R. Meldrum Robertson

Inhibition of the sodium pump in the locust central nervous system (CNS) causes repetitive spreading depolarization (SD) and a negative ultraslow potential (NUP) providing a model for investigation of phenomena relevant to human health. We show that impairment of the blood-brain barrier accelerates the NUP but has no impact on the trajectory of SD events. Hence, rapid mechanisms of onset and recovery of ion homeostasis occur against a background of slowly increasing neural damage.

Thermal acclimation induces variation in the temperatures leading to spreading depolarization at the critical thermal limits in invertebrates, but mechanistic inferences based on electrophysiology might be skewed by thermodynamic effects. Here, we quantify the thermal dependence of spreading depolarization parameters in fruit flies, use it to infer mechanisms, and provide perspectives for future comparative research. In addition, we propose Drosophila as a model system to understand this event in vertebrates, including humans.

Insects provide useful models for investigating evolutionarily conserved mechanisms underlying electrical events associated with brain injury and death. Spreading depolarizations (SD) are transient events that propagate through neuropil whereas the negative ultraslow potential (NUP) is sustained and reflects accumulating damage in the tissue. We used the locust, Locusta migratoria , to investigate ion homeostasis at the blood brain barrier (BBB) during SD and NUP induced by treatment with the Na ⁺ /K ⁺ -ATPase inhibitor, ouabain. We found that sustained SD caused by the metabolic inhibitor, sodium azide, was associated with a large reduction of K ⁺ efflux through the BBB at ganglia (= grey matter) but not at connectives (= white matter). This was accompanied by a large increase in tissue resistivity but no conductance changes of identified motoneuron dendrites in the neuropil. Males recovered more slowly from ouabain-induced SD, as previously described for anoxic SD. Impairment of barrier functions of the BBB pharmacologically with cyclosporin A or DIDS, or by cutting nerve roots, accelerated the NUP, thus promoting earlier and more frequent SD, but had no effect on the temporal parameters of SD. We conclude that the mechanisms underlying onset and recovery of SD are minimally affected by the damage associated with the NUP. We suggest that future research using tissue-specific genetic approaches in Drosophila to target identified molecular structures of the BBB are likely to be fruitful. New and Noteworthy: Inhibition of the sodium pump in the locust CNS causes repetitive spreading depolarization (SD) and a negative ultraslow potential (NUP) providing a model for investigation of phenomena relevant to human health. We show that impairment of the blood brain barrier accelerates the NUP but has no impact on the trajectory of SD events. Hence, rapid mechanisms of onset and recovery of ion homeostasis occur against a background of slowly increasing neural damage.

Anoxia and similar energetic crises trigger a shutdown of central neural processing in a process of spreading depolarization (SD) that is generally pathological in mammals and protective in insects. We show that older animals are slower to recover from SD in an insect model. Moreover, preventing oxidative stress with an antioxidant speeds recovery. These findings demonstrate the role of oxidative stress in contributing to the vulnerability of the aging insect central nervous system (CNS) in energetic emergencies.

Spreading depolarization (SD) is a critical mechanism underlying central nervous system (CNS) shutdown and injury under stress, yet the initiating ion channel remains unknown. Here, we used the marine poison palytoxin (PLTX), which converts the sodium-potassium ATPase (NKA) into an open channel, to initiate SD in intact locust CNS. We show for the first time that PLTX-induced SD is rapid and recoverable in vivo, providing support that NKA conversion to a channel may be the SD-initiating mechanism.

