Differential roles of auditory and visual cortex for sensory detection in mice.

Sensory cortex encompasses the regions of the cerebral cortex that receive primary sensory inputs and is crucial for conscious sensory perception in humans. Yet, some forms of perception are possible without sensory cortex. For example in animal models, the association of a sound detection to a simple behavior resists to the inactivation of auditory cortex. In contrast, post-training inactivation experiments conducted in visual or somatosensory cortex led to much stronger effects. Here we show that muscimol inactivation of visual or auditory cortex in the same detection protocol transiently abolishes visual but not auditory detection. We also observe that cortex-dependency correlates with longer reaction times. This suggests that auditory cortex is more easily bypassed by other circuits for stimulus detection than other primary sensory areas, which may be due to timing differences between auditory and visual associations.

Le cortex sensoriel englobe les régions du cortex cérébral qui reçoivent les entrées sensorielles primaires et il est crucial pour la perception sensorielle consciente chez les humains. Pourtant, certaines formes de perception sont possibles sans cortex sensoriel. Par exemple, chez des modèles animaux, l'association d'une détection sonore à un comportement simple résiste à l'inactivation du cortex auditif. En revanche, des expériences d'inactivation post-entraînement menées dans le cortex visuel ou somatosensoriel ont conduit à des effets beaucoup plus forts. Nous montrons ici que l'inactivation par le muscimol du cortex visuel ou auditif dans le même protocole de détection abolit transitoirement la détection visuelle mais pas la détection auditive. Nous observons également que la dépendance au cortex est corrélée à des temps de réaction plus longs. Cela suggère que le cortex auditif est plus facilement contourné par d'autres circuits pour la détection des stimuli que d'autres zones sensorielles primaires, ce qui peut être dû à des différences de timing entre les associations auditives et visuelles.

Targeted Cortical Manipulation of Auditory Perception.

Driving 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.

MitoToxy assay: A novel cell-based method for the assessment of metabolic toxicity in a multiwell plate format using a lactate FRET nanosensor, Laconic.

Mitochondrial toxicity is a primary source of pre-clinical drug attrition, black box warning and post-market drug withdrawal. Methods that detect mitochondrial toxicity as early as possible during the drug development process are required. Here we introduce a new method for detecting mitochondrial toxicity based on MDA-MB-231 cells stably expressing the genetically encoded FRET lactate indicator, Laconic. The method takes advantage of the high cytosolic lactate accumulation observed during mitochondrial stress, regardless of the specific toxicity mechanism, explained by compensatory glycolytic activation. Using a standard multi-well plate reader, dose-response curve experiments allowed the sensitivity of the methodology to detect metabolic toxicity induced by classical mitochondrial toxicants. Suitability for high-throughput screening applications was evaluated resulting in a Z'-factor > 0.5 and CV% < 20 inter-assay variability. A pilot screening allowed sensitive detection of commercial drugs that were previously withdrawn from the market due to liver/cardiac toxicity issues, such as camptothecin, ciglitazone, troglitazone, rosiglitazone, and terfenadine, in ten minutes. We envisage that the availability of this technology, based on a fluorescent genetically encoded indicator, will allow direct assessment of mitochondrial metabolism, and will make the early detection of mitochondrial toxicity in the drug development process possible, saving time and resources.

Context-dependent signaling of coincident auditory and visual events in primary visual cortex.

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.

Cortical recruitment determines learning dynamics and strategy.

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 can be associated with a conditioned event. Here, we identify sounds of equal intensity and perceptual detectability, which due to their spectro-temporal content recruit 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. We test an essential prediction of this model by training mice to discriminate light-sculpted optogenetic activity patterns in auditory cortex, and verify that cortical recruitment causally determines association or overshadowing of the stimulus components. This demonstrates that cortical recruitment underlies major aspects of stimulus salience during reinforcement learning.

Imaging mitochondrial flux in single cells with a FRET sensor for pyruvate.

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.

Single-cell imaging tools for brain energy metabolism: a review.

Neurophotonics comes to light at a time in which advances in microscopy and improved calcium reporters are paving the way toward high-resolution functional mapping of the brain. This review relates to a parallel revolution in metabolism. We argue that metabolism needs to be approached both in vitro and in vivo, and that it does not just exist as a low-level platform but is also a relevant player in information processing. In recent years, genetically encoded fluorescent nanosensors have been introduced to measure glucose, glutamate, ATP, NADH, lactate, and pyruvate in mammalian cells. Reporting relative metabolite levels, absolute concentrations, and metabolic fluxes, these sensors are instrumental for the discovery of new molecular mechanisms. Sensors continue to be developed, which together with a continued improvement in protein expression strategies and new imaging technologies, herald an exciting era of high-resolution characterization of metabolism in the brain and other organs.

A genetically encoded FRET lactate sensor and its use to detect the Warburg effect in single cancer cells.

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.