The effects of doxapram and its potential interactions with K2P channels in experimental model preparations.

The channels commonly responsible for maintaining cell resting membrane potentials are referred to as K2P (two-P-domain K+ subunit) channels. These K+ ion channels generally remain open but can be modulated by their local environment. These channels are classified based on pharmacology, pH sensitivity, mechanical stretch, and ionic permeability. Little is known about the physiological nature of these K2P channels in invertebrates. Acidic conditions depolarize neurons and muscle fibers, which may be caused by K2P channels given that one subtype can be blocked by acidic conditions. Doxapram is used clinically as a respiratory aid known to block acid-sensitive K2P channels; thus, the effects of doxapram on the muscle fibers and synaptic transmission in larval Drosophila and crawfish were monitored. A dose-dependent response was observed via depolarization of the larval Drosophila muscle and an increase in evoked synaptic transmission, but doxapram blocked the production of action potentials in the crawfish motor neuron and had a minor effect on the resting membrane potential of the crawfish muscle. This indicates that the nerve and muscle tissues in larval Drosophila and crawfish likely express different K2P channel subtypes. Since these organisms serve as physiological models for neurobiology and physiology, it would be of interest to further investigate what types of K2P channel are expressed in these tissues. (212 words).

Examining the effect of iron (ferric) on physiological processes: Invertebrate models.

Iron is a common and essential element for maintaining life in bacteria, plants and animals and is found in soil, fresh waters and marine waters; however, over exposure is toxic to organisms. Iron is used in electron transport complexes within mitochondria as well as a co-factor in many essential proteins. It is also established that iron accumulation in the central nervous system in mammals is associated with various neurological disorders. Ample studies have investigated the long-term effects of iron overload in the nervous system. However, its acute effects in nervous tissue and additional organ systems warrant further studies. This study investigates the effects of iron overload on development, behavior, survival, cardiac function, and glutamatergic synaptic transmission in the Drosophila melanogaster. Additionally, physiological responses in crayfish were examined following Fe3+ exposure. Fe3+ reduced neuronal excitability in proprioceptive neurons in a crayfish model. Thus, Fe3+ may block stretch activated channels (SACs) as well as voltage-gated Na+ channels. Exposure also rapidly reduces synaptic transmission but does not block ionotropic glutamatergic receptors, suggesting a blockage of pre-synaptic voltage-gated Ca2+ channels in both crustacean and Drosophila models. The effects are partly reversible with acute exposure, indicating the cells are not rapidly damaged. This study is relevant in demonstrating the effects of Fe3+ on various physiological functions in different organisms in order to further understand the acute and long-term consequences of overload.

The Effects of Doxapram Blocking the Response of Gram-Negative Bacterial Toxin (LPS) at Glutamatergic Synapses.

Lipopolysaccharides (LPS) associated with Gram-negative bacteria are one factor responsible for triggering the mammalian immune response. Blocking the action of LPS is key to reducing its downstream effects. However, the direct action of LPS on cells is not yet fully addressed. LPS can have rapid, direct effects on cells in the absence of a systemic immune response. Recent studies have shown that doxapram, a blocker of a subset of K2P channels, also blocks the acute actions of LPS. Doxapram was evaluated to determine if such action also occurs at glutamatergic synapses in which it is known that LPS can increase synaptic transmission. Doxapram at 5 mM first enhanced synaptic transmission, then reduced synaptic response, while 10 mM rapidly blocked transmission. Doxapram at 5 mM blocked the excitatory response induced by LPS. Enhancing synaptic transmission with LPS and then applying LPS combined with doxapram also resulted in retarding the response of LPS. It is possible doxapram and LPS are mediated via a similar receptor or cellular responses. The potential of designing pharmacological compounds with a similar structure to doxapram and determining the binding of such compounds can aid in addressing the acute, direct actions by LPS on cells.

The effects of Gram-positive and Gram-negative bacterial toxins (LTA & LPS) on cardiac function in Drosophila melanogaster larvae.

