Bag Cell Research Articles

Measuring Retrograde Actin Flow in Neuronal Growth Cones.

Actin flow refers to the motion of the F-actin cytoskeleton and has been observed in many different cell types, especially in motile cells including neuronal growth cones. The direction of the actin flow is generally retrograde from the periphery toward the center of the cell. Actin flow can be harnessed for forward movement of the cell through substrate-cytoskeletal coupling; thus, a key function of actin flow is in cell locomotion. In this chapter, we illustrate three different methods of quantifying retrograde F-actin flow in growth cones derived from cultured Aplysia bag cell neurons. These methods include tracking the movement of surface marker beads as well as kymograph analysis of time-lapse sequences acquired by differential interference contrast (DIC) imaging or fluorescent speckle microscopy (FSM). Due to their large size, Aplysia neuronal growth cones are uniquely suited for these methods; however, they can also be applied to any other growth cones with clear F-actin-rich peripheral domains.

Neuronal NADPH oxidase is required for neurite regeneration of Aplysia bag cell neurons.

NADPH oxidase (Nox), a major source of reactive oxygen species (ROS), is involved in neurodegeneration after injury and disease. Nox is expressed in both neuronal and non-neuronal cells and contributes to an elevated ROS level after injury. Contrary to the well-known damaging effect of Nox-derived ROS in neurodegeneration, recently a physiological role of Nox in nervous system development including neurogenesis, neuronal polarity, and axonal growth has been revealed. Here, we tested a role for neuronal Nox in neurite regeneration following mechanical transection in cultured Aplysia bag cell neurons. Using a novel hydrogen peroxide (H2 O2 )-sensing dye, 5'-(p-borophenyl)-2'-pyridylthiazole pinacol ester (BPPT), we found that H2 O2 levels are elevated in regenerating growth cones following injury. Redistribution of Nox2 and p40phox in the growth cone central domain suggests Nox2 activation after injury. Inhibiting Nox with the pan-Nox inhibitor celastrol reduced neurite regeneration rate. Pharmacological inhibition of Nox is correlated with reduced activation of Src2 tyrosine kinase and F-actin content in the growth cone. Taken together, these findings suggest that Nox-derived ROS regulate neurite regeneration following injury through Src2-mediated regulation of actin organization in Aplysia growth cones.

Use-dependent facilitation of electrical transmission involves changes to postsynaptic K+ current.

Activity-dependent modulation of electrical transmission typically involves Ca2+ influx acting directly on gap junctions or initiating Ca2+-dependent pathways that in turn modulate coupling. We now describe short-term use-dependent facilitation of electrical transmission between bag cell neurons from the hermaphroditic snail, Aplysia californica, that is instead mediated by changes in postsynaptic responsiveness. Bag cell neurons secrete reproductive hormone during a synchronous afterdischarge of action potentials coordinated by electrical coupling. Here, recordings from pairs of coupled bag cell neurons in culture showed that nonjunctional currents influence electrical transmission in a dynamic manner. Under a dual whole cell voltage-clamp, the junctional current was linear and largely voltage-independent, while in current-clamp, the coupling coefficient was similar regardless of the extent of presynaptic hyperpolarization. Moreover, a train stimulus of action potential-like waveforms, in a voltage-clamped presynaptic neuron, elicited electrotonic potentials, in a current-clamped postsynaptic neuron, that facilitated over time when delivered at a frequency approximating the afterdischarge. Junctional current remained constant over the train stimulus, as did postsynaptic voltage-gated Ca2+ current. However, postsynaptic voltage-gated K+ current underwent cumulative inactivation, suggesting that K+ current run-down facilitates the electrotonic potential by boosting the response to successive junctional currents. Accordingly, preventing run-down by blocking postsynaptic K+ channels occluded facilitation. Finally, stimulation of bursts in coupled pairs resulted in synchronous firing, where active neurons could recruit silent partners through short-term use-dependent facilitation. Thus, potentiation of electrical transmission may promote synchrony in bag cell neurons and, by extension, reproductive function.NEW & NOTEWORTHY The understanding of how activity can facilitate electrical transmission is incomplete. We found that electrotonic potentials between electrically coupled neuroendocrine bag cell neurons facilitated in a use-dependent fashion. Rather than changes to the junctional current, facilitation was associated with cumulative inactivation of postsynaptic K+ current, presumably augmenting responsiveness. When made to burst, neurons synchronized their spiking, in part by use-dependent facilitation bringing quiescent cells to the threshold. Facilitation may foster en masse firing and neurosecretion.

