Bag Cell Neurons Of Aplysia Research Articles

Persistent Ca2+Current Contributes to a Prolonged Depolarization inAplysiaBag Cell Neurons

Neurons may initiate behavior or store information by translating prior activity into a lengthy change in excitability. For example, brief input to the bag cell neurons of Aplysia results in an approximate 30-min afterdischarge that induces reproduction. Similarly, momentary stimulation of cultured bag cells neurons evokes a prolonged depolarization lasting many minutes. Contributing to this is a voltage-independent cation current activated by Ca(2+) entering during the stimulus. However, the cation current is relatively short-lived, and we hypothesized that a second, voltage-dependent persistent current sustains the prolonged depolarization. In bag cell neurons, the inward voltage-dependent current is carried by Ca(2+); thus we tested for persistent Ca(2+) current in primary culture under voltage clamp. The observed current activated between -40 and -50 mV exhibited a very slow decay, presented a similar magnitude regardless of stimulus duration (10-60 s), and, like the rapid Ca(2+) current, was enhanced when Ba(2+) was the permeant ion. The rapid and persistent Ca(2+) current, but not the cation current, were Ni(2+) sensitive. Consistent with the persistent current contributing to the response, Ni(2+) reduced the amplitude of a prolonged depolarization evoked under current clamp. Finally, protein kinase C activation enhanced the rapid and persistent Ca(2+) current as well as increased the prolonged depolarization when elicited by an action potential-independent stimulus. Thus the prolonged depolarization arises from Ca(2+) influx triggering a cation current, followed by voltage-dependent activation of a persistent Ca(2+) current and is subject to modulation. Such synergy between currents may represent a common means of achieving activity-dependent changes to excitability.

Detecting intrinsic scattering changes correlated to neuron action potentials using optical coherence imaging.

We demonstrate how optical coherence imaging techniques can detect intrinsic scattering changes that occur during action potentials in single neurons. Using optical coherence tomography (OCT), an increase in scattering intensity from neurons in the abdominal ganglion of Aplysia californica is observed following electrical stimulation of the connective nerve. In addition, optical coherence microscopy (OCM), with its superior transverse spatial resolution, is used to demonstrate a direct correlation between scattering intensity changes and membrane voltage in single cultured Aplysia bag cell neurons during evoked action potentials. While intrinsic scattering changes are small, OCT and OCM have potential use as tools in neuroscience research for non-invasive and non-contact measurement of neural activity without electrodes or fluorescent dyes. These techniques have many attractive features such as high sensitivity and deep imaging penetration depth, as well as high temporal and spatial resolution. This study demonstrates the first use of OCT and OCM to detect functionally-correlated optical scattering changes in single neurons.

Ca2+ entry through a non-selective cation channel in Aplysia bag cell neurons

Ca2+ influx through voltage-gated Ca2+ channels is a fundamental signaling event in neurons; however, non-traditional routes, such as non-selective cation channels, also permit Ca2+ entry. The present study examines the Ca2+ permeability of a cation channel that drives an afterdischarge in Aplysia bag cell neurons. The firing of these neurons induces peptide release and reproduction. Single channel-containing inside-out patches excised from cultured bag cell neurons, with the cytoplasmic face bathed in K+-aspartate and the extracellular face bathed in artificial seawater (11 mM Ca2+), had a reversal potential near +50 mV. In keeping with Ca2+ permeability, this was right-shifted to approximately +60 mV in high Ca2+ (substituted for Mg2+) and left-shifted to around +40 mV in zero Ca2+ (replaced with Mg2+). The current showed inward rectification between +30 and +90 mV, and a conductance of 29 pS in normal Ca2+, 30 pS in high Ca2+, 32 pS in Ba2+ (substituted for Ca2+), but only 21 pS in zero Ca2+. Despite a greater conductance in Ba2+, the channel did not display anomalous mol fraction in an equimolar Ca2+–Ba2+ mix. Eliminating internal Mg2+ lowered activity, but did not alter inward rectification, suggesting intracellular Mg2+ is a fast, voltage-independent blocker. Imaging bag cell neurons in Mn2+ saline (substituted for Ca2+) revealed enhanced fura-quench following cation channel activation, consistent with Mn2+ permeating as a Ca2+ surrogate. Finally, triggering the cation channel while tracking capacitance revealed a Ca2+-dependent increase in membrane surface area, consistent with vesicle fusion. Thus, the cation channel not only drives the afterdischarge, but also passes Ca2+ to potentially initiate secretion. In general, this may represent an alternate means by which neurons elicit neuropeptide release.

