Chick Forebrain Research Articles

Src-NADH dehydrogenase subunit 2 complex and recognition memory of imprinting in domestic chicks.

Src is a non-receptor tyrosine kinase participating in a range of neuronal processes, including synaptic plasticity. We have recently shown that the amounts of total Src and its two phosphorylated forms, at tyrosine-416 (activated) and tyrosine-527 (inhibited), undergoes time-dependent, region-specific learning-related changes in the domestic chick forebrain after visual imprinting. These changes occur in the intermediate medial mesopallium (IMM), a site of memory formation for visual imprinting, but not the posterior pole of the nidopallium (PPN), a control brain region not involved in imprinting. Src interacts with mitochondrial genome-coded NADH dehydrogenase subunit 2 (NADH2), a component of mitochondrial respiratory complex I. This interaction occurs at brain excitatory synapses bearing NMDA glutamate receptors. The involvement of Src-NADH2 complexes in learning and memory is not yet explored. We show for the first time that, independently of changes in total Src or total NADH2, NADH2 bound to Src immunoprecipitated from the P2 plasma membrane-mitochondrial fraction: (i) is increased in a learning-related manner in the left IMM 1 h after the end of training; (ii), is decreased in the right IMM in a learning-related way 24 h after training. These changes occurred in the IMM but not the PPN. They are attributable to learning occurring during training rather than a predisposition to learn. Learning-related changes in Src-bound NADH2 are thus time- and region-dependent.

Development of artificial synapse organizers liganded with a peptide tag for molecularly inducible neuron-microelectrode interface

It has been proposed that cell-type-specific bioelectronic interfaces for neuronal circuits could be established by utilizing the function of synapse organizers. For this purpose, using neurexin-1β and a peptide tag, we engineered compact synapse organizers that do not interact with the naturally occurring receptors but induce presynaptic differentiation upon contact with nanobody-decorated objects in cultured mammalian and chick forebrain neurons. In chick neurons, the engineered organizer exerted synaptogenesis typically in ∼4 h after the contact, even under an air atmosphere at room temperature, thereby providing a useful cellular model for establishing the molecularly inducible neuron-microelectrode interface.

CNKSR2, a downstream mediator of retinoic acid signaling, modulates the Ras/Raf/MEK pathway to regulate patterning and invagination of the chick forebrain roof plate.

During embryonic development, the forebrain roof plate undergoes invagination, leading to separation of the cerebral hemispheres. Any defects in this process, in humans, lead to middle interhemispheric holoprosencephaly (MIH-HPE). In this study, we have identified a previously unreported downstream mediator of retinoic acid (RA) signaling, CNKSR2, which is expressed in the forebrain roof plate in the chick embryo. Knockdown of CNKSR2 affects invagination, cell proliferation and patterning of the roof plate, similar to the phenotypes observed upon inhibition of RA signaling. We further demonstrate that CNKSR2 functions by modulating the Ras/Raf/MEK signaling. This appears to be crucial for patterning of the forebrain roof plate and its subsequent invagination, leading to the formation of the cerebral hemispheres. Thus, a set of novel molecular players have been identified that regulate the morphogenesis of the avian forebrain.

Filial imprinting in domestic chicks; cytoplasmic polyadenylation element binding protein 3, predispositions and learning.

Visual imprinting is a learning process, whereby young animals come to prefer a visual stimulus after exposure to it (training). Available evidence indicates that the intermediate medial mesopallium (IMM) in the domestic chick forebrain is a site of memory formation during visual imprinting. We have found previously that cytoplasmic polyadenylation element binding protein 3 in the P2 plasma membrane-mitochondrial fraction (CPEB3-P2) is upregulated in a learning-dependent way in the left IMM 24 h after training. CPEB3 has two forms, soluble and aggregated. Soluble CPEB3 represses translation; the aggregated form (CPEB3-AF) is amyloid-like and can promote translation. Our previous study did not show which of these two forms is increased after imprinting. We have now resolved this matter by measuring, 24 h after training, CPEB3-P2 and CPEB3-AF in the IMM and a control brain region, the posterior pole of nidopallium (PPN). The methods include imprinting training with a visual stimulus, behavioral measurement of preference, preparation of aggregated CPEB3, western immunoblotting, quantitation of proteins, statistical linear modeling. Only in the left IMM were the level of CPEB3-AF and learning strength correlated, increased CPEB3-AF level reflecting a predisposition to learn readily. CPEB3-P2 level also increased with learning strength in the left IMM, but as a result of learning. No correlations were detected in the right IMM or PPN. We propose two separate systems, both modulating synaptic strength through control of local translation. They are represented by CPEB3-AF (associated with a predisposition to learn) and soluble CPEB3 (associated with learning itself).

