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).

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.

Effects of bacterial endotoxin on regulation of the heart, a sensory-CNS-motor nerve circuit and neuromuscular junctions: Crustacean model.

Eatable crustaceans are susceptible to bacterial septicemia from injury or a compromised immune defense, which can possibly have detrimental effects in mammals that consume them. Since many crustaceans (i.e., crabs, lobsters and crayfish) are used for animal food and human consumption, it is of interest to understand the effects potential bacterial infections can have on their health as well as ours, including effects on cardiovascular and neuromuscular activities. The Red Swamp crayfish (Procambarus clarkii) was used as a model crustacean to investigate the effect of direct exposure to isolated endotoxin lipopolysaccharide (LPS) and the associated peptidoglycans from gram-negative bacteria (Serratia marcescens). S. marcescens is a common strain identified to cause septicemia in mammals and is prevalently found in nature. LPS injection into the hemolymph of crayfish revealed acute changes in heart rate and effects on survival. Direct LPS exposure on an in situ sensory-CNS-motor circuit produces a decrease in recruiting of the motor nerve at 500 µg/ml but has no significant effect at 100 µg/ml. At the isolated neuromuscular junction, the direct action of the LPS endotoxin (500 µg/ml) enhances evoked synaptic transmission, while not altering facilitation. Also, the amplitude and the frequency of spontaneous vesicle fusion events was not altered by LPS exposure. However, the resting membrane potential of the muscle transiently hyperpolarizes. These direct actions on tissues appear to be independent of innate immune responses and suggest the LPS targets on these tissues have a role in excitability of cellular function. {242 words}.

Using optogenetics to assess neuroendocrine modulation of heart rate in Drosophila melanogaster larvae.

The Drosophila melanogaster heart has become a principal model in which to study cardiac physiology and development. While the morphology of the heart in Drosophila and mammals is different, many of the molecular mechanisms that underlie heart development and function are similar and function can be assessed by similar physiological measurements, such as cardiac output, rate, and time in systole or diastole. Here, we have utilized an intact, optogenetic approach to assess the neural influence on heart rate in the third instar larvae. To simulate the release of modulators from the nervous system in response to environmental influences, we have directed expression of channel-rhodopsin variants to targeted neuronal populations to assess the role of these neural ensembles in directing release of modulators that may affect heart rate in vivo. Our observations show that the activation of targeted neurons, including cholinergic, dopaminergic, and serotonergic neurons, stimulate the release of cardioactive substances that increase heart rate after the initial activation at both room temperature and in a cold environment. This parallels previous studies suggesting these modulators play a crucial role in altering heart rate when applied to exposed hearts and adds to our understanding of chemical modulation of heart rate in intact Drosophila larvae.

Haddenham easywrap as part of self-management in lymphoedema and lipoedema: The patient perspective.

Self-management and the use of adjustable velcro compression wraps are not new concepts and quite often both can form part of the maintenance phase of treatment in those with lymphoedema or lipoedema, as well as those diseases in which compression therapy is advised as long-term management. The aim of this article is to identify some aspects that contribute to effective self-management and how the use of easywrap adjustable velcro compression wraps have improved quality of life for those with lymphoedema, chronic oedema and lipoedema. Case studies are given from patients to demonstrate the individual experience of living with lymphoedema or lipoedema, how this has impacted on daily life, and how using easywrap has helped as part of self-management.

Modulatory Action by the Serotonergic System: Behavior and Neurophysiology in Drosophila melanogaster.

Serotonin modulates various physiological processes and behaviors. This study investigates the role of 5-HT in locomotion and feeding behaviors as well as in modulation of sensory-motor circuits. The 5-HT biosynthesis was dysregulated by feeding Drosophila larvae 5-HT, a 5-HT precursor, or an inhibitor of tryptophan hydroxylase during early stages of development. The effects of feeding fluoxetine, a selective serotonin reuptake inhibitor, during early second instars were also examined. 5-HT receptor subtypes were manipulated using RNA interference mediated knockdown and 5-HT receptor insertional mutations. Moreover, synaptic transmission at 5-HT neurons was blocked or enhanced in both larvae and adult flies. The results demonstrate that disruption of components within the 5-HT system significantly impairs locomotion and feeding behaviors in larvae. Acute activation of 5-HT neurons disrupts normal locomotion activity in adult flies. To determine which 5-HT receptor subtype modulates the evoked sensory-motor activity, pharmacological agents were used. In addition, the activity of 5-HT neurons was enhanced by expressing and activating TrpA1 channels or channelrhodopsin-2 while recording the evoked excitatory postsynaptic potentials (EPSPs) in muscle fibers. 5-HT2 receptor activation mediates a modulatory role in a sensory-motor circuit, and the activation of 5-HT neurons can suppress the neural circuit activity, while fluoxetine can significantly decrease the sensory-motor activity.

Managing chronic oedema in a patient with arterial disease and leg ulceration.

Treating lymphoedema in patients with critical arterial disease can be contraindicated. This case study describes current methods of managing lymphoedema in a patient with arterial disease and leg ulcers. The patient, a 65-year-old male, had paraplegia and lower-limb lymphoedema with leg ulceration for 18 years, as well as arterial disease. The patient was referred to the lymphoedema/vascular service in 2013. Duplex ultrasound indicated superficial femoral occlusion. The arterial disease was treated with an angiogram and angioplasty, and when the blood supply was improved, the lymphoedema was treated. Emphasis was placed on self-care and reducing the need for community nurse involvement. Selfcare included compression bandaging, use of FarrowWrap, low-level light therapy, and ulcer dressings. Outcomes were measured using a telemedicine software programme. The patient's lymphoedema was reduced, leg ulcers healed, and quality of life transformed.

Analysis of various physiological salines for heart rate, CNS function, and synaptic transmission at neuromuscular junctions in Drosophila melanogaster larvae.

Drosophila serves as a playground for examining the effects of genetic mutations on development, physiological function and behavior. Many physiological measures that address the effects of mutations require semi-intact or cultured preparations. To obtain consistent physiological recordings, cellular function needs to remain viable. Numerous physiological salines have been developed for fly preparations, with emphasis on nervous system viability. The commonly used saline drifts in pH and will cause an alteration in the heart rate. We identify a saline that maintains a stable pH and physiological function in the larval heart, skeletal neuromuscular junction, and ventral nerve cord preparations. Using these common assays, we screened various pH buffers of differing concentrations to identify optimum conditions. Buffers at 25 mM produce a stable heart rate with minimal variation in pH. Excitatory junction potentials evoked directly on larval muscles or through sensory-CNS-motor circuits were unaffected by at buffers at 25 mM. The salines examined did not impede the modulatory effect of serotonin on heart rate or neural activity. Together, our results indicate that the higher buffer concentrations needed to stabilize pH in HL3 hemolymph-like saline do not interfere with the acute function of neurons or cardiac myocytes.

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.

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).

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.

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.

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.

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.

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.