Post-hatching Day Research Articles

Critical role of the neural pathway from the intermediate medial mesopallium to the intermediate hyperpallium apicale in filial imprinting of domestic chicks (Gallus gallus domesticus)

Filial imprinting in precocial birds is a useful model for studying early learning and cognitive development, as it is characterized by a well-defined sensitive or critical period. We recently showed that the thyroid hormone 3,5,3′-triiodothyronine (T3) determines the onset of the sensitive period. Moreover, exogenous injection of T3 into the intermediate medial mesopallium (IMM) region (analogous to the associative cortex in mammals) enables imprinting even on post-hatch day 4 or 6 when the sensitive period has been terminated. However, the neural mechanisms downstream from T3 action in the IMM region remain elusive. Here, we analyzed the functional involvement of the intermediate hyperpallium apicale (IMHA) in T3 action. Bilateral excitotoxic ablation of the IMHA prevented imprinting in newly hatched chicks, and also suppressed the recovery of the sensitive period by systemic intra-venous or localized intra-IMM injection of T3 in day-4 chicks. In contrast to the effect in the IMM, direct injection of T3 into the IMHA did not enable imprinting in day-4 chicks. Moreover, bilateral ablation of IMHA after imprinting training impaired recall. These results suggest that the IMHA is critical for memory acquisition downstream following T3 action in the IMM and further, that it receives and retains information stored in the IMM for recall. Furthermore, both an avian adeno-associated viral construct containing an anterograde tracer (wheat-germ agglutinin) and a retrograde tracer (cholera toxin subunit B) revealed neural connections from the IMM to the IMHA. Taken together, our findings suggest that hierarchical processes from the primary area (IMM) to the secondary area (IMHA) are required for imprinting.

Ontogeny of the corticotrophin-releasing hormone system in slow- and fast-growing chicks (Gallus gallus)

Selective breeding has caused striking phenotypic differences among chickens. For example, the broiler, a fast-growing phenotype, is a relatively heavy bird selected for meat production while the layer, a slower-growing, lighter bird, was developed for egg production. Broilers are prone to early obesity and show physiological and behavioural differences in response to stressors compared with layers. However, the genetic causes of the differences in the responses to stressors between them have not been determined. The purpose of the present study was to compare the ontogeny of the corticotropin-releasing hormone (CRH) system between broilers and layers, because the CRH system plays a major role in regulating stress. We found that fast-growing broilers showed significantly lower levels of diencephalic CRH mRNA expression at post-hatch days 8 and 15 and pituitary CRH receptor 1 mRNA expression from the embryonic to post-hatch stage than slower-growing layers. However, a significantly higher level of CRH-binding protein (CRH-BP), which inactivates CRH and prevents pituitary–adrenal stimulation, was found in broilers compared with layers. Indeed, broilers showed significantly lower levels of plasma corticosterone (CORT) than layers. Subjecting birds to isolation stress did not alter the CORT level of broilers, but increased that of layers. Collectively, the stress-coping actions of the CRH system via the hypothalamic–pituitary–adrenal (HPA) axis might be less responsive in broilers than in layers due to differential gene expression. Together, the present results provide evidence that genetic selection has altered gene expression in the CRH system of the fast-growing broiler, causing blunted HPA axis activity in response to stressors.

Differential Expression of Cell Cycle Regulators During Hyperplastic and Hypertrophic Growth of Broiler Subcutaneous Adipose Tissue.

Hyperplastic growth and hypertrophic growth within adipose tissue is tightly associated with cell cycle activity. In this study, CCNG2 and CDKN2C were found to be correlated with cell cycle inhibition during fat cell differentiation, whereas CCND3, CCNA1, and ANAPC5 were positively associated with cell cycle activity during fat cell proliferation after selection based on GEO datasets available on the NCBI website. The findings were validated through comparison of expressions of these genes among different tissues/fractions in broiler chickens and time points during primary cell culture using quantitative real-time PCR. Development of broiler subcutaneous adipose tissue was investigated on embryonic days 15 and 17 and on post-hatch days 0, 5, 11, and 33 using H&E staining and PCNA immunostaining with DAPI counter stain. In addition, mRNA expressions of five cell cycle regulators as well as precursor cell and adipocyte markers were measured at those time points. The results suggest that cellular proliferation activity decreased as the fat pad grows, but a population of precursor cells seemed to be maintained until post-hatch day 5 despite increasing differentiation activity. Hypertrophic growth gradually intensified despite a slight cessation on post-hatch day 0 due to increased energy expenditure during hatching and delayed food access. From post-hatch day 5 to day 11, most of the precursor cells may become differentiated. After post-hatch day 11, hyperplastic growth seemed to slow, while hypertrophic growth may become dominant. This study provides further understanding about broiler fat tissue development which is imperative for effective control of fat deposition.