Functional development of olfactory nerve‐related neural circuits in the embryonic chick forebrain revealed by voltage‐sensitive dye imaging

Multiple-site optical recordings with NK2761, a voltage-sensitive absorption dye, were applied to the embryonic chick olfactory system, and the functional development of olfactory nerve (N.I)-related neural circuits was examined in the forebrain. The stimulation of the N. I elicited neural responses in N.I-olfactory bulb (OB)-forebrain preparations at the embryonic 8-12 day (E8-E12) stages. At the E11 stage, we functionally identified two circuits projecting from the OB to the forebrain. The first circuit passed through the ventral side of the forebrain and spread in the dorso-caudal direction, whereas the second circuit passed through the dorsal side to the first circuit. Pharmacological experiments showed that N-methyl-D -aspartate (NMDA) receptor function was more significant for the transfer of sensory information in these circuits. The functional development of N.I-related circuits was investigated, and the results obtained revealed that the ventral circuit was generated earlier than the dorsal circuit. Neural responses in the ventral circuit were detected from the E9 stage in normal physiological solution and the E8 stage in Mg2+ -free solution, which activated NMDA receptor function. At the E10 stage, neural responses in the dorsal circuit were clearly recognised in addition to ventral responses. We attempted to identify possible candidates for relay nuclei in the forebrain by comparing contour line maps of the optical signal amplitude with previously reported neuroanatomical data. The results suggest that N.I-related neural circuits from the periphery to the subpallium functionally mature earlier than those to the pallium during ontogenesis.

Suppressive Modulation of the Chick Forebrain Network for Imprinting by Thyroid Hormone: An in Vitro Study.

The thyroid hormone 3,5,3′-triiodothyronine (T3) is considered to act acutely in the chick forebrain because focal infusion of T3 to the intermediate medial mesopallium (IMM) causes 4 to 6-day-old hatchlings to become imprintable approximately 30 min after the infusion. To understand the mechanism of this acute T3 action, we examined synaptic responses of IMM neurons in slice preparations in vitro. Extracellular field potential responses to local electrical stimulation were pharmacologically dissociated to synaptic components mediated by AMPA and NMDA receptors, as well as GABA-A and -B receptors. Bath-applied T3 (20–40 μM) enhanced the positive peak amplitude of the field potential, which represented the GABA-A component. Bicuculline induced spontaneous epileptic bursts by NMDA receptor activation, and subsequent application of T3 suppressed the bursting frequency. Pretreatment of slices with T3 failed to influence the synaptic potentiation caused by tetanic stimulation. Intracellular whole-cell recording using a patch electrode confirmed the T3 actions on the GABA-A and NMDA components. T3 enhanced the GABA-A response and suppressed the NMDA plateau potential without changes in the resting membrane potential or the threshold of action potentials. Contrary to our initial expectation, T3 suppressed the synaptic drives of IMM neurons, and did not influence activity-dependent synaptic potentiation. Imprinting-associated T3 influx may act as an acute suppressor of the IMM network.

