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NEUROBIOLOGY:
Enhanced: Dissecting Dendrite Dynamics

The functioning of the brain depends on the interconnections of billions of neurons through trillions of synapses[HN1]. But what developmental processes could possibly guide the correct formation of such vast numbers of synaptic connections? This question is at the heart of understanding brain development and the storage and processing of information throughout life. Although the surface of this problem has barely been scratched, some of the scratches have become a little deeper with new observations by Maletic-Savatic and colleagues [HN2] reported on page 1923 of this issue (1). A confluence of new optical imaging methods enabled these investigators to take a much closer look at the dynamics of neuronal structure in developing brain tissue[HN3]. They conclude that patterns of electrical activity may shape the morphology of developing neurons by promoting new dendritic extensions called filopodia [HN4], which may in turn initiate the formation of new synapses (synaptogenesis). The authors go on to show that activation of the N-methyl-D-aspartate (NMDA) receptor by electrical activity may be the event that triggers filopodial extension and synapse formation.

Two decades of experiments have demonstrated that the electrical activity of neurons can shape patterns of synaptic interconnections during early development (2) [HN5]. For instance, nerve impulses in the pathways that carry sensory information influence the functional maps of the brain areas that receive these impulses[HN6]. But it is not just the total number of impulses that count; temporal or spatial patterning of impulse activity may critically influence the shaping of such brain maps. The formation of memory [HN7] in the mature brain also may involve activity-dependent morphological changes in neurons similar to those seen in early development (3). Although there are numerous well-documented examples of electrical activity driving neuronal morphogenesis, there have been few clues to indicate how this comes about.

One molecular lead implicates the NMDA receptor, which binds the neurotransmitter glutamate at excitatory synapses in the central nervous system (4) [HN8]. Compared to other types of glutamate receptor found at excitatory synapses, the NMDA receptor plays a minor role in generating postsynaptic electrical responses. Rather, upon activation by glutamate, the NMDA receptor promotes a local influx of calcium ions (5). The effects of NMDA receptor activation are thus much more localized than those of other glutamate receptors whose electrical signals are conducted over much greater distances. Another property peculiar to the NMDA receptor is that it must be activated both by glutamate and by membrane depolarization [HN9] to permit local influxes of calcium ions. This "associative" property enables NMDA receptors in the postsynaptic membrane to potentially discriminate between temporal and spatial patterns of impulses arriving at a given neuron. Evidence that NMDA receptors affect the neuronal morphogenesis of early development primarily comes from experiments with highly selective antagonists of these receptors such as APV [D,L(-)-2-amino-5-phosphonovaleric acid]. But, there have been no clues to indicate which steps in neuronal morphogenesis are affected by NMDA receptor activation and calcium influx.

Provocative ideas about how electrical activity and neurotransmitter release might affect the morphology of neurons come from dynamic optical microscopy[HN10]. For example, in living, dissociated neurons in culture the growing tips (growth cones) and filopodia of both axons and dendrites exhibit motility (in the form of membrane protrusions) that can initiate cell-cell contact and synaptogenesis (6-8). This form of motility may be modulated by neurotransmitters and secondary messengers (9). Unfortunately, cell culture is a condition far removed from the environment of the neuron in vivo, so the physiological relevance of these findings is still in doubt.

More recently, techniques combining newer fluorescent dyes (such as the lipophilic carbocyanine membrane stains, DiI and DiO) [HN11] with confocal microscopy [HN12] have enabled time-lapse observations of single cells within whole embryos and brain tissue slices (10-13). These studies show that extension and retraction of filopodia occur in tissue slices as well as in cell culture and provide evidence that this motility is modulated by neurotransmitters such as glutamate and electrical activity at synapses. Although these experiments provide access to neurons in appropriate tissue environments, interpretation of the results is restricted by the limits of detection inherent in confocal microscopy and the possible effects of excessive dye and light exposure.

Maletic-Savatic and co-workers have taken advantage of sophisticated new techniques in fluorescence microscopy to observe the dynamics of individual neurons in rat brain hippocampal slices at substantially higher resolutions than achieved before. They used a benign virus that infects neurons to shuttle the gene encoding a soluble green fluorescent protein (GFP) [HN13] marker into hippocampal neurons. The principal advantage offered by GFP is that it provides excellent fluorescent signals with few toxic side effects (14). Finally, these investigators measured fluorescence with a two-photon laser scanning microscope[HN14]. This new imaging method uses nonlinear fluorescence photoexcitation to achieve optical sectioning and three-dimensional imaging that is far more efficient (in terms of reducing noise and photodamage) than that achieved with the older confocal microscope.

