Angeles B. Ribera - US grants

This proposal addresses questions about the development of modulation by neurotransmitters of the action potential of dorsal root ganglion cells of Xenopus tadpoles. My goals are (a) to determine the extent of modulation by GABA, serotonin or enkephalin of the action potential of dorsal root ganglion cells, (b) to determine the timing of the acquisition of neuromodulation by examining the correleation of neuromodulation with (i) the type of action potential expressed by the neuron, (ii) the age of the cell examined, (iii) the developmental stage of the tadpole, (c) to identify the membrane current that is sensitive to modulation, and (d) to investigate the mechanism of this modulation with respect both to developmental properties and roles of likely molecular intracellular components (GTP binding proteins, protein phosphorylation). Blockers and stimulators of these regulatory mechanisms will be used. The studies will be carried out on in vivo preparations of Xenopus tadpoles over a range of ages. Intracellular recording under current clamp conditions will be used to measure membrane potentials, action potentials, input resistance, and membrane potential and conductance changes in response to application of neurotransmitter. The effects of modulators on individual membrane currents (e.g., Na+, Ca2+, or K+ current) from neurons whose intracellular contents from cell- attached patches will be recorded using patch clamp techniques. Cell birthdating will be accomplished by 3H-thymidine labeling and autoradiographic analysis of dorsal root ganglion cells in Xenopus tadpoles whose exposures to label are begun at different stages between 1.5 and 45 days of embryonic development.

Developmental changes in the expression of voltage-dependent potassium channels have significant consequences for transmission of electrical signals in the nervous system. The phenotype of the action potential of embryonic amphibian (Xenopus laevis) spinal neurons is determined to a large extent by the balance between calcium and potassium currents. This balance is normally altered during development to permit an early transient period of impulses that have long duration calcium-dependent plateaus. As potassium currents mature and dominate the balance of current, the calcium-dependent plateaus are suppressed and the action potential becomes a brief principally sodium-dependent spike. Recent work has demonstrated that the genes encoding potassium channels in Drosophila and mammals comprise a gene family. A Xenopus potassium channel gene (XSha2) has been cloned by reducing stringency screening with a Drosophila potassium channel clone (Shaker). Its coding region is contained within a single uninterrupted exon. Southern analysis of Xenopus genomic DNA suggests the presence of a gene family. Three specific aims are focussed to identify the members of a potassium channel gene family and their tissue specific expression; their functional properties; and their roles in the developing nervous system as deduced by overexpressing or by blocking their expression in embryos. The research plan consists of applying both molecular and physiological analyses to the study of the development of potassium current expression and its role in regulating neuronal excitability. Techniques to be employed include use of the polymerase chain reaction and standard genomic library screening for the identification of potassium channel clones, RNase protection assays to determine levels of transcript expression, functional expression in oocytes followed by two electrode voltage clamp recordings, and molecular manipulations leading to misexpression of potassium channel transcripts in the developing embryo. If the developmental program of excitability is established at the level of transcription, overexpression and block of potassium channel transcripts should be sufficient to eliminate and prolong, respectively, the early period of calcium dependent impulses. These genetic manipulations will thus alter the amount of intracellular calcium provided to immature neurons by an excitable membrane. Subsequent aspects of neuronal development may be directed by early transient elevations in intracellular calcium. These studies may point to regulatory mechanisms linking electrical activity and neuronal differentiation.

This proposal addresses questions about the development of modulation by neurotransmitters of the action potential of dorsal root ganglion cells of Xenopus tadpoles. My goals are (a) to determine the extent of modulation by GABA, serotonin or enkephalin of the action potential of dorsal root ganglion cells, (b) to determine the timing of the acquisition of neuromodulation by examining the correleation of neuromodulation with (i) the type of action potential expressed by the neuron, (ii) the age of the cell examined, (iii) the developmental stage of the tadpole, (c) to identify the membrane current that is sensitive to modulation, and (d) to investigate the mechanism of this modulation with respect both to developmental properties and roles of likely molecular intracellular components (GTP binding proteins, protein phosphorylation). Blockers and stimulators of these regulatory mechanisms will be used. The studies will be carried out on in vivo preparations of Xenopus tadpoles over a range of ages. Intracellular recording under current clamp conditions will be used to measure membrane potentials, action potentials, input resistance, and membrane potential and conductance changes in response to application of neurotransmitter. The effects of modulators on individual membrane currents (e.g., Na+, Ca2+, or K+ current) from neurons whose intracellular contents from cell- attached patches will be recorded using patch clamp techniques. Cell birthdating will be accomplished by 3H-thymidine labeling and autoradiographic analysis of dorsal root ganglion cells in Xenopus tadpoles whose exposures to label are begun at different stages between 1.5 and 45 days of embryonic development.