Src and Memory: A Study of Filial Imprinting and Predispositions in the Domestic Chick

Visual imprinting is a learning process whereby young animals come to prefer a visual stimulus after exposure to it (training). The available evidence indicates that the intermediate medial mesopallium (IMM) in the domestic chick forebrain is a site of memory formation during visual imprinting. We have studied the role of Src, an important non-receptor tyrosine kinase, in memory formation. Amounts of total Src (Total-Src) and its two phosphorylated forms, tyrosine-416 (activated, 416P-Src) and tyrosine-527 (inhibited, 527P-Src), were measured 1 and 24 h after training in the IMM and in a control brain region, the posterior pole of nidopallium (PPN). One hour after training, in the left IMM, we observed a positive correlation between the amount of 527P-Src and learning strength that was attributable to learning, and there was also a positive correlation between 416P-Src and learning strength that was attributable to a predisposition to learn readily. Twenty-four hours after training, the amount of Total-Src increased with learning strength in both the left and right IMM, and amount of 527P-Src increased with learning strength only in the left IMM; both correlations were attributable to learning. A further, negative, correlation between learning strength and 416P-Src/Total-Src in the left IMM reflected a predisposition to learn. No learning-related changes were found in the PPN control region. We suggest that there are two pools of Src; one of them in an active state and reflecting a predisposition to learn, and the second one in an inhibited condition, which increases as a result of learning. These two pools may represent two or more signaling pathways, namely, one pathway downstream of Src activated by tyrosine-416 phosphorylation and another upstream of Src, keeping the enzyme in an inactivated state via phosphorylation of tyrosine-527.

Physical understanding of axonal growth patterns on grooved substrates: groove ridge crossing versus longitudinal alignment

Surface topographies such as micrometric edges and grooves have been widely used to improve neuron outgrowth. However, finding the mechanism of neuron–surface interactions on grooved substrates remains a challenge. In this work, PC12 cells and chick forebrain neurons (CFNs) were cultured on grooved and smooth polyacrylonitrile substrates. It was found that CFNs showed a tendency of growing across groove ridges; while PC12 cells were only observed to grow in the longitudinal direction of grooves. To further investigate these observations, a 3D physical model of axonal outgrowth was developed. In this model, axon shafts are simulated as elastic 3D beams, accounting for the axon outgrowth as well as the focal contacts between axons and substrates. Moreover, the bending direction of axon tips during groove ridge crossing is governed by the energy minimization principle. Our physical model predicts that axonal groove ridge crossing is contributed by the bending compliance of axons, caused by lower Young’s modulus and smaller diameters. This work will aid the understanding of the mechanisms involved in axonal alignment and elongation of neurons guided by grooved substrates, and the obtained insights can be used to enhance the design of instructive scaffolds for nerve tissue engineering and regeneration applications.

Do chick and rodent neuron biosensors function similarly?

AbstractA growing number of research papers report similarities in cell types, neuronal connections and information‐processing principles between chick and rodent cortical tissues, which have very different architectures. This paper extends these comparisons beyond the cortical tissues. Using microelectrode array technology, we report three remarkable functional similarities between our original chick data and rodent data from the literature: (a) the pattern of spontaneous spiking activity from chick spinal cord neuron biosensors is very similar to that of rodent spinal cord neuron biosensors (i.e. rodent counterparts). (b) The spontaneous spiking activity pattern of the chick forebrain neuron biosensors is very similar to that of the rodent cortical neuron biosensors, but chick forebrain neuron biosensors contain not only cortical neurons, but also diencephalic neurons. In other words, chick forebrain neuron biosensors cannot be considered the counterparts of rodent cortical neuron biosensors. (c) Chick forebrain neuron biosensors respond to several classical neuroactive agents in a way similar to rodent cortical neuron biosensors as reflected in their agent‐specific concentration–response curves and their values of EC50 (the effective concentration that causes 50% of the maximal effect of an agent). These preliminary findings provide both direct and indirect support for a positive answer to the big research question in the title: ‘Do chick and rodent neuron biosensors function similarly’ if the sources of the neurons are homologous between chick and rodent? Our findings are of particular value to comparative biology/physiology, pharmacology, neurotoxicology and bioengineering and to research on the more cost‐effective extended application of chick neuron biosensors.

Chicken cognin interactome and the memory of filial imprinting.

