Cell ratcheting through the Sbf RabGEF directs force balancing and stepped apical constriction.

During Drosophila melanogaster gastrulation, the invagination of the prospective mesoderm is driven by the pulsed constriction of apical surfaces. Here, we address the mechanisms by which the irreversibility of pulsed events is achieved while also permitting uniform epithelial behaviors to emerge. We use MSD-based analyses to identify contractile steps and find that when a trafficking pathway initiated by Sbf is disrupted, contractile steps become reversible. Sbf localizes to tubular, apical surfaces and associates with Rab35, where it promotes Rab GTP exchange. Interestingly, when Sbf/Rab35 function is compromised, the apical plasma membrane becomes deeply convoluted, and nonuniform cell behaviors begin to emerge. Consistent with this, Sbf/Rab35 appears to prefigure and organize the apical surface for efficient Myosin function. Finally, we show that Sbf/Rab35/CME directs the plasma membrane to Rab11 endosomes through a dynamic interaction with Rab5 endosomes. These results suggest that periodic ratcheting events shift excess membrane from cell apices into endosomal pathways to permit reshaping of actomyosin networks and the apical surface.

Serotonergic modulation of a voltage-gated calcium current in parapodial swim muscle from Aplysia brasiliana.

Here we describe the effects of serotonin (5-HT) on dissociated parapodial muscle fibers from Aplysia brasiliana. 5-HT has previously been implicated as a modulatory transmitter at the parapodial neuromuscular junction. Exogenously applied or endogenously released 5-HT increases the amplitude of motoneuron-induced excitatory junctional potentials and contractions in parapodial muscle. Exogenously applied 5 microM 5-HT increases the amplitude of a voltage-gated inward calcium current in isolated muscle fibers by an average of 42% in response to a voltage step from -70 to -10 mV. The amplitude of the inward current was increased at all voltages tested, with the peak increase occurring between -30 and -20 mV. The dihydropyridine calcium channel antagonist nifedipine (10 microM) blocked this effect of 5-HT. The data indicate that 5-HT increases a previously identified calcium current in parapodial muscle fibers that is similar to the vertebrate L-type current. Although several types of K+ channels exist in these fibers, including Ca(2+)-dependent K+ channels, the results suggest that 5-HT has little effect on these currents. Parapodial muscle contractions during swimming behavior occur in response to bursts of motoneuron action potentials that produce graded muscle depolarizations that occur over a 1- to 2-s period rather than being instantaneous or rapid responses as might be produced by one or two action potentials or a brief voltage step. With the use of 1-s voltage ramps, we attempted to mimic physiological depolarization and demonstrate that 5-HT is able to increase the amplitude of the inward calcium current. The data presented in this paper provide evidence that 5-HT increases the Ca2+ current, which may be one mechanism by which 5-HT modulates muscle contractions during swim behavior.

Parapodial swim muscle in Aplysia brasiliana. I. Voltage-gated membrane currents in isolated muscle fibers.

