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Genetic schemes and schemata in neurophysiology
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Genetic schemes and schemata in neurophysiology Boris V Zemelman and Gero Miesenböck* Information in nervous systems is often carried by neural ensembles — groups of neurons in transient functional linkage — and written in a code that involves the spatial locations of active neurons or synapses and the times at which activity occurs. Even in favorable neuroanatomical circumstances, studying neural ensemble function presents a serious experimental challenge. One recent strategy to overcome this challenge relies on protein-based sensors that provide direct optical images of neural activity, and on proteinbased effectors that interfere with it. Because these molecules are encodable in DNA, they can be introduced into intact animals by genetic manipulation, and their expression pattern can be tailored to include — exclusively and at the same time comprehensively — the neurons of interest. Circumscribed populations of neurons can thus be studied in virtual isolation at defined stages of intact neural pathways. Addresses Laboratory of Neural Systems, Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, Box 205, 1275 York Avenue, New York, NY 10021, USA *e-mail: [email protected] Current Opinion in Neurobiology 2001, 11:409–414 0959-4388/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved. Abbreviations 2HG second-harmonic generation GFP green fluorescent protein YFP yellow fluorescent protein
Introduction Information is inevitably tied to a physical carrier — be it a pencil mark on paper or a neurotransmitter molecule bound to its receptor, the orientation of a dipole in a magnetic domain or the polarity of a voltage across a neuronal membrane. It is the physical carrier of a message rather than its content that is observed directly, and it is the carrier rather than the content on which physically implemented computations operate. Neurophysiologists rely on this fundamental link between information and its physical substrate when they explore how information is encoded and processed in the brain. What makes their task technically difficult is the way nervous systems are organized. Information tends to be distributed over large numbers of processing elements, and analyzing their function or interfering with it requires ways to monitor or manipulate many elements at once. To make matters worse, the typical neuroanatomy does not segregate different classes of processing elements, but rather interleaves them in arrangements that combine regularity on a large scale with a significant degree of local variability. Isolating the function of a specific class would
depend on methods with both a built-in selectivity for these processing elements and an ability to distinguish them reliably from neighboring ones belonging to different classes. Here, we review recent developments in experimental approaches that use genetics to achieve such selectivity. The shared premise of these approaches is that gene expression patterns can be used to single out functionally related groups of neurons for analysis or intervention. To distinguish this brand of genetics from more traditional ones that emphasize individual gene products and use mutations as their chief analytical tool, we refer to it as ‘schema genetics’: the genetics of organized patterns.
Schemata and schemes In the simplest genetic schemata, the choice of marker gene follows immediately from function. All neurons communicating through a specific neurotransmitter, for example, will express the cognate biosynthetic enzymes, and these can serve to define the class genetically. Will genetic markers also exist for groups of neurons not bound by an obvious functional characteristic, such as, for instance, the pyramidal cells of one cortical layer? The answer is likely to be affirmative. Shotgun searches for genetic regulatory elements in Drosophila [1,2] often reveal genes whose expression patterns map precisely to identified processing stages in layered neural pathways (see the online databases of FlyBase, http://flybase.bio.indiana.edu/, and Flybrain, http://flybrain.neurobio.arizona.edu, for examples). Anecdotal evidence in vertebrates points in the same direction [3,4•,5•]. We do not consider this a surprising coincidence. Biological structures are products of developmental programs encoded, to a large extent, genetically, and the gene expression patterns discernible within them may be viewed as an imprint that the generative programs have left behind. As such, they are likely to encapsulate the organizing principles. The genetic schemata that pattern nervous systems thus provide an experimental platform on which schemes to analyze or perturb defined populations of neurons can be built. Once the regulatory elements responsible for a particular gene expression pattern have been isolated, they can be used to drive the expression of natural or engineered proteins capable of reporting or interfering with neural physiology. These genetically encoded tools are the focus of this review.
Genetic schemes for analyzing neural function Genetic schemes for analyzing neural function unite the selectivity of genetics with the resolution of optical imaging to highlight the activities of circumscribed classes of
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neurons in virtual isolation. To this end, all current schemes use derivatives of green fluorescent protein (GFP), whose light-emitting properties have been engineered to report changes in an electrical or chemical variable that is used by neurons to convey information. Older schemes based on luminescent light emitters [6,7] have become obsolete; they typically generate photon fluxes several orders of magnitude below those of fluorescent probes, depend on exogenous substrates (which impedes their application in intact systems) and lack depth resolution because they operate without an exciting light beam and/or scanning optics. The main genetically encoded sensors report changes in membrane potential and intracellular calcium concentration, or signal the release of neurotransmitter (but see also [8–12]). The three major types of probes have reached varying levels of maturity. Membrane-potential indicators ‘Electromechanical’ sensors
The first and only genetically encoded fluorescent membrane-potential sensor, called FlaSh [13], must be considered a prototype. FlaSh comprises two modules: the Shaker potassium channel of Drosophila, which is rendered non-conducting by a point mutation in its pore [14], serves as the voltage-sensing device; GFP, inserted after the integral membrane portion of Shaker, provides the optical read-out. When expressed in Xenopus oocytes, the fluorescence of FlaSh decreases by 5% after a voltage step that moves the entire gating charge of Shaker [13]. The kinetics of the optical signal are sluggish, and a mechanistic link with the slow process of C-type inactivation [15] is likely. The conformational change associated with inactivation probably creates or rearranges physical contacts among the four GFP modules crowded at the inner pore of the tetrameric channel, and this reduces their fluorescence output. In the three years since the first description of FlaSh, its use in cell types other than oocytes has not been reported. A likely explanation is that FlaSh subunits are incorporated into endogenous voltage-gated potassium channels, which are absent in oocytes, and that subunit mixing disrupts the function of these channels or of FlaSh itself. The former would be an expected consequence of the conductanceblocking mutation in FlaSh; the latter, a result of the loss of interacting partners required to couple channel inactivation to fluorescence changes. We presume that the potential solution to these problems — linking four FlaSh coding regions in a single transcript — has been tried and found unsatisfactory. Because the mechanism of FlaSh remains fundamentally obscure, it is difficult to base design improvements on it. Therefore, in a number of laboratories efforts are under way to develop membrane-potential sensors that retain the
modularity and even the basic building blocks of FlaSh, but that combine them in mechanistically more transparent designs that can be iteratively optimized. ‘Solid-state’ sensors
The most elegant voltage probes would operate without moving parts. Rather than relying on conformational rearrangements to transduce membrane-potential changes into optical signals, they would report the direct effects of the transmembrane electric field on the electronic structures of their chromophores. Fluorescence, unfortunately, provides a poor read-out in these circumstances: even the large electric fields (about 100,000 V/cm) experienced by fluorophores placed directly into lipid bilayers typically cause only small fluorescence changes (generally less than 0.5% per 10 mV). More sensitive to applied electric forces than fluorescence is the nonlinear optical phenomenon of second-harmonic generation (2HG) [16], in which a membrane-associated chromophore scatters the energy of two incident fundamental photons into one frequency-doubled harmonic photon. Even the attenuated electric field at some distance from the plasma membrane may suffice to modulate 2HG. The fluorescence of GFP fused to an integral membrane protein, for example, is unaffected by membrane potential, but the same protein gives rise to an apparent harmonic signal with a steep voltage dependence of about 10% per 10 mV [17•]. As a second-order reaction in the concentration of incident photons, 2HG possesses the same intrinsic resolving power as two-photon fluorescence excitation. Unlike fluorescent light, however, which is radiated isotropically, harmonic light propagates only in the forward direction and at a particular off-axis angle with respect to the driving beam. ‘Harmonic’ microscopes (and the samples imaged on them) must therefore accommodate special detection pathways to capture the off-axis photons [18•]. Calcium indicators
The classical alternative to detecting changes in membrane potential is to record the changes in intracellular calcium concentration that follow them. The advantages of this indirect measurement include strong signals from large populations of soluble indicator molecules with wide dynamic ranges. The disadvantage is that rapid membrane-potential changes appear temporally filtered when viewed through intracellular calcium dynamics. One temporal filter reflects the kinetics of cellular calcium clearance, another the association–dissociation kinetics of indicator and calcium ion. This disadvantage can, however, be turned into an advantage: a signal that is expanded in time will register more reliably in imaging systems unable to survey continuously every point in space. Dual-fluorophore sensors
Genetically encoded dual-emission calcium sensors, called cameleons, comprise four elements in linear arrangement