Visual imprinting is a learning process whereby young animals come to prefer a visual stimulus after exposure to it (training). The intermediate medial mesopallium in the domestic chick forebrain is critical for visual imprinting and contributes to molecular regulation of memory formation. Criteria used to infer that a change following training is learning-related have been formulated and published. Cognin (protein disulphide isomerase) is one of several identified plasma membrane and mitochondrial proteins that are upregulated in a learning-related way 24 hours after training. Since virtually nothing is known about the cognin interactome, we have used immunoaffinity chromatography and mass spectrometry to identify proteins that interact with cognin in the cytoplasmic and plasma membrane-mitochondrial fractions. As the learning-related upregulation of cognin has been shown to occur in the plasma membrane-mitochondrial fraction and not in the cytoplasmic fraction, we studied the effect of training on three cognin-interacting partners in the plasma membrane-mitochondrial fraction: the b5 subunit of mitochondrial ATP synthase and the alpha-2 and alpha-3 subunits of sodium-potassium ATPase. Learning-related upregulation was found in the left intermediate medial mesopallium 24 hours after training for the b5 subunit of mitochondrial ATP synthase and the alpha-2 subunit of sodium-potassium ATPase. The hemispheric asymmetry revealed here is consistent with the predominance of many other learning-related effects in the left intermediate medial mesopallium. The alpha-2 subunit of sodium-potassium ATPase is mainly expressed in astrocytes, supporting a role for these glial cells in memory.

In Vitro Differentiation of Chicken Astrocytes: Growth, Morphology, and Protein Expression of Astrocytes in Primary Cultures.

Astrocytes regulate synaptic transmission in the central nervous system. Astrocytes in vivo have "stems" that express glial fibrillary acidic protein (GFAP), intermediate filaments, and peripheral astrocyte processes (PAPs), which contain actin-rich cytoskeletal structures. At the PAPs, the perisynaptic glia contacts and enwraps synapses, and modulates glia-neuronal communication. Cultured astrocytes have been an invaluable tool for studying roles of astrocytes; however, the morphology of mammalian primary astrocytes cultured in conventional medium containing fetal bovine serum (FBS) was similar to that of fibroblasts, and many culture conditions have been developed to generate stellate astrocytes observed in vivo. Avian astrocytes have been prepared from embryonic chick forebrain and maintained at a high cell density in conventional FBS-containing medium as mammalian astrocytes, thus the morphological analysis of chicken astrocytes has not yet been performed. In the present study, we report that the morphology of astrocytes freshly harvested from the forebrain of a chicken embryo in serum-free Neurobasal medium with B-27 supplement and basic fibroblast growth factor (bFGF) is similar to that of the astrocyte morphology in vivo. We also find that astrocytes in this medium express similar levels of GFAP and two actin-binding proteins as astrocytes in conventional FBS-containing medium, although they have different morphologies. Furthermore, we confirmed that cryopreserved astrocytes differentiate faster than freshly harvested astrocytes.

Investigating Novel Roles for Hedgehog Co‐Receptors During Craniofacial Development

Hedgehog (HH) signaling is essential for correct patterning of vertebrate embryos, including during craniofacial development. HH ligands signal through the canonical receptor Patched1 (PTCH1), and three co‐receptors–growth arrest specific 1 (GAS1), cell adhesion molecule‐related/down‐regulated by oncogenes (CDON), and brother of CDON (BOC). Together, these co‐receptors are required during embryogenesis to mediate proper HH signaling. However, recent genetic studies suggest tissue‐specific, antagonistic roles for these proteins. For example, Gas1 restricts the HH pathway during mouse tooth development, while Boc antagonizes HH signaling during zebrafish jaw formation. Here I explored possible antagonistic roles for GAS1, CDON and BOC in HH‐dependent mammalian craniofacial development. Genetic deletion of these co‐receptors results in variable holoprosencephaly (HPE) phenotypes (failure to divide the forebrain into two cerebral hemispheres). Gas1 mutant mice display a spectrum of HPE phenotypes, and significant patterning defects in the neural tube and limb. In contrast, genetic deletion of Boc does not perturb patterning in any of these tissues. Strikingly, the combined deletion of Boc and Gas1 results in more severe neural tube and limb patterning defects than those observed in Gas1 single mutants. However, the Gas1 craniofacial phenotype is partially ameliorated in Gas1;Boc double mutant embryos as determined by the levels of NKX2.1, Gli1, Ptch1 and internasal distance quantitation. Micro‐computed tomography and skeletal analyses reveal that the rescue of the craniofacial defects in Gas1;Boc double mutants is restricted to the nasal bone and nasal capsule; other craniofacial bones are more severely affected, similar to what is observed in the neural tube and the limb. This effect does not appear to be due to differential GAS1 and BOC function in the forebrain neuroepithelium as overexpression of these co‐receptors in the developing chicken forebrain both promote HH signaling. Instead, I hypothesize that these co‐receptors have distinct, tissue‐specific functions in the craniofacial mesenchyme or the surface ectoderm. To further explore an antagonistic role for BOC during craniofacial development, I am currently deleting Boc in a sensitized HH genetic background through breeding with Ptch1 and Shh heterozygous mutant animals. These findings suggest that GAS1 and BOC do not function simply as redundant HH co‐receptors, but instead display distinct, tissue‐specific functions, particularly during craniofacial development.Support or Funding InformationNational Sciences Foundation Graduate Research Fellowship Program (DGE 1256260), Rackham Pre‐Candidate Graduate Student Research Grant, Bradley Merill Patten Fellowship and Organogenesis Training Grant (NIH T32HD007505)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Micro-RNAs, their target proteins, predispositions and the memory of filial imprinting