Spreading depolarization (SD) temporarily shuts down neural processing in nervous systems with effective blood brain barriers. In mammals this is usually pathological in response to energetic stress. In insects a very similar process is induced by abiotic environmental stressors and can be beneficial by conserving energy. Age is a critical factor for predicting the consequences of SD in humans. We investigated the effect of aging on SD in an insect model of SD and explored the contribution of oxidative stress. Aging slowed the recovery of intact locusts from asphyxia by water submersion. In semi-intact preparations we monitored SD by recording the DC potential across the blood brain barrier in response to bath application of the Na ⁺ /K ⁺ -ATPase inhibitor, ouabain. Treatment with ouabain induced changes to the DC potential that could be separated into two distinct components: a slow, permanent negative shift, similar to the negative ultraslow potential recorded in mammals and human patients, as well as rapid, reversible negative DC shifts (SD events). Aging had no effect on the slow shift but increased the duration of SD events from ∼0.6 minutes in young locusts to ∼0.9 minutes in old ones. This was accompanied by a decrease in the rate of recovery of DC potential at the end of the SD event, from ∼1.5 mV/s (young) to ∼0.6 mV/s (old). An attempt to generate oxidative stress using rotenone was unsuccessful, but pretreatment with the antioxidant, N-acetylcysteine amide, had opposite effects to those of aging, reducing duration (control ∼1.1 minutes, NACA ∼0.7 minutes) and increasing rate of recovery (control ∼0.5 mV/s, NACA ∼1.0 mV/s) suggesting that it prevented oxidative damage occurring during the ouabain treatment. The antioxidant also reduced the rate of the slow negative shift. We propose that the aging locust nervous system is more vulnerable to stress due to a prior accumulation of oxidative damage. Our findings also strengthen the notion that insects provide useful models for the investigation of cellular and molecular mechanisms underlying SD in mammals. Significance Statement: Anoxia and similar energetic crises trigger a shutdown of central neural processing in a process of spreading depolarization (SD) which is generally pathological in mammals and protective in insects. We show that some variability in the consequences of SD in an insect model can be attributed to age, such that older animals are slower to recover. Moreover, preventing oxidative stress with an antioxidant speeds recovery. These findings demonstrate a role for oxidative stress in contributing to the vulnerability of the aging insect CNS in energetic emergencies. Abstract Figure: Graphical Abstract of Robertson and Wang - Locust Spreading Depolarization:

Severe abiotic stress causes insects to lose nervous function and enter a state of paralytic coma. Central to this loss of function is a spreading depolarization (SD), where a characteristic collapse of ion gradients depolarizes neuronal and glial membranes and rapidly shuts down the CNS. Despite representing a critical limit to CNS function, the stress threshold that elicits SD can be altered by the process of acclimation, though the mechanisms underlying this response remain largely unknown. Here, we made electrophysiological measurements of SD and investigated the role of K ⁺ channels in acclimation of the CNS stress response of Drosophila melanogaster . First, we demonstrate that improved cold tolerance in the CNS elicited by cold acclimation was abolished by pharmacological blockade of K ⁺ channels with voltage-gated K ⁺ channels representing most of this effect. Next, we used the UAS/Gal4 model system to screen for candidate genes encoding glial voltage-gated K ⁺ channels and found that knockdown of sei - and Shaw -encoded channels mimicked the effect of K ⁺ blockade in cold-acclimated flies. Furthermore we show that the knockdown of glial sei -encoded channels also impair tolerance to anoxia and heat stress. These findings suggest that voltage-gated K ⁺ channels, especially those encoded by sei , are integral to the CNS stress- and acclimation-response and we posit that this is elicited through mechanisms involving glial spatial buffering and barrier function. Establishing such causal links between tissue-specific expression of candidate genes and SD mechanisms will inevitably aid our understanding of insect ecophysiology and SD-related neuropathologies. New and Noteworthy: Using thermal acclimation and pharmacology, we demonstrate that voltage-gated K ⁺ channels are involved in setting the threshold for cold-induced spreading depolarization (SD) in the Drosophila melanogaster CNS. Glial knockdown of channels encoded by sei and Shaw reduced the resistance to cold-induced SD, highlighting their importance in acclimation of the CNS. Glia-specific sei -knockdown also reduced tolerance to anoxia and heat. We posit that sei -channels are involved the CNS stress- and acclimation-responses through glial spatial buffering mechanisms.