With the improved image quality provided by the combination of these methods, the authors confirm and extend the results of confocal studies (12, 13), including the observation of abundant filopodial protrusions (13). They then forged on to investigate the effects of the NMDA receptor blocker APV. Intriguingly, image analysis demonstrated that the firing of action potentials [HN15] resulted in an increased rate of filopodial formation, which was abrogated by APV. Thus, they demonstrated that the possible morphological target of NMDA receptor activation is the initiation of filopodial extension. This provides an exciting link between NMDA receptor activation and neuronal morphogenesis because filopodial formation has been implicated in synaptogenesis (7) (see the figure). In this case, NMDA receptor activation might stimulate the extension of filopodia, which could then contact neighboring axons to initiate cell-cell adhesion and synaptogenesis. Alternatively, NMDA receptor activation might stabilize existing or nascent synapses, preventing their loss. As evidence accumulates that filopodial sprouting is important in synapse formation, it seems that both of these possibilities should be taken seriously. Of course, they are not mutually exclusive. Maletic-Savatic et al. note that the very localized control of dendritic filopodial activity by presynaptic action potentials could lead to an associative or "Hebbian" [HN16] characteristic of synapse formation--that is, axons that fire and release glutamate, thereby triggering local filopodial protrusions, would seem to be more likely targets for synaptogenesis than nonfiring axons at other, more distant sites. These new findings bring to the fore many fascinating questions. What are the mechanisms by which dendritic filopodia protrude, and how are these processes linked to NMDA receptors and calcium influx? Almost certainly, the process involves the actin [HN17] cytoskeleton (8, 15), but the accessory proteins that generate mechanical force remain to be determined. In addition, filopodial protrusions are obviously accompanied by local rearrangements at the cell surface; these may include exocytotic delivery [HN18] of membrane vesicles or clustering of molecules specialized for protrusion or adhesion. How do the basic cytomechanical and regulatory schemes for dendritic filopodial formation relate to those governing neuronal filopodial dynamics in other studies of neuronal (6, 8, 13, 16) and nonneuronal cells (17)? Finally, this work only hints at the part played by filopodial protrusion in synaptogenesis. Much more work is required to establish a definite connection between these two events, and to identify alternative developmental or functional consequences. For instance, the motive forces evident in filopodial protrusion may have their major consequences in more subtle rearrangements of existing synapses rather than in the birth of new synapses (18).

References

1. M. Maletic-Savatic, R. Malinow, K. Svoboda, Science 283, 1923 (1999).
2. L. C. Katz and C. J. Shatz, ibid. 274, 1133 (1996).
3. C. H. Bailey and E. R. Kandel, Annu. Rev. Physiol. 55, 397 (1993) [Medline].
4. M. L. Vallano, Crit. Rev. Neurobiol. 12, 177 (1998) [Medline].
5. A. B. MacDermott et al., Nature 321, 519 (1986) [Medline].
6. M. W. Cooper and S. J. Smith, J. Neurobiol. 23, 814 (1992) [Medline].
7. N. E. Ziv and S. J. Smith, Neuron 17, 91 (1996) [Medline].
8. M. Fischer, S. Kaech, D. Knutti, A. Matus, ibid. 20, 847 (1998) [Medline].
9. S. B. Kater and V. Rehder, Curr. Opin. Neurobiol. 5, 68 (1995) [Medline].
10. N. A. O'Rourke and S. E. Fraser, Neuron 5, 159 (1990) [Medline].
11. M. E. Dailey, J. Buchanan, D. E. Bergles, S. J. Smith, J. Neurosci. 14, 1060 (1994) [Medline].
12. T. Hosokawa, D. A. Rusakov, T. V. Bliss, A. Fine, ibid. 15, 5560 (1995) [Medline].
13. M. E. Dailey and S. J. Smith, ibid. 16, 2983 (1996) [Medline].
14. B. Ludin and A. Matus, Trends Cell Biol. 8, 72 (1998) [Medline].
15. A. Matus, M. Ackermann, G. Pehling, H. R. Byers, K. Fujiwara, Proc. Natl. Acad. Sci. U.S.A. 79, 7590 (1982) [Medline].
16. S. J. Smith and C. E. Jahr, in The Nerve Growth Cone, P. C. Letourneau, S. B. Kater, E. R. Macagno, Eds. (Raven, New York, 1991), pp. 19-26.
17. H. Miki, T. Sasaki, Y. Takai, T. Takenawa, Nature 391, 93 (1998) [Medline].
18. F. Crick, Trends Neurosci. 5, 44 (1982).

