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.

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.

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.

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.

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.

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.

Tyrosine Phosphorylation Determines Afterdischarge Initiation by Regulating an Ionotropic Cholinergic Receptor.

Changes to neuronal activity often involve a rapid and precise transition from low to high excitability. In the marine snail, Aplysia, the bag cell neurons control reproduction by undergoing an afterdischarge, which begins with synaptic input releasing acetylcholine to open an ionotropic cholinergic receptor. Gating of this receptor causes depolarization and a shift from silence to continuous action potential firing, leading to the neuroendocrine secretion of egg-laying hormone and ovulation. At the onset of the afterdischarge, there is a rise in intracellular Ca2+, followed by both protein kinase C (PKC) activation and tyrosine dephosphorylation. To determine whether these signals influence the acetylcholine ionotropic receptor, we examined the bag cell neuron cholinergic response both in culture and isolated clusters using whole-cell and/or sharp-electrode electrophysiology. The acetylcholine-induced current was not altered by increasing intracellular Ca2+ via voltage-gated Ca2+ channels, clamping intracellular Ca2+ with exogenous Ca2+ buffers, or activating PKC with phorbol esters. However, lowering phosphotyrosine levels by inhibiting tyrosine kinases both reduced the cholinergic current and prevented acetylcholine from triggering action potentials or afterdischarge-like bursts. In other systems, acetylcholine receptors are often modulated by multiple signals, but bag cell neurons appear to be more restrictive in this regard. Prior work finds that, as the afterdischarge proceeds, tyrosine dephosphorylation leads to biophysical alterations that promote persistent firing. Because this firing is subsequent to the cholinergic input, inhibiting the acetylcholine receptor may represent a means of properly orchestrating synaptically induced changes in excitability.

Diacylglycerol-mediated regulation of Aplysia bag cell neuron excitability requires protein kinase C.

KEY POINTS: In Aplysia, reproduction is initiated by the bag cell neurons and a prolonged period of enhanced excitability known as the afterdischarge. Phosphoinositide turnover is upregulated during the afterdischarge resulting in the hydrolysis of phosphatidylinositol-4,5-bisphosphate by phospholipase C (PLC) and the release of diacylglycerol (DAG) and inositol trisphosphate (IP3 ). In whole-cell voltage-clamped cultured bag cell neurons, 1-oleoyl-2-acetyl-sn-glycerol (OAG), a synthetic DAG analogue, activates a dose-dependent, transient, inward current (IOAG ) that is enhanced by IP3 , mimicked by PLC activation and dependent on basal protein kinase C (PKC) activity. OAG depolarizes bag cell neurons and triggers action potential firing in culture, and prolongs electrically stimulated afterdischarges in intact bag cell neuron clusters ex vivo. Although PKC alone cannot activate the current, it is required for IOAG ; this is the first description of required obligate PKC activity working in concert with PLC, DAG and IP3 to maintain the depolarization required for prolonged excitability in Aplysia reproduction. ABSTRACT: Following synaptic input, the bag cell neurons of Aplysia undergo a long-term afterdischarge of action potentials to secrete egg-laying hormone and initiate reproduction. Early in the afterdischarge, phospholipase C (PLC) hydrolyses phosphatidylinositol-4,5-bisphosphate into inositol trisphosphate (IP3 ) and diacylglycerol (DAG). In Aplysia, little is known about the action of DAG, or any interaction with IP3 ; thus, we examined the effects of a synthetic DAG analogue, 1-oleoyl-2-acetyl-sn-glycerol (OAG), on whole-cell voltage-clamped cultured bag cell neurons. OAG induced a large, prolonged, Ca(2+) -permeable, concentration-dependent inward current (IOAG ) that reversed at ∼-20 mV and was enhanced by intracellular IP3 . A similar current was evoked by either another DAG analogue, 1,2-dioctanoyl-sn-glycerol (DOG), or activating PLC with N-(3-trifluoromethylphenyl)-2,4,6-trimethylbenzenesulfonamide (m-3M3FBS). IOAG was reduced by the general cation channel blockers Gd(3+) or flufenamic acid. Work in other systems indicated that OAG activates channels independently of protein kinase C (PKC); however, we found pretreating bag cell neurons with any of the PKC inhibitors bisindolylmaleimide, sphinganine, or H7, attenuated IOAG . However, stimulating PKC with phorbol 12-myristate 13-acetate (PMA) did not evoke current or enhance IOAG ; moreover, unlike PMA, OAG failed to trigger PKC, as confirmed by an independent bioassay. Finally, OAG or m-3M3FBS depolarized cultured neurons, and while OAG did not provoke afterdischarges from bag cell neurons in the nervous system, it did double the duration of synaptically elicited afterdischarges. To our knowledge, this is the first report of obligate PKC activity for IOAG gating. An interaction between phosphoinositol metabolites and PKC could control the cation channel to influence afterdischarge duration.

