Elsevier

Neuroscience

Volume 224, 8 November 2012, Pages 116-124
Neuroscience

Inhibition of endoplasmic reticulum Ca2+ ATPase in preBötzinger complex of neonatal rat does not affect respiratory rhythm generation

https://doi.org/10.1016/j.neuroscience.2012.08.016Get rights and content

Abstract

PreBötzinger complex (preBötC) neurons in the brainstem underlie respiratory rhythm generation in vitro. As a result of network interactions, preBötC neurons burst synchronously to produce rhythmic premotor inspiratory activity. Each inspiratory neuron has a characteristic 10–20 mV, 0.3–0.8 s synchronous depolarization known as the inspiratory drive potential or inspiratory envelope, topped by action potentials (APs). Mechanisms involving Ca2+ fluxes have been proposed to underlie the initiation of the inspiratory drive potential. An important source of intracellular Ca2+ is the endoplasmic reticulum (ER) in which active Ca2+ sequestration is mediated by a class of transporters termed sarco/endoplasmic reticulum Ca2+ ATPases (SERCAs). We aim to test the hypothesis that disruption of Ca2+ sequestration into the ER affects respiratory rhythm generation. We examined the effect of inhibiting SERCA on respiratory rhythm generation in an in vitro slice preparation. Bath application of the potent SERCA inhibitors thapsigargin or cyclopiazonic acid (CPA) for up to 90 min did not significantly affect the period or amplitude of respiratory-related motor output or integral and duration of inspiratory drive in preBötC neurons. We promoted the depletion of intracellular Ca2+ stores by a transient bath application of 30 mM K+ (high K+) in the continuous presence of thapsigargin or CPA. After washing out the high K+, respiratory rhythm period and amplitude returned to baseline values. These results show that after inhibition of SERCA and depletion of intracellular Ca2+ stores, respiratory rhythm remains substantially the same, suggesting that this source of Ca2+ does not significantly contribute to rhythm generation in the preBötC in vitro.

Highlights

► We examined the effect of blocking SERCA on respiratory rhythm generation in an in vitro preparation. ► Thapsigargin and CPA do not affect period or amplitude of respiratory-related motor output. ► SERCA blockade and depletion of Ca2+ stores do not affect the mechanisms for rhythm generation. ► We suggest that Ca2+ from intracellular stores does not play a relevant role for rhythm generation.

Introduction

PreBötzinger complex (preBötC) neurons in the brainstem underlie respiratory rhythm generation in vitro (Smith et al., 1991). The inspiratory phase of the respiratory cycle results from preBötC neurons firing action potentials (APs) on top of inspiratory drive potentials. The mechanisms underlying the initiation and termination of the inspiratory drive potential are unclear. The critical role of the persistent sodium current in bursting activity in the preBötC and its contribution to shaping the inspiratory drive potential remain debated (Butera et al., 1999a, Butera et al., 1999b, Del Negro et al., 2001, Pace et al., 2007a, Rybak et al., 2007, Koizumi and Smith, 2008). A mechanism dependent on a Ca2+-activated non-specific cationic current (ICAN) has been proposed as the main current underlying inspiratory drive potential initiation (Pace et al., 2007b). In this mechanism, ICAN activation requires Ca2+ flux, so three sources of Ca2+ have been proposed: NMDA receptor-mediated Ca2+ influx, Ca2+ influx through voltage-gated Ca2+ channels (VGCC) and inositol 1,4,5-trisphosphate receptor (InsP3R)-mediated intracellular Ca2+ release (Pace et al., 2007b, Pace and Del Negro, 2008).

Evidence suggests that NMDAR is not essential for respiratory rhythm generation or drive transmission (Funk et al., 1997, Morgado-Valle and Feldman, 2007). In non-neuronal systems, Ca2+ flux through l-type VGCCs has been suggested as a source for ICAN activation (Wu et al., 1998). In neurons however, the membrane potential necessary to open VGCCs is mainly achieved during APs. In active preBötC neurons Ca2+ enters to the soma only during APs, several milliseconds after initiation of the inspiratory drive (Morgado-Valle et al., 2008).

The ER accumulates and releases Ca2+ via the activity-dependent mechanism Ca2+-induced Ca2+ release (CICR) or after the activation of InsP3R. CICR is proposed as relevant for dendritic Ca2+ transients associated with inspiratory activity (Mironov, 2008); however, there is no evidence showing that CICR plays an obligatory role in respiratory rhythm generation.

To investigate how disruption of ER Ca2+ sequestration affects respiratory rhythm generation, we examined the effects of the potent, lipid soluble, cell-permeable SERCA inhibitors thapsigargin and CPA on respiratory-related rhythmic activity in a neonatal rat medullary slice preparation. These drugs deplete Ca2+ stores by preventing SERCA from counterbalancing the passive and activity-dependent Ca2+ fluxes from stores to the cytosol (Seidler et al., 1989, Plenge-Tellechea et al., 1997, Treiman et al., 1998).

We tested the hypothesis that depletion of intracellular Ca2+ stores by inhibiting SERCA affects respiratory rhythm generation in vitro. We bath applied thapsigargin or CPA while recording respiratory-related motor-output. Neither period nor amplitude of respiratory-related motor output was affected by thapsigargin or CPA. 30 mM K+ (high K+) applied in the presence of thapsigargin or CPA as a method to deplete intracellular Ca2+ stores (Friel, 2004) transiently decreased respiratory period, which returned to the baseline after removal of high K+. These results show that inhibition of SERCA and subsequent depletion of intracellular Ca2+ stores do not alter respiratory rhythm, suggesting that this source of intracellular Ca2+ does not play a relevant role for rhythm generation.

Section snippets

Medullary slice preparation

Experiments were performed on neonatal rat transverse brainstem slices that generate respiratory-related motor output (Smith et al., 1991). The Ethics Committee of the Universidad Veracruzana approved all protocols. Neonatal rats (0–3 days old) were anesthetized with isoflurane and decerebrated. The neuroaxis was isolated, the cerebellum was removed and the brainstem block mounted with the rostral side up. Under a microscope the brainstem was sectioned serially in the transverse plane using a

Effects of inhibiting SERCA on respiratory-related motor output

To explore the effects of SERCA inhibition on respiratory-related motor output, we analyzed the effect of chronic exposure (90 min) to 20 μM thapsigargin or 30 μM CPA on the period and amplitude of ∫XIIn bursts. Based on the pharmacological properties of thapsigargin and CPA described in previous reports (see Discussion) we assumed that during chronic exposure, thapsigargin and CPA concentrations would reach equilibrium in the slice. We averaged the ∫XIIn bursts occurring in 1-min segments before

Discussion

In neonatal rat medullary slices containing the preBötC, inhibition of SERCA and subsequent depletion of intracellular Ca2+ stores did not interfere with the mechanisms underlying respiratory rhythm generation. Bath application of thapsigargin or CPA, pressure ejection of thapsigargin in the preBötC, or high K+-induced CICR in the presence of thapsigargin or CPA did not change inspiratory-related ∫XIIn activity with respect to control conditions. These results have important implications for

Acknowledgements

This research was supported by IBRO Returning Home Fellowship; PROMEP SEP UV PTC-487 and PTC-419; CONACYT-128392 and CONACYT-153627.

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