Flufenamic Acid Affects Multiple Currents and Causes Intracellular Ca2+Release inAplysiaBag Cell Neurons

Flufenamic acid (FFA) is a nonsteroidal antiinflammatory agent, commonly used to block nonselective cation channels. We previously reported that FFA potentiated, rather than inhibited, a cation current in Aplysia bag cell neurons. Prompted by this paradoxical result, the present study examined the effects of FFA on membrane currents and intracellular Ca2+ in cultured bag cell neurons. Under whole cell voltage clamp, FFA evoked either outward (I out) or inward (I in) currents. I out had a rapid onset, was inhibited by the K+ channel blocker, tetraethylammonium, and was associated with both an increase in membrane conductance and a negative shift in the whole cell current reversal potential. I in developed more slowly, was inhibited by the cation channel blocker, Gd3+, and was concomitant with both an increased conductance and positive shift in reversal potential. FFA also enhanced the use-dependent inactivation and caused a positive-shift in the activation curve of the voltage-dependent Ca2+ current. Furthermore, as measured by ratiometric imaging, FFA produced a rise in intracellular Ca2+ that persisted in the absence of extracellular Ca2+ and was reduced by depleting either the endoplasmic reticulum and/or mitochondrial stores. Ca2+ appeared to be involved in the activation of I in, as strong intracellular Ca2+ buffering effectively eliminated I in but did not alter I out. Finally, the effects of FFA were likely not due to block of cyclooxygenase given that the general cyclooxygenase inhibitor, indomethacin, failed to evoke either current. That FFA influences a number of neuronal properties needs to be taken into consideration when employing it as a cation channel antagonist.

Repetitive Firing Triggers Clustering of Kv2.1 Potassium Channels in Aplysia Neurons

The Kv2.1 gene encodes a highly conserved delayed rectifier potassium channel that is widely expressed in neurons of the central nervous system. In the bag cell neurons of Aplysia, Kv2.1 channels contribute to the repolarization of action potentials during a prolonged afterdischarge that triggers a series of reproductive behaviors. Partial inactivation of Aplysia Kv2.1 during repetitive firing produces frequency-dependent broadening of action potentials during the afterdischarge. We have now found that, as in mammalian neurons, Kv2.1 channels in bag cell neurons are localized to ring-like clusters in the plasma membrane of the soma and proximal dendrites. Either elevation of cyclic AMP levels or direct electrical stimulation of afterdischarge rapidly enhanced formation of these clusters on the somata of these neurons. In contrast, injection of a 13-amino acid peptide corresponding to a region in the C terminus that is required for clustering of Kv2.1 channels produced disassociation of the clusters, resulting in a more uniform distribution over the somata. Voltage clamp recordings demonstrated that peptide-induced dissociation of the Kv2.1 clusters is associated with an increase in the amplitude of delayed rectifier current and a shift of activation toward more negative potentials. In current clamp recording, injection of the unclustering peptide reduced the width of action potentials and reduced frequency-dependent broadening of action potentials. Our results suggest that rapid redistribution of Kv2.1 channels occurs during physiological changes in neuronal excitability.