DESCRIPTION (provided by applicant): Normal formation and function of spinal cord circuits requires differentiation of sensory, motor and interneuron subtypes. Our work addresses the role that voltage-gated potassium (Kv) channels play in neuronal subtype differentiation in the embryonic vertebrate spinal cord. We propose that a select subset of Kv channels regulate neuronal differentiation by determining electrical membrane properties that control spontaneous elevations of intracellular calcium, known as calcium (Ca) spikes. Long-duration action potentials occur spontaneously and trigger Ca spikes during a restricted ~6 hr period prior to synapse formation (Stages [St] 20-28). The frequency of Ca spikes determines downstream effects on differentiation programs. We have found that a subset of Kv channels show patterned expression in the embryonic spinal cord and regulate Ca spike properties differently in the dorsal versus ventral embryonic spinal cord. We test the roles of dorsally-expressed Kv channel (Kv1.1) in differentiation of spinal interneurons (Aim 1) and ventrally-expressed Kv channel (Kv2.2) in differentiation of motor neurons (Aim 2). For Aim 3, we focus on subtypes within the motor neuron population and test whether differences in Kv currents account for subtype-specific Ca spike properties and encoded developmental signals Our studies take advantage of the experimental strengths of the Xenopus and zebrafish embryo models and our experience using (1) antisense (AS), morpholino (MO) and dominant negative (DN) overexpression methods in Xenopus and zebrafish embryos, (2) zebrafish genetic mutants and transgenic lines, (3) electrophysiological recording and Ca imaging from neurons in vivo, and (4) morphological methods to analyze spinal cord development. The results of the proposed experiments will provide new insights into molecular mechanisms that allow early spinal cord neuron activity to direct generation of the diverse neuronal identities required for formation of functional circuits.

neurogenetics; embryogenesis; membrane channels; nonmammalian vertebrate embryology; developmental genetics; gene mutation; developmental neurobiology; neurogenesis; species difference; animal genetic material tag; immunocytochemistry; zebrafish;

Electrical activity plays key roles during neuronal development including regulation of process outgrowth, synapse formation and patterns of gene expression. Our long-term goal is to define the mechanisms and molecules that direct cell-type specific and developmentally regulated expression of ion channel genes that are essential for rapid signaling of electrically excitable membranes. Our recent work has demonstrated that despite uniformity in the biophysical properties of voltage-dependent potassium current in spinal neurons, expression of voltage-dependent potassium (Kv) genes is heterogeneous. The hypothesis underlying this proposal is that members of 3 different potassium channel gene subfamilies (Kv1 alpha, Kv2 alpha and Kv beta) contribute to the voltage- dependent potassium current of different spinal neuron subtypes, Kv1 alpha and Kv2 alpha are pore-forming subunits, whereas Kv beta is an auxiliary subunit associated with Kv1 channels. There are 4 Specific Aims: 1) Identify the Kv1 alpha subunit isotypes that are expressed in Xenopus embryonic primary spinal neurons. 2) Examine the role of auxiliary Kv beta subunits in developmental regulation of Kv1 currents. 3) Determine the contribution of Kv2 alpha subunits to the endogenous voltage-dependent potassium current. 4) Identify the potassium channel Kv1 alpha, Kv2 alpha and Kv beta subunits that contribute to voltage-dependent potassium current in specific neuronal subtypes. A combination of embryological, electrophysiological, molecular biological and immunological techniques will be used to fulfill these Aims. The experimental preparation - the Xenopus embryo provides an ideal system for analysis of gene function in embryonic neurons, because manipulation of gene expression is easily achieved by microinjection of RNA. The information provided by these studies will allow us to define the role of electrical excitability in differentiation and function of the emerging nervous system, leading to the potential treatment of developmental disorders of the brain.

