Goodman MB et al. (1998) East Coast Worm Meeting "In situ patch-clamp recording from neurons in C. elegans"

Goodman MB, Chalfie M

[East Coast Worm Meeting, 1998]

We have extended recently described methods for in situ patch-clamp recording from C. elegans neurons (1, 2) to permit recording in adult animals. The main improvement is the use of a fixed-stage, upright microscope mounted on an x-y translation stage. In this way, worms can be visualized from above using a high N.A. water immersion objective (Zeiss 63X/0.9 or 100X/1.0). This arrangement gives superior optics compared to viewing worms on an inverted microscope and makes it possible to expose neurons in adult animals. In addition, methods for exposing neuronal cell bodies in the head were modified to expose neuronal cell bodies in the tail for in situ patch-clamp recording. With this apparatus, we plan to record the response of PLM cells to light touch. 1. Lockery, S. R. & Goodman, M. B. (1998) Tight-seal whole-cell patch clamping of C. elegans neurons. Methods in Enzymology (in press). 2. Goodman, M. B., Hall, D. H., Avery, L. & Lockery, S. R. (1998). Active currents regulate sensitivity and dynamic range in C. elegans neurons. Neuron (in press).

Dickson B (1998) Nature "Axon guidance: A roundabout way of avoiding the midline."

Dickson B

[Nature, 1998]

Bilaterally symmetrical animals must be able to integrate sensory inputs and coordinate motor control on both sides of the body. Thus, many neurons in the central nervous system (CNS) project their axons to the opposite side of the body, whereas others project axons that remain on the same side. In the latest issues of Cell and Neuron, the groups of Corey Goodman, Guy Tear, Marc Tessier-Lavigne and Cori Bargmann report that, from worms and flies to rats and humans, a common mechanism determines which axons cross the midline and which do not.

Anand Y Mehta et al. (2005) International Worm Meeting "AMP Kinase signaling mediates growth and fat metabolism in C. elegans."

Dan Goodman, Maria Alexander-Bridges, Kaveh Ashrafi, Nargis Nasrin, Anand Y Mehta

[International Worm Meeting, 2005]

AMP kinase is a highly conserved sensor of cellular and systemic energy status in eukaryotes. AMP kinase is activated by stressors that increase the cellular AMP:ATP ratio such as osmotic shock, hypoxia, oxidative damage, glucose deprivation, and exercise. AMP kinase can also be activated by the synthetic AMP analogue, 5 aminoimidazole-4-carboxamide riboside (AICAR). When activated, AMP kinase phosphorylates downstream signals that up-regulate ATP producing pathways and down-regulate ATP consuming pathways. The downstream effects of activated AMP kinase include both the regulation of cell-cycle progression and the maintenance of energy stores by increasing fatty acid oxidation and muscle glucose transport via distinct mechanisms. To understand the role of AMP kinase in C. elegans, we used AICAR to activate AMP kinase. We show that the addition of AICAR results in growth suppression and a rapid and reversible fat reduction. Using RNA inactivation we show that these effects are mediated through the catalytic subunit of AMP Kinase. Worms exposed to RNAi of the AMP kinase catalytic subunit are resistant to the effects of AICAR. Furthermore, our data confirm that the critical components of the AMP kinase signaling cascade to fatty acid oxidation are conserved in C. elegans. Using AICAR and RNA inactivation, we are currently exploring novel components of the AMP kinase signaling pathway.

Johnson, Christina K. et al. (2021) MicroPubl Biol

Miller, David D., Johnson, Christina K., Bianchi, Laura

[MicroPubl Biol, 2021]

MEC-4 and UNC-8 are subunits of the DEG/ENaC family of voltage-independent Na+ channels in C. elegans (Driscoll and Chalfie 1991, Canessa, Horisberger et al. 1993, Waldmann, Champigny et al. 1996, Waldmann, Champigny et al. 1997, de Weille, Bassilana et al. 1998, Waldmann and Lazdunski 1998). While MEC-4 is expressed in body touch neurons where it mediates the transduction of gentle touch sensation (Driscoll and Chalfie 1991, O'Hagan, Chalfie et al. 2005), UNC-8 is primarily expressed in motoneurons where it is involved in synaptic remodeling during development (Tavernarakis, Shreffler et al. 1997, Miller-Fleming, Petersen et al. 2016). Both MEC-4 and UNC-8 can be hyperactivated by genetic mutations that hinder channel closing, called (d) mutations (Driscoll and Chalfie 1991, Shreffler, Magardino et al. 1995, Goodman, Ernstrom et al. 2002, Wang, Matthewman et al. 2013). C. elegans neurons and Xenopus oocytes expressing these hyperactive variants of MEC-4 and UNC-8 undergo cell death due to uncontrolled flux of ions into the cell. Cell death in Xenopus oocytes and in cultured C. elegans neurons can be prevented by incubation with the DEG/ENaC channel blocker amiloride (Goodman, Ernstrom et al. 2002, Suzuki, Kerr et al. 2003, Wang, Matthewman et al. 2013).