Aplysia Bag Cells Function as a Distributed Neurosecretory Network

The anatomical organization of many neuroendocrine systems implies multiple sites of hormone release in areas mediating multiple aspects of physiology and behavior, yet this neurosecretory complexity has not often been verified. Here we probe the well-characterized hormonal model, the reproductive bag cell neuroendocrine system of the sea slug Aplysia californica. The bag cell neurons of Aplysia mediate egg-laying behavior through the coordinated secretion of a suite of peptides derived from a single gene product, the egg-laying prohormone (proELH). Although the majority of bag cell neurons are located within two major abdominal bag cell clusters, smaller groups of egg-laying hormone-expressing cells have been observed in specific pleural and cerebral ganglia regions, some of which have been reported to be electrically connected to the abdominal bag cell clusters. Releasates are sampled from discrete locations within the Aplysia CNS before and during stimulation of afterdischarge activity and subsequently analyzed with matrix assisted laser desorption/ionization time-of-flight mass spectrometry. Site-specific release profiles are observed at bag cell cluster, pleural, and genital ganglion sites after site-specific electrophysiological activation of bag cell afterdischarges. These data demonstrate that the bag cell network has multiple neurohemal release sites, exhibits descending activation that travels from the cerebral and pleural ganglia down to the abdominal bag cell clusters, and releases spatially distinct profiles of proELH-derived peptides within the Aplysia nervous system. Such distributed neurosecretory organization may be a common feature of neuroendocrine systems across higher order organisms linking multiple behavioral aspects to a single neuronal network.

Monitoring Activity-Dependent Peptide Release from the CNS Using Single-Bead Solid-Phase Extraction and MALDI TOF MS Detection

To investigate dynamic peptidergic cell-cell communication, single micrometer-sized solid-phase extraction (SPE) beads were used to collect peptides from specific locations of well-characterized neurosecretory structures and even individual neuronal processes for off-line MALDI MS analyses. Peptide binding parameters of single SPE beads, including limits of collection, detection, and saturation capacity, were tested with 14C-labeled cytochrome c as well as with mixtures of multiple neuropeptides (bradykinin, Aplysia acidic peptide 1-20, and insulin). MALDI MS detection of secreted peptides was demonstrated in two well-characterized neurosecretory structures, the rat pituitary gland and single cultured Aplysia bag cell neurons. With cultured cells, precise placement of SPE beads allowed peptide collection from distinct neurites with spatial localization on the order of 200 microm, and SPE beads could be replaced within time frames that allowed analyte collection before and after cell stimulation paradigms. Comparison between pre- and poststimulation peptide profiles in both model systems allowed a directed strategy to determine which compounds were released with neuronal activity. Single SPE bead MALDI MS offers a novel approach to investigate peptide signaling that allows the detection and discovery of unknown intercellular signals secreted from a large variety of biological tissues.

The Appearance of a Protein Kinase A-regulated Splice Isoform of slo Is Associated with the Maturation of Neurons That Control Reproductive Behavior

In response to brief synaptic stimulation that activates protein kinase A (PKA), the bag cell neurons of Aplysia trigger the onset of reproductive behaviors by generating a prolonged afterdischarge. In juvenile animals, such afterdischarges are inhibited by a high density of Ca2+ -activated K+ (BK) channels, encoded by the slo gene. An increase in this current also follows an afterdischarge in mature animals, contributing to a subsequent refractory state that limits reproductive behaviors. Using a bag cell cDNA library, we have isolated two alternative transcripts of the slo gene, differing in the presence (slo-a) or absence (slo-b) of a consensus phosphorylation site for PKA. Expression of either isoform in Chinese hamster ovary cells produced Ca2+ - and voltage-dependent channels with macroscopic and unitary properties matching those in bag cell neurons. The isoforms differed, however, in their response to application of the catalytic subunit of PKA, which reduced the open probability of Slo-a, an effect that was reversed by a PKA inhibitor. In contrast, PKA had no effect on Slo-b. By immunocytochemistry, we determined that the PKA-regulated Slo-a subunit is present in adult, but not juvenile, bag cell neurons. Patch clamp recordings from adult and juvenile bag cell neurons confirmed that PKA decreases BK channel activity only in adults. Our findings suggest that a change in the identity of Slo isoforms expressed during development allows mature neurons to generate afterdischarges that are required for reproduction.

Activation of a calcium entry pathway by sodium pyrithione in the bag cell neurons of Aplysia.