[unreadable] DESCRIPTION (provided by applicant): Our research seeks to identify the essential roles of specific sodium channels in directing nervous system development. Our studies exploit the experimental advantages of zebrafish for genetic and molecular manipulations, embryological analyses, in vivo live imaging and physiological study. Multigene families (SCNA/scna) encode voltage-gated sodium channel a-subunits in both mammals (Nav1) and zebrafish (nav1). Each sodium channel gene (SCNA/scna) displays a specific expression pattern with respect to space and time. Although several isotypes are expressed during development of the nervous system, little is known about the developmental roles that sodium channels play in the embryonic vertebrate nervous system. Our studies focus on the scn8aa gene, because it is expressed during vertebrate nervous system development. Further, knock-down of its encoded protein, nav1.6a, perturbs development of several neuronal populations in the spinal cord and periphery. In mammals, genetic elimination of the homologous gene (SCN8A) leads to severe neurological deficits as assessed at late postnatal stages. However, the extent to which embryonic events contribute to the phenotypes has not been investigated. The results of the proposed studies are expected to provide important new information about the role(s) of voltage-gated sodium channels in directing key aspects of spinal cord and peripheral sensory neuron development. We propose two specific Aims that will test the roles of nav1.6a in development of motor neurons (Aim 1) and neural crest-derived dorsal root ganglia (Aim 2). Importantly, our preliminary data indicate that nav1.6a acts both cell-autonomously as well as non-cell-autonomously in the embryonic nervous system. Overall, the proposed studies are expected to identify essential roles of nav1.6a in the developing vertebrate nervous system. [unreadable] [unreadable] [unreadable]

This application is for support of collaborative research conducted by a U.S. Principal Investigator (Angeles B. Ribera) and a Foreign Scientist (Manuel Kukuljan; Chile). The principal goals of the proposed research are to isolate cDNAs corresponding to the Xenopus orthologue of Slo (xSlo). The Slo gene encodes the pore-forming "-subunit of large conductance potassium (BK) channels. In Xenopus embryonic spinal neurons, BK channels undergo a developmentally regulated change in their sensitivity to calcium. By isolating variants of Slo expressed in embryonic spinal neurons during the developmental period of interest, the investigators will examine the possibility that alternatively spliced variants of xSlo are expressed and that these xSlo variants determine the observed developmentally regulated calcium sensitivity of BK channels. There are 3 Specific Aims: 1) isolate the Xenopus laevis orthologue of the Slo gene (xSlo), 2) analyze expression of xSlo and alternatively spliced forms in Xenopus embryonic spinal neurons, and 3) determine the functional properties of xSlo and variants. The proposed experiments combine embryological, molecular and electrophysiological techniques. The majority of the experiments will be carried out at the foreign site. The long-term goal of this research is to obtain molecular entry into developmental pathways that regulate ion channel expression and function.

Overall Component ? Project Summary The primary goal of the Rocky Mountain Neurological Disorders Center Core (RMNDC) is to provide resources that will allow neuroscientists on the Anschutz Medical Campus to incorporate transformative technologies into their research. Here, we target cutting edge neuroscience tools that are relatively new and considered to be those that will have the highest impact on our on-going research, allow investigators to incorporate powerful new approaches into their research programs and catalyze new collaborations. We propose three Research Cores (A, Optogenetics & Neural Engineering; B, Nanoscopy; C, Behavioral and In Vivo Neurophysiology) that will provide powerful tools that advance strategically the impact of the research and productivity of our neuroscience community. The new Cores will provide services not available elsewhere on campus. Moreover, these Cores focus on research tools that are of high impact and use to our neuroscience community. We prioritize staffing the Cores with experts in the equipment and tools so that investigators without prior experience will be in a position to incorporate new methods into their research programs appropriately and to the most exacting standards. Overall, the Cores will open up new avenues of research that are essential for on-going work of the current 44 RMNDC PIs. Thus, the Cores will promote research regarding fundamental mechanisms underlying nervous system development and function that is required to find innovative and more effective treatments for neurological disease.

ABSTRACT NOT PROVIDED

DESCRIPTION (provided by applicant): This is the second resubmission of a competing renewal for a neurobiology training grant now in its 26th year. The program is called BNAT, which stands for Basic Neuroscience Advanced Training. This reflects our goal to provide training for advanced pre-doctoral students and postdoctoral fellows in basic neuroscience, especially molecular, cellular, and developmental neuroscience. In the previous submission, we proposed significant new changes in the program in response to the initial review. We have begun to implement those changes. In particular, we have strengthened the administrative structure of the program, and we have created a new programmatic focus on fluorescence imaging, involving the creation of genetically-encoded fluorophores and the use of advanced fluorescence microscopes. In this revised application, we have further strengthened the BNAT administrative structure, as suggested by Reviewer 2, particularly in terms of creating a database of pre- and postdoctoral applicants. In addition, we have proactively made a major new commitment to minority training, by establishing a formal collaborative partnership with the Denver School of Science and Technology. Finally, our fifteen trainers remain very active researchers, being principal investigators on 31 extramural research grants, 17 of which are NIH R01s.

