Low Glycolysis Is Neuroprotective during Anoxic Spreading Depolarization (SD) and Reoxygenation in Locusts.

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 CO2 output during the early phase of reoxygenation. Conversely, 2DG resulted in a bimodal response, initially dampening CO2 output 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.

Rapid cold hardening modifies ion regulation to delay anoxia-induced spreading depolarization in the CNS of the locust.

Insects experience different kinds of environmental stresses that can impair neural performance, leading to spreading depolarization (SD) of nerve cells and neural shutdown underlying coma. SD is associated with a sudden loss of ion, notably K+, homeostasis in the central nervous system. The sensitivity of an insect's nervous system to stress (e.g., anoxia) can be modulated by acute pre-treatment. Rapid cold hardening (RCH) is a form of preconditioning, in which a brief exposure to low temperature can enhance the stress tolerance of insects. We used a pharmacological approach to investigate whether RCH affects anoxia-induced SD in the locust, Locusta migratoria, via one or more of the following homeostatic mechanisms: (1) Na+/K+-ATPase (NKA), (2) Na+/K+/2Cl- co-transporter (NKCC), and (3) voltage-gated K+ (Kv) channels. We also assessed abundance and phosphorylation of NKCC using immunoblotting. We found that inhibition of NKA or Kv channels delayed the onset of anoxia-induced SD in both control and RCH preparations. However, NKCC inhibition preferentially abrogated the effect of RCH. Additionally, we observed a higher abundance of NKCC in RCH preps but no statistical difference in its phosphorylation level, indicating the involvement of NKCC expression or degradation as part of the RCH mechanism.

A cold and quiet brain: mechanisms of insect CNS arrest at low temperatures.

Exposure to cold causes insects to enter a chill coma at species-specific temperatures and such temperature sensitivity contributes to geographic distribution and phenology. Coma results from abrupt spreading depolarization (SD) of neural tissue in the integrative centers of the central nervous system (CNS). SD abolishes neuronal signaling and the operation of neural circuits, like an off switch for the CNS. Turning off the CNS by allowing ion gradients to collapse will conserve energy and may offset negative consequences of temporary immobility. SD is modified by prior experience via rapid cold hardening (RCH) or cold acclimation that alter properties of Kv channels, Na+/K+-ATPase, and Na+/K+/2Cl- cotransporter. The stress hormone octopamine mediates RCH. Future progress depends on developing a more complete understanding of ion homeostasis in and of the insect CNS.

Plasticity in Na+/K+-ATPase thermal kinetics drives variation in the temperature of cold-induced neural shutdown of adult Drosophila melanogaster.

Most insects can acclimate to changes in their thermal environment and counteract temperature effects on neuromuscular function. At the critical thermal minimum, a spreading depolarization (SD) event silences central neurons, but the temperature at which this event occurs can be altered through acclimation. SD is triggered by an inability to maintain ion homeostasis in the extracellular space in the brain and is characterized by a rapid surge in extracellular K+ concentration, implicating ion pump and channel function. Here, we focused on the role of the Na+/K+-ATPase specifically in lowering the SD temperature in cold-acclimated Drosophila melanogaster. After first confirming cold acclimation altered SD onset, we investigated the dependency of the SD event on Na+/K+-ATPase activity by injecting the inhibitor ouabain into the head of the flies to induce SD over a range of temperatures. Latency to SD followed the pattern of a thermal performance curve, but cold acclimation resulted in a left-shift of the curve to an extent similar to its effect on the SD temperature. With Na+/K+-ATPase activity assays and immunoblots, we found that cold-acclimated flies have ion pumps that are less sensitive to temperature, but do not differ in their overall abundance in the brain. Combined, these findings suggest a key role for plasticity in Na+/K+-ATPase thermal sensitivity in maintaining central nervous system function in the cold, and more broadly highlight that a single ion pump can be an important determinant of whether insects can respond to their environment to remain active at low temperatures.

Rapid cold hardening increases axonal Na+/K+-ATPase activity and enhances performance of a visual motion detection circuit in Locusta migratoria.

