Neuron, Vol. 39, 1005���1017, September 11, 2003, Copyright ���2003 by Cell Press In Vivo Imaging of C. elegans Mechanosensory Neurons Demonstrates a Specific Role for the MEC-4 Channel in the Process of Gentle Touch Sensation (Driscoll and Chalfie, 1991 Huang and Chalfie, 1994 Welsh et al., 2002) that are coexpressed in the touch neurons (Huang and Chalfie, 1994 Mitani et al., 1993) and are required for sensation of gentle touch. The sto- matin-related MEC-2 protein (Huang et al., 1995) and Hiroshi Suzuki,1,4 Rex Kerr,1,4 Laura Bianchi,2 Christian Fr��kjaer-Jensen,1,3 Dan Slone,2 Jian Xue,2 Beate Gerstbrein,2 Monica Driscoll,2 and William R. Schafer1,* 1Division of Biology University of California, San Diego paraoxonase-related MEC-6, also essential for touch, associate with MEC-4 to enhance current conducted by La Jolla, California 92093 2 Department of Molecular Biology and MEC-4 channels expressed in Xenopus oocytes (Good- man et al., 2002 Chelur et al., 2002). In vivo, a MEC Biochemistry Nelson Biological Laboratories channel composed of these subunits has been hypothe- sized to make critical contacts to the extracellular matrix Rutgers, The State University of New Jersey Piscataway, New Jersey 08855 and to a specialized intracellular cytoskeleton that ex- erts the tension required for mechanical gating. 3 Niels Bohr Institute University of Copenhagen However, direct evidence for this model for MEC channel function has been difficult to obtain. The pheno- Copenhagen Denmark types of mec-4 and mec-2 loss-of-function mutants are identical to those caused by touch neuron ablation, leav- ing the nature of MEC channel���s role in touch transduc- tion an open question. Although the MEC channel might Summary be specifically involved in mechanotransduction, an identical mutant phenotype would result if this MEC-4 In the nematode C. elegans, genes encoding compo- nents of a putative mechanotransducing channel com- channel were generally required for normal touch neuron physiology, as has been shown for a Mirp K channel plex have been identified in screens for light-touch- insensitive mutants. A long-standing question, however, that maintains the touch neurons��� transmembrane po- tential (Bianchi et al., 2003). Since human disorders in- is whether identified MEC proteins act directly in touch transduction or contribute indirectly by maintaining volving stomatin defects are correlated with leaky plasma membrane channels (Stewart and Fricke, 2003), basic mechanoreceptor neuron physiology. In this study, we used the genetically encoded calcium indi- a role for the MEC proteins in general touch neuron physiology is not an unreasonable possibility. Although cator cameleon to record cellular responses of mech- anosensory neurons to touch stimuli in intact, behav- it has been possible to reconstitute sodium channels in heterologous expression systems using mutant forms ing nematodes. We defined a gentle touch sensory modality that adapts with a time course of approxi- of MEC-4 (Goodman et al., 2002), these heterologously expressed MEC channels are not mechanically gated, mately 500 ms and primarily senses motion rather than pressure. The DEG/ENaC channel subunit MEC-4 and perhaps because the reconstituted system does not recapitulate specific forces required for mechanical gat- channel-associated stomatin MEC-2 are specifically required for neural responses to gentle mechanical ing. Since dissection protocols that expose tiny C. ele- gans neurons rupture the worm���s cuticle and destroy stimulation, but do not affect the basic physiology of touch neurons or their in vivo responses to harsh me- the hydrostatic skeleton, in vivo electrophysiological re- cording from neurons of behaving C. elegans is not chanical stimulation. These results distinguish a spe- cific role for the MEC channel proteins in the process technically feasible. As a consequence, it has not yet been possible to directly assay the effects of the mec of gentle touch mechanosensation. genes on the activity of touch neurons in response to natural touch stimuli. Introduction To address this critical question, we established a Studies in organisms ranging from bacteria to mammals protocol for measuring physiological neuronal re- indicate that mechanotransduction can be mediated by sponses to touch in living animals through in vivo optical specialized ion channels that open or close in response imaging. Our strategy was to use the genetically en- to mechanical stimuli such as stretch or pressure (Hamill coded calcium indicator cameleon (Miyawaki et al., and Martinac, 2001). In C. elegans, screens for mecha- 1997) to monitor the activity of touch neurons in re- nosensory defective (Mec) mutants (Chalfie and Sulston, sponse to controlled mechanosensory stimuli. Camel- 1981 Chalfie and Au, 1989) have identified several genes eons are multidomain proteins that include YFP and CFP that encode candidate subunits of a mechanically gated moieties linked by calmodulin and a calmodulin binding ion channel responsible for sensing light touch in six peptide when Ca2 binds the calmodulin domain, con- body touch sensory neurons (Tavernarakis and Driscoll, formational changes allow fluorescence resonance en- 1997). In particular, mec-4 and mec-10 genes encode ergy transfer (FRET) between YFP and CFP such that members of the DEG/ENaC sodium channel superfamily ratios of fluorescence signals reflect intracellular Ca2 changes (Miyawaki and Tsien, 2000). We had previously recorded calcium transients in muscles and electrically *Correspondence: wschafer@biomail.ucsd.edu 4These authors contributed equally to this work. stimulated neurons (Kerr et al., 2000), suggesting that

