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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 (V(te)) and the rate of acid secretion as indicated by the color change of m-cresol purple during intervals of perfusion stop. The lumen-positive V(te) and reacidification were significantly increased by serotonin (0.2 mumol l(-1)). The V-type H(+)-ATPase inhibitor concanamycin A (10 mumol l(-1)) on the luminal side inhibited acidification and decreased V(te). On the hemolymph side the carbonic anhydrase (CA) inhibitor acetazolamide (1 mmol l(-1)) almost abolished V(te), 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 V(te), whereas Ba(2+) (5 mmol l(-1)) was without effect. DPC and amiloride also reduced V(te) when applied to the luminal side of the epithelium. Unilateral substitution of gluconate for Cl(-) affected V(te) in a way consistent with a greater permeability for Cl(-) versus Na(+). Cl(-) replacement in the lumen decreased V(te), whereas replacement on the hemolymph side increased it. Bilateral replacement left the control voltage unaffected. Na(+) replacement on either side of the tissue reduced V(te) to different degrees. Omission of luminal amino acids was followed by a significant decrease in V(te). 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.

Isolated anterior midguts of larval Aedes aegypti were bathed in aerated mosquito saline containing serotonin (0.2 micromol L(-1)) and perfused with NaCl (100 mmol L(-1)). The lumen negative transepithelial voltage (V(te)) was measured and luminal alkalinization was determined through the color change of luminal m-cresol purple from yellow to purple after luminal perfusion stops. Addition of 10 mmol L(-1) amino acids (arginine, glutamine, histidine or proline) or dicarboxylic acids (malate or succinate) to the luminal perfusate resulted in more negative V(te) values, whereas addition of glucose was without effect. In the presence of TRIS chloride as luminal perfusate, addition of nutrients did not change V(te). These results are consistent with Na(+)-dependent absorption of amino acids and dicarboxylic acids. Effects of serotonin withdrawal indicated that nutrient absorption is stimulated by this hormone. Strong luminal alkalinization was observed with mosquito saline containing serotonin on the hemolymph-side and 100 mmol L(-1) NaCl in the lumen, indicating that alkalinization does not depend on luminal nutrients. Omission of glucose or dicarboxylic acids from the hemolymph-side solution had no effect on luminal alkalinization, whereas omission of amino acids significantly decelerated it. Re-addition of amino acids restored alkalinization, suggesting the involvement of amino acid metabolism in luminal alkalinization.

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.

These studies focus on the pupal Aedes aegypti midgut muscularis for the first 26 h following larval-pupal transition. The midgut muscularis of Ae. aegypti pupae during this first half of the pupal stadium is a grid of both circularly and longitudinally oriented muscle bands, arranged in a manner resembling that of the larvae. While many muscle bands exhibit signs of degeneration during the time period studied, not all bands degrade, nor is this degradation simultaneous. Band deterioration involves destruction of internal elements while the muscle fiber plasma membrane remains intact. Deterioration of contractile elements may involve proteosome-like structures and associated enzymes. Many features of the larval muscularis including cruciform cells, bifurcating circular bands, and bifurcating longitudinal bands of muscle are retained during the time period investigated. Neuromuscular junctions along some muscle bands are retained through at least 16 h into the pupal stadium. The selective nature of muscle fiber degradation, coupled with the retention of larval features and neural input, may allow for limited functionality of the muscularis during metamorphosis. Evidence of sexual dimorphism in the midgut muscularis of male and female Ae. aegypti pupae was not observed during the time period studied.

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 micromol 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 micromol l(-1)) on the hemolymph side almost abolished V(te) and inhibited luminal alkalinization. The carbonic anhydrase inhibitor, methazolamide (50 micromol 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 V(te) or alkalinization. Cl(-) substitution in the lumen or on both sides of the tissue affected V(te), 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 micromol l(-1)), reduced V(te) and luminal alkalinization. Luminal amiloride (200 micromol l(-1)) was without effects on V(te) or alkalinization. High K(+) (60 mmol l(-1)) in the lumen reduced V(te) 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(-)/HCO(3)(-) exchange or apical K(+)/2H(+) antiport.

The midgut muscularis of larvae of the mosquito Aedes aegypti takes the form of a grid of longitudinal and circular muscle bands. The longitudinal and circular bands overlap at near right angles at many areas of intersection. The longitudinal bands run the length of the midgut. However, some bands of circular muscle, located in the anterior midgut, pass only partway around the gut. An unusual feature was observed at some regions where longitudinal and circular bands of muscle intersect: filaments oriented at near right angles to one another were present in the same membrane-bound fiber. These cruciform regions send contractile elements into both circular and longitudinal bands. The muscularis was fixed in a contracted state, so most of the sarcomeres are represented by complete overlap of myosin and lighter staining actin filaments. Features characteristic of supercontracting muscle, including perforated Z-lines, were seen in sarcomeres of circular muscle bands. Small invaginations resembling transverse tubules were present but a sarcoplasmic reticulum was not observed. While occasional cells that may be neurons or neurosecretory cells were observed, a network that might serve to coordinate the segmentation and peristaltic movement of the muscularis was not apparent.