The ability of sodium pyrithione (NaP), an agent that produces delayed neuropathy in some species, to alter neuronal physiology was accessed using ratiometric imaging of cytosolic free Ca(2+) concentration ([Ca(2+)](i)) in fura PE-filled cultured Aplysia bag cell neurons. Bath-application of NaP evoked a [Ca(2+)](i) elevation in both somata and neurites with an EC(50) of approximately 300 nM and a Hill coefficient of approximately 1. The response required the presence of external Ca(2+), had an onset of 3-5 min, and generally reached a maximum within 30 min. 2-Methyl-sulfonylpyridine, a metabolite and close structural analog of NaP, did not elevate [Ca(2+)](i). Under whole-cell current-clamp recording, NaP produced a approximately 14 mV depolarization of resting membrane potential that was dependent on external Ca(2+). These data suggested that NaP stimulates Ca(2+) entry across the plasma membrane. To minimize the possibility that a change in cytosolic pH was the basis for NaP-induced Ca(2+) entry, bag cell neuron intracellular pH was estimated with the dye 2',7'-bis(carboxyethyl-5(6)-carboxy-fluorescein acetoxy methylester. Exposure of the neurons to NaP did not alter intracellular pH. The slow onset and sustained nature of the NaP response suggested that a cation exchange mechanism coupled either directly or indirectly to Ca(2+) entry could underlie the phenomenon. However, neither ouabain, a Na(+)/K(+) ATPase inhibitor, nor removal of extracellular Na(+), which eliminates Na(+)/Ca(2+) exchanger activity, altered the NaP-induced [Ca(2+)](i) elevation. Finally, the possibility that NaP gates a Ca(2+)-permeable ion channel in the plasma membrane was examined. NaP did not appear to activate two major forms of bag cell neuron Ca(2+)-permeable ion channels, as Ca(2+) entry was unaffected by inhibition of voltage-gated Ca(2+) channels using nifedipine or by inhibition of a voltage-dependent, nonselective cation channel using a high concentration of tetrodotoxin. In contrast, two potential store-operated Ca(2+) entry current inhibitors, SKF-96365 and Ni(2+), attenuated NaP-induced Ca(2+) entry. We conclude that NaP activates a slow, persistent Ca(2+) influx in Aplysia bag cell neurons.

Synaptic stimulation of Aplysia peptidergic neurons can activate hormone secretion in the absence of an afterdischarge

For over 20 years, the bag cell neurons of the marine mollusk Aplysia have been used to investigate second-messenger pathways that mediate effects of synaptic stimulation on ion currents and membrane excitability, presumably leading to exocytotic release of the neuropeptide egg-laying hormone (ELH). It is widely cited that a train of action potentials, called an afterdischarge, is necessary for activating cellular events leading to ELH secretion. Using a combination of electrophysiology, optical imaging of calcium signaling, and radioimmunoassay of ELH secretion, we show that an afterdischarge is not required for ELH secretion. Electrical stimulation that failed to produce afterdischarges but did lead to prolonged membrane depolarization and a rise in intracellular calcium concentration was sufficient to stimulate significant ELH release.

Synaptic stimulation of Aplysia peptidergic neurons can activate hormone secretion in the absence of an afterdischarge

For over 20 years, the bag cell neurons of the marine mollusk Aplysia have been used to investigate second-messenger pathways that mediate effects of synaptic stimulation on ion currents and membrane excitability, presumably leading to exocytotic release of the neuropeptide egg-laying hormone (ELH). It is widely cited that a train of action potentials, called an afterdischarge, is necessary for activating cellular events leading to ELH secretion. Using a combination of electrophysiology, optical imaging of calcium signaling, and radioimmunoassay of ELH secretion, we show that an afterdischarge is not required for ELH secretion. Electrical stimulation that failed to produce afterdischarges but did lead to prolonged membrane depolarization and a rise in intracellular calcium concentration was sufficient to stimulate significant ELH release.

Post-afterdischarge depolarization does not stimulate prolonged neurohormone secretion but is required for activity-dependent stimulation of neurohormone biosynthesis from peptidergic neurons.

