Gary A. B. Armstrong

Stress preconditioning of spreading depression in the locust CNS.

Cortical spreading depression (CSD) is closely associated with important pathologies including stroke, seizures and migraine. The mechanisms underlying SD in its various forms are still incompletely understood. Here we describe SD-like events in an invertebrate model, the ventilatory central pattern generator (CPG) of locusts. Using K+ -sensitive microelectrodes, we measured extracellular K+ concentration ([K+]o) in the metathoracic neuropile of the CPG while monitoring CPG output electromyographically from muscle 161 in the second abdominal segment to investigate the role K+ in failure of neural circuit operation induced by various stressors. Failure of ventilation in response to different stressors (hyperthermia, anoxia, ATP depletion, Na+/K+ ATPase impairment, K+ injection) was associated with a disturbance of CNS ion homeostasis that shares the characteristics of CSD and SD-like events in vertebrates. Hyperthermic failure was preconditioned by prior heat shock (3 h, 45°C) and induced-thermotolerance was associated with an increase in the rate of clearance of extracellular K+ that was not linked to changes in ATP levels or total Na+/K+ ATPase activity. Our findings suggest that SD-like events in locusts are adaptive to terminate neural network operation and conserve energy during stress and that they can be preconditioned by experience. We propose that they share mechanisms with CSD in mammals suggesting a common evolutionary origin.

Coma in response to environmental stress in the locust: A model for cortical spreading depression

Spreading depression (SD) is an interesting and important phenomenon due to its role in mammalian pathologies such as migraine, seizures, and stroke. Until recently investigations of the mechanisms involved in SD have mostly utilized mammalian cortical tissue, however we have discovered that SD-like events occur in the CNS of an invertebrate model, Locusta migratoria. Locusts enter comas in response to stress during which neural and muscular systems shut down until the stress is removed, and this is believed to be an adaptive strategy to survive extreme environmental conditions. During stress-induced comas SD-like events occur in the locust metathoracic ganglion (MTG) that closely resemble cortical SD (CSD) in many respects, including mechanism of induction, extracellular potassium ion changes, and propagation in areas equivalent to mammalian grey matter. In this review we describe the generation of comas and the associated SD-like events in the locust, provide a description of the similarities to CSD, and show how they can be manipulated both by stress preconditioning and pharmacologically. We also suggest that locust SD-like events are adaptive by conserving energy and preventing cellular damage, and we provide a model for the mechanism of SD onset and recovery in the locust nervous system.

Suppression of Spreading Depression-Like Events in Locusts by Inhibition of the NO/cGMP/PKG Pathway

Despite considerable research attention focused on mechanisms underlying neural spreading depression (SD), because of its association with important human CNS pathologies, such as stroke and migraine, little attention has been given to explaining its occurrence and regulation in invertebrates. In the locust metathoracic ganglion (MTG), an SD-like event occurs during heat and anoxia stress, which results in cessation of neuronal output for the duration of the applied stress. SD-like events were characterized by an abrupt rise in extracellular potassium ion concentration ([K+]o) from a baseline concentration of ∼8 to >30 mm, which returned to near baseline concentrations after removal of the applied stress. After return to baseline [K+]o, neuronal output (ventilatory motor pattern activity) from the MTG recovered. Unlike mammalian neurons, which depolarize almost completely during SD, locust neurons only partially depolarized. SD-like events in the locust CNS were suppressed by pharmacological inhibition of the nitric oxide/cyclic guanosine monophosphate/protein kinase G (NO/cGMP/PKG) pathway and were exacerbated by its activation. Also, environmental stressors such as heat and anoxia increased production of nitric oxide in the locust CNS. Finally, for the intact animal, manipulation of the pathway affected the speed of recovery from suffocation by immersion under water. We propose that SD-like events in locusts provide an adaptive mechanism for surviving extreme environmental conditions. The highly conserved nature of the NO/cGMP/PKG signaling pathway suggests that it may be involved in modulating SD in other organisms, including mammals.