Sexual Differences in Cell Loss during the Post-Hatch Development of Song Control Nuclei in the Bengalese Finch

Birdsongs and the regions of their brain that control song exhibit obvious sexual differences. However, the mechanisms underlying these sexual dimorphisms remain unknown. To address this issue, we first examined apoptotic cells labeled with caspase-3 or TUNEL in Bengalese finch song control nuclei - the robust nucleus of the archopallium (RA), the lateral magnocellular nucleus of the anterior nidopallium (LMAN), the high vocal center (HVC) and Area X from post-hatch day (P) 15 to 120. Next, we investigated the expression dynamics of pro-apoptotic (Bid, Bad and Bax) and anti-apoptotic (Bcl-2 and Bcl-xL) genes in the aforementioned nuclei. Our results revealed that the female RA at P45 exhibited marked cell apoptosis, confirmed by low densities of Bcl-xL and Bcl-2. Both the male and female LMAN exhibited apoptotic peaks at P35 and P45, respectively, and the observed cell loss was more extensive in males. A corresponding sharp decrease in the density of Bcl-2 after P35 was observed in both sexes, and a greater density of Bid was noted at P45 in males. In addition, we observed that RA volume and the total number of BDNF-expressing cells decreased significantly after unilateral lesion of the LMAN or HVC (two areas that innervate the RA) and that greater numbers of RA-projecting cells were immunoreactive for BDNF in the LMAN than in the HVC. We reasoned that a decrease in the amount of BDNF transported via HVC afferent fibers might result in an increase in cell apoptosis in the female RA. Our data indicate that cell apoptosis resulting from different pro- and anti-apoptotic agents is involved in generating the differences between male and female song control nuclei.

Divergent selection for shape of the growth curve in Japanese quail. 8. Effect of long-term selection on embryonic development and growth

1. Changes in embryonic development and growth were analysed in relation to direct changes in postnatal growth and correlated responses in egg parameters using Japanese quail lines selected for more than 30 generations for high (HG) and low (LG) relative gain of body weight (BW) between 11 and 28 d of age, and constant BW at 49 d of age.2. During the first 42 h as well as at the end of incubation, LG embryos were developmentally accelerated in comparison with their HG counterparts. An expected increase of line divergence across generations was observed only in traits analysed at the end of incubation.3. In contrast to early generations, LG embryos continuously exhibited a higher BW than HG embryos and this difference temporarily disappeared only around incubation d 8. Analogous to early generations, the HG compared with LG embryos showed two periods of transient growth retardation compensated subsequently by a higher growth rate (incubation d 3–8 and 8–16).4. More pronounced growth retardation of HG versus LG embryos in late versus early generations corresponded to more distinct decrease of HG versus LG growth rate during the first post-hatch days. Likewise, a disappearance of line BW differences on incubation d 8 characterising the late generations corresponded to the elimination of line differences in adult BW.5. Alterations of growth pattern were associated with changes of egg size. While HG quail maintained a relatively constant adult BW and egg size across generations, the gradually increasing incidence of large eggs in the LG line allowed selection of birds with higher growth potential, which in turn amplified the line differences in the embryonic BW and eliminated the line differences in adult BW. Line differences in egg composition (larger albumen with lower density in LG compared with HG eggs) apparently contributed to the strengthening of line developmental divergence during incubation.6. Transient lack of nutrient supply to HG embryos due to their developmental delay is probably responsible for a higher HG versus LG embryo mortality.

Upregulation of inorganic pyrophosphatase 1 as a JNK phosphatase in hypothyroid embryonic chick cerebellum.

