Simultaneous quantitative respirometry and fluorometric assays in dissected hippocampal tissue from mice.

Respirometry is a technique for studying mitochondrial function that has proven compatibility with ≥0.5 mg of brain tissue. Here, we present a protocol for assessing oxygen consumption and H2O2 production rates in hippocampal tissue using the Oroboros O2k system. We describe steps for brain harvesting, tissue preparation, hippocampal microdissection, and respirometry assays. This approach has been valuable to study the metabolism of dentate granule cells of the hippocampus and could be applicable to other brain subregions. For complete details on the use and execution of this protocol, please refer to Rose et al.1.

MLKL overexpression leads to Ca2+ and metabolic dyshomeostasis in a neuronal cell model.

The necroptotic effector molecule MLKL accumulates in neurons over the lifespan of mice, and its downregulation has the potential to improve cognition through neuroinflammation, and changes in the abundance of synaptic proteins and enzymes in the central nervous system. Notwithstanding, direct evidence of cell-autonomous effects of MLKL expression on neuronal physiology and metabolism are lacking. Here, we tested whether the overexpression of MLKL in the absence of cell death in the neuronal cell line Neuro-2a recapitulates some of the hallmarks of aging at the cellular level. Using genetically-encoded fluorescent biosensors, we monitored the cytosolic and mitochondrial Ca2+ levels, along with the cytosolic concentrations of several metabolites involved in energy metabolism (lactate, glucose, ATP) and oxidative stress (oxidized/reduced glutathione). We found that MLKL overexpression marginally decreased cell viability, however, it led to reduced cytosolic and mitochondrial Ca2+ elevations in response to Ca2+ influx from the extracellular space. On the contrary, Ca2+ signals were elevated after mobilizing Ca2+ from the endoplasmic reticulum. Transient elevations in cytosolic Ca2+, mimicking neuronal stimulation, lead to higher lactate levels and lower glucose concentrations in Neuro-2a cells when overexpressing MLKL, which suggest enhanced neuronal glycolysis. Despite these alterations, energy levels and glutathione redox state in the cell bodies remained largely preserved after inducing MLKL overexpression for 24-48 h. Taken together, our proof-of-concept experiments are consistent with the hypothesis that MLKL overexpression in the absence of cell death contributes to both Ca2+ and metabolic dyshomeostasis, which are cellular hallmarks of brain aging.

Brain energy metabolism: A roadmap for future research.

Although we have learned much about how the brain fuels its functions over the last decades, there remains much still to discover in an organ that is so complex. This article lays out major gaps in our knowledge of interrelationships between brain metabolism and brain function, including biochemical, cellular, and subcellular aspects of functional metabolism and its imaging in adult brain, as well as during development, aging, and disease. The focus is on unknowns in metabolism of major brain substrates and associated transporters, the roles of insulin and of lipid droplets, the emerging role of metabolism in microglia, mysteries about the major brain cofactor and signaling molecule NAD+, as well as unsolved problems underlying brain metabolism in pathologies such as traumatic brain injury, epilepsy, and metabolic downregulation during hibernation. It describes our current level of understanding of these facets of brain energy metabolism as well as a roadmap for future research.

Mitochondrial calcium uniporter deficiency in dentate granule cells remodels neuronal metabolism and impairs reversal learning.

The mitochondrial calcium uniporter (MCU) is the main route of calcium (Ca2+ ) entry into neuronal mitochondria. This channel has been linked to mitochondrial Ca2+ overload and cell death under neurotoxic conditions, but its physiologic roles for normal brain function remain poorly understood. Despite high expression of MCU in excitatory hippocampal neurons, it is unknown whether this channel is required for learning and memory. Here, we genetically down-regulated the Mcu gene in dentate granule cells (DGCs) of the hippocampus and found that this manipulation increases the overall respiratory activity of mitochondrial complexes I and II, augmenting the generation of reactive oxygen species in the context of impaired electron transport chain. The metabolic remodeling of MCU-deficient neurons also involved changes in the expression of enzymes that participate in glycolysis and the regulation of the tricarboxylic acid cycle, as well as the cellular antioxidant defenses. We found that MCU deficiency in DGCs does not change circadian rhythms, spontaneous exploratory behavior, or cognitive function in middle-aged mice (11-13 months old), when assessed with a food-motivated working memory test with three choices. DGC-targeted down-regulation of MCU significantly impairs reversal learning assessed with an 8-arm radial arm water maze but does not affect their ability to learn the task for the first time. Our results indicate that neuronal MCU plays an important physiologic role in memory formation and may be a potential therapeutic target to develop interventions aimed at improving cognitive function in aging, neurodegenerative diseases, and brain injury.