HyperNotes
Related Resources on the World Wide Web

General Hypernotes

The WWW Virtual Library of Neuroscience is maintained by the Department of Neurology and Neuroscience, Cornell University Medical College.

Neuroscience on the Internet is a searchable and browsable index of neuroscience resources available on the Internet.

Neuroanatomy and Neuropathology on the Internet, maintained by K. Hegedüs, Department of Neurology, University Medical School, Debrecen, Hungary, is a well-organized collection of links to neuroscience Web resources.

Cell & Molecular Biology Online, maintained by P. Gannon, is a well-organized collection of annotated links to Internet resources.

A neuroscience glossary is provided for a course on computational neuroscience at the University of Wisconsin.

A Brief Tour of the Brain is part of the Mind and Machine Module from the Department of Physics, Syracuse University.

An exploration of the nervous system is offered by E. Chudler of the University of Washington on his Neuroscience for Kids Web site. His home page features an extensive list of links to neuroscience Web resources.

R. M. Robertson, Department of Biology, Queen's University, Kingston, Canada, provides extensive lecture notes for a course on integrative neurobiology and neuroethology.

D. Atkins, Department of Biological Sciences, George Washington University, Washington, DC, offers a Web tutorial on neurobiology.

The MIT Encyclopedia of Cognitive Sciences (MITECS), a comprehensive reference work edited by R. Wilson and F. Keil, includes a section of articles on neuroscience topics. The developmental Web site for MITECS is available to everyone at this time but may require user registration.

The 10 Jan 1997 issue of Science had an Enhanced Perspective by T. Sejnowski titled "The year of the dendrite."

The 15 November 1996 issue of Science included a news article by Marcia Barinaga titled "Guiding neurons to the cortex."

A review article by M. Tessier-Lavigne and C. Goodman titled "The molecular biology of axon guidance" appeared in the 15 November 1996 issue of Science.

A 13 October 1996 article by R. Hotz titled "Deciphering the miracles of the mind" is featured in a special report on the brain from the Los Angeles Times.

Numbered Hypernotes

1. The On-Line Biology Book from the Estrella Mountain Community College, Avondale, AZ, presents illustrated sections on the neuron and the nerve message. Brain & Mind, an electronic magazine about neuroscience, provides an introduction to the parts of the nerve cell and their functions. E. Chudler offers a presentation on the synapse. J. Clothier, Department of Psychiatry, University of Arkansas for Medical Sciences, provides lecture notes on synaptic biology for a behavioral sciences course. The Society for Neuroscience presents a backgrounder titled "How do nerve cells communicate messages."

2. M. Maletic-Svatic, R. Malinow, and K. Svoboda are at the Cold Spring Harbor Laboratory, NY.

3. A Brief Tour of the Brain includes a section on organization that reviews the development and operation of neurons. For a course on developmental biology, a discussion of how neurons grow is provided by G. Podgorsk, Department of Biology, Utah State University, Logan, in lecture notes on neurulation and development of the nervous system. R. M. Robertson provides lecture notes titled "Axonal pathfinding: Forming the circuits." The Society for Neuroscience provides an article on axonal guidance in its Brain Briefings collection.

4. Filopodium is defined in the On-line Medical Dictionary. A video of growth cone filopodia is presented by the Center for Biomedical Imaging Technology, University of Connecticut Health Center. Filopodia are shown in an illustration of a growth cone in a presentation titled "How does a neuron know where to grow?" by D. Pataky, Department of Zoology, University of British Columbia. The Laboratory of Developmental Neurobiology, Rockefeller University, presents a movie of a neuron migrating that shows filopodia extending and retracting. Filopodial activity and the role of filopodia are sections of a research report by P. Myers and M. Bastiani titled "Growth cone dynamics during the migration of an identified commissural growth cone."

5. The Neuroscience Program of the Howard Hughes Medical Institute provides articles about the research of L. Katz titled "Development and modification of local circuits in the mammalian cortex" and the research of C. Shatz titled "Development of connections in the mammalian central nervous system." G. Wallis reviews briefly the research on neural activity and interconnectivity in the section on neural plasticity in his 1994 thesis for a D.Phil. degree in the Department of Experimental Psychology, Oxford University.

6. Brain Mapping Web, maintained by Chenyang Xu, provides a collection of links to groups and individuals involved in brain mapping. The NeuroInformatics--Human Brain Project Web page from the National Institute of Mental Health provides information about the project and links to research centers. The Research Imaging Center, University of Texas Health Science Center at San Antonio, presents information on the BrainMap project; the BrainMap Database is available on the Web. The Laboratory of Neuro-Imaging, University of California, Los Angeles, presents a news article about the brain mapping project.