Distribution, cellular localization, and colocalization of several peptide neurotransmitters in the central nervous system of Aplysia.

Neuropeptides are widely used as neurotransmitters in vertebrates and invertebrates. In vertebrates, a detailed understanding of their functions as transmitters has been hampered by the complexity of the nervous system. The marine mollusk Aplysia, with a simpler nervous system and many large, identified neurons, presents several advantages for addressing this question and has been used to examine the roles of tens of peptides in behavior. To screen for other peptides that might also play roles in behavior, we observed immunoreactivity in individual neurons in the central nervous system of adult Aplysia with antisera raised against the Aplysia peptide FMRFamide and two mammalian peptides that are also found in Aplysia, cholecystokinin (CCK) and neuropeptide Y (NPY), as well as serotonin (5HT). In addition, we observed staining of individual neurons with antisera raised against mammalian somatostatin (SOM) and peptide histidine isoleucine (PHI). However, genomic analysis has shown that these two peptides are not expressed in the Aplysia nervous system, and we have therefore labeled the unknown peptides stained by these two antibodies as XSOM and XPHI There was an area at the anterior end of the cerebral ganglion that had staining by antisera raised against many different transmitters, suggesting that this may be a modulatory region of the nervous system. There was also staining for XSOM and, in some cases, FMRFamide in the bag cell cluster of the abdominal ganglion. In addition, these and other studies have revealed a fairly high degree of colocalization of different neuropeptides in individual neurons, suggesting that the peptides do not just act independently but can also interact in different combinations to produce complex functions. The simple nervous system of Aplysia is advantageous for further testing these ideas.

Cholinergic depolarization recruits a persistent Ca2+ current in Aplysia bag cell neurons.

Many behaviors and types of information storage are mediated by lengthy changes in neuronal activity. In bag cell neurons of the hermaphroditic sea snail Aplysia californica, a transient cholinergic synaptic input triggers an ∼30-min afterdischarge. This causes these neuroendocrine cells to release egg laying hormone and elicit reproductive behavior. When acetylcholine is pressure-ejected onto a current-clamped bag cell neuron, the evoked depolarization is far longer than the current evoked by acetylcholine under voltage clamp, suggesting recruitment of another conductance. Our earlier studies found bag cell neurons to display a voltage-dependent persistent Ca2+ current. Hence, we hypothesized that this current is activated by the acetylcholine-induced depolarization and sought a selective Ca2+ current blocker. Rapid Ca2+ current evoked by 200-ms depolarizing steps in voltage-clamped cultured bag cell neurons demonstrated a concentration-dependent sensitivity to Ni2+, Co2+, Zn2+, and verapamil but not Cd2+ or ω-conotoxin GIVa. Leak subtraction of Ca2+ current evoked by 10-s depolarizing steps using the IC100 (concentration required to eliminate maximal current) of Ni2+, Co2+, Zn2+, or verapamil revealed persistent Ca2+ current, demonstrating persistent current block. Only Co2+ and Zn2+ did not suppress the acetylcholine-induced current, although Zn2+ appeared to impact additional channels. When Co2+ was applied during an acetylcholine-induced depolarization, the amplitude was reduced; furthermore, protein kinase C activation, previously established to enhance the persistent Ca2+ current, extended the depolarization. Therefore, the persistent Ca2+ current sustains the acetylcholine-induced depolarization and may translate brief cholinergic input into afterdischarge initiation. This could be a general mechanism of triggering long-term change in activity with a short-lived input.NEW & NOTEWORTHY Ionotropic acetylcholine receptors mediate brief synaptic communication, including in bag cell neurons of the sea snail Aplysia. However, this study demonstrates that cholinergic depolarization can open a voltage-gated persistent Ca2+ current, which extends the bag cell neuron response to acetylcholine. Bursting in these neuroendocrine cells results in hormone release and egg laying. Thus, this emphasizes the role of ionotropic signaling in reaching a depolarized level to engage Ca2+ influx and perpetuating the activity necessary for behavior.

