Spatiotemporal Imaging of Zinc Ions in Zebrafish Live Brain Tissue Enabled by Fluorescent Bionanoprobes.

The zebrafish is a powerful model organism to study the mechanisms governing transition metal ions within whole brain tissue. Zinc is one of the most abundant metal ions in the brain, playing a critical pathophysiological role in neurodegenerative diseases. The homeostasis of free, ionic zinc (Zn2+) is a key intersection point in many of these diseases, including Alzheimer's disease and Parkinson's disease. A Zn2+ imbalance can eventuate several disturbances that may lead to the development of neurodegenerative changes. Therefore, compact, reliable approaches that allow the optical detection of Zn2+ across the whole brain would contribute to our current understanding of the mechanisms that underlie neurological disease pathology. We developed an engineered fluorescence protein-based nanoprobe that can spatially and temporally resolve Zn2+ in living zebrafish brain tissue. The self-assembled engineered fluorescence protein on gold nanoparticles was shown to be confined to defined locations within the brain tissue, enabling site specific studies, compared to fluorescent protein-based molecular tools, which diffuse throughout the brain tissue. Two-photon excitation microscopy confirmed the physical and photometrical stability of these nanoprobes in living zebrafish (Danio rerio) brain tissue, while the addition of Zn2+ quenched the nanoprobe fluorescence. Combining orthogonal sensing methods with our engineered nanoprobes will enable the study of imbalances in homeostatic Zn2+ regulation. The proposed bionanoprobe system offers a versatile platform to couple metal ion specific linkers and contribute to the understanding of neurological diseases.

Are superficial neuromasts proprioceptors underlying fast copulatory behavior?

In male Poeciliid fishes, the modified anal fin (i.e., gonopodium) and its axial and appendicular support are repositioned within the axial skeleton, creating a novel sexually dimorphic ano-urogenital region. During copulation, the relative location of the gonopodium is crucial for successful insemination. Therefore, the repositioning of these structures and organ relied on the reorganization of the efferent circuitry that controls spinal motor neurons innervating appendicular muscles critical for the movement of the gonopodium, including the fast and synchronous torque-trust motion during insemination attempts. Copulation occurs when a male positions himself largely outside a female's field of view, circumducts his gonopodium, and performs a rapid, complex maneuver to properly contact the female urogenital sinus with the distal tip of the gonopodium and transfers sperm. Although understanding of the efferent circuitry has significantly increased in the last 24 years, nothing is known about the cutaneous receptors involved in gonopodium movement, or how the afferent signals are processed to determine the location of this organ during copulation. Using Western mosquitofish, Gambusia affinis, as our model, we attempt to fill this gap in knowledge. Preliminary data showed cutaneous nerves and sensory neurons innervating superficial neuromasts surrounding the base of adult male gonopodium; those cutaneous nerves projected ventrally from the spinal cord through the 14th dorsal root ganglion and its corresponding ventral root towards the base and fin rays of the gonopodium. We asked what role the cutaneous superficial neuromasts play in controlling the positioning and timing of the gonopodium's fast and synchronous movements for effective sperm transfer. First, we found a greater number of superficial neuromasts surrounding the base of the male's gonopodium compared to the base of the female's anal fin. Second, we systemically removed superficial neuromasts surrounding the gonopodium base and observed significant impairment of the positioning and timing of gonopodial movements. Our findings provide a first step to supporting the following hypothesis: during radical reorganization of the Poeciliid body plan, superficial neuromasts have been partially co-opted as proprioceptors that allow the gonopodium to control precise positioning and timing during copulatory attempts.

Accelerating Biological Insight for Understudied Genes.

The rapid expansion of genome sequence data is increasing the discovery of protein-coding genes across all domains of life. Annotating these genes with reliable functional information is necessary to understand evolution, to define the full biochemical space accessed by nature, and to identify target genes for biotechnology improvements. The majority of proteins are annotated based on sequence conservation with no specific biological, biochemical, genetic, or cellular function identified. Recent technical advances throughout the biological sciences enable experimental research on these understudied protein-coding genes in a broader collection of species. However, scientists have incentives and biases to continue focusing on well documented genes within their preferred model organism. This perspective suggests a research model that seeks to break historic silos of research bias by enabling interdisciplinary teams to accelerate biological functional annotation. We propose an initiative to develop coordinated projects of collaborating evolutionary biologists, cell biologists, geneticists, and biochemists that will focus on subsets of target genes in multiple model organisms. Concurrent analysis in multiple organisms takes advantage of evolutionary divergence and selection, which causes individual species to be better suited as experimental models for specific genes. Most importantly, multisystem approaches would encourage transdisciplinary critical thinking and hypothesis testing that is inherently slow in current biological research.