Genetic schemes and schemata in neurophysiology Zemelman and Miesenböck
[19,20]: a blue-shifted variant of GFP; the calcium-binding protein calmodulin; a calmodulin target; and a native GFP or red-shifted variant. At resting calcium concentrations, cameleons adopt a conformation with a large average distance and a wide range of dipole angles between the two fluorophores; fluorescence resonance energy transfer between them is only moderately more efficient than if they were physically uncoupled. As calcium concentrations rise, calmodulin engages its target and locks the cameleon into a more rigid and tighter arrangement. The efficiency of fluorescence resonance energy transfer consequently increases. Although cameleons display only modest fluorescence changes in response to calcium, they have proven adequate for detecting spontaneous calcium transients in muscle, as well as calcium transients evoked by intense electrical stimulation in neurons of Caenorhabditis elegans [21•]. Single-fluorophore sensors
Single-emission probes termed camgaroos [22] combine calmodulin and yellow fluorescent protein (YFP) in an unusual topology: the YFP sequence is interrupted at a strategic location to accommodate calmodulin. The calciumdriven conformational changes of calmodulin are thus transmitted directly to the protein shell surrounding the chromophore of YFP, which switches from a protonated non-fluorescent state in the absence of calcium to a deprotonated fluorescent state in the presence of calcium. The strengths as well as the weaknesses of camgaroos are rooted in this peculiar structure. The tight conformational coupling between calcium sensor and fluorophore allows for dynamic ranges that match those of small-molecule calcium indicators (about a sevenfold increase in fluorescence at saturating compared with basal calcium concentrations). However, drawbacks include the role of protons in the calcium-driven fluorescent switch, which makes camgaroos sensitive to pH interference; the lack of a calmodulin target peptide, which lowers their calcium affinity to about 7 µM; and the crack in the shell of YFP (necessary to accept the calmodulin insert), which causes the protein to misfold at temperatures above 28°C. Recent variations on the camgaroo topology, called G-CaMP [23•] and pericams [24•], overcome one of these drawbacks: they incorporate a calmodulin target. As expected, the calcium affinity of these probes rises to < 1 µM; however, pH interference and stability problems tend to persist. The latter, in particular, may continue to stand in the way of an eagerly anticipated first test of these probes in a vertebrate neuron. Indicators of neurotransmitter release
The third type of genetically encoded indicator signals the release of neurotransmitter [25] and thus complements membrane potential and calcium sensors, which reveal cellular activity. To convert synaptic transmission into an
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optical signal, a switchable light emitter is tethered to the inner surface of synaptic vesicles. Light emission switches ‘on’ the instant that a vesicle fuses with the presynaptic plasma membrane, and ‘off’ the instant that the vesicle membrane is retrieved via endocytosis. The light emitter is a variant of GFP, called pHluorin, the fluorescence of which is reversibly quenched by protonation [25]. Vacuolar proton ATPases maintain the interior of synaptic vesicles at a pH of about 5.7 [25], whereas the extracellular fluid has a more alkaline pH of about 7.4. A roughly 50-fold difference in proton concentration thus exists between the two compartments. pHluorins rather efficiently translate this difference into about a 20-fold increase in fluorescence associated with exocytosis [26]. The magnitude and waveform of the fluorescent signal arising from vesicle fusion at a synapse are determined by a number of synaptic properties. The first is the size of the resting vesicle pool: larger pools will increase background fluorescence and make fusion events more difficult to detect. The second factor is how cleanly the reporter partitions between synaptic vesicles and the presynaptic membrane. When the v-SNARE VAMP/synaptobrevin-2 is used as an anchor, about 10% of the indicator is found in the plasma membrane even under resting conditions [26,27]. Although this fraction may appear small, it is typically the main source of background fluorescence, as surface-located pHluorins tend to be in the deprotonated, fluorescent state. Finally, because synaptic vesicle membrane is re-internalized only after a delay following exocytosis (re-acidification of the vesicle interior, in contrast, occurs virtually instantaneously after the vesicle lumen is sealed off [27]), the pHluorin signal represents the convolution of each synaptic impulse with the time course of the associated vesicle turnover. This time course is in itself subject to short-term modulation: by the magnitude of the load on the endocytic machinery [27], for instance, or by the concentration of calcium in the nerve terminal [28•]. The pHluorin trace therefore represents a running average of synaptic activity, with an integration window of several to several tens of seconds. Of the three main types of genetically encoded sensors, indicators of synaptic transmission have moved the farthest beyond the prototype stage. They have generated new insights into the physiology of nerve terminals [27,28•], and may soon make their debut in areas of systems physiology where their two fundamental limitations can be tolerated or bypassed. These limitations are the small optical signal associated with a single synaptic impulse (owing to only a handful of light emitters that can be packed onto the surface of a vesicle) and their poor temporal resolution (owing to the synaptic vesicle cycle). Their forte, accordingly, will be analyses of spatial activity patterns in topographic projections and measurements of aggregate synaptic strength. While the potential of analyzing neural physiology with genetically encoded optical probes has been instantly and
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widely recognized [7,29,30], the volume of scientific knowledge that the probes have produced up until March 2001 is disappointing. The indicators have been applied exclusively in single cells in culture (but see [17•,21•]), and here mostly for purposes of validation rather than discovery (but see [27,28•]). We can only speculate as to what has precluded applications in multicellular systems — maybe cultural barriers or skepticism about the performance of some of the first-generation probes. Or, perhaps, the lead times to generate the necessary organisms and imaging platfoms are so long that the most interesting work is still in progress. In any case, the study that unleashes the full potential of the approach has yet to be published.
Genetic schemes for perturbing neural function Compared with the genetically encoded indicators, the use of which requires imaging expertise, the easily accessible genetic tools for interfering with populations of neurons have a more extensive record of accomplishment. In the past year alone, genetic interference with neural function has been used in flies and mice to address a broad range of questions from the specification of neuronal cell types to the consolidation and generalization of memories [31,32•–34•]. Irreversible effectors
All existing tools silence defined groups of neurons. The simplest schemes do so unconditionally and irreversibly: the light chain of tetanus toxin cleaves the v-SNARE VAMP/synaptobrevin-2 and blocks synaptic transmission [35]; the inwardly rectifying potassium channel Kir2.1 clamps the neuronal membrane potential below its resting value and suppresses excitability [36]; the A chain of diphtheria toxin ADP-ribosylates elongation factor 2 to inhibit protein synthesis and cause neuronal cell death [37,38]; cell-death genes such as reaper block caspase inhibitors and trigger apoptosis [39]. To achieve some temporal control over their effect, silencing genes are often expressed from promoters controlled by ligand-dependent transcription factors without endogenous targets. In these refined genetic circuits, patterned expression of the transcription factor determines which neurons are capable of synthesizing an effector protein, whereas adding or removing a ligand (usually tetracycline [40,41]) controls the actual onset of synthesis [32•]. Because many toxins are effective in copy numbers as low as a single molecule per cell, measures must be taken to ensure tight transcriptional regulation [41], or to decrease the toxicity of the effector if transcription remains persistently leaky [32•,38]. A different way to time an intervention, and to add a layer of spatial control, is to express the non-toxic bacterial enzyme β-galactosidase in designated neurons and use its activity to accumulate a non-toxic reaction product, fluorescein, from a synthetic
substrate. The dye-loaded neurons can then be photoablated when desired [42]. While the onset of neuronal quiescence can be controlled by regulating transcription, the result is, for all practical purposes, permanent even if the targeted neurons survive the intervention. For example, the effect of tetanus toxin light chain will not subside when transcription of the gene encoding the toxin is shut down, but only after all mRNA and toxin molecules have been degraded and all proteolyzed v-SNAREs have been replaced. This will only occur over a period of many hours or days. In many instances, however, one would prefer to switch neurons between silent and active states repeatedly and rapidly in the course of a single experiment. This requires that effector activity is controlled at a level other than gene expression. Reversible effectors
The first genetic scheme to accomplish control at a level other than gene expression uses a temperature-sensitive allele of shibire in Drosophila [34•,43•]. The shibire gene encodes dynamin, a homo-oligomeric GTPase required for fission of recycling synaptic vesicles from the plasma membrane [44]. Some mutant forms of dynamin act as dominant inhibitors of wild-type function [45]; oligomers which incorporate a sizeable proportion of them cannot sustain vesicle recycling at a sufficient rate. As a result, affected synapses cease to transmit once the available pool of synaptic vesicles has been consumed. The two properties of dominance and temperature-sensitivity ensure spatial and temporal control, respectively, over the synaptic blockade. Susceptible neurons (those which express the mutant shibire allele along with the endogenous wild-type gene) are designated genetically, while shifts between permissive and restrictive temperatures are used to impose and relieve the block on synaptic vesicle recycling [34•,43•]. Effects on vesicle release tend to trail those on recycling by about a minute [43•], the time typically required to deplete or refill the synaptic vesicle pool.