Ca2+ removal by the plasma membrane Ca2+-ATPase influences the contribution of mitochondria to activity-dependent Ca2+ dynamics in Aplysia neuroendocrine cells.

After Ca(2+) influx, mitochondria can sequester Ca(2+) and subsequently release it back into the cytosol. This form of Ca(2+)-induced Ca(2+) release (CICR) prolongs Ca(2+) signaling and can potentially mediate activity-dependent plasticity. As Ca(2+) is required for its subsequent release, Ca(2+) removal systems, like the plasma membrane Ca(2+)-ATPase (PMCA), could impact CICR. Here we examine such a role for the PMCA in the bag cell neurons of Aplysia californica CICR is triggered in these neurons during an afterdischarge and is implicated in sustaining membrane excitability and peptide secretion. Somatic Ca(2+) was measured from fura-PE3-loaded cultured bag cell neurons recorded under whole cell voltage clamp. Voltage-gated Ca(2+) influx was elicited with a 5-Hz, 1-min train, which mimics the fast phase of the afterdischarge. PMCA inhibition with carboxyeosin or extracellular alkalization augmented the effectiveness of Ca(2+) influx in eliciting mitochondrial CICR. A Ca(2+) compartment model recapitulated these findings and indicated that disrupting PMCA-dependent Ca(2+) removal increases CICR by enhancing mitochondrial Ca(2+) loading. Indeed, carboxyeosin augmented train-evoked mitochondrial Ca(2+) uptake. Consistent with their role on Ca(2+) dynamics, cell labeling revealed that the PMCA and mitochondria overlap with Ca(2+) entry sites. Finally, PMCA-dependent Ca(2+) extrusion did not impact endoplasmic reticulum-dependent Ca(2+) removal or release, despite the organelle residing near Ca(2+) entry sites. Our results demonstrate that Ca(2+) removal by the PMCA influences the propensity for stimulus-evoked CICR by adjusting the amount of Ca(2+) available for mitochondrial Ca(2+) uptake. This study highlights a mechanism by which the PMCA could impact activity-dependent plasticity in the bag cell neurons.

Nicotine inhibits potassium currents in Aplysia bag cell neurons.

Acetylcholine and the archetypal cholinergic agonist, nicotine, are typically associated with the opening of ionotropic receptors. In the bag cell neurons, which govern the reproductive behavior of the marine snail, Aplysia californica, there are two cholinergic responses: a relatively large acetylcholine-induced current and a relatively small nicotine-induced current. Both currents are readily apparent at resting membrane potential and result from the opening of distinct ionotropic receptors. We now report a separate current response elicited by applying nicotine to cultured bag cell neurons under whole cell voltage-clamp. This current was ostensibly inward, best resolved at depolarized voltages, presented a noncooperative dose-response with a half-maximal concentration near 1.5 mM, and associated with a decrease in membrane conductance. The unique nicotine-evoked response was not altered by intracellular perfusion with the G protein blocker GDPßS or exposure to classical nicotinic antagonists but was occluded by replacing intracellular K(+) with Cs(+) Consistent with an underlying mechanism of direct inhibition of one or more K(+) channels, nicotine was found to rapidly reduce the fast-inactivating A-type K(+) current as well as both components of the delayed-rectifier K(+) current. Finally, nicotine increased bag cell neuron excitability, which manifested as reduction in spike threshold, greater action potential height and width, and markedly more spiking to continuous depolarizing current injection. In contrast to conventional transient activation of nicotinic ionotropic receptors, block of K(+) channels could represent a nonstandard means for nicotine to profoundly alter the electrical properties of neurons over prolonged periods of time.