Metabolic regulation of species-specific developmental rates.

Animals display substantial inter-species variation in the rate of embryonic development despite a broad conservation of the overall sequence of developmental events. Differences in biochemical reaction rates, including the rates of protein production and degradation, are thought to be responsible for species-specific rates of development1-3. However, the cause of differential biochemical reaction rates between species remains unknown. Here, using pluripotent stem cells, we have established an in vitro system that recapitulates the twofold difference in developmental rate between mouse and human embryos. This system provides a quantitative measure of developmental speed as revealed by the period of the segmentation clock, a molecular oscillator associated with the rhythmic production of vertebral precursors. Using this system, we show that mass-specific metabolic rates scale with the developmental rate and are therefore higher in mouse cells than in human cells. Reducing these metabolic rates by inhibiting the electron transport chain slowed down the segmentation clock by impairing the cellular NAD+/NADH redox balance and, further downstream, lowering the global rate of protein synthesis. Conversely, increasing the NAD+/NADH ratio in human cells by overexpression of the Lactobacillus brevis NADH oxidase LbNOX increased the translation rate and accelerated the segmentation clock. These findings represent a starting point for the manipulation of developmental rate, with multiple translational applications including accelerating the differentiation of human pluripotent stem cells for disease modelling and cell-based therapies.

The Na+/K+ pump dominates control of glycolysis in hippocampal dentate granule cells.

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 Ca2+ influx, but is not activated by feedforward Ca2+ signaling (Díaz-García et al., 2021a). Since ATP-powered Na+ and Ca2+ 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 Ca2+ could only increase NADH production if both forward Na+/Ca2+ exchange and Na+/K+ pump activity were intact. Additionally, antidromic stimulation-induced intracellular [Na+] increases were reduced >50% by blocking Ca2+ 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+/Ca2+ exchanger during extrusion of Ca2+ following stimulation.

A high-throughput multiparameter screen for accelerated development and optimization of soluble genetically encoded fluorescent biosensors.

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.

The distinct roles of calcium in rapid control of neuronal glycolysis and the tricarboxylic acid cycle.

When neurons engage in intense periods of activity, the consequent increase in energy demand can be met by the coordinated activation of glycolysis, the tricarboxylic acid (TCA) cycle, and oxidative phosphorylation. However, the trigger for glycolytic activation is unknown and the role for Ca2+ in the mitochondrial responses has been debated. Using genetically encoded fluorescent biosensors and NAD(P)H autofluorescence imaging in acute hippocampal slices, here we find that Ca2+ uptake into the mitochondria is responsible for the buildup of mitochondrial NADH, probably through Ca2+ activation of dehydrogenases in the TCA cycle. In the cytosol, we do not observe a role for the Ca2+/calmodulin signaling pathway, or AMPK, in mediating the rise in glycolytic NADH in response to acute stimulation. Aerobic glycolysis in neurons is triggered mainly by the energy demand resulting from either Na+ or Ca2+ extrusion, and in mouse dentate granule cells, Ca2+ creates the majority of this demand.

Delivery of AAV for Expression of Fluorescent Biosensors in Juvenile Mouse Hippocampus.

Genetically encoded fluorescent biosensors are versatile tools for studying brain metabolism and function in live tissue. The genetic information for these biosensors can be delivered into the brain by stereotaxic injection of engineered adeno-associated viruses (AAVs), which can selectively target different cell types depending on the capsid serotype and/or the viral promoter. Here, we describe a protocol for intracranial injections of two viral vectors encoding the metabolic biosensor Peredox and the calcium biosensor RCaMP1h. When combined with 2-photon microscopy and fluorescence lifetime imaging, this protocol allows the simultaneous quantitative assessment of changes in the cytosolic NADH/NAD+ ratio and the intracellular Ca2+ levels in individual dentate granule cells from acute hippocampal slices. Graphic abstract: Workflow diagram for biosensor expression in the mouse hippocampus using intracranial injections of adeno-associated viruses.

Fluorescent Biosensors for Neuronal Metabolism and the Challenges of Quantitation.

Over the past decade, genetically encoded fluorescent biosensors that report metabolic changes have become valuable tools for understanding brain metabolism. These sensors have been targeted to specific brain regions and cell types in different organisms to track multiple metabolic processes at single cell (and subcellular) resolution. Here, we review genetically encoded biosensors used to study metabolism in the brain. We particularly focus on the principles needed to use these sensors quantitatively while avoiding false inferences from variations in sensor fluorescence that arise from differences in expression level or environmental influences such as pH or temperature.