Conclusions and perspectives Genetics and protein engineering are invading what not long ago was the exclusive domain of synthetic chemistry and pharmacology. Protein-based sensors of neural activity are emerging opposite synthetic indicator dyes; drugs that silence neurons or synapses are finding genetically encoded counterparts. A conspicuous void, however, exists in the genetic line-up at the position that photostimulants and caged neurotransmitters (that is, inactive precursors activated by light) occupy in the synthetic arsenal [46,47]. We feel that filling this void constitutes a challenging but particularly rewarding area for future developments. Whereas conventional photostimulation must localize the stimulus in the form of the uncaging light beam, genetic schemes would localize the response (in the form of an
Genetic schemes and schemata in neurophysiology Zemelman and Miesenböck
encoded sensitivity to light), and illumination could then be broad. Patterns of distributed activity might be fed directly into a genetically circumscribed population of neurons, irrespective of the anatomical location of its members or their connection to sensory input. Like metabolic pathways, neural pathways are arranged as graphs of interconnected nodes. The ability to probe individual nodes with pure, often synthetic substrates proved instrumental for the dissection of metabolism. Perhaps the ability to probe defined groups of neurons with synthetic information will hold the key to an understanding of neural systems.
Acknowledgements Gero Miesenböck is an Alfred P Sloan and Klingenstein Fellow, a Beckman Young Investigator and a Searle Scholar.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest 1.
O’Kane CJ, Gehring WJ: Detection in situ of genomic regulatory elements in Drosophila. Proc Natl Acad Sci USA 1987, 84:9123-9127.
2.
Bier E, Vaessin H, Shepherd S, Lee K, McCall K, Barbel S, Ackerman L, Carretto R, Uemura T, Grell E et al.: Searching for pattern and mutation in the Drosophila genome with a P-lacZ vector. Genes Dev 1989, 3:1273-1287.
3.
Tsien JZ, Chen DF, Gerber D, Tom C, Mercer EH, Anderson DJ, Mayford M, Kandel ER, Tonegawa S: Subregion- and cell typerestricted gene knockout in mouse brain. Cell 1996, 87:1317-1326.
4. •
Feng G, Mellor RH, Bernstein M, Keller-Peck C, Nguyen QT, Wallace M, Nerbonne JM, Lichtman JW, Sanes JR: Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Neuron 2000, 28:41-51. GFP variants are expressed in mouse projection neurons under the control of regulatory elements derived from the thy1 gene. Multiple independent mouse lines differ in the numbers and types of neurons labeled; within any one line, the pattern of gene expression is constant and heritable, and often confined to a particular anatomical structure. 5. •
Leighton PA, Mitchell KJ, Goodrich LV, Lu X, Pinson K, Scherz P, Skames WC, Tessier-Lavigne M: Defining brain wiring patterns and mechanisms through gene trapping in mice. Nature 2001, 410:174-179. Gene expression and wiring patterns in transgenic mouse lines generated by a ‘secretory trap’ method display remarkable variety and anatomical specificity. 6.
Shimomura O, Inouye S, Musicki B, Kishi Y: Recombinant aequorin and recombinant semi-synthetic aequorins. Cellular Ca2+ ion indicators. Biochem J 1990, 270:309-312.
7.
Miesenböck G, Rothman JE: Patterns of synaptic activity in neural networks recorded by light emission from synaptolucins. Proc Natl Acad Sci USA 1997, 94:3402-3407.
8.
Kuner T, Augustine GJ: A genetically encoded ratiometric indicator for chloride: capturing chloride transients in cultured hippocampal neurons. Neuron 2000, 27:447-459.
9.
Jayaraman S, Haggie P, Wachter RM, Remington SJ, Verkman AS: Mechanism and cellular applications of a green fluorescent protein-based halide sensor. J Biol Chem 2000, 275:6047-6050.
10. Zaccolo M, De Giorgi F, Cho CY, Feng L, Knapp T, Negulescu PA, Taylor SS, Tsien RY, Pozzan T: A genetically encoded, fluorescent indicator for cyclic AMP in living cells. Nat Cell Biol 2000, 2:25-29. 11. Sato M, Hida N, Ozawa T, Umezawa Y: Fluorescent indicators for cyclic GMP based on cyclic GMP-dependent protein kinase I alpha and green fluorescent proteins. Anal Chem 2000, 72:5918-5924.
413
12. Honda A, Adams SR, Sawyer CL, Lev-Ram V, Tsien RY, Dostmann WR: Spatiotemporal dynamics of guanosine 3′′,5′′-cyclic monophosphate revealed by a genetically encoded, fluorescent indicator. Proc Natl Acad Sci USA 2001, 98:2437-2442. 13. Siegel MS, Isacoff EY: A genetically encoded optical probe of membrane voltage. Neuron 1997, 19:735-741. 14. Perozo E, MacKinnon R, Bezanilla F, Stefani E: Gating currents from a nonconducting mutant reveal open-closed conformations in Shaker K+ channels. Neuron 1993, 11:353-358. 15. Hoshi T, Zagotta WN, Aldrich RW: Two types of inactivation in Shaker K+ channels: effects of alterations in the carboxy-terminal region. Neuron 1991, 7:547-556. 16. Bouevitch O, Lewis A, Pinevsky I, Wuskell JP, Loew LM: Probing membrane potential with nonlinear optics. Biophys J 1993, 65:672-679. 17. •
Khatchatouriants A, Lewis A, Rothman Z, Loew L, Treinin M: GFP is a selective non-linear optical sensor of electrophysiological processes in Caenorhabditis elegans. Biophys J 2000, 79:2345-2352. GFP is attached to two integral membrane proteins in C. elegans: an acetylcholine receptor subunit and the stomatin homologue MEC-2, which is expressed in mechanosensory neurons. The proteins give rise to optical signals which are detected at exactly half the fundamental wavelength (and therefore attributed to 2HG) and modulated by choline and touch, respectively. Animals lacking functional acetylcholine receptors lack the choline-responsive optical signal. Many mechanistic details remain mysterious, in particular, how flexible linkage with a membrane protein creates an ordered GFP array in which the phased emissions from individual dipoles can sum constructively, a requirement for 2HG. 18. Moreaux L, Sandre O, Charpak S, Blanchard-Desce M, Mertz J: • Coherent scattering in multi-harmonic light microscopy. Biophys J 2001, 80:1568-1574. A ‘harmonic’ laser-scanning microscope, and examples of its use for imaging natural and artificial membranes. 19. Miyawaki A, Llopis J, Heim R, McCaffery JM, Adams JA, Ikura M, Tsien RY: Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 1997, 388:882-887.
