Anirvan Ghosh
e-mail: aghosh@ucsd.edu |
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Wiring the Brain: The Specification and Refinement of Cortical Circuits
Perhaps the most remarkable feature of the developing brain is its ability to self-organize into functional circuits. We know that the formation of appropriate connections requires the targeting of axons and dendrites to specific regions of the brain and the selection of appropriate synaptic partners within those regions. Our lab is interested in understanding the mechanisms that mediate these decisions. In the past few years we have identified a number of extracellular factors as well as transcription factors that regulate different aspects of cortical connectivity. In addition, in recently initiated projects we are investigating the molecular basis of synapse specificity and the refinement of cortical circuits. A brief history of our investigation of these problems and current research efforts are described below.
Molecular Control of Cortical Neuron Morphology
Although the importance of neuronal morphology in the establishment of neural circuits has been long recognized, little is known about the molecular mechanisms that regulate neuronal morphology. To investigate the influence of local environmental signals on neuronal morphology we developed an assay in which dissociated cortical neurons from GFP-expressing mice are cultured over cortical slices (slice overlay assay). Using this assay we were able to image the behavior of neurons in their native microenvironment and identify several signals that regulate their development.
Our initial investigations were on the mechanisms that regulate the polarized growth of axons and dendrites in cortical pyramidal neurons. These neurons, which are the principal excitatory cells of the cortex, are characterized by an apical dendrite that extends towards the surface of the brain, and an axon that grows in the opposite direction towards the white matter. The apical dendrite allows the neurons to integrate cortical input, which is the conveyed to subcortical targets by the axon. Using the slice overlay assay we discovered that the polarized growth of dendrites and axons in pyramidal neurons was regulated by the secreted factor Sema3A, which was present at high levels near the surface of the brain and acted as a chemoattractant for the dendrites, and as a chemorepellant for axons.
The fact that Sema3A had opposite effects on axons and dendrites led us to explore the intracellular mechanisms by which different cellular compartments can respond differentially to the same extracellular signal. We found that soluble guanylate cyclase (sGC), an enzyme that regulates cGMP production, was localized asymmetrically in immature cortical neurons and was preferentially targeted to the emerging apical dendrite. Pharmacological inhibition of sGC activity abolished the ability of Sema3A to attract apical dendrites, but did not affect axonal responses. Thus the basis of the differential response of axons and dendrites to Sema3A appears to be asymmetric targeting of sGC to the emerging dendrite.
Following the identification of Sema3A as a dendrite orientation signal, we identified a number of other signals that regulate other aspects of dendritic patterning. We found that Slit1 regulates dendritic growth and branching and that Notch signaling restricts dendritic growth and promoted branching. While exploring control of dendritic development, we also studied the role of the extracellular environment in regulating other aspects of differentiation. We found that the sequential generation of neurons and glia was regulated by developmentally regulated extracellular signals. These signals regulate cell-type-specific gene expression by regulating Histone methylation at specific genomic promoters. We also discovered that the tangential migration of interneurons to the cortex was regulated by neurotrophins, which engaged the PI3-kinase signaling pathway to regulate motility. These studies revealed the identity of the signals that regulate various aspects of early cortical development.
Regulation of Cortical Neuron Development by Calcium-dependent Transcription
While extracellular factors play a critical role in regulating the initial differentiation of neurons, denditic development during postnatal development is influenced by sensory input and neuronal activity. We found that calcium influx via voltage sensitive calcium channels (VSCCs) induces dendritic growth via a CaM kinase IV- and CREB-dependent mechanism. CaM kinase IV, a nuclear CaM kinase, phosphorylates the transcription factor CREB at Ser-133 and drives expression of CREB and CBP-dependent genes.
Although CREB function is necessary for dendritic growth, we found that activation of CREB is not sufficient to induce dendritic growth. This suggests that calcium-induced changes in neuronal morphology may require activation of other transcription factors (Redmond and Ghosh, unpublished observation). To explore this possibility we developed a strategy called ‘Transactivator Trap’ to identify calcium regulated transcription factors in neurons.
Transactivator Trap takes advantage of the modular nature of transcription factors such that the transactivation domain of a transcription factor can function when fused to a heterologous DNA-binding domain. To identify genes that encode calcium-dependent transcription factors we generated a cDNA library in which the GAL4 DNA binding domain was cloned upstream of neonatal rat brain cDNAs. These constructs should encode fusion proteins that contain a GAL4 DNA binding domain. We transfected pools of these cDNAs into cortical neurons together with a reporter, in which expression of the CAT reporter gene was driven by the GAL4 upstream activating sequence (UAS-CAT). In transfected neurons the GAL4 DNA binding sequence targets fusion proteins to the UAS promoter. If the transfected cDNA pool contains a calcium-inducible transcription factor, then the UAS-CAT reporter gets expressed in a stimulus-dependent manner and can be detected by CAT immunocytochemistry.