1. We describe voltage-gated membrane currents present in single muscle fibers dissociated from the parapodia (swim appendages) of the marine gastropod mollusk Aplysia brasiliana. These muscles are utilized in swimming behavior and their activity is modulated by serotonin. It is necessary to characterize the innate membrane properties of these fibers before defining the mechanism of action of serotonin in facilitating muscle fiber responses to motoneuron input. 2. Freshly dissociated parapodial muscle fibers appear by morphological criteria to be a uniform population with an average length of 240 microns and width of 15 microns. The average resting potential of all fibers is -56 mV and the fibers contract in response to elevated extracellular K+ concentration or intracellular depolarization. 3. Muscle membrane currents were studied by single-electrode voltage clamp with the use of intracellular microelectrodes. The muscle fibers were found to fall into one of two groups, which we have classified as type I and type II, the former having two voltage-gated outward K+ currents and a small, less frequently seen Ca2+ current. Type II fibers display the same two K+ currents, a prominent Ca2+ current and, in addition, two Ca(2+)-dependent K+ currents, the latter described in a companion paper. 4. Membrane currents were characterized using 1-s voltage ramps and several voltage step protocols, including ones for analyzing K+ tail currents. Both fiber types had similar current-voltage relationships and input resistance of > or = 60 - 300 M omega. The current-voltage curves were quite flat at potentials more negative than resting potential, with no evidence of a voltage-gated, inwardly rectified (anomalous) potassium current. Outward K+ currents and a Ca2+ current were seen to appear at a threshold of near -40 mV. 5. Because type I fibers had no apparent Ca(2+)-activated K+ currents, the two voltage-gated outward K+ currents were most conveniently studied in these fibers. Compared with type II fibers, type I fibers display a relatively slowly rising total outward current with depolarization comprised of a delayed rectifier current and a transient A current (IA). These two currents were distinguished by slightly different thresholds for activation, by inactivation properties of IA, and by their partially selective sensitivity to tetraethylammonium and 4-aminopyridine. 6. Although contraction of all parapodial muscle fibers is dependent on extracellular Ca2+, an inward Ca2+ current was detected in only about one third of type I fibers, and the current was small. A similar and more prominent Ca2+ current was observed in all type II fibers and was analyzed more fully in these cells. This current had an activation threshold near -40 mV and peaked between -10 and 0 mV. It displayed little inactivation with depolarization steps of 80-200 ms, was blocked in the absence of Ca2+ or in the presence of Co2+, and was present, although not enhanced, when Ba2+ was substituted for Ca2+. This current was completely blocked by the dihydropyridine nifedipine (10 microM), and is therefore similar to an L-type Ca2+ current. 7. The voltage-gated membrane currents described in parapodial muscle fibers provide a framework for analyzing possible mechanisms by which serotonin facilitates neuromuscular output. This facilitatory mechanism will provide a better understanding of the role of serotonin in controlling locomotion.

Parapodial swim muscle in Aplysia brasiliana. II. Ca(2+)-dependent K+ currents in isolated muscle fibers and their blockade by chloride substitutes.

1. We describe here the properties of two Ca(2+)-dependent K+ currents found in type II muscle fibers dissociated from the parapodia (swim appendages) of the marine snail Aplysia brasiliana. 2. Type II parapodial muscle fibers display three voltage-dependent currents that are also seen in type I fibers, a delayed rectifier current [IK(V)], a transient A current (IA), and a prominent L-type Ca2+ current. In addition, type II fibers also have two outward K+ currents, a transient, inactivating one and a slower, noninactivating one [IK(Ca,t) and IK(Ca,s), respectively], that are Ca2+ dependent. The expression of these currents in normal type II fibers generally produces a waveform of total outward current that is faster to peak than the total outward current seen in response to voltage steps in type I fibers and that does not inactivate at the end of an 80-ms voltage step. 3. Both IK(Ca,t) and IK(Ca,s) are absent when external Ca2+ is eliminated or when extracellular Ca2+ concentration ([Ca2+]o) is substituted with 10 mM Co2+ or Ba2+. Their threshold for activation is around -40 mV. IK(Ca,t) peaks rapidly and then inactivates, but IK(Ca,s) rises slowly and does not inactivate for as long as 200 ms. Both currents, like IK(V) and IA, are sensitive to tetraethylammonium and 4-aminopyridine and are not readily separated from either the voltage-gated currents or from one another by these pharmacological agents. 4. Tail current analysis from depolarized voltage steps in varying (K+]o demonstrates that these currents are carried by K+ ions and not by Cl-. 5. An unexpected finding, however, is that these Ca(2+)-dependent K+ currents are blocked by standard Cl- ion substitutes, such as methanesulfonate, isethionate, and propionate. IK(Ca,s) is slightly more sensitive to these Cl- substitutes than is IK(Ca,t). The chloride blocker 4,4'-diisothiocyantastilbene-2,2'disulfonic acid also partially blocked the Ca(2+)-dependent K+ currents. 6. The presence of these Ca(2+)-dependent K+ currents in type II fibers may contribute to a more rapid repolarization following depolarization-induced contractions. In contrast to type I fibers, which have smaller calcium current and no Ca(2+)-activated K+ currents, type II muscle cells may function more like "fast" fibers and relax more rapidly.

