de nacer, pero presentaban una pobre condición física y murieron a los 4 días tras el nacimiento (PH) mostrando nefritis y enteritis hemorrágica del ganso (HNEG). La excreción vírica se evaluó mediante PCR. La capacidad de algunos de los embriones infectados para nacer puede indicar la potencia de GHPV para transmitirse verticalmente, pero este hecho requerirá estudios confirmatorios.

Developmental increases in hypothalamic neuropeptide Y content with the embryonic age of meat- and layer-type chicks

We determined central neuropeptide Y (NPY) content of meat- and layer-type chicks at embryonic days 7, 14, 20, and at post-hatching day 1. The central NPY was detectable at day 7; hypothalamic NPY content developmentally increased with a similar pattern but a different level between both types of chicks. These results were discussed with respect to feeding behavior early period after hatching.

In ovo exposure to monochromatic green light promotes skeletal muscle cell proliferation and affects myofiber growth in posthatch chicks

Our previous studies demonstrated that illumination of chicken embryos with monochromatic green light results in enhanced body and muscle weight at later posthatch stages. In the present study, we investigated the cellular and molecular basis of this phenomenon. First, we showed that on day 6 posthatch, myofibers were more uniform in the in ovo illuminated group than in the control group incubated in the dark, with respect to the number of myofibers displaying diameter values within the range of the mean value. Second, we tested the hypothesis that in ovo illumination causes an increase in the number of myoblasts; this in turn can promote posthatch muscle growth. Indeed, a significant increase in the number of skeletal muscle cells isolated from pectoralis muscle was observed in the in ovo illuminated group on days 1 and 3 posthatch relative to the control group. This increased cell number was accompanied by higher expression levels of Pax7 and myogenin proteins on posthatch days 1 and 3, respectively. A parallel analysis of proliferating cells in the intact muscle further demonstrated a significant increase in the number of cells positive for proliferating cell nuclear antigen in muscle from the in ovo illuminated group. Third, we demonstrated that the transition from fetal- to adult-type myoblasts, normally occurring in late stages of chicken embryogenesis, is initiated earlier in embryos subjected to in ovo green-light illumination. We suggest that the stimulatory effect of in ovo illumination on posthatch muscle growth is the result of enhanced proliferation and differentiation of adult myoblasts and myofiber synchronization.

Distribution and onset of retinaldehyde dehydrogenase (zRalDH) expression in zebra finch brain: Lack of sex difference in HVC and RA at early posthatch ages

Using in situ hybridization to detect the expression of the retinoic acid synthesizing enzyme (retinaldehyde dehydrogenase: zRalDH) mRNA, we mapped the distribution of its expression in adult zebra finch brain. In the neural song circuit, strong expression was found in high vocal center (HVC), para-HVC, and at a very low level in the robust nucleus of the arcopallium (RA). The expression in HVC and RA was found in both males and females. Outside of the song system, major areas of expression were in medial nidopallium (N), hyperpallium apicale (HA), mesopallium ventrale (MV), taenial amygdala (TnA), cerebellar Purkinje cells, and nucleus isthmo-opticus (IO). In nestlings, we found zRalDH mRNA expression in HVC and RA as early as posthatch day 4 or 5 (P4-5), although the expression varied among individuals. Thus, retinoic acid synthesis in HVC and RA could participate in song system formation and development. However, we found no sex difference in volume or intensity of zRalDH and androgen receptor (AR) expression in HVC and RA at P11 prior to the development of significant size dimorphisms in these nuclei. The size of HVC in females at P11 defined by zRalDH expression was greater than that in adult females, suggesting that HVC might experience net cell loss between P11 and adulthood.

Development of Defense Mechanisms in the Digestive Tract of the Chick

The intestinal lumen contains food and bacteria in close contact with the mucus layer, which lines the absorptive surface. This matrix contains IgA and other antibacterial substances and acts as a barrier to harmful components and organisms. The underlying epithelial layer is protected by intraepithelial lymphocytes at the junctions between enterocytes and by the action of IgA within the enterocytes. The luminal contents are continuously sampled by M cells and dendritic cells in lymphoid tissue, which then present antigens to the complete array of immune cells present in the lamina propria, which can then mount appropriate defenses.This system is immature at hatch with the intestinal lymphoid tissues developing in the first posthatch days, mainly in the distal intestines. Lymphocyte colonization begins in the first posthatch week, whereas functional T lymphocytes and the presence of IgA secreting cells are only observed in the second posthatch week, and maturation occurs later. Thus, the hatchling appears to be susceptible to pathogens in the immediate posthatch period.