The first gene we cloned using this screen was CREST (11). We found that calcium influx via NMDA receptors as well as VSCCs can induce CREST-mediated transcription. To characterize the CREST complex, we carried out a yeast 2-hybrid screen, and identified BAF150, a chromatin remodeling protein as a CREST interactor. We found that the core chromatin remodeling proteins BRM and BRG1 are also part of the CREST complex, as is the histone acetyl transferase, CBP. To assess the in vivo function of CREST, we generated mice that have a targeted disruption of the crest gene. These mice are normal at birth, but their brains fail to develop normally. Crest null mice have a defect in cortical dendrite development, and consistent with the possibility that CREST is an important mediator of activity-dependent dendritic growth, we found that calcium-induced dendrite growth is compromised in crest-null neurons. We are currently investigating the mechanisms by which calcium regulates CREST function and are making a conditional crest mutant so that we can define the developmental window when CREST function is required for dendritic growth.
We are also investigating the function of NeuroD2 and LMO4, which were also identified in the screen. Both of these genes are expressed in the somatosensory cortex, and we have discovered that mutations in these genes lead to defects in segregation of thalamocortical axons in barrel cortex. The NeuroD2 phenotype is particularly interesting since it also has a defect in synaptic maturation. The cortex-specific phenotypes of the first three genes cloned using the screen indicate that this is an effective approach for identifying transcription factors that regulate various aspect of cortical connectivity.
Molecular Mechanisms of Synapse Specificity
While there has been impressive progress in understanding the molecular control of axonal and dendritic development during the last decade, we know very little about the mechanisms that allow neurons to select appropriate synaptic partners. This problem, which may be the most fundamental unsolved problem in neural development, is the focus of a newly initiated project in the lab. We are using the hippocampus as a model system to study the mechanisms of synaptic specificity because of its highly structured connectivity. The main goal of this project is to use biochemical and expression cloning strategies to identify molecular signals that regulate synaptic specificity.
Subunit-specific Roles of NMDA Receptors in Cortical Development
Excitatory synaptic transmission in the brain is mediated by AMPA and NMDA receptors. A wealth of pharmacological and molecular studies indicate that NMDA receptors play an essential role in cellular forms of plasticity and are involved in activity-dependent cortical development. NMDA receptor signaling leads to rapid changes in synaptic function by regulating AMPA receptor trafficking to the cell surface, and also leads to long-term changes in synaptic function by regulating gene expression. In a new series of studies we are using molecular and genetic approaches to determine how signaling via specific NMDA receptor subunits contributes to these diverse effects of NMDA receptor signaling.
Activity-dependent Refinement of Cortical Circuits
Another area of recent investigation in the lab is to use molecular genetic approaches to study the role of specific cell types in the development of cortical circuits. We are exploring the possibility that GABAergic inputs, which regulate postsynaptic depolarization with great spatial and temporal precision, may play a critical role in determining cortical connectivity by influencing input selectivity. GABAergic inputs are ideally suited to mediate input selectivity since different classes of GABAergic neurons innervate different parts of the principal (pyramidal) neurons and serve different functions. While the dendritic inhibitory inputs locally regulate the amplitude of excitatory postsynaptic potentials (EPSPs), the perisomatic inhibitory inputs regulate the overall firing rates of neurons. In principle each of these inputs could influence which excitatory inputs are stabilized, but whether they serve such a function is not known. By selectively silencing the activity of different GABAergic subpopulations we should be able to assess their contribution to the development and refinement of cortical circuits.
Summary
The areas of investigation outlined above address major aspects of how the brain wires itself during development. In order to make functional circuits neurons must extend axons and dendrites towards potential synaptic partners, they must select the neurons with which to make synapses, and finally they must eliminate those connections that are behaviorally inappropriate or irrelevant. We expect that our efforts will provide critical insight into these central questions of mammalian brain development.Polleux, F., T. Morrow and A. Ghosh (2000). Semaphorin 3A is a chemoattractant for developing cortical dendrites. Nature (lead article) 404:567-573.