Role for electrical synapses in shaping the output of coupled peptidergic neurons from Lymnaea.

Electrically coupled neurons communicate through channel assemblies called gap junctions, which mediate the transfer of current from one cell to another. Electrical synapses ensure spike synchronization and reliable transmission, which influences bursting patterns and firing frequency. The present study concerns an electrically coupled two-neuron network in the gastropod mollusc, Lymnaea stagnalis. The neurons, designated Visceral Dorsal 1 (VD1) and Right Parietal Dorsal 2 (RPD2), are peptidergic, innervate aspects of the cardio-respiratory system, and show strong coupling, such that they fire synchronously. Using dual sharp-electrode current-clamp recording and morphological staining in isolated brain preparations, the hypothesis that the electrical synapse is necessary for accurate network output was tested. We found that both cells make extensive projections within and out of the brain, including across the visceral-parietal connective, which links VD1 and RPD2. Cutting this connective uncoupled the neurons and disrupted the firing rate and pattern of RPD2 more than VD1, consistent with VD1 being the master and RPD2 the follower. The electrical synapse was inhibited by select gap junction blockers, with niflumic acid and 5-nitro-2-(3-phenylpropylamino) benzoic acid decreasing the VD1→RPD2 and RPD2→VD1 coupling coefficients, whereas carbenoxolone, α-glycyrrhetinic acid, meclofenamic acid, and quinine were ineffective. There was little-to-no impact on VD1↔RPD2 firing synchrony or frequency when coupling was reduced pharmacologically. However, in the presence of gap junction blockers, suppressing the activity of VD1 by prolonged hyperpolarization revealed a distinct, low-frequency firing pattern in RPD2. This suggests that strong electrical coupling is key to maintaining a synchronous output and proper firing rate.

PKC enhances the capacity for secretion by rapidly recruiting covert voltage-gated Ca2+ channels to the membrane.

It is unknown whether neurons can dynamically control the capacity for secretion by promptly changing the number of plasma membrane voltage-gated Ca(2+) channels. To address this, we studied peptide release from the bag cell neurons of Aplysia californica, which initiate reproduction by secreting hormone during an afterdischarge. This burst engages protein kinase C (PKC) to trigger the insertion of a covert Ca(2+) channel, Apl Cav2, alongside a basal channel, Apl Cav1. The significance of Apl Cav2 recruitment to secretion remains undetermined; therefore, we used capacitance tracking to assay secretion, along with Ca(2+) imaging and Ca(2+) current measurements, from cultured bag cell neurons under whole-cell voltage-clamp. Activating PKC with the phorbol ester, PMA, enhanced Ca(2+) entry, and potentiated stimulus-evoked secretion. This relied on channel insertion, as it was occluded by preventing Apl Cav2 engagement with prior whole-cell dialysis or the cytoskeletal toxin, latrunculin B. Channel insertion reduced the stimulus duration and/or frequency required to initiate secretion and strengthened excitation-secretion coupling, indicating that Apl Cav2 accesses peptide release more readily than Apl Cav1. The coupling of Apl Cav2 to secretion also changed with behavioral state, as Apl Cav2 failed to evoke secretion in silent neurons from reproductively inactive animals. Finally, PKC also acted secondarily to enhance prolonged exocytosis triggered by mitochondrial Ca(2+) release. Collectively, our results suggest that bag cell neurons dynamically elevate Ca(2+) channel abundance in the membrane to ensure adequate secretion during the afterdischarge.

Ca2+-induced uncoupling of Aplysia bag cell neurons.

