REGULATION OF NEURONAL GENE EXPRESSION BY ACTION POTENTIALS
     

R. Douglas Fields, Ph.D., Principal Investigator
Serena Dudek, Ph.D., Senior Staff Fellow
Feleke Eshete, Ph.D., Postdoctoral Fellow
Laure Haak, Ph.D., Postdoctoral Fellow
Philip Lee, Ph.D., Postdoctoral Fellow
Elisabetta Tendi, Ph.D., Postdoctoral Fellow
Beth Stevens, B.S., Biologist
Kevin Becker, Ph.D., collaborator, NIA, Baltimore, MD
George DeVries, Ph.D., collaborator, Loyola University, Chicago, IL
Vittorio Gallo, Ph.D., collaborator, NICHD, Bethesda, MD
Michael Iadarola, Ph.D., collaborator, NIMH, Bethesda, MD

For More Information
R. Douglas Fields
 
Research in the Unit on Neurocytology and Physiology is concerned with understanding how the brain develops and modifies its structure and function through experience. Functional activity in the brain during late stages of fetal development and in early postnatal life is essential for normal development of the nervous system of higher vertebrates. The unit investigates the molecular mechanisms that enable neural impulse activity to regulate major developmental processes of both neurons and glia. The main objectives of the research program are to understand how the expression of genes controlling the developing structure and function of the nervous system are regulated by patterned neural impulse activity; to determine the functional consequences of neural impulse activity on major developmental processes, including cell proliferation, survival, differentiation, growth cone motility, axon bundling (fasciculation), neurite outgrowth, synaptogenesis and synapse remodeling, myelination, interactions with glia, and the mechanisms of learning and memory in postnatal animals; and to understand how information contained in the temporal pattern of neural impulse activity is transduced and integrated within the intracellular signaling networks of neurons to activate specific genes and control appropriate adaptive responses.

Action Potential Regulation of Glial Development
Stevens, Haak, Lee, Fields, Gallo, Becker, DeVries
Nerve axons are known to regulate glial development and myelination, but little is known about how impulse activity in premyelinated axons could be detected by non-neuronal cells or have functional effects on their development and myelination. Using an in vitro culture preparation equipped with stimulating electrodes and confocal calcium imaging, the unit has tested whether impulse activity stimulated in mouse dorsal root ganglion (DRG) axons would have an effect on the intracellular signaling pathways of myelinating glia in the PNS (Schwann cells) and CNS (oligodendrocyte progenitor cells). Fifteen to 90 seconds after stimulating action potentials in DRG axons, we observed a large rise in intracellular calcium in Schwann cells. We determined that the response could be blocked by degrading extracellular ATP enzymatically or blocking P2Y (ATP receptors) on Schwann cells. Using immunocytochemistry and RT-PCR, we found that a number of key genes controlling Schwann cell development were regulated by action potentials in DRG axons or by ATP application, and we identified the signal transduction pathways responsible for such regulation. Our research has determined that impulse activity in premyelinated axons can inhibit proliferation and differentiation of Schwann cells. This neuron-glial signaling is mediated by adenosine triphosphate (ATP) acting through P2Y receptors on Schwann cells and through intracellular signaling pathways involving calcium, calcium-calmodulin kinase, mitogen-activated protein kinase, CREB and expression of c-fos and krox-24. ATP arrests maturation of Schwann cells at an immature morphological stage and prevents expression of O4, myelin basic protein, and the formation of myelin. Through this mechanism, functional activity in the developing nervous system could delay terminal differentiation of Schwann cells until exposure to appropriate axon-derived signals. We are expanding our research to investigate oligodendrocyte progenitor cells to determine if similar mechanisms are involved in neuron-glial communication and myelination in the CNS.

Activity-Dependent Regulation of Gene Expression
Eshete, Tendi, Fields, Becker
Using cDNA arrays for gene expression profiling, the unit has investigated how gene expression is regulated by patterned action potential and synaptic activity in cell culture and hippocampal slice. Our work indicates that expression of large sets of genes depends on the pattern of neural impulse firing. Our work is revealing signaling pathways and gene regulatory networks that respond selectively to appropriate temporal patterns of action potential and synaptic firing. Similarly, gene expression profiling has provided novel insights into signaling pathways in Schwann cells responding to extracellular ATP and into the molecular basis for neurofibromatoma type 1 formation in human Schwann cells caused by mutations in the NF-1 gene.