20. Miyawaki A, Griesbeck O, Heim R, Tsien RY: Dynamic and quantitative Ca2+ measurements using improved cameleons. Proc Natl Acad Sci USA 1999, 96:2135-2140. 21. Kerr R, Lev-Ram V, Baird G, Vincent P, Tsien RY, Schafer WR: Optical • imaging of calcium transients in neurons and pharyngeal muscle of C. elegans. Neuron 2000, 26:583-594. Cameleons are expressed in pharyngeal muscle and neurons of C. elegans. Serotonin-stimulated feeding is correlated with calcium transients in muscle. Electrically evoked neural activity results in calcium transients and muscle contractions. Significantly, cameleons reveal predictably altered calcium transients in animals carrying mutant calcium channels. 22. Baird GS, Zacharias DA, Tsien RY: Circular permutation and receptor insertion within green fluorescent proteins. Proc Natl Acad Sci USA 1999, 96:11241-11246. 23. Nakai J, Ohkura M, Imoto K: A high signal-to-noise Ca2+ probe • composed of a single green fluorescent protein. Nat Biotechnol 2001, 19:137-141. The M13 fragment of myosin light chain kinase is linked to the newly created amino terminus of a circularly permuted GFP, and calmodulin is attached to the carboxyl terminus. The resulting fluorescent calcium sensor, with a dissociation constant for calcium of 235 nM, is expressed in cultured mouse myotubes grown at 28°C and used to monitor calcium transients caused by pharmacological and electrical stimulation. 24. Nagai T, Sawano A, Park ES, Miyawaki A: Circularly permuted green • fluorescent proteins engineered to sense Ca2+. Proc Natl Acad Sci USA 2001, 98:3197-3202. Pericams are single-fluorophore calcium sensors with a topology similar to that of G-CaMP [23•], except that YFP is used instead of GFP and coupled in a slightly different arrangement to calmodulin and the M13 peptide. The dissociation constant for calcium of the prototypical 'flash-pericam' is approximately 700 nM. Additional mutations in YFP give rise to pericams with ratiometric and ‘inverse’ calcium responses and possibly improved stability. 25. Miesenböck G, De Angelis DA, Rothman JE: Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature 1998, 394:192-195. 26. Sankaranarayanan S, De Angelis D, Rothman JE, Ryan TA: The use of pHluorins for optical measurements of presynaptic activity. Biophys J 2000, 79:2199-2208.
414
27.
Sensory systems
Sankaranarayanan S, Ryan TA: Real-time measurements of vesicleSNARE recycling in synapses of the central nervous system. Nat Cell Biol 2000, 2:197-204.
28. Sankaranarayanan S, Ryan TA: Calcium accelerates endocytosis of • vSNAREs at hippocampal synapses. Nat Neurosci 2001, 4:129-136. Synaptic vesicle endocytosis and exocytosis are monitored optically in hippocampal neurons expressing synapto-pHluorin, the genetically encoded indicator of synaptic transmission. Both exo- and endocytic rates are affected by the concentration of calcium ion in the nerve terminal. Calcium thus seems to couple the rate of vesicle recycling to the rate of release. 29. Siegel MS: Genetic probes: new ways to watch cells in action. Curr Biol 1997, 7:R556-R557. 30. Crick F: The impact of molecular biology on neuroscience. Philos Trans R Soc London Ser B 1999, 354:2021-2025. 31. Liu L, Wolf R, Ernst R, Heisenberg M: Context generalization in Drosophila visual learning requires the mushroom bodies. Nature 1999, 400:753-756. 32. Gogos JA, Osborne J, Nemes A, Mendelsohn M, Axel R: Genetic • ablation and restoration of the olfactory topographic map. Cell 2000, 103:609-620. In contrast to many other types of neurons, olfactory sensory neurons are continuously but asynchronously shed and replaced. The authors express diphtheria toxin in P2 olfactory neurons (that is, sensory neurons expressing the olfactory receptor P2) under the control of tetracycline-dependent transcriptional activator. After selective, synchronous ablation of all P2 neurons, growth and targeting of newly generated neurons restores the topographic projections observed during normal development. 33. Lee KJ, Dietrich P, Jessell TM: Genetic ablation reveals that the roof • plate is essential for dorsal interneuron specification. Nature 2000, 403:734-740. Roof-plate cells in mouse embryos are destroyed by selective expression of diphtheria toxin to assess the contributions of the roof plate and epidermal ectoderm to dorsal neuronal patterning. The authors conclude that the roof plate is essential for specifying several classes of neurons in the mammalian central nervous system. 34. Waddell S, Armstrong JD, Kitamoto T, Kaiser K, Quinn WG: The • amnesiac gene product is expressed in two neurons in the Drosophila brain that are critical for memory. Cell 2000, 103:805-813. See also Kitamoto [43•]. Expression of a temperature-sensitive allele of shibire is restricted to the dorsal paired medial cells in Drosophila to analyze the role of the secreted amnesiac gene product, which is synthesized preferentially in these cells, in memory formation. Inactivation of the dorsal paired medial cells abolishes olfactory memory, demonstrating the importance of the amnesiac gene product. The authors remark that the ablation of dorsal paired medial cells via expression of cell death genes proved lethal, emphasizing the benefit of a conditional approach.
35. Sweeney ST, Broadie K, Keane J, Niemann H, O’Kane CJ: Targeted expression of tetanus toxin light chain in Drosophila specifically eliminates synaptic transmission and causes behavioral defects. Neuron 1995, 14:341-351. 36. Johns DC, Marx R, Mains RE, O’Rourke B, Marban E: Inducible genetic suppression of neuronal excitability. J Neurosci 1999, 19:1691-1697. 37.
Palmiter RD, Behringer RR, Quaife CJ, Maxwell F, Maxwell IH, Brinster RL: Cell lineage ablation in transgenic mice by cell-specific expression of a toxin gene. Cell 1987, 50:435-443.
38. Breitman ML, Rombola H, Maxwell IH, Klintworth GK, Bernstein A: Genetic ablation in transgenic mice with an attenuated diphtheria toxin A gene. Mol Cell Biol 1990, 10:474-479. 39. White K, Tahaoglu E, Steller H: Cell killing by the Drosophila gene reaper. Science 1996, 271:805-807. 40. Gossen M, Freundlieb S, Bender G, Muller G, Hillen W, Bujard H: Transcriptional activation by tetracyclines in mammalian cells. Science 1995, 268:1766-1769. 41. Urlinger S, Baron U, Thellmann M, Hasan MT, Bujard H, Hillen W: Exploring the sequence space for tetracycline-dependent transcriptional activators: novel mutations yield expanded range and sensitivity. Proc Natl Acad Sci USA 2000, 97:7963-7968. 42. Nirenberg S, Cepko C: Targeted ablation of diverse cell classes in the nervous system in vivo. J Neurosci 1993, 13:3238-3251. 43. Kitamoto T: Conditional modification of behavior in Drosophila by • targeted expression of a temperature-sensitive shibire allele in defined neurons. J Neurobiol 2001, 47:81-92. The temperature-sensitive ts-1 allele of shibire is expressed in genetically defined neuronal subsets in Drosophila. When flies are shifted to elevated temperatures, the genetically tagged neurons are reversibly inactivated: expression in photoreceptors leads to conditional blindness; expression in cholinergic neurons to conditional paralysis. When combined with ‘enhancer trapping’, the method raises the possibility of screening for behavioral phenotypes caused by the loss of function of specific circuits. 44. Hinshaw JE: Dynamin and its role in membrane fission. Annu Rev Cell Dev Biol 2000, 16:483-519. 45. Grant D, Unadkat S, Katzen A, Krishnan KS, Ramaswami M: Probable mechanisms underlying interallelic complementation and temperature-sensitivity of mutations at the shibire locus of Drosophila melanogaster. Genetics 1998, 149:1019-1030. 46. Farber IC, Grinvald A: Identification of presynaptic neurons by laser photostimulation. Science 1983, 222:1025-1027. 47.
Callaway EM, Katz LC: Photostimulation using caged glutamate reveals functional circuitry in living brain slices. Proc Natl Acad Sci USA 1993, 90:7661-7665.