Shawn Lockery et al. (2001) Worm Breeder's Gazette "Tight-seal whole-cell patch clamping of C. elegans neurons: erratum"

Miriam Goodman, Shawn Lockery

[Worm Breeder's Gazette, 2001]

We regret to inform the C. elegans community that the published recipe for internal saline for whole-cell recordings[1,2] from neurons was incorrect. The published recipe was (in mM): KGluconate 125, KCl 18, NaCl 0, CaCl2 0.7, MgCl2 1, HEPES 10, EGTA 10. The recipe actually used was (in mM): KGluconate 125, KCl 18, NaCl 4, CaCl2 0.6, MgCl2 1, HEPES 10, EGTA 10. The main effect of this error resides in the difference in NaCl concentration. The correct saline will produce a predicted Na reversal potential of 90 mV with the published external saline, while the erroneous published saline has an undefined ENa. Because C. elegans lacks voltage-gated Na channels, this difference in salines may have little or no effect on recordings of voltage-gated currents. It may, however, affect measurements of currents carried by ligand-gated currents and currents carried by DEG/ENaC channels. We apologize for any inconvenience this error may have caused. 1. Goodman, M.B., Hall, D.H., Avery, L., and Lockery, S.R. (1998) Active Currents Regulate Sensitivity and Dynamic Range in C. elegans Neurons. Neuron 20:763-772. 2. Lockery, S.R. and Goodman, M.B. (1998) Tight-seal whole-cell patch clamping of C. elegans neurons. Methods in Enzymology 295:201-217.

Chen, Lili et al. (2020) MicroPubl Biol

Wang, Ya, Chen, Lili, Gao, Shangbang

[MicroPubl Biol, 2020]

The action potential (AP) is the basic signaling unit in various crucial physiological processing, for instance, in neurotransmission, muscle contraction, and glandular secretion (Koch, 1990). The classic model animal, Caenorhabditis elegans (or C. elegans), with a simple and compact nervous system, conservatively employs the calcium-mediated all-or-none APs for odor response in AWA olfactory neurons (Liu et al., 2018), as well as for muscle contraction in either body wall muscles (Gao and Zhen, 2011; Liu et al., 2011) and pharyngeal muscles (Davis et al., 1999). Plateau potentials were also observed in ASE and RMD neurons (Goodman et al., 1998; Mellem et al., 2008; Lockery et al., 2009; Lockery and Goodman, 2009), though the underlying roles in specific behavior are still elusive. Either in neurons or in muscles, the action potential firing is dependent on the excitatory pre-synaptic vesicles release. The minimum number of the presynaptic vesicles to elicit a single action potential in C. elegans has not been reported before. Here, by the combination of optogenetics with in-vivo patch clamping technology, we demonstrated that at least approximately 37 excitatory acetylcholinergic vesicles are required for the initiation of an action potential at post-synaptic body wall muscles.

Shindou, Tomomi et al. (2015) International Worm Meeting "Electrophysiological properties of ASE neurons."

Wickens, Jeffery, MARUYAMA, Ichiro, Shindou, Tomomi, Shindou, Mayumi, Murayama, Takashi

[International Worm Meeting, 2015]

A pair of ASE chemosensory neurons, ASEL and ASER, are major salt sensors, and play critical roles in chemotaxis to NaCl. Calcium imaging has previously revealed that ASEL and ASER are activated by an increase and decrease in NaCl concentrations, respectively (Suzuki et al., 2008; Ortiz et al., 2009). These asymmetric responses by ASEL and ASER to changes of NaCl concentrations are crucial to efficient chemotaxis of C. elegans toward higher concentrations of NaCl. While Goodman et al. (1998) reported in situ whole-cell patch-clump recording of ASER, electrophysiological characterisation of ASE neurons is still required to understand how the neurons respond to the NaCl concentration changes.Toward the goal, we have investigated electrophysiological properties of ASE neurons in wild-type C. elegans by in vivo whole-cell patch-clamp recordings, and have found that both of ASE neurons showed resting membrane potentials of approximately -60 mV and membrane resistances of about 2 Gomega. In both of ASE neurons, voltage responses to current injections showed solitary action potentials. Depolarization of wild-type ASEL was observed when a puff of 150 mM NaCl was applied to the animal's nose in bath solution containing 50 mM NaCl. On the other hand, a puff of NaCl-free buffer induced ASER depolarization. These results are consistent with those of calcium imaging. To understand roles of the action potentials in ASE, we are currently trying to analyse electrophysiological properties of ASE neurons in various mutants.References1. Suzuki et al., Nature 454: 114-118 (2008)2. Ortiz et al., Current Biology 19: 996-1004 (2009)3. Goodman et al., Neuron 20: 763-772 (1998).

Moon, K. et al. (2019) International Worm Meeting "Gait selection of Caenorhabditis elegans is regulated by mechanosensitive DEG/ENaC channels."

Moon, K., Kim, K., Kim, J., Yeon, J.