Octopamine-MAPK-SKN-1 signaling suppresses mating-induced oxidative stress in Caenorhabditis elegans gonads to protect fertility

Sexual conflict over mating is costly to female physiology. Caenorhabditis elegans hermaphrodites generally produce self-progeny, but they can produce cross-progeny upon successfully mating with a male. We have uncovered that C.elegans hermaphrodites experience sexual conflict over mating, resulting in severe costs in terms of their fertility and longevity. We show that reactive oxygen species (ROS) accumulate on the apical surfaces of spermathecal bag cells after successful mating and induce cell damage, leading to ovulation defects and fertility suppression. To counteract these negative impacts, C.elegans hermaphrodites deploy the octopamine (OA) regulatory pathway to enhance glutathione (GSH) biosynthesis and protect spermathecae from mating-induced ROS. We show that the SER-3 receptor and mitogen-activated protein kinase (MAPK) KGB-1 cascade transduce the OA signal to transcription factor SKN-1/Nrf2 in the spermatheca to upregulate GSH biosynthesis.

Erythematous, telangiectatic nodule within the melomental fold

Erythematous, telangiectatic nodule within the melomental fold

Hydrogen peroxide and phosphoinositide metabolites synergistically regulate a cation current to influence neuroendocrine cell bursting.

In various neurons, including neuroendocrine cells, non-selective cation channels elicit plateau potentials and persistent firing. Reproduction in the marine snail Aplysia californica is initiated when the neuroendocrine bag cell neurons undergo an afterdischarge, that is, a prolonged period of enhanced excitability and spiking during which egg-laying hormone is released into the blood. The afterdischarge is associated with both the production of hydrogen peroxide (H2 O2 ) and activation of phospholipase C (PLC), which hydrolyses phosphatidylinositol-4,5-bisphosphate into diacylglycerol (DAG) and inositol trisphosphate (IP3 ). We previously demonstrated that H2 O2 gates a voltage-dependent cation current and evokes spiking in bag cell neurons. The present study tests if DAG and IP3 impact the H2 O2 -induced current and excitability. In whole-cell voltage-clamped cultured bag cell neurons, bath-application of 1-oleoyl-2-acetyl-sn-glycerol (OAG), a DAG analogue, enhanced the H2 O2 -induced current, which was amplified by the inclusion of IP3 in the pipette. A similar outcome was produced by the PLC activator, N-(3-trifluoromethylphenyl)-2,4,6-trimethylbenzenesulfonamide. In current-clamp, OAG or OAG plus IP3 , elevated the frequency of H2 O2 -induced bursting. PKC is also triggered during the afterdischarge; when PKC was stimulated with phorbol 12-myristate 13-acetate, it caused a voltage-dependent inward current with a reversal potential similar to the H2 O2 -induced current. Furthermore, PKC activation followed by H2 O2 reduced the onset latency and increased the duration of action potential firing. Finally, inhibiting nicotinamide adenine dinucleotide phosphate oxidase with 3-benzyl-7-(2-benzoxazolyl)thio-1,2,3-triazolo[4,5-d]pyrimidine diminished evoked bursting in isolated bag cell neuron clusters. These results suggest that reactive oxygen species and phosphoinostide metabolites may synergize and contribute to reproductive behaviour by promoting neuroendocrine cell firing. KEY POINTS: Aplysia bag cell neurons secrete reproductive hormone during a lengthy burst of action potentials, known as the afterdischarge. During the afterdischarge, phospholipase C (PLC) hydrolyses phosphatidylinositol-4,5-bisphosphate into diacylglycerol (DAG) and inositol trisphosphate (IP3 ). Subsequent activation of protein kinase C (PKC) leads to H2 O2 production. H2 O2 evokes a voltage-dependent inward current and action potential firing. Both a DAG analogue, 1-oleoyl-2-acetyl-sn-glycerol (OAG), and IP3 enhance the H2 O2 -induced current, which is mimicked by the PLC activator, N-(3-trifluoromethylphenyl)-2,4,6-trimethylbenzenesulfonamide. The frequency of H2 O2 -evoked afterdischarge-like bursting is augmented by OAG or OAG plus IP3 . Stimulating PKC with phorbol 12-myristate 13-acetate shortens the latency and increases the duration of H2 O2 -induced bursts. The nicotinamide adenine dinucleotide phosphate oxidase inhibitor, 3-benzyl-7-(2-benzoxazolyl)thio-1,2,3-triazolo[4,5-d]pyrimidine, attenuates burst firing in bag cell neuron clusters.