The effects of Gram negative and positive bacterial sepsis depend on the type of toxins released, such as lipopolysaccharides (LPS) or lipoteichoic acid (LTA). Previous studies show LPS to rapidly hyperpolarize larval Drosophila skeletal muscle, followed by desensitization and return to baseline. In larvae, heart rate increased then decreased with exposure to LPS. However, responses to LTA, as well as the combination of LTA and LPS, on the larval Drosophila heart have not been previously examined. This study examined the effects of LTA and a cocktail of LTA and LPS on heart rate. The combined effects were examined by first treating with either LTA or LPS only, and then with the cocktail. The results showed a rapid increase in heart rate upon LTA application, followed by a gradual decline over time. When applying LTA followed by the cocktail, an increase in the rate occurred. However, if LPS was applied before the cocktail, the rate continued declining. These responses indicate the receptors or cellular cascades responsible for controlling heart rate within seconds and the rapid desensitization are affected by LTA or LPS and a combination of the two. The mechanisms for rapid changes which are not regulated by gene expression by exposure to LTA or LPS or associated bacterial peptidoglycans have yet to be identified in cardiac tissues of any organism.

Bacterial lipopolysaccharide hyperpolarizes the membrane potential and is antagonized by the K2p channel blocker doxapram.

Exposure of Drosophila skeletal muscle to bacterial lipopolysaccharides (LPS) rapidly and transiently hyperpolarizes membrane potential. However, the mechanism responsible for hyperpolarization remains unclear. The resting membrane potential of the cells is maintained through multiple mechanisms. This study investigated the possibility of LPS activating calcium-activated potassium channels (KCa) and/or K2p channels. 2-Aminoethyl diphenylborinate (2-APB), blocks uptake of Ca2+ into the endoplasmic reticulum (ER); thus, limiting release from ryanodine-sensitive internal stores to reduce the function of KCa channels. Exposure to 2-APB produces waves of hyperpolarization even during desensitization of the response to LPS and in the presence of doxapram. This finding in this study suggests that doxapram blocked the acid-sensitive K2p tandem-pore channel subtype known in mammals. Doxapram blocked LPS-induced hyperpolarization and depolarized the muscles as well as induced motor neurons to produce evoked excitatory junction potentials (EJPs). This was induced by depolarizing motor neurons, similar to the increase in extracellular K+ concentration. The hyperpolarizing effect of LPS was not blocked by decreased extracellular Ca2+or the presence of Cd2+. LPS appears to transiently activate doxapram sensitive K2p channels independently of KCa channels in hyperpolarizing the muscle. Septicemia induced by gram-negative bacteria results in an increase in inflammatory cytokines, primarily induced by bacterial LPS. Currently, blockers of LPS receptors in mammals are unknown; further research on doxapram and other K2p channels is warranted. (220 words).

An invertebrate model in examining the effect of acute ferric iron exposure on proprioceptive neurons.

Iron is an essential element for plant and animal life and is found in soil, fresh waters and marine waters. The Fe3+ ion is a vital prosthetic group and cofactor to mitochondrial electron transport complexes and numerous proteins involved in normal functioning. Despite its importance to life-sustaining processes, overexposure results in toxicity. For example, ferric iron (Fe3+) accumulation in the mammalian central nervous system is associated with various neurological disorders. Although current literature addresses the long-term effects of Fe3+ overload, fewer studies exist examining the effects of acute exposure. Using the blue crab (Callinectes sapidus), we investigate the effects of acute Fe3+ overload on proprioception within the propodite-dactylopodite (PD) nerve. For proprioceptive studies, 10- and 20-mM ferric chloride and ferric ammonium citrate solutions were used at 5- and 20- min exposure times. Exposure to 20 mM concentrations of ferric chloride and ferric ammonium citrate reduced excitability in proprioceptive neurons. Thus, Fe3+ likely blocks stretch-activated channels or voltage-gated Na+ channels. The depressive effects of Fe3+ are partly reversible following saline washout, indicating cells are not acutely damaged. Gadolinium (GdCl3, 1 and 10 mM) was used to examine the effects of an additional trivalent ion comparator. Gd3+ depressed PD nerve compound action potential amplitude while increasing the compound action potential duration. This study is relevant in demonstrating the dose-dependent effects of acute Fe3+ and Gd3+ exposure on proprioception and provides a model system to further investigate the mechanisms by which metals act on the nervous system.

The effects of doxapram (blocker of K2p channels) on resting membrane potential and synaptic transmission at the Drosophila neuromuscular junction.