Membrane depolarization plays a critical role in stimulating secretion of neuropeptides and can also be important in regulating transcriptional and translational events that control peptide biosynthesis. The purpose of this study was to test the hypothesis that persistent membrane depolarization after the end of an electrical afterdischarge plays an important role in stimulating both prolonged secretion of egg-laying hormone (ELH) and ELH synthesis from peptidergic bag cell neurons of Aplysia. Experimental preparations were treated with a low Na(+) solution to rapidly repolarize membrane potential (Vm). Compared with control preparations, the low Na(+) solution caused rapid membrane repolarization to resting levels, significant shortening of the duration of the afterdischarge, and significant decrease in the decay time constant of cytosolic Ca(2+) ([Ca(2+)](i)) concentrations, but no effect on peak [Ca(2+)](i), total [Ca(2+)](i) above baseline, or duration of elevated [Ca(2+)](i). Contrary to both theoretical expectations and findings in other cell types, low Na(+) treatment and the resulting premature repolarization of Vm did not inhibit ELH secretion. On the other hand, low Na(+) treatment that blocked prolonged depolarization, as well as inhibition of Ca(2+) influx, prevented the afterdischarge-induced stimulation of ELH synthesis. These findings provide support for membrane depolarization acting as a trigger mechanism, rather than a sustained driving force, for cellular events that control ELH secretion. It also demonstrates, for the first time, a critical role for postdischarge Vm in regulating an important aspect of neuroendocrine cell function-that of hormone synthesis.

Prolonged Activation of Ca2+-Activated K+Current Contributes to the Long-Lasting Refractory Period ofAplysiaBag Cell Neurons

Stimulation of the bag cell neurons of Aplysia activates several biochemical pathways, including protein kinase C (PKC), and alters their excitability for many hours. After an approximately 30 min afterdischarge, these neurons enter an approximately 18 hr inhibited state during which additional stimulation fails to evoke discharges. In vivo, this refractory period limits the frequency of reproductive behaviors associated with egg laying. We have now examined the role of Ca2+-activated K+ (BK) currents in the refractory period. Outward currents gated by both intracellular Ca2+ and depolarization, with pharmacological characteristics of BK currents, were recorded in isolated bag cell neurons. These currents were enhanced by the BK channel activators phloretin and 1,3-dihydro-1-[2-hydroxy-5-(trifluoro-methyl)phenyl]-5-trifluoromethyl-2H-benzimidazol-2-one and inhibited by the BK blocker paxilline. The BK component of K+ current was enhanced by 12-O-tetradecanoyl-phorbol-13-acetate, an activator of PKC, and this effect was blocked by sphinganine and PKC(19-36), inhibitors of PKC in bag cell neurons. To test whether the BK current is altered during the refractory period, intact clusters were stimulated to afterdischarge, and neurons were isolated after the clusters had entered the refractory period. Compared with unstimulated cells, current density was almost doubled in refractory neurons. This increase in current was inhibited by preincubating clusters in sphinganine. Treatment of refractory clusters with paxilline significantly restored the ability of stimulation to evoke afterdischarges. Conversely, application of phloretin to previously unstimulated clusters inhibited the onset of afterdischarges. These results indicate that a prolonged increase in BK channel activity contributes to the prolonged refractory period of the bag cell neurons.

The development of excitatory capability in Aplysia californica bag cells observed in cohorts

