Projects in Cellular and Molecular Neurobiology
- Project 1
- Project 2
- Project 3
- Project 4
- Project 5
- Project 6
- Project 7
- Project 8
- Project 9
- Project 10
- Project 11
Molecular mechanisms of nicotine adaptation in Caenorhabditis elegans
We use C. elegans as a model system to investigate the molecular mechanisms of the adaptation of the neuro(muscular) system(s) to nicotine. Work in vertebrate systems showed that chronic exposure to low levels of nicotine leads to long-lasting changes in both the abundance and the functional properties of nicotinic acetylcholine receptors (nAChRs) in the brain. The molecular mechanisms underlying these changes, which are believed to cause initially tolerance and ultimately addiction to the drug, remain poorly understood. Even though the simple C. elegans nervous system can not serve as a model for the psychological component of nicotine addiction, the molecules and genetic pathways that govern the initial molecular changes in nAChRs in response to nicotine are likely to be well conserved. We thus make use of the extensive repertoire of methods available in C. elegans to investigate these mechanisms, using a combination of behavioural studies, genetics, cell biology and electrophysiology.
This project is currently dormant.
Proteomic identification of factors associated with nicotinic acetylcholine receptors (nAChRs)
Proteins that act as regulators of nAChR function, for example through direct post-translational modification (like protein kinases), must physically interact with nAChR subunits. Thus they can be identified through biochemical co-purification with nAChRs. We have purified the so-called levamisole-sensitive nAChR from C. elegans, which is expressed in both muscles and many neurons (including motorneurons). To do this, we used the tandem affinity purification (TAP; see below). Proteins that were co-purified with TAP-tagged levamisole receptor subunits were identified in the whole sample, using 2D-HPLC of trypsin-digested peptides and nanoelectrospray-tandem mass spectrometry. In addition to abundant nAChR subunits, we could identify proteins that interact with the nAChR with very low affinity, and thus were highly underrepresented in the sample. To distinguish those from co-isolated contaminating proteins, we use a functional assay. Each of the proteins is depleted in vivo using doublestranded RNA interference (RNAi), and the responses of these animals to nicotine are then assayed. Thus we could identify a number of proteins whose depletion either caused nicotine resistance or hypersensitivity, making them prime candidates for factors affecting nAChR biology. We are characterizing these proteins in detail, and two of these proteins were found to act in the ER to influence the subunit composition of nAChRs. Gottschalk et al., 2005, EMBO J. 24, p 2566-2578. Almedom RB, Liewald J, Hernando G, Schultheis C, Rayes D, Pan J, Schedletzky T, Hutter H, Bouzat C, Gottschalk A. 2009. EMBO J. 28:2636-49.
Light-activation or -inhibition of excitable cells in live nematodes, using Channelrhodopsin-2 and Halorhodopsin
In collaboration with Georg Nagel and Ernst Bamberg (Würzburg University and Max Planck Institute for Biophysics), we have established a directly light-gated cation channel, channelrhodopsin-2 (ChR2), from a green alga, as a tool to evoke activity in muscles and neurons of C. elegans, simply by illumination with blue light. This allows to trigger neuronal activity in an essentially non-invasive manner in live and behaving animals. Thus, we could cause simultaneous contraction of all body muscles in response to light. When expressed in mechanosensory neurons, ChR2 could evoke (reversal) responses, as if the animals had been touched. This was even possible in animals that bear a mutation of the mechanosensory ion-channel and thus lack normal activity of these cells. The activity of ChR2 in situ could also be shown by direct electrophysiology of C. elegans muscle. This work has recently been published: Nagel et al., 2005, Curr. Biol. 15, 2279-2284. Recently, we also introduced the yellow-light driven chloride pump Halorhodopsin from the Archaeon Natronomonas pharaonis (NpHR), as a photo-hyperpolarizing agent, to acutely inhibit neurons and muscles. NpHR can be combined with ChR2 to allow acute, bidirectional control of neurons using yellow and blue light. This work was performed in collaboration with Karl Deisseroth's lab in Stanford (USA): Zhang et al. (2007) Nature 446: 633-39
Collaborations: Georg Nagel, Würzburg University and Ernst Bamberg, Max Planck Institute for Biophysics; Karl Deisseroth, Stanford University, USA
Direct electrophysiology in C. elegans muscle cells
Jana Liewald, a postdoc in our lab, has established methods for direct electrophysiology in muscle cells of C. elegans, following previously published methods (see Richmond et al. (1999) Nat. Neurosci. 2, 959-64; Richmond and Jorgensen (1999) Nat. Neurosci. 2, 791-797). This allows us to directly measure the effects of certain mutants (for example in the proteins we co-purified with nicotinic receptors, see project #2) on the function of post-synaptic nAChRs, as well as to unravel defects in pre-synaptic neurotransmission. Furthermore, Jana could measure the currents mediated by light-activation of ChR2 (see project #3, and the Figure below).
