Dynamic changes in the localization of synapse associated proteins during development and differentiation of the mammalian retina

We have examined the developmental distribution and differential localization of presynaptic terminal associated proteins in the mammalian retina. We have used antibodies specific for synaptic vesicle associated proteins Synaptotagmin, Rab 3A, Synaptophysin and Synaptobrevin, and presynaptic terminal membrane associated proteins SNAP-25 and Syntaxin, to characterize their spatio-temporal distribution during retinal differentiation and in the mature retina;The vertebrate retina has a laminar organization consisting of three cellular layers separated by two synaptic layers, the inner piexiform layer and the outer plexiform layer. In general, immunoreactivity for presynaptic terminal associated proteins was first observed in cellular layers, and as differentiation progressed, immunoreactivity was localized to the synaptic or plexiform layers. However, there were distinct differences in the relative intensities and localization of patterns of immunoreactivity for these proteins;This analysis revealed a temporal difference in the onset of detectable immunoreactivity for Synaptotagmin and Rab 3A compared with Synaptophysin and Synaptobrevin, suggesting they may have additional roles in vesicle trafficking during neural development. Immunoreactivity for presynaptic terminal membrane associated protein SNAP-25 is at relatively high levels in cholinergic amacrine cells during their differentiation, but not at maturity. This transient expression of high levels of SNAP-25 may contribute to the functional role these cells play in propagation of spontaneous retinal activity. Spatial and temporal differences in localization of SNARE complex proteins (Synaptophysin, SNAP-25 and Syntaxin), were also observed during development and at maturity. Detectable SNAP-25- and Syntaxin-immunoreactivity preceded Synaptobrevin-immunoreactivity during differentiation of synaptic layers. In addition, immunoreactivity for each protein had a distinct pattern of differential intensities within the inner plexiform or synaptic layer during development and in the mature retina, and differential localization in the OPL;This analysis was the first to systematically characterize the distribution of multiple presynaptic terminal associated proteins, including proteins of the SNARE complex, during development of an organized tissue like the retina. The dynamic, differential patterns of immunoreactivity for these proteins suggests several of them may have additional roles in the development of the nervous system

Semaphorin Signaling in Vertebrate Neural Circuit Assembly

Neural circuit formation requires the coordination of many complex developmental processes. First, neurons project axons over long distances to find their final targets and then establish appropriate connectivity essential for the formation of neuronal circuitry. Growth cones, the leading edges of axons, navigate by interacting with a variety of attractive and repulsive axon guidance cues along their trajectories and at final target regions. In addition to guidance of axons, neuronal polarization, neuronal migration, and dendrite development must be precisely regulated during development to establish proper neural circuitry. Semaphorins consist of a large protein family, which includes secreted and cell surface proteins, and they play important roles in many steps of neural circuit formation. The major semaphorin receptors are plexins and neuropilins, however other receptors and co-receptors also mediate signaling by semaphorins. Upon semaphorin binding to their receptors, downstream signaling molecules transduce this event within cells to mediate further events, including alteration of microtubule and actin cytoskeletal dynamics. Here, I review recent studies on semaphorin signaling in vertebrate neural circuit assembly, with the goal of highlighting how this diverse family of cues and receptors imparts exquisite specificity to neural complex connectivity