Identification and characterization of cerebral ganglion neurons that induce swimming and modulate swim-related pedal ganglion neurons in Aplysia brasiliana.

1. We have identified and characterized a family of several pairs of neurons in the cerebral ganglion of Aplysia brasiliana that are capable of inducing, maintaining, or modulating a motor program that underlies swim locomotion in this marine mollusk. We have operationally defined these cells as command neurons (CNs) for swimming. 2. The command cells occur in bilateral pairs in the cerebral ganglion and make direct and indirect outputs to neurons in the pedal ganglia, including motor neurons, a central pattern generator circuit, and modulatory neurons that enhance muscle contractions during swimming. Several of the CNs are sufficient individually to induce the swim motor program (SMP), all receive sensory feedback from the periphery, and several interconnect with other swim-related CNs. 3. Tonic discharges of approximately 10 Hz in CN types 1-3 (CN1-CN3) are capable of eliciting the oscillatory, phasic SMP as recorded in peripheral nerves that innervate the swim appendages, the parapodia. CN1, CN2, and CN3 make monosynaptic excitatory connections onto ipsilateral, contralateral, and bilateral pedal swim-modulatory neurons [parapodial opener-phase (POP) cells], respectively; and each command cell type activates the pedal central pattern generator (CPG), leading to sustained phasic output of motor neurons and POP cells. 4. Tonic firing of CN4 causes weak activation of the SMP contralaterally. These neurons occur as two pairs of neurons in each cerebral hemiganglion, with mutual electrical and chemical synaptic interconnections. CN4 cells also excite CN1 and CN2 cells. Thus CN4 is classified as a higher-order swim command cell type. 5. Command cells classified as types 5-8 (CN5-CN8), although not capable of inducing the SMP individually, nonetheless have strong synaptic connections with pedal POP cells and/or with other command neurons. These command cells may excite or inhibit follower cells on the same or opposite sides of the preparation and modulate the swim output. 6. All the command cells tested received strong input from mechanical stimulation, either stretch or pinching, of either parapodium. Mechanosensory input from the parapodia was shown to depend on the presence of the pedal ganglion, but not the pleural. Sensory stimulation activated command cells and motor neurons, but POP cells received input from sensory stimuli only through the cerebral ganglion, probably via command cells. The effects of applied mechanosensory stimuli could be entirely mimicked by motor neuron-induced contractions of the parapodia.

Neural control of swimming in Aplysia brasiliana. I. Innervation of parapodial muscle by pedal ganglion motoneurons.

1. Swimming is an oscillatory locomotor behavior in Aplysia accomplished by rhythmic undulating movements of the parapodia, winglike flaps that cover the dorsum of the body. As part of an analysis of the neural basis of this behavior, we have identified and characterized motoneurons in the pedal ganglia that directly innervate parapodial muscle and fire phasically during fictive swimming. 2. Parapodial musculature is organized into at least eight discrete layers. Fibers of adjacent layers are directed orthogonally. 3. Motoneurons were localized to the middle and rostral portions of the dorsal surface of each pedal ganglion by the use of backfill staining and intracellular dyes. These neurons were defined as motoneurons on the basis of additional physiological evidence for peripheral axons and their ability to cause excitatory junction potentials (EJPs; average amplitude, 2-5 mV) in muscle fibers and discrete contractions of parapodial muscles. Muscle fibers are polyneuronally innervated. Fibers had an average resting potential of -79 mV and no over-shooting action potentials. 4. There are probably at least 50 motoneurons. Their average resting potential was -48 mV, and they do not appear to be directly connected synaptically to one another. One identifiable motoneuron is described in detail. It participates in the opener (downstroke) phase of swimming and causes contraction of one of the described muscle layers. 5. Divalent ion concentrations were altered centrally and peripherally during motoneuron activity to demonstrate that the motoneurons directly innervate muscle fibers. Blockage of EJPs by hexamethonium and the presence of specific anticholinesterase staining in parapodial nerves and muscle fibers strongly suggest that many of the motoneurons are cholinergic. 6. Studies of excitation-contraction coupling showed that single or a few spikes in motoneurons rarely cause an EJP. Bursts of motoneuron spikes produced facilitating EJPs. With approximately 10 spikes in a 1-s motoneuron burst, adequate depolarization occurred in muscle fibers to initiate a small, slow contraction. Increased spike frequency led to greater depolarization, because of EJP summation, and larger contractions. Contraction requires depolarization of the muscle above a threshold, beyond which the force of contraction depends on both the duration and degree of depolarization. 7. Although dozens of motoneurons appear to be involved in the complex control of parapodial movements during swimming, preliminary evidence indicates that these neurons are probably not participating directly in the circuitry of the central pattern generator for swimming, which has been shown by others also to reside in the pedal ganglia.