Uniformity of Flat Li-Ion Batteries Studied by Diffraction and Imaging of X-rays and Neutrons

A combination of high-resolution neutron powder diffraction, energy-dependent cold-neutron radiography, and synchrotron-based X-ray diffraction radiography on two pouch bag cells of different dimen...

Implementation of Collodion Bag Protocol to Improve Whole-slide Imaging of Scant Gynecologic Curettage Specimens

Background: Digital pathology has been increasingly implemented for primary surgical pathology diagnosis. In our institution, digital pathology was recently deployed in the gynecologic (GYN) pathology practice. A notable challenge encountered in the digital evaluation of GYN specimens was high rates of scanning failure of specimens with fragmented as well as scant tissue. To improve tissue detection failure rates, we implemented a novel use of the collodion bag cell block preparation method. Materials and Methods: In this study, we reviewed 108 endocervical curettage (ECC) specimens, representing specimens processed with and without the collodion bag cell block method (n = 56 without collodion bag, n = 52 with collodion bag). Results: Tissue detection failure rates were reduced from 77% (43/56) in noncollodion bag cases to 23/52 (44%) of collodion bag cases, representing a 42% reduction. The median total area of tissue detection failure per level was 0.35 mm2 (interquartile range [IQR]: 0.14, 0.70 mm2) for noncollodion bag cases and 0.08 mm2 (IQR: 0.03, 0.20 mm2) for collodion bag cases. This represents a greater than fourfold reduction in the total area of tissue detection failure per level (P < 0.001). In addition, there were no out-of-focus levels among collodion bag cases, compared to 6/56 (11%) of noncollodion bag cases (median total area = 4.9 mm2). Conclusions: The collodion bag method significantly improved the digital image quality of fragmented/scant GYN curettage specimens, increased efficiency and accuracy of diagnostic evaluation, and enhanced identification of tissue contamination during processing. The logistical challenges and labor cost of deploying the collodion bag protocol are important considerations for feasibility assessment at an institutional level.

A Co-purification Method for Efficient Production and Src Kinase-mediated Phosphorylation of Aplysia Cortactin.

Cortactin is an actin-binding protein that regulates processes like cell migration, endocytosis, and tumor cell metastasis. Although cortactin is associated with actin-cytoskeletal dynamics in non-neuronal cells and cell-free systems, the exact mechanisms underlying its fundamental roles in neuronal growth cones are not fully explored. Recent reports show that Aplysia Src2 tyrosine kinase induces phosphorylation of cortactin as a mechanism to control lamellipodia protrusion and filopodia formation in cultured Aplysia bag cell neurons ( He et al., 2015 ; Ren et al., 2019 ). In order to provide in vitro evidence for Src2-mediated phosphorylation of cortactin, we developed a robust and cost-effective method for the efficient expression and purification of Aplysia cortactin and Src2 kinase that can be used for biochemical studies including phosphorylation assays. By co-purifying cortactin and Src kinase with a phosphatase (YopH) from Yersinia enterocolitica, we eliminated the problem of non-specific phosphorylation of induced proteins by bacterial kinases and also reduced costs by bypassing the need for commercial enzymatic treatments. This protocol is reproducible and can be modified to produce homogenous non-phosphorylated proteins during recombinant protein expression in Escherichia coli.