Neuron 1006 Figure 1. Detection of Touch-Activated Cal- cium Transients in Intact C. elegans (A) An adult hermaphrodite C. elegans pre- pared for calcium imaging and mechanical stimulation. Worms are glued (black arrow) to a 2% agarose pad and immersed in extra- cellular saline. Mechanical stimulation is de- livered by moving the probe (white arrow) against the worm by means of a computer- controlled motorized stage. Scale bar, 50 m. (B) Pseudocolor ratio image of mechanosen- sory neuron ALM before, during, and after a 1.4 s mechanical stimulation. Blue to red represents a ratio of 1.25���1.95. Scale bar, 5 m. (C) Response of ALM to mechanical stimula- tion. Mechanical stimulation (black line) con- sisting of a 10 m deflection of the worm���s body lasting 200 ms was delivered at 5 s inter- vals. The fluorescence ratio (red line) in the ALM touch neuron cell body responded with characteristic rises. CFP and YFP intensities (top two lines) changed reciprocally (black arrowheads), indicating that the ratio change was due to a change in fluorescence resonance energy transfer. Matched upwards or downwards spikes in YFP and CFP intensities reflect lamp instability and are effectively canceled out in the ratio. Motion of the cell was moderate (brown line) and had a different profile from ratio changes, indicating that motion was insufficient to cause substantial artifacts. Although the baseline increased, potentiation was not observed during closely repeated stimuli. calcium transients could serve as a reliable indicator of stimuli in which the probe is kept continually moving against the cuticle. neuronal depolarization in C. elegans. Here we document the in vivo physiological responses We first recorded from the ALM touch neuron. Images of CFP and YFP emission were captured simultaneously to touch stimuli in C. elegans mechanoreceptor neurons. Using a cameleon-based reporter of transient calcium at 25 Hz, and custom software was used to compute the fluorescence ratio at each pixel and over the entire influx, we deduced the basic sensory/response capaci- ties of the body touch neurons, distinguished a specific cell body. Long buzz stimuli produced clear and rela- tively uniform ratio changes across the entire cell body role for the MEC-4/MEC-2 channel in the sensation of gentle touch, and unexpectedly discovered that body as shown in Figure 1B for a 1.4 s buzz. Short poke stimuli also generated reliable increases in the fluorescence touch receptor neurons also have the capacity to re- spond to harsh touch stimuli using molecular machinery ratio averaged over the ALM cell body (Figure 1C). The yellow and cyan intensities showed reciprocal changes distinct from the MEC-4/MEC-2 channel. These data significantly extend understanding of molecular mecha- (Figure 1C), as expected for a FRET change caused by an increase in calcium. All three classes of touch neu- nisms of touch transduction and establish feasibility of using cameleon reporters for analyses of nematode neu- rons, ALM, AVM, and PLM (Chalfie et al., 1985), exhibited reliable calcium responses when we delivered stimuli to ronal responses in a native context. the appropriate sensory region (anterior body for ALM and AVM, posterior body for PLM) and responded less Results reliably or not at all when the inappropriate body region was stimulated (data not shown see also Figure 3F). In Vivo Detection of Touch-Evoked Neural Activity in C. elegans Mechanoreceptors Thus, we could robustly detect calcium transients in the touch neurons in response to mechanical stimulation. We generated transgenic lines that expressed chame- leon YC2.12 (Nagai et al., 2002) in the touch neurons To verify that our stimuli corresponded to gentle touch, we assayed the behavioral responses of partially under the control of the mec-4 promoter (Mitani et al., 1993). In the lines assayed, we found touch sensitivity immobilized wild-type and mutant nematodes under im- aging conditions. Pokes and buzzes elicited vigorous to be normal (for example, for transgenic line bzIs18 [pmec-4YC2.12 lin-15( )], 82%, and for wild-type, 84% thrashing from wild-type animals (7/8 responded to poke, 8/9 to buzz), but mec mutants failed to respond of animals respond to the first three successive touches), indicating that expression of cameleon in the (0/9 mec-4(u253) animals responded to poke, 0/9 to buzz), indicating that our stimuli correspond to gentle touch receptors did not disrupt their function. To test whether we could detect calcium transients in response touch typically delivered in behavioral tests by an eye- lash stroke. We also noted an unexpectedly slow recov- to mechanical stimulation, we glued individual adult her- maphrodites to 2% agarose pads and stimulated them ery of calcium levels to baseline, so we examined cell body responses with the lower affinity YC3.12 and ob- with a round-tipped glass probe connected to a motor- ized stage (Figure 1A). Various types of gentle stimula- served a more rapid return to baseline (Figure 2B). This difference may be due to rapid reduction of calcium tion were given (Figure 2A): pokes, which represent brief stimuli in which the probe transiently pushes in and pulls levels to below YC3.12 but not YC2.12 detection limits, or due to the higher affinity calcium buffering action of out of the cuticle presses, which give a longer stimulus of constant displacement and buzzes, which are longer YC2.12 impeding the removal of calcium from the cell.