Investigating lactate dynamics in brain tissue is challenging, partly because in vivo data at cellular resolution are not available. We monitored lactate in cortical astrocytes and neurons of mice using the genetically encoded FRET sensor Laconic in combination with two-photon microscopy. An intravenous lactate injection rapidly increased the Laconic signal in both astrocytes and neurons, demonstrating high lactate permeability across tissue. The signal increase was significantly smaller in astrocytes, pointing to higher basal lactate levels in these cells, confirmed by a one-point calibration protocol. Trans-acceleration of the monocarboxylate transporter with pyruvate was able to reduce intracellular lactate in astrocytes but not in neurons. Collectively, these data provide in vivo evidence for a lactate gradient from astrocytes to neurons. This gradient is a prerequisite for a carrier-mediated lactate flux from astrocytes to neurons and thus supports the astrocyte-neuron lactate shuttle model, in which astrocyte-derived lactate acts as an energy substrate for neurons.

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Highlights d Reperfusion failure despite clot retrieval leads to unfavorable outcome in stroke d In the thrombin model of stroke, z30% capillaries remain occluded after thrombolysis d Capillaries are plugged by neutrophils, hindering blood flow d Neutrophil depletion facilitates capillary reperfusion and stroke recovery

Excitatory synaptic transmission is accompanied by a local surge in interstitial lactate that occurs despite adequate oxygen availability, a puzzling phenomenon termed aerobic glycolysis. In addition to its role as an energy substrate, recent studies have shown that lactate modulates neuronal excitability acting through various targets, including NMDA receptors and G-protein-coupled receptors specific for lactate, but little is known about the cellular and molecular mechanisms responsible for the increase in interstitial lactate. Using a panel of genetically encoded fluorescence nanosensors for energy metabolites, we show here that mouse astrocytes in culture, in cortical slices, and in vivo maintain a steady-state reservoir of lactate. The reservoir was released to the extracellular space immediately after exposure of astrocytes to a physiological rise in extracellular K ϩ or cell depolarization. Cell-attached patch-clamp analysis of cultured astrocytes revealed a 37 pS lactate-permeable ion channel activated by cell depolarization. The channel was modulated by lactate itself, resulting in a positive feedback loop for lactate release. A rapid fall in intracellular lactate levels was also observed in cortical astrocytes of anesthetized mice in response to local field stimulation. The existence of an astrocytic lactate reservoir and its quick mobilization via an ion channel in response to a neuronal cue provides fresh support to lactate roles in neuronal fueling and in gliotransmission.

Little is known about the functional role of axotomized cortical neurons that survive spinal cord injury. Large thoracic spinal cord injuries in adult rats result in impairments of hindlimb function. Using retrograde tracers, we found that axotomized corticospinal axons from the hindlimb sensorimotor cortex sprouted in the cervical spinal cord. Mapping of these neurons revealed the emergence of a new forelimb corticospinal projection from the rostral part of the former hindlimb cortex. Voltage-sensitive dye (VSD) imaging and blood-oxygen-level-dependent functional magnetic resonance imaging (BOLD fMRI) revealed a stable expansion of the forelimb sensory map, covering in particular the former hindlimb cortex containing the rewired neurons. Therefore, axotomized hindlimb corticospinal neurons can be incorporated into the sensorimotor circuits of the unaffected forelimb.

It has been suggested that, in states of arousal, release of noradrenaline and -adrenergic signalling affect long-term memory formation by stimulating astrocytic lactate production from glycogen. However, the temporal relationship between cortical activity and cellular lactate fluctuations upon changes in arousal remains to be fully established. Also, the role of -adrenergic signalling and brain glycogen metabolism on neural lactate dynamics in vivo is still unknown. Here, we show that an arousalinduced increase in cortical activity triggers lactate release into the extracellular space, and this correlates with a fast and prominent lactate dip in astrocytes. The immediate drop in astrocytic lactate concentration and the parallel increase in extracellular lactate levels underline an activity-dependent lactate release from astrocytes. Moreover, when -adrenergic signalling is blocked or the brain is depleted of glycogen, the arousal-evoked cellular lactate surges are significantly reduced. We provide in vivo evidence that cortical activation upon arousal triggers lactate release from astrocytes, a rise in intracellular lactate levels mediated by -adrenergic signalling and the mobilization of lactate from glycogen stores.

Laser speckle imaging (LSI) based on the speckle contrast analysis is a simple and robust technique for imaging of heterogeneous dynamics. LSI finds frequent application for dynamical mapping of cerebral blood flow, as it features high spatial and temporal resolution. However, the quantitative interpretation of the acquired data is not straightforward for the common case of a speckle field formed by both by moving and localized scatterers such as blood cells and bone or tissue. Here we present a novel processing scheme, we call dynamic laser speckle imaging (dLSI), that can be used to correctly extract the temporal correlation parameters from the speckle contrast measured in the presence of a static or slow-evolving background. The static light contribution is derived from the measurements by cross-correlating sequential speckle images. In-vivo speckle imaging experiments performed in the rodent brain demonstrate that dLSI leads to improved results. The cerebral hemodynamic response observed through the thinned and intact skull are more pronounced in the dLSI images as compared to the standard speckle contrast analysis. The proposed method also yields benefits with respect to the quality of the speckle images by suppressing contributions of non-uniformly distributed specular reflections.Dynamic laser speckle imaging of cerebral blood flow Abstract: Laser speckle imaging (LSI) based on the speckle contrast analysis is a simple and robust technique for imaging of heterogeneous dynamics. LSI finds frequent application for dynamical mapping of cerebral blood flow, as it features high spatial and temporal resolution. However, the quantitative interpretation of the acquired data is not straightforward for the common case of a speckle field formed by both by moving and localized scatterers such as blood cells and bone or tissue. Here we present a novel processing scheme, we call dynamic laser speckle imaging (dLSI), that can be used to correctly extract the temporal correlation parameters from the speckle contrast measured in the presence of a static or slow-evolving background. The static light contribution is derived from the measurements by cross-correlating sequential speckle images. In-vivo speckle imaging experiments performed in the rodent brain demonstrate that dLSI leads to improved results. The cerebral hemodynamic response observed through the thinned and intact skull are more pronounced in the dLSI images as compared to the standard speckle contrast analysis. The proposed method also yields benefits with respect to the quality of the speckle images by suppressing contributions of non-uniformly distributed specular reflections. ©2009 Optical Society of America

Lactate has been shown to offer neuroprotection in several pathologic conditions. This beneficial effect has been attributed to its use as an alternative energy substrate. However, recent description of the expression of the HCA1 receptor for lactate in the central nervous system calls for reassessment of the mechanism by which lactate exerts its neuroprotective effects. Here, we show that HCA1 receptor expression is enhanced 24 hours after reperfusion in an middle cerebral artery occlusion stroke model, in the ischemic cortex. Interestingly, intravenous injection of L-lactate at reperfusion led to further enhancement of HCA1 receptor expression in the cortex and striatum. Using an in vitro oxygen-glucose deprivation model, we show that the HCA1 receptor agonist 3,5-dihydroxybenzoic acid reduces cell death. We also observed that D-lactate, a reputedly non-metabolizable substrate but partial HCA1 receptor agonist, also provided neuroprotection in both in vitro and in vivo ischemia models. Quite unexpectedly, we show D-lactate to be partly extracted and oxidized by the rodent brain. Finally, pyruvate offered neuroprotection in vitro whereas acetate was ineffective. Our data suggest that L- and D-lactate offer neuroprotection in ischemia most likely by acting as both an HCA1 receptor agonist for non-astrocytic (most likely neuronal) cells as well as an energy substrate.