Isoflurane Exposure in Juvenile Caenorhabditis elegans Causes Persistent Changes in Neuron Dynamics

Abstract

Background Animal studies demonstrate that anesthetic exposure during neurodevelopment can lead to persistent behavioral impairment. The changes in neuronal function underlying these effects are incompletely understood. Caenorhabditis elegans is well suited for functional imaging of postanesthetic effects on neuronal activity. This study aimed to examine such effects within the neurocircuitry underlying C. elegans locomotion. Methods C. elegans were exposed to 8% isoflurane for 3 h during the neurodevelopmentally critical L1 larval stage. Locomotion was assessed during early and late adulthood. Spontaneous activity was measured within the locomotion command interneuron circuitry using confocal and light-sheet microscopy of the calcium-sensitive fluorophore GCaMP6s. Results C. elegans exposed to isoflurane demonstrated attenuation in spontaneous reversal behavior, persisting throughout the animal’s lifespan (reversals/min: untreated early adulthood, 1.14 ± 0.42, vs. isoflurane-exposed early adulthood, 0.83 ± 0.55; untreated late adulthood, 1.75 ± 0.64, vs. isoflurane-exposed late adulthood, 1.14 ± 0.68; P = 0.001 and 0.006, respectively; n > 50 animal tracks/condition). Likewise, isoflurane exposure altered activity dynamics in the command interneuron AVA, which mediates crawling reversals. The rate at which AVA transitions between activity states was found to be increased. These anesthetic-induced effects were more pronounced with age (off-to-on activity state transition time (s): untreated early adulthood, 2.5 ± 1.2, vs. isoflurane-exposed early adulthood, 1.9 ± 1.3; untreated late adulthood, 4.6 ± 3.0, vs. isoflurane-exposed late adulthood, 3.0 ± 2.4; P = 0.028 and 0.008, respectively; n > 35 traces acquired from more than 15 animals/condition). Comparable effects were observed throughout the command interneuron circuitry, indicating that isoflurane exposure alters transition rates between behavioral crawling states of the system overall. These effects were modulated by loss-of-function mutations within the FoxO transcription factor daf-16 and by rapamycin-mediated mechanistic Target of Rapamycin (mTOR) inhibition. Conclusions Altered locomotive behavior and activity dynamics indicate a persistent effect on interneuron dynamics and circuit function in C. elegansafter developmental exposure to isoflurane. These effects are modulated by a loss of daf-16 or mTOR activity, consistent with a pathologic activation of stress-response pathways. Editor’s Perspective What We Already Know about This Topic What This Article Tells Us That Is New

Quantitative behavioural phenotyping to investigate anaesthesia induced neurobehavioural impairment

“…We find that C. elegans larvae exposed to anaesthesia shortly after hatching display later behavioural differences when compared to controls. This is consistent with results from previous studies which exposed C. elegans to isoflurane alone 27,28 . Our research further establishes that the effect is agent-and concentration-dependent, with combinations of isoflurane and ketamine at higher doses consistently being correlated with significant differences.…”

“…In C. elegans there is evidence to suggest that isoflurane may cause the neurotoxic effect by pathological activation of stress-response pathways. These effects have been shown to be blocked by of loss-of-function mutations in the insulin-like growth factor receptor gene daf-2 27 , the FoxO transcription factor daf-16 28 , and rapamycin-mediated inhibition of the mechanistic Target of Rapamycin (mTOR) 27,28 , consistent with the stress pathway hypothesis. However, no single theory unifies these mechanisms, or accounts for all reported histopathological and neurobehavioral phenotypes.…”

“…This timepoint was chosen because it is a period of extensive neurogenesis and neural rewiring (Fig. 1a); exposure to isoflurane at this stage has previously been showed to result in defective behaviour 26,28 . Agents were chosen because of previous vertebrate literature suggesting a neurotoxic effect (ketamine 8 , isoflurane 9 ), lack of effect (morphine 33 , dexmedetomidine 34 ) or potential to rescue the neurotoxic deficit (lithium 35 ).…”

“…Key anaesthetic targets including GABA and NMDA receptors are conserved in C. elegans and neuronal cells in the worm undergo apoptosis through conserved mechanisms first discovered in C. elegans 25 . Larval worms have previously been shown to develop neuronal and behavioural changes after exposure to isoflurane anaesthesia [26][27][28] . However, the potential of C. elegans goes further: its behaviours are amenable to quantitative aggregation and analysis, which yields a phenotypic 'signature' .…”

Beyond Anesthesia Apoptosis

“…In this issue of Anesthesiology, an elegant experimental work conducted on the nematode Caenorhabditis elegans seems to emphasize this latter possibility. 3 C. elegans is an appealing model for studying the mechanistic and functional neuronal correlates to anesthesia-induced behavioral impairments since morphological, genetic, and functional characteristics of neuronal networks are well-characterized in this species. An additional advantage of the model is that, since this nematode does not have a cardiovascular system, systemic hemodynamic effects of anesthetic drugs need not to be considered as potential confounding factors.…”