Decoding Action Potential Frequency
Eshete, Lee, Fields
A fundamental problem in cellular neuroscience is to understand how action potential firing patterns are transmitted and integrated within intracellular signaling cascades to regulate neuronal responses to appropriate patterns of action potential firing. It has been proposed that the special autoregulatory properties of CaMKII, which depends on phosphorylation of Thr-286, enable the enzyme to decode the frequency and number of cytoplasmic calcium transients into graded levels of autocatalytic activity. This important theory has had a major influence in the fields of synaptic plasticity and neuronal gene expression, but it has not been tested experimentally in neurons. Using confocal calcium imaging in “xy” and single line-scan modes, in combination with biochemical measurements of CaMKII autocatalytic activity, the unit determined that the spike frequency decoding of CaMKII in response to action potentials is limited to a range of between 0.1 and 1 Hz in mouse DRG neurons. The experiments show that the frequency of calcium pulses rather than the amplitude of the calcium pulse can be more crucial in autonomous activation of CaMKII in response to repetitive stimulation. Consistent with in vitro simulations, both the duration of the calcium spike and the state of autophosphorylation before stimulation act to reduce the sensitivity of the enzyme to this low range of action potential frequencies. An unexpected finding was that the autonomous activity of the enzyme was sensitive to changes in extracellular calcium as well as to the intracellular calcium concentration. Extracellular calcium causes sustained phosphorylation of the enzyme, which is not due to secondary effects on neural impulse activity or specialized synaptic receptors. In DRG neurons, CaMKII may participate in decoding spike frequencies of less than 1 Hz, but responses to higher frequency stimulation are not explained by this mechanism.

Gene Expression in Hippocampal Long-Term Potentiation
Dudek, Fields
The conversion of short-term memory into long-term memory requires CREB-dependent gene expression and protein synthesis. The unit is investigating the intracellular signaling pathways involved in a cellular model of long-term memory: hippocampal long-term potentiation (LTP) in CA1 neurons of hippocampal brain slices. Of particular interest are the calcium-dependent intracellular signaling cascades that are critical for synaptic plasticity and gene expression in response to afferent stimulation. Mitogen-activated protein kinase (MAPK) has been identified as a potential element in regulating LTP, gene expression, and excitability in hippocampal neurons. Experiments using pharmacological inhibitors of calcium channels and NMDA channels show that different patterns of stimulation activate MAPK via calcium influx through different subcellular domains of CA1 hippocampal neurons. High-frequency and theta-burst stimulation (stimulus patterns that can induce LTP) activated MAPK via calcium influx through NMDA and L-type calcium channels. Low-frequency stimulation (5 Hz) caused phosphorylation of MAPK via calcium influx through NMDA receptors, but pharmacological blockade of L-type calcium channels had no effect on activation of MAPK by this stimulus. Activation of MAPK correlated well with the spread of the population spike across CA1 and could be blocked by suppressing the postsynaptic spike with a GABA receptor agonist. The work shows that afferent stimulation is differentially effective in activating MAPK via frequency-dependent effects on postsynaptic action potential firing and the mode of calcium entry in somatic and dendritic compartments of the neuron. Research in progress is comparing the relative contribution of somatic action potentials and synaptic potentials in e-LTP and l-LTP (a model of conversion of short-term to long-term memory) by using gene arrays, immunocytochemistry, and long-term electrophysiological recording.
 

PUBLICATIONS

  1. Dudek SM, Fields RD. Mitogen-activated protein kinase/extracellular signal regulated kinase activation in somatodendritic compartments: roles of action potentials, frequency and mode of calcium entry. J Neurosci 2001;21:RC122:1-5.
  2. Eshete F, Fields RD. Spike frequency decoding by autonomous activation of CaMKII in DRG neurons. J Neurosci 2001;21: 6694-6705.
  3. Fields, RD. Development of the vertebrate nervous system. In: The encyclopedia of the neurological sciences. San Diego: Academic Press, 2002, in press.
  4. Fields RD, Eshete F, Dudek S, Ozsarac N, Stevens B. Regulation of gene expression by action potentials: Complexity in cellular information processing. Novartis Found Symp 2001;239:160-176.
  5. Fields RD, Ozsarac N. Gene chips: applications to neuroscience. The Neuroscientist 2000;6:310-314.
  6. Fields RD, Stevens B. ATP in signaling between neurons and glia. Trends Neurosci 2000;23:625-633.
  7. Fields RD, Stevens B. Glial-neuronal culture models--do we need to change the paradigms? Trends Neurosci 2001;24:205-206.
  8. Olah Z, Szabo T, Karai L, Hough C, Fields RD, Caudle RM, Blumberg PM, Iadarola MJ. Ligand-induced dynamic membrane changes and cell deletion conferred by vanilloid receptor 1. J Biol Chem 2001;276:11021-11030.
  9. Prabhakar NR, Fields RD, Baker T, Fletcher EC. Intermittent hypoxia: cell to system. Am J Physiol 2001;281:L524-L528.
  10. Stevens B, Fields RD. Action potentials regulate Schwann cell proliferation and development. Science 2000;287:2267-2271.
  11. Stevens B, Fields RD. Regulation of the cell cycle in normal and pathological glia. The Neuroscientist 2001, in press.
  12. Zalc B, Fields RD. Do action potentials regulate myelination? The Neuroscientist 2000;6:5-13.