Non-invasive Brain Delivery and Efficacy of BDNF in APP/PS1 Transgenic Mice as a Model of Alzheimer's Disease.

Neurotrophic factors such as brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) have been demonstrated for their potential as a neuroregenerative treatment of Alzheimer's disease (AD). Unfortunately, most proteins cannot be effectively delivered into the brain from the blood stream due to the presence of the blood-brain barrier (BBB). In this study, we delivered BDNF using ADTC5 as BBB modulator (BBBM) into the brains of transgenic APP/PS1 mice, a mouse model for AD. As controls, two groups of APP/PS1 mice were treated with BDNF alone and vehicle, respectively. All three groups were subjected to behavioral/cognitive assessments in Y-maze and novel object recognition (NOR) tests as well as evaluation of the brain markers activated by BDNF. The results showed that BDNF + ADTC5 group performed significantly better in both the Y-maze and NOR assessments compared to mice that received BDNF alone or vehicle. In addition, significant upregulations of NG2 receptors as well as EGR1 and ARC mRNA transcripts were observed in the brain cortex of mice treated with BDNF + ADTC5, further indicating the efficacy of delivered BDNF in the brain. There were high plaque loads in all groups of mice, suggesting no influence of BDNF on the plaque formation. In summary, ADTC5 can deliver BDNF into the brains of APP/PS1 mice and the activity of BDNF in improving cognitive function was likely due to improvement in synaptic plasticity via NG2 glia cells and not by reducing the plaque load.

Noninvasive Brain Delivery and Efficacy of BDNF to Stimulate Neuroregeneration and Suppression of Disease Relapse in EAE Mice.

The number of FDA-approved protein drugs (biologics), such as antibodies, antibody-drug conjugates, hormones, and enzymes, continues to grow at a rapid rate; most of these drugs are used to treat diseases of the peripheral body. Unfortunately, most of these biologics cannot be used to treat brain diseases such as Alzheimer's disease (AD), multiple sclerosis (MS), and brain tumors in a noninvasive manner due to their inability to permeate the blood-brain barrier (BBB). Therefore, there is a need to develop an effective method to deliver protein drugs into the brain. Here, we report a proof of concept to deliver a recombinant brain-derived neurotrophic factor (BDNF) to the brains of healthy and experimental autoimmune encephalomyelitis (EAE) mice via intravenous (iv) injections by co-administering BDNF with a BBB modulator (BBBM) peptide ADTC5. Western blot evaluations indicated that ADTC5 enhanced the brain delivery of BDNF in healthy SJL/elite mice compared to BDNF alone and triggered the phosphorylation of TrkB receptors in the brain. The EAE mice treated with BDNF + ADTC5 suppressed EAE relapse compared to those treated with BDNF alone, ADTC5 alone, or vehicle. We further demonstrated that brain delivery of BDNF induced neuroregeneration via visible activation of oligodendrocytes, remyelination, and ARC and EGR1 mRNA transcript upregulation. In summary, we have demonstrated that ADTC5 peptide modulates the BBB to permit noninvasive delivery of BDNF to exert its neuroregeneration activity in the brains of EAE mice.

Glatiramer acetate persists at the injection site and draining lymph nodes via electrostatically-induced aggregation.

Glatiramer acetate (GA) is widely prescribed for the treatment of relapsing-remitting multiple sclerosis, however, the mechanism of action is still not fully understood. We investigated the structural properties of GA and examined alterations to the drug upon injection into the subcutaneous space. First, a variety of biophysical characterization techniques were employed to characterize GA in solution. GA was found to exist as alpha helices in solution with a hydrodynamic radius of ~3 nm in size. To simulate GA behavior at the site of injection, GA was injected into a solution of 1.5 MDa hyaluronic acid (HA). Visible aggregates were observed immediately upon injection and subsequent testing indicated aggregation was driven by electrostatic interactions between the positively-charged GA and negatively-charged HA. In vivo testing confirmed GA formed spherical particles in the nano- to micrometer size range, suggesting this mechanism contributes to persistence at the injection site and in draining lymph nodes. The aggregates were found to associate with glycosaminoglycans, suggesting an electrostatic mechanism of induced aggregation like the simulated injection. These novel observations may help explain the complex immunomodulatory mechanisms of GA and adverse injection site reactions seen in patients.

Controls for immunohistochemistry: the Histochemical Society's standards of practice for validation of immunohistochemical assays.