Clean plasma modification for recycling waste plastic bags: From improving interfacial adhesion with wood towards fabricating formaldehyde-free plywood

It remains a great challenge to manufacture formaldehyde-free plywood, because of the wide application and cost advantage of formaldehyde-based adhesives. Herein, the waste plastic bag was recycled as an alternative to formaldehyde-based adhesives for manufacturing formaldehyde-free plywood. An industrial atmospheric dielectric-barrier-discharge plasma system was used to treat the plastic bag, in order to enhance the interfacial adhesion of the resultant plywood. The results suggested that plasma modification would excite oxygen and nitrogen into energetic particles and further ruptured the raw chemical bonds of plastic bag. Oxygen- and nitrogen-containing functional groups can be grafted, thus enhancing plastic bags’ surface polarity and wettability. As further supported by nanoindentation, the adhesion energy of plasma-modified plastic bag and poplar cell wall was elevated by 19.13% when compared to the unmodified sample, thus suggesting the improved interfacial adhesion and interfacial compatibility. Benefiting from that, formaldehyde-free plywood with a high bonding strength (0.82 MPa) was successfully fabricated. Shear strain in plywood distributed uniformly after plasma modification, suggesting the even distribution of adhesive and the enhanced bearing-load capacity of interface. This study paves a new way to effectively recycle plastic waste as an alternative to traditional adhesives for formaldehyde-free plywood manufacture.

Transdermal delivery enhancement of carvacrol from Origanum vulgare L. essential oil by microemulsion.

Carvacrol has been reported for analgesic and anti-inflammatory activity by cyclooxygenase inhibition but it could induce gastrointestinal toxicity because of its non-selective inhibition. Therefore, the present study aimed to develop transdermal microemulsion from Origanum vulgare essential oil to deliver carvacrol into and through the skin which would overwhelm the gastrointestinal problems. O. vulgare essential oil was extracted by hydrodistillation and its carvacrol content was determined using high performance liquid chromatography. Pseudoternary phase diagrams were constructed using water dilution method to investigate the suitable microemulsion components. Microemulsions were then characterized for external appearance, particle size, size distribution, zeta potential, electrical conductivity, refractive index, viscosity, transmittance, pH, and stability. Additionally, the irritation property of microemulsions were investigated by hen's egg on the chorioallantoic membrane assay. The release profile, percutaneous absorption, and skin retention were investigated using dialysis bag and Franz diffusion cell, respectively. The interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were investigated using the enzyme-linked immunosorbent assay. The results remarked that carvacrol was a major component of O. vulgare essential oil with high concentration of 83.7%. The most suitable microemulsion (ME 1), composing of 5% w/w O. vulgare essential oil, 25%w/w Tween 60, 25%w/w butylene glycol, and 45%w/w deionized water, had the smallest internal droplet size (179.5±27.9nm), the narrowest polydispersity index (0.30±0.07), the highest transmittance (93.13±0.04%), and Newtonian flow behavior with low viscosity of 0.30±0.07 Pas. ME 1 could reduce the irritation effect of O. vulgare essential oil since ME 1 (IS=3.1±0.10) exhibited significantly lower irritation effect than its blank formulation (IS=4.8±0.02) and O. vulgare oil solution (IS=5.0±0.01) (p<0.05). Furthermore, ME 1 sustain released carvacrol from the formulation, remarkedly deliver more carvacrol through the skin layer (2.6±2.2%) and significantly retained carvacrol in the skin layer (2.60±1.25%). Additionally, ME 1 significantly enhanced IL-6 inhibition of O. vulgaris oil and carvacrol (p<0.05). Therefore, O. vulgaris oil microemulsion was suggested to be used for the transdermal delivery and anti-inflammatory activities enhancement of carvacrol.

Hydrogen Peroxide Gates a Voltage-Dependent Cation Current in Aplysia Neuroendocrine Cells.