Genetic schemes and schemata in neurophysiology Boris V Zemelman and Gero Miesenböck* Information in nervous systems is often carried by neural ensembles — groups of neurons in transient functional linkage — and written in a code that involves the spatial locations of active neurons or synapses and the times at which activity occurs. Even in favorable neuroanatomical circumstances, studying neural ensemble function presents a serious experimental challenge. One recent strategy to overcome this challenge relies on protein-based sensors that provide direct optical images of neural activity, and on proteinbased effectors that interfere with it. Because these molecules are encodable in DNA, they can be introduced into intact animals by genetic manipulation, and their expression pattern can be tailored to include — exclusively and at the same time comprehensively — the neurons of interest. Circumscribed populations of neurons can thus be studied in virtual isolation at defined stages of intact neural pathways. Addresses Laboratory of Neural Systems, Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, Box 205, 1275 York Avenue, New York, NY 10021, USA *e-mail: [email protected] Current Opinion in Neurobiology 2001, 11:409–414 0959-4388/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved. Abbreviations 2HG second-harmonic generation GFP green fluorescent protein YFP yellow fluorescent protein
Introduction Information is inevitably tied to a physical carrier — be it a pencil mark on paper or a neurotransmitter molecule bound to its receptor, the orientation of a dipole in a magnetic domain or the polarity of a voltage across a neuronal membrane. It is the physical carrier of a message rather than its content that is observed directly, and it is the carrier rather than the content on which physically implemented computations operate. Neurophysiologists rely on this fundamental link between information and its physical substrate when they explore how information is encoded and processed in the brain. What makes their task technically difficult is the way nervous systems are organized. Information tends to be distributed over large numbers of processing elements, and analyzing their function or interfering with it requires ways to monitor or manipulate many elements at once. To make matters worse, the typical neuroanatomy does not segregate different classes of processing elements, but rather interleaves them in arrangements that combine regularity on a large scale with a significant degree of local variability. Isolating the function of a specific class would
depend on methods with both a built-in selectivity for these processing elements and an ability to distinguish them reliably from neighboring ones belonging to different classes. Here, we review recent developments in experimental approaches that use genetics to achieve such selectivity. The shared premise of these approaches is that gene expression patterns can be used to single out functionally related groups of neurons for analysis or intervention. To distinguish this brand of genetics from more traditional ones that emphasize individual gene products and use mutations as their chief analytical tool, we refer to it as ‘schema genetics’: the genetics of organized patterns.
Schemata and schemes In the simplest genetic schemata, the choice of marker gene follows immediately from function. All neurons communicating through a specific neurotransmitter, for example, will express the cognate biosynthetic enzymes, and these can serve to define the class genetically. Will genetic markers also exist for groups of neurons not bound by an obvious functional characteristic, such as, for instance, the pyramidal cells of one cortical layer? The answer is likely to be affirmative. Shotgun searches for genetic regulatory elements in Drosophila [1,2] often reveal genes whose expression patterns map precisely to identified processing stages in layered neural pathways (see the online databases of FlyBase, http://flybase.bio.indiana.edu/, and Flybrain, http://flybrain.neurobio.arizona.edu, for examples). Anecdotal evidence in vertebrates points in the same direction [3,4•,5•]. We do not consider this a surprising coincidence. Biological structures are products of developmental programs encoded, to a large extent, genetically, and the gene expression patterns discernible within them may be viewed as an imprint that the generative programs have left behind. As such, they are likely to encapsulate the organizing principles. The genetic schemata that pattern nervous systems thus provide an experimental platform on which schemes to analyze or perturb defined populations of neurons can be built. Once the regulatory elements responsible for a particular gene expression pattern have been isolated, they can be used to drive the expression of natural or engineered proteins capable of reporting or interfering with neural physiology. These genetically encoded tools are the focus of this review.
Genetic schemes for analyzing neural function Genetic schemes for analyzing neural function unite the selectivity of genetics with the resolution of optical imaging to highlight the activities of circumscribed classes of
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neurons in virtual isolation. To this end, all current schemes use derivatives of green fluorescent protein (GFP), whose light-emitting properties have been engineered to report changes in an electrical or chemical variable that is used by neurons to convey information. Older schemes based on luminescent light emitters [6,7] have become obsolete; they typically generate photon fluxes several orders of magnitude below those of fluorescent probes, depend on exogenous substrates (which impedes their application in intact systems) and lack depth resolution because they operate without an exciting light beam and/or scanning optics. The main genetically encoded sensors report changes in membrane potential and intracellular calcium concentration, or signal the release of neurotransmitter (but see also [8–12]). The three major types of probes have reached varying levels of maturity. Membrane-potential indicators ‘Electromechanical’ sensors
The first and only genetically encoded fluorescent membrane-potential sensor, called FlaSh [13], must be considered a prototype. FlaSh comprises two modules: the Shaker potassium channel of Drosophila, which is rendered non-conducting by a point mutation in its pore [14], serves as the voltage-sensing device; GFP, inserted after the integral membrane portion of Shaker, provides the optical read-out. When expressed in Xenopus oocytes, the fluorescence of FlaSh decreases by 5% after a voltage step that moves the entire gating charge of Shaker [13]. The kinetics of the optical signal are sluggish, and a mechanistic link with the slow process of C-type inactivation [15] is likely. The conformational change associated with inactivation probably creates or rearranges physical contacts among the four GFP modules crowded at the inner pore of the tetrameric channel, and this reduces their fluorescence output. In the three years since the first description of FlaSh, its use in cell types other than oocytes has not been reported. A likely explanation is that FlaSh subunits are incorporated into endogenous voltage-gated potassium channels, which are absent in oocytes, and that subunit mixing disrupts the function of these channels or of FlaSh itself. The former would be an expected consequence of the conductanceblocking mutation in FlaSh; the latter, a result of the loss of interacting partners required to couple channel inactivation to fluorescence changes. We presume that the potential solution to these problems — linking four FlaSh coding regions in a single transcript — has been tried and found unsatisfactory. Because the mechanism of FlaSh remains fundamentally obscure, it is difficult to base design improvements on it. Therefore, in a number of laboratories efforts are under way to develop membrane-potential sensors that retain the
modularity and even the basic building blocks of FlaSh, but that combine them in mechanistically more transparent designs that can be iteratively optimized. ‘Solid-state’ sensors
The most elegant voltage probes would operate without moving parts. Rather than relying on conformational rearrangements to transduce membrane-potential changes into optical signals, they would report the direct effects of the transmembrane electric field on the electronic structures of their chromophores. Fluorescence, unfortunately, provides a poor read-out in these circumstances: even the large electric fields (about 100,000 V/cm) experienced by fluorophores placed directly into lipid bilayers typically cause only small fluorescence changes (generally less than 0.5% per 10 mV). More sensitive to applied electric forces than fluorescence is the nonlinear optical phenomenon of second-harmonic generation (2HG) [16], in which a membrane-associated chromophore scatters the energy of two incident fundamental photons into one frequency-doubled harmonic photon. Even the attenuated electric field at some distance from the plasma membrane may suffice to modulate 2HG. The fluorescence of GFP fused to an integral membrane protein, for example, is unaffected by membrane potential, but the same protein gives rise to an apparent harmonic signal with a steep voltage dependence of about 10% per 10 mV [17•]. As a second-order reaction in the concentration of incident photons, 2HG possesses the same intrinsic resolving power as two-photon fluorescence excitation. Unlike fluorescent light, however, which is radiated isotropically, harmonic light propagates only in the forward direction and at a particular off-axis angle with respect to the driving beam. ‘Harmonic’ microscopes (and the samples imaged on them) must therefore accommodate special detection pathways to capture the off-axis photons [18•]. Calcium indicators
The classical alternative to detecting changes in membrane potential is to record the changes in intracellular calcium concentration that follow them. The advantages of this indirect measurement include strong signals from large populations of soluble indicator molecules with wide dynamic ranges. The disadvantage is that rapid membrane-potential changes appear temporally filtered when viewed through intracellular calcium dynamics. One temporal filter reflects the kinetics of cellular calcium clearance, another the association–dissociation kinetics of indicator and calcium ion. This disadvantage can, however, be turned into an advantage: a signal that is expanded in time will register more reliably in imaging systems unable to survey continuously every point in space. Dual-fluorophore sensors
Genetically encoded dual-emission calcium sensors, called cameleons, comprise four elements in linear arrangement
Genetic schemes and schemata in neurophysiology Zemelman and Miesenböck
