Control of Neuronal Structure and Function In Vivo

Hollis T. Cline, Ph.D.

Cold Spring Harbor Laboratory

Funded in September, 2003: $100000 for 2 years
LAY SUMMARY . ABSTRACT . HYPOTHESIS . FINDINGS . SELECTED PUBLICATIONS .

ABSTRACT

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Control of Neuronal Structure and Function In Vivo

Normal brain development requires the elaboration of neuronal structures and formation of synaptic connections in order to assemble the neuronal circuits required for cognitive function. Increasing evidence suggests that disorders in circuit formation during brain development contribute to the etiology of several neurological diseases, including schizophrenia, autism, epilepsy, and bipolar disease, as well as X-linked mental retardation syndromes. An initial analysis of Fragile X, an X-linked form of mental retardation in which the Fragile X mental retardation protein (FMRP) is not made, suggests that local protein synthesis within neuronal dendrites is essential for brain development. FMRP is an RNA binding protein that associates with ribonucleoprotein (RNP) granules and ribosomes in neuronal dendrites.

Anatomical studies of brains of Fragile X patients and mouse knockouts of FMRP indicate that their neurons do not development a normal dendritic arbor structure. Consequently, neuronal circuits governing cognitive and behavioral functions do not develop normally. The function of local protein synthesis in regulating the development of dendritic structure and neuronal circuits is not known. Using a combination of molecular biology, gene transfection and in vivo time-lapse imaging with the 2-photon microscope, the proposed experiments will test whether dendritic protein synthesis is required for the elaboration of the neuronal dendritic arbor and formation of neuronal circuits in vivo.

 We will perform the experiments using in vivo time-lapse 2-photon imaging of neurons in the visual system of Xenopus tadpoles. These animals are transparent, so that the structure of single fluorescently labeled neurons can be imaged with high resolution over periods up to 2 weeks in the intact animal. The time-lapse movies of dendritic arbor development generated from these images have allowed us to determine many of the cellular mechanisms and molecular components required for normal neuronal development. Furthermore, we have recently shown that a brief period of enhanced visual experience increases dendritic arbor development and increases visual responsiveness in the intact animal. This type of plasticity in the visual responsiveness may require protein synthesis. We now propose to use this versatile system to test whether transport of mRNA into dendrites and activity-regulated local protein synthesis are required for the development of dendritic arbors and the formation of the neuronal circuits in the optic tectum that are required for receiving and processing visual information.

HYPOTHESIS

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Hypothesis:
Normal brain development requires the elaboration of neuronal structures and formation of synaptic connections in order to assemble the neuronal circuits required for cognitive function. The function of local protein synthesis in regulating the development of dendritic structure and neuronal circuits is not known. The proposed experiments will test whether dendritic protein synthesis is required for the elaboration of the neuronal dendritic arbor and formation of neuronal circuits in vivo.

Goals:
We propose to test whether dendritic arbor development in the neurons of the Xenopus optic tectum, requires dendritic protein synthesis. The aims of the proposal are to:

1. Test whether RNP granules containing the mRNA binding protein, cytoplasmic polyadenylation element binding protein (CPEB), are located in dendrites;
2. Test whether dendritic protein synthesis is necessary and sufficient for dendritic arbor growth in vivo.

Methods:
We will perform the experiments using in vivo time-lapse 2-photon imaging of neurons in the visual system of Xenopus tadpoles. We will visualize by expression of CFP-tagged RNP components in neurons expressing the cytosolic marker, YFP. To test whether dendritic protein synthesis is required for activity-dependent dendritic arbor development, CFP-fusion proteins of dominant negative mutant forms of CPEB will be co-expressed with YFP using single cell electroporation. Time-lapse images of optic tectal neurons will be collected over different time intervals and analyzed quantitatively to determine dendritic arbor growth rates.

FINDINGS

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Scientific Results:
We compared neurons in the Xenopus laevis optic tectum transfected with full-length cytoplasmic polyadenylation element binding protein (CPEB) and those expressing CPEB deletion mutation covering the protein’s phosphorylation/activation site (dnCPEB) to YFP-expressing control cells.

Imaging single neurons over 3 days in vivo revealed that dnCPEB expressing cells reach less than half of the control cell’s size and complexity. High frequency time-lapse imaging reveals that full-length CPEB and dnCPEB puncta are highly motile, able to invade newly formed branches within minutes of their extension. Compared to the neurons expressing full-length CPEB, the terminal branches of the dnCPEB neurons had greater puncta numbers and overall intensity levels. Previously, Sin et al. (2002) showed that exposing freely swimming tadpoles to an enhanced visual environment for four hours produced an increase in the growth of the dendritic arbor. Using this protocol, we tested the full-length and dnCPEB neurons and found that compared to control cells, dnCPEB cells did not respond to the increased visual drive with dendritic arbor growth. These results suggest that CPEB plays a role in the activity-dependent processes that govern dendritic arbor development.

SELECTED PUBLICATIONS

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Sin W-C., Haas K., Ruthazer E.S., and Cline H.T. Dendrite growth increased by visual activity requires NMDA receptor and Rho GTPases. Nature. 2002 Oct 3;419(6906):475-80.

Haas H., Sin W-C., Javaherian A., Li Z., and Cline H.T. Single-cell electroporation for gene transfer in vivo. Neuron. 2001 Mar; 29(3):583-91.

Cline H.T. Dendritic arbor development and synaptogenesis. Curr Opin Neurobiol 2001 Feb; 11(1):118-126.

Rajan I. and Cline H.T. Glutamate receptor activity is required for normal development of tectal cell dendrites in vivo. J. Neurosci. 1998 Oct 1;18(19):7836-7846.