The anterior stomach of larval Aedes aegypti was isolated and perfused via two pipettes. For transepithelial voltage (V(te)) measurement, the inflow pipette and the bath were connected via agar bridges to calomel electrodes. For voltage-clamping, the lumen of the tissue contained an Ag/AgCl wire held by the outflow pipette, and the preparation was placed in a bath within a spiral of Ag/AgCl wire. After equilibrating the tissue in mosquito saline on both sides, a V(te) of -8+/-1 mV was measured (+/-S.E.M., N=32). Current-voltage curves (+/-100 mV) demonstrated ohmic behaviour of the epithelium. Short-circuiting resulted in a current (I(sc)) of 103+/-16 microA cm(-2) and a mean transepithelial conductance (G(te)) of 11.8+/-1.3 mS cm(-2) (+/-S.E.M., N=32). A Yonath-Civan plot of G(te) of individual preparations over the corresponding I(sc) resulted in a straight line (r(2)=0.8422), indicating that the difference in I(sc) of individual preparations is mainly based on different transcellular conductances (G(c)). This analysis allowed to estimate the mean leak conductance (G(l) approximately 3.9 mS cm(-2)) and the mean transcellular electromotive force (E(c) approximately 13 mV). After administering 0.2 micromol L(-1) serotonin, I(sc) and G(te) significantly increased, to 457+/-49 microA cm(-2) and to 21.3+/-2.3 mS cm(-2) (+/-S.E.M., N=31, P<0.05), respectively. The Yonath-Civan plot after serotonin resulted again in a straight line (r(2)=0.8219), indicating a mean G(l) of about 1 mS cm(-2) and a mean E(c) of about 22 mV. Dinitrophenol (2.5 mmol L(-1)) almost abolished I(sc) and significantly reduced G(te) (N=6). Concanamycin A (100 micromol L(-1)) reduced I(sc) by more than 90% without significantly affecting G(te).

Analysis of larval Aedes aegypti midgut using scanning electron microscopy, nuclear and mitochondrial dyes, response to Bacillus thuringiensis israelensis CryIVB toxin, and electrophysiology is described. The anterior ventriculus ("stomach") region is found to have much lower mitochondrial densities than other midgut regions. The transitional region is distinguished by apical surface architecture, and by region-specific effects of CryIVB endotoxin. In this region CryIVB causes holes ranging from 1.0 to 7.0 microm in diameter (mean 3.3+/-0.53 microm, N=12), blisters 16.9+/-1.54 microm in diameter (N=10), and separation of adjacent cells. The holes are not consistent with damage due to the colloid osmotic lysis model of delta-endotoxin activity. The posterior ventriculus possesses a distinctive cellular architecture consisting of hemispherical, domed apical membranes surrounded by deep clefts. Functional and morphological heterogeneity is revealed within the posterior ventriculus, with the anterior end dominating the electrical profile of isolated, perfused preparations and showing the greatest response to serotonin. Hyperpolarization of the transepithelial potential by serotonin occurred in conjunction with a decrease in the space constant lambda, ruling out closure of ion channels as the mechanism of action of serotonin.

In all life stages, the gut of the mosquito is innervated by a small number (typically 4) of central neurons immunoreactive to serotonin (SI). The serotonergic system appears to pass through metamorphosis largely intact, despite extensive remodeling of the gut. Axons immunoreactive to antibodies raised against molluscan FMRFamide (RF-I) constitute peptidergic innervation that anatomically parallels the serotonergic system. In the larva, two clusters of 3 neurons project to the anterior regions of the gut, whereas in the pupa and adult, typically two large RF-I neurons located next to the esophagus send several processes posteriorly. In adults, these neurons branch throughout the diverticula and anterior stomach. In pupae, but not in larvae or adults, the gut RF-l system coexpresses reactivity to antibodies raised against a member of another peptide family, molluscan small cardioactive peptide b (SCP-I). SCP-I immunoreactivity is localized independently of RF-l immunoreactivity in the ganglia of all stages and in neurons that project along the gut of the adult. We did not find any colocalization of S-I and the peptide markers. Distinct populations of enteroendocrine cells populate different regions of the gut at different life stages. Changes in staining pattern suggest that these cells are replaced at metamorphosis along with the other gut cells during the extensive remodeling of the tract. Distributed in the gut epithelium are subpopulations that express either RF-I or SCP-I; a small fraction of these cells bind antibodies to both peptides. The stomachs of adult females are larger than those of males, and the numbers of SCP-I and RF-I enteroendocrine cells are proportionately greater in females. In all the life stages, the junctions between different regions of the gut are the focus of regulatory input. The larval cardiac valve possesses a ring of cells, the necklace cells, which appear to receive extensive synaptic inputs from both the serotonergic system and the peptidergic system. Another focus of control is the pyloric valve, which is encircled by axon-like processes. The immunoreactive pattern of this region differs across life stages, expressing SCP-I in larvae, S-I in pupae, and both SCP-I and RF-I in adults.