Thyroid hormones play important roles in vertebrate neuronal development and differentiation. In our previous study, we showed that fetal thyroid dysfunction led to impaired social behaviors of hatchlings on post-hatch day 3, as well as to impaired learning and memory determined by the imprinting preference. However, little is known about the mechanisms underlying the direct adverse effects of fetal thyroid dysfunction on neuronal development. We used a chick embryo as a fetal model to investigate the effects of prenatal exposure to antithyroid drugs on neuronal development in the chick cerebellum. Methimazole (MMI) at a dose of 20μmol/egg was administered to eggs on day 14, while the control was given only a vehicle. In order to address the underlying mechanisms of the impaired behavior, proteomic approaches were employed in the chick cerebellum two days after MMI treatment. In this experiment, we found that inorganic pyrophosphatase 1 (PPA1) was upregulated in the chick cerebellum treated with MMI, and we confirmed this upregulation of PPA1 by Western blot analysis as well as by RT-PCR analysis. Concomitant with the upregulation of PPA1, a marked reduction in JNK activity, as well as of phospho-JNK level, was detected in the MMI-treated chick cerebellum. Since PPA1 can dephosphorylate JNK, these results suggest that the upregulation of PPA1 during neuronal development in the hypothyroid chick cerebellum may lead to impaired social behaviors as well as to impaired learning and memory via JNK dephosphorylation and inactivation in the chick cerebellum.

Effects of pre-incubation laser irradiation on hatchability and small intestine enzymes activity in post-hatched broiler chickens

The aim of this study was to investigate effects of short-term laser application on hatchability, died embryo in eggs and effects on post-hatch enzyme activity in different sections of small intestine (1, 10, 30, 50, 70 and 90%) in broiler chickens at 21 and 42 days of age. Two experiments were carried out. In the first experiment, 900 fertilised eggs (Ross 308) were randomly divided into three experimental groups (three replications and 100 eggs per group), which were irradiated with laser (HeliumNeon) at 0, 6 and 10 mW power respectively for 90 s; 12 h prior to incubation. The eggs were randomly incubated at industrial hatcheries. At hatch day, rates of unfertilised eggs, hatched, unhatched, dead unhatched embryos were determined in different phase of incubation (1–6 and 7–18 days). In the second experiment, 234 one-day-old chickens where allocated into three experimental groups based on irradiation laser levels (three replications and 26 birds per group). At post hatch days 21 and 42, six birds were randomly selected from each group, slaughtered and various sections of small intestine (1, 10, 30, 50, 70 and 90%) were sampled to evaluate alkaline phosphates (ALP), leucine aminopeptidase (LAP) and sucrase activities. According to the results, laser irradiation had no significant difference on egg hatchability, unfertilised eggs, unhatched died and died embryos (P>0.05). A significant difference was observed in small intestine LAP and ALP levels at days 21 and 42 (P<0.05). These results suggested that laser irradiation altered small intestine enzyme activity in broilers.

Early-life nutritional stress affects associative learning and spatial memory but not performance on a novel object test

Developmental environments can have long-term effects on cognition. Multiple aspects of cognition may be affected by unfavourable conditions during development if underlying neuronal structures are maturing simultaneously. We investigated the effects of nutritional stress at different stages of development on adult associative learning, spatial memory, and behaviours towards a novel object in a songbird. Zebra finches (Taeniopygia guttata) were raised in consistently high or low food conditions until post-hatch day (PHD) 36 (i.e., nutritional independence), where half of the birds from each condition were switched to the other condition until PHD 61. Subjects were then tested as adults with two associative learning tasks, a hippocampus-dependent spatial memory task, and for responses toward a novel object. Surprisingly, low food treatment before independence facilitated spatial associative learning but impaired an aspect of hippocampus-dependent spatial memory. Responses to a novel object were not altered by treatments. This study demonstrates that developmental stress is able to influence learning without simultaneously affecting a measure of personality.

