Jesudason MV et al. (2006) Indian J Med Res "Toxigenicity testing of clinical isolates of non-typhoidal salmonellae in Vero cell culture & Caenorhabditis elegans model."

V Balaji V, Jesudason MV, Densibai S

[Indian J Med Res, 2006]

The non-typhoidal salmonellae (NTS) are recognized agents of gastroenteritis worldwide. Some of the NTS do not produces cytotoxic changes in tissue culture and not much is known about the endotoxicity of the clinical isolates of NTS (mostly Salmonella enterica serotype Typhimurium and Salmonella enterica serotype Enteritidis). We examined the exotoxic (cytotoxin) and endotoxic activity of clinical isolates of NTS in two assay models namely Vero cell culture and the nematode, Caenorhabditis elegans. Bacteria-free culture supernatants of 40 isolates NTS were tested in 96 well microtitre plate containing confluent monolayers of Vero cells. For the effects on C. elegans, the worms were exposed to bacteria free culture supernatants in 24 well microtitre plate for 24 h and then transferred to OP50 Escherichia coli lawn culture. The endotoxic activity of the live bacterium was studied by feeding the worms in the lawn culture of NTS separately. No cytopathic effect was observed with NTS tested in Vero cell culture assay. Likewise, the worms exposed to the bacteria-free culture supernatants were found active up to 7 days. In the co-culture killing assay, worms were found dead with characteristic stiff and straight appearance by 16(th) day. The worms were alive up to 21 days in OP50 E. coli. Bacteriafree culture supernatants did not have any deleterious effect on worms or in Vero cell culture, suggesting that there is no soluble toxic factor (diffusible toxin) in the culture supernatants. However, live NTS were found to be lethal to the worms; indicating that direct interaction between viable NTS and C. elegans is necessary for killing.

Christensen M et al. (1999) Worm Breeder's Gazette "Identification of a Novel Mechanosensitive Anion Current in Embryonic Cells of C. elegans*"

Strange K, Christensen M

[Worm Breeder's Gazette, 1999]

Mechanotransduction plays a central role in fundamental physiologic processes such as detection of touch and sound, regulation of cell volume, and control of motility. Mechanosensitive channels have been studied extensively using electrophysiology. Little is known, however, about the molecular structure of mechanosensitive channels or about how mechanical stress is transduced into altered channel gating. We developed techniques to patch-clamp and study ion channels in C. elegans embryonic cells. In cell-attached and inside-out patches, application of gentle suction activated a mechanosensitive current. Suction caused an immediate increase in current amplitude of 6.4 2.8 fold (n = 20) at +100 mV in inside-out patches. The current rapidly inactivated when suction was discontinued and could be repeatedly reactivated by additional suction. When membrane voltage was ramped from +100 to -100 mV at 100 mV/second, the current showed moderate outward rectification (outward:inward current = 2.0 0.05 at 100 mV). Current amplitude was largely unaffected when bath Na+ was replaced with NMDG+ (n = 10). However, replacement of bath Cl- with either gluconate or glutamate reduced inward and outward currents by 46 10% and 39 7%, respectively (n = 9). Replacement of 120 mM bath Cl- with a mixture of 60 mM Cl- and 60 mM SCN- increased the inward current at -100 mV by 3.0 0.4 fold and shifted Erev by 16 2 mV (n=7). We conclude that membrane stretch activates a novel mechanosensitive anion current in inside-out patches from C. elegans embryonic cells.

Goodman MB et al. (1996) West Coast Worm Meeting "TOWARD LINKING MOLECULES TO NEURONS TO BEHAVIOR: A PROGRESS REPORT ON C. ELEGANS ELECTROPHYSIOLOGY"

Lockery SR, Goodman MB

[West Coast Worm Meeting, 1996]