The resting membrane potential of most cells is maintained by potassium K2p channels. The pharmacological profile and distribution of various K2p channel subtypes in organisms are still being investigated. The Drosophila genome contains 11 subtypes; however, their function and expression profiles have not yet been determined. Doxapram is clinically used to enhance respiration in humans and blocks the acid-sensitive K2p TASK subtype in mammals. The resting membrane potential of larval Drosophila muscle and synaptic transmission at the neuromuscular junction are pH sensitive. The present study investigated the effects of doxapram on membrane potential and synaptic transmission using intracellular recordings of larval Drosophila muscles. Doxapram (1 mM and 10 mM) depolarizes the muscle and appears to depolarize motor neurons, causing an increase in the frequency of spontaneous quantal events and evoked excitatory junction potentials. Verapamil (1 and 10 mM) paralleled the action of doxapram. These changes were matched by an extracellular increase in KCl (50 mM) and blocked by Cd2+. It is assumed that the motor nerve depolarizes to open voltage-gated Ca2+ channels in presynaptic nerve terminals because of exposure to doxapram. These findings are significant for building models to better understand the function of pharmacological agents that affect K2p channels and how K2p channels contribute to the physiology of tissues. Drosophila offers a genetically amenable model that can alter the tissue-specific expression of K2p channel subtypes to simulate known human diseases related to this family of channels.

Hyperpolarization Induced by Lipopolysaccharides but Not by Chloroform Is Inhibited by Doxapram, an Inhibitor of Two-P-Domain K+ Channel (K2P).

Bacterial septicemia is commonly induced by Gram-negative bacteria. The immune response is triggered in part by the secretion of bacterial endotoxin lipopolysaccharide (LPS). LPS induces the subsequent release of inflammatory cytokines which can result in pathological conditions. There is no known blocker to the receptors of LPS. The Drosophila larval muscle is an amendable model to rapidly screen various compounds that affect membrane potential and synaptic transmission such as LPS. LPS induces a rapid hyperpolarization in the body wall muscles and depolarization of motor neurons. These actions are blocked by the compound doxapram (10 mM), which is known to inhibit a subtype of the two-P-domain K+ channel (K2P channels). However, the K2P channel blocker PK-THPP had no effect on the Drosophila larval muscle at 1 and 10 mM. These channels are activated by chloroform, which also induces a rapid hyperpolarization of these muscles, but the channels are not blocked by doxapram. Likewise, chloroform does not block the depolarization induced by doxapram. LPS blocks the postsynaptic glutamate receptors on Drosophila muscle. Pre-exposure to doxapram reduces the LPS block of these ionotropic glutamate receptors. Given that the larval Drosophila body wall muscles are depolarized by doxapram and hyperpolarized by chloroform, they offer a model to begin pharmacological profiling of the K2P subtype channels with the potential of identifying blockers for the receptors to mitigate the actions of the Gram-negative endotoxin LPS.

Building bridges: mycelium-mediated plant-plant electrophysiological communication.

Whether through root secretions or by emitting volatile organic compounds, plant communication has been well-documented. While electrical activity has been documented in plants and mycorrhizal bodies on the individual and ramet, electrical propagation as a means of communication between plants has been hypothesized but understudied. This study aimed to test the hypothesis that plants can communicate with one another electrically via conductively isolated mycelial pathways. We created a bio-electric circuit linking two plants using a mycelial network grown from a blend of mycorrhizal fungi which was directly inoculated onto potato dextrose agar, or onto the host plants placed on the agar. The mycelium that grew was forced to cross, or "bridge," an air gap between the two islands of agar - thus forming the isolated conductive pathway between plants. Using this plant-fungal biocircuit we assessed electrical propagation between Pisum sativum and Cucumis sativus. We found that electrical signals were reliably conducted across the mycelial bridges from one plant to another upon the induction of a wound response. Our findings provide evidence that mechanical input can be communicated between plant species and opens the door to testing how this information can affect plant and fungal physiology.

Most plants form underground relationships with fungi. These relationships are mutually beneficial. The plants and fungi share, trade, and distribute resources between themselves, their neighbors, and their offspring. Plants employ diverse methods to detect and respond to their environment and the production of electric signals is one of these methods. It would be favorable to a plant's survival and the survival of their neighbors, if this plant could transmit and share the information these electrical signals contain. Possible avenues of transmission exist in the roots, and the fungi these roots are in contact with. If a fungal mass is in contact with the roots of multiple plants, it could propagate electrical signals throughout the plant network. We found that electric signals were reliably transmitted from one plant to another via fungal pathways upon the induction of a wound response. Our findings provide evidence that mechanical input can be communicated between plant species and opens the door to testing how this information can affect plant and fungal physiology.

Impedance Measures for Detecting Electrical Responses during Acute Injury and Exposure of Compounds to Roots of Plants.