Rapid cold hardening (RCH) is a type of phenotypic plasticity that delays the occurrence of chill coma in insects. Chill coma is mediated by a spreading depolarization of neurons and glia in the CNS, triggered by a failure of ion homeostasis. We used biochemical and electrophysiological approaches in the locust, Locusta migratoria, to test the hypothesis that the protection afforded by RCH is mediated by activation of the Na+/K+-ATPase (NKA) in neural tissue. RCH did not affect NKA activity measured in a biochemical assay of homogenized thoracic ganglia. However, RCH hyperpolarized the axon of a visual interneuron (DCMD) and increased the amplitude of an activity-dependent hyperpolarization (ADH) shown previously to be blocked by ouabain. RCH also improved performance of the visual circuitry presynaptic to DCMD to minimize habituation and increase excitability. We conclude that RCH enhances in situ NKA activity in the nervous system but also affects other neuronal properties that promote visual processing in locusts.

Knockdown of a Cyclic Nucleotide-Gated Ion Channel Impairs Locomotor Activity and Recovery From Hypoxia in Adult Drosophila melanogaster.

Cyclic guanosine monophosphate (cGMP) modulates the speed of recovery from anoxia in adult Drosophila and mediates hypoxia-related behaviors in larvae. Cyclic nucleotide-gated channels (CNG) and cGMP-activated protein kinase (PKG) are two cGMP downstream targets. PKG is involved in behavioral tolerance to hypoxia and anoxia in adults, however little is known about a role for CNG channels. We used a CNGL (CNG-like) mutant with reduced CNGL transcripts to investigate the contribution of CNGL to the hypoxia response. CNGL mutants had reduced locomotor activity under normoxia. A shorter distance travelled in a standard locomotor assay was due to a slower walking speed and more frequent stops. In control flies, hypoxia immediately reduced path length per minute. Flies took 30-40 min in normoxia for >90% recovery of path length per minute from 15 min hypoxia. CNGL mutants had impaired recovery from hypoxia; 40 min for ∼10% recovery of walking speed. The effects of CNGL mutation on locomotor activity and recovery from hypoxia were recapitulated by pan-neuronal CNGL knockdown. Genetic manipulation to increase cGMP in the CNGL mutants increased locomotor activity under normoxia and eliminated the impairment of recovery from hypoxia. We conclude that CNGL channels and cGMP signaling are involved in the control of locomotor activity and the hypoxic response of adult Drosophila.

The Critical Role of Spreading Depolarizations in Early Brain Injury: Consensus and Contention.

BACKGROUND: When a patient arrives in the emergency department following a stroke, a traumatic brain injury, or sudden cardiac arrest, there is no therapeutic drug available to help protect their jeopardized neurons. One crucial reason is that we have not identified the molecular mechanisms leading to electrical failure, neuronal swelling, and blood vessel constriction in newly injured gray matter. All three result from a process termed spreading depolarization (SD). Because we only partially understand SD, we lack molecular targets and biomarkers to help neurons survive after losing their blood flow and then undergoing recurrent SD. METHODS: In this review, we introduce SD as a single or recurring event, generated in gray matter following lost blood flow, which compromises the Na+/K+ pump. Electrical recovery from each SD event requires so much energy that neurons often die over minutes and hours following initial injury, independent of extracellular glutamate. RESULTS: We discuss how SD has been investigated with various pitfalls in numerous experimental preparations, how overtaxing the Na+/K+ ATPase elicits SD. Elevated K+ or glutamate are unlikely natural activators of SD. We then turn to the properties of SD itself, focusing on its initiation and propagation as well as on computer modeling. CONCLUSIONS: Finally, we summarize points of consensus and contention among the authors as well as where SD research may be heading. In an accompanying review, we critique the role of the glutamate excitotoxicity theory, how it has shaped SD research, and its questionable importance to the study of early brain injury as compared with SD theory.

Questioning Glutamate Excitotoxicity in Acute Brain Damage: The Importance of Spreading Depolarization.