Neural control of swimming in Aplysia brasiliana. II. Organization of pedal motoneurons and parapodial motor fields.

1. We have examined the locations and functional properties of a large number of motoneurons in the pedal ganglia of Aplysia brasiliana. These neurons control movement of the parapodia and body during swimming. We have grouped the motoneurons into classes based on several criteria, including the topology of the cells and their axons, the properties of their peripheral motor fields, and their phasic activity during an induced swim motor program. 2. A total of 410 motoneurons were analyzed. There are at least 16 distinguishable motor fields in the parapodia, based on the region affected, direction of contraction, and phase of neuronal activity during fictive swimming. 3. Motoneurons for each motor field tend to appear in the same region of the ganglion in different preparations. 4. Most motoneurons have only ipsilateral effects. About 1% cause contralateral contraction, and they project directly to the contralateral parapodium. 5. Three types of motoneuron are described that cause parapodial expansion. 6. Two other groups of motoneurons were found that innervate either the columellar muscle or longitudinal foot muscles. 7. Almost all motoneurons fired rhythmically during fictive swimming, including those controlling foot and columellar muscle.

Neural control of swimming in Aplysia brasiliana. III. Serotonergic modulatory neurons.

1. We describe a group of serotonergic neurons in the pedal ganglia of Aplysia brasiliana and characterize their modulatory effects on the motoneuron input to swimming muscles of the parapodia. Each pedal ganglion contains one cluster of large neurons near its dorsomedial surface that fires in phase with opening (downstroke) of the parapodia; these have been designated parapodial opener-phase (POP) cells. 2. POP cells are large, number 15-20 per ganglion, have peripheral axons in parapodial nerves, have distinctively shaped action potentials, and fire in bursts phasically with motoneurons during the opening, or downstroke portion, of parapodial movement during fictive swimming. Firing individual POP cells with intracellular current indicates that they have no direct detectable effect on muscle, causing neither junction potentials nor contractions. 3. 5,7-Dihydroxytryptamine (5,7-DHT) staining, immunocytochemistry using serotonin (5-HT) antibodies, and direct biochemical measurements revealed that POP cells are serotonergic. Serotonergic nerve endings were also seen in parapodial muscle. 4. Simultaneous intracellular recordings and use of altered divalent concentrations revealed that no detectable direct synaptic interactions exist between POP cells and motor neurons. 5. When POP cells and motoneurons were simultaneously recorded while measuring muscle contractions, it was found that POP cell activity enhances motoneuron-induced tension by 120-900%, averaging around 300%. Variability in the efficacy of individual POP cells suggests that they may influence specific regions or groups of muscle fibers. 6. POP cell activity also significantly increased the rate of relaxation of parapodial muscle contractions, averaging about a 40% reduction in the time required to relax to one-half peak tension. Increased relaxation rate implies a postsynaptic change in muscle behavior. 7. The effectiveness of POP cells to increase contraction tension and relaxation rate was positively correlated with POP cell spike frequency. These effects were slow (seconds) in onset and could persist for a minute or more after cessation of POP firing. Concurrent motoneuron activity is not required for modulation by POP cells. 8. Simultaneous intracellular recording from a POP cell, motoneuron, and muscle fiber revealed that POP cell activity enhanced the amplitude of motoneuron-induced excitatory junction potentials (EJPs). Activity of POP cells did not alter muscle fiber membrane potential, but the experiments left open the possibility that EJP enhancement is presynaptic, postsynaptic, or a combination.(ABSTRACT TRUNCATED AT 400 WORDS)

The bag cell egg-laying hormones of Aplysia brasiliana and Aplysia californica are identical.

