The brain's energy demands are remarkable both in their intensity and in their moment-to-moment dynamic range. This perspective considers the evidence for Warburg-like aerobic glycolysis during the ...transient metabolic response of the brain to acute activation, and it particularly addresses the cellular mechanisms that underlie this metabolic response. The temporary uncoupling between glycolysis and oxidative phosphorylation led to the proposal of an astrocyte-to-neuron lactate shuttle whereby during stimulation, lactate produced by increased glycolysis in astrocytes is taken up by neurons as their primary energy source. However, direct evidence for this idea is lacking, and evidence rather supports that neurons have the capacity to increase their own glycolysis in response to stimulation; furthermore, neurons may export rather than import lactate in response to stimulation. The possible cellular mechanisms for invoking metabolic resupply of energy in neurons are also discussed, in particular the roles of feedback signaling via adenosine diphosphate and feedforward signaling by calcium ions.
A dietary therapy for pediatric epilepsy known as the ketogenic diet has seen a revival in its clinical use during the past decade. Although the underlying mechanism of the diet remains unknown, ...modern scientific approaches, such as the genetic disruption of glucose metabolism, are allowing for more detailed questions to be addressed. Recent work indicates that several mechanisms may exist for the ketogenic diet, including disruption of glutamatergic synaptic transmission, inhibition of glycolysis, and activation of ATP-sensitive potassium channels. Here, we describe on-going work in these areas that is providing a better understanding of metabolic influences on brain excitability and epilepsy.
•Two-photon imaging of biosensors in intact tissue is challenging to calibrate.•Ratiometric or fluorescence lifetime imaging can provide the needed normalization.•Calibrated ratiometric imaging ...requires depth and power correction.•Fluorescence lifetime imaging provides a calibrated readout of sensor occupancy.
Fluorescent biosensors are now routinely imaged using two-photon microscopy in intact tissue, for instance, in brain slices and brains in living animals. But most studies measure temporal variation—for example, calcium transients in response to neuronal activity—rather than calibrated levels of biosensor occupancy (and thus levels of the sensed analyte). True quantitative measurements are challenging, since it is difficult or impossible to calibrate a sensor's dose–response in situ, and difficult to compare the optical signals from tissue to those during in vitro calibration. Ratiometric measurements (at two wavelengths) are complicated by variations in laser power and by wavelength-dependent attenuation in tissue. For some biosensors, fluorescence lifetime imaging microscopy (FLIM) provides a valuable alternative that gives well-calibrated measurements of analyte levels.
Cytosolic NADH-NAD(+) redox state is central to cellular metabolism and a valuable indicator of glucose and lactate metabolism in living cells. Here we sought to quantitatively determine NADH-NAD(+) ...redox in live cells and brain tissue using a fluorescence lifetime imaging of the genetically-encoded single-fluorophore biosensor Peredox.
We show that Peredox exhibits a substantial change in its fluorescence lifetime over its sensing range of NADH-NAD(+) ratio. This allows changes in cytosolic NADH redox to be visualized in living cells using a two-photon scanning microscope with fluorescence lifetime imaging capabilities (2p-FLIM), using time-correlated single photon counting.
Because the lifetime readout is absolutely calibrated (in nanoseconds) and is independent of sensor concentration, we demonstrate that quantitative assessment of NADH redox is possible using a single fluorophore biosensor.
Imaging of the sensor in mouse hippocampal brain slices reveals that astrocytes are typically much more reduced (with higher NADH:NAD(+) ratio) than neurons under basal conditions, consistent with the hypothesis that astrocytes are more glycolytic than neurons. Antioxid. Redox Signal. 25, 553-563.
Genetically encoded fluorescent biosensors are powerful tools used to track chemical processes in intact biological systems. However, the development and optimization of biosensors remains a ...challenging and labor-intensive process, primarily due to technical limitations of methods for screening candidate biosensors. Here we describe a screening modality that combines droplet microfluidics and automated fluorescence imaging to provide an order of magnitude increase in screening throughput. Moreover, unlike current techniques that are limited to screening for a single biosensor feature at a time (e.g. brightness), our method enables evaluation of multiple features (e.g. contrast, affinity, specificity) in parallel. Because biosensor features can covary, this capability is essential for rapid optimization. We use this system to generate a high-performance biosensor for lactate that can be used to quantify intracellular lactate concentrations. This biosensor, named LiLac, constitutes a significant advance in metabolite sensing and demonstrates the power of our screening approach.
Certain neuron types fire spontaneously at high rates, an ability that is crucial for their function in brain circuits. The spontaneously active GABAergic neurons of the substantia nigra pars ...reticulata (SNr), a major output of the basal ganglia, provide tonic inhibition of downstream brain areas. A depolarizing 'leak' current supports this firing pattern, but its molecular basis remains poorly understood. To understand how SNr neurons maintain tonic activity, we used single-cell RNA sequencing to determine the transcriptome of individual mouse SNr neurons. We discovered that SNr neurons express the sodium leak channel, NALCN, and that SNr neurons lacking NALCN have impaired spontaneous firing. In addition, NALCN is involved in the modulation of excitability by changes in glycolysis and by activation of muscarinic acetylcholine receptors. Our findings suggest that disruption of NALCN could impair the basal ganglia circuit, which may underlie the severe motor deficits in humans carrying mutations in NALCN.
Aerobic glycolysis or the Warburg Effect (WE) is characterized by the increased metabolism of glucose to lactate. It remains unknown what quantitative changes to the activity of metabolism are ...necessary and sufficient for this phenotype. We developed a computational model of glycolysis and an integrated analysis using metabolic control analysis (MCA), metabolomics data, and statistical simulations. We identified and confirmed a novel mode of regulation specific to aerobic glycolysis where flux through GAPDH, the enzyme separating lower and upper glycolysis, is the rate-limiting step in the pathway and the levels of fructose (1,6) bisphosphate (FBP), are predictive of the rate and control points in glycolysis. Strikingly, negative flux control was found and confirmed for several steps thought to be rate-limiting in glycolysis. Together, these findings enumerate the biochemical determinants of the WE and suggest strategies for identifying the contexts in which agents that target glycolysis might be most effective.
Cellular ATP that is consumed to perform energetically expensive tasks must be replenished by new ATP through the activation of metabolism. Neuronal stimulation, an energetically demanding process, ...transiently activates aerobic glycolysis, but the precise mechanism underlying this glycolysis activation has not been determined. We previously showed that neuronal glycolysis is correlated with Ca
influx, but is not activated by feedforward Ca
signaling (Díaz-García et al., 2021a). Since ATP-powered Na
and Ca
pumping activities are increased following stimulation to restore ion gradients and are estimated to consume most neuronal ATP, we aimed to determine if they are coupled to neuronal glycolysis activation. By using two-photon imaging of fluorescent biosensors and dyes in dentate granule cell somas of acute mouse hippocampal slices, we observed that production of cytoplasmic NADH, a byproduct of glycolysis, is strongly coupled to changes in intracellular Na
, while intracellular Ca
could only increase NADH production if both forward Na
/Ca
exchange and Na
/K
pump activity were intact. Additionally, antidromic stimulation-induced intracellular Na
increases were reduced >50% by blocking Ca
entry. These results indicate that neuronal glycolysis activation is predominantly a response to an increase in activity of the Na
/K
pump, which is strongly potentiated by Na
influx through the Na
/Ca
exchanger during extrusion of Ca
following stimulation.
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