OptIoN: Optogenetic analysis of synaptic function
We recently introduced optogenetic investigation of neurotransmission (OptIoN) for time-resolved and quantitative assessment of synaptic function via behavioral and electrophysiological analyses. Release of acetylcholine (ACh) or gamma-aminobutyric acid (GABA) at Caenorhabditis elegans neuromuscular junctions can be photo-triggered using targeted expression of Channelrhodopsin-2 (ChR2). In intact ChR2 transgenic worms, photostimulation instantly induces body elongation (for GABA) or contraction (for ACh), which can be analyzed acutely, or during sustained activation with automated image analysis, to assess synaptic efficacy. In dissected worms, photostimulation evokes neurotransmitter-specific postsynaptic currents that can be triggered repeatedly and at various frequencies. Light-evoked behaviors and postsynaptic currents are significantly altered in mutants with pre- or postsynaptic defects. OptIoN facilitates the analysis of neurotransmission with high temporal precision, in a neurotransmitter-selective manner, likely allowing investigation of synaptic plasticity in C. elegans.
For more information, see our publication in Nature Methods: Liewald, Brauner, et al. 14 Sep. 2008; DOI:10.1038/NMETH.1252"
Optogenetic analysis of neuronal networks that generate behavior
Neuronal networks underlie behavior in every animal. C. elegans has a very simple and anatomically well-defined nervous system, allowing to decipher how neurons generate a particular behavior, and how they interact in networks. Such approaches are largely supported by optogenetic methods, as they allow to acutely stimulate, inhibit or otherwise modify neuronal function in live, freely behaving animals. We used these methods to decipher a nociceptive neuronal network of C. elegans, which involves the multidendritic neurons FLP and PVD, that cover head and body of the worm with fine processes and detect harsh touch, among other modalities. We investigated how these neurons interact with command interneurons to evoke backward or forward escape responses. The PVD-evoked forward escape behavior was further used to analyze the cell-autonomous function of genes specifically expressed in PVD. We found that the TRPM channel GTL-1 acts as a general amplifier of signals within PVD, and that the DEG/ENaC ASIC-1 potentiates the signal output of PVD and might extend its dynamic range.
Husson S, Steuer Costa W, Wabnig S, Stirman JN, Watson JD, Spencer WC, Akerboom J, Looger LL, Treinin M, Miller III DM, Lu H and Gottschalk A. (2012)
Optogenetic analysis of a nociceptor neuron and network reveals ion channels acting downstream of primary sensors.
Current Biology 22: 743-752.
Collaboration: David Miller III (Vanderbilt University), Hang Lu (Georgia Tech), Millet treinin (Hebrew University)
Photoactivated Adenylyl Cyclase (PAC) is a BLUF (blue-light sensing using flavin) domain protein from the flagellate Euglena gracilis. It generates cAMP in response to blue light and proved to be a useful optogenetic tool. Increasing cAMP in neurons causes enhanced synaptic transmission, however, unlike Channelrhodopsin, it does not override the intrinsic activity of the neuron or neuronal circuit, but simply enhances its transmitter output. Thus, intrinsic programs of neural circuits are upregulated. In C. elegans cholinergic motor neurons, PAC activation caused increased transmitter output and faster locomotion rates.
Weissenberger S, Schultheis C, Liewald J, Erbguth K, Nagel G, Gottschalk A.
PACα - an optogenetic tool for in vivo manipulation of cellular cAMP levels, neurotransmitter release, and behavior in Caenorhabditis elegans.
J Neurochem. 2011 Feb;116(4):616-25.
Colaboration: Georg Nagel, Würzburg University
Methods I: Tandem affinity purification (TAP) in C. elegans, a multicellular organism
The straightforward creation of transgenic animals, the ease to grow them in large amounts and the availability of a loss-of-function mutants as well as a sequenced genome make C. elegans an ideal system for proteomic approaches to the elucidation of protein networks. A generic method for the purification of a protein (complex) of interest is the tandem affinity purification, which had originally been developed in the yeast system (Rigaut et al., 1999, Nature Biotech 17 pp 1030-2). The TAP method makes use of a genetically encoded double affinity tag that allows the purification of the tagged protein (and interacting partners) through two subsequent chromatographies, under native conditions. We have adapted this method for C. elegans, in order to get access to (membrane-) protein networks especially in the nervous system. Our first approach was the identification of proteins interacting with nicotinic acetylcholine receptors.