The bag cells of Aplysia release egg laying hormone in sexually mature animals. Bag cells cannot sustain the long-lasting excitatory afterdischarge (AD) required for hormone release prior to sexual maturity (T.A. Nick, L.K. Kaczmarek, T.J. Carew, Ionic currents underlying developmental regulation of repetitive firing in Aplysia bag cell neurons, J. Neurosci. 1996;16:7583–7598; L.A. Fieber, Characterization of Na + and Ca 2+ currents in bag cells of sexually immature Aplysia californica, J. Exp. Biol. 1998;201:745–754). To investigate the development of bag cell excitability, whole-cell voltage-clamp experiments were executed in dissociated bag cells from four cohorts (batches) of hatchery-reared A. californica maintained at 13–15°C. K + current densities, representing the sum of at least four different outward K + currents (Nick et al., 1996), declined significantly as a function of age, beginning at least 2–3 months before sexual maturity. The K + current decreases coincided with the first appearance of Na + and Ca 2+ currents in bag cells, which occurred at ages 6–7 months. Whole cell K + currents were not decreased significantly by a cAMP analog earlier than 1 month prior to the onset of reproductive activity. The frequency of observing Na + currents in whole cell recordings was low for developmental times earlier than sexual maturity. In one winter batch, both control and PMA-treated Na + currents increased significantly with age, and PMA-treated current densities were significantly greater than controls, but the other two batches studied had significant differences in Na + current frequency only at sexual maturity. Ca 2+ currents were reliably measured in more cells than were Na + currents. The Ca 2+ current frequency increased significantly with maturity in one winter batch. Ca 2+ currents were significantly increased by phorbol ester treatment beginning 6–8 weeks before reproductive activity in the two winter batches. These observations support the hypothesis that bag cell excitability is not fully developed until shortly before sexual maturity.

Persistent activation of calcium-activated and calcium-independent protein kinase C in response to electrical afterdischarge from peptidergic neurons of Aplysia

The purpose of this work was to investigate the effects of electrical afterdischarge on protein kinase C (PKC) activity from bag cell neurons (BCNs) of Aplysia. Bilateral clusters of BCNs were divided: one cluster was stimulated to afterdischarge, the other was a control. Clusters were processed for PKC activity assay 5–120 min after electrical stimulation. Afterdischarge triggered a rapid and persistent increase in both calcium-activated and calcium-independent PKC activity.

Growth Factor Modulation of Substrate-Specific Morphological Patterns in Aplysia Bag Cell Neurons

Components of the extracellular matrix (ECM) can act not only as passive substrates for neuronal attachment and outgrowth but also as active sites for signal transduction. Thus, specific ECM components may modulate effects of growth factors (GFs) that play an important role in structural changes in development and adult neuronal plasticity. In this study we examined the interaction of cultured Aplysia bag cell neurons (BCNs) with components of ECM and different GFs. Different ECM substrata induce a substrate-specific BCN morphology: BCNs grown on collagen or poly-L-lysine have larger soma diameter and more extensive neurite outgrowth than BCNs grown on laminin or fibronectin. BCNs also interact in a substrate-dependent way with GFs: BDNF treatment leads to a reduction of outgrowth on poly-L-lysine but an enhancement on fibronectin and laminin. CNTF reduces the soma diameter on collagen IV but enlarges it on laminin or fibronectin. In contrast, NGF induces a reduction of both soma diameter and outgrowth, on all substrata. Plating of BCNs in the presence of anti-beta1-integrin reduces adhesion to fibronectin but does not change outgrowth. In contrast, RGD peptides block adhesion to laminin and poly-L-lysine and, additionally, reduce outgrowth on laminin. These data suggest that BCNs use different beta1-integrin-dependent as well as RGD-dependent mechanisms for adhesion and outgrowth on different ECM substrata, providing possible sites of modulation by specific GFs.

The Roles of Transcription and Translation in Mediating the Effect of Electrical Afterdischarge on Neurohormone Synthesis in Aplysia Bag Cell Neurons

The molecular links between membrane excitability and neurohormone synthesis were investigated using a simple model system: the bag cell neurons of Aplysia. We tested the hypothesis that the electrical afterdischarge, which leads to depletion of egg-laying hormone (ELH) by triggering secretion, rapidly stimulates ELH synthesis to replenish bag cell stores of hormone. Newly synthesized peptides were radiolabeled, and ELH peptide was immunoprecipitated. Within 4 h of afterdischarge, there was a 100% increase in ELH synthesis compared with that in unstimulated controls. Northern blot analysis showed that ELH messenger RNA (mRNA) levels were not altered for at least 8 h after the onset of afterdischarge. ELH mRNA levels were also not affected by 4-h treatments that inhibited either transcription (with actinomycin D) or translation (with anisomycin). Further work revealed that ELH mRNA is stable, with a half-life exceeding 32 h. Notably, the stimulatory effect of afterdischarge on ELH synthesis was blocked in response to treatment with the transcription inhibitor actinomycin D. These results suggest that the afterdischarge-induced increase in ELH synthesis is mediated by an increase in the rate of translation of already existing ELH mRNA, and that transcription of a non-ELH gene(s) is required for this effect to occur.