7. J. Clothier, Department of Psychiatry, University of Arkansas for Medical Sciences, presents lecture notes on the neurobiology of memory and learning. The chapter on brain evolution and development in Without Miracles by G. Cziko includes a discussion on learning and memory. R. M. Robertson discusses the synaptic basis of learning and memory and the possible role of dendritic spines in changes associated with memory.

8. Receptor, NMDA receptor, neurotransmitter, and glutamate are defined in the neuroscience glossary from the University of Wisconsin. The Addiction Science Research and Education Center, University of Texas College of Pharmacy, offers a presentation on neurotransmitters. E. Chudler's Exploring the Nervous System provides an illustrated introduction to neurotransmitters and neuroactive peptides. The 5 June 1998 issue of Science had a Research commentary by C. Miller titled "Glutamate receptor activation: A four-step program." The Molecular & Cellular Classifications Web page from the Neuromuscular Disease Center, Washington University School of Medicine, St. Louis, includes an entry for glutamate. The HotMolecBase from the Bioinformatics Unit, Weizmann Institute of Science, Israel, has an entry for NMDA receptor. The Society for Neuroscience presents a briefing on NMDA receptor blockers. A review titled "Glutamate receptors and long-term potentiation in CA1 and CA3 of the hippocampus" is made available by D. Gellerman, Department of Psychiatry, University of California, Davis.

9. Depolarization is defined in the neuroscience glossary from the University of Wisconsin.

10. The Center for Toxicology, University of Arizona College of Pharmacy, Tucson, presents a collection of links to microscopy and imaging resources on the Web. A Microscopy Primer with a section devoted to fluorescence microscopy is offered by the Molecular Expressions Website, a collection of photo galleries that explore the worlds of optical microscopy.

11. Molecular Probes, Inc., offers an online Handbook of Fluorescent Probes and Research Chemicals, which includes a section on fluorescent lipophilic tracers; also available is a guide titled "Reagents for Cell Biology and Imaging."

12. The Confocal Microscope Facility at the School of Biological Sciences, University of Manchester, provides introductions to confocal microscopy and fluorescent imaging. The 3-D Laser Scanning Confocal Microscopy Web site, provided by L. Ladic, Department of Physiology, University of British Columbia, Vancouver, includes links to Web sites that have sample images and animations related to confocal microscopy. J. Marelius includes a section on confocal laser scanning microscopy in his M.Sc. thesis (for the Uppsala University School of Engineering, Sweden) titled "Autofluorescence imaging of living cells." A confocal microscopy image of a growth cone is provided by the Department of Cell Biology and Neuroanatomy, University of Minnesota; other images are available on the sampler page.

13. W. Marshall, Department of Molecular, Cellular, and Developmental Biology, Yale University, maintains a Green Fluorescent Protein Web site. The Spring 1998 issue of Inquiry from the University of Oregon had an article about the structure of green fluorescent protein titled "Cracking the code: Researcher's know-how turns jellyfish protein into tool for medical science." An article titled "The molecular structure of green fluorescent protein" by F. Yang, L. Moss, and G. Phillips Jr. is available from G. Phillips at the W.M. Keck Center for Computational Biology, Rice University, Houston.

14. The Integrated Microscopy Resource, an NIH-supported resource center at the University of Wisconsin, provides information on confocal microscopy and multiple-photon excitation microscopy. The Discover Microscopy Web presentation from Bio-Rad Laboratories offers a technical note on multi-photon fluorescence microscopy and an application note by J. White and A. Dixon titled "Applications of multi-photon fluorescence microscopy in cell and developmental biology using a mode-locked all-solid-state Nd:YLF laser." S. Potter and S. Fraser, Biological Imaging Center, California Institute of Technology, Pasadena, present an introduction to the two-photon laser-scanning microscope. The Center for Biomedical Imaging Technology at the University of Connecticut Health Center offers a presentation about the two-photon laser scanning microscope.

15. Action potential is defined in the University of Wisconsin neuroscience glossary.

16. A biographical note about Donald Hebb is provided by the Great Canadian Scientists Web page. The Neuroscience Section of MITECS includes an entry about D. O. Hebb and his work. R. Spencer includes a brief discussion of Hebbian learning in the Neuroscience & Neural Networks Notes section of his Neural Networks Web site.

17. Actin is defined in the On-line Medical Dictionary.

18. Exocytosis is defined in the On-line Medical Dictionary.

19. S. J. Smith is in the Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA.

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