PhD

dissertationThe mammalian retina is comprised of 55-60 cell types mediating transduction of photic information through visual preprocessing channels. These cell types fall into six major cell superclasses including photoreceptors, horizontal, amacrine, Muller and ganglion cells. Through computational molecular phenotyping, using amino acids as discriminands, this dissertation shows that the major cellular superclasses of the murine retina are subdivisible into the following natural classes; 1 retinal pigment epithelium class, 2 photoreceptor, 2 bipolar cell, 1 horizontal cell, 15 amacrine cell, 1 Muller cell, and 7 ganglion cell classes. Retinal degenerative diseases like retinitis pigmentosa result in loss of photoreceptors, which constitutes deafferentation of the neural retina. This deafferentation, when complete, is followed by retinal remodeling, which is the common fate of all retinal degenerations that trigger photoreceptor loss. The same strategy used to visualize cell classes in wild type murine retina was applied to examples of retinal degenerative disease in human tissues and naturally and genetically engineered models, examining all cell types in 17 human cases of retinitis pigmentosa (RP) and 85 cases of rodent retinal degenerations encompassing 13 different genetic models. Computational molecular phenotyping concurrently visualized glial transformations, neuronal translocations, and the emergence of novel synaptic complexes, achievements not possible with any other method. The fusion of phenotyping and anatomy at the ultrastructure level also enabled the modeling of synaptic connections, illustrating that the degenerating retina produces new synapses with vigor with the possibility that this phenomenon might be exploited to rescue vision. However, this circuitry is likely corruptive of visual processing and reflects, we believe, attempts by neurons to find synaptic excitation, demonstrating that even minor rewiring seriously corrupts signal processing in retinal pathways leaving many current approaches to bionic and biological retinal rescue unsustainable. The ultimate conclusion is that the sequelae of retinal degenerative disease are far more complex than previously believed, and schemes to rescue vision via bionic implants or stem/engineered cells are based on presumed beliefs in preservation of normal wiring and cell population patterning after photoreceptor death. Those beliefs are incorrect: retinal neurons die, migrate, and create new circuitries. Vision rescue strategies will need to be refined

PhD

dissertationThe mammalian retina is comprised of 55-60 cell types mediating transduction of photic information through visual preprocessing channels. These cell types fall into six major cell superclasses including photoreceptors, horizontal, amacrine, Muller and ganglion cells. Through computational molecular phenotyping, using amino acids as discriminands, this dissertation shows that the major cellular superclasses of the murine retina are subdivisible into the following natural classes; 1 retinal pigment epithelium class, 2 photoreceptor, 2 bipolar cell, 1 horizontal cell, 15 amacrine cell, 1 Muller cell, and 7 ganglion cell classes. Retinal degenerative diseases like retinitis pigmentosa result in loss of photoreceptors, which constitutes deafferentation of the neural retina. This deafferentation, when complete, is followed by retinal remodeling, which is the common fate of all retinal degenerations that trigger photoreceptor loss. The same strategy used to visualize cell classes in wild type murine retina was applied to examples of retinal degenerative disease in human tissues and naturally and genetically engineered models, examining all cell types in 17 human cases of retinitis pigmentosa (RP) and 85 cases of rodent retinal degenerations encompassing 13 different genetic models. Computational molecular phenotyping concurrently visualized glial transformations, neuronal translocations, and the emergence of novel synaptic complexes, achievements not possible with any other method. The fusion of phenotyping and anatomy at the ultrastructure level also enabled the modeling of synaptic connections, illustrating that the degenerating retina produces new synapses with vigor with the possibility that this phenomenon might be exploited to rescue vision. However, this circuitry is likely corruptive of visual processing and reflects, we believe, attempts by neurons to find synaptic excitation, demonstrating that even minor rewiring seriously corrupts signal processing in retinal pathways leaving many current approaches to bionic and biological retinal rescue unsustainable. The ultimate conclusion is that the sequelae of retinal degenerative disease are far more complex than previously believed, and schemes to rescue vision via bionic implants or stem/engineered cells are based on presumed beliefs in preservation of normal wiring and cell population patterning after photoreceptor death. Those beliefs are incorrect: retinal neurons die, migrate, and create new circuitries. Vision rescue strategies will need to be refined

