Stacia B. Moffett

The anterior stomach of larval mosquitoes (Aedes aegypti): effects of neuropeptides on transepithelial ion transport and muscular motility

SUMMARY The present investigation studied the influence of a number of neuropeptides on semi-open preparations of the isolated and perfused anterior stomach of larval Aedes aegypti. Effects of peptides were observed on the lumen negative transepithelial voltage (Vte) that is present with serotonin in the bath; this voltage most likely reflects active HCO3– secretion involved in alkalization of the larval anterior stomach. The five different A. aegypti allatostatins (allatostatin A 1–5) all affected Vte in almost identical ways, causing a 10–15% reduction of the voltage at 10–7 mol l–1. A. aegypti neuropeptide F and proctolin reduced Vte at submicromolar concentrations. At 10–6 mol l–1, neuropeptide F reduced Vte by 30% and proctolin reduced Vte by 50%. In contrast, A. aegypti allatotropin, A. aegypti head peptides I and III and A. aegypti short neuropeptide F were without effect on Vte. During the investigation it was observed that the peristaltic contractions of the preparations caused a dynamic component of Vte. Peristaltic contractions and the correlated voltage fluctuations depended on the presence of serotonin. Peristaltic activity and Vte deflections were progressively inhibited by A. aegypti head peptides I and III by A. aegypti short neuropeptide F and by A. aegypti neuropeptide F when the peptide concentrations were increased from 10–8 to 10–6 mol l–1. These observations show that physiological concentrations of some of the tested neuropeptides affect two processes that require coordination: ion transport and motility of the larval anterior stomach.

Neural regeneration in gastropod molluscs.

Snails recover function following a variety of neural injuries. They grow new tentacles with associated tentacle ganglia, selectively reinnervate peripheral targets, repair central connections and may even replace lost neurons and ganglia. The plasticity revealed in their responses to neural injury is an extreme expression of the adaptability observed in studies of learning and age-related changes in the nervous system. Recent information on neurogenesis in gastropods is providing a basis for comparing developmental events with neural regeneration. Studies of neural regeneration in gastropods have capitalized on our ability to identify many gastropod neurons individually and characterize the cellular properties and network properties that generate output patterns that underlie behaviors. The robustness of the model systems formed by cultured gastropod neurons is apparent in the similarity of the activity patterns in circuits formed in vitro and in vivo. Cell membrane repair, activation of an altered pattern of protein synthesis, and observation of the searching action of the growth cones can be studied under defined conditions that promote or inhibit the processes. Basic properties of growth cones, the molecular binding and second messenger systems underlying adhesion, sprouting and pathfinding, and events in synaptogenesis are accessible to analysis. Rules that govern selection of synaptic partners are being evaluated on the basis of cellular characteristics such as transmitter and receptor expression and ganglion of origin. The conservation of the molecular language that governs growth and communication between cells suggest that information gained in such studies may some day be applied to promote neural regeneration in mammals.

Intracellular K+ activities and cell membrane potentials in a K+-transporting epithelium, the midgut of tobacco hornoworm (Manduca sexta)

SummaryTransbasal electrical potential (Vb) and intraepithelial potassium chemical activity ((K+)i) were measured in isolated midgut epithelium of tobacco hornworm (Manduca sexta) using double-barrelled glass microelectrodes. Values ofVb ranging from +8 to −48 mV (relative to blood side) were recorded. For all sites, (K+)i is within a few millivolts of electrochemical equilibrium with the blood side bathing solution. Sites more negative than −20 mV show relatively high sensitivity ofVb to changes in blood side K+ concentration: 43% of these sites can be marked successfully with iontophoresed Lucifer yellow CH dye and shown to represent epithelial cells of all three types present in the midgut. In about half of successful marks, “dye-coupling” of several adjacent cells is seen. Low potential sites — those withVb less negative than −20 mV —typically do not show high sensitivity ofVb to changes of external K+, but rather (K+)i rapidly approaches the K+ activity of blood side bathing solution. These sites can seldom be marked with Lucifer yellow (4% success). The mean (K+)i of the high potential sites is 95±29 (sd)mm under standard conditions, a value which is in accord with published values for the whole tissue.