Immunohistochemistry is widely used in biomedical research to localize specific epitopes of molecules in cells and tissues. The validity of interpretations based on immunohistochemistry requires appropriate positive and negative controls that are often not reported in publications. This omission may lead to incorrect interpretations and irreproducible results in the literature and contribute to wasted time, effort, and resources as well as erosion of confidence in scientific investigation by the general public, legislative bodies and funding agencies. The present article summarizes essential controls required for validation of immunohistochemical findings and represents a standard of practice for the use of immunohistochemistry in research and diagnostic investigations. Adherence to the guidelines described in the present article can be cited by authors as support for the validity of interpretations of the immunohistochemistry reported in their publications.

Abundance of gap junctions at glutamatergic mixed synapses in adult Mosquitofish spinal cord neurons.

"Dye-coupling", whole-mount immunohistochemistry for gap junction channel protein connexin 35 (Cx35), and freeze-fracture replica immunogold labeling (FRIL) reveal an abundance of electrical synapses/gap junctions at glutamatergic mixed synapses in the 14th spinal segment that innervates the adult male gonopodium of Western Mosquitofish, Gambusia affinis (Mosquitofish). To study gap junctions' role in fast motor behavior, we used a minimally-invasive neural-tract-tracing technique to introduce gap junction-permeant or -impermeant dyes into deep muscles controlling the gonopodium of the adult male Mosquitofish, a teleost fish that rapidly transfers (complete in <20 mS) spermatozeugmata into the female reproductive tract. Dye-coupling in the 14th spinal segment controlling the gonopodium reveals coupling between motor neurons and a commissural primary ascending interneuron (CoPA IN) and shows that the 14th segment has an extensive and elaborate dendritic arbor and more gap junctions than do other segments. Whole-mount immunohistochemistry for Cx35 results confirm dye-coupling and show it occurs via gap junctions. Finally, FRIL shows that gap junctions are at mixed synapses and reveals that >50 of the 62 gap junctions at mixed synapses are in the 14th spinal segment. Our results support and extend studies showing gap junctions at mixed synapses in spinal cord segments involved in control of genital reflexes in rodents, and they suggest a link between mixed synapses and fast motor behavior. The findings provide a basis for studies of specific roles of spinal neurons in the generation/regulation of sex-specific behavior and for studies of gap junctions' role in regulating fast motor behavior. Finally, the CoPA IN provides a novel candidate neuron for future studies of gap junctions and neural control of fast motor behaviors.

A male poecillid's sexually dimorphic body plan, behavior, and nervous system.

Here we review the literature of a male poecillid's sexually dimorphic body plan, behavior, and nervous system, including work dating from the mid 1800s to the mid 1990s as well as work in press or in preparation for publication. Rosa-Molinar described the remodeling of the sexually dimorphic anal fin appendicular support, confirmed earlier claims about the development of the male and female secondary sex characteristics in the Western Mosquitofish, Gambusia affinis and provided for the first time direct embryonic evidence suggesting that remodeling of the sexually dimorphic anal fin appendicular support is biphasic. The first process begins in embryos and proceeds similarly in immature males and females; the second process occurs only in males and results in the anterior transposition of the anal fin and its appendicular support to the level of vertebra 11 [Rosa-Molinar E, Hendricks SE, Rodriguez-Sierra JF, Fritzsch B. 1994. Development of the anal fin appendicular support in the western mosquitofish, Gambusia affinis (Baird and Girard, 1854): a reinvestigation and reinterpretation. Acta Anat 151:20-35.] and the formation of a gonopodium used for internal fertilization. Studies using high-speed video cameras confirmed and extended Peden's and others' observations of copulatory behavior. The cameras showed that circumduction is a complex movement combining in a very fast sequence abduction, extension and pronation, S-start-type fast-start (defined as torque-thrust), and adduction movements. Recent work on the nervous system demonstrated dye-coupling between motor neurons and interneurons via gap junctions, suggesting an attractive substrate for the rapid motions involved in poecillid copulatory reflexes.

Drug encapsulation within self-assembled microglobules formed by thermoresponsive supramolecules.

An 8-(phenyl)-2'-deoxyguanosine derivative self-assembles in aqueous media into discrete hexadecamers that further self-assemble above 32 °C into microglobules that encapsulate the drug doxorubicin.

The western mosquitofish (Gambusia affinis affinis): a new laboratory animal resource for the study of sexual dimorphism in neural circuits.

The western mosquitofish (Gambusia affinis affinis) is a useful model for the study of sexual dimorphism and the neural circuits associated with sexual differentiation. This is largely because of its anal fin, which undergoes radical postnatal transformation in males. Understanding the neural mechanisms involved in this process may also help elucidate basic principles of the nervous system. The authors describe the mosquitofish as a model for research and present guidelines for the care and use of this species.

The development of the hindbrain afferent projections in the axolotl: evidence for timing as a specific mechanism of afferent fiber sorting.

