Lactate is shuttled between and inside cells, playing metabolic and signaling roles in healthy tissues. Lactate is also a harbinger of altered metabolism and participates in the pathogenesis of inflammation, hypoxia/ischemia, neurodegeneration and cancer. Many tumor cells show high rates of lactate production in the presence of oxygen, a phenomenon known as the Warburg effect, which has diagnostic and possibly therapeutic implications. In this article we introduce Laconic, a genetically-encoded Forster Resonance Energy Transfer (FRET)-based lactate sensor designed on the bacterial transcription factor LldR. Laconic quantified lactate from 1 µM to 10 mM and was not affected by glucose, pyruvate, acetate, betahydroxybutyrate, glutamate, citrate, α-ketoglutarate, succinate, malate or oxalacetate at concentrations found in mammalian cytosol. Expressed in astrocytes, HEK cells and T98G glioma cells, the sensor allowed dynamic estimation of lactate levels in single cells. Used in combination with a blocker of the monocarboxylate transporter MCT, the sensor was capable of discriminating whether a cell is a net lactate producer or a net lactate consumer. Application of the MCT-block protocol showed that the basal rate of lactate production is 3–5 fold higher in T98G glioma cells than in normal astrocytes. In contrast, the rate of lactate accumulation in response to mitochondrial inhibition with sodium azide was 10 times lower in glioma than in astrocytes, consistent with defective tumor metabolism. A ratio between the rate of lactate production and the rate of azide-induced lactate accumulation, which can be estimated reversibly and in single cells, was identified as a highly sensitive parameter of the Warburg effect, with values of 4.1 ± 0.5 for T98G glioma cells and 0.07 ± 0.007 for astrocytes. In summary, this article describes a genetically-encoded sensor for lactate and its use to measure lactate concentration, lactate flux, and the Warburg effect in single mammalian cells.

Mitochondrial flux is currently accessible at low resolution. Here we introduce a genetically-encoded FRET sensor for pyruvate, and methods for quantitative measurement of pyruvate transport, pyruvate production and mitochondrial pyruvate consumption in intact individual cells at high temporal resolution. In HEK293 cells, neurons and astrocytes, mitochondrial pyruvate uptake was saturated at physiological levels, showing that the metabolic rate is determined by intrinsic properties of the organelle and not by substrate availability. The potential of the sensor was further demonstrated in neurons, where mitochondrial flux was found to rise by 300% within seconds of a calcium transient triggered by a short theta burst, while glucose levels remained unaltered. In contrast, astrocytic mitochondria were insensitive to a similar calcium transient elicited by extracellular ATP. We expect the improved resolution provided by the pyruvate sensor will be of practical interest for basic and applied researchers interested in mitochondrial function.

SummaryDriving perception by direct activation of neural ensembles in cortex is a necessary step for achieving a causal understanding of the neural code for auditory perception and developing central sensory rehabilitation methods. Here, using optogenetic manipulations during an auditory discrimination task in mice, we show that auditory cortex can be short-circuited by coarser pathways for simple sound identification. Yet when the sensory decision becomes more complex, involving temporal integration of information, auditory cortex activity is required for sound discrimination and targeted activation of specific cortical ensembles changes perceptual decisions, as predicted by our readout of the cortical code. Hence, auditory cortex representations contribute to sound discriminations by refining decisions from parallel routes.

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Detecting rapid, coincident changes across sensory modalities is essential for recognition of sudden threats or events. Using two-photon calcium imaging in identified cell types in awake, head-fixed mice, we show that, among the basic features of a sound envelope, loud sound onsets are a dominant feature coded by the auditory cortex neurons projecting to primary visual cortex (V1). In V1, a small number of layer 1 interneurons gates this cross-modal information flow in a context-dependent manner. In dark conditions, auditory cortex inputs lead to suppression of the V1 population. However, when sound input coincides with a visual stimulus, visual responses are boosted in V1, most strongly after loud sound onsets. Thus, a dynamic, asymmetric circuit connecting AC and V1 contributes to the encoding of visual events that are coincident with sounds.

Brain tissue is highly dynamic in terms of electrical activity and energy demand. Relevant energy metabolites have turnover times ranging from milliseconds to seconds and are rapidly exchanged between cells and within cells. Until recently these fast metabolic events were inaccessible, because standard isotopic techniques require use of populations of cells and/or involve integration times of tens of minutes. Thanks to fluorescent probes and recently available genetically-encoded optical nanosensors, this Technology Report shows how it is now possible to monitor the concentration of metabolites in real-time and in single cells. In combination with ad hoc inhibitor-stop protocols, these probes have revealed a key role for K+ in the acute stimulation of astrocytic glycolysis by synaptic activity. They have also permitted detection of the Warburg effect in single cancer cells. Genetically-encoded nanosensors currently exist for glucose, lactate, NADH and ATP, and it is envisaged that other metabolite nanosensors will soon be available. These optical tools together with improved expression systems and in vivo imaging, herald an exciting era of single-cell metabolic analysis.

No abstract

Abstract. Salience is a broad and widely used concept in neuroscience whose neuronal correlates, however, remain elusive. In behavioral conditioning, salience is used to explain various effects, such as stimulus overshadowing, and refers to how fast and strongly a stimulus is associated to a conditioned event. Here, we show that sounds of diverse quality, but equally intense and perceptually detectable, recruit surprisingly different levels of population activity in mouse auditory cortex. When using these sounds as cues in a Go/NoGo discrimination task, the degree of cortical recruitment matches the salience parameter of a reinforcement learning model used to analyze learning speed. Moreover, we confirm a generic prediction of the model by training mice to discriminate light-sculpted optogenetic activity patterns in auditory cortex verifying that cortical recruitment causally determines association or overshadowing of the stimulus components. This demonstrates that cortical recruitment captures major aspects of stimulus salience during reinforcement learning.peer-reviewed)