NERVOUS SYSTEM DEVELOPMENT AND PLASTICITY
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R. Douglas Fields, PhD, Head, Section on Nervous System Development and Plasticity Jonathan Cohen, PhD, Postdoctoral Fellow |
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| Our research is concerned with understanding the molecular mechanisms by which functional activity in the brain regulates development of the nervous system during late stages of fetal development and early postnatal life. The main objectives of our research program are (1) to understand how the expression of genes controlling the developing structure and function of the nervous system are regulated by patterned neural impulse activity and (2) 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, synapse remodeling, myelination, interactions with glia, and the mechanisms of learning and memory in postnatal animals. Action potential regulation of glial development and myelination Stevens, Dakin, Weinberg, Fields; in collaboration with Gallo The importance of neural impulse activity in regulating development of neurons is widely appreciated, though not in the case of the development of non-neuronal cells (glia). Our recent work has examined the possible influence of axonal impulse activity on developing glia, which form myelin in the peripheral (PNS) and central nervous system (CNS). Myelin is the spiral wrapping of membrane around axons that provides electrical insulation essential for rapid impulse conduction. Curiously, published evidence suggests that action potentials may inhibit myelination in the PNS but stimulate myelination in the CNS; however, the results are controversial. The mechanism for this activity-dependent axon-glial communication is unknown, as are the molecular mechanisms that would mediate effects of action potentials on myelination. We investigated this apparent paradox by taking advantage of a preparation of DRG neurons that can be stimulated in cell cultures fitted with platinum electrodes. The central axons of DRG neurons are myelinated by oligodendrocytes, and their peripheral axons are myelinated by Schwann cells. Our findings confirm the opposite effects of impulse activity in myelination of CNS and PNS and show that different purinergic signaling molecules released by axons in an activity-dependent manner control differentiation of myelinating glia in the PNS and CNS. Our studies on Schwann cells show that, in the PNS, extracellular ATP released from axon ring action potentials acts on purinergic receptors on Schwann cells (P2Y receptors), causing an increase in intracellular calcium, activation of transcription factors, and regulation of genes involved in Schwann cell differentiation. The result is that several developmental processes in Schwann cells are regulated by the release of ATP from electrically active axons, including inhibiting Schwann cell proliferation, arresting cellular differentiation at an immature stage, and inhibiting myelination. Our studies on CNS myelination reveal that oligodendrocyte
progenitor cells (OPCs) can detect impulse activity in premyelinated
axons and respond with an increase in intracellular calcium. We identified
the axon-glial signaling molecules involved and found that adenosine,
a metabolite of ATP, is of primary importance. Using RT-PCR, we detected
all four types of adenosine receptor in purified cultures of OPCs
and in acutely dissociated OPCs from transgenic mice. Impulse activity
inhibited cell proliferation of myelinating glia in both the PNS and
CNS, but adenosine derived from electrically active axons, rather
than ATP as in the PNS, was responsible for inhibiting proliferation
of myelinating glia in the CNS. Moreover, the effects of impulse activity
on differentiation and myelination were opposite in Schwann cells
and OPCs. In contrast to Schwann cells, in which our experiments showed
that electrical activity arrests differentiation, we found that action
potentials acting through adenosine receptors promoted differentiation
of OPCs to the mature stage. When cultures of DRG neurons were myelinated
by oligodendrocytes, we found that the addition of adenosine significantly
increased myelination. Even a transient exposure of OPCs to adenosine
stimulates differentiation and increases myelination by oligodendrocytes. Fields RD, Stevens-Graham B. New insight into neuron-glia communication. Science 2002;298:556-562. Stevens B, Porta S, Haak LL, Gallo V, Fields RD. Adenosine: a neuron-glial transmitter promoting myelination in the CNS in response to action potentials. Neuron 2002;36:855-868. Intracellular signaling in conversion of short-term memory into long-term memory Dudek, Fields It is widely appreciated that there are two types of memory: short term and long term. It has been well known for decades that gene expression is necessary for converting short-term into long-term memory, but it is not known how signals reach the nucleus to initiate the conversion process. Current theory holds that, in response to ring a synapse in the appropriate manner to generate a short-term memory, a signaling molecule is generated from the submembrane region of the synapse, which travels up the dendritic tree to the cell body. After entering the nucleus, the signaling molecule is presumed to initiate gene transcription by activating the transcription factor CREB. The gene product is synthesized and distributed throughout the dendritic tree, but the protein can recognize synapses that have been temporarily strengthened, rendering the change permanent. We proposed an alternative hypothesis based on our