William J. Moody - US grants

This project will examine the development of electrophysiological properties in neural-lineage cells of the early Xenopus embryo. Although much attention has focussed on the modulation of populations of ion channels during the terminal differentiation of specific sets of post-mitotic neurons, very little information is available about the development of ion channel populations in neural-lineage cells at earlier stages, when initial commitment to neural phenotypes occurs. Dr. Moody will use patch clamp techniques to study this problem in the Xenopus embryo, at stages between the unfertilized oocyte and the neurula. He will examine the stages at which cells involved in the formation of the embryonic nervous system first express functional ion channel populations distinct from cells of other fates, and then investigate the developmental processes by which the electrical properties of the oocyte are transformed into those of cells committed to neuronal phenotypes. Particular attention will be paid to the roles of ion channel synthesis, the modulation of pre-existing channels, and the spatial distribution of ion channels over the surface of single cells in the early electrophysiological differentiation of the nervous system.

To be adaptive, an animal's behavior must change depending on the organism's physiological or developmental state. Dr. Truman's research is directed towards understanding both external (e.g. circadian rhythms) factors and internal (e.g. steroid and peptide hormones) factors that influence the central nervous system to regulate behavior. Using a relatively simple invertebrate model system, he is unraveling the mechanisms by which ecdysteroids, a class of steroid hormones, affect the release of eclosion hormone, a neuropeptide, to orchestrate changes in behavior. Dr. Truman has identified four neurons that are responsible for all of the eclosion hormone found in the central nervous system. He will now focus on the cellular properties of these neurons. Using state of the art electrophysiological and anatomical techniques, Dr Truman will define the mechanisms that control the release of this peptide, and then determine how it acts on the central nervous system to evoke the stereotyped behavior changes. In addition, he will employ a different invertebrate model system to examine the importance of circadian control in controlling behavior. The results from this research will provide novel insights into the relationship between the neuroendocrine system and behavior. These data serve as the foundation for the development and application of practical procedures in controlling agricultural and medical insect pests.

IBN-9708656 PI: MOODY The neurons that populate the mammalian brain are generated during embryonic development from cells that line the ventricles. These cells divide repeatedly, and then migrate away from the ventricles to populate the brain itself. It is critically important that the processes of division and migration occur correctly in order for the brain to develop normally. Although it is well known that mature neurons in the brain use electrical signalling to process information, there is also some evidence that modified forms of electrical signalling are used during the processes of division and migration that create the brain during early embryogenesis. Dr. Moody will use quantitative techniques to record electrical signals from individual cells lining the ventricles of the embryonic mouse brain to determine how such signals are used in early brain development. By using living slices of embryonic brain tissue, it will be possible to identify individual cells according to their state of division and migration, and to then detemine the exact nature of the electrical signals they can generate. Once this is understood, specific drugs that block different aspects of those signals will be used to determine whether normal patterns of division and migration require this type of signalling. Results from this project will allow us to understand whether the normal adult forms of electrical communication between neurons in the brain are also used in modified form to mediate critical aspects of neural development.