BACKGROUND: Within 2 min of severe ischemia, spreading depolarization (SD) propagates like a wave through compromised gray matter of the higher brain. More SDs arise over hours in adjacent tissue, expanding the neuronal damage. This period represents a therapeutic window to inhibit SD and so reduce impending tissue injury. Yet most neuroscientists assume that the course of early brain injury can be explained by glutamate excitotoxicity, the concept that immediate glutamate release promotes early and downstream brain injury. There are many problems with glutamate release being the unseen culprit, the most practical being that the concept has yielded zero therapeutics over the past 30 years. But the basic science is also flawed, arising from dubious foundational observations beginning in the 1950s METHODS: Literature pertaining to excitotoxicity and to SD over the past 60 years is critiqued. RESULTS: Excitotoxicity theory centers on the immediate and excessive release of glutamate with resulting neuronal hyperexcitation. This instigates poststroke cascades with subsequent secondary neuronal injury. By contrast, SD theory argues that although SD evokes some brief glutamate release, acute neuronal damage and the subsequent cascade of injury to neurons are elicited by the metabolic stress of SD, not by excessive glutamate release. The challenge we present here is to find new clinical targets based on more informed basic science. This is motivated by the continuing failure by neuroscientists and by industry to develop drugs that can reduce brain injury following ischemic stroke, traumatic brain injury, or sudden cardiac arrest. One important step is to recognize that SD plays a central role in promoting early neuronal damage. We argue that uncovering the molecular biology of SD initiation and propagation is essential because ischemic neurons are usually not acutely injured unless SD propagates through them. The role of glutamate excitotoxicity theory and how it has shaped SD research is then addressed, followed by a critique of its fading relevance to the study of brain injury. CONCLUSIONS: Spreading depolarizations better account for the acute neuronal injury arising from brain ischemia than does the early and excessive release of glutamate.

Rapid cold hardening delays the onset of anoxia-induced coma via an octopaminergic pathway in Locusta migratoria.

Rapid cold hardening (RCH) is a short-term hormesis that occurs in many invertebrate species, especially in insects. Although RCH is best known as enhancing cold tolerance, it can also enhance anoxic tolerance. When exposed to prolonged anoxia, insects enter a reversible coma, which is associated with spreading depolarization (SD) in the central nervous system (CNS). In this study, we investigated the effects of RCH and octopamine (OA) on anoxia-induced SD in L. migratoria. OA is an insect stress hormone that has roles in many physiological processes. Thus, we hypothesized that OA is involved in the mechanism of RCH. First, we found that RCH affects the K+ sensitivity of the locust blood brain barrier (BBB) in a way similar to the previously described effects of OA. Next, using SD as an indicator of anoxia-induced coma, we took a pharmacological approach to investigate the effects of OA and epinastine (EP), an octopaminergic receptor (OctR) antagonist. We found that OA mimics, whereas EP blocks, the effect of RCH on anoxia-induced SD. This study demonstrates that OA is involved in the mechanism of RCH in delaying the onset of anoxia-induced locust coma and contributes to determining the mechanism of RCH that modulates insect stress tolerances.

Motor patterning, ion regulation and spreading depolarization during CNS shutdown induced by experimental anoxia in Locusta migratoria.

Anoxia induces a reversible coma in insects. Coma onset is triggered by the arrest of mechanisms responsible for maintaining membrane ion homeostasis in the CNS, resulting in a wave of neuronal and glial depolarization known as spreading depolarization (SD). Different methods of anoxia influence the behavioural response but their effects on SD are unknown. We investigated the effects of CO2, N2, and H2O on the characteristics of coma induction and recovery in Locusta migratoria. Water immersion delayed coma onset and recovery, likely due to involvement of the tracheal system and the nature of asphyxiation but otherwise resembled N2 delivery. The main difference between N2 and CO2 was that CO2 hastened onset of neural failure and SD and delayed recovery. In the CNS, this was associated with CO2 inducing an abrupt and immediate decrease of interstitial pH and increase of extracellular [K+]. Recording of the transperineurial potential showed that SD propagation and a postanoxic negativity (PAN) were similar with both gases. The PAN increased with ouabain treatment, likely due to removal of the counteracting electrogenic effect of Na+/K+-ATPase, and was inhibited by bafilomycin, a proton pump inhibitor, suggesting that it was generated by the electrogenic effect of a Vacuolar-type ATPase (VA). Muscle fibres depolarized by ~20 mV, which happened more rapidly with CO2 compared with N2. Wing muscle motoneurons depolarized nearly completely in two stages, with CO2 causing more rapid onset and slower recovery than N2. Other parameters of SD onset and recovery were similar with the two gases. Electrical resistance across the ganglion sheath increased during anoxia and at SD onset. We provisionally attribute this to cell swelling reducing the dimensions of the interstitial pathway from neuropil to the bathing saline. Neuronal membrane resistance decreased abruptly at SD onset indicating opening of an unidentified membrane conductance. Consideration of the intracellular recording relative to the saline suggests that the apical membrane of perineurial glia depolarizes prior to neuron depolarization. We propose that SD is triggered by events at the perineurial sheath and then propagates laterally and more deeply into the neuropil. We conclude that the fundamental nature of SD is not dependent on the method of anoxia however the timing of onset and recovery are influenced; water immersion is complicated by the tracheal system and CO2 delivery has more rapid and longer lasting effects, associated with severe interstitial acidosis.