Alkalinization in the isolated and perfused anterior midgut of the larval mosquito, Aedes aegypti.

Abstract In the present study, isolated midguts of larval Aedes aegypti L. (Diptera: Culicidae) were mounted on perfusion pipettes and bathed in high buffer mosquito saline. With low buffer perfusion saline, containing m-cresol purple, transepithelial voltage was monitored and luminal alkalinization became visible through color changes of m-cresol purple after perfusion stop. Lumen negative voltage and alkalinization depended on metabolic energy and were stimulated in the presence of serotonin (0.2 µmol l-1). In some experiments a pH microelectrode in the lumen recorded pH values up to 10 within minutes after perfusion stop. The V-ATPase inhibitor concanamycin (50 µmol l-1) on the hemolymph side almost abolished Vte and inhibited luminal alkalinization. The carbonic anhydrase inhibitor, methazolamide (50 µmol l-1), on either the luminal or hemolymph-side, or the inhibitor of anion transport, DIDS (1 mmol l-1) on the luminal side, had no effect on Vte or alkalinization. Cl- substitution in the lumen or on both sides of the tissue affected Vte, but the color change of m-cresol purple was unchanged from control conditions. Hemolymph-side Na+ substitution or addition of the Na+/H+ exchange inhibitor, amiloride (200 µmol l-1), reduced Vte and luminal alkalinization. Luminal amiloride (200 µmol l-1) was without effects on Vte or alkalinization. High K+ (60 mmol l-1) in the lumen reduced Vte without affecting alkalinization. These results indicate that strong luminal alkalinization in isolated and perfused anterior midgut of larval A. aegypti depends on basolateral V-ATPase, but is apparently independent of carbonic anhydrase, apical Cl-/HCO3- exchange or apical K+/2H+ antiport.

Cellular mechanisms of acid secretion in the posterior midgut of the larval mosquito (Aedes aegypti).

SUMMARY The gut contents of larval mosquitoes are alkalinized by the anterior midgut and reacidified by the posterior midgut. In the present study the cellular mechanisms of reacidification were studied in isolated, perfused posterior midgut by measuring the transepithelial voltage (Vte) and the rate of acid secretion as indicated by the color change of m-cresol purple during intervals of perfusion stop. The lumen-positive Vte and reacidification were significantly increased by serotonin (0.2 μmol l−1). The V-type H+-ATPase inhibitor concanamycin A (10 μmol l−1) on the luminal side inhibited acidification and decreased Vte. On the hemolymph side the carbonic anhydrase (CA) inhibitor acetazolamide (1 mmol l−1) almost abolished Vte, but had no effect on acidification. Similarly, hemolymph-side DIDS (0.1 mmol l−1), DPC (0.5 mmol l−1), amiloride (1 mmol l−1) and ouabain (2.5 mmol l−1) significantly reduced Vte, whereas Ba2+ (5 mmol l−1) was without effect. DPC and amiloride also reduced Vte when applied to the luminal side of the epithelium. Unilateral substitution of gluconate for Cl− affected Vte in a way consistent with a greater permeability for Cl− versus Na+. Cl− replacement in the lumen decreased Vte, whereas replacement on the hemolymph side increased it. Bilateral replacement left the control voltage unaffected. Na+ replacement on either side of the tissue reduced Vte to different degrees. Omission of luminal amino acids was followed by a significant decrease in Vte. Except for concanamycin A, none of the above manipulations impaired acidification, indicating that acidification requires only the apical proton pump. However, the chemical source of secreted H+ is still unknown and needs to be investigated.

Neuronal events underlying rhythmic behaviors in invertebrates

Abstract 1. 1. Neuronal mechanisms underlying rhythmic behaviors: cockroach walking, ventilatory movements, food-grinding movements and heartbeat in crustaceans, leech swimming, gastropod feeding and swimming and bivalve burrowing are discussed. 2. 2. The oscillators which set the repeat cycle timing for these behaviors are treated under two major categories, endogenous oscillators and connectivity oscillators. Behaviors that possess elements of both types of oscillator are discussed. 3. 3. Strategies that have been used in analyzing oscillator mechanisms are described.

