Neil S. Magoski

Activation of a Ca2+-permeable cation channel produces a prolonged attenuation of intracellular Ca2+ release in Aplysia bag cell neurones

1 Brief synaptic stimulation, or exposure to Conus textile venom (CtVm), triggers an afterdischarge in the bag cell neurones of Aplysia. This is associated with an elevation of intracellular calcium ([Ca2+]i) through Ca2+ release from intracellular stores and Ca2+ entry through voltage‐gated Ca2+ channels and a non‐selective cation channel. The afterdischarge is followed by a prolonged (∼18 h) refractory period during which the ability of both electrical stimulation and CtVm to trigger afterdischarges or elevate [Ca2+]i is severely attenuated. By measuring the response of isolated cells to CtVm, we have now tested the contribution of different sources of Ca2+ elevation to the onset of the prolonged refractory period. 2 CtVm induced an increase in [Ca2+]i in both normal and Ca2+‐free saline, in part by liberating Ca2+ from a store sensitive to thapsigargin or cyclopiazonic acid, but not sensitive to heparin. 3 In the presence of extracellular Ca2+, the neurones became refractory to CtVm after a single application but recovered following ∼24 h, when CtVm could again elevate [Ca2+]i. However, this refractoriness did not develop if CtVm was applied in Ca2+‐free saline. Thus, elevation of [Ca2+]i alone does not induce refractoriness to CtVm‐induced [Ca2+]i elevation, but Ca2+ influx triggers this refractory‐like state. 4 CtVm produces a depolarization of isolated bag cell neurones. To determine if Ca2+ influx through voltage‐gated Ca2+ channels, activated during this depolarization, caused refractoriness to CtVm‐induced [Ca2+]i elevation, cells were depolarized with high external potassium (60 mm), which produced a large increase in [Ca2+]i. Nevertheless, subsequent exposure of the cells to CtVm produced a normal response, suggesting that Ca2+ influx through voltage‐gated Ca2+ channels does not induce refractoriness. 5 As a second test for the role of voltage‐gated Ca2+ channels, these channels were blocked with nifedipine. This drug failed to prevent the onset of refractoriness to CtVm‐induced [Ca2+]i elevation, providing further evidence that Ca2+ entry through voltage‐gated Ca2+ channels does not initiate refractoriness. 6 To examine if Ca2+ entry through the CtVm‐activated, non‐selective cation channel caused refractoriness, neurones were treated with a high concentration of TTX, which blocks the cation channel. TTX protected the neurones from the refractoriness to [Ca2+]i elevation produced by CtVm in Ca2+‐containing medium. 7 Using clusters of bag cell neurones in intact abdominal ganglia, we compared the ability of nifedipine and TTX to protect the cells from refractoriness to electrical stimulation. Normal, long‐lasting afterdischarges could be triggered by stimulation of an afferent input after a period of exposure to CtVm in the presence of TTX. In contrast, exposure to CtVm in the presence of nifedipine resulted in refractoriness. 8 Our data indicate that Ca2+ influx through the non‐selective cation channel renders cultured bag cell neurones refractory to repeated stimulation with CtVm. Moreover, the refractory period of the afterdischarge itself may also be initiated by Ca2+ entry through this cation channel.

Flufenamic Acid Affects Multiple Currents and Causes Intracellular Ca2+ Release in Aplysia Bag 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.

Ca2+-dependent regulation of a non-selective cation channel from Aplysia bag cell neurones

Ca2+‐activated, non‐selective cation channels feature prominently in the regulation of neuronal excitability, yet the mechanism of their Ca2+ activation is poorly defined. In the bag cell neurones of Aplysia californica, opening of a voltage‐gated, non‐selective cation channel initiates a long‐lasting afterdischarge that induces egg‐laying behaviour. The present study used single‐channel recording to investigate Ca2+ activation in this cation channel. Perfusion of Ca2+ onto the cytoplasmic face of channels in excised, inside‐out patches yielded a Ca2+ activation EC50 of 10 μm with a Hill coefficient of 0.66. Increasing Ca2+ from 100 nm to 10 μm caused an apparent hyperpolarizing shift in the open probability (Po) versus voltage curve. Beyond 10 μm Ca2+, additional changes in voltage dependence were not evident. Perfusion of Ba2+ onto the cytoplasmic face did not alter Po; moreover, in outside‐out recordings, Po was decreased by replacing external Ca2+ with Ba2+ as a charge carrier, suggesting Ca2+ influx through the channel may provide positive feedback. The lack of Ba2+ sensitivity implicated calmodulin in Ca2+ activation. Consistent with this, the application to the cytoplasmic face of calmodulin antagonists, calmidazolium and calmodulin‐binding domain, reduced Po, whereas exogenous calmodulin increased Po. Overall, the data indicated that the cation channel is activated by Ca2+ through closely associated calmodulin. Bag cell neurone intracellular Ca2+ rises markedly at the onset of the afterdischarge, which would enhance channel opening and promote bursting to elicit reproduction. Cation channels are essential to nervous system function in many organisms, and closely associated calmodulin may represent a widespread mechanism for their Ca2+ sensitivity.