Genetic analyses of behavior have identified gene products important for neuronal function in C. elegans, including putative sensory and neurotransmitter receptors. As a first step in forging a link between these proteins and cellular physiology, we have recorded from neurons in the heads of semi-intact, L1 worms. The technique was modified from that reported previously [WBG 13(5): 32] and regularly yields tight-seal, whole-cell recordings. In current clamp, neurons responded to current steps with graded potentials whose time course depended on the injected current (n=24). Action potentials could not be elicited. Some cells (n=3) exhibited spontaneous, sustained shifts in membrane potential from approximately -60 to -20 mV. At -60 mV, smaller amplitude, transient depolarizations were frequently observed. Voltage pulses to greater than -10 mV elicited an outward current with transient and sustained components. The rate of decay of the transient component varied between cells. The range (tau = 10-100 ms) is similar to that reported for the superfamily of cloned, voltage-gated potassium currents. Thus, the variation in decay rate could reflect heterogeneity in potassium channel expression among different classes of neurons. Hyperpolarizing pulses to less than -80 mV elicited a voltage-dependent inward current with no obvious time dependence. Between -80 and -10 mV, most cells had a region of high resistance. We are currently characterizing the main outward current and investigating how it might contribute to temporal processing in individual C. elegans neurons.

Balaji V et al. (2004) Indian J Med Res "Detection of virulence attributes of Burkholderia pseudomallei."

Jesudason MV, Subramanian K, Balaji V, Sridharan G

[Indian J Med Res, 2004]

Background & objectives: Melioidosis caused by Burkholderia pseudomallei is an emerging disease in India. This study examined the toxin activity of bacteria-free culture filtrate in three different cell lines (cytotoxic assay) and its effect on Caenorhabditis elegans (nematode toxicity assay). Endotoxic activity of the viable bacteria was also studied in C elegans (co-culture killing assay). Methods: For toxin studies, serial doubling dilutions of unheated, heated crude and ultra filtrate of bacteria-free culture supernatants of B. pseudomallei were tested in 96-well microtitre plate containing confluent mono layers of McCoy, Hep-2 and HeLa cell lines. For the effects on C. elegans, the worms were exposed to heated and unheated bacteria-free culture supernatants in 24-well microtitre plate for 24 h and then transferred to OP50 Escherichia coli lawn culture. The endotoxic activity of the live bacterium was studied by feeding the worms in the lawn culture of B. pseudomallei. Results: All the clinical isolates (n=38) produced cytotoxic changes in all the cell lines. No difference was observed in the cytotoxicity of unheated, heated and ultra-filtered culture supernatant. The septicaemic isolates were observed to produce cytotoxic changes in high dilutions (1:160) of culture filtrate. None of the unheated and heated crude filtrate had deleterious effect on C. elegans, while all the live bacteria were found to be lethal to the nematode. Interpretation & conclusion: The culture supernatants, though produced cytopathic effect in various tissue cultures, failed to have any deleterious effect on the worms. However, live bacteria were lethal to the worms B. pseudomallei. Use of C elegans model to detect virulence attributes of B. pseudomallei is recommended as an alternative to tissue culture methods as this can be carried out in laboratories where a tissue culture set up is not available.

Tom Nickell et al. (2002) Mid-west Worm Meeting "Calcium-activated Potassium Channels in C. elegans chemosensory neurons"

Ray Pun, Steve Kleene, Tom Nickell

[Mid-west Worm Meeting, 2002]

Calcium-activated potassium channels are present in the C. elegans nervous system, including many neurons of the nerve ring. Properties of these channels have been reported in a heterologous expression system; however, it was of interest to determine whether channels of similar properties could be recorded from C. elegans neurons. We recorded single channels in excised patches from the chemosensory neuron AWA, which were labeled with GFP. Neurons were exposed by cutting the worm with a scalpel blade just behind the terminal bulb. Excised patches were formed in conventional manner. Channels corresponding to BK potassium channels were present in AWA neurons. In patches containing the channels, channels were absent or only activated at high positive potentials (+100 mV). Addition of 100 mM calcium markedly shifted the activation of the channels toward more negative potentials, with channel openings visible at 0 mV and more positive potentials. Mean conductance of these channels was about 65 pS, with a reversal potential of -50 mV.

I Vinogradova et al. (2002) European Worm Meeting "Patch-clamp study of the cationic currents in the pharyngeal muscle cells of the adult nematode C.elegans"

R Walker, I Vinogradova, L Holden-Dye

[European Worm Meeting, 2002]