[19,20]: a blue-shifted variant of GFP; the calcium-binding protein calmodulin; a calmodulin target; and a native GFP or red-shifted variant. At resting calcium concentrations, cameleons adopt a conformation with a large average distance and a wide range of dipole angles between the two fluorophores; fluorescence resonance energy transfer between them is only moderately more efficient than if they were physically uncoupled. As calcium concentrations rise, calmodulin engages its target and locks the cameleon into a more rigid and tighter arrangement. The efficiency of fluorescence resonance energy transfer consequently increases. Although cameleons display only modest fluorescence changes in response to calcium, they have proven adequate for detecting spontaneous calcium transients in muscle, as well as calcium transients evoked by intense electrical stimulation in neurons of Caenorhabditis elegans [21•]. Single-fluorophore sensors
Single-emission probes termed camgaroos [22] combine calmodulin and yellow fluorescent protein (YFP) in an unusual topology: the YFP sequence is interrupted at a strategic location to accommodate calmodulin. The calciumdriven conformational changes of calmodulin are thus transmitted directly to the protein shell surrounding the chromophore of YFP, which switches from a protonated non-fluorescent state in the absence of calcium to a deprotonated fluorescent state in the presence of calcium. The strengths as well as the weaknesses of camgaroos are rooted in this peculiar structure. The tight conformational coupling between calcium sensor and fluorophore allows for dynamic ranges that match those of small-molecule calcium indicators (about a sevenfold increase in fluorescence at saturating compared with basal calcium concentrations). However, drawbacks include the role of protons in the calcium-driven fluorescent switch, which makes camgaroos sensitive to pH interference; the lack of a calmodulin target peptide, which lowers their calcium affinity to about 7 µM; and the crack in the shell of YFP (necessary to accept the calmodulin insert), which causes the protein to misfold at temperatures above 28°C. Recent variations on the camgaroo topology, called G-CaMP [23•] and pericams [24•], overcome one of these drawbacks: they incorporate a calmodulin target. As expected, the calcium affinity of these probes rises to < 1 µM; however, pH interference and stability problems tend to persist. The latter, in particular, may continue to stand in the way of an eagerly anticipated first test of these probes in a vertebrate neuron. Indicators of neurotransmitter release
The third type of genetically encoded indicator signals the release of neurotransmitter [25] and thus complements membrane potential and calcium sensors, which reveal cellular activity. To convert synaptic transmission into an
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optical signal, a switchable light emitter is tethered to the inner surface of synaptic vesicles. Light emission switches ‘on’ the instant that a vesicle fuses with the presynaptic plasma membrane, and ‘off’ the instant that the vesicle membrane is retrieved via endocytosis. The light emitter is a variant of GFP, called pHluorin, the fluorescence of which is reversibly quenched by protonation [25]. Vacuolar proton ATPases maintain the interior of synaptic vesicles at a pH of about 5.7 [25], whereas the extracellular fluid has a more alkaline pH of about 7.4. A roughly 50-fold difference in proton concentration thus exists between the two compartments. pHluorins rather efficiently translate this difference into about a 20-fold increase in fluorescence associated with exocytosis [26]. The magnitude and waveform of the fluorescent signal arising from vesicle fusion at a synapse are determined by a number of synaptic properties. The first is the size of the resting vesicle pool: larger pools will increase background fluorescence and make fusion events more difficult to detect. The second factor is how cleanly the reporter partitions between synaptic vesicles and the presynaptic membrane. When the v-SNARE VAMP/synaptobrevin-2 is used as an anchor, about 10% of the indicator is found in the plasma membrane even under resting conditions [26,27]. Although this fraction may appear small, it is typically the main source of background fluorescence, as surface-located pHluorins tend to be in the deprotonated, fluorescent state. Finally, because synaptic vesicle membrane is re-internalized only after a delay following exocytosis (re-acidification of the vesicle interior, in contrast, occurs virtually instantaneously after the vesicle lumen is sealed off [27]), the pHluorin signal represents the convolution of each synaptic impulse with the time course of the associated vesicle turnover. This time course is in itself subject to short-term modulation: by the magnitude of the load on the endocytic machinery [27], for instance, or by the concentration of calcium in the nerve terminal [28•]. The pHluorin trace therefore represents a running average of synaptic activity, with an integration window of several to several tens of seconds. Of the three main types of genetically encoded sensors, indicators of synaptic transmission have moved the farthest beyond the prototype stage. They have generated new insights into the physiology of nerve terminals [27,28•], and may soon make their debut in areas of systems physiology where their two fundamental limitations can be tolerated or bypassed. These limitations are the small optical signal associated with a single synaptic impulse (owing to only a handful of light emitters that can be packed onto the surface of a vesicle) and their poor temporal resolution (owing to the synaptic vesicle cycle). Their forte, accordingly, will be analyses of spatial activity patterns in topographic projections and measurements of aggregate synaptic strength. While the potential of analyzing neural physiology with genetically encoded optical probes has been instantly and
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widely recognized [7,29,30], the volume of scientific knowledge that the probes have produced up until March 2001 is disappointing. The indicators have been applied exclusively in single cells in culture (but see [17•,21•]), and here mostly for purposes of validation rather than discovery (but see [27,28•]). We can only speculate as to what has precluded applications in multicellular systems — maybe cultural barriers or skepticism about the performance of some of the first-generation probes. Or, perhaps, the lead times to generate the necessary organisms and imaging platfoms are so long that the most interesting work is still in progress. In any case, the study that unleashes the full potential of the approach has yet to be published.
Genetic schemes for perturbing neural function Compared with the genetically encoded indicators, the use of which requires imaging expertise, the easily accessible genetic tools for interfering with populations of neurons have a more extensive record of accomplishment. In the past year alone, genetic interference with neural function has been used in flies and mice to address a broad range of questions from the specification of neuronal cell types to the consolidation and generalization of memories [31,32•–34•]. Irreversible effectors
All existing tools silence defined groups of neurons. The simplest schemes do so unconditionally and irreversibly: the light chain of tetanus toxin cleaves the v-SNARE VAMP/synaptobrevin-2 and blocks synaptic transmission [35]; the inwardly rectifying potassium channel Kir2.1 clamps the neuronal membrane potential below its resting value and suppresses excitability [36]; the A chain of diphtheria toxin ADP-ribosylates elongation factor 2 to inhibit protein synthesis and cause neuronal cell death [37,38]; cell-death genes such as reaper block caspase inhibitors and trigger apoptosis [39]. To achieve some temporal control over their effect, silencing genes are often expressed from promoters controlled by ligand-dependent transcription factors without endogenous targets. In these refined genetic circuits, patterned expression of the transcription factor determines which neurons are capable of synthesizing an effector protein, whereas adding or removing a ligand (usually tetracycline [40,41]) controls the actual onset of synthesis [32•]. Because many toxins are effective in copy numbers as low as a single molecule per cell, measures must be taken to ensure tight transcriptional regulation [41], or to decrease the toxicity of the effector if transcription remains persistently leaky [32•,38]. A different way to time an intervention, and to add a layer of spatial control, is to express the non-toxic bacterial enzyme β-galactosidase in designated neurons and use its activity to accumulate a non-toxic reaction product, fluorescein, from a synthetic
substrate. The dye-loaded neurons can then be photoablated when desired [42]. While the onset of neuronal quiescence can be controlled by regulating transcription, the result is, for all practical purposes, permanent even if the targeted neurons survive the intervention. For example, the effect of tetanus toxin light chain will not subside when transcription of the gene encoding the toxin is shut down, but only after all mRNA and toxin molecules have been degraded and all proteolyzed v-SNAREs have been replaced. This will only occur over a period of many hours or days. In many instances, however, one would prefer to switch neurons between silent and active states repeatedly and rapidly in the course of a single experiment. This requires that effector activity is controlled at a level other than gene expression. Reversible effectors
The first genetic scheme to accomplish control at a level other than gene expression uses a temperature-sensitive allele of shibire in Drosophila [34•,43•]. The shibire gene encodes dynamin, a homo-oligomeric GTPase required for fission of recycling synaptic vesicles from the plasma membrane [44]. Some mutant forms of dynamin act as dominant inhibitors of wild-type function [45]; oligomers which incorporate a sizeable proportion of them cannot sustain vesicle recycling at a sufficient rate. As a result, affected synapses cease to transmit once the available pool of synaptic vesicles has been consumed. The two properties of dominance and temperature-sensitivity ensure spatial and temporal control, respectively, over the synaptic blockade. Susceptible neurons (those which express the mutant shibire allele along with the endogenous wild-type gene) are designated genetically, while shifts between permissive and restrictive temperatures are used to impose and relieve the block on synaptic vesicle recycling [34•,43•]. Effects on vesicle release tend to trail those on recycling by about a minute [43•], the time typically required to deplete or refill the synaptic vesicle pool.
