Calcium clearance and its energy requirements in cerebellar neurons

Abstract

Quick cytosolic calcium clearance is essential for the effective modulation of various cellular functions. An excess of cytosolic calcium after influx is largely removed via ATP-dependent mechanisms located in the plasma membrane and the endoplasmic reticulum. Therefore, calcium clearance depends critically on the adequate supply of ATP, which may come from either glycolysis or mitochondria, or both. However, it presently remains unknown the degree to which individual ATP generating pathways -glycolysis and mitochondria power ATP-dependent calcium as well as other vital ion clearance mechanisms in neurons. In this study, we explored the relationship between the energy generating pathways and ion clearance mechanisms in neurons by characterizing the effects of glycolytic and mitochondrial inhibitors of ATP synthesis on calcium clearance kinetics in the soma, dendrites and spines. Stimulation of cultured cerebellar granule cells by brief pulses of 60 mM potassium ACSF, and electrical stimulation of purkinje cells in acutely prepared slices led to a transient calcium influx, whose clearance was largely mediated by the plasma membrane Ca 2+ -ATPase pump. Inhibition of glycolysis by deoxyglucose or iodoacetic acid resulted in a marked slowing in calcium clearance in the soma, dendrites, and spines with the spines affected the most. However, inhibition of the mitochondrial citric acid cycle with fluoroacetate and arsenite, or mitochondrial ATP-synthase with oligomycin did not produce any immediate effects on calcium clearance kinetics in any of those neuronal regions. Although cytosolic calcium clearance was not affected by the inhibition of mitochondria, the magnitude of the calcium clearance delay induced by glycolytic inhibitors in different neuronal compartments was related to their mitochondrial density. Conversely, the endoplasmic reticulum Ca 2+ -ATPase pump activity is fuelled by both glycolytic and mitochondrial ATP, as evidenced by a minimal change in the endoplasmic reticulum calcium contents in cells treated with either deoxyglucose supplemented with lactate or arsenite. Taken together, these data suggest that calcium clearance in cerebellar granule and purkinje cells relies on the plasma membrane Ca 2+ -ATPase, and is powered by glycolysis.

Brain Lactate Metabolism: The Discoveries and the Controversies

“…Postsynaptic densities contain glycolytic enzymes that synthesize ATP (Wu et al, 1997), and GLUT3 is localized in synaptic endings and postsynaptic structures (Leino et al, 1997). Calcium clearance in activated cultured cerebellar granule neurons and in Purkinje cells in brain slices relies on glycolysis to power the plasma membrane Ca 2 + -ATPase in the soma, dendrites, and spines, and inhibition of mitochondrial ATP generation does not affect operation of this pump (Ivannikov et al, 2010). These findings underscore the importance of glycolysis in neuronal dendritic spines and show that diffusion of ATP from the dendritic shaft into the spine cannot support calcium pumping at the plasma membrane of spines.…”

“…Strong Ca 2 + signals in neuronal mitochondrial reduce MAS activity, which would increase neuronal lactate production and reduce any neuronal lactate utilization (Bak et al, 2009;Contreras and Satrú stegui, 2009). Specific neuronal structures and activities depend on glycolysis, including dendritic spines that lack mitochondria (Li et al, 2004;Bourne and Harris, 2008), the plasma membrane calcium pump (Ivannikov et al, 2010), and glutamate loading into synaptic vesicles (Ikemoto et al, 2003). The cost for a neuron to package one glutamate is one ATP, which is half that required by astrocytes for glutamate-glutamine cycling (one ATP for sodium extrusion and one for glutamine synthesis).…”

Cerebral Energy Metabolism and the Brain’s Functional Network Architecture: An Integrative Review

“…55 The rate of ATP production is tightly coupled to neuronal activity by both intracellular calcium concentrations and the ATP/adenosine diphosphate (ADP) ratio. 56,57 To counterbalance local ATP consumption during g-oscillations, PV neurons likely rely on oxidative phosphorylation in the mitochondria; the metabolic process underlying most of the brain's ATP production. 58,59 Accordingly, the maintenance of g-oscillations is expected to require strong performance of the mitochondria, and correspondingly elevated local oxygen and glucose consumption.…”

Fluorescence lifetime microscopy of NADH distinguishes alterations in cerebral metabolism in vivo

“…The concentrations of pharmacological agents are provided in Fig. 1(b) and were chosen to match, or slightly exceed, concentrations utilized in published reports to disrupt metabolism in cultured cells and isolated mitochondria [4,[23][24][25][26][27][28].…”