Spontaneous electrical activity plays a major role in nerve and muscle development. In the vertebrate central nervous system, spontaneous activity is required for the establishment of correct neuronal position, morphology, and connectivity. This spontaneous activity is distinct from later experience-dependent activity: it occurs without sensory input, and in some cases in completely isolated cells. It must therefore be controlled by the ion channels present in individual cells at early stages of development. Supporting this idea are findings that the properties of ion channels present early in development are often markedly different from those in the mature cell. Studies of ion channel development that have provided these ideas have primarily been done in invertebrates and lower vertebrates, whereas many studies of the developmental roles of spontaneous activity have been done in mammalian brain. In fact, there is very little information about the development of ion channel properties in the prenatal mammalian neocortex. The neurons of the cortex arise from divisions of ventricular one progenitor cells, which then migrate into the cortical plate. In mouse, these divisions occur between embryonic days 11 and 17. Our preliminary patch clamp experiments during this neurogenic interval have shown interesting differences in voltage-gated ion channel populations between neural progenitor cells and the radial glia along which they migrate, as well as marked change in Na and K currents as migrating neurons leave the ventricular zone. The proposed experiments use patch clamp to measure ion channel properties, dye fills to determine cell morphology, and immunocytochemistry to determine cell identity, to study the development of voltage-gated ion channels in ventricular zone cells and migrating neurons during the neuronogenic interval in the embryonic mouse. This data will be used to detect periods of development during which the populations of channels present mediate spontaneous activity, and then to develop strategies to block that activity and thus determine its developmental functions. Diseases that disrupt electrical activity in the developing fetal brain, such as epilepsy, are likely to have profound effects, because activity serves a developmental role at early stages, in contrast to its information processing role in the adult. The studies proposed here bear on the mechanisms by which such disruptions occur.

Spontaneous electrical activity plays central roles in nervous system development, helping to control how neurons migrate, make synaptic connections with one another, and develop their ability to generate electrical signals appropriate for their mature functions. How spontaneous activity is generated during nervous system development is only poorly understood.
The present project results from the discovery in the last year of a striking form of spontaneous electrical activity in the mouse cerebral cortex near the time of birth. Brain slice calcium imaging methods revealed that on the day of birth (P0) neurons of the cortex generate spontaneous transient increases in intracellular calcium concentration ([Ca2+]i transients) that are highly synchronized across very large populations of nerve cells. These [Ca2+]i transients result from electrical activity, and occur synchronously in 60-80% of all cortical neurons in all cortical layers and regions. This activity is seen almost exclusively on the day of birth. The onset of this activity just before birth seems to be regulated by a large increase in Na+ current density, and the cessation of activity by a large increase in resting conductance just after P0.
This project investigates the role of voltage-gated Ca2+ currents in admitting Ca2+ ions into the neurons during this spontaneous activity. Ca2+ is first step in the mechanism by which spontaneous activity carries out its developmental functions, and both the frequency of [Ca2+]i transients and the exact ion channels through which Ca2+ enters the cell are crucial parameters. The recent experiments have shown that there is a dramatic increase in total Ca2+ current density in cortical neurons near P0, suggesting that Ca2+ current development is coordinated with the activity. Patch clamp and Ca2+ imaging methods will be used to determine what Ca2+ channel subtypes are present in cortical neurons at P0, what the patterns of their development in the perinatal period are, and whether and to what extent they participate in Ca2+ entry leading to [Ca2+]i transients during spontaneous activity.

Intellectual merit. These experiments will elucidate how the intrinsic ion channel properties of cortical neurons, and the patterns of development of those channels, help shape developmentally critical spontaneous activity in the brain. The laboratory is one of the first to discover such activity in cortex, and to map the ion channel development leading up to it. The laboratory has also carried out extensive prior studies of ion channel development and the roles of spontaneous activity in invertebrate and amphibian preparations. The mechanism by which Ca2+ enters neurons during spontaneous activity is a primary determinant of how electrical activity carries out its important roles in nervous system development.

Broader impacts. In addition to adding fundamentally to our understanding of nervous system development, this project will occur in an environment particularly suited to integrating research and undergraduate education. As director of the undergraduate Neurobiology Major, Dr. Moody regularly advises undergraduate researchers in his own laboratory. In fact, the discovery of spnontaneous activity in mouse cortex was made by one such undergraduate, who presented her findings at the most recent Society for Neuroscience meeting. Experiments on Ca2+ current development in cortical neurons are being carried out by another undergraduate who has just started in the lab. In teaching the introductory course in the major, The Lab's research is brought into the classroom as an example of integrating biophysical and developmental studies of neurons.