Hsp90, Hsp60 and HSF-1 genes expression in muscle, heart and brain of thermally manipulated broiler chicken

The effect of thermal manipulation (TM) during embryogenesis (ED 12–18) on mRNA expressions of heat shock proteins (Hsp90, Hsp60 and HSF-1) in muscle, heart and brain tissues during thermal challenge (TC) at post-hatching days 10 and 28 was investigated. Fertile chicken eggs were randomly divided into four groups: Control group (37.8 °C), TM1 (39 °C for 9 h), TM2 (39 °C for 12 h) and TM3 (39 °C for 18 h). At days 10 and 28 of age, chicks in TC groups were subjected to thermal challenge (TC) at 43.0 °C for 6 h while naïve chicks were kept under regular conditions. When compared with the control, TM resulted in a significant increase in mRNA levels of Hsp90, Hsp60 and HSF-1in muscle, heart and brain tissues during embryogenesis and during TC at days 10 and 28 post-hatching. These results indicate a long-term enhancement of Hsp90, Hsp60 and HSF-1 gene expressions associated with improved thermotolerance acquisition in thermally manipulated chicks.

No Trade-Offs between Lipid Stores and Structural Growth in Juvenile Zebra Finches Undergoing Nutritional Stress during Development.

Nutritional conditions during development can affect both structural growth and body fat deposition. Body size and body fat each have significant consequences for fitness, yet few studies have investigated how young birds balance resource allocation between structural growth and fat reserves. We raised zebra finches (Taeniopygia guttata) in consistently high- or low-food conditions until posthatch day 35 (PHD 35). From this age until PHD 62, half of the birds in each condition were switched to the other treatment, while the rest were maintained on the same conditions. Body mass, lean mass, body fat, and tarsus length were measured before (PHD 25) and after (PHD 55) nutritional independence. Precise measures of body composition were obtained noninvasively at both ages using quantitative magnetic resonance analysis. At PHD 25, birds in the high treatment had more body mass and lean mass than birds in the low treatment, but nutritional treatments did not affect body fat at this age. Unexpectedly, the strategic response of birds that experienced deteriorating food availability was to maintain body mass by increasing body fat and decreasing lean mass. Birds that experienced an improvement in food availability significantly increased body mass by increasing lean mass and not body fat. Birds maintained on a low diet throughout did not significantly increase body mass, lean mass, or body fat. Tarsus length was not affected by nutritional manipulations. These findings indicate that nutritional stress did not affect the relationship between skeletal growth and body fat deposition because lean mass, body fat, and tarsus length can be independently regulated at different developmental periods depending on nutritional conditions.

Tracheosyringeal nerve transection in juvenile male zebra finches decreases BDNF in HVC and RA and the projection between them

This study investigated relationships among disruption of normal vocal learning, brain-derived neurotrophic factor (BDNF), and the morphology of song nuclei in juvenile male zebra finches. The tracheosyringeal nerves were bilaterally transected at post-hatching day 20–25, so that the animals could not properly develop species-typical vocalizations. BDNF protein and the projection from HVC to the robust nucleus of the arcopallium (RA) were quantified during the sensorimotor integration phase of song development. The manipulation decreased the number of BDNF cells in HVC and RA, the volume of these areas defined by BDNF labeling, and the projection from HVC to RA. BDNF was not affected in Area X or the lateral magnocellular nucleus of the anterior nidopallium (LMAN). Thus, inhibition of a bird's ability to practice and/or to hear its own typically developing song specifically diminishes BDNF expression in cortical motor regions required for song production.

High resolution 1H NMR-based metabonomic study of the auditory cortex analogue of developing chick (Gallus gallus domesticus) following prenatal chronic loud music and noise exposure