Pax-6 Expression in Posthatch Chick Retina during and Recovery from Form-Deprivation Myopia

The expression of Pax-6 in fully-differentiated chick retina remains largely confined to the amacrine and ganglion cell layers. In the developing posthatch chick retina, Pax-6 expression shows a biphasic pattern; a decrease by posthatch day 17 followed by a steady increase in the adult eye. Interestingly, we find that this biphasic expression of Pax-6 is reflected in the biphasic growth pattern of the posthatch chick eye, which is disrupted by form-deprivation myopia (FDM). We have now examined the pattern of Pax-6 accumulation in 3-day-old chick eyes subjected to 2 weeks of FDM followed by 2 weeks of recovery from FDM. Quantitative RT-PCR (with a homologous internal control) revealed that after 2 weeks of occlusion the contralateral non occluded eyes, the occluded eyes and the normal nonexperimental chick eyes did not show any drastic changes in the number of Pax-6 transcripts. The data obtained suggests that the contralateral eye does not represent a ‘normal’ control eye; similar but nonidentical changes are seen, at the molecular level, in both the contralateral and the occluded eyes. Comparisons with the control nonexperimental animals, however, are meaningful. Even after 2 weeks of recovery under normal light conditions, the occluded eyes do not seem to reach the same level of Pax-6 expression (number of molecules per mg tissue) as seen in normal control eyes, suggesting that exposure of the posthatch chick eye to FDM impedes developmental progression that normally culminates in emmetropia.

Development of the neuromuscular junction in extraocular muscles of white Leghorn chicks

Relatively little is known about the development of the neuromuscular junction of extraocular muscles (EOMs). In recent years, chicks have been increasingly used as a developmental model in ophthalmological research. To utilize this model system for understanding the development and plasticity of the extraocular motor system, we investigated the structural changes that occur at the developing neuromuscular junction in the chick between embryonic day 14 (E14) and posthatch day 2 (P2). Axons and nerve terminals were visualized with fluorescent neurofilament antibodies and motor endplates with rhodamine-conjugated alpha-bungarotoxin. Nerve fibers and endplates were colabeled within the same tissue samples. Motor endplates (density, length, width, and area) were measured and numbers of axons per neuromuscular junction were counted using confocal and conventional microscopy. In P2 chicks, densities of motor endplates were significantly greater in the superior oblique muscle when compared with the superior rectus and lateral gastrocnemius muscle. EOMs showed a two- to threefold larger area of motor endplate size as compared to gastrocnemius muscle. Motor endplate size also differed among EOMs with the superior oblique muscle having endplates with a larger area than those of the superior rectus muscle. The period of synapse elimination was similar between EOM and gastrocnemius muscle. Synapse elimination began at about E18 and was completed by P2. By describing the normal morphological changes in developing EOMs, this study provides a baseline for future work to elucidate underlying molecular mechanisms that regulate EOM innervation and strength.

Dissociation between extension of the sensitive period for avian vocal learning and dendritic spine loss in the song nucleus lMAN

Several instances of early learning coincide with significant rearrangements of neural connections in regions contributing to these behaviors. In fact developmentally restricted learning may be constrained temporally by the opportunity for experience to selectively maintain appropriate synapses amidst the elimination of exuberant connections. Consistent with this notion, during the normal sensitive period for vocal learning in zebra finches ( Taenopygia guttata), there is a decline in the density of dendritic spines within a region essential for song development, the lateral magnocellular nucleus of the anterior nidopallium (lMAN). Moreover, in birds isolated from conspecific song shortly after hatching, both the closure of the sensitive period for vocal learning and the pruning of spines from lMAN neurons is delayed. Here, we employed a more subtle form of deprivation to delay the close of the sensitive period for song learning, and found that late song learning occurred without obvious alterations in the pruning of dendritic spines on lMAN neurons. At posthatch day (PHD) 65 (beyond the end of the normal sensitive period for song memorization in zebra finches), birds isolated from song beginning on PHD30 did not differ from normally reared birds in measures of dendritic spine density on Golgi-Cox stained lMAN neurons. Moreover, tutor exposure from PHD65 to 90 did not increase spine elimination in these isolates (who memorized new song material) relative to controls (who did not). Thus, we conclude that the extent of normally occurring lMAN spine loss is not sufficient to account for the timing of the sensitive period for zebra finch song learning.

Oxygen consumption of the chicken embryo: interaction between temperature and oxygenation

We measured the effects of hypoxia and changes in ambient temperature (T) on the oxygen consumption (VO2) of chicken embryos at embryonic days 11, 16 and 20 (E11, E16 and E20, respectively), and post-hatching day 1 (H1). Between 30 and 39 degrees C, at E11 and E16, VO2 changed linearly with T, as in ectothermic animals, with a Q10 of about 2.1. At E20, VO2 did not significantly change with T, indicating the onset of endothermy. At H1, a drop in T increased VO2, a clear thermogenic response. Hypoxia (11% O2 for 30 min) decreased VO2, by an amount that varied with T and age. At H1, hypoxia lowered VO2 especially at low T. At E20, hypoxic hypometabolism was similar at all T. At E11 and E16, hypoxia lowered VO2 only at the higher T. In fact, at E11, with T=39 degrees C even a modest hypoxia (15-18% O2) decreased VO2. Upon return to normoxia after 40 min of 11% O2, VO2 did not rise above the pre-hypoxic level, indicating that the hypometabolism during hypoxia did not generate an O2 debt. At E11, during modest hypoxia (16% O2) at 36 degrees C, the drop in VO2 was lifted by raising the T to 39 degrees C, suggesting that the hypoxic hypometabolism at 36 degrees C was not due to O2-supply limitation. In conclusion, the hypometabolic effects of hypoxia on the chicken embryo's VO2 depend on the development of the thermogenic ability, occurring predominantly at high T during the early (ectothermic phase) and at low T during the late (endothermic) phase. At E11, both low T and low oxygen force VO2 to drop. However, at a near-normal T, modest hypoxia promotes a hypometabolic response with the characteristics of regulated O2 conformism.

