Peter Kozulin

Analysis of Complement Expression in Light-Induced Retinal Degeneration: Synthesis and Deposition of C3 by Microglia/Macrophages Is Associated with Focal Photoreceptor Degeneration

PURPOSE To investigate the expression and localization of complement system mRNA and protein in a light-induced model of progressive retinal degeneration. METHODS Sprague-Dawley (SD) rats were exposed to 1000 lux of bright continuous light (BCL) for up to 24 hours. At time points during (1-24 hours) and after (3 and 7 days) exposure, the animals were euthanatized and the retinas processed. Differential expression of complement genes at 24 hours of exposure was assessed using microarray analysis. Expression of complement genes was validated by quantitative PCR, and expression of selected genes was investigated during and after BCL exposure. Photoreceptor apoptosis was assessed using TUNEL and C3 was further investigated by spatiotemporal analysis using in situ hybridization and immunohistochemistry. RESULTS Exposure to 24 hours of BCL induced differential expression of a suite of complement system genes, including classic and lectin components, regulators, and receptors. C1qr1, MCP, Daf1, and C1qTNF6 all modulated in concert with photoreceptor death and AP-1 expression, which reached a peak at 24 hours exposure. C1s and C4a reached peak expression at 3 days after exposure, while expression of C3, C3ar1, and C5r1 were maximum at 7 days after exposure. C3 mRNA was detected in ED1- and IBA1-positive microglia/macrophages, in the retinal vessels and optic nerve head and in the subretinal space, particularly at the margins of the emerging lesion. CONCLUSIONS The data indicate that BCL induces the prolonged expression of a range of complement genes and show that microglia/macrophages synthesize C3 and deposit it in the ONL after BCL injury. These findings have relevance to the role of complement in progressive retinal degeneration, including atrophic AMD.

The Cellular Expression of Antiangiogenic Factors in Fetal Primate Macula

PURPOSE To characterize the cellular expression patterns of antiangiogenic factors differentially regulated in the fetal human macula. METHODS RNA was extracted from macular, nasal, and surround biopsies of three human fetal retinas at midgestation. Relative levels of expression of pigment epithelium-derived factor (PEDF), brain natriuretic peptide (BNP), collagen type IValpha2 (COL4A2), and natriuretic peptide receptors A and C (NPRA and NPRC) were determined with quantitative PCR. Cellular expression of PEDF and BNP was investigated by in situ hybridization on retinal sections from monkeys aged between fetal day 55 and 11 years. BNP, COL4A2, and NPRA proteins were localized by immunohistochemistry. Labeling was imaged and quantified by confocal microscopy and optical densitometry. RESULTS Quantitative PCR confirmed higher levels of PEDF and BNP and lower levels of COL4A2 in the macula at midgestation. PEDF mRNA was detected in ganglion cells (GCs) and the pigment epithelium (RPE). BNP mRNA was detected in GCs and macroglia, although BNP immunoreactivity (IR) was predominantly perivascular. COL4A2-IR was detected in large blood vessels and NPRA-IR on the retinal vascular endothelium, GC axons in fetal retinas, and cone axons at all ages. Optical densitometry showed a graded expression of PEDF and BNP at all ages, with highest levels of expression in GCs in the developing fovea. CONCLUSIONS Because the retinal vessels initially form in the GC layer, it is likely that PEDF has a key role in defining and maintaining the foveal avascular area. The precise role of BNP is unclear, but it may include both antiangiogenic and natriuretic functions.

Effects of a Head-Mounted Display on the Oculomotor System of Children

Purpose. This study evaluated the effects of short term and extended viewing of virtual imagery using the Binocular Viewer (new generation bi-ocular viewer) on the visual system of children, and compared these effects with that of viewing a high definition television (HDTV) display. Methods. Sixty children aged 5 to 16 years viewed 30 min of virtual imagery using the Binocular Viewer and a HDTV display on two occasions. Sixteen subjects, aged 13 to 16 years, completed a third session of extended viewing (80 min) with the Binocular Viewer. Oculomotor function and symptoms were assessed previewing, immediately postviewing, and 10 min postviewing. Results. Thirty minutes of Binocular Viewer use resulted in symptom increases (p < 0.05) immediately postviewing (“feeling tired,” “feeling sleepy,” “difficulty concentrating,” and “sore/aching eyes”) however most symptoms had dissipated by 10-min postviewing. There were no significant symptom differences between viewing with the Binocular Viewer and the HDTV display at either time point. An increase in symptoms (p < 0.05) immediately postviewing was recorded after 80 min of Binocular Viewer use (“feeling tired,” “feeling bored,” “feeling sleepy,” and “tired eyes”), however only “feeling tired” and “feeling bored” remained significantly increased (p < 0.05) 10-min postviewing. Near unaided visual acuity demonstrated a significant and consistent reduction immediately (p < 0.01) and at 10 min (p < 0.05) following 30 min of Binocular Viewer use and immediately following 80 min of use (p < 0.01). Near unaided VA was also significantly reduced (p < 0.01) immediately after 30 min of HDTV display use. Conclusions. Virtual imagery viewing with the Binocular Viewer in children aged 5 to 16 years had few additional adverse effects when compared to viewing a more conventional HDTV display. The Binocular Viewer was comfortable to wear for up to 80 min of viewing. The consistent reduction in near vision for both viewing durations with the Binocular Viewer requires further investigation.