Natural Variation in the Thermotolerance of Neural Function and Behavior due to a cGMP-Dependent Protein Kinase

Although it is acknowledged that genetic variation contributes to individual differences in thermotolerance, the specific genes and pathways involved and how they are modulated by the environment remain poorly understood. We link natural variation in the thermotolerance of neural function and behavior in Drosophila melanogaster to the foraging gene (for, which encodes a cGMP-dependent protein kinase (PKG)) as well as to its downstream target, protein phosphatase 2A (PP2A). Genetic and pharmacological manipulations revealed that reduced PKG (or PP2A) activity caused increased thermotolerance of synaptic transmission at the larval neuromuscular junction. Like synaptic transmission, feeding movements were preserved at higher temperatures in larvae with lower PKG levels. In a comparative assay, pharmacological manipulations altering thermotolerance in a central circuit of Locusta migratoria demonstrated conservation of this neuroprotective pathway. In this circuit, either the inhibition of PKG or PP2A induced robust thermotolerance of neural function. We suggest that PKG and therefore the polymorphism associated with the allelic variation in for may provide populations with natural variation in heat stress tolerance. fors function in behavior is conserved across most organisms, including ants, bees, nematodes, and mammals. PKGs role in thermotolerance may also apply to these and other species. Natural variation in thermotolerance arising from genes involved in the PKG pathway could impact the evolution of thermotolerance in natural populations.

Loss and gain of FUS function impair neuromuscular synaptic transmission in a genetic model of ALS

Amyotrophic lateral sclerosis (ALS) presents clinically in adulthood and is characterized by the loss of motoneurons in the spinal cord and cerebral cortex. Animal models of the disease suggest that significant neuronal abnormalities exist during preclinical stages of the disease. Mutations in the gene fused in sarcoma (FUS) are associated with ALS and cause impairment in motor function in animal models. However, the mechanism of neuromuscular dysfunction underlying pathophysiological deficits causing impairment in locomotor function resulting from mutant FUS expression is unknown. To characterize the cellular pathophysiological defect, we expressed the wild-type human gene (wtFUS) or the ALS-associated mutation R521H (mutFUS) gene in zebrafish larvae and characterized their motor (swimming) activity and function of their neuromuscular junctions (NMJs). Additionally, we tested knockdown of zebrafish fus with an antisense morpholino oligonucleotide (fus AMO). Expression of either mutFUS or knockdown of fus resulted in impaired motor activity and reduced NMJ synaptic fidelity with reduced quantal transmission. Primary motoneurons expressing mutFUS were found to be more excitable. These impairments in neuronal function could be partially restored in fus AMO larvae also expressing wtFUS (fus AMO+wtFUS) but not mutFUS (fus AMO+mutFUS). These results show that both a loss and gain of FUS function result in defective presynaptic function at the NMJ.

Homology Directed Knockin of Point Mutations in the Zebrafish tardbp and fus Genes in ALS Using the CRISPR/Cas9 System

The methodology for site-directed editing of single nucleotides in the vertebrate genome is of considerable interest for research in biology and medicine. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 type II (Cas9) system has emerged as a simple and inexpensive tool for editing genomic loci of interest in a variety of animal models. In zebrafish, error-prone non-homologous end joining (NHEJ) has been used as a simple method to disrupt gene function. We sought to develop a method to easily create site-specific SNPs in the zebrafish genome. Here, we report simple methodologies for using CRISPR/Cas9-mediated homology directed repair using single-stranded oligodeoxynucleotide donor templates (ssODN) for site-directed single nucleotide editing, for the first time in two disease-related genes, tardbp and fus.

Cold hardening modulates K+ homeostasis in the brain of Drosophila melanogaster during chill coma.

Environmental temperature is one of the most important abiotic factors affecting insect behaviour; virtually all physiological processes, including those which regulate nervous system function, are affected. At both low and high temperature extremes insects enter a coma during which individuals do not display behaviour and are unresponsive to stimulation. We investigated neurophysiological correlates of chill and hyperthermic coma in Drosophila melanogaster. Coma resulting from anoxia causes a profound loss of K(+) homeostasis characterized by a surge in extracellular K(+) concentration ([K(+)](o)) in the brain. We recorded [K(+)](o) in the brain during exposure to both low and high temperatures and observed a similar surge in [K(+)](o) which recovered to baseline concentrations following return to room temperature. We also found that rapid cold hardening (RCH) using a cold pretreatment (4°C for 2h; 2h recovery at room temperature) increased the peak brain [K(+)](o) reached during a subsequent chill coma and increased the rates of accumulation and clearance of [K(+)](o). We conclude that RCH preserves K(+) homeostasis in the fly brain during exposure to cold by reducing the temperature sensitivity of the rates of homeostatic processes.

