Summary:The objective of this program has been to explore the self-organizing properties and computational architecture of the brain with an eye to the development of new architectures for machine computation.
Our labs approach has been based on observation of the early development of the brain at the level of individual neurons and synapses. In the first year (beginning 7/98), we developed an advanced two photon microscope optimized for the study of living embryos of intensively studied model organisms, the fruit fly Drosophila and the zebrafish.
In May of 1999, we started to concentrate on the development of genetically encoded fluorescent probes and on substantive applications in the study of biological self-organization. Elements of this work are summarized below.
Biological work on Drosophila embryos was carried
out primarily by Eric Anderson and Eileen Furlong from the Scott lab, using
the Smith lab’s two-photon microscopy facilities, developed jointly by the Harris and Smith groups.
Synapse formation in the central nervous system of zebrafish embryos:We used time-lapse fluorescence microscopy to observe the growth of Mauthner cell axons (see movies: Zfish_growth_cone.avi (19.5MBytes!) and Zfish_growth_cone_pan.avi (19.7MBytes!) ) and their postsynaptic targets (see movie: Zfish_dendrite.mov ), the primary motor neurons, in spinal cords of developing zebrafish embryos. Upon reaching successive motor neurons, the Mauthner growth cone paused briefly before continuing along its path. Varicosities containing organelles resembling synaptic vesicle and dense-core vesicles formed at regular intervals and were preferentially associated with the target regions of the primary motor neurons. In addition, the postsynaptic motor neurons showed highly dynamic filopodia, which transiently interacted with both the growth cone and the axon. Both Mauthner cell and motor neurons were highly active, each showing motility sufficient to initiate synaptogenesis.
See movies: zfish contact 1, zfish contact 2, zfish contact 3 (Zfish_contact_3.avi 9MBytes!).
Primary work published this year, Reference
1; Reviewed along with other relevant work in Reference
2.
Development of GFP probes for study of neural circuit formation 1:A new molecular biology postdoc in the lab, Martin Meyer, has made excellent progress in development of new GFP probes for studies of zebrafish neural circuit generation.
Cell-specific promoters and genetically encoded pathway tracing markers.
A construct where GFP is expressed under the control of an islet1 promoter was shown to permit detailed studies of axon outgrowth by Rohon-Beard cell of the zebrafish, see movie: Rohon-Beard Axon 1 (Rohon-Beard_Axon_1.avi 4.7MBytes!).
In addition, Martin has very exciting preliminary results with a novel construct in which a barley lectin construct, see Reference 4, is fused to a DsRed (fluorescent protein) gene.
The trans-synaptically transported marker expressed should
allow tracing of nascent neural circuits in real time in intact embryos.
Development of GFP probes for study of neural circuit formation 2:Fast neurotransmitter secretion occurs at active zones comprising a conspicuous specialization of the presynaptic plasma membrane and a cytoplasmic cluster of synaptic vesicles.
Dynamics of synaptic vesicles and precursors during presynaptic active zone assembly.
Little is known about how such active zones are assembled during central nervous system (CNS) synaptogenesis
We characterized a chimera of the synaptic vesicle protein VAMP and GFP and used time-lapse fluorescence imaging, along with immunocytochemistry, and electron microscopy, and identified protein precursors of the presynaptic active zone being transported along developing axons together as discrete packets.
These packets range up to 1 mm in maximum extent and translocate
at rates up to 0.5 mm/sec.
The transport packets are composed of varied vesicular
and tubulovesicular membrane structures and often contain a 1a subunits
of calcium channels, the synaptic vesicle protein SV2, synapsin Ia, and
amphiphysin I, in addition to VAMP.
Packets containing these heterogeneous structures were observed to stabilize specifically at new sites of cell-cell contact: within less than one hour, evoked vesicle recycling was observed at these putative nascent synapses. These adhesions were initiated by both axons and dendrites; packet trapping was most evident at sites of dendrite-initiated contacts. These observations indicate that synapses may be rapidly formed at specific contact areas via stabilization of pre-assembled active zones.
We expect that this VAMP-GFP construct will be extremely
valuable as a vital probe for synaptogenesis in future studies of CNS synaptogenesis
Work published this year, Reference
3.
Use of a GFP-actin chimera to study morphogenesis in Drosophila embryo (Scott lab).All of these movies and images were taken of transgenic Drosophilia embryos that have a functional GFP-actin fusion specially expressed in the developing mesoderm. MDCK_contact_actin-GFP.avi (13.8MBytes!)
Therefore the GFP expression is present in all of the somatic muscle cells, visceral muscle cells that lines the gut and the cardioblasts.
The difficulty with taking these movies with a normal
scope is that these are not only thick sections but some of the cells are
moving into the interior of the embryo.
Fly movie 1 (Fly_movie_1.avi 253MBytes!):Begins with an embryo about 2 hours after gastrulation and therefore after the invagination of the mesoderm.
Fly movie 2 (Fly_movie_2.avi 155MBytes!):This movie is made as a dorsal view (looking down on the dorsal side).
Fly heart and somatic muscle:This is a nice image of the heart after it has fused at the dorsal midline.
References:
1.
Jontes, J.D., Buchanan, J. and Smith, S.J (2000)
Growth cone and dendrite dynamics in zebrafish embryos:
in vivo imaging of early events in synaptogenesis.
Nature Neuroscience 3: 231-237.
2.
Jontes, J.D. and Smith, S.J (2000)
Filopodia, spines and the generation of synaptic diversity.
Neuron (in press).
3.
Ahmari, S.E., Buchanan, J. and Smith, S.J (2000)
Assembly of presynaptic active zones from cytoplasmic
transport packets.
Nature Neuroscience 3: 445-451.
4.
Horowitz LF, Montmayeur JP, Echelard Y, Buck LB (1999)
A genetic approach to trace neural circuits.
Proc Natl Acad Sci U S A 96:3194-9.