Whitford, K.L., V. Marillat, E. Stein, C. S. Goodman, M. Tessier-Lavigne, A. Chedotal and A. Ghosh (2002). Regulation of cortical dendrite development by Slit-Robo interactions. Neuron 33:47-61.
Redmond, L.J., S.-R. Oh, C. Hicks, G. Weinmaster, and A. Ghosh (2000). Nuclear Notch1 signaling and the regulation of dendritic development. Nature Neuroscience 3:30-40.
Morrow, T., M.-R. Song and A. Ghosh (2001). Sequential specification of neurons and glia by developmentally regulated extracellular factors. Development 128:3585-3594.
Song, M.-R. and A. Ghosh (2004). FGF2-induced chromatin remodeling regulates CNTF-mediated gene expression and astrocyte differentiation. Nature Neuroscience 7:229-235.
Polleux, F., Whitford, K.L., Dijkhuizen, P.A., Vitalis, T., and A. Ghosh (2002). Control of cortical interneuron migration by neurotrophins and PI 3-kinase signaling. Development 129:3147-3160.
Redmond, L., Kashani, A., and A. Ghosh (2002). Calcium regulation of dendritic growth via Cam kinase IV and CREB-mediated transcription. Neuron 34:999-1010.
Aizawa, H., Hu, S-C, Bobb, K., Balakrishnan, K., Ince, G., Gurevich, I., Cowan, M., and A. Ghosh (2004). Dendrite development regulated by CREST, a calcium-regulated transcription activator. Science (lead article) 303:197-202.
Song, M.-R. and A. Ghosh (2004). FGF2-induced chromatin remodeling regulates CNTF-mediated gene expression and astrocyte differentiation. Nature Neuroscience 7(3):229-235
Dijkhuizen, P.A. and A. Ghosh (2005). BDNF regulates primary dendrite formation in cortical neurons via the PI3-Kinase and MAP Kinase signaling pathways J. Neurobiol. 62(2):278-288..
Chen, Y. and A. Ghosh (2005). Regulation of cortical dendrite development by Rap1 signaling. Mol. Cell. Neurosci. 28(2):215-228
Ince-Dunn, G., Hall, B.H., Hu, S-C., Ripley, B., Huganir, R.L., Olson, J.M., and A. Ghosh (2006). Regulation of thalamocortical patterning and synaptic maturation by NeuroD2. Neuron (cover) 49:683-695.
Kashani, A.H., Qiu, Z., Jurata, L., Lee, S.-K., Pfaff, S, Goebbels, S., Nave, K.-A., and A. Ghosh (2006). Calcium activation of the LMO4 transcription complex and its role in the patterning of thalamocortical connections. J. Neurosci. 26:8398-8408.
Polleux, F., Ince-Dunn, G., and A. Ghosh (2007). Transcriptional regulation of axon guidance and synapse formation. Nature Reviews Neuroscience 8:331-340.
Davis, E. and A. Ghosh (2007). Should I stay or should I go: Wnt signals at the synapse. Cell 130:593-596.
Wu JI, Lessard J, Olave IA, Qiu Z, Ghosh A, Graef IA, Crabtree GR (2007). Regulation of dendritic development by neuron-specific chromatin remodeling complexes. Neuron. 56(1):94-108.
Hall, BJ, Ripley B, A. Ghosh (2007). NR2B signaling regulates the development of synaptic AMPA receptor current. J Neurosci. 27(49):13446-56
Ultanir SK, Kim JE, Hall BJ, Deerinck T, Ellisman M, A. Ghosh (2007). Regulation of spine morphology and spine density by NMDA receptor signaling in vivo. Proc Natl Acad Sci U S A. 104(49):19553-8. Epub 2007 Nov 28.
Polleux, F., A. Ghosh (2008). Molecular determinants of dendrite and spine development. In Dendrites, 2nd Ed. Oxford University Press.
Cline, H., Ghosh, A., Jan, Y-N. (2008) Dendritic Development. In Fundamental Neuroscience, 3rd Ed. Elsevier
Anirvan Ghosh received his B.S. in Physics from Caltech in 1985 and his Ph.D. in Neurobiology from Stanford University in 1991. He was a postdoctoral fellow at Harvard Medical School from 1991-1995, and was on the faculty at Johns Hopkins University School of Medicine from 1995-2003. Dr. Ghosh joined UCSD as Stephen Kuffler Professor of Biology in 2003. He is the recipient of numerous awards including the Society for Neuroscience Young Investigator Award, and the Presidential Early Career Award for Scientists and Engineers.
