Bag Cell Neurons Of Aplysia Californica Research Articles

An ALS-Associated Mutant SOD1 Rapidly Suppresses KCNT1 (Slack) Na+-Activated K+ Channels in Aplysia Neurons.

Mutations that alter levels of Slack (KCNT1) Na+-activated K+ current produce devastating effects on neuronal development and neuronal function. We now find that Slack currents are rapidly suppressed by oligomers of mutant human Cu/Zn superoxide dismutase 1 (SOD1), which are associated with motor neuron toxicity in an inherited form of amyotrophic lateral sclerosis (ALS). We recorded from bag cell neurons of Aplysia californica, a model system to study neuronal excitability. We found that injection of fluorescent wild-type SOD1 (wt SOD1YFP) or monomeric mutant G85R SOD1YFP had no effect on net ionic currents measured under voltage clamp. In contrast, outward potassium currents were significantly reduced by microinjection of mutant G85R SOD1YFP that had been preincubated at 37°C or of cross-linked dimers of G85R SOD1YFP. Reduction of potassium current was also seen with multimeric G85R SOD1YFP of ∼300 kDa or >300 kDa that had been cross-linked. In current clamp recordings, microinjection of cross-linked 300 kDa increased excitability by depolarizing the resting membrane potential, and decreasing the latency of action potentials triggered by depolarization. The effect of cross-linked 300 kDa on potassium current was reduced by removing Na+ from the bath solution, or by knocking down levels of Slack using siRNA. It was also prevented by pharmacological inhibition of ASK1 (apoptosis signal-regulating kinase 1) or of c-Jun N-terminal kinase, but not by an inhibitor of p38 mitogen-activated protein kinase. These results suggest that soluble mutant SOD1 oligomers rapidly trigger a kinase pathway that regulates the activity of Na+-activated K+ channels in neurons.SIGNIFICANCE STATEMENT Slack Na+-activated K+ channels (KCNT1, KNa1.1) regulate neuronal excitability but are also linked to cytoplasmic signaling pathways that control neuronal protein translation. Mutations that alter the amplitude of these currents have devastating effects on neuronal development and function. We find that injection of oligomers of mutant superoxide dismutase 1 (SOD1) into the cytoplasm of invertebrate neurons rapidly suppresses these Na+-activated K+ currents and that this effect is mediated by a MAP kinase cascade, including ASK1 and c-Jun N-terminal kinase. Because amyotrophic lateral sclerosis is a fatal adult-onset neurodegenerative disease produced by mutations in SOD1 that cause the enzyme to form toxic oligomers, our findings suggest that suppression of Slack channels may be an early step in the progression of the disease.

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.

Identification of a Vesicular Pool of Calcium Channels in the Bag Cell Neurons ofAplysia californica

To study the molecular mechanism of calcium current modulation in the bag cell neurons of Aplysia californica, we have identified calcium channel subtypes expressed in these cells and analyzed their distribution using channel-specific antibodies. Using PCR to amplify reverse-transcribed RNA from bag cell clusters, we identified two classes of calcium channel alpha1 subunit. One, BCCa-I, belongs to the ABE subfamily of calcium channels, whereas the other, BCCa-II, belongs to the SCD subfamily. Antibodies generated against the bag cell calcium channels recognize membrane proteins of approximately 210 and 280 kDa on immunoblots. Both channels are expressed in the bag cell clusters as well as in other parts of the Aplysia nervous system. BCCa-II also localizes to glia and muscle. The subcellular distribution of the two channel types is strikingly different. Antibody staining of bag cell neurons in primary culture shows that BCCa-II is present on the plasma membrane, whereas BCCa-I has a punctate, intracellular distribution consistent with a vesicular localization. The BCCa-I-containing vesicles are found in bag cell neuron somata and growth cones and occasionally in neuritic hotspots. Their distribution is similar but not identical to that of LysoTracker Red, a marker for acidic organelles, but unlike that of dense-core vesicles containing egg-laying hormone. The vesicular channels may represent the protein kinase C-sensitive calcium channels of bag cell neurons that are believed to enhance hormonal release during electrical activity.

Ionic Currents Underlying Developmental Regulation of Repetitive Firing inAplysiaBag Cell Neurons

We have investigated the developmental regulation of the ability to fire repetitively in the bag cell neurons of Aplysia californica, a neuronal system in which the behavioral effects of repetitive firing are well characterized. Adult bag cell neurons exhibit an afterdischarge, consisting of prolonged depolarization and repetitive firing, which causes the release of several peptides from these neurons that induce egg-laying behaviors. Afterdischarge can be triggered in vitro by a variety of stimuli, including electrical stimulation and exposure to the potassium channel blocker tetraethyl ammonium chloride (TEA). In contrast to adults, juvenile neurons did not exhibit afterdischarge in response to pleural-abdominal connective shock or TEA. Juvenile neurons did exhibit, however, prolonged depolarizations in the presence of TEA, perhaps reflecting the anlage of the mechanism responsible for afterdischarge in the adult. To investigate developmental mechanisms underlying the regulation of repetitive firing, we compared ionic currents in adult and juvenile bag cell neurons. We found that during the period in which these neurons acquire the capacity to fire repetitively, a number of currents are regulated: (1) three K+ currents decrease (Ca2+)-dependent K+ and two components of voltage-dependent delayed-rectifier K+ current); (2) A-type K+ current increases; and (3) two Ca2+ currents increase (basal and PKC-activated). This pattern is consistent with the increase in the ability to fire repetitively that we observe during maturation: our results indicate that developmental control of repetitive firing in this system is accompanied by selective regulation of specific ionic currents which, after maturation, play important roles in generating the afterdischarge and triggering egg-laying behaviors.

Regulated release of multiple peptides from the bag cell neurons ofAplysia californica

The bag cell neurons of Aplysia californica synthesize and store large amounts of peptides derived from the egg-laying-hormone (ELH) neuropeptide precursor. Different sets of peptides derived from the amino- and carboxyl-terminal regions of the prohormone possess unique biological activities, and are packaged in distinct sets of secretory granules. We report here quantitative measurements of the amounts of the peptide products stored in and released from the bag cell neurons using high pressure liquid chromatography (HPLC) and amino acid composition analysis. These studies demonstrate that both the autocrine acting bag cell peptides (BCPs) and ELH are released coincident with electrical activity in the bag cell cluster. The composition of the released peptide mixture is similar to that stored in the bag cells. ELH and other carboxy-terminal derived peptides are most often present at 5-fold greater levels than the BCPs. These results provide further insight into the use of multiple chemical messengers by the bag cell neurons.