Electrical activity is widely used for assessing a plant's response to an injury or environmental stimulus. Commonly, a differential electrode recording between silver wire leads with the reference wire connected to the soil, or a part of the plant, is used. One method uses KCl-filled glass electrodes placed into the plant, similar to recording membrane/cell potentials in animal tissues. This method is more susceptible to artifacts of equipment noise and photoelectric effects than an impedance measure. An impedance measure using stainless steel wires is not as susceptible to electrically induced noises. Impedance measurements are able to detect injury in plants as well as exposure of the roots to environmental compounds (glutamate). The impedance measures were performed in 5 different plants (tomato, eggplant, pepper, liverwort, and Coleus scutellarioides), and responses to mechanical movement of the plant, as well as injury, were recorded. Monitoring electrical activity in a plant that arises in a distant plant was also demonstrated using the impedance method. The purpose of this report is to illustrate the ease in using impedance measures for monitoring electrical signals from individual plants or aggregates of plants for potentially scaling for high throughput and monitoring controlled culturing and outdoor field environments.

Measuring Electrical Responses during Acute Exposure of Roots and Rhizoids of Plants to Compounds Using a Flow-Through System.

Monitoring electrical signals in plants allows the examination of their acute and chronic physiological changes and responses to stimuli. Understanding how plant roots/rhizoids respond to chemical cues in their environment will provide insight into how these structures acquire resources. Chronic exposure to L-glutamate alters root growth and is known to alter Ca2+ flux inside roots. The ionic flux can be detected by electrical changes. A rapid and relatively easy approach is presented to screen the electrical sensitivity of roots/rhizoids to compounds such as amino acids and known agonists/antagonists to receptors and ion channels. The approach uses a background-flow system of basal salt or water; then, the administered compounds are added to the roots/rhizoids while monitoring their electrical responses. As a proof of concept, the response to flow-through of glutamate (1 mM) was targeted at the root/rhizoids of three plants (Arabidopsis thaliana, Pisum sativum and Marchantia inflexa). Both Arabidopsis thaliana and Pisum sativum produced rapid depolarization upon exposure to glutamate, while M. inflexa did not show an electrical response. In some experiments, simultaneous recordings with impedance measures for acute changes and glass electrodes for chronic electrical potential changes were used. The effect of potassium chloride (300 mM) as a depolarizing stimulus produced responses in both P. sativum and M. inflexa. The protocol presented can be used to screen various compounds in a relatively rapid manner for responsiveness by the roots/rhizoids of plants.

Molecular physiology of manganese in insects.

Manganese is an essential element for maintaining life. Overexposure to the metal, however, can be toxic to organisms. Given the significant function of manganese in insects, agriculture, and human disease, as well as in the healthy ecology of the planet, the biological activities of manganese in insects needs consideration. Because of the role of manganese as a cofactor for essential enzymes present in different organelles, both over and underexposure to manganese has a multifaceted effect on organisms. At the physiological level, the effects of insect exposure to the metal on enzymatic activities and consequent alteration of insect behaviors are best explained through the metal's role in modulating the dopaminergic system. Despite numerous examples that alterations in manganese homeostasis have profound effects on insects, the cellular mechanisms that ensure homeostasis of this essential metal remain presently unknown, calling for further research in this area.

Examining the effect of manganese on physiological processes: Invertebrate models.

Manganese (Mn2+ as MnSO4 &/or MnCl2) is a common and essential element for maintaining life in plants and animals and is found in soil, fresh waters and marine waters; however, over exposure is toxic to organisms. MnSO4 is added to soil for agricultural purposes and people are exposed to Mn2+ in the mining industry. Hypermanganesemia in mammals is associated with neurological issues mimicking Parkinson's disease (PD) and appears to target dopaminergic neural circuits. However, it also seems that hypermanganesemia can affect many aspects of health besides dopaminergic synapses. We examined the effect on development, behavior, survival, cardiac function, and glutamatergic synaptic transmission in the Drosophila melanogaster. In addition, we examined the effect of Mn2+ on a sensory proprioceptive organ and nerve conduction in a marine crustacean and synaptic transmission at glutamatergic neuromuscular junctions of freshwater crayfish. A dose-response effect of higher Mn2+ retards development, survival and cardiac function in larval Drosophila and survival in larvae and adults. MnSO4 as well as MnCl2 blocks stretch activated responses in primary proprioceptive neurons in a dose-response manner. Mn2+ blocks glutamatergic synaptic transmission in Drosophila as well as crayfish via presynaptic action. This study is relevant in demonstrating the effects of Mn2+ on various physiological functions in order to learn more about acute and long-term consequences Mn2+ exposure.