Methods II: In vivo labeling of cell surface exposed proteins in
The most frequently used method to study expression patterns of proteins in C. elegans is to tag the protein of interest with GFP and express such a transgene in vivo. Especially for proteins expressed in the nervous system (for example neurotransmitter receptors), it is often desireable to visualize only those proteins that are inserted in the plasma membrane and thus exposed on the cell surface in a functional manner (for example as part of post-synaptic receptor clusters). However, GFP-tagged transgenes often hamper such investigations, because the protein is also found intracellularly and its fluorescence masks the signal from proteins that are inserted in the plasma membrane. Also, with GFP one cannot be sure if a spot of fluorescence belongs to proteins that are inserted in the plasma membrane, or to proteins that are present in vesicles close to, but not as part of the plasma membrane. We thus developed a method for specific immunolabeling of cell-surface exposed proteins and other extracellular epitopes. The protein of choice, for example a nAChR subunit, is expressed with a short peptide epitope tag (or GFP) that is placed extracellularly, if the protein is correctly inserted in the plasma membrane.
Fluorophore-labeled antibodies specific for this epitope tag are then injected into the body cavity of C. elegans, from where they get access to those epitopes on cell surfaces. Excess antibody is filtered from the extracellular fluid by the six scavenger cells, that constantly take up this fluid to remove metabolic breakdown products, thus leaving a clean staining patterns, essentially without background fluorescence. Alternatively, one can also use natural ligands of the protein of interest, labeled with a fluorophore, to label this protein, for example α-bungarotoxin, that binds to the nAChR. Examples of such staining have been published in McKay et al., 2004, Genetics 166, p. 161-9 and Zheng et al., 2004, Nature 427 pp 451-7, Gottschalk and Schafer, 2006 J. Neurosci. Meth. in press.
Panel D: The levamisole receptor subunit UNC-38, tagged with 3 cMYC epitopes at its C-terminus, was co-expressed with GFP-tagged synaptobrevin (VAMP::GFP, green signal), a pre-synaptic marker and stained with AlexaFluor594-labeled anti-cMyc antibodies (red signal). Antibody signal (corresponding to UNC-38 on cell surfaces) can be seen at post-synaptic sites (juxtaposed to green VAMP::GFP, arrows) and extrasynaptic sites (arrowheads) on nose muscles. Panels I, J: UNC-38::3xMYC was co-expressed with GFP-tagged UNC-29, another levamisole receptor subunit, and labeled with anti-Myc antibodies (red signal). Antibody-labeled UNC-38 can be seen along the nervecords and on muscle cell edges (arrowheads in I), as well as on neuronal cell bodies (arrowheads in J). In contrast, green signal originating from UNC-29::GFP can be seen throughout the cells (muscle and neuron), masking the signal of plasma membrane inserted UNC-29::GFP.
Methods III: Multicolor, selective photostimulation of neurons in freely moving animals
Specific photostimulation or inhibition of specific or even single neurons in C. elegans using microbial rhodopsins requires specific expression in just this cell, such that it can be activated by wide-field illumination. As such specific expression is hard to achieve, one could also restrict the light to just the region of the body that contains the neuron of interest. If this can be done in freely moving animals, one could influence the behavior in an acute fashion, while it occurs. Furthermore, multiple rhodopsins that are responsive to different colors of light could be combined in cells of a circuit, to probe different nodes in their effects on the behavior. Such multicolor illumination of distinct regions of the animal was achieved by our collaborators Jeff Stirman and Hang Lu (Georgia Tech). They devised a system that tracks an individual animal, and then projects a predefined multicolor light pattern on the animal, faithfully, while it is moving. We designed key proof of principle experiments to develop and test this system, and are now using it routinely in our lab.
Stirman J, Crane M, Husson S, Gottschalk A and Lu H. (2012)
A multispectral optical illumination system with precise spatiotemporal control for the manipulation of optogenetic reagents.
Nature Protocols 7: 207-220.
Stirman J, Crane M, Husson S, Wabnig S, Schultheis C, Gottschalk A*, Lu H*.
Real-time multimodal optical control of neurons and muscles in freely behaving Caenorhabditis elegans.
Nat Methods. 2011 Feb; 8 (2):153-8.
Electron Microscopy of Photostimulated Chemical Synapses
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