The Roles of Transcription and Translation in Mediating the Effect of Electrical Afterdischarge on Neurohormone Synthesis inAplysiaBag Cell Neurons1

The molecular links between membrane excitability and neurohormone synthesis were investigated using a simple model system: the bag cell neurons of Aplysia. We tested the hypothesis that the electrical afterdischarge, which leads to depletion of egg-laying hormone (ELH) by triggering secretion, rapidly stimulates ELH synthesis to replenish bag cell stores of hormone. Newly synthesized peptides were radiolabeled, and ELH peptide was immunoprecipitated. Within 4 h of afterdischarge, there was a 100% increase in ELH synthesis compared with that in unstimulated controls. Northern blot analysis showed that ELH messenger RNA (mRNA) levels were not altered for at least 8 h after the onset of afterdischarge. ELH mRNA levels were also not affected by 4-h treatments that inhibited either transcription (with actinomycin D) or translation (with anisomycin). Further work revealed that ELH mRNA is stable, with a half-life exceeding 32 h. Notably, the stimulatory effect of afterdischarge on ELH synthesis was blocked in response to treatment with the transcription inhibitor actinomycin D. These results suggest that the afterdischarge-induced increase in ELH synthesis is mediated by an increase in the rate of translation of already existing ELH mRNA, and that transcription of a non-ELH gene(s) is required for this effect to occur.

Expression of a foreign G-protein coupled receptor modulates the excitability of the peptidergic bag cell neurons of Aplysia

Brief afferent stimulation of the bag cell neurons can trigger a sustained period of spontaneous firing (the `afterdischarge'). Pharmacological activation of a number of second messenger pathways has previously been shown to partially replicate this behavior. One strategy to identify additional pathways may be to heterologously express a variety of G-protein coupled receptors in bag cell neurons, then test whether receptor activation can induce an afterdischarge. We find that expression of the 1 α metabotropic glutamate receptor in single bag cell neurons mimics some features of the afterdischarge. This approach is thus feasible and may allow the reconstruction of different components of afterdischarge in the absence of the afferent input.

Protein kinase C regulates a vesicular class of calcium channels in the bag cell neurons of aplysia.

Protein kinase C (PKC) acutely increases calcium currents in Aplysia bag cell neurons by recruiting calcium channels different from those constitutively active in the plasma membrane. To study the mechanism of PKC regulation we previously identified two calcium channel alpha1-subunits expressed in bag cell neurons. One of these, BC-alpha1A, is localized to vesicles concentrated primarily in somata and growth cones. We used antibodies to BC-alpha1A to analyze its expression in the bag cell neurons of juvenile Aplysia at a developmental stage at which PKC-sensitive calcium currents have previously been shown to be low. We find that vesicular BC-alpha1A staining is generally reduced in juvenile bag cell neurons but that its expression level can vary among juvenile animals. In 17 bag cell clusters examined, the percentage of neurons that displayed punctate alphaBC-alpha1A staining ranged from 0 to 85%. Sampling of calcium currents from cells of the same clusters by whole cell patch-clamp techniques revealed that the PKC-sensitive calcium current density is significantly correlated with the degree of vesicular staining. In contrast, no correlation of basal calcium current levels with aBC-alpha1A staining was found. These results strongly suggest that BC-alpha1A, a member of the ABE-subfamily of calcium channels, carries the PKC-sensitive calcium current in bag cell neurons. They are consistent with a model in which PKC recruits channels from the vesicular pool to the plasma membrane.