Differential gene expression in the developing human macula: microarray analysis using rare tissue samples

The macula is a unique and important region in the primate retina that achieves high resolution and color vision in the central visual field. We recently reported data obtained from microarray analysis of gene expression in the macula of the human fetal retina (Kozulin et al., Mol Vis 15:45–59, 1). In this paper, we describe the preliminary analyses undertaken to visualize differences and verify comparability of the replicates used in that study, report the differential expression of other gene families obtained from the analysis, and show the reproducibility of our findings in several gene families by quantitative real-time PCR.

Development of body, head and brain features in the Australian fat-tailed dunnart (Sminthopsis crassicaudata; Marsupialia: Dasyuridae); A postnatal model of forebrain formation

Most of our understanding of forebrain development comes from research of eutherian mammals, such as rodents, primates, and carnivores. However, as the cerebral cortex forms largely prenatally, observation and manipulation of its development has required invasive and/or ex vivo procedures. Marsupials, on the other hand, are born at comparatively earlier stages of development and most events of forebrain formation occur once attached to the teat, thereby permitting continuous and non-invasive experimental access. Here, we take advantage of this aspect of marsupial biology to establish and characterise a resourceful laboratory model of forebrain development: the fat-tailed dunnart (Sminthopsis crassicaudata), a mouse-sized carnivorous Australian marsupial. We present an anatomical description of the postnatal development of the body, head and brain in dunnarts, and provide a staging system compatible with human and mouse developmental stages. As compared to eutherians, the orofacial region develops earlier in dunnarts, while forebrain development is largely protracted, extending for more than 40 days versus ca. 15 days in mice. We discuss the benefits of fat-tailed dunnarts as laboratory animals in studies of developmental biology, with an emphasis on how their accessibility in the pouch can help address new experimental questions, especially regarding mechanisms of brain development and evolution.

Multiple events of gene manipulation via in pouch electroporation in a marsupial model of mammalian forebrain development

BACKGROUND The technique of in utero electroporation has been widely used in eutherians, such as mice and rats, to investigate brain development by selectively manipulating gene expression in specific neuronal populations. A major challenge, however, is that surgery is required to access the embryos, affecting animal survival and limiting the number of times it can be performed within the same litter. NEW METHOD Marsupials are born at an early stage of brain development as compared to eutherians. Forebrain neurogenesis occurs mostly postnatally, allowing electroporation to be performed while joeys develop attached to the teat. Here we describe the method of in pouch electroporation using the Australian marsupial fat-tailed dunnart (Sminthopsis crassicaudata, Dasyuridae). RESULTS In pouch electroporation is minimally invasive, quick, successful and anatomically precise. Moreover, as no surgery is required, it can be performed several times in the same individual, and littermates can undergo independent treatments. COMPARISON WITH EXISTING METHOD As compared to in utero electroporation in rodents, in pouch electroporation in marsupials offers unprecedented opportunities to study brain development in a minimally invasive manner. Continuous access to developing joeys during a protracted period of cortical development allows multiple and independent genetic manipulations to study the interaction of different systems during brain development. CONCLUSIONS In pouch electroporation in marsupials offers an excellent in vivo assay to study forebrain development and evolution. By combining developmental, functional and comparative approaches, this system offers new avenues to investigate questions of biological and medical relevance, such as the precise mechanisms of brain wiring and the organismic and environmental influences on neural circuit formation.

Astroglial-mediated remodeling of the interhemispheric midline during telencephalic development is exclusive to eutherian mammals

The corpus callosum forms the major interhemispheric connection in the human brain and is unique to eutherian (or placental) mammals. The developmental events associated with the evolutionary emergence of this structure, however, remain poorly understood. A key step in callosal formation is the prior remodeling of the interhemispheric fissure by embryonic astroglial cells, which then subsequently act as a permissive substrate for callosal axons, enabling them to cross the interhemispheric midline. However, whether astroglial-mediated interhemispheric remodeling is unique to eutherian mammals, and thus possibly associated with the phylogenetic origin of the corpus callosum, or instead is a general feature of mammalian brain development, is not yet known. To investigate this, we performed a comparative analysis of interhemispheric remodeling in eutherian and non-eutherian mammals, whose lineages branched off before the evolution of the corpus callosum. Whole brain MRI analyses revealed that the interhemispheric fissure is retained into adulthood in marsupials and monotremes, in contrast to eutherians (mice), in which the fissure is significantly remodeled throughout development. Histological analyses further demonstrated that, while midline astroglia are present in developing marsupials, these cells do not intercalate with one another through the intervening interhemispheric fissure, as they do in developing mice. Thus, developing marsupials do not undergo astroglial-mediated interhemispheric remodeling. As remodeling of the interhemispheric fissure is essential for the subsequent formation of the corpus callosum in eutherians, our data highlight the role of astroglial-mediated interhemispheric remodeling in the evolutionary origin of the corpus callosum.