Electrical transmission is a dynamically regulated form of communication and key to synchronizing neuronal activity. The bag cell neurons of Aplysia are a group of electrically coupled neuroendocrine cells that initiate ovulation by secreting egg-laying hormone during a prolonged period of synchronous firing called the afterdischarge. Accompanying the afterdischarge is an increase in intracellular Ca2+ and the activation of protein kinase C (PKC). We used whole cell recording from paired cultured bag cell neurons to demonstrate that electrical coupling is regulated by both Ca2+ and PKC. Elevating Ca2+ with a train of voltage steps, mimicking the onset of the afterdischarge, decreased junctional current for up to 30 min. Inhibition was most effective when Ca2+ entry occurred in both neurons. Depletion of Ca2+ from the mitochondria, but not the endoplasmic reticulum, also attenuated the electrical synapse. Buffering Ca2+ with high intracellular EGTA or inhibiting calmodulin kinase prevented uncoupling. Furthermore, activating PKC produced a small but clear decrease in junctional current, while triggering both Ca2+ influx and PKC inhibited the electrical synapse to a greater extent than Ca2+ alone. Finally, the amplitude and time course of the postsynaptic electrotonic response were attenuated after Ca2+ influx. A mathematical model of electrically connected neurons showed that excessive coupling reduced recruitment of the cells to fire, whereas less coupling led to spiking of essentially all neurons. Thus a decrease in electrical synapses could promote the afterdischarge by ensuring prompt recovery of electrotonic potentials or making the neurons more responsive to current spreading through the network.

Electrical coupling between Aplysia bag cell neurons: characterization and role in synchronous firing.

In neuroendocrine cells, hormone release often requires a collective burst of action potentials synchronized by gap junctions. This is the case for the electrically coupled bag cell neurons in the reproductive system of the marine snail, Aplysia californica. These neuroendocrine cells are found in two clusters, and fire a synchronous burst, called the afterdischarge, resulting in neuropeptide secretion and the triggering of ovulation. However, the physiology and pharmacology of the bag cell neuron electrical synapse are not completely understood. As such, we made dual whole cell recordings from pairs of electrically coupled cultured bag cell neurons. The junctional current was nonrectifying and not influenced by postsynaptic voltage. Furthermore, junctional conductance was voltage independent and, not surprisingly, strongly correlated with coupling coefficient magnitude. The electrical synapse also acted as a low-pass filter, although under certain conditions, electrotonic potentials evoked by presynaptic action potentials could drive postsynaptic spikes. If coupled neurons were stimulated to spike simultaneously, they presented a high degree of action potential synchrony compared with not-coupled neurons. The electrical synapse failed to pass various intracellular dyes, but was permeable to Cs(+), and could be inhibited by niflumic acid, meclofenamic acid, or 5-nitro-2-(3-phenylpropylamino)benzoic acid. Finally, extracellular and sharp-electrode recording from the intact bag cell neuron cluster showed that these pharmacological uncouplers disrupted both electrical coupling and afterdischarge generation in situ. Thus electrical synapses promote bag cell neuron firing synchrony and may allow for electrotonic spread of the burst through the network, ultimately contributing to propagation of the species.

A potentially novel nicotinic receptor in Aplysia neuroendocrine cells.