Quantitative in vivo imaging of neuronal glucose concentrations with a genetically encoded fluorescence lifetime sensor.

Glucose is an essential source of energy for the brain. Recently, the development of genetically encoded fluorescent biosensors has allowed real time visualization of glucose dynamics from individual neurons and astrocytes. A major difficulty for this approach, even for ratiometric sensors, is the lack of a practical method to convert such measurements into actual concentrations in ex vivo brain tissue or in vivo. Fluorescence lifetime imaging provides a strategy to overcome this. In a previous study, we reported the lifetime glucose sensor iGlucoSnFR-TS (then called SweetieTS) for monitoring changes in neuronal glucose levels in response to stimulation. This genetically encoded sensor was generated by combining the Thermus thermophilus glucose-binding protein with a circularly permuted variant of the monomeric fluorescent protein T-Sapphire. Here, we provide more details on iGlucoSnFR-TS design and characterization, as well as pH and temperature sensitivities. For accurate estimation of glucose concentrations, the sensor must be calibrated at the same temperature as the experiments. We find that when the extracellular glucose concentration is in the range 2-10 mM, the intracellular glucose concentration in hippocampal neurons from acute brain slices is ~20% of the nominal external glucose concentration (~0.4-2 mM). We also measured the cytosolic neuronal glucose concentration in vivo, finding a range of ~0.7-2.5 mM in cortical neurons from awake mice.

Neurons rely on glucose rather than astrocytic lactate during stimulation.

Brain metabolism increases during stimulation, but this increase does not affect all energy metabolism equally. Briefly after stimulation, there is a local increase in cerebral blood flow and in glucose uptake, but a smaller increase in oxygen uptake. This indicates that temporarily the rate of glycolysis is faster than the rate of oxidative metabolism, with a corresponding temporary increase in lactate production. This minireview discusses the long-standing controversy about which cell type, neurons or astrocytes, are involved in this increased aerobic glycolysis. Recent biosensor studies measuring metabolic changes in neurons, in acute brain slices or in vivo, are placed in the context of other data bearing on this question. The most direct measurements indicate that, although both neurons and astrocytes may increase glycolysis after stimulation, neurons do not rely on import of astrocytic-produced lactate, and instead they increase their own glycolytic rate and become net exporters of lactate. This temporary increase in neuronal glycolysis may provide rapid energy to meet the acute energy demands of neurons.

Calcium Channels in Postnatal Development of Rat Pancreatic Beta Cells and Their Role in Insulin Secretion.

Pancreatic beta cells during the first month of development acquire functional maturity, allowing them to respond to variations in extracellular glucose concentration by secreting insulin. Changes in ionic channel activity are important for this maturation. Within the voltage-gated calcium channels (VGCC), the most studied channels are high-voltage-activated (HVA), principally L-type; while low-voltage-activated (LVA) channels have been poorly studied in native beta cells. We analyzed the changes in the expression and activity of VGCC during the postnatal development in rat beta cells. We observed that the percentage of detection of T-type current increased with the stage of development. T-type calcium current density in adult cells was higher than in neonatal and P20 beta cells. Mean HVA current density also increased with age. Calcium current behavior in P20 beta cells was heterogeneous; almost half of the cells had HVA current densities higher than the adult cells, and this was independent of the presence of T-type current. We detected the presence of α1G, α1H, and α1I subunits of LVA channels at all ages. The Cav 3.1 subunit (α1G) was the most expressed. T-type channel blockers mibefradil and TTA-A2 significantly inhibited insulin secretion at 5.6 mM glucose, which suggests a physiological role for T-type channels at basal glucose conditions. Both, nifedipine and TTA-A2, drastically decreased the beta-cell subpopulation that secretes more insulin, in both basal and stimulating glucose conditions. We conclude that changes in expression and activity of VGCC during the development play an important role in physiological maturation of beta cells.

Rat Pancreatic Beta-Cell Culture.

In this chapter, we describe the methods used to culture mainly rat pancreatic beta cells. We consider necessary to use this approach to get more information about physiological, biophysical, and molecular biology characteristics of primary beta cells. Most of the literature published has been developed in murine and human beta-cell lines. However, there are many differences between tumoral cell lines and native cells because, in contrast to cell lines, primary cells do not divide. Moreover, cell lines can be in various stages of the cell cycle and thus have a different sensitivity to glucose, compared to primary cells. Finally, for these reasons, cell lines can be heterogeneous, as the primary cells are. The main problem in using primary beta cells is that despite that they are a majority within a culture they appear mixed with other kinds of pancreatic islet cells. If one needs to identify single cells or has an only beta-cell composition, it is necessary to process the sample further. For example, one may obtain an enriched population of beta cells using fluorescence-activated cell sorting or identify single cells with the reverse hemolytic plaque assay. The other problem is that cells change with time in culture, becoming old and losing some characteristics, and so must be used preferentially during the first week. The development of human beta-cell cultures is of importance in medicine because we hope one day to be able to transplant viable beta cells to patients with diabetes mellitus type 1.

