Role for protein kinase C in controlling Aplysia bag cell neuron excitability

White SH, Carter CJ, Magoski NS. A potentially novel nicotinic receptor in Aplysia neuroendocrine cells. J Neurophysiol 112: 446-462, 2014. First published April 16, 2014 doi:10.1152/jn.00796.2013.-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 EC 50 near 500 M, readily recovered from desensitization, showed Ca 2ϩ permeability, and was blocked by mecamylamine, dihydro-␤-erythroidine, or strychnine, but not by ␣-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.

Section: Nicotinic Agonists Depolarize Cultured Bag Cell Neuronsmentioning

confidence: 99%

A potentially novel nicotinic receptor inAplysianeuroendocrine cells

White

Carter

Magoski

2014

“…Phorbol 12-myristate 13-acetate (PMA; P-8139, SigmaAldrich), cyclopiazonic acid (CPA; C-1530, Sigma-Aldrich), carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP; 21857, SigmaAldrich), KN-62 (I-2142, Sigma-Aldrich), KN-92 (422709, Calbiochem/EMD Millipore, Billerica, MA), KN-93 (422708, Calbiochem), and ryanodine (559276, Calbiochem) were dissolved as stocks in dimethyl sulfoxide (DMSO; BP231-1, Fisher). The maximal final concentration of DMSO ranged from 0.05% to 0.5% (vol/vol), which in control experiments both here and in prior work from our laboratory had no effect on holding current, membrane conductance, or junctional current (Dargaei et al 2014;Hickey et al 2010Hickey et al , 2013Kachoei et al 2006;Tam et al 2011).…”

Section: Methodsmentioning

confidence: 61%

“…PKC activation enhances voltage-gated Ca 2ϩ current and increases the overall amount of Ca 2ϩ during the afterdischarge (DeRiemer et al 1985). Application of the phorbol ester PMA activates Aplysia PKC, as seen in both biochemical assays of nervous system ganglia and bag cell neurons in the intact cluster or primary culture (DeRiemer et al 1985;Sossin et al 1993;Sossin and Schwartz 1992;Tam et al 2011). The ability of PKC to close gap junctions has been shown in a number of cell types (Cruciani et al 2001;Lampe et al 2000;Spray and Bennett 1985).…”

Section: Cytosolic Camentioning

confidence: 99%

“…This may serve to elevate excitability by increasing system input resistance in the intact cluster. For instance, neurons could become more responsive to the cholinergic synaptic input that drives the start of the afterdischarge or the cation and persistent Ca 2ϩ currents that maintain the depolarization (Hung and Magoski 2007;Magoski et al 2000;Tam et al 2009Tam et al , 2011White and Magoski 2012).…”

Section: Cytosolic Camentioning

confidence: 99%

See 1 more Smart Citation

Ca²⁺-induced uncoupling ofAplysiabag cell neurons

Dargaei

Standage

Groten

et al. 2015

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.

“…The maximal final concentration of DMSO or ethanol ranged from 0.05 to 0.5% (vol/vol), which in control experiments had no effect on holding current, membrane conductance, or junctional current. Prior work from our laboratory has found that these vehicles do not alter bag cell neuron macroscopic or single-channel currents, membrane potential, membrane capacitance, or intracellular Ca 2ϩ (Magoski et al 2000;Magoski and Kaczmarek 2005;Kachoei et al 2006;Lupinsky and Magoski 2006;Hung and Magoski 2007;Gardam et al 2008;Geiger and Magoski 2008;Tam et al 2009Tam et al , 2011Hickey et al 2010Hickey et al , 2013. We also attempted to employ carbenoxolone (C4790; Sigma-Aldrich) but were unable to dissolve it into nASW from either a DMSO-or ethanol-based stock.…”

mentioning

confidence: 97%

Electrical coupling betweenAplysiabag cell neurons: characterization and role in synchronous firing

Dargaei

Colmers

Hodgson

et al. 2014

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.