Spreading depolarization (SD) describes the near-complete depolarization of CNS neural cells as a consequence of chemical, electrical, and metabolic perturbations. It is well-established as the central mechanism underlying insect coma and various mammalian neurological dysfunctions. Despite significant progress in our understanding, the question remains: which cation channel, if any, generates SD in the CNS? Previously, we speculated that the sodium-potassium ATPase (NKA) might function as a large-conductance ion channel to initiate SD in insects, potentially mediated by a palytoxin (PLTX)-like endogenous activator. In the current study, we evaluate the effectiveness and properties of PLTX as an SD initiator in L. migratoria . Whereas bath-applied PLTX failed to ignite SD, direct injection into the neuropil triggered SD in 57% of the preparations. Notably, PLTX-induced SD onset was significantly more rapid compared to ouabain injection and azide controls, though their electrophysiological features remained similar. Furthermore, PLTX-induced SD was recoverable and resulted in a greater frequency of repetitive SD events compared to ouabain. Surprisingly, prior PLTX treatment disrupted the onset and recovery of subsequent SD evoked by other means. PLTX injection could attenuate the amplitude and hasten the onset time of azide-induced SD. Such an effect is associated with a complete inhibition at higher doses of subsequent anoxic SD induced through azide treatment or submersion. These results show that PLTX can trigger repetitive and reversible SD-like events in locusts and simultaneously interfere with anoxic SD occurrence. We suggest that the well-documented NKA pump conversion into an open non-selective cationic channel is a plausible mechanism of SD activation in the locust CNS, warranting additional investigations.

During exposure to extreme stress, the CNS of mammals and insects fails through a phenomenon known as spreading depolarization (SD). SD is characterized by an abrupt disruption of ion gradients across neural and glial membranes that spreads through the CNS, silencing neural activity. In humans, SD is associated with neuropathological conditions like migraine and stroke. In insects, it is coincident with critical thermal limits for activity and can be conveniently monitored by observing the transperineurial potential (TPP). We used the TPP to explore the temperature-dependence and plasticity of SD thresholds and SD-induced changes to the TPP in fruit flies ( Drosophila melanogaster ) acclimated to different temperatures. Specifically, we characterized the effects of thermal acclimation on the TPP characteristics of cold-induced SD, after which we induced SD via anoxia at different temperatures in both acclimation groups to examine the interactive effects of temperature and acclimation status. Lastly, we investigated these effects on the rate of SD propagation across the fruit fly CNS. Cold acclimation enhanced resistance to both cold- and anoxic SD and our TPP measurements revealed independent and interactive effects of temperature and acclimation on the TPP and SD propagation. This suggests thermodynamic processes and physiological mechanisms interact to modulate the threshold for activity through SD and its electrophysiological phenomenology. These findings are discussed in relation to conceptual models for SD and established mechanisms for variation in the thermal threshold for SD.

Migratory locusts enter a reversible hypometabolic coma to survive environmental anoxia, wherein the cessation of CNS activity is driven by spreading depolarization (SD). While glycolysis is recognized as a crucial anaerobic energy source contributing to animal anoxia tolerance, its influence on the anoxic SD trajectory and recovery outcomes remains poorly understood. We investigated the effects of varying glycolytic capacity on adult female locust anoxic SD parameters, using glucose or the glycolytic inhibitors 2-deoxy-d-glucose (2DG) or monosodium iodoacetate (MIA). Surprisingly, 2DG treatment shared similarities with glucose yet had opposite effects compared with MIA. Specifically, although SD onset was not affected, both glucose and 2DG expedited the recovery of CNS electrical activity during reoxygenation, whereas MIA delayed it. Additionally, glucose and MIA, but not 2DG, increased tissue damage and neural cell death following anoxia-reoxygenation. Notably, glucose-induced injuries were associated with heightened CO₂output during the early phase of reoxygenation. Conversely, 2DG resulted in a bimodal response, initially dampening CO₂output and gradually increasing it throughout the recovery period. Given the discrepancies between effects of 2DG and MIA, the current results require cautious interpretations. Nonetheless, our findings present evidence that glycolysis is not a critical metabolic component in either anoxic SD onset or recovery and that heightened glycolysis during reoxygenation may exacerbate CNS injuries. Furthermore, we suggest that locust anoxic recovery is not solely dependent on energy availability, and the regulation of metabolic flux during early reoxygenation may constitute a strategy to mitigate damage.