Rapid and Direct Action of Lipopolysaccharide (LPS) on Skeletal Muscle of Larval Drosophila.

The endotoxin lipopolysaccharide (LPS) from Gram-negative bacteria exerts a direct and rapid effect on tissues. While most attention is given to the downstream actions of the immune system in response to LPS, this study focuses on the direct actions of LPS on skeletal muscle in Drosophila melanogaster. It was noted in earlier studies that the membrane potential rapidly hyperpolarizes in a dose-dependent manner with exposure to LPS from Pseudomonas aeruginosa and Serratia marcescens. The response is transitory while exposed to LPS, and the effect does not appear to be due to calcium-activated potassium channels, activated nitric oxide synthase (NOS), or the opening of Cl- channels. The purpose of this study was to further investigate the mechanism of the hyperpolarization of the larval Drosophila muscle due to exposure of LPS using several different experimental paradigms. It appears this response is unlikely related to activation of the Na-K pump or Ca2+ influx. The unknown activation of a K+ efflux could be responsible. This will be an important factor to consider in treatments of bacterial septicemia and cellular energy demands.

Whakarongorau Aotearoa: insight into the delivery of New Zealand's national telehealth services.

Whakarongorau Aotearoa/New Zealand Telehealth Services, formerly known as Homecare Medical, is New Zealand's largest digital healthcare service. It originated as a house call doctor service about 20 years ago and now delivers free 24/7 telehealth services to the New Zealand public 365 days a year. Whakarongorau Aotearoa changed its name in April 2021 to reflect the growing kaupapa and was gifted this whakatauki: He reo marohirohi ka taringa rongohia-A brave voice deserves a listening ear. This viewpoint sets out to address a number of public and professional misconceptions about Whakarongorau Aotearoa and provide a more detailed description of the depth, breadth and complexity of the organisation, how it is structured, the range of services available to the public and its clinical governance, leadership and oversight.

Something Old, Something New, Something Borrowed, Something Taboo: Interaction and Creativity in Humour.

In this paper we treat humorous situations as a series of events underpinned by topoi, principles of reasoning recognised within a socio-cultural community. We claim that humorous effect in jokes and other discourse is often created by the juxtaposition of topoi evoked. A prerequisite for this is that there is a shift where the interpreter of the discourse updates their information state with regard to a second topos being evoked. This view of humour is consistent with an incremental analysis of dialogue, and we therefore argue that interaction is central both for humour creation and interpretation. We point out some different ways in which topoi are juxtaposed in humorous dialogues as well as in jokes published in social media or in joke books, and take jokes from the coronavirus pandemic as an example because this makes lots of new topoi available and therefore offers the opportunity of creating novel jokes based on the juxtaposition of the new and existing topoi. We explore how the mechanisms of inference in dialogue can be applied to humour through the four elements from our title: old (existing), new (not previously existing), borrowed (associated with a different situation) and taboo (inappropriate in the context).

Effect of Temperature on Heart Rate for Phaenicia sericata and Drosophila melanogaster with Altered Expression of the TrpA1 Receptors.

The transient receptor potential (TrpA-ankyrin) receptor has been linked to pathological conditions in cardiac function in mammals. To better understand the function of the TrpA1 in regulation of the heart, a Drosophila melanogaster model was used to express TrpA1 in heart and body wall muscles. Heartbeat of in intact larvae as well as hearts in situ, devoid of hormonal and neural input, indicate that strong over-expression of TrpA1 in larvae at 30 or 37 °C stopped the heart from beating, but in a diastolic state. Cardiac function recovered upon cooling after short exposure to high temperature. Parental control larvae (UAS-TrpA1) increased heart rate transiently at 30 and 37 °C but slowed at 37 °C within 3 min for in-situ preparations, while in-vivo larvae maintained a constant heart rate. The in-situ preparations maintained an elevated rate at 30 °C. The heartbeat in the TrpA1-expressing strains could not be revived at 37 °C with serotonin. Thus, TrpA1 activation may have allowed enough Ca2+ influx to activate K(Ca) channels into a form of diastolic stasis. TrpA1 activation in body wall muscle confirmed a depolarization of membrane. In contrast, blowfly Phaenicia sericata larvae increased heartbeat at 30 and 37 °C, demonstrating greater cardiac thermotolerance.