Optical reversal of halothane-induced immobility in C. elegans

Abstract

Volatile anesthetics (VAs) cause profound neurological effects, including reversible loss of consciousness and immobility. Despite their widespread use, the mechanism of action of VAs remains one of the unsolved puzzles of neuroscience [1, 2]. Genetic studies in Caenorhabditis elegans [3, 4], Drosophila [3, 5], and mice [6-9] indicate that ion channels controlling the neuronal resting membrane potential (RMP) also control anesthetic sensitivity. Leak channels selective for K(+) [10-13] or permeable to Na(+) [14] are critical for establishing RMP. We hypothesized that halothane, a VA, caused immobility by altering the neuronal RMP. In C. elegans, halothane-induced immobility is acutely and completely reversed by channelrhodopsin-2 based depolarization of the RMP when expressed specifically in cholinergic neurons. Furthermore, hyperpolarizing cholinergic neurons via halorhodopsin activation increases sensitivity to halothane. The sensitivity of C. elegans to halothane can be altered by 25-fold by either manipulation of membrane conductance with optogenetic methods or generation of mutations in leak channels that set the RMP. Immobility induced by another VA, isoflurane, is not affected by these treatments, thereby excluding the possibility of nonspecific hyperactivity. The sum of our data indicates that leak channels and the RMP are important determinants of halothane-induced general anesthesia.

Figure 1. K2P channels alter anesthetic sensitivity in C. elegans

a) K2P(lf_sup-9(n180)), shows a moderate resistance to halothane. (N2, EC₅₀=3.08±0.06%, K2P(lf), EC₅₀=3.35±0.07%, P<0.006) b) K2P(gf_unc-92(n200)),shows an increased sensitivity to halothane (EC₅₀ =1.43±0.07%, P< 0.0001 vs. N2). c) Loss of function of K2P channels partially rescues anesthetic sensitivity seen in the nca (lf) mutants (nca(lf) EC₅₀=0.68±0.05%, K2P(lf);nca(lf) EC₅₀=0.98±0.05%, P=0.0002). d) Animals carrying both K2P(gf) and nca (lf) are extremely hypersensitive to halothane (EC₅₀ =0.18±0.03%, vs. nca(lf), P<0.0001 and vs. K2P(gf), P<0.0001).

Figure 2. Rescue of nca(lf) and K2P(gf) movement defects

Swimming is moderately and severely impaired in K2P(gf) and nca(lf) mutants respectively, as measured by the number of thrashes/sec. Activation of ChR2 significantly improves swimming in retinal fed nca(lf);Punc-17::ChR2 compared to the same worms before ChR2 activation by blue light, and when compared to blue light exposed non retinal fed nca(lf);Punc-17::ChR2 or nca(lf). Upon cessation of blue light stimulation, these worms became paralyzed again. Activation of ChR2 in retinal fed K2P(gf);Punc-17::ChR2 worms lead to more thrashes compared to the non retinal fed and non blue light exposed controls.

Figure 3. Reversal of immobility in C. elegans expressing channelrhodopsin-2 in cholinergic neurons (Punc-17::ChR2)

a) Stills taken every 5 seconds from a movie of a retinal fed Punc-17::ChR2 C. elegans immobilized by 4.85% halothane responding to blue light. Exposure to blue light causes the worm to resume sinuous motion again. Also see Supplemental video 3. b) Dose response curve showing anesthetic sensitivity of light activated Punc-17::ChR2 animals under halothane. EC₅₀ is shifted to 4.29%±0.03 under blue light for retinal fed worms from 3.62±0.04% for non retinal fed control worms, p< 0.0001. c) Dose response curve for the Punc-17::ChR2 animals under isoflurane (EC₅₀ retinal fed, blue light 6.4±0.45% vs. EC₅₀ non retinal fed, no blue light 6.82±0.1%, p=0.3603, not significant). d) Pmyo-3::ChR2 worms fed with retinal when anesthetized under 4.8% halothane only contract their body wall muscles during blue light stimulation, resulting in no net movement.

Figure 4. Mechanism of anesthetic induced immobility

a) In nca(lf);Punc-17::ChR2 depolarizing cholinergic neurons causes a significant rescue of anesthetic sensitivity (EC₅₀ retinal fed worms 3.79±0.04%, non retinal fed and without blue light exposure, EC₅₀<1%). b) K2P(gf);Punc-17::ChR2 and K2P(gf);nca(lf);Punc-17::ChR2 become mobile upon exposure to blue light in halothane (where the K2P(gf) and K2P(gf);nca(lf) are immobile). gas-1 Punc-17::ChR2, and gas-1 Punc-17::ChR2;Pmyo-3::ChR2 mutants did not become mobile when ChR2 was activated in either cholinergic neurons, or cholinergic neurons and muscle. c) In halothane anesthetized Punc-47::halorhodopsin worms, hyperpolarization of GABAergic neurons by halorhodopsin activation, did not reverse immobility at any concentration of halothane (at 3.18% halothane-No green light, no retinal=41.6% immobile, Green light, retinal fed=48.9% immobile). d) In Punc-17::halorhodopsin worms, hyperpolarization of cholinergic neurons by halorhodopsin activation reduced the amount of halothane required for immobilization. 68% of animals were immobile at 1.49% halothane. Less than 7% of the non retinal fed, and non green light exposed animals were immobile at the same halothane concentration. A similar pattern is seen at 2.16% and 2.26% halothane. See also Supplemental video 6.