Neuronal Stimulation Triggers Neuronal Glycolysis and Not Lactate Uptake.

Proper brain function requires a substantial energy supply, up to 20% of whole-body energy in humans, and brain activation produces large dynamic variations in energy demand. While local increases in cerebral blood flow are well known, the cellular responses to energy demand are controversial. During brain excitation, glycolysis of glucose to lactate temporarily exceeds the rate of mitochondrial fuel oxidation; although the increased energy demand occurs mainly within neurons, some have suggested this glycolysis occurs mainly in astrocytes, which then shuttle lactate to neurons as their primary fuel. Using metabolic biosensors in acute hippocampal slices and brains of awake mice, we find that neuronal metabolic responses to stimulation do not depend on astrocytic stimulation by glutamate release, nor do they require neuronal uptake of lactate; instead they reflect increased direct glucose consumption by neurons. Neuronal glycolysis temporarily outstrips oxidative metabolism, and provides a rapid response to increased energy demand.

Neuropathy-specific alterations in a Mexican population of diabetic patients.

BACKGROUND: Neuropathy is one of the major complications of type 2 diabetes mellitus. Our first aim was to determine the clinical characteristics of a population of diabetic patients with different types of neuropathy. Our next goal was to characterize the cytokine profile (IL-6 and IL-10), nerve growth factor (NGF) and circulating cell-adhesion molecules in these patients. Finally, we aimed to compare the renal function among the groups of neuropathic patients. METHODS: In a cross-sectional study, we included 217 diabetic patients classified in three groups: sensory polyneuropathy with hypoesthesia (DShP) or hyperesthesia (DSHP), and motor neuropathy (DMN). Two control groups were included: one of 26 diabetic non-neuropathic patients (DNN), and the other of 375 non-diabetic (ND) healthy subjects. The participants were attending to the Mexican Institute of Social Security. RESULTS: The circulating levels of NGF were significantly lower in diabetic patients, compared to healthy subjects. The range of IL-6 and IL-10 levels in neuropathic patients was higher than the control groups; however, several samples yielded null measurements. Neuropathic patients also showed increased circulating levels of the adhesion molecules ICAM, VCAM, and E-Selectin, compared to the ND group. Moreover, neuropathic patients showed reduced glomerular filtration rates compared to healthy subjects (82-103 ml/min per 1.73 m2, data as range from 25th-75th percentiles), especially in the group with DMN (45-76 ml/min per 1.73 m2). CONCLUSIONS: Some particular alterations in neuropathic patients included -but were not limited to- changes in circulating NGF, cell adhesion molecules, inflammation, and the worsening of the renal function. This study supports the need for further clinical surveillance and interventions considering a neuropathy-related basis.

Modulation of Ionic Channels and Insulin Secretion by Drugs and Hormones in Pancreatic Beta Cells.

Pancreatic beta cells, unique cells that secrete insulin in response to an increase in glucose levels, play a significant role in glucose homeostasis. Glucose-stimulated insulin secretion (GSIS) in pancreatic beta cells has been extensively explored. In this mechanism, glucose enters the cells and subsequently the metabolic cycle. During this process, the ATP/ADP ratio increases, leading to ATP-sensitive potassium (KATP) channel closure, which initiates depolarization that is also dependent on the activity of TRP nonselective ion channels. Depolarization leads to the opening of voltage-gated Na(+) channels (Nav) and subsequently voltage-dependent Ca(2+) channels (Cav). The increase in intracellular Ca(2+) triggers the exocytosis of insulin-containing vesicles. Thus, electrical activity of pancreatic beta cells plays a central role in GSIS. Moreover, many growth factors, incretins, neurotransmitters, and hormones can modulate GSIS, and the channels that participate in GSIS are highly regulated. In this review, we focus on the principal ionic channels (KATP, Nav, and Cav channels) involved in GSIS and how classic and new proteins, hormones, and drugs regulate it. Moreover, we also discuss advances on how metabolic disorders such as metabolic syndrome and diabetes mellitus change channel activity leading to changes in insulin secretion.