Proper functional development of the auditory cortex (ACx) critically depends on early relevant sensory experiences. Exposure to high intensity noise (industrial/traffic) and music, a current public health concern, may disrupt the proper development of the ACx and associated behavior. The biochemical mechanisms associated with such activity dependent changes during development are poorly understood. Here we report the effects of prenatal chronic (last 10days of incubation), 110dB sound pressure level (SPL) music and noise exposure on metabolic profile of the auditory cortex analogue/field L (AuL) in domestic chicks. Perchloric acid extracts of AuL of post hatch day 1 chicks from control, music and noise groups were subjected to high resolution (700MHz) 1H NMR spectroscopy. Multivariate regression analysis of the concentration data of 18 metabolites revealed a significant class separation between control and loud sound exposed groups, indicating a metabolic perturbation. Comparison of absolute concentration of metabolites showed that overstimulation with loud sound, independent of spectral characteristics (music or noise) led to extensive usage of major energy metabolites, e.g., glucose, β-hydroxybutyrate and ATP. On the other hand, high glutamine levels and sustained levels of neuromodulators and alternate energy sources, e.g., creatine, ascorbate and lactate indicated a systems restorative measure in a condition of neuronal hyperactivity. At the same time, decreased aspartate and taurine levels in the noise group suggested a differential impact of prenatal chronic loud noise over music exposure. Thus prenatal exposure to loud sound especially noise alters the metabolic activity in the AuL which in turn can affect the functional development and later auditory associated behaviour.

Hsp90, hsp60 and hsf‐1 genes expression in muscle, heart and brain of thermal manipulated chicken (539.12)

The aim of this study was to investigate the effect of thermal manipulation (TM during embryonic days ED12 ‐ 18) on the gene expressions of Hsp90 and Hsp60 and HSF‐1 on hatchability, growth performance and thermotolerance acquisition during thermal challenge (TC) in three different thermal manipulated groups and also to investigate their ability to cope with thermal challenged (TC) during post hatching days (10 and 28) by using real time RT‐PCR and ELISA techniques. One thousand five hundred fertile chicken eggs were divided randomly into four groups: control group (37.8°C), TM1 (39°C for 9h), TM2 (39°C for 12h) and TM3 (39°C for 18h). Chicks from each treatment group were then randomly divided into 2 sub‐treatment groups (Naive and TC). Chicks in TC groups were subjected to thermal challenge at 43.0°C for 6h on days 10 and 28 of age while naïve chicks were kept under regular conditions. Chick’s response to TC was evaluated by determination of body temperature, plasma T3 and T4 levels and mRNA levels of Hsp90, Hsp60 &amp; Hsf‐1 of muscle, heart and brain. There was a significant differences in muscle, heart and brain mRNA levels of Hsp90, Hsp60 &amp; Hsf‐1 during embryogenesis and during TC in post‐hatch chicks that indicates tissue responds differently to heat stress. While hatchability was adversely affected, the body weight in TM3 chicks was significantly higher at the end of the study period (35 days). Results of this study indicated a long‐term enhancement of Hsp90, Hsp60 &amp; Hsf‐1 gene expressions associated with thermotolerance acquisition in treated chicks without adversely affecting body parameters.

Prenatal music stimulation facilitates the postnatal functional development of the auditory as well as visual system in chicks (Gallus domesticus)

Rhythmic sound or music is known to improve cognition in animals and humans. We wanted to evaluate the effects of prenatal repetitive music stimulation on the remodelling of the auditory cortex and visual Wulst in chicks. Fertilized eggs (0 day) of white leghorn chicken (Gallus domesticus) during incubation were exposed either to music or no sound from embryonic day 10 until hatching. Auditory and visual perceptual learning and synaptic plasticity, as evident by synaptophysin and PSD-95 expression, were done at posthatch days (PH) 1, 2 and 3. The number of responders was significantly higher in the music stimulated group as compared to controls at PH1 in both auditory and visual preference tests. The stimulated chicks took significantly lesser time to enter and spent more time in the maternal area in both preference tests. A significantly higher expression of synaptophysin and PSD-95 was observed in the stimulated group in comparison to control at PH1-3 both in the auditory cortex and visual Wulst. A significant inter-hemispheric and gender-based difference in expression was also found in all groups. These results suggest facilitation of postnatal perceptual behaviour and synaptic plasticity in both auditory and visual systems following prenatal stimulation with complex rhythmic music.