Conclusions and perspectives Genetics and protein engineering are invading what not long ago was the exclusive domain of synthetic chemistry and pharmacology. Protein-based sensors of neural activity are emerging opposite synthetic indicator dyes; drugs that silence neurons or synapses are finding genetically encoded counterparts. A conspicuous void, however, exists in the genetic line-up at the position that photostimulants and caged neurotransmitters (that is, inactive precursors activated by light) occupy in the synthetic arsenal [46,47]. We feel that filling this void constitutes a challenging but particularly rewarding area for future developments. Whereas conventional photostimulation must localize the stimulus in the form of the uncaging light beam, genetic schemes would localize the response (in the form of an
Genetic schemes and schemata in neurophysiology Zemelman and Miesenböck
encoded sensitivity to light), and illumination could then be broad. Patterns of distributed activity might be fed directly into a genetically circumscribed population of neurons, irrespective of the anatomical location of its members or their connection to sensory input. Like metabolic pathways, neural pathways are arranged as graphs of interconnected nodes. The ability to probe individual nodes with pure, often synthetic substrates proved instrumental for the dissection of metabolism. Perhaps the ability to probe defined groups of neurons with synthetic information will hold the key to an understanding of neural systems.
Acknowledgements Gero Miesenböck is an Alfred P Sloan and Klingenstein Fellow, a Beckman Young Investigator and a Searle Scholar.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest 1.
O’Kane CJ, Gehring WJ: Detection in situ of genomic regulatory elements in Drosophila. Proc Natl Acad Sci USA 1987, 84:9123-9127.
2.
Bier E, Vaessin H, Shepherd S, Lee K, McCall K, Barbel S, Ackerman L, Carretto R, Uemura T, Grell E et al.: Searching for pattern and mutation in the Drosophila genome with a P-lacZ vector. Genes Dev 1989, 3:1273-1287.
3.
Tsien JZ, Chen DF, Gerber D, Tom C, Mercer EH, Anderson DJ, Mayford M, Kandel ER, Tonegawa S: Subregion- and cell typerestricted gene knockout in mouse brain. Cell 1996, 87:1317-1326.
4. •
Feng G, Mellor RH, Bernstein M, Keller-Peck C, Nguyen QT, Wallace M, Nerbonne JM, Lichtman JW, Sanes JR: Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Neuron 2000, 28:41-51. GFP variants are expressed in mouse projection neurons under the control of regulatory elements derived from the thy1 gene. Multiple independent mouse lines differ in the numbers and types of neurons labeled; within any one line, the pattern of gene expression is constant and heritable, and often confined to a particular anatomical structure. 5. •
Leighton PA, Mitchell KJ, Goodrich LV, Lu X, Pinson K, Scherz P, Skames WC, Tessier-Lavigne M: Defining brain wiring patterns and mechanisms through gene trapping in mice. Nature 2001, 410:174-179. Gene expression and wiring patterns in transgenic mouse lines generated by a ‘secretory trap’ method display remarkable variety and anatomical specificity. 6.
Shimomura O, Inouye S, Musicki B, Kishi Y: Recombinant aequorin and recombinant semi-synthetic aequorins. Cellular Ca2+ ion indicators. Biochem J 1990, 270:309-312.
7.
Miesenböck G, Rothman JE: Patterns of synaptic activity in neural networks recorded by light emission from synaptolucins. Proc Natl Acad Sci USA 1997, 94:3402-3407.
8.
Kuner T, Augustine GJ: A genetically encoded ratiometric indicator for chloride: capturing chloride transients in cultured hippocampal neurons. Neuron 2000, 27:447-459.
9.
Jayaraman S, Haggie P, Wachter RM, Remington SJ, Verkman AS: Mechanism and cellular applications of a green fluorescent protein-based halide sensor. J Biol Chem 2000, 275:6047-6050.
10. Zaccolo M, De Giorgi F, Cho CY, Feng L, Knapp T, Negulescu PA, Taylor SS, Tsien RY, Pozzan T: A genetically encoded, fluorescent indicator for cyclic AMP in living cells. Nat Cell Biol 2000, 2:25-29. 11. Sato M, Hida N, Ozawa T, Umezawa Y: Fluorescent indicators for cyclic GMP based on cyclic GMP-dependent protein kinase I alpha and green fluorescent proteins. Anal Chem 2000, 72:5918-5924.
413
12. Honda A, Adams SR, Sawyer CL, Lev-Ram V, Tsien RY, Dostmann WR: Spatiotemporal dynamics of guanosine 3′′,5′′-cyclic monophosphate revealed by a genetically encoded, fluorescent indicator. Proc Natl Acad Sci USA 2001, 98:2437-2442. 13. Siegel MS, Isacoff EY: A genetically encoded optical probe of membrane voltage. Neuron 1997, 19:735-741. 14. Perozo E, MacKinnon R, Bezanilla F, Stefani E: Gating currents from a nonconducting mutant reveal open-closed conformations in Shaker K+ channels. Neuron 1993, 11:353-358. 15. Hoshi T, Zagotta WN, Aldrich RW: Two types of inactivation in Shaker K+ channels: effects of alterations in the carboxy-terminal region. Neuron 1991, 7:547-556. 16. Bouevitch O, Lewis A, Pinevsky I, Wuskell JP, Loew LM: Probing membrane potential with nonlinear optics. Biophys J 1993, 65:672-679. 17. •
Khatchatouriants A, Lewis A, Rothman Z, Loew L, Treinin M: GFP is a selective non-linear optical sensor of electrophysiological processes in Caenorhabditis elegans. Biophys J 2000, 79:2345-2352. GFP is attached to two integral membrane proteins in C. elegans: an acetylcholine receptor subunit and the stomatin homologue MEC-2, which is expressed in mechanosensory neurons. The proteins give rise to optical signals which are detected at exactly half the fundamental wavelength (and therefore attributed to 2HG) and modulated by choline and touch, respectively. Animals lacking functional acetylcholine receptors lack the choline-responsive optical signal. Many mechanistic details remain mysterious, in particular, how flexible linkage with a membrane protein creates an ordered GFP array in which the phased emissions from individual dipoles can sum constructively, a requirement for 2HG. 18. Moreaux L, Sandre O, Charpak S, Blanchard-Desce M, Mertz J: • Coherent scattering in multi-harmonic light microscopy. Biophys J 2001, 80:1568-1574. A ‘harmonic’ laser-scanning microscope, and examples of its use for imaging natural and artificial membranes. 19. Miyawaki A, Llopis J, Heim R, McCaffery JM, Adams JA, Ikura M, Tsien RY: Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 1997, 388:882-887.