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.

The lumen-negative transepithelial voltage (V(te)) of the isolated and perfused anterior stomach of mosquito larvae (Aedes aegypti) was studied with a 'semi-open' preparation in which one end of the gut was ligated onto a perfusion pipette and the other end remained open to the bath. All experiments were performed with serotonin-stimulated preparations. V(te) was abolished after addition of 2.5 mmol l(-1) dinitrophenol and depended on the presence of Cl(-). Na(+) substitution experiments showed that a major part of V(te) depended on the presence of this cation in the hemolymph side of the epithelium. Addition of 10 micro mol l(-1) concanamycin (78+/-6% inhibition) or 2.5 mmol l(-1) ouabain (15+/-2% inhibition) to the bath partially inhibited V(te). DPC (0.5 mmol l(-1)) or DIDS (0.1 mmol l(-1)) reduced V(te) when applied to the hemolymph side of the epithelium (to 49+/-8% or 78+/-3% of the control, respectively). When present on both sides of the epithelium, these inhibitors caused further V(te) reductions (to 23+/-4% or 35+/-4% of the control, respectively). Hemolymph-side furosemide (0.1 mmol l(-1)) or BaCl(2) (5 mmol l(-1)) reduced V(te) by 13+/-3% or 23+/-4% of the control, respectively. When applied to the hemolymph side of the epithelium, amiloride (0.2 mmol l(-1)) significantly decreased V(te) by 35+/-6% of the control, whereas the drug caused no further effect when it was subsequently also applied to the luminal side of the epithelium. The above results are the basis for an extended model for the cellular mechanisms of NaHCO(3) secretion/HCl absorption involved in alkalization of the anterior stomach of mosquito larvae.

Immunohistochemical and ultrastructural techniques were used to study sequelae of nerve injury in the pulmonate snail Melampus bidentatus. Either pedal or tentacle nerves were crushed, severing all axons, and recovery was monitored over 15 days. The axons regenerated from the segment attached to the soma, with no evidence of fusion of proximal and distal segments. The medium to large axons of central neurons, including those monitored with serotonin immunohistochemistry, grow distally across the path of smaller axons extending centrally from peripheral somata. The regions into which the growing axons projected were a focus of phagocytic activity. Cells previously labeled by PKH-26PCL, a fluorescent marker for phagocytic activity, were attracted to the crushed nerve within 6 h and were a consistent feature in the vicinity of the injury for at least 9 days, gradually extending their range as repair progressed in both directions from the crush. Repair proceeded within an intact sheath, and many sheath cells survived the crush, although the nuclear dye Hoechst 33258 revealed an initial distortion of their nuclei. The concentration of cells in the sheath in the crushed region increases after the crush, with the packing of nuclei peaking at 3 days and gradually returning to control conditions; this probably reflects migration of resident sheath cells. Cell division is rare in the sheath of intact nerves, but labeling with bromodeoxyuridine increases at the crush site between 4 and 9 days, indicating that cell replacement also occurs at the site.

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., (Copyright 2001 Wiley-Liss, Inc.)

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., (Copyright 2000 Wiley-Liss, Inc.)

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.

Previous anatomical observations have established that the penial complex of adult Melampus bidentatus is specifically reinnervated and that the penial complex itself can regenerate. This review describes experiments to determine whether a reinnervated penial complex and a regenerated penial complex can function in mating. The snails are not self-fertile, so the ability to transfer sperm, evaluated by production of fertile eggs, was the test of successful regeneration. Snails with nerve transections produced fertile eggs within the time-span expected for axonal regeneration to the target organ. Penial complex ablation interfered with sperm transfer for two egg-laying cycles but fertile eggs were laid within a time-span consistent with regeneration of a penial complex.

1. The patterned neural activity that drives muscular locomotor movements in Melampus is generated within the central nervous system. 2. In the transition from quiescent state to crawling, the pattern recorded in nerves and connectives changes from short-duration bursts in many units to the 60-100 sec cycle of events recorded during tethered crawling in the semi-intact snail. 3. Extracellularly recorded bursts and individually recognizable spikes in pedal nerves are correlated with movements that occur at each stage of the cyclically repeated crawl-steps. 4. Intracellularly recorded pedal neurons involved in locomotion receive excitatory drive, inhibitory drive, or alternating excitatory and inhibitory drive during the step cycle.

1. When one pedal ganglion is removed, snails first crawl using the unoperated side of the foot, but in 4-8 weeks the operated side exhibits an anterior-to-posterior gradient of recovery. 2. A ganglion bud bridges the site of the missing ganglion and axons project from intact central ganglia into the foot. 3. Rhythmic activity in right and left pedal nerve pairs is correlated during locomotion in the regenerated snails. 4. The oscillator in the remaining pedal ganglion drives bilaterally coordinated activity. Regenerated projections from the cerebral ganglia through the bud to the remaining pedal ganglion suffice to initiate locomotion.

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.