Effects of nutritional stress during different developmental periods on song and the hypothalamic–pituitary–adrenal axis in zebra finches

In songbirds, developmental stress affects song learning and production. Altered hypothalamic–pituitary–adrenal (HPA) axis function resulting in elevated corticosterone (CORT) may contribute to this effect. We examined whether developmental conditions affected the association between adult song and HPA axis function, and whether nutritional stress before and after nutritional independence has distinct effects on song learning and/or vocal performance. Zebra finches (Taeniopygia guttata) were raised in consistently high (HH) or low (LL) food conditions until post-hatch day (PHD) 62, or were switched from high to low conditions (HL) or vice versa (LH) at PHD 34. Song was recorded in adulthood. We assessed the response of CORT to handling during development and to dexamethasone (DEX) and adrenocorticotropic hormone (ACTH) challenges during adulthood. Song learning and vocal performance were not affected by nutritional stress at either developmental stage. Nutritional stress elevated baseline CORT during development. Nutritional stress also increased rate of CORT secretion in birds that experienced stress only in the juvenile phase (HL group). Birds in the LL group had lower CORT levels after injection of ACTH compared to the other groups, however there was no effect of nutritional stress on the response to DEX. Thus, our findings indicate that developmental stress can affect HPA function without concurrently affecting song.

Impaired imprinting and social behaviors in chicks exposed to mifepristone, a glucocorticoid receptor antagonist, during the final week of embryogenesis

The effects of glucocorticoid receptor dysfunction during embryogenesis on the imprinting abilities and social behaviors of hatchlings were examined using “fertile hen's egg-embryo-chick” system. Methods and resultsOf embryos treated with mifepristone (0.4μmol/egg) on day 14, over 75% hatched a day later than the controls (day 22) without external anomalies. The mifepristone-treated hatchlings were assayed for imprinting ability on post-hatching day 2 and for social behaviors on day 3. The findings were as follows: imprinting ability (expressed as preference score) was significantly lower in mifepristone-treated hatchlings than in controls (0.65±0.06 vs. 0.92±0.02, P<0.005). Aggregation tests to evaluate the speed (seconds) required for four chicks, individually isolated with cardboard dividers in a box, to form a group after removal of the barriers showed that aggregation was significantly slower in mifepristone-treated hatchlings than in controls (8.7±1.1 vs. 2.6±0.3, P<0.001). In belongingness tests to evaluate the speed (seconds) for a chick isolated at a corner to join a group of three chicks placed at the opposite corner, mifepristone-treated hatchlings took significantly longer than controls (4.5±0.4/40cm vs. 2.4±0.08/40cm, P<0.001). In vocalization tests, using a decibel meter to measure average decibel level/30s (chick vocalization), mifepristone-treated hatchlings had significantly weaker vocalizations than controls (14.2±1.9/30s vs. 26.4±1.3/30s P<0.001). In conclusion, glucocorticoid receptor dysfunction during the last week embryogenesis altered the programming of brain development, resulting in impaired behavioral activities in late life.

Sequential accumulation of iron in glial cells during chicken cerebellar development

Iron is an essential, but potentially harmful, metal in the brain. In normal brain, iron has been reported to accumulate mainly in glial cells and occasionally in neurons in some particular nuclei. However, the majority of investigations have targeted the adult brain. Here, we investigated spatiotemporal localization of iron in developing and adult chicken cerebellum using iron histochemistry. Iron reactivity was not detected in the chick cerebellum until embryonic day 12. Iron accumulation was first found in mature myelinating oligodendrocytes located in the inner part of the cerebellar folium at embryonic day 14. From embryonic day 20, iron-positive mature myelinating oligodendrocytes were localized in the white matter and the granular layer. From post-hatching day 2, iron accumulation was observed in Bergmann glia in the Purkinje cell layer as well as in mature myelinating oligodendrocytes. Iron accumulation in microglia was observed in the granular and molecular layers at post-hatching month 12. Our data indicate that during cerebellar development iron is accumulated in a unique sequence according to individual requirements or microenvironmental demands.