Ca2+-induced Ca2+ release in Aplysia bag cell neurons requires interaction between mitochondrial and endoplasmic reticulum stores.

Intracellular Ca2+ is influenced by both Ca2+ influx and release. We examined intracellular Ca2+ following action potential firing in the bag cell neurons of Aplysia californica. Following brief synaptic input, these neuroendocrine cells undergo an afterdischarge, resulting in elevated Ca2+ and the secretion of neuropeptides to initiate reproduction. Cultured bag cell neurons were injected with the Ca2+ indicator, fura-PE3, and subjected to simultaneous imaging and electrophysiology. Delivery of a 5-Hz, 1-min train of action potentials (mimicking the fast phase of the afterdischarge) produced a Ca2+ rise that markedly outlasted the initial influx, consistent with Ca2+-induced Ca2+ release (CICR). This response was attenuated by about half with ryanodine or depletion of the endoplasmic reticulum (ER) by cyclopiazonic acid. However, depletion of the mitochondria, with carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone, essentially eliminated CICR. Dual depletion of the ER and mitochondria did not reduce CICR further than depletion of the mitochondria alone. Moreover, tetraphenylphosphonium, a blocker of mitochondrial Ca2+ release, largely prevented CICR. The Ca2+ elevation during and subsequent to a stimulus mimicking the full afterdischarge was prominent and enhanced by protein kinase C activation. Traditionally, the ER is seen as the primary Ca2+ source for CICR. However, bag cell neuron CICR represents a departure from this view in that it relies on store interaction, where Ca2+ released from the mitochondria may in turn liberate Ca2+ from the ER. This unique form of CICR may be used by both bag cell neurons, and other neurons, to initiate secretion, activate channels, or induce gene expression.

Association/Dissociation of a Channel–Kinase Complex Underlies State-Dependent Modulation

Although ion channels are regulated by protein kinases, it has yet to be established whether the behavioral state of an animal may dictate whether or not modulation by a kinase can occur. Here, we describe behaviorally relevant changes in the ability of a nonselective cation channel from Aplysia bag cell neurons to be regulated by protein kinase C (PKC). This channel drives a prolonged afterdischarge that triggers the release of egg-laying hormone and a series of reproductive behaviors. The afterdischarge is followed by a lengthy refractory period, during which additional bursting cannot be elicited. Previously, we reported that, in excised inside-out patches, the cation channel is closely associated with PKC, which increases channel activity. We now show that this channel–kinase association is plastic, because channels excised from certain neurons lack PKC-dependent modulation. Although direct application of PKC-activating phorbol ester to these patches had no effect, exposing the neurons themselves to phorbol ester reinstated modulation, suggesting that an absence of modulation was attributable to a lack of associated kinase. Furthermore, modulation was restored by pretreating neurons with either PP1 [4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine] or SU6656, inhibitors of Src tyrosine kinase, an enzyme whose Src homology 3 domain is required for channel–PKC association. Neurons that were stimulated to afterdischarge and had entered the prolonged refractory period were found to have more phosphotyrosine staining and less channel–PKC association than unstimulated neurons. These findings suggest that Src-dependent regulation of the association between the cation channel and PKC controls both the long-term excitability of these neurons and their ability to induce reproduction.

Regulation of an Aplysia bag-cell neuron cation channel by closely associated protein kinase A and a protein phosphatase.

Ion channel regulation by closely associated kinases or phosphatases has emerged as a key mechanism for orchestrating neuromodulation. An exemplary case is the nonselective cation channel that drives the afterdischarge in Aplysia bag cell neurons. Initial studies showed that this channel is modulated by both a closely associated PKC and a serine/threonine protein phosphatase (PP). In excised, inside-out patches, the addition of ATP (a phosphate source) increases open probability (PO) through PKC, and this is reversed by the PP. Previous work also reported that, in certain cases, ATP can decrease cation channel PO. The present study characterizes and provides a mechanism for this decreased PO ATP response. The kinetic change for channels inhibited by ATP was identical to the previously reported effect of exogenously applied protein kinase A (PKA) (i.e., a lengthening of the third closed-state time constant). The decreased PO ATP response was blocked by the PKA inhibitor peptide PKA6-22, and its reversal was prevented by the PP inhibitor microcystin-LR. Furthermore, PKA6-22 did not alter the increased PO ATP response. This suggests that both PKA and a PP are closely associated with these cation channels, but PKA and PKC are not simultaneously targeted. After an afterdischarge, the bag cell neurons are refractory and fail to respond to subsequent stimulation. The association of PKA with the cation channel may contribute to this decrease in excitability. Altering the constituents of a regulatory complex, such as exchanging PKA for PKC, may represent a general mechanism to precisely control ion channel function and excitability.

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.