20. Miyawaki A, Griesbeck O, Heim R, Tsien RY: Dynamic and quantitative Ca2+ measurements using improved cameleons. Proc Natl Acad Sci USA 1999, 96:2135-2140. 21. Kerr R, Lev-Ram V, Baird G, Vincent P, Tsien RY, Schafer WR: Optical • imaging of calcium transients in neurons and pharyngeal muscle of C. elegans. Neuron 2000, 26:583-594. Cameleons are expressed in pharyngeal muscle and neurons of C. elegans. Serotonin-stimulated feeding is correlated with calcium transients in muscle. Electrically evoked neural activity results in calcium transients and muscle contractions. Significantly, cameleons reveal predictably altered calcium transients in animals carrying mutant calcium channels. 22. Baird GS, Zacharias DA, Tsien RY: Circular permutation and receptor insertion within green fluorescent proteins. Proc Natl Acad Sci USA 1999, 96:11241-11246. 23. Nakai J, Ohkura M, Imoto K: A high signal-to-noise Ca2+ probe • composed of a single green fluorescent protein. Nat Biotechnol 2001, 19:137-141. The M13 fragment of myosin light chain kinase is linked to the newly created amino terminus of a circularly permuted GFP, and calmodulin is attached to the carboxyl terminus. The resulting fluorescent calcium sensor, with a dissociation constant for calcium of 235 nM, is expressed in cultured mouse myotubes grown at 28°C and used to monitor calcium transients caused by pharmacological and electrical stimulation. 24. Nagai T, Sawano A, Park ES, Miyawaki A: Circularly permuted green • fluorescent proteins engineered to sense Ca2+. Proc Natl Acad Sci USA 2001, 98:3197-3202. Pericams are single-fluorophore calcium sensors with a topology similar to that of G-CaMP [23•], except that YFP is used instead of GFP and coupled in a slightly different arrangement to calmodulin and the M13 peptide. The dissociation constant for calcium of the prototypical 'flash-pericam' is approximately 700 nM. Additional mutations in YFP give rise to pericams with ratiometric and ‘inverse’ calcium responses and possibly improved stability. 25. Miesenböck G, De Angelis DA, Rothman JE: Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature 1998, 394:192-195. 26. Sankaranarayanan S, De Angelis D, Rothman JE, Ryan TA: The use of pHluorins for optical measurements of presynaptic activity. Biophys J 2000, 79:2199-2208.
414
27.
Sensory systems
Sankaranarayanan S, Ryan TA: Real-time measurements of vesicleSNARE recycling in synapses of the central nervous system. Nat Cell Biol 2000, 2:197-204.
28. Sankaranarayanan S, Ryan TA: Calcium accelerates endocytosis of • vSNAREs at hippocampal synapses. Nat Neurosci 2001, 4:129-136. Synaptic vesicle endocytosis and exocytosis are monitored optically in hippocampal neurons expressing synapto-pHluorin, the genetically encoded indicator of synaptic transmission. Both exo- and endocytic rates are affected by the concentration of calcium ion in the nerve terminal. Calcium thus seems to couple the rate of vesicle recycling to the rate of release. 29. Siegel MS: Genetic probes: new ways to watch cells in action. Curr Biol 1997, 7:R556-R557. 30. Crick F: The impact of molecular biology on neuroscience. Philos Trans R Soc London Ser B 1999, 354:2021-2025. 31. Liu L, Wolf R, Ernst R, Heisenberg M: Context generalization in Drosophila visual learning requires the mushroom bodies. Nature 1999, 400:753-756. 32. Gogos JA, Osborne J, Nemes A, Mendelsohn M, Axel R: Genetic • ablation and restoration of the olfactory topographic map. Cell 2000, 103:609-620. In contrast to many other types of neurons, olfactory sensory neurons are continuously but asynchronously shed and replaced. The authors express diphtheria toxin in P2 olfactory neurons (that is, sensory neurons expressing the olfactory receptor P2) under the control of tetracycline-dependent transcriptional activator. After selective, synchronous ablation of all P2 neurons, growth and targeting of newly generated neurons restores the topographic projections observed during normal development. 33. Lee KJ, Dietrich P, Jessell TM: Genetic ablation reveals that the roof • plate is essential for dorsal interneuron specification. Nature 2000, 403:734-740. Roof-plate cells in mouse embryos are destroyed by selective expression of diphtheria toxin to assess the contributions of the roof plate and epidermal ectoderm to dorsal neuronal patterning. The authors conclude that the roof plate is essential for specifying several classes of neurons in the mammalian central nervous system. 34. Waddell S, Armstrong JD, Kitamoto T, Kaiser K, Quinn WG: The • amnesiac gene product is expressed in two neurons in the Drosophila brain that are critical for memory. Cell 2000, 103:805-813. See also Kitamoto [43•]. Expression of a temperature-sensitive allele of shibire is restricted to the dorsal paired medial cells in Drosophila to analyze the role of the secreted amnesiac gene product, which is synthesized preferentially in these cells, in memory formation. Inactivation of the dorsal paired medial cells abolishes olfactory memory, demonstrating the importance of the amnesiac gene product. The authors remark that the ablation of dorsal paired medial cells via expression of cell death genes proved lethal, emphasizing the benefit of a conditional approach.
35. Sweeney ST, Broadie K, Keane J, Niemann H, O’Kane CJ: Targeted expression of tetanus toxin light chain in Drosophila specifically eliminates synaptic transmission and causes behavioral defects. Neuron 1995, 14:341-351. 36. Johns DC, Marx R, Mains RE, O’Rourke B, Marban E: Inducible genetic suppression of neuronal excitability. J Neurosci 1999, 19:1691-1697. 37.
Palmiter RD, Behringer RR, Quaife CJ, Maxwell F, Maxwell IH, Brinster RL: Cell lineage ablation in transgenic mice by cell-specific expression of a toxin gene. Cell 1987, 50:435-443.
38. Breitman ML, Rombola H, Maxwell IH, Klintworth GK, Bernstein A: Genetic ablation in transgenic mice with an attenuated diphtheria toxin A gene. Mol Cell Biol 1990, 10:474-479. 39. White K, Tahaoglu E, Steller H: Cell killing by the Drosophila gene reaper. Science 1996, 271:805-807. 40. Gossen M, Freundlieb S, Bender G, Muller G, Hillen W, Bujard H: Transcriptional activation by tetracyclines in mammalian cells. Science 1995, 268:1766-1769. 41. Urlinger S, Baron U, Thellmann M, Hasan MT, Bujard H, Hillen W: Exploring the sequence space for tetracycline-dependent transcriptional activators: novel mutations yield expanded range and sensitivity. Proc Natl Acad Sci USA 2000, 97:7963-7968. 42. Nirenberg S, Cepko C: Targeted ablation of diverse cell classes in the nervous system in vivo. J Neurosci 1993, 13:3238-3251. 43. Kitamoto T: Conditional modification of behavior in Drosophila by • targeted expression of a temperature-sensitive shibire allele in defined neurons. J Neurobiol 2001, 47:81-92. The temperature-sensitive ts-1 allele of shibire is expressed in genetically defined neuronal subsets in Drosophila. When flies are shifted to elevated temperatures, the genetically tagged neurons are reversibly inactivated: expression in photoreceptors leads to conditional blindness; expression in cholinergic neurons to conditional paralysis. When combined with ‘enhancer trapping’, the method raises the possibility of screening for behavioral phenotypes caused by the loss of function of specific circuits. 44. Hinshaw JE: Dynamin and its role in membrane fission. Annu Rev Cell Dev Biol 2000, 16:483-519. 45. Grant D, Unadkat S, Katzen A, Krishnan KS, Ramaswami M: Probable mechanisms underlying interallelic complementation and temperature-sensitivity of mutations at the shibire locus of Drosophila melanogaster. Genetics 1998, 149:1019-1030. 46. Farber IC, Grinvald A: Identification of presynaptic neurons by laser photostimulation. Science 1983, 222:1025-1027. 47.
Callaway EM, Katz LC: Photostimulation using caged glutamate reveals functional circuitry in living brain slices. Proc Natl Acad Sci USA 1993, 90:7661-7665.