ABSTRACT NOT PROVIDED

Zebrafish Core C3.1 Rationale. Core C maintains wild type, transgenic and mutant zebrafish strains for RMNDC investigators. The unique developmental and optical imaging properties of the zebrafish embryo makes it an ideal model to address questions at the heart of the research programs RMNDC investigators. UCD NINDS researchers are internationally recognized and expert in the fields of neurophysiology, neurodevelopment and optical imaging. During the previous award period. Core C removed technical barriers for RMNDC investigators who were interested in using the model system (e.g., Betz, Caldwell, Clouthier, Niswander, Restrepo, Taylor, Williams) but did not have experience or means for maintaining lines. We propose three Specific Aims for Core C that will allow RMNDC members to take advantage of the zebrafish system for neurodevelopmental and behavioral studies. In addition to the 13 current Core C users, the facility will allow access to the zebrafish model to investigators who have not considered its use because of the pragmatic limitations associated with maintaining and breeding zebrafish. In particular, there has been growing interest in complementing rodent studies (e.g.. Core B) with ones done in zebrafish (e.g., Appel, Artinger, Barlow, Clouthier, Macklin, Niswander, Restrepo, Williams), and vice versa. The core will also be a resource for the larger scientific community because transgenic lines will be described on the RMNDC website and made available to the Zebrafish International Resource Center (ZIRC) by the time of the first publication.

DESCRIPTION (provided by applicant): Although adult neurogenesis provides new neurons to some regions of the central nervous system, whether this also occurs in the peripheral nervous system is not known. Our recent in vivo studies in zebrafish larvae suggest that another mechanism might serve as a source of new neurons for the peripheral nervous system. We have found that a subset of differentiated dorsal root ganglion neurons to migrate new ventral locations and acquire new morphologies and molecular properties. The novel characteristics are all indicative of adoption of a new identity as a sympathetic ganglion neuron. Importantly, dorsal root ganglion neurons can acquire sympathetic ganglion neuron-like properties in wild type larvae under control conditions. However, sensory deprivation and blockade of a specific sodium channel, Nav1.6a, increases the number of dorsal root ganglion neurons that acquire a new identity, i.e., transdifferentiate. Even though Nav1.6a is normally expressed in several neurons, the channel's activity is required in only one cell type for maintenance of dorsal root ganglion identity. Interestingly, that cell type is not the dorsal root ganglion neuron but rather n earlier appearing sensory neuron, known as the Rohon-Beard cell. Our previous work has also shown that the neurotrophin BDNF mediates that Nav1.6a-activity-dependent signal. Recently, we have found that differentiated dorsal root ganglion neurons migrate to yet other locations besides the sympathetic ganglion, raising the possibility that migratory dorsal root ganglion neurons might assume other fates than that of the sympathetic ganglion neuron. Here we propose three Specific Aims that will provide important information about the underlying mechanisms involved in the relevant BDNF- dependent signaling. In Aim 1, we identify the cell types that secrete and respond to the relevant BDNF. In Aim 2, we determine the full range of cellular identities that migratory dorsal root ganglion neurons may adopt. Aim 3 experiments identify gene expression changes that underlie BDNF's regulation of the migratory phenotype of differentiated DRG neurons. The results will provide information that could lead to alternative strategies for treatment of neurodegenerative conditions.

Administrative Core Summary The Administrative Core provides the leadership, oversight and management of the Rocky Mountain Neurological Disorders Center Core (RMNDC). This Core collaborates with the three Research Cores to monitor Core use and the impact of the Research Cores, and to maintain a detailed Core user base. The Administrative Core will also collaborate with the three Research Cores in informational and educational efforts and conduct surveys designed to provide feedback about the value and ?user-friendliness? of Core services. These functions are essential for assessment of the effectiveness of the Center Core, its alignment with the NINDS mission, and to plan strategically for development of future innovative services.