[International Worm Meeting, 2019]

Animals change their locomotion or gaits in response to environmental condition. In vertebrates, gait transition has been shown to be mediated by monoamines, which is conserved across many species including the nematode Caenorhabditis elegans (Vidal-Gadea et al., 2011). However, molecular mechanisms of gait transition are still unclear. C. elegans exhibits two gaits, swimming in liquids and crawling on dense gels. C. elegans genome contains evolutionarily conserved 28 DEG/ENaC channels, of which functions may be involved in mechanosensory transduction and locomotion (Goodman and Schwarz, 2003). We first hypothesized that mechanosensitive channels could act as a gait transition initiator and examined crawl-to-swim transition phenotype in DEG/ENaC mutants. We found that while acd-5 mutants show normal crawling, transition from crawling to swimming upon liquid exposure is defective, suggesting roles of ACD-5 in gait transition. We are currently generating acd-5 rescue lines and examining expression pattern of acd-5.

Austin L Brown et al. (2007) International Worm Meeting "The Accessory Proteins MEC-2 and MEC-6 Promote an Active State of the MEC-4 Channel Complex."

Austin L Brown, Miriam B Goodman

[International Worm Meeting, 2007]

The stomatin-related protein MEC-2 and the paraoxonase-related protein MEC-6 are required for mechanical activation of native force detection channels in C. elegans touch receptor neurons [1]. Prior work indicates that such channels contain at least two pore-forming degenerin subunits, MEC-4 and MEC-10. Both MEC-4 and MEC-10 belong to the DEG/ENaC superfamily of amiloride-sensitive Na+ channel subunits. Wild-type copies of all four proteins are needed for touch sensation in vivo. Co-expressing MEC-2 and MEC-6 with constitutively-active isoforms of MEC-4 and MEC-10 in Xenopus oocytes leads to a dramatic and synergistic increase in amiloride-sensitive whole-cell Na+ current. This effect is not explained by a MEC-2- or MEC-6-dependent increase in the amount of MEC-4 or MEC-10 protein in the plasma membrane [2, 3], suggesting that MEC-2 and MEC-6 alter the activity of individual channels in the plasma membrane. To test this, we analyzed single, amiloride-sensitive Na+ channels in membrane patches drawn from oocytes expressing MEC-4 and MEC-10 in the presence or absence of MEC-2, MEC-6 or both MEC-2 and MEC-6. We found that while MEC-2 coexpression leads to a modest, but significant increase in single channel conductance, <font face=symbol>g</font>, the effect is too small to explain the MEC-2-dependent increase in whole-cell current. Otherwise, <font face=symbol>g</font> and steady-state open probability (P_o) are not significantly altered by the presence or absence of MEC-2 and MEC-6. We conclude that, in the absence of MEC-2 and MEC-6, the vast majority of channels occupy a non-conducting state despite correct expression at the plasma membrane. 1.O''Hagan, R., M. Chalfie, and M.B. Goodman, Nat Neurosci, 2005. 8(1):43 2.Chelur, D.S., et al., Nature, 2002. 420(6916):669 3.Goodman, M.B., et al., Nature, 2002. 415(6875):1039.

Brown AL et al. (2007) J Gen Physiol "Gain-of-function mutations in the MEC-4 DEG/ENaC sensory mechanotransduction channel alter gating and drug blockade."

Brown AL, Goodman MB, Liao Z, Fernandez-Illescas SM

[J Gen Physiol, 2007]

MEC-4 and MEC-10 are the pore-forming subunits of the sensory mechanotransduction complex that mediates touch sensation in Caenorhabditis elegans (O''Hagan, R., M. Chalfie, and M.B. Goodman. 2005. Nat. Neurosci. 8:43-50). They are members of a large family of ion channel proteins, collectively termed DEG/ENaCs, which are expressed in epithelial cells and neurons. In Xenopus oocytes, MEC-4 can assemble into homomeric channels and coassemble with MEC-10 into heteromeric channels (Goodman, M.B., G.G. Ernstrom, D.S. Chelur, R. O''Hagan, C.A. Yao, and M. Chalfie. 2002. Nature. 415:1039-1042). To gain insight into the structure-function principles that govern gating and drug block, we analyzed the effect of gain-of-function mutations using a combination of two-electrode voltage clamp, single-channel recording, and outside-out macropatches. We found that mutation of A713, the d or degeneration position, to residues larger than cysteine increased macroscopic current, open probability, and open times in homomeric channels, suggesting that bulky residues at this position stabilize open states. Wild-type MEC-10 partially suppressed the effect of such mutations on macroscopic current, suggesting that subunit-subunit interactions regulate open probability. Additional support for this idea is derived from an analysis of macroscopic currents carried by single-mutant and double-mutant heteromeric channels. We also examined blockade by the diuretic amiloride and two related compounds. We found that mutation of A713 to threonine, glycine, or aspartate decreased the affinity of homomeric channels for amiloride. Unlike the increase in open probability, this effect was not related to size of the amino acid side chain, indicating that mutation at this site alters antagonist binding by an independent mechanism. Finally, we present evidence that amiloride block is diffusion limited in DEG/ENaC channels, suggesting that variations in amiloride affinity result from variations in binding energy as opposed to accessibility. We conclude that the d position is part of a key region in the channel functionally and structurally, possibly representing the beginning of a pore-forming domain.