Calcium Channel Agonists Protect against Neuromuscular Dysfunction in a Genetic Model of TDP-43 Mutation in ALS

TAR DNA binding protein (TDP-43, encoded by the TARDBP gene) has recently been shown to be associated with amyotrophic lateral sclerosis (ALS), but the early pathophysiological deficits causing impairment in motor function are unknown. Here we expressed the wild-type human gene (wtTARDBP) or the ALS mutation G348C (mutTARDBP) in zebrafish larvae and characterized their motor (swimming) activity and the structure and function of their neuromuscular junctions (NMJs). Of these groups only mutTARDBP larvae showed impaired swimming and increased motoneuron vulnerability with reduced synaptic fidelity, reduced quantal transmission, and more orphaned presynaptic and postsynaptic structures at the NMJ. Remarkably, all behavioral and cellular features were stabilized by chronic treatment with either of the L-type calcium channel agonists FPL 64176 or Bay K 8644. These results indicate that expression of mutTARDBP results in defective NMJs and that calcium channel agonists could be novel therapeutics for ALS.

Octopamine Mediates Thermal Preconditioning of the Locust Ventilatory Central Pattern Generator via a cAMP/Protein Kinase A Signaling Pathway

We investigated the role of biogenic amines in generating thermoprotection of the ventilatory motor pattern circuitry in Locusta migratoria. Levels of octopamine (OA) and dopamine (DA) in the metathoracic ganglion decreased during heat stress. We measured the thermosensitivity of central pattern generation in response to a ramped increase of temperature in semi-intact preparations. OA, DA, and tyramine (TA) were either bath applied or injected into the locust hemocoel 4–8 h before testing. Neither TA nor DA modified the thermotolerance of ventilatory motor pattern generation. However, OA treatment by bath applications (10−4 m OA) or by injections into the hemocoel (2 μg/10 μl OA) mimicked heat shock preconditioning and improved the thermotolerance of the motor pattern by increasing the failure temperature and by decreasing the time taken to recover operation after a return to room temperature. Heat shock-induced thermoprotection was eradicated in locusts preinjected with epinastine (OctβR antagonist). Neuropil injections of the cAMP agonist and protein kinase A (PKA) activator, Sp-cAMPs, both conferred thermoprotection in control locusts and rescued thermoprotection in epinastine-treated HS locusts. Similar injections of the PKA inhibitor Rp-cAMPs blocked the thermoprotective effect of bath-applied OA. Octopamine-mediated thermoprotection was also abolished with neuropil injections of cycloheximide or actinomycin D, indicating a requirement for transcription and translation. We conclude that OA has a crucial role in triggering protein synthesis-dependent physiological adaptations to protect CNS function during heat stress by activating a cAMP/PKA pathway.

Fishing for causes and cures of motor neuron disorders.

Motor neuron disorders (MNDs) are a clinically heterogeneous group of neurological diseases characterized by progressive degeneration of motor neurons, and share some common pathological pathways. Despite remarkable advances in our understanding of these diseases, no curative treatment for MNDs exists. To better understand the pathogenesis of MNDs and to help develop new treatments, the establishment of animal models that can be studied efficiently and thoroughly is paramount. The zebrafish (Danio rerio) is increasingly becoming a valuable model for studying human diseases and in screening for potential therapeutics. In this Review, we highlight recent progress in using zebrafish to study the pathology of the most common MNDs: spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS) and hereditary spastic paraplegia (HSP). These studies indicate the power of zebrafish as a model to study the consequences of disease-related genes, because zebrafish homologues of human genes have conserved functions with respect to the aetiology of MNDs. Zebrafish also complement other animal models for the study of pathological mechanisms of MNDs and are particularly advantageous for the screening of compounds with therapeutic potential. We present an overview of their potential usefulness in MND drug discovery, which is just beginning and holds much promise for future therapeutic development.