PhD

dissertationThe mammalian retina is comprised of 55-60 cell types mediating transduction of photic information through visual preprocessing channels. These cell types fall into six major cell superclasses including photoreceptors, horizontal, amacrine, Muller and ganglion cells. Through computational molecular phenotyping, using amino acids as discriminands, this dissertation shows that the major cellular superclasses of the murine retina are subdivisible into the following natural classes; 1 retinal pigment epithelium class, 2 photoreceptor, 2 bipolar cell, 1 horizontal cell, 15 amacrine cell, 1 Muller cell, and 7 ganglion cell classes. Retinal degenerative diseases like retinitis pigmentosa result in loss of photoreceptors, which constitutes deafferentation of the neural retina. This deafferentation, when complete, is followed by retinal remodeling, which is the common fate of all retinal degenerations that trigger photoreceptor loss. The same strategy used to visualize cell classes in wild type murine retina was applied to examples of retinal degenerative disease in human tissues and naturally and genetically engineered models, examining all cell types in 17 human cases of retinitis pigmentosa (RP) and 85 cases of rodent retinal degenerations encompassing 13 different genetic models. Computational molecular phenotyping concurrently visualized glial transformations, neuronal translocations, and the emergence of novel synaptic complexes, achievements not possible with any other method. The fusion of phenotyping and anatomy at the ultrastructure level also enabled the modeling of synaptic connections, illustrating that the degenerating retina produces new synapses with vigor with the possibility that this phenomenon might be exploited to rescue vision. However, this circuitry is likely corruptive of visual processing and reflects, we believe, attempts by neurons to find synaptic excitation, demonstrating that even minor rewiring seriously corrupts signal processing in retinal pathways leaving many current approaches to bionic and biological retinal rescue unsustainable. The ultimate conclusion is that the sequelae of retinal degenerative disease are far more complex than previously believed, and schemes to rescue vision via bionic implants or stem/engineered cells are based on presumed beliefs in preservation of normal wiring and cell population patterning after photoreceptor death. Those beliefs are incorrect: retinal neurons die, migrate, and create new circuitries. Vision rescue strategies will need to be refined

Same same but different: plasticity of a 'conserved' reflex

Transformation of sensory percepts into motor output form a core element of how any animal interacts with their environment. While some such sensorimotor transformations can be very elaborate and depend on the lifestyle of a species, others serve basic functions and are ubiquitous across vertebrates. Among the latter ones are gaze-stabilizing reflexes, which serve to maintain stable vision during head motion through compensatory eye movements. Despite this conservation throughout evolution, these reflexive behaviors must remain plastic depending on context or past experience to maintain functionality after e.g. impairments of motor or sensory systems through compensation, or to changes in the environment through adaptation. In this thesis, I employ tadpoles of the frog Xenopus laevis to investigate how neuronal circuits contribute to either adaptive or compensatory plasticity on otherwise conserved gaze-stabilizing reflexes. My first study centers on the role of bilateral visual pathways in the development of the optokinetic reflex (OKR). In early embryos, I unilaterally remove the precursor of the eye, the optic vesicle. Tadpoles that develop under such monocular conditions display pathfinding errors of retinal ganglion cells at the optic chiasm. Tadpoles with near normal contralateral projections functionally compensate for the loss of one eye and show consistent responses to both leftward and rightward moving stimuli. In animals with an induced aberrant ipsilateral projection, compensation is increasingly impaired with more pathfinding errors. Combined, this study shows that binocular eyes are required for appropriate visual circuit formation, and that resulting anatomical aberrations impose limitations on compensatory plasticity. In my second study I focus on the role of the cerebellum in plasticity. Combinations of prolonged, repetitive stimulation with lesions of the cerebellum revealed adaptive plasticity of the OKR, where initially very variable OKR responses converge towards a homeostatic motor output by selective increase and decrease of response magnitude. The cerebellum is specifically associated only with response increases, and only starts to exert this influence well after initial OKR onset. This study therefore shows that multiple brain areas differentially contribute to plasticity of eye movements, leading to heterogenous appearance of different modes of plasticity throughout development. Combined, these studies contribute to the understanding of development and purpose of plasticity in Xenopus OKR. Multiple brain areas are involved with plasticity, and their formation depends on canonical, bilateral visual input. Once functional, plasticity mechanisms serve to maintain homeostasis of the OKR response in response to both adaptation and compensation