Nonselective cation channels promote persistent spiking in many neurons from a diversity of animals. In the hermaphroditic marine-snail, Aplysia californica, synaptic input to the neuroendocrine bag cell neurons triggers various cation channels, causing an ∼30 min afterdischarge of action potentials and the secretion of egg-laying hormone. During the afterdischarge, protein kinase C is also activated, which in turn elevates hydrogen peroxide (H2O2), likely by stimulating nicotinamide adenine dinucleotide phosphate oxidase. The present study investigated whether H2O2 regulates cation channels to drive the afterdischarge. In single, cultured bag cell neurons, H2O2 elicited a prolonged, concentration- and voltage-dependent inward current, associated with an increase in membrane conductance and a reversal potential of ∼+30 mV. Compared with normal saline, the presence of Ca2+-free, Na+-free, or Na+/Ca2+-free extracellular saline, lowered the current amplitude and left-shifted the reversal potential, consistent with a nonselective cationic conductance. Preventing H2O2 reduction with the glutathione peroxidase inhibitor, mercaptosuccinate, enhanced the H2O2-induced current, while boosting glutathione production with its precursor, N-acetylcysteine, or adding the reducing agent, dithiothreitol, lessened the response. Moreover, the current generated by the alkylating agent, N-ethylmaleimide, occluded the effect of H2O2 The H2O2-induced current was inhibited by tetrodotoxin as well as the cation channel blockers, 9-phenanthrol and clotrimazole. In current-clamp, H2O2 stimulated burst firing, but this was attenuated or prevented altogether by the channel blockers. Finally, H2O2 evoked an afterdischarge from whole bag cell neuron clusters recorded ex vivo by sharp-electrode. H2O2 may regulate a cation channel to influence long-term changes in activity and ultimately reproduction.SIGNIFICANCE STATEMENT Hydrogen peroxide (H2O2) is often studied in a pathological context, such as ischemia or inflammation. However, H2O2 also physiologically modulates synaptic transmission and gates certain transient receptor potential channels. That stated, the effect of H2O2 on neuronal excitability remains less well defined. Here, we examine how H2O2 influences Aplysia bag cell neurons, which elicit ovulation by releasing hormones during an afterdischarge. These neuroendocrine cells are uniquely identifiable and amenable to recording as individual cultured neurons or a cluster from the nervous system. In both culture and the cluster, H2O2 evokes prolonged, afterdischarge-like bursting by gating a nonselective voltage-dependent cationic current. Thus, H2O2, which is generated in response to afterdischarge-associated second messengers, may prompt the firing necessary for hormone secretion and procreation.

It is all in the bag: collodion bag versus HistoGel cell block method

We performed a comparison of cell blocks prepared with the collodion bag and HistoGel to evaluate the ease of embedding and cutting, performance with low cellularity specimens, time and cost per specimen, and value to support immunohistochemistry and molecular diagnostics. We processed 11 fresh, unfixed effusions using both the collodion bag and the HistoGel cell block preparation methods. Six immunohistochemistry stains were tested on 2 of the body fluids. DNA was extracted and quantified, and polymerase chain reaction cycle thresholds were evaluated from cell blocks prepared from 5 of the body fluids. The comparison parameters included embedding difficulty, cutting resistance, adequacy, cell yield, cell preservation, immunohistochemistry stain quality, DNA quantity, integrity, and purity. The time and cost to prepare each specimen was compared using normalized values for preparation of specimen, cost per year, and cost per specimen. Each parameter was assessed for both cell block preparation methods. All 3 of the samples with moderate or poor cell yield were low-volume (5-mL) samples prepared with the HistoGel method. In contrast, the collodion bag method produced a good yield with all three 5-mL samples. DNA recovery was greater in the collodion bag method. Similar crossing threshold values in purity reactions indicated equally high-quality matrix properties for the collodion bag and HistoGel preparations. Preparation of the specimen was 10 minutes faster with the collodion bag method, and the cost for the collodion bag method was $0.24 more expensive per cell block than using the HistoGel. The collodion bag method produced superior cell blocks for both morphologic and molecular studies more consistently, with lower volume specimens and with less time per specimen.