DESCRIPTION (provided by applicant): Spontaneous waves of electrical activity propagate across many structures of the central nervous system during critical stages of early development. It is now known that specialized pacemaker neurons are responsible for initiating these waves, but it is not clear how such pacemakers operate, or what properties determine some neurons to initiate the waves and other neurons to propagate the waves once they are initiated. A great deal of attention has been paid to how the synaptic interactions between neurons serve to initiate and propagate spontaneous waves. Very little is known, however, about how these immature neurons transform their synaptic inputs to spike train outputs, and how this computation is involved in wave initiation and propagation. Recent collaborative experiments of our two laboratories have used white noise current stimuli delivered to single neurons in the developing mouse cortex to try to understand how these neurons extract features from their synaptic inputs and compute their outputs. This work has shown that near the end of the first postnatal week, cortical neurons acquire the ability to scale their output function to the amplitude of their inputs, thus reducing the gain between input amplitude and spike frequency output. At late embryonic and early postnatal stages, however, many cortical neurons lack this gain scaling ability. These early stages correspond to those at which spontaneous waves of activity are generated in the cortex. Recent experiments in one of our laboratories have shown that cortical waves are driven by a pacemaker population in the ventrolateral quadrant of the cortex. We propose here to test the hypothesis that the pacemaker neurons in this region are the neurons that show the most pronounced lack of gain scaling, and that this inability to scale output to input amplitude effectively is one of the properties required for their pacemaking function. High-speed calcium imaging will be used to identify the location of the pacemaker in individual slices, and then whole-cell recordings and white noise stimuli will be applied to neurons in that region to measure their gain scaling ability. This will be compared to neurons in follower regions to see whether lack of gain scaling correlates with pacemaker function. These data will be combined with recordings of synaptic inputs in the pacemaker neurons to create neuronal models to test whether the inability to scale spike train outputs to synaptic input amplitudes is the computational property that determines pacemaker function. PUBLIC HEALTH RELEVANCE: Waves of electrical activity cross the brain during early development and are essential for the correct wiring of brain circuitry. The present experiments study how the properties of single neurons cause them to trigger this activity. The work will provide insights into defects in brain development that occur in humans, and in particular how abnormal electrical activity, such as seizures, can disrupt brain development.

Neurons that are being formed in the brain of a developing animal send out electrical signals that spread like waves over large parts of the brain. The waves are essential for normal brain development. The goal of this project is to find out how this spontaneous electrical activity controls brain development. Recently, a specific type of neuron has been identified as being the pacemaker, or trigger, for these waves. This project will study how these neurons trigger the spontaneous waves. The project will take advantage of a mouse that allows these neurons to be stimulated using light. The responses of the neurons to these stimuli will be monitored, and the results will be used to make computer models of the neurons. These models will reveal the properties of these neurons that allow them to produce the waves. The project will offer opportunities for undergraduate and graduate students to be trained in interdisciplinary research that involves both theoretical and experimental biology, under the guidance of two collaborating principal investigators with unique expertise in these areas. Emphasis will be placed on actively recruiting women into full participation in the computational aspects of the project.

This project investigates how the intrinsic electrical properties of developing GABAergic interneurons in the cerebral cortex allow them to initiate spontaneous waves of electrical activity during early development. Previous genetic and pharmacological data indicate that these neurons, which are excitatory during early development, are the primary pacemakers for waves of spontaneous activity in the mouse cortex between embryonic day 18 and and postnatal day 3. The detailed input:output relations of GABAergic neurons will be determined by using calibrated optical stimulation in a dlx5/6 channel-rhodopsin mouse and by recording the outputs of the neurons with extracellular electrode arrays. Conductance-based models of the neurons will be constructed that reflect the diversity of intrinsic properties that are encountered in the population. The model neurons will be connected into synaptic circuits to determine whether the measured properties lead to pacemaking activity in the circuit. By systematically varying the intrinsic properties in the model, aspects of the intrinsic properties that are critical for pacemaking function will be determined. If successful, this project will provide a new high-throughput method for determining how the intrinsic properties of neurons determine circuit output in the brain.