NERVOUS SYSTEM DEVELOPMENT AND PLASTICITY
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R.
Douglas Fields, PhD, Chief,
Section on Nervous System Development and Plasticity Philip
Lee, PhD, Research Fellow Jonathan
Cohen, PhD, Postdoctoral Fellow Tomoko
Ishibashi, PhD, Postdoctoral Fellow Kelly
Dakin, BS, Predoctoral fellow Brian
Weinberg, BS, Technician Beth Stevens, BS,
Biologist |
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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 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 to determine the
functional consequences of neural impulse activity on major developmental
processes. Areas of study include cell proliferation, survival,
differentiation, neurite outgrowth, synaptogenesis and synapse remodeling,
myelination, interactions with glia, and the mechanisms of learning and
memory. Activity-dependent
neuron-glia interactions Stevens, Dakin,a
Ishibashi, Fields; in collaboration with Gallo While
the importance of neural impulse activity in regulating development of
neurons is widely appreciated, such is not the case for development of glia
cells, which are non-neuronal. Our recent work has examined the possible
influence of axonal impulse activity on developing glia, which form myelin in
the peripheral nervous system (PNS) and central nervous system (CNS). Myelin
is the spiral wrapping of membrane around axons that provides electrical
insulation essential for rapid impulse conduction. Our findings show that
distinct purinergic signaling molecules released by axons in an
activity-dependent manner control differentiation of myelinating glia in the
PNS and CNS. In
the PNS, our studies on Schwann cells show that extracellular ATP released
from axons firing 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, processes that include inhibiting Schwann cell proliferation,
arresting cellular differentiation at an immature stage, and inhibiting
myelination. In
studying CNS myelination, our research reveals that oligodendrocyte
progenitor cells (OPCs) can detect impulse activity in premyelinated axons
and respond with an increase in intracellular calcium. We identified many of
the axon-glial signaling molecules involved in impulse activity and found
that adenosine, a breakdown product 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 stimulated OPC
differentiation to a promyelinating stage and increased myelination. These
findings open new avenues of research into oligodendrocyte development with
potential for new therapeutic approaches to treating demyelinating disease. Fields RD. Opposite effects of impulse activity on myelination
in the PNS and CNS via differential axon-glial signaling molecules. Neurochem
Int 2003;45:503-509. Stevens B, Ishibashi T, Chen J-F, Fields RD. Adenosine: an
activity-dependent axonal signal regulating MAP kinase and proliferation in
developing Schwann cells. Neuron Glia Biol 2004;1:23-34. 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,b
Cohen, 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 posits that, in response to firing a synapse in the appropriate manner
to generate a short-term memory, a signaling molecule generated from the
submembrane region of the synapse 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. The identity of this putative
synapse-to-nucleus signaling molecule remains unknown. 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 studied
in DRG neurons. We tested the hypothesis by causing neurons to fire somatic
action potentials in the absence of all excitatory synaptic input in brain
slice preparations. Specifically, we backfired axons from CA1 hippocampal
neurons in slice preparations in the presence of glutamate neurotransmitter
blockers. Following stimulation, we analyzed the slices by
immunocytochemistry for activation of CREB and other signaling enzymes
involved in long-term potentiation (LTP) of synapses and for expression of
genes closely associated with LTP induction. The results showed that
backfiring the axons by antidromic stimulation was sufficient to activate
CREB and the signaling enzyme MAPK in CA1 neurons in hippocampal slices in
the absence of all excitatory synaptic stimulation. When slices were
stimulated antidromically, synaptic stimulation that normally results in a transient
strengthening of the synapse (e-LTP) resulted instead in long-lasting
strengthening of the synapse (l-LTP). The results support our hypothesis and
eliminate the necessity of a synapse-to-nucleus signaling molecule for l-LTP
and are consistent with the original theory of Hebb, which states that the
firing of a neuron determines whether it forms a stronger synaptic connection
with another neuron. 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 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. Our objective
is to understand 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. Neuroscientist 2003;9:12-17. Regulation
of gene expression by action potential firing patterns Lee, 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 firing 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, mRNA and protein expression are measured 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 firing. Results thus far reveal signaling pathways and
gene-regulatory networks that respond selectively to appropriate temporal
patterns of action potential firing in neurons. The findings provide a better
understanding of how nervous system development and plasticity may be
regulated by information encoded in the temporal pattern of impulse firing in
the brain. Klein JP, Tendi EA, Black JA, Neurofibromatosis Lee, Tendi,c
Cohen; in collaboration with Becker, De Vries Neurofibromatosis
(NF-1) is an autosomal dominant condition leading to nerve sheath tumors that
can become malignant. 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 relying
on three types of custom 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
neurofibromatosis. Our studies identify hundreds of genes that are
dysregulated in the malignant Schwann cells, including those associated with
all aspects of cellular function connected with malignancy, such as, for
example, those regulating cell proliferation, motility, growth factor, and
immune system responses. Lee PR, Cohen JE, Tendi EA, Farrer R, De
Vries GH, Becker KG, Fields RD. Transcriptional profiling in an MPNST-derived
cell line and normal human Schwann cells. Neuron Glia Biology 2004, in
press. Stevens B, Fields RD. Regulation of the
cell cycle in normal and pathological glia. Neuroscientist
2002;8:93-97. Stem
cells derived from bone marrow for transplantation Ishibashi Stem
cells can be obtained from several sources in postnatal animals, but the
means of controlling their differentiation into the appropriate cell type is a
major problem in their use for therapeutic purposes. In an effort to
understand the fundamental biological processes controlling differentiation
of stem cells into nervous system cells, we are focusing our research on
factors controlling differentiation of nervous system cells. We will apply
the results in cell culture in animal studies to promote recovery following
nervous system injury. aKelly Dakin, BS, former
Postbaccalaureate Fellow bSevena Dudek, PhD, former
Senior Staff Fellow cElisabetta
Tendi, PhD, former Postdoctoral Fellow COLLABORATORS Kevin Becker, PhD, Research Resources
Branch, NIA, Jiang-Fan Chen, MD, PhD, Boston University
School of Medicine, George De Vries, PhD, Loyola University, Vittorio Gallo, PhD, George Washington
University, Washington, DC Michael Schwarzschild, MD, PhD, Massachusetts
General Hospital, Stephen Waxman, MD, PhD, Yale University,
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