Morphological features of chick retinal ganglion cells

On average, in chicks, the total number of retinal ganglion cells is 4.9 x 10(6) and the cell density is 10400 cells/mm2. Two high-density areas, namely the central area (CA) and the dorsal area (DA), are located in the central and dorsal retinas, respectively, in post-hatching day 8 (P8) chicks (19000 cells/mm2 in the CA; 12800 cells/mm2 in the DA). Thirty percent of total cells in the ganglion cell layer are resistant to axotomy of the optic nerve. The distribution of the axotomy resistant cells shows two high-density areas in the central and dorsal retinas, corresponding to the CA (5800 cells/mm2) and DA (3200 cells/mm2). The number of presumptive ganglion cells in P8 chicks is estimated to be 4 x 10(6) (8600 cells/mm2 on average) and the density is 13500 and 10200 cells/mm2 in the CA and DA, respectively, and 4300 cell/mm2 in the temporal periphery (TP). The somal area of presumptive ganglion cells is small in the CA and DA (mean (+/- SD) 35.7 +/- 9.1 and 40.0 +/- 11.3 microm2, respectively) and their size increases towards the periphery (63.4 +/- 29.7 microm2 in the TP), accompanied by a decrease in cell density. Chick ganglion cells are classified according to dendritic field, somal size and branching density of the dendrites as follows: group Ic, Is, IIc, IIs, Ills, IVc. The density of branching points of dendrites is approximately 10-fold higher in the complex type (c) than in the simple type (s) in each group. The chick inner plexiform layer is divided into eight sublayers according to the dendritic strata of retinal ganglion cells and 26 stratification patterns are discriminated.

Post-hatching syrinx development in the zebra finch: an analysis of androgen receptor, aromatase, estrogen receptor ? and estrogen receptor ? mRNAs

In zebra finches, the vocal organ (syrinx) is larger in males than in females. Specific details about the mechanisms responsible for this dimorphism are not known, but may involve sex differences in steroid hormone action early in post-hatching development. The distribution of androgen receptor (AR), aromatase (AROM), estrogen receptor alpha (ERalpha), and estrogen receptor beta (ERbeta) mRNAs was examined at post-hatching days 3, 10 and 17. A low level of AR was equivalently expressed in the syrinx muscles of both sexes at all three ages. We detected no specific expression of AROM or ERalpha mRNAs. In contrast, ERbeta mRNA was detected in chondrocytes of the forming bone. The density of this expression increased with age as the chondrocytes hypertrophied, but did not differ between the sexes. Taken together, these data suggest that estrogens may act on cartilage/bone, and androgens may act on muscle fibers in early post-hatching development to influence syrinx morphology. However, the lack of a sex difference in steroid receptor mRNA expression in the syrinx suggests that, similar to the forebrain regions that control song, the interaction of androgens and estrogens with their receptors is not sufficient to induce full sexual differentiation of this organ.

Sequential Learning From Multiple Tutors and Serial Retuning of Auditory Neurons in a Brain Area Important to Birdsong Learning

Songbirds hear many vocal models during a juvenile sensitive period, transiently imitating some while retaining imitations of others in their repertoires. Despite subsequent conflicting experiences, early experience can exert lasting effects on neural structure and function, raising the possibility that transiently expressed vocalizations or their relevant models are stored in the adult songbird's brain. One site where learned song representations could be stored is the lateral magnocellular nucleus of the anterior nidopallium (LMAN), which in the adult songbird contains neurons responsive to playback of the bird's own song (BOS) and the tutor song (TS). To test whether LMAN neurons develop and retain responses to transiently learned songs, we exposed zebra finch hatchlings [posthatch day 0 (PHD0)] to a first TS (TS1) for about 30 days, isolated them for about 30 days, then exposed them to a second TS (TS2) for 30 days starting at PHD 60. Behavioral analysis showed that PHD 60 juveniles had started to copy TS1, although this copying was transient, because the adult BOS resembled TS2 and not TS1. We found that LMAN auditory responses paralleled these behavioral changes: LMAN neurons at PHD 60 responded strongly and selectively to both the juvenile BOS and TS1, whereas LMAN neurons in adults responded to the adult BOS and TS2, but not to the transiently learned song or its model. Therefore LMAN auditory responses can be lost or overwritten as the juvenile copies a new song, suggesting that the adult LMAN does not store information about transiently learned songs or their models.