Measuring enzyme activities in crude homogenates: Na+/K+-ATPase as a case study in optimizing assays.

In this review of assays of Na+/K+-ATPase (NKA), we explore the choices made by researchers assaying the enzyme to investigate its role in physiological regulation. We survey NKA structure and function in the context of how it is typically assayed, and how technical choices influence what can be said about the enzyme. In comparing different methods for extraction and assay of NKA, we identified a series of common pitfalls that compromise the veracity of results. We include experimental work to directly demonstrate how choices in detergents, salts and substrates influence NKA activities measured in crude homogenates. Our review of assay approaches integrates what is known from enzymology, biomedical physiology, cell biology and evolutionary biology, offering a more robust method for assaying the enzyme in meaningful ways, identifying caveats and future directions to explore its structure and function. The goal is to provide the sort of background on the enzyme that should be considered in exploring the function of the enzyme in comparative physiology.

Inhibition of ATP-sensitive potassium channels exacerbates anoxic coma in Locusta migratoria.

Under extreme environmental conditions, many insects enter a protective coma associated with a spreading depolarization (SD) of neurons and glia in the central nervous system (CNS). Recovery depends on the restoration of ion gradients by mechanisms that are not well understood. We investigated the effects of glybenclamide, an ATP-sensitive K+ (KATP) channel inhibitor, and pinacidil, a KATP activator, on the mechanisms involved in anoxic coma induction and recovery in Locusta migratoria. KATP channels allow for the efflux of K+ when activated, thereby linking cellular metabolic state to membrane potential. In intact locusts, we measured the time to enter a coma after water immersion and the time to recover the righting reflex after returning to normoxia. In semi-intact preparations, we measured the time to SD in the metathoracic ganglion after flooding the preparation with saline or exposing it to 100% N2 gas, and the time for the transperineurial potential to recover after removal of the saline or return to air. Glybenclamide decreased the time to coma induction, whereas pinacidil increased induction times. Glybenclamide also lengthened the time to recovery and decreased the rate of recovery of transperineurial potential after SD. These results were not the same as the effects of 10-2 M ouabain on N2-induced SD. We conclude that glybenclamide affects the CNS response to anoxia via inhibition of KATP channels and not an effect on the Na+/K+-ATPase.NEW & NOTEWORTHY We demonstrate the involvement of ATP-sensitive K+ (KATP) channels during recovery from spreading depolarization (SD) induced via anoxic coma in locusts. KATP inhibition using glybenclamide impaired ion homeostasis across the blood-brain barrier resulting in a longer time to recovery of transperineurial potential following SD. Comparison with ouabain indicates that the effects of glybenclamide are not mediated by the Na+/K+-ATPase but are a result of KATP channel inhibition.

Effects of brief chilling and desiccation on ion homeostasis in the central nervous system of the migratory locust, Locusta migratoria.

In insects, chilling, anoxia, and dehydration are cues to trigger rapid physiological responses enhancing stress tolerance within minutes. Recent evidence suggests that responses elicited by different cues are mechanistically distinct from each other, though these differences have received little attention. Further, the effects are not well studied in neural tissue. In this study, we examined how brief exposure to desiccation and chilling affect ion homeostatic mechanisms in metathoracic ganglion of the migratory locust, Locusta migratoria. Both desiccation and chilling enhanced resistance to anoxia, though only chilling hastened recovery from anoxic coma. Similarly, only chilling enhanced resistance to pharmacological perturbation of neuronal ion homeostasis. Our results indicate that chilling and desiccation trigger mechanistically distinct responses and, while both may be important for neuronal ion homeostasis, chilling has a larger effect on this tissue. SUMMARY STATEMENT: This is one of few studies to demonstrate the importance of the central nervous system in rapid acclimatory responses in insects.