Nicotinic receptors form a diverse group of ligand-gated ionotropic receptors with roles in both synaptic transmission and the control of excitability. In the bag cell neurons of Aplysia, acetylcholine activates an ionotropic receptor, which passes inward current to produce a long-lasting afterdischarge and hormone release, leading to reproduction. While testing the agonist profile of the cholinergic response, we observed a second current that appeared to be gated only by nicotine and not acetylcholine. The peak nicotine-evoked current was markedly smaller in magnitude than the acetylcholine-induced current, cooperative (Hill value of 2.7), had an EC50 near 500 µM, readily recovered from desensitization, showed Ca(2+) permeability, and was blocked by mecamylamine, dihydro-ß-erythroidine, or strychnine, but not by α-conotoxin ImI, methyllycaconitine, or hexamethonium. Aplysia transcriptome analysis followed by PCR yielded 20 full-length potential nicotinic receptor subunits. Sixteen of these were predicted to be cation selective, and real-time PCR suggested that 15 of the 16 subunits were expressed to varying degrees in the bag cell neurons. The acetylcholine-induced current, but not the nicotine current, was reduced by double-strand RNA treatment targeted to both subunits ApAChR-C and -E. Conversely, the nicotine-evoked current, but not the acetylcholine current, was lessened by targeting both subunits ApAChR-H and -P. To the best of our knowledge, this is the first report suggesting that a nicotinic receptor is not gated by acetylcholine. Separate receptors may serve as a means to differentially trigger plasticity or safeguard propagation by assuring that only acetylcholine, the endogenous agonist, initiates large enough responses to trigger reproduction.

Separate Ca2+ sources are buffered by distinct Ca2+ handling systems in aplysia neuroendocrine cells.

Although the contribution of Ca(2+) buffering systems can vary between neuronal types and cellular compartments, it is unknown whether distinct Ca(2+) sources within a neuron have different buffers. As individual Ca(2+) sources can have separate functions, we propose that each is handled by unique systems. Using Aplysia californica bag cell neurons, which initiate reproduction through an afterdischarge involving multiple Ca(2+)-dependent processes, we investigated the role of endoplasmic reticulum (ER) and mitochondrial sequestration, as well as extrusion via the plasma membrane Ca(2+)-ATPase (PMCA) and Na(+)/Ca(2+) exchanger, to the clearance of voltage-gated Ca(2+) influx, Ca(2+)-induced Ca(2+)-release (CICR), and store-operated Ca(2+) influx. Cultured bag cell neurons were filled with the Ca(2+) indicator, fura-PE3, to image Ca(2+) under whole-cell voltage clamp. A 5 Hz, 1 min train of depolarizing voltage steps elicited voltage-gated Ca(2+) influx followed by EGTA-sensitive CICR from the mitochondria. A compartment model of Ca(2+) indicated the effect of EGTA on CICR was due to buffering of released mitochondrial Ca(2+) rather than uptake competition. Removal of voltage-gated Ca(2+) influx was dominated by the mitochondria and PMCA, with no contribution from the Na(+)/Ca(2+) exchanger or sarcoplasmic/endoplasmic Ca(2+)-ATPase (SERCA). In contrast, CICR recovery was slowed by eliminating the Na(+)/Ca(2+) exchanger and PMCA. Last, store-operated influx, evoked by ER depletion, was removed by the SERCA and depended on the mitochondrial membrane potential. Our results demonstrate that distinct buffering systems are dedicated to particular Ca(2+) sources. In general, this may represent a means to differentially regulate Ca(2+)-dependent processes, and for Aplysia, influence how reproductive behavior is triggered.

Decreased response to acetylcholine during aging of aplysia neuron R15.

How aging affects the communication between neurons is poorly understood. To address this question, we have studied the electrophysiological properties of identified neuron R15 of the marine mollusk Aplysia californica. R15 is a bursting neuron in the abdominal ganglia of the central nervous system and is implicated in reproduction, water balance, and heart function. Exposure to acetylcholine (ACh) causes an increase in R15 burst firing. Whole-cell recordings of R15 in the intact ganglia dissected from mature and old Aplysia showed specific changes in burst firing and properties of action potentials induced by ACh. We found that while there were no significant changes in resting membrane potential and latency in response to ACh, the burst number and burst duration is altered during aging. The action potential waveform analysis showed that unlike mature neurons, the duration of depolarization and the repolarization amplitude and duration did not change in old neurons in response to ACh. Furthermore, single neuron quantitative analysis of acetylcholine receptors (AChRs) suggested alteration of expression of specific AChRs in R15 neurons during aging. These results suggest a defect in cholinergic transmission during aging of the R15 neuron.

Acetylcholine-evoked afterdischarge in Aplysia bag cell neurons.