studies of action potential-dependent intracellular signaling and CREB activation in DRG neurons. The hypothesis proposed that, rather than generating a specialized synapse-to-nucleus signaling molecule, somatic action potentials could be responsible for activating gene transcription to convert short-term into long-term memory via the same intracellular signaling pathway that we previously studied in DRG neurons. We tested the hypothesis by backring axons from CA1 hippocampal neurons in slice preparations in the absence of glutamate neurotransmitter blockers, causing neurons to re somatic action potentials in the absence of all excitatory synaptic input in brain slice preparations. Following stimulation, we analyzed the slices by immunocyochemistry for activation of CREB and other signaling enzymes involved in long-term potentiation (LTP) of synapses as well as for expression of genes closely associated with LTP induction. The results showed that backring the axons by antidromic stimulation was sufficient to activate CREB and the signaling enzyme MAPK in CA1 neurons in the absence of all excitatory synaptic stimulation. When slices were stimulated antidromically, the outcome was synaptic stimulation that normally results in a transient strengthening of the synapse (e-LTP) rather than a long-lasting strengthening of the synapse (l-LTP). These results, which support our hypothesis and eliminate the necessity of a synapse-to-nucleus signaling molecule for l-LTP, are consistent with the original theory of Hebb that states that the ring of a neuron determines whether or not it forms a stronger synaptic connection with another neuron. Dudek MS, 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 Neuroscience 2001;21,RC122:1-5. Dudek S, Fields RD. Somatic action potentials are sufficient for late-phase LTP-related cell signaling. Proc Natl Acad Sci USA 2002;99:3962-3967. Gene expression in hippocampal synaptic plasticity Cohen, Lee, Fields; in collaboration with Becker Long-term potentiation (LTP) and long-term depression (LTD) are two widely studied forms of synaptic plasticity that can be recorded electrophysiologically in the hippocampus; these phenomena are believed to represent a cellular basis for memory. We are using custom-made cDNA microarrays to investigate the signaling pathways, genes, and proteins involved in these forms of synaptic plasticity. Our research has identified sets of transcription factors, structural genes, and signaling pathways that are regulated specifically by activity patterns leading selectively to different types of synaptic plasticity. The objective of our research is to understand the how gene-regulatory networks are controlled by the appropriate patterns of impulses leading to different forms of synaptic plasticity and to identify new molecular mechanisms regulating synaptic strength. Cohen JE, Fields RD. Extracellular calcium depletion in synaptic transmission. The Neuroscientist 2003; in press. Regulation of gene expression by action potential ring patterns Lee, Tendi, Cohen, Fields; in collaboration with Becker, Waxman Using custom cDNA arrays for gene expression profiling, we have investigated how gene expression is regulated by patterned action potential ring in neuronal cell culture. The pattern of impulse activity is regulated by electrical stimulation through platinum electrodes in specially designed cell culture dishes. After stimulation, we measure mRNA and protein expression by gene arrays, quantitative RT-PCR, Western blot, and immunocytochemistry. Our work tests the hypothesis that gene transcription can be regulated by the pattern of action potential ring. The results obtained thus far reveal signaling pathways and gene regulatory networks that respond selectively to appropriate temporal patterns of action potential ring in neurons. Our findings provide a better understanding of how nervous system development and plasticity may be regulated by information coded in the temporal pattern of impulse ring in the brain.
Eshete F, Fields RD. Spike frequency decoding by autonomous activation of CaMKII in DRG neurons. J Neurosci 2001;21:6694-6705. Klein JP, Tendi EA, Black JA, Fields RD, Waxman SG. Patterned electrical activity modulates sodium channel expression in sensory neurons. J. Neurophysiology 2003;74:192-198. Neurobromatosis Lee, Tendi, Cohen; in collaboration with Becker, DeVries Neurobromatosis (NF-1) is an autosomal dominant condition leading to nerve sheath tumors that can develop into malignant cancer. We are using gene expression profiling in combination with quantitative RT-PCR and biochemical methods to investigate the processes involved in the transition from a benign to a malignant tumor. We are using three types of custom-made microarrays of genes involved in cell signaling, immune system function, and nervous system expression to compare mRNA expression in normal human Schwann cells and a Schwann cell line derived originally from an NF-1 patient who died from a highly malignant form of neurobromatosis. Our studies identify hundreds of genes that are dysregulated in the malignant Schwann cells, including those associated with all aspects of cellular function associated with malignancy, such as, for example, those regulating cell proliferation, motility, growth factor, and immune system responses. COLLABORATORS Kevin Becker, PhD, Research Resources Branch, NIA, Baltimore MD Vittorio Gallo, PhD, George Washington University, Washington DC *Departed NICHD in 2002 For further information, contact fields@helix.nih.gov |
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