Visual imprinting is a learning process whereby young animals come to prefer a visual stimulus after exposure to it (training). The intermediate medial mesopallium (IMM) in the domestic chick forebrain is critical for visual imprinting and contributes to molecular regulation of memory formation. We investigated the role of micro-RNAs (miRNAs) in such regulation. Twenty-four hours after training, miRNA spectra in the left IMM were compared between chicks with high preference scores (strong memory for imprinting stimulus), and chicks with low preference scores (weak memory for imprinting stimulus). Using criteria of significance and expression level, we chose gga-miR-130b-3p for further study and found that down-regulation correlated with learning strength. No effect was detected in posterior nidopallium, a region not involved in imprinting. We studied two targets of gga-miR-130b-3p, cytoplasmic polyadenylation element binding proteins 1 (CPEB-1) and 3 (CPEB-3), in two subcellular fractions (P2 membrane-mitochondrial and cytoplasmic) of IMM and posterior nidopallium. Only in the left IMM was a learning-related effect observed, in membrane CPEB-3. Variances from the regression with preference score and untrained chicks suggest that, in the IMM, gga-miR-130b-3p level reflects a predisposition, i.e. capacity to learn, whereas P2 membrane-mitochondrial CPEB-3 is up-regulated in a learning-specific way.

Morphometric staging of organ development based on cross sectional images

An objective, continuous, and robust method for staging developing embryos or organs is essential for providing a common measure of time when studying quantitative/systems developmental biology. However, classical methods based on factors such as somite number or qualitative visual attributes are discrete and/or ambiguous due to observers’ subjectivity. Thus, an alternative staging method based on an explicit and continuous description of developmental states over time, such as anatomy/morphology, is needed. Here, we briefly propose a novel staging method as a natural extension of classical staging based on cross sectional images of organs, which are more accessible than full 3D structures. The contours are represented as 2D closed curves and quantified using elliptic Fourier descriptors. Treating the ambiguity in classical staging as a statistical model, the relationship between the novel morphometric staging and classical staging can be determined. This method was validated by applying it to two different sets of data: chick forebrain and Xenopus hindlimb development. Using this method, it is also possible to reconstruct the time evolution of the average morphology, which would be useful for quantitatively comparing morphologies between embryos or between normal and abnormal conditions.

Fabrication of Inner Grooved Hollow Fiber Membranes Using Microstructured Spinneret for Nerve Regeneration

Nerve conduits with topographical guidance have been recognized as the efficient repair of damaged peripheral nerves. In this study, polymeric hollow fiber membranes (HFMs) with grooved inner surface have been fabricated from a microstructured spinneret using a dry-jet wet spinning process for nerve regeneration studies. The effectiveness of HFM inner grooves has been demonstrated during an in vitro study of chick forebrain neuron outgrowth. It is of great importance that the groove geometry can be controllable to meet various needs in promoting nerve regeneration performance. While the overall groove geometry is determined by the spinneret design, fabrication conditions are also indispensable in fine-tuning the final groove geometry such as the groove height and width on the order of 10 μm or less. It is found that the bore fluid flow rate can be utilized to effectively adjust the resulting groove height by at most 52% and groove width by at most 61%, respectively, without modifying the spinneret geometry. This enables a new approach to fabricate different grooved HFMs using the same spinneret. By comparing to the influences of bore fluid flow rate, the dope fluid flow rate is less effective in regulating the groove height and width when using the same microstructured spinneret. Both bore and dope fluid flow rates should be carefully selected for fine groove width tuning.