The aim of this study is to reveal the timing and growth pattern of central octavolateral projection development in the Mexican axolotl, Ambystoma mexicanum. In this amphibian species the development of the inner ear occurs first, followed by mechanosensory lateral line organs, and finally by ampullary electroreceptors. Several hypotheses have been proposed about how the development of peripheral organs, including differential projections of the ear, might relate to the development of central projections. Our data suggest that the sequence of maturation of the ear, mechanosensory lateral line, and ampullary electroreceptive organs is closely accompanied by the timed development of the trigeminal, inner ear, mechanosensory lateral line organs, and the ampullary electroreceptor afferent projections in the axolotl. Our data suggest that segregation of central termination within the alar plate is a function of time and space: later forming organs are likely innervated by later forming ganglia that project centrally later and to more dorsal areas of the alar plate that have not yet received any other afferents. Later forming ganglia of the same type may grow along existing pathways of earlier formed neurons.

Starting from fins: parallelism in the evolution of limbs and genitalia. The fin-to-limb transition.

The March/April 2002 issue of Evolution and Development focused on three presentations made at the Starting from Fins: Parallelism in the Evolution of Limbs and Genitalia symposium held as part of the 2001 Chicago meeting of the Society of Integrative and Comparative Biology. The intention of the symposium and the publication of the presentations was to extend discussion of the potential and the limits of using serial homologues to understand developmental aspects of morphological evolution. The March/April 2002 issue concentrated on unpaired fin to genitalia transitions. This issue focuses on paired fins to limbs and highlights the need for developmental data to be integrated with data from fossil materal, phylogenetic analysis, and explicitly comparative studies. Coates et al. use phylogenetic methods to explore the limb/fin characters of taxa, but their analysis departs somewhat from the usual in that the reference group for organisms includes sister group taxa not usually considered true tetrapods. They state that including finned taxa from the stem group permits an attempt to distinguish the primitive condition of the characteristics demonstrated by the crown group, that is, "limbed tetrapods." In focusing on limb characters specifically and including aspects of the appendicular girdles, Coates et al. highlight morphological details and trends within a given phylogeny. They also demonstrate the degree of relevance of limb characters during the establishment of lineages and their branching patterns by using only limb characters to generate a tree and use a direct comparison of serial versus special homologies to explore the degree of evolutionary parallelism between fore-and hindlimbs. The preliminary conclusions indicate a high level of independence between the serially homologous fore-and hindlimb. Innes et al. present outcomes from the use of cutting edge molecular genetic approaches to understand developmental aspects of limb morphology. In a manner conceptually similar to Coates et al.'s use of fossil characters, Innes et al. use the serial analysis of gene expression to sort differences from similarities in the gene expression profiles of fore-and hindlimbs of the same embryos. Although these gene expression pattems are likely to reflect the serial homology of the paired limbs, they are silent in terms of our understanding both the profound and subtle differences between fore- and hindlimbs in any given species. Innes et al. point out the volume of data generated by SAGE far exceeds our ability to interpret its biological meaning. The studies presented here and in the March/April issue are excellent examples of the need to interpret complex data in light of collective knowledge of evolutionary history. We hope the insights gained from the symposium and papers contribute to a dialogue on how to integrate different approaches and assist in moving forward the field of Evolution and Development.

Starting from fins: parallelism in the evolution of limbs and genitalia: the fin-to-genitalia transition.

Organizers of the symposium Starting from Fins: Parallelism in the Evolution of Limbs and Genitalia intended it 1) to begin debates and discussions about parallelism, serial homology and transitions in development, as well as evolution of gene function and theories of origins and 2) to examine closely the potential significance of serial homology in understanding the evolution of morphology. This issue of Evolution and Development focuses on unpaired fin to genitalia transitions; the July-August issue will focus on paired fins to limbs, revisit the issues raised in the symposium, and point to future directions. Minelli's opening presentation introduced the central theme of the symposium by suggesting that body appendages such as arthropod and vertebrate limbs, chordate tails and external genitalia are evolutionarily divergent duplicates (paramorphs) of the main body axis. Suzuki's and Podlasek's presentations focused on the development of mammalian genitalia. Suzuki presented Suzuki et al investigations of the role of fibroblast growth factor (Fgf) and Sonic hedgehog (Shh) as signaling molecules during murine external genitalia formation, and Podlasek presented Podlasek et al investigations to elucidate a rudimentary pathway of essential developmental genes and transcriptional regulators such as Hox genes, Sonic hedgehog (Shh), and Bone morphogenetic proteins 2 and 4 (BMP-2; BMP-4), found in the limb. Discussions and questions emerging from the symposium point to the need to recognize that claims of phylogenetic cause must be based on something more than similarities; research must focus on the extent to which comparisons can be taken as well as on the evolutionary significance of similarities.