Tranexamic acid is an active site inhibitor of urokinase plasminogen activator

Key PointsTXA is an active-site inhibitor of uPA. TXA attenuates MDA-MB-231 BAG cell migration and inhibits endogenous uPA activity.

Distribution of abalone egg-laying hormone-like peptide in the central nervous system and reproductive tract of the male mud crab, Scylla olivacea

The mud crab, Scylla olivacea, is a high value economic marine animal in Thailand. However, collection of these crabs from natural habitat for local consumption and export has caused rapid population decline. Hence, aquaculture of this species is required and to this measure understanding of endocrine control of their reproduction must be understood. Egg laying hormone (ELH) is a neuropeptide synthesized by the bag cells (neurons) in the abdominal ganglia of Aplysia gastropods. It plays a critical role in controlling egg production and laying in gastropods, and its possible homolog (ELH-like peptide) was reported in the neural and ovarian tissues of prawns and recently in female reproductive tract of the blue swimming crab, Portunus pelagicus. In this study, we have studied the histology of the male reproductive tract in Scylla olivacea which are comprised of anterior testis, posterior testis, early proximal spermatic duct (ePSD), proximal spermatic duct (PSD), middle spermatic duct (MSD) and distal spermatic duct (DSD), by immunohistochemistry, detected an abalone ELH- immunoreactivity (aELH-ir) in epithelium of ducts in posterior testis and epithelium of all parts of spermatic duct. Furthermore, we could detect aELH-ir in neurons of cluster 9, 11, olfactory neuropil (ON) in the brain and in the small neurons located between the third and the fourth thoracic neuropils (T3-T4) and between the fourth and the fifth thoracic neuropils (T4-T5) of thoracic ganglia. Thus, the presence of aELH in male S. olivacea was designated the role of female egg laying behavior in the male mud crab.

A Closely Associated Phospholipase C Regulates Cation Channel Function through Phosphoinositide Hydrolysis.

In the hemaphroditic sea snail, Aplysia californica, reproduction is initiated when the bag cell neurons secrete egg-laying hormone during a protracted afterdischarge. A source of depolarization for the afterdischarge is a voltage-gated, nonselective cation channel, similar to transient receptor potential (TRP) channels. Once the afterdischarge is triggered, phospholipase C (PLC) is activated to hydrolyze phosphatidylinositol-4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol trisphosphate (IP3). We previously reported that a DAG analog, 1-oleoyl-2-acetyl-sn-glycerol (OAG), activates a prominent, inward whole-cell cationic current that is enhanced by IP3 To examine the underlying mechanism, we investigated the effect of exogenous OAG and IP3, as well as PLC activation, on cation channel activity and voltage dependence in excised, inside-out patches from cultured bag cell neurons. OAG transiently elevated channel open probability (PO) when applied to excised patches; however, coapplication of IP3 prolonged the OAG-induced response. In patches exposed to OAG and IP3, channel voltage dependence was left-shifted; this was also observed with OAG, but not to the same extent. Introducing the PLC activator, m-3M3FBS, to patches increased channel PO, suggesting PLC may be physically linked to the channels. Accordingly, blocking PLC with U-73122 ablated the m-3M3FBS-induced elevation in PO Treatment with m-3M3FBS left-shifted cation channel voltage dependence to a greater extent than exogenous OAG and IP3 Finally, OAG and IP3 potentiated the stimulatory effect of PKC, which is also associated with the channel. Thus, the PLC-PKC signaling system is physically localized such that PIP2 breakdown products liberated during the afterdischarge modulate the cation channel and temporally influence neuronal activity.SIGNIFICANCE STATEMENT Using excised patches from Aplysia bag cell neurons, we present the first evidence of a nonselective cation channel physically associating with phospholipase C (PLC) at the single-channel level. PLC-mediated breakdown of phospholipids generates diacylglycerol and inositol trisphosphate, which activate the cation channel. This is mimicked by exogenous lipids; furthermore, these second messengers left-shift channel voltage dependence and enhance the response of the channel to protein kinase C. PLC-mediated lipid signaling controls single-channel currents to ensure depolarization is maintained for an extended period of firing, termed the afterdischarge, when the bag cell neurons secrete egg-laying hormone to trigger reproduction.