Investigating Early Formation of the Cerebral Cortex by In Utero Electroporation: Methods and Protocols

Cortical development requires a strict balance between neuronal proliferation, differentiation, and cellular migration within restricted developmental stages. The precise spatiotemporal gene manipulation used in developmental studies can be achieved by in vitro or ex vivo experiments or by the generation of transgenic animals. However, these approaches have significant limitations when trying to investigate the origin and molecular regulation of early cortical neurons. In utero electroporation (IUE) is an informative cell labeling technique that provides the ability to label neural progenitor cells and their progeny in vivo with promoter-specific reporter constructs as well as to induce or repress gene expression in a spatially and temporally specific manner. Technical improvements of this method have allowed the targeting of multiple neural cell types, as well as the precise transfection of subpopulations of neurons at increasingly earlier embryonic stages. Furthermore, neuronal projection studies and the use of multiple electroporations in the same embryo have made it possible to examine processes occurring at different developmental stages and/or cortical areas and link their anatomy to their function. In this chapter, we present the latest advances of the in utero electroporation technique for the study of early formation of the cerebral cortex, together with a description of the necessary tools.

A pan-mammalian map of interhemispheric brain connections predates the evolution of the corpus callosum

Significance The neocortex is a hallmark of mammalian evolution, and connections between both hemispheres integrate bilateral functions. In eutherians (e.g., rodents and humans), interhemispheric circuits course via the corpus callosum and share a similar connectome throughout species. Noneutherian mammals (i.e., monotremes and marsupials), however, did not evolve a corpus callosum; therefore, whether the eutherian connectome arose as consequence of callosal evolution or instead reflects ancient connectivity principles remains unknown. We studied monotreme and marsupial interhemispheric neocortical connectomes and compared these with eutherian datasets. This revealed interhemispheric connectivity features shared across mammals, with or without a corpus callosum, suggesting that an ancient connectome originated at least 80 million years before callosal evolution. The brain of mammals differs from that of all other vertebrates, in having a six-layered neocortex that is extensively interconnected within and between hemispheres. Interhemispheric connections are conveyed through the anterior commissure in egg-laying monotremes and marsupials, whereas eutherians evolved a separate commissural tract, the corpus callosum. Although the pattern of interhemispheric connectivity via the corpus callosum is broadly shared across eutherian species, it is not known whether this pattern arose as a consequence of callosal evolution or instead corresponds to a more ancient feature of mammalian brain organization. Here we show that, despite cortical axons using an ancestral commissural route, monotremes and marsupials share features of interhemispheric connectivity with eutherians that likely predate the origin of the corpus callosum. Based on ex vivo magnetic resonance imaging and tractography, we found that connections through the anterior commissure in both fat-tailed dunnarts (Marsupialia) and duck-billed platypus (Monotremata) are spatially segregated according to cortical area topography. Moreover, cell-resolution retrograde and anterograde interhemispheric circuit mapping in dunnarts revealed several features shared with callosal circuits of eutherians. These include the layered organization of commissural neurons and terminals, a broad map of connections between similar (homotopic) regions of each hemisphere, and regions connected to different areas (heterotopic), including hyperconnected hubs along the medial and lateral borders of the cortex, such as the cingulate/motor cortex and claustrum/insula. We therefore propose that an interhemispheric connectome originated in early mammalian ancestors, predating the evolution of the corpus callosum. Because these features have been conserved throughout mammalian evolution, they likely represent key aspects of neocortical organization.

Axonal guidance: making connections

The vertebrate brain contains millions of neuronal and glial cells arranged in a highly organized manner forming functional neural circuits. To form these circuits during brain development, neurons extend an axon from the cell body to make connections with neurons in target brain areas, which can be a considerable distance away from the neuronal cell body. To ensure that axons accurately elongate toward the correct target field over such a distance, specific guidance cues are used to navigate the axons through their environment in a reproducible pattern of growth. These cues involve guidance molecules that can elicit attractive or repellent guidance on the extending axon and can act over long distances by secretion into extracellular space or over short distances through direct cell contact. In response to the guidance cues, the distal tip of the axon, known as the growth cone, undergoes dynamic structural changes to ensure that it is continually growing in the correct direction. In this chapter, we will discuss the range of highly conserved mechanisms and molecules involved in axon guidance, the biological changes that occur in axons during guidance, and the major assays used to measure the guidance of neuronal axons in vitro.