Regeneration as an application of gastropod neural plasticity.

Gastropod research is providing many insights into mechanisms of neural regeneration. These observations were made possible by the pioneering work of individuals who described the nervous systems of gastropods, mapped prominent neurons and determined their roles and connections, and developed the techniques for culturing them. This information has allowed questions about injury responses, target selection, and pathway cues to be explored at the level of individually identified neurons. Because of gastropod studies, more is known about axon sealing, growth cone formation and behavior, signals that travel from the site of axotomy to the soma, and the second messengers that are activated there. The responses in neurons and non‐neuronal cells during neural development and injury are coordinated by chemical messenger systems that are highly conserved, including neurotransmitters, cytokines, and neurotrophins. The nervous system is modified in learning paradigms by some of the same messenger systems activated by injury, because learning and injury both challenge neurons to change. The conservation of basic mechanisms that coordinate neuronal plasticity allows us to approach basic questions in relatively simple nervous systems with reasonable confidence that the findings will be relevant for other nervous systems, including possible applications to the mammalian nervous system. Microsc. Res. Tech. 49:579–588, 2000.

The common inhibitor innervates muscles proximal to the autotomy fracture plane in Carcinus maenas

The neuron common to proximal leg muscles of the crab Carcinus maenas projects to the propus opener nerve. Inhibitory effects include hyperpolarizing postsynaptic potentials, prolonged muscle fiber membrane hyperpolarization upon repetitive stimulation and suppression of force development by excitatory motorneurons of the anterior levator. The soma of the common inhibitor of fifth legs is located ventrally along the ganglion midline, contralateral to its axonal projections.

Strong Alkalinization in the Anterior Midgut of Larval Yellow Fever Mosquitoes (Aedes aegypti): Involvement of Luminal Na + /K + - ATPase

Recently, Na(+)/K(+)-ATPase has been detected in the luminal membrane of the anterior midgut of larval yellow fever mosquitoes (Aedes aegypti) with immunohistochemical techniques. In this study, the possible involvement of this ATPase in strong alkalinization was investigated on the level of whole larvae, isolated and perfused midgut preparations and on the molecular level of the Na(+)/K(+)-ATPase protein. Ouabain (5 mM) did not inhibit the capability of intact larval mosquitoes to alkalinize their anterior midgut. Also in isolated and perfused midgut preparations the perfusion of the lumen with ouabain (5 mM) did not result in a significant change of the transepithelial voltage or the capacity of luminal alkalinization. Na(+)/K(+)-ATPase activity was completely abolished when KCl was substituted with choline chloride, suggesting that the enzyme cannot act as an ATP-driven Na(+)/H(+)-exchanger. Altogether the results of the present investigation indicate that apical Na(+)/K(+)-ATPase is not of direct importance for strong luminal alkalinization in the anterior midgut of larval yellow fever mosquitoes.

Muscle Phenotype Remains Unaltered After Limb Autotomy and Unloading

Loss of chelipeds in crustaceans results in severe atrophy of the major muscle responsible for lifting the limb, the anterior levator. We decided to test if this loss of mechanical load altered muscle phenotype as measured by SDS-PAGE analysis of levator total protein and actomyosin fractions. Levator muscles of adult crayfish, Procambarus clarkii, with either functional regenerate limbs or lack of limb buds (papilla stage) were compared with those from normal contralateral limbs and those from pristine animals. We find that there is no difference in protein profiles among the three conditions. However, the total protein profile for the dually excited levator muscle is unique compared to those of fast or slow muscles of the abdomen (L and SEL, respectively), which receive only phasic or tonic excitatory innervation. The levator myosin heavy chain profile is similar to that of slow phenotype muscles such as the SEL and opener. We conclude that load does not influence levator phenotype. This is likely due either to the intact innervation and continued activation of the levator during atrophy or to the maintenance of passive tension on the muscle. J. Exp. Zool. 289:10-22, 2001.