Neural dysfunction correlates with heat coma and CTmax in Drosophila but does not set the boundaries for heat stress survival.

When heated, insects lose coordinated movement followed by the onset of heat coma (critical thermal maximum, CTmax). These traits are popular measures to quantify interspecific and intraspecific differences in insect heat tolerance, and CTmax correlates well with current species distributions of insects, including Drosophila Here, we examined the function of the central nervous system (CNS) in five species of Drosophila with different heat tolerances, while they were exposed to either constant high temperature or a gradually increasing temperature (ramp). Tolerant species were able to preserve CNS function at higher temperatures and for longer durations than sensitive species, and similar differences were found for the behavioural indices (loss of coordination and onset of heat coma). Furthermore, the timing and temperature (constant and ramp exposure, respectively) for loss of coordination or complete coma coincided with the occurrence of spreading depolarisation (SD) events in the CNS. These SD events disrupt neurological function and silence the CNS, suggesting that CNS failure is the primary cause of impaired coordination and heat coma. Heat mortality occurs soon after heat coma in insects; to examine whether CNS failure could also be the proximal cause of heat death, we used selective heating of the head (CNS) and abdomen (visceral tissues). When comparing the temperature causing 50% mortality (LT50) of each body part versus that of the whole animal, we found that the head was not particularly heat sensitive compared with the abdomen. Accordingly, it is unlikely that nervous failure is the principal/proximate cause of heat mortality in Drosophila.

Role of adenosine in functional recovery following anoxic coma in Locusta migratoria.

When exposed to prolonged anoxia insects enter a reversible coma during which neural and muscular systems temporarily shut down. Nervous system shut down is a result of spreading depolarization throughout neurons and glial cells. Upon return to normoxia, recovery occurs following the restoration of ion gradients. However, there is a delay in the functional recovery of synaptic transmission following membrane repolarization. In mammals, the build-up of extracellular adenosine following spreading depolarization contributes to this delay. Adenosine accumulation is a marker of metabolic stress and it has many downstream effects through the activation of adenosine receptors, including the inhibition of cAMP production. Here we demonstrate that adenosine lengthens the time to functional recovery following anoxic coma in locusts. Caffeine, used as an adenosine receptor antagonist, decreased the time to recovery in intact animals and lengthened the time to recovery in semi-intact animals. A cAMP inhibitor, NKH 477, delayed recovery time in male animals. Our results show that the rate of recovery in insect systems is affected by the presence of adenosine.

Neural shutdown under stress: an evolutionary perspective on spreading depolarization.

Neural function depends on maintaining cellular membrane potentials as the basis for electrical signaling. Yet, in mammals and insects, neuronal and glial membrane potentials can reversibly depolarize to zero, shutting down neural function by the process of spreading depolarization (SD) that collapses the ion gradients across membranes. SD is not evident in all metazoan taxa with centralized nervous systems. We consider the occurrence and similarities of SD in different animals and suggest that it is an emergent property of nervous systems that have evolved to control complex behaviors requiring energetically expensive, rapid information processing in a tightly regulated extracellular environment. Whether SD is beneficial or not in mammals remains an open question. However, in insects, it is associated with the response to harsh environments and may provide an energetic advantage that improves the chances of survival. The remarkable similarity of SD in diverse taxa supports a model systems approach to understanding the mechanistic underpinning of human neuropathology associated with migraine, stroke, and traumatic brain injury.

Which Spreading Depolarizations Are Deleterious To Brain Tissue?