A low-cost microwell device for high-resolution imaging of neurite outgrowth in 3D

Objective. Current neuronal cell culture is mostly performed on two-dimensional (2D) surfaces, which lack many of the important features of the native environment of neurons, including topographical cues, deformable extracellular matrix, and spatial isotropy or anisotropy in three dimensions. Although three-dimensional (3D) cell culture systems provide a more physiologically relevant environment than 2D systems, their popularity is greatly hampered by the lack of easy-to-make-and-use devices. We aim to develop a widely applicable 3D culture procedure to facilitate the transition of neuronal cultures from 2D to 3D. Approach. We made a simple microwell device for 3D neuronal cell culture that is inexpensive, easy to assemble, and fully compatible with commonly used imaging techniques, including super-resolution microscopy. Main results. We developed a novel gel mixture to support 3D neurite regeneration of Aplysia bag cell neurons, a system that has been extensively used for quantitative analysis of growth cone dynamics in 2D. We found that the morphology and growth pattern of bag cell growth cones in 3D culture closely resemble the ones of growth cones observed in vivo. We demonstrated the capability of our device for high-resolution imaging of cytoskeletal and signaling proteins as well as organelles. Significance. Neuronal cell culture has been a valuable tool for neuroscientists to study the behavior of neurons in a controlled environment. Compared to 2D, neurons cultured in 3D retain the majority of their native characteristics, while offering higher accessibility, control, and repeatability. We expect that our microwell device will facilitate a wider adoption of 3D neuronal cultures to study the mechanisms of neurite regeneration.

Protein Kinase C Enhances Electrical Synaptic Transmission by Acting on Junctional and Postsynaptic Ca2+ Currents.

By synchronizing neuronal activity, electrical transmission influences the coordination, pattern, and/or frequency of firing. In the hemaphroditic marine-snail, Aplysia calfornica, the neuroendocrine bag cell neurons use electrical synapses to synchronize a 30 min afterdischarge of action potentials for the release of reproductive hormone. During the afterdischarge, protein kinase C (PKC) is activated, although its impact on bag cell neuron electrical transmission is unknown. This was investigated here by monitoring electrical synapses between paired cultured bag cell neurons using dual whole-cell recording. Voltage clamp revealed a largely voltage-independent junctional current, which was enhanced by treating with a PKC activator, PMA, before recording. We also examined the transfer of presynaptic action potential-like waveforms (generated in voltage clamp) to the postsynaptic cell (measured in current clamp). For control pairs, the presynaptic spike-like waveforms mainly evoked electrotonic potentials; however, when PKC was triggered, these stimuli consistently produced postsynaptic action potentials. To assess whether this involved changes to postsynaptic responsiveness, single bag cell neurons were injected with junctional-like current mimicking that evoked by a presynaptic action potential. Unlike control neurons, which were less likely to spike, cells in PMA always fired action potentials to the junctional-like current. Furthermore, PKC activation increased a postsynaptic voltage-gated Ca2+ current, which was recruited even by modest depolarization associated with an electrotonic potential. Whereas PKC inhibits gap junctions in most systems, bag cell neurons are rather unique, as the kinase potentiates the electrical synapse; in turn, this synergizes with augmented postsynaptic Ca2+ current to promote synchronous firing.SIGNIFICANCE STATEMENT Electrical coupling is a fundamental form of communication. For the bag cell neurons of Aplysia, electrical synapses coordinate a prolonged burst of action potentials known as the afterdischarge. We looked at how protein kinase C, which is upregulated with the afterdischarge, influences information transfer across the synapse. The kinase activation increased junctional current, a remarkable finding given that this enzyme is largely considered inhibitory for gap junctions. There was also an augmentation in the ability of a presynaptic neuron to provoke postsynaptic action potentials. This increased excitability was, in part, due to enhanced postsynaptic voltage-dependent Ca2+ current. Thus, protein kinase C improves the fidelity of electrotonic transmission and promotes synchronous firing by modulating both junctional and membrane conductances.