Glutamate as a putative neurotransmitter in the mollusc, Lymnaea stagnalis

Bath-applied glutamate (10-1000 microM) produced excitatory and inhibitory responses on numerous identified neurons of the mollusc Lymnaea stagnalis. Using both in situ and in vitro preparations, glutamate or glutamate agonists produced a depolarization in identified neurons right pedal dorsal 1 and right pedal dorsal 2 and 3. However, attempts to block glutamate-evoked responses with glutamate antagonists were unsuccessful. We examined a potential glutamatergic neuron, visceral dorsal 4. Exogenous application of the peptides (GDPFLRFamide and SDPFLRFamide) could mimic the inhibitory, but not the excitatory effects of visceral dorsal 4 on its postsynaptic cells, implying the presence of a second transmitter. We tested the possibility that glutamate is this second neurotransmitter by using excitatory synapses between visceral dorsal 4 and postsynaptic cells right pedal dorsal 2 and 3, right pedal dorsal 1, visceral F group and right parietal B group neurons. Of all the putative neurotransmitters tested, only glutamate had consistent excitatory effects on these postsynaptic cells. Also, the amplitude of the right pedal dorsal 2 and 3 excitatory postsynaptic potentials was reduced in the presence of N-methyl-D-aspartate and other glutamate agonists, suggesting desensitization of the endogenous transmitter receptor. In conclusion, some identified Lymnaea neurons respond to glutamate via a receptor with novel pharmacological properties. Furthermore, a Lymnaea interneuron may employ glutamate as a transmitter at excitatory synapses.

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

Ca(2+) influx through voltage-gated Ca(2+) channels is a fundamental signaling event in neurons; however, non-traditional routes, such as non-selective cation channels, also permit Ca(2+) entry. The present study examines the Ca(2+) 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 Ca(2+)), had a reversal potential near +50 mV. In keeping with Ca(2+) permeability, this was right-shifted to approximately +60 mV in high Ca(2+) (substituted for Mg(2+)) and left-shifted to around +40 mV in zero Ca(2+) (replaced with Mg(2+)). The current showed inward rectification between +30 and +90 mV, and a conductance of 29 pS in normal Ca(2+), 30 pS in high Ca(2+), 32 pS in Ba(2+) (substituted for Ca(2+)), but only 21 pS in zero Ca(2+). Despite a greater conductance in Ba(2+), the channel did not display anomalous mol fraction in an equimolar Ca(2+)-Ba(2+) mix. Eliminating internal Mg(2+) lowered activity, but did not alter inward rectification, suggesting intracellular Mg(2+) is a fast, voltage-independent blocker. Imaging bag cell neurons in Mn(2+) saline (substituted for Ca(2+)) revealed enhanced fura-quench following cation channel activation, consistent with Mn(2+) permeating as a Ca(2+) surrogate. Finally, triggering the cation channel while tracking capacitance revealed a Ca(2+)-dependent increase in membrane surface area, consistent with vesicle fusion. Thus, the cation channel not only drives the afterdischarge, but also passes Ca(2+) to potentially initiate secretion. In general, this may represent an alternate means by which neurons elicit neuropeptide release.

Localization, physiology, and modulation of a molluskan dopaminergic synapse

We investigated the location, physiology, and modulation of an identified synapse from the central nervous system (CNS) of the mollusk Lymnaea stagnalis. Specifically, the excitatory synapse from interneuron right pedal dorsal one (RPeD1) to neurons visceral dorsal two and three (VD2/3) was examined. The gross and fine morphology of these neurons was determined by staining with Lucifer yellow or sulforhodamine. In preparations where RPeD1 was stained with Lucifer yellow and VD2/3 with sulforhodamine, the axon collaterals occupied similar regions, suggesting that these neurons make physical contact in the CNS. Digital confocal microscopy of these preparations revealed that presynaptic varicosities made apparent contact (synapses) with smooth postsynaptic axon collaterals. The number of putative synapses per preparation was about five to 10. Regarding physiology, the synaptic latency was moderately rapid at 24.1 +/- 5.2 ms. Previous work indicated that RPeD1 uses dopamine as a neurotransmitter. The RPeD1 --> VD2/3 excitatory postsynaptic potential (EPSP) and the VD2/3 bath-applied dopamine (100-microM) response displayed a similar decrease in input resistance and a similar predicted reversal potential (-31 vs. -26 mV), indicating that the synapse and exogenous dopamine activate the same conductance. Finally, bath-applied serotonin (10 microM) rapidly and reversibly depressed the RPeD1 --> VD2/3 synapse but did not affect the VD2/3 bath-applied dopamine (100-microM) response, suggesting a presynaptic locus of action for serotonin. The effect of serotonin was not associated with any changes to the pre- or postsynaptic membrane potential and input resistance, or the presynaptic action potential half-width. The RPeD1 --> VD2/3 synapse provides an opportunity to examine the anatomy and physiology of transmission, and is amenable to the study of neuromodulation.