Spreading depolarizations (SDs) are profound disruptions of cellular homeostasis that slowly propagate through gray matter and present an extraordinary metabolic challenge to brain tissue. Recent work has shown that SDs occur commonly in human patients in the neurointensive care setting and have established a compelling case for their importance in the pathophysiology of acute brain injury. The International Conference on Spreading Depolarizations (iCSD) held in Boca Raton, Florida, in September of 2018 included a discussion session focused on the question of "Which SDs are deleterious to brain tissue?" iCSD is attended by investigators studying various animal species including invertebrates, in vivo and in vitro preparations, diseases of acute brain injury and migraine, computational modeling, and clinical brain injury, among other topics. The discussion included general agreement on many key issues, but also revealed divergent views on some topics that are relevant to the design of clinical interventions targeting SDs. A draft summary of viewpoints offered was then written by a multidisciplinary writing group of iCSD members, based on a transcript of the session. Feedback of all discussants was then formally collated, reviewed and incorporated into the final document. It is hoped that this report will stimulate collection of data that are needed to develop a more nuanced understanding of SD in different pathophysiological states, as the field continues to move toward effective clinical interventions.

Expression of Heat Shock Protein 70 Is Insufficient To Extend Drosophila melanogaster Longevity.

It has been known for over 20 years that Drosophila melanogaster flies with twelve additional copies of the hsp70 gene encoding the 70 kD heat shock protein lives longer after a non-lethal heat treatment. Since the heat treatment also induces the expression of additional heat shock proteins, the biological effect can be due either to HSP70 acting alone or in combination. This study used the UAS/GAL4 system to determine whether hsp70 is sufficient to affect the longevity and the resistance to thermal, oxidative or desiccation stresses of the whole organism. We observed that HSP70 expression in the nervous system or muscles has no effect on longevity or stress resistance but ubiquitous expression reduces the life span of males. We also observed that the down-regulation of hsp70 using RNAi did not affect longevity.

Experimental evolution of response to anoxia in Drosophilamelanogaster: recovery of locomotion following CO2 or N2 exposure.

Many insects enter coma upon exposure to anoxia, a feature routinely exploited by experimentalists to handle them. But the genetic and physiological bases of anoxic coma induction and recovery are only partially understood, as are the long-term consequences for the animal's performance. We examined three populations of Drosophila melanogaster (designated B) that have been inadvertently under selection for rapid recovery from CO2 exposure for nearly 40 years (around 1000 generations) resulting from routine maintenance practices. We contrasted CO2 and N2 (presumed a less reactive gas) knockdown and recovery times of these B flies with six populations of common ancestry (A and C populations) that were not exposed to CO2 over the same period. We found that B populations showed faster and more consistent locomotor recovery than A or C populations after CO2 knockdown, a result also observed with N2 knockdown. A and C populations showed much higher variance in recovery time after CO2 exposure than after N2 exposure, suggesting gas-specific effects on pathways associated with locomotor recovery. Although these selection treatments result in considerable variation in life history attributes and body size, with the characteristic intermediacy of B populations, their superiority in resistance to gas exposure and locomotor recovery suggests that this is a direct consequence of prior repeated exposure to anoxia, broadly, and CO2, specifically. Hence we describe a powerful new evolutionary model for the genetic and physiological investigation of anoxic coma in insects.

Rapid cold hardening and octopamine modulate chill tolerance in Locusta migratoria.

Temperature has profound effects on the neural function and behaviour of insects. When exposed to low temperature, chill-susceptible insects enter chill coma, a reversible state of neuromuscular paralysis. Despite the popularity of studying the effects of low temperature on insects, we know little about the physiological mechanisms controlling the entry to, and recovery from, chill coma. Spreading depolarization (SD) is a phenomenon that causes a neural shutdown in the central nervous system (CNS) and it is associated with a loss of K+ homeostasis in the CNS. Here, we investigated the effects of rapid cold hardening (RCH) on chill tolerance of the migratory locust. With an implanted thermocouple in the thorax, we determined the temperature associated with a loss of responsiveness (i.e. the critical thermal minimum - CTmin) in intact male adult locusts. In parallel experiments, we recorded field potential (FP) in the metathoracic ganglion (MTG) of semi-intact preparations to determine the temperature that would induce neural shutdown. We found that SD in the CNS causes a loss of coordinated movement immediately prior to chill coma and RCH reduces the temperature that evokes neural shutdown. Additionally, we investigated a role for octopamine (OA) in the locust chill tolerance and found that OA reduces the CTmin and mimics the effects of prior stress (anoxia) in locust.