Ouabain inhibition of Na/K-ATPase across the retina prevents signed refractive compensation to lens-induced defocus, but not default ocular growth in young chicks

Purpose: The relevance of retinal integrity and energy pathways to ocular growth and induction of refractive errors has seldom been investigated. Thus, we used ouabain to target the channels that are essential for the maintenance of membrane potentials in cells, sodium potassium ATPase (Na/K-ATPase), to examine refractive compensation and ocular growth in response to lens-induced defocus in the chick. Methods: A single intravitreal injection of 1 mM ouabain in dimethyl sulfoxide (DMSO) carrier or DMSO alone was followed by monocular defocus with positive or negative 10 D lens (or no lens) from post-hatching days 5-9 under 12/12 hr light/dark conditions. Biometry and dark-adapted flash and electroretinography (ERG) were conducted on day 9, followed by immunohistological analyses. Results: Ouabain inhibited differential ocular growth and refractive compensation to signed defocus compared to DMSO. By 4-days post-ouabain injection all components of the typical ERG responses to light had been eliminated, and widespread histological damage was apparent, though some ‘default state’ ocular growth was measurable. Immunohistochemistry demonstrated reduction in the specialized water channel Aquaporin 4 (AQP4) expression and increased evidence of caspase 3 expression (a cell death associated protein) in ouabain-treated eyes compared with DMSO alone. Conclusion: The current study demonstrates that blockade of photoreceptor and inner retinal responses to light onset and offset by ouabain inhibits differential refractive compensation to optical blur, but does not prevent ocular growth.

GABAergic Agents Modify the Response of Chick Scleral Fibroblasts to Myopic and Hyperopic Eye Cup Tissues

Purpose: GABA antagonists inhibit experimental myopia in chick and GABA receptors have been localized to chick sclera and the retinal pigment epithelium (RPE). The RPE and the choroid alter scleral DNA and glycosaminoglycan (GAG) content in vitro; opposite effects have been observed for tissues from myopic and hyperopic eyes. The aim was to determine the effect of GABAergic agents on the DNA and GAG content of chick scleral fibroblasts directly and in co-culture with ocular tissues from myopic and hyperopic chick eyes.Materials and Methods: Primary cultures of fibroblastic cells expressing vimentin and α-smooth muscle actin were established. GABAergic agents were added separately (i) to the culture medium of the scleral cells and (ii) to the culture medium of the scleral cells with the addition of posterior eye cup tissue (retina, RPE, retina + RPE, choroid + RPE) to cell culture inserts. Ocular tissues were obtained from chick eyes wearing + 15D (lens-induced hyperopia, LIH) or −15D lenses (lens-induced myopia, LIM) for three days (post-hatch day 5–8) (n = 12). GAG and DNA content of scleral fibroblasts were measured.Results: GABA agents had a small direct effect on scleral cell GAG and DNA content but a larger effect was measured when GABA agents were added to the culture medium with myopic and hyperopic RPE and choroid + RPE tissues. GABA agonists increased (p = 0.002) whereas antagonists decreased (p = 0.0004) DNA content of scleral cells; effects were opposite for scleral GAG content. GABA agents significantly altered the effect of both LIM and LIH tissues (p = 0.0005) compared to control; the effects were greater for LIM tissue versus LIH tissue co-culture (p = 0.0004).Conclusion: GABAergic agents affect the DNA and GAG content of scleral fibroblasts both directly and when co-cultured with ocular tissues. GABA antagonists that prevent myopia development in chick model could act via a scleral mechanism utilizing the RPE/choroid.

Juvenile Nutritional Stress Affects Growth Rate, Adult Organ Mass, and Innate Immune Function in Zebra Finches (Taeniopygia guttata)

Developmental conditions may influence many aspects of adult phenotype, including growth and immune function. Whether poor developmental environments impair both growth and immune function or induce a trade-off between the two processes is inconclusive, and the impact of the timing of stress in determining this relationship has so far been overlooked. We tested the hypothesis that the long-term effects of nutritional stress on growth, body composition, and immune function in zebra finches (Taeniopygia guttata) are different depending on whether stress is experienced during an early or a juvenile phase (i.e., before or after nutritional independence, respectively). We raised birds on high (H) or low (L) food conditions until posthatch day (PHD) 35 and switched treatments for half of the birds in each of the H and L groups from PHD 36 to 61. We found that unfavorable juvenile conditions (PHD 36-61) increased somatic growth rates and liver mass, body fat, and some aspects of immune function. We also observed a positive relationship between growth and immune function, as individuals that grew faster as juveniles also had better innate immune responses as adults. There was no effect of treatment on basal metabolic rate. These findings demonstrate the importance of juvenile developmental conditions in shaping multiple aspects of the adult phenotype.