Continuing contractions and extensive intercellular coupling prevent reliable whole-cell current recording from the enzymatically cleaned muscle cells of the C.elegans pharynx. However, confirming data previously obtained with intracellular recording with sharp microelectrodes (Pemberton et al., 2002), it has been shown that sodium omission from the Dent's physiological saline blocks inward currents in response to depolarisations from the holding potential of 120 mV to 40 mV both in wild type worms and egl 19 (n582). In outside-out patches tested with Cs-internal solution and Dent's saline and held at 80 mV channels with very low opening probability were observed. Amplitude of the unitary currents in these patches was reduced in 0Na+ Dent's saline but not in 0Ca2+ solution. Currents were further reduced and then abolished in 0Na+ 0Ca2+ solution. Exposure of the patches to the Dent's saline with 10-times reduced concentration of Na+ caused a shift in reversal potential smaller that expected in accordance with the Nernst equation. Channels can be blocked by gadolinium ions and their mean opening time increased by veratridine. In inside out patches tested in symmetrical 150 mM Na+ solution containing no Ca2+ and K+ ions opening probability of the recorded channels at 80 mV was 3 times higher than in outside-out patches but was reduced to the level observed in outside-out patches when 3 mM Ca2+ were added to the solution contacting the external surface of the membrane patches. Taken together these data suggest the presence of some sort of sodium permeable channels in pharyngeal muscle cells of the adult worms.

Byerly L et al. (1978) Worm Breeder's Gazette "voltage-clamp studies of the electrical currents of the Ascaris pharyngeal muscle."

Masuda M, Byerly L

[Worm Breeder's Gazette, 1978]

It has proven difficult to study the currents that generate the electrical activity in the somatic muscle cells of Ascaris due to the inability to control the voltage across the excitable membrane. Therefore, we have directed our attention to the pharyngeal muscle, where it is possible to directly measure the voltage and pass large currents across the excitable membrane. We have developed a system which allows us to do current-clamp and voltage-clamp experiments on an isolated segment of the pharyngeal membrane. We find that this membrane has no pacemaker activity. In the absence of nervous input the membrane potential is flat at a level near -40 mV. Two types of spontaneous postsynaptic potentials are frequently seen; one type has a reversal potential near -40 mV and the other has a reversal potential near -10 mV. When the membrane is at the resting level, this second type PSP triggers a positive-going action potential, which reaches a level between +30 and +50 mV. The membrane potential then falls to a plateau near O mV, where it remains until a negative-going PSP triggers a negative-going action potential that reaches about -50 mV (the potassium reversal potential). The membrane potential remains at the plateau level for periods ranging from 100 msec to several minutes. The positive-going action potential is produced by an inward current that appears to be carried by both Na+ and Ca++. This current is prolonged, showing little inactivation by 200 msec after a positive voltage step. Clamping the membrane to positive potentials elicits essentially no delayed-rectification K current, the current that normally repolarizes active membranes. However, stepping the membrane potential back to the resting level after a large positive pulse elicits a strong, transient outward K+ current; this is the current that produces the negative-going action potential. We have done a detailed analysis of this current and have shown that it is a voltage- inverted analogue of the Hodgkin-Huxley Na+ current. It is activated by negative steps in potential. It shows inactivation, being completely inactivated at the resting potential. Conditioning pulses to levels more positive than -10 mV are necessary to remove the inactivation from the channel. This is a new K+ conductance that has not been found in any other animal. This demonstration of unique mechanisms in nematode physiology should serve as a caution in trying to interpret the function of the nematode nervous system. The pharyngeal nervous system must not only generate the signal that triggers the pharynx to contract (open the lumen), but also the signal to relax (close the lumen). Since the relaxation of the pharynx is the 'power stroke' of this muscle, it is not surprising that the membrane has developed a special K+ current to produce a fast, separately-triggerable repolarization.

Jospin M et al. (2002) J Physiol "Characterization of K(+) currents using an in situ patch clamp technique in body wall muscle cells from Caenorhabditis elegans."

Jospin M, Allard B, Segelat L, Mariol MC, Segalat L

[J Physiol, 2002]

The properties of K+ channels in body wall muscle cells acutely dissected from the nematode Caenorhabditis elegans were investigated at the macroscopic and unitary level using an in situ patch clamp technique. In the whole-cell configuration, depolarizations to potentials positive to -40 mV gave rise to outward currents resulting from the activation of two kinetically distinct voltage-dependent K+ currents: a fast activating and inactivating 4-aminopyridine-sensitive component and a slowly activating and maintained tetraethylammonium-sensitive component. In cell-attached patches, voltage-dependent K+ channels, with unitary conductances of 34 and 80 pS in the presence of 5 and 140 mm external K+, respectively, activated at membrane potentials positive to -40 mV. Excision revealed that these channels corresponded to Ca2+-activated K+ channels exhibiting an unusual sensitivity to internal Cl- and whose activity progressively decreased in inside-out conditions. After complete run-down of these channels, one third of inside-out patches displayed activity of another Ca2+-activated K+ channel of smaller unitary conductance (6 pS at 0 mV in the presence of 5 mm external K+). In providing a detailed description of native K+ currents in body wall muscle cells of C. elegans, this work lays the basis for further comparisons with mutants to assess the function of K+ channels in this model organism that is highly amenable to molecular and classical genetics.

