Modulation of Low-Voltage-Activated Inward Current Permeable to Sodium and Calcium by DARPP-32 Drives Spontaneous Firing of Insect Octopaminergic Neurosecretory Cells.

Bruno Lapied,Valérie Raymond,Eléonore Moreau,Antoine Defaix,Maria Stankiewicz

doi:10.3389/fnsys.2017.00031

Abstract

Identification of the different intracellular pathways that control phosphorylation/dephosphorylation process of ionic channels represents an exciting alternative approach for studying the ionic mechanisms underlying neuronal pacemaker activity. In the central nervous system of the cockroach Periplaneta americana, octopaminergic neurons, called dorsal unpaired median (DUM; DUM neurons), generate spontaneous repetitive action potentials. Short-term cultured adult DUM neurons isolated from the terminal abdominal ganglion (TAG) of the nerve cord were used to study the regulation of a tetrodotoxin-sensitive low-voltage-activated (LVA) channel permeable to sodium and calcium (Na/Ca), under whole cell voltage- and current-clamp conditions. A bell-shaped curve illustrating the regulation of the amplitude of the maintained current vs. [ATP]i was observed. This suggested the existence of phosphorylation mechanisms. The protein kinase A (PKA) inhibitor, H89 and elevating [cyclic adenosine 3′, 5′ monophosphate, cAMP]i, increased and decreased the current amplitude, respectively. This indicated a regulation of the current via a cAMP/PKA cascade. Furthermore, intracellular application of PP2B inhibitors, cyclosporine A, FK506 and PP1/2A inhibitor, okadaic acid decreased the current amplitude. From these results and because octopamine (OA) regulates DUM neuron electrical activity via an elevation of [cAMP]i, we wanted to know if, like in vertebrate dopaminergic neurons, OA receptor (OAR) stimulation could indirectly affect the current via PKA-mediated phosphorylation of Dopamine- and cAMP-regulated Phosphoprotein-32 (DARPP-32) known to inhibit PP1/2A. Experiments were performed using intracellular application of phospho-DARPP-32 and non-phospho-DARPP-32. Phospho-DARPP-32 strongly reduced the current amplitude whereas non-phospho-DARPP-32 did not affect the current. All together, these results confirm that DARPP-32-mediated inhibition of PP1/2A regulates the maintained sodium/calcium current, which contributes to the development of the pre-depolarizing phase of the DUM neuron pacemaker activity.

Highlights

Pacemaker neurons are well characterized by their intrinsic ability to generate spontaneous beating or bursting overshooting action potentials
All experiments were performed on isolated adult dorsal unpaired median (DUM) neuron cell body exhibiting OA-like immunoreactivity (Figure 1A) and known to generate endogenous pacemaker activity even in the absence of rhythmic somatic input, which is dependent on multiple different voltage-gated currents and background currents (Figure 1B; Grolleau and Lapied, 2000; Wicher et al, 2001)
DUM neurons may be activated by sensory stimuli (e.g., Baudoux and Burrows, 1998; Field et al, 2008; Pflüger et al, 2011; Rand et al, 2012), they are defined by the absence of common somatic synaptic inputs from presynaptic neurons and by their uncommon intrinsic property allowing adequate beating pacemaker activity (Grolleau and Lapied, 2000; Wicher et al, 2001; Defaix and Lapied, 2005; Heidel and Pflüger, 2006; Lavialle-Defaix et al, 2006; Gautier et al, 2008)

Summary

Introduction

Pacemaker neurons are well characterized by their intrinsic ability to generate spontaneous beating or bursting overshooting action potentials. The molecular nature of the channels carrying persistent sodium current seems unclear, the persistent sodium current could be carried by fraction of sodium channels that fails to inactivate (Taverna et al, 1999) or the persistent sodium current could arise from the incomplete inactivation of the fast sodium current (Crill, 1996; Taddese and Bean, 2002) Besides this myriad of LVA currents, another less known LVA maintained inward current permeable to both sodium and calcium (Na/Ca) has been characterized in DUM neurons (Defaix and Lapied, 2005). These findings lead us to propose a novel control of the neuronal pacemaker mechanism

Methods

Results

Conclusion