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Our research is concerned with understanding the molecular and cellular mechanisms by which activity in the brain regulates development of the nervous system during late stages of fetal development and early postnatal life. This work has three main areas of emphasis. 1) We strive to determine how different patterns of neural impulses regulate specific genes controlling development and plasticity of the nervous system. This includes effects of impulse activity on neurons and glia and the molecular signaling pathways regulating gene expression in these cells in response to neural impulses. 2) We investigate how neurons and non-neuronal cells (glia) interact, communicate, and cooperate functionally. A major emphasis of this research is to understand how myelin (white matter in the brain) may be regulated by functional activity, which could implicate myelin in learning, cognition, child development, and psychiatric disorders. The research is exploring how glia sense neural impulse activity at synapses and non-synaptic regions and the functional and developmental consequences of activity-dependent regulation of neurons and glia. 3) We aim to determine the molecular mechanisms converting short-term memory into long-term memory, and in particular, how gene expression necessary for long-term memory is controlled. We use cellular, molecular, and electrophysiological studies in hippocampal brain slices.
Regulation of myelination by neural impulse activity
Myelin, the multilayered membrane of insulation wrapped around nerve fibers by glial cells (oligodendrocytes), is essential for nervous system function, increasing conduction velocity at least 50-fold. Myelination is essential for brain development. The processes controlling myelination of appropriate axons are not well understood. Myelination begins late in fetal life and continues through childhood and adolescence, but myelination of some brain regions is not completed until the early twenties. Our research shows that neurotransmitters are released along axons firing action potentials. The neurotransmitters activate receptors on myelinating glia (Schwann cells in the peripheral nervous system and oligodendrocytes in the central nervous system) as well as astrocytes and other cells that, in turn, release growth factors or cytokines that regulate the development of myelinating glia.
In addition to effects of impulse activity on proliferation and differentiation of myelinating glia, we determined that release of the neurotransmitter glutamate from vesicles along axons promotes the initial events in myelin induction. The process involves stimulating the formation of cholesterol-rich signaling domains between oligodendrocytes and axons and increasing the local synthesis of the major protein in the myelin sheath, myelin basic protein, through Fyn kinase–dependent signaling. Such axon-oligodendrocyte signaling would promote myelination of electrically active axons to regulate neural development and function according to environmental experience. The findings are also relevant to demyelinating disorders, such as multiple sclerosis, and to remyelination after axon injury.
We also found that other signaling molecules released from axons, notably ATP, act to stimulate differentiation of oligodendrocytes with increases myelination. In collaboration with colleagues in Italy, we found that a new membrane receptor on oligodendrocyte progenitor cells, GPR-17, regulates oligodendrocyte differentiation.
The release of neuronal messengers outside synapses has broad biological implications, particularly with regard to communication between axons and glia. We identified a mechanism for nonsynaptic, nonvesicular release of ATP from axons through volume-activated anion channels (VAACs)—activated by microscopic axon swelling during action potential firing. The studies combine imaging of single photons to measure ATP release in a luciferin/luciferase assay with imaging of intrinsic optical signals and intracellular calcium, time-lapse video, and confocal microscopy. Microscopic axon swelling accompanying electrical depolarization of axons activates the VAACs to release ATP. Such nonvesicular, nonsynaptic communication could mediate various activity-dependent interactions between axons and nervous system cells in normal conditions, development, and disease.
Homeostatic mechanisms are required to control formation and maintenance of synaptic connections to maintain the general level of neural impulse activity within normal limits. How genes controlling these processes are coordinately regulated during homeostatic synaptic plasticity is unknown. Micro RNAs (miRNAs) exert regulatory control over mRNA stability and translation and may contribute to local activity-dependent post-transcriptional control of synapse-associated mRNAs. Using a bioinformatics screen to identify sequence motifs enriched in the 3′UTR of mRNAs that are rapidly destabilized after increasing impulse activity in hippocample neurons in culture, we identified a developmentally and activity-regulated miRNA (miR-485); we found that miR-485 controls dendritic spine number and synapse formation in an activity-dependent, homeostatic manner. Many plasticity-associated genes contain predicted miR-485 binding sites, including the presynaptic protein SV2A. We found that miR-485 reduces SV2A abundance and negatively regulates dendritic spine density, postsynaptic density protein (PSD-95) clustering, and surface expression of GluR2. Over-expression of miR-485 reduces spontaneous synaptic responses and transmitter release, as measured by miniature excitatory postsynaptic current analysis and FM 1-43 staining. The findings show that miRNAs participate in homeostatic synaptic plasticity with possible implications for neurological disorders such as Huntington's and Alzheimer's disease, in which miR-485 has been found to be dysregulated.
Hippocampal synaptic plasticity
It is widely appreciated that there are two types of memory: short-term and long-term. It has been known for decades that gene expression is required to convert short-term into long-term memory, but it is not known how signals reach the nucleus to initiate this process or which genes make memories permanent. 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, and these processes are believed to represent a cellular basis for memory. We use cDNA microarrays to investigate the signaling pathways, genes, and proteins involved in LTP and LTD. We work to understand how 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.
We identified sets of transcription factors, structural genes, and signaling pathways that are regulated by activity patterns leading selectively to different types of synaptic plasticity. One finding of particular interest is that expression of the gene BDNF, which encodes a growth factor, depends on whether firing of postsynaptic hippocampal CA1 neurons is coincident with excitatory synaptic firing from presynaptic neurons.
Regulation of gene expression by action-potential firing patterns
All information in the nervous system is encoded in the pattern of neural impulse activity. Given that experience regulates nervous system structure and function, gene activity in neurons must be regulated by the pattern of neural impulse activity. We tested this hypothesis by using cDNA arrays for gene expression profiling. We stimulated nerve cells to fire impulses in different patterns by delivering electrical stimulation through platinum electrodes in specially designed cell culture dishes. After stimulation, we measured mRNA and protein expression by gene arrays, quantitative RT-PCR (reverse transcriptase–polymerase chain reaction), Western blot, and immunocytochemistry. Our results confirm our hypothesis that precise patterns of impulse activity can turn specific genes on or off. The experiments revealed signaling pathways and gene-regulatory networks that respond selectively to appropriate temporal patterns of action potential firing in neurons. Temporal aspects of intracellular calcium signaling are particularly important in regulating gene expression according to neural impulse firing patterns in normal and pathological conditions. We are also analyzing the role of post-transcriptional gene regulation mediated by mRNA stability and transport in hippocampal and dorsal root ganglion neurons. Our findings provide a deeper understanding of how nervous system development and plasticity may be regulated by information coded in the temporal pattern of impulse firing in the brain, which has relevance to chronic pain and regulating nervous system development by functional activity.