A brief synaptic input to the bag cell neurons of Aplysia evokes a lengthy afterdischarge and the secretion of peptide hormones that trigger ovulation. The input transmitter is unknown, although prior work has shown that afterdischarges are prevented by strychnine. Because molluscan excitatory cholinergic synapses are blocked by strychnine, we tested the hypothesis that acetylcholine acts on an ionotropic receptor to initiate the afterdischarge. In cultured bag cell neurons, acetylcholine induced a short burst of action potentials followed by either return to near baseline or, like a true afterdischarge, transition to continuous firing. The current underlying the acetylcholine-induced depolarization was dose dependent, associated with increased membrane conductance, and sensitive to the nicotinic antagonists hexamethonium, mecamylamine, and α-conotoxin ImI. Whereas nicotine, choline, carbachol, and glycine did not mimic acetylcholine, tetramethylammonium did produce a similar current. Consistent with an ionotropic receptor, the response was not altered by intracellular dialysis with the G protein blocker guanosine 5'-(ß-thio)diphosphate. Recording from the intact bag cell neuron cluster showed acetylcholine to evoke prominent depolarization, which often led to extended bursting, but only in the presence of the acetylcholinesterase inhibitor neostigmine. Extracellular recording confirmed that exogenous acetylcholine caused genuine afterdischarges, which, as per those generated synaptically, rendered the cluster refractory to further stimulation. Finally, treatment with a combination of mecamylamine and α-conotoxin ImI blocked synaptically induced afterdischarges in the intact bag cell neuron cluster. Acetylcholine appears to elicit the afterdischarge through an ionotropic receptor. This represents an expedient means for transient stimulation to elicit prolonged firing in the absence of ongoing synaptic input.

Mitochondrial Ca2+ activates a cation current in Aplysia bag cell neurons.

Ion channels may be gated by Ca(2+) entering from the extracellular space or released from intracellular stores--typically the endoplasmic reticulum. The present study examines how Ca(2+) impacts ion channels in the bag cell neurons of Aplysia californica. These neuroendocrine cells trigger ovulation through an afterdischarge involving Ca(2+) influx from Ca(2+) channels and Ca(2+) release from both the mitochondria and endoplasmic reticulum. Liberating mitochondrial Ca(2+) with the protonophore, carbonyl cyanide-4-trifluoromethoxyphenyl-hydrazone (FCCP), depolarized bag cell neurons, whereas depleting endoplasmic reticulum Ca(2+) with the Ca(2+)-ATPase inhibitor, cyclopiazonic acid, did not. In a concentration-dependent manner, FCCP elicited an inward current associated with an increase in conductance and a linear current/voltage relationship that reversed near -40 mV. The reversal potential was unaffected by changing intracellular Cl(-), but left-shifted when extracellular Ca(2+) was removed and right-shifted when intracellular K(+) was decreased. Strong buffering of intracellular Ca(2+) decreased the current, although the response was not altered by blocking Ca(2+)-dependent proteases. Furthermore, fura imaging demonstrated that FCCP elevated intracellular Ca(2+) with a time course similar to the current itself. Inhibiting either the V-type H(+)-ATPase or the ATP synthetase failed to produce a current, ruling out acidic Ca(2+) stores or disruption of ATP production as mechanisms for the FCCP response. Similarly, any involvement of reactive oxygen species potentially produced by mitochondrial depolarization was mitigated by the fact that dialysis with xanthine/xanthine oxidase did not evoke an inward current. However, both the FCCP-induced current and Ca(2+) elevation were diminished by disabling the mitochondrial permeability transition pore with the alkylating agent, N-ethylmaleimide. The data suggest that mitochondrial Ca(2+) gates a voltage-independent, nonselective cation current with the potential to drive the afterdischarge and contribute to reproduction. Employing Ca(2+) from mitochondria, rather than the more common endoplasmic reticulum, represents a diversification of the mechanisms that influence neuronal activity.

Persistent Ca2+ current contributes to a prolonged depolarization in Aplysia bag 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.