Molecular and Cellular Mechanisms Underlying Somatostatin-Based Signaling in Two Model Neural Networks, the Retina and the Hippocampus

Neural inhibition plays a key role in determining the specific computational tasks of different brain circuitries. This functional \u201cbraking\u201d activity is provided by inhibitory interneurons that use different neurochemicals for signaling. One of these substances, somatostatin, is found in several neural networks, raising questions about the significance of its widespread occurrence and usage. Here, we address this issue by analyzing the somatostatinergic system in two regions of the central nervous system, the retina and the hippocampus. By comparing the available information on these structures, we have identified common motifs in the action of somatostatin that may explain its involvement in such diverse circuitries. The emerging concept is that somatostatin-based signaling, through conserved molecular and cellular mechanisms, allows neural networks to operate correctly

GABA Expression and Regulation by Sensory Experience in the Developing Visual System

The developing retinotectal system of the Xenopus laevis tadpole is a model of choice for studying visual experience-dependent circuit maturation in the intact animal. The neurotransmitter gamma-aminobutyric acid (GABA) has been shown to play a critical role in the formation of sensory circuits in this preparation, however a comprehensive neuroanatomical study of GABAergic cell distribution in the developing tadpole has not been conducted. We report a detailed description of the spatial expression of GABA immunoreactivity in the Xenopus laevis tadpole brain at two key developmental stages: stage 40/42 around the onset of retinotectal innervation and stage 47 when the retinotectal circuit supports visually-guided behavior. During this period, GABAergic neurons within specific brain structures appeared to redistribute from clusters of neuronal somata to a sparser, more uniform distribution. Furthermore, we found that GABA levels were regulated by recent sensory experience. Both ELISA measurements of GABA concentration and quantitative analysis of GABA immunoreactivity in tissue sections from the optic tectum show that GABA increased in response to a 4 hr period of enhanced visual stimulation in stage 47 tadpoles. These observations reveal a remarkable degree of adaptability of GABAergic neurons in the developing brain, consistent with their key contributions to circuit development and function

The potential of neural progenitor cells: an investigation into their fate in vitro and after transplantation

Neuronal loss within the central nervous system (CNS) has been considered an irreversible process, as cells within the mammalian CNS fail to regenerate after degeneration and trauma. Neural stem (and progenitor) cells have been proposed as a potential source of new neurons. This investigation describes the fate of neural progenitor cells both in vitro and after transplantation. Progenitor cells were isolated from the brains of neonatal green fluorescent protein (GFP) expressing mice thereby allowing cells to be easily visualized both in culture and after transplantation. We have shown brain-derived progenitor cells (BPCs) can be maintained in defined conditions and are capable of neurogenesis and gliogenesis. Cells adopted characteristic morphologies and phenotypes of both neurons and glia as determined by immunocytochemistry. To determine the potential of BPCs to undergo neurogenesis and specifically retinogenesis we adopted three approaches: (1) transplantation, (2) coculture and (3) induction/treatment with a developmental cue. We have shown brain progenitor cells can incorporate into the developing retina and adopt characteristic morphologies and phenotypes. Furthermore, using immunocytochemistry we show BPCs expressed proteins typical of surrounding retinal neurons. We further analyzed the potential of BPCs to adopt retinal fates using P1 mouse retina in a coculture system with BPCs. When cocultured with P1 retina but not P7 retina BPCs expressed rhodopsin a protein restricted to photoreceptors. Furthermore, rhodopsin expression was induced by a soluble factor and not cell-contact mediated. BPCs have been shown to adopt neuronal fates, yet not synaptic structures. Using cholesterol as a potential differentiation cue and developmental factor we treated BPCs and analyzed their fates in vitro. Cholesterol induced dramatic morphological differentiation and pre and post synaptic protein expression among BPCs. This investigation provides the first description of brain-derived cells undergoing retinalization and illustrates the most dramatic integration of stem/progenitor cells within the CNS. The ability to produce \u27neurons\u27 from neural progenitor cells both in vitro and in vivo has tremendous implications for treating numerous conditions