Egg laying in the marine molluscan genus Aplysia is elicited by an egg-laying hormone (ELH) which induces ovulation and acts on central neurons to effect egg-laying behavior. ELH, isolated from the A. californica bag cells, and three ELH-related peptides, isolated from the A. californica atrial gland, have been chemically characterized, yet relatively little is known about homologous peptides in other Aplysia species. In these studies, the primary structure of A. brasiliana ELH was determined. Bag cell clusters were extracted in an acidic solution, and the peptides purified by sequential gel filtration and reversed-phase HPLC; ELH was identified by bioassay. Amino acid compositional and sequence analyses demonstrated that the neurohormone was a 36-residue peptide whose sequence was identical to that of A. californica ELH: NH2-Ile-Ser-Ile-Asn-Gln-Asp-Leu-Lys-Ala-Ile-Thr-Asp-Met-Leu-Leu-Thr-Glu- Gln-Ile- Arg-Glu-Arg-Gln-Arg-Tyr-Leu-Ala-Asp-Leu-Arg-Gln-Arg-Leu-Leu-Glu-Lys-COOH .

Effect of microwave heating of soybeans on protein quality.

The purpose of this study was to determine the protein quality of microwave cooked soybeans by the rat growth and protein efficiency ratio method (PER). It was found that properly cooked, dry, hulled, whole soybean seeds had a PER of 2.4 +/- .06 (mean +/- standard error) and a mean weekly weight gain of 21.2 +/- 1.1 which were equivalent to 2.53 +/- .10 and 18.3 +/- 1.0 g for casein, respectively. These data demonstrate the value of microwave cooked soybeans and suggest further research on the possible economical and biological advantages of microwave cooked soybeans.

Effect of microwave heating of soybeans on protein quality.

El proposito de este estudio fue determinar la calidad de la proteina del frijol de soya cocido por microonda, usando los metodos de crecimiento y del indice de eficiencia proteinica (PER) en ratas. Se encontro que la coccion adecuada de frijol de soya en su estado seco y entero tenia un PER de 2.40 mas o menos .06 (promedio mas o menos error estandar) y un promedio de crecimiento de 21.2 mas o menos 1.1 g, los cuales son equivalentes a 2.53 mas o menos .10 y 18.3 mas o menos 1.0 g respectivamente, para la caseina. Estos datos demuestran el valor del frijol de soya cocido por microondas y, ademas, sugieren la conveniencia de que se investigue las ventajas economicas y biologicas del uso de microondas para la coccion del frijol de soya

The abdominal ganglion of Aplysia brasiliana: a comparative morphological and electrophysiological study, with notes on A. dactylomela.

The ultrastructure and electrophysiological properties of neurons in the abdominal (visceral) ganglion of the marine opisthobranch gastropod Aplysia brasiliana have been investigated to determine whether this preparation compares favorably with the well studied A. californica for neurobiological research. In general, the topography, morphology and physiological characteristics, including synaptic connections, of neurons in this ganglion are quite similar to those of A. californica. There is close correspondence between the two animals in terms of each of the identified cells or neuronal clusters in the ganglion, including the presence of the cell L10 (interneuron I) in A. brasiliana which makes synaptic connections comparable with those in A. californica. New follower cells of this interneuron have been found in A. brasiliana. This species offers some advantages in that the connective tissue surrounding the ganglion is thinner and more transparent, making cell identification and penetration easier. A. brasiliana appears to exhibit the behaviors of A. californica that have been used in previous functional analyses of neural circuits. In addition, this species swims and exhibits a "burrowing" activity less commonly seen in A. californica. The rich repertoire of behaviors and accessibility of large identifiable and functionally interconnected neurons makes this species of Aplysia an excellent model preparation for future neurobiological studies. Similar, less thorough, investigations of the abdominal ganglion of A. dactylomela indicate that this species is also very similar to A. californica in terms of the identified cells in the abdominal ganglion.