Shifting the optimal stiffness for cell migration

Cell migration, which is central to many biological processes including wound healing and cancer progression, is sensitive to environmental stiffness, and many cell types exhibit a stiffness optimum, at which migration is maximal. Here we present a cell migration simulator that predicts a stiffness optimum that can be shifted by altering the number of active molecular motors and clutches. This prediction is verified experimentally by comparing cell traction and F-actin retrograde flow for two cell types with differing amounts of active motors and clutches: embryonic chick forebrain neurons (ECFNs; optimum ∼1 kPa) and U251 glioma cells (optimum ∼100 kPa). In addition, the model predicts, and experiments confirm, that the stiffness optimum of U251 glioma cell migration, morphology and F-actin retrograde flow rate can be shifted to lower stiffness by simultaneous drug inhibition of myosin II motors and integrin-mediated adhesions.

Reconstructing 3D deformation dynamics for curved epithelial sheet morphogenesis from positional data of sparsely-labeled cells

Quantifying global tissue deformation patterns is essential for understanding how organ-specific morphology is generated during development and regeneration. However, due to imaging difficulties and complex morphology, little is known about deformation dynamics for most vertebrate organs such as the brain and heart. To better understand these dynamics, we propose a method to precisely reconstruct global deformation patterns for three-dimensional morphogenesis of curved epithelial sheets using positional data from labeled cells representing only 1–10% of the entire tissue with limited resolution. By combining differential-geometrical and Bayesian frameworks, the method is applicable to any morphology described with arbitrary coordinates, and ensures the feasibility of analyzing many vertebrate organs. Application to data from chick forebrain morphogenesis demonstrates that our method provides not only a quantitative description of tissue deformation dynamics but also predictions of the mechanisms that determine organ-specific morphology, which could form the basis for the multi-scale understanding of organ morphogenesis.

Tug of War at the Cell-Matrix Interface

Mechanical interactions between a cell and its environment, or between cells, influence key developmental and physiologic processes as well as many aspect of disease (1,2). Indeed the ability of a cell to sense, produce, and respond to mechanical cues has emerged as a fundamental regulator of cell behaviors such as differentiation, proliferation, survival, and migration. In mechanical terms the interactions governing these behaviors are regulated by intracellular and extracellular physical events that are orchestrated by complex biochemical and mechanical signals.

In Vitro Synapse Development in the Cultured Neuronal Network Formed from Dissociated Chick Forebrain Neurons

In Vitro Synapse Development in the Cultured Neuronal Network Formed from Dissociated Chick Forebrain Neurons

An Integrated Stochastic Model of Matrix-Stiffness-Dependent Filopodial Dynamics

The ways that living cells regulate their behavior in response to their local mechanical environment underlie growth, development, and healing and are important to critical pathologies such as metastasis and fibrosis. Although extensive experimental evidence supports the hypothesis that this regulation is governed by the dependence of filopodial dynamics upon extracellular matrix stiffness, the pathways for this dependence are unclear. We therefore developed a model to relate filopodial focal adhesion dynamics to integrin-mediated Rho signaling kinetics. Results showed that focal adhesion maturation, i.e., focal adhesion links reinforcement and integrin clustering, dominates over filopodial dynamics. Downregulated focal adhesion maturation leads to the biphasic relationship between extracellular matrix stiffness and retrograde flow that has been observed in embryonic chick forebrain neurons, whereas upregulated maturation leads to the monotonically decreasing relationship that has been observed in mouse embryonic fibroblasts. When integrin-mediated Rho activation and stress-dependent focal adhesion maturation are combined, the model shows how filopodial dynamics endows cells with exquisite mechanosensing. Taken together, the results support the hypothesis that mechanical and structural factors combine with signaling kinetics to enable cells to probe their environments via filopodial dynamics.