Azimi Hashemi N et al. (2019) Proc Natl Acad Sci U S A "Rhodopsin-based voltage imaging tools for use in muscles and neurons of Caenorhabditis elegans."

Liewald JF, Bach M, Azimi Hashemi N, Bergs ACF, Schuler C, Gottschalk A, Scheiwe AR, Steuer Costa W

[Proc Natl Acad Sci U S A, 2019]

Genetically encoded voltage indicators (GEVIs) based on microbial rhodopsins utilize the voltage-sensitive fluorescence of all-<i>trans</i> retinal (ATR), while in electrochromic FRET (eFRET) sensors, donor fluorescence drops when the rhodopsin acts as depolarization-sensitive acceptor. In recent years, such tools have become widely used in mammalian cells but are less commonly used in invertebrate systems, mostly due to low fluorescence yields. We systematically assessed Arch(D95N), Archon, QuasAr, and the eFRET sensors MacQ-mCitrine and QuasAr-mOrange, in the nematode <i>Caenorhabditis elegans</i> ATR-bearing rhodopsins reported on voltage changes in body wall muscles (BWMs), in the pharynx, the feeding organ [where Arch(D95N) showed approximately 128% F/F increase per 100 mV], and in neurons, integrating circuit activity. ATR fluorescence is very dim, yet, using the retinal analog dimethylaminoretinal, it was boosted 250-fold. eFRET sensors provided sensitivities of 45 to 78% F/F per 100 mV, induced by BWM action potentials, and in pharyngeal muscle, measured in simultaneous optical and sharp electrode recordings, MacQ-mCitrine showed approximately 20% F/F per 100 mV. All sensors reported differences in muscle depolarization induced by a voltage-gated Ca²⁺-channel mutant. Optogenetically evoked de- or hyperpolarization of motor neurons increased or eliminated action potential activity and caused a rise or drop in BWM sensor fluorescence. Finally, we analyzed voltage dynamics across the entire pharynx, showing uniform depolarization but compartmentalized repolarization of anterior and posterior parts. Our work establishes all-optical, noninvasive electrophysiology in live, intact <i>C. elegans</i>.

Faumont S et al. (2000) West Coast Worm Meeting "Voltage-dependent currents in homologous chemosensory neurons with different functions in C. elegans"

Lockery SR, Faumont S

[West Coast Worm Meeting, 2000]

In C. elegans, chemotaxis to the attractant NaCl is largely controlled by a pair of left-right homologous chemosensory neurons ASER and ASEL. Although the two neurons are bilaterally symetric in their morphology and pattern of connectivity , recent work argues for functional asymetry between these neurons in chemotaxis (Pierce-Shinomura et al., abstr. WCWM). Laser ablation of ASER greatly reduces chemotaxis to Cl- but not Na+; conversely, ablation of ASEL greatly reduces chemotaxis to Na+ but not Cl-. To determine if this functional asymetry reflects a difference in the ionic currents expressed in the two neurons, we made whole-cell voltage clamp recordings fron ASEL (n=20) for comparison with previous recording from ASER (Goodman et al., Neuron 20: 763-72, 1998). We found that ASEL is similar to ASER in three main respects. First, ASEL exhibits an outward current activated by depolarisation (-30 to 100 mV) and an inward current activated by hyperpolarisation below -70 mV. Little or no current is activated in the region between -70 and -30 mV. Second, the outward current comprises inactivating and sustained components which are probably carried by K+ ions, because both components are eliminated by substitution of N-methyl-glutamine (NMG) for K+ in the recording pipette. Third, outward current inactivation is voltage-dependent, because prepulses more positive than -70mV decreases peak amplitude of the outward current. These results suggest that the functional asymetry reflect differences in the response to chemical stimuli rather than differences in voltage dependent ionic current. Support: NIMH 51383 and NSF IBN-9458102.