Rapid Fluorescence Lifetime Imaging Reveals That TRPV4 Channels Promote Dysregulation of Neuronal Na+ in Ischemia.

Fluorescence imaging is an indispensable method for analysis of diverse cellular and molecular processes, enabling, for example, detection of ions, second messengers, or metabolites. Intensity-based approaches, however, are prone to artifacts introduced by changes in fluorophore concentrations. This drawback can be overcome by fluorescence lifetime imaging (FLIM) based on time-correlated single-photon counting. FLIM often necessitates long photon collection times, resulting in strong temporal binning of dynamic processes. Recently, rapidFLIM was introduced, exploiting ultra-low dead-time photodetectors together with rapid electronics. Here, we demonstrate the applicability of rapidFLIM, combined with new and improved correction schemes, for spatiotemporal fluorescence lifetime imaging of low-emission fluorophores in a biological system. Using tissue slices of hippocampi of mice of either sex, loaded with the Na+ indicator ING2, we show that improved rapidFLIM enables quantitative, dynamic imaging of neuronal Na+ signals at a full-frame temporal resolution of 0.5 Hz. Induction of transient chemical ischemia resulted in unexpectedly large Na+ influx, accompanied by considerable cell swelling. Both Na+ loading and cell swelling were dampened on inhibition of TRPV4 channels. Together, rapidFLIM enabled the spatiotemporal visualization and quantification of neuronal Na+ transients at unprecedented speed and independent from changes in cell volume. Moreover, our experiments identified TRPV4 channels as hitherto unappreciated contributors to neuronal Na+ loading on metabolic failure, suggesting this pathway as a possible target to ameliorate excitotoxic damage. Finally, rapidFLIM will allow faster and more sensitive detection of a wide range of dynamic signals with other FLIM probes, most notably those with intrinsic low-photon emission.SIGNIFICANCE STATEMENT FLIM is an indispensable method for analysis of cellular processes. FLIM often necessitates long photon collection periods, requiring the sacrifice of temporal resolution at the expense of spatial information. Here, we demonstrate the applicability of the recently introduced rapidFLIM for quantitative, dynamic imaging with low-emission fluorophores in brain slices. RapidFLIM, combined with improved correction schemes, enabled intensity-independent recording of neuronal Na+ transients at unprecedented full-frame rates of 0.5 Hz. It also allowed quantitative imaging independent from changes in cell volume, revealing a surprisingly strong and hitherto uncovered contribution of TRPV4 channels to Na+ loading on energy failure. Collectively, our study thus provides a novel, unexpected insight into the mechanisms that are responsible for Na+ changes on energy depletion.

Activation of TRPV4 channels promotes the loss of cellular ATP in organotypic slices of the mouse neocortex exposed to chemical ischemia.

The vertebrate brain has an exceptionally high energy need. During ischemia, intracellular ATP concentrations decline rapidly, resulting in the breakdown of ion gradients and cellular damage. Here, we employed the nanosensor ATeam1.03YEMK to analyse the pathways driving the loss of ATP upon transient metabolic inhibition in neurons and astrocytes of the mouse neocortex. We demonstrate that brief chemical ischemia, induced by combined inhibition of glycolysis and oxidative phosphorylation, results in a transient decrease in intracellular ATP. Neurons experienced a larger relative decline and showed less ability to recover from prolonged (>5 min) metabolic inhibition than astrocytes. Blocking voltage-gated Na+ channels or NMDA receptors ameliorated the ATP decline in neurons and astrocytes, while blocking glutamate uptake aggravated the overall reduction in neuronal ATP, confirming the central role of excitatory neuronal activity in the cellular energy loss. Unexpectedly, pharmacological inhibition of transient receptor potential vanilloid 4 (TRPV4) channels significantly reduced the ischemia-induced decline in ATP in both cell types. Imaging with Na+ -sensitive indicator dye ING-2 furthermore showed that TRPV4 inhibition also reduced ischemia-induced increases in intracellular Na+ . Altogether, our results demonstrate that neurons exhibit a higher vulnerability to brief metabolic inhibition than astrocytes. Moreover, they reveal an unexpected strong contribution of TRPV4 channels to the loss of cellular ATP and suggest that the demonstrated TRPV4-related ATP consumption is most likely a direct consequence of Na+ influx. Activation of TRPV4 channels thus provides a hitherto unacknowledged contribution to the cellular energy loss during energy failure, generating a significant metabolic cost in ischemic conditions. KEY POINTS: In the ischemic brain, cellular ATP concentrations decline rapidly, which results in the collapse of ion gradients and promotes cellular damage and death. We analysed the pathways driving the loss of ATP upon transient metabolic inhibition in neurons and astrocytes of the mouse neocortex. Our results confirm the central role of excitatory neuronal activity in the cellular energy loss and demonstrate that neurons experience a larger decline in ATP and are more vulnerable to brief metabolic stress than astrocytes. Our study also reveals a new, previously unknown involvement of osmotically activated transient receptor potential vanilloid 4 (TRPV4) channels to the reduction in cellular ATP in both cell types and indicates that this is a consequence of TRPV4-mediated Na+ influx. We conclude that activation of TRPV4 channels provides a considerable contribution to the cellular energy loss, thereby generating a significant metabolic cost in ischemic conditions.

Novel algorithms for improved detection and analysis of fluorescent signal fluctuations.

Fluorescent dyes and genetically encoded fluorescence indicators (GEFI) are common tools for visualizing concentration changes of specific ions and messenger molecules during intra- as well as intercellular communication. Using advanced imaging technologies, fluorescence indicators are a prerequisite for the analysis of physiological molecular signaling. Automated detection and analysis of fluorescence signals require to overcome several challenges, including correct estimation of fluorescence fluctuations at basal concentrations of messenger molecules, detection, and extraction of events themselves as well as proper segmentation of neighboring events. Moreover, event detection algorithms need to be sensitive enough to accurately capture localized and low amplitude events exhibiting a limited spatial extent. Here, we present two algorithms (PBasE and CoRoDe) for accurate baseline estimation and automated detection and segmentation of fluorescence fluctuations.

Exacerbation of Epilepsy by Astrocyte Alkalization and Gap Junction Uncoupling.

Seizures invite seizures. At the initial stage of epilepsy, seizures intensify with each episode; however, the mechanisms underlying this exacerbation remain to be solved. Astrocytes have a strong control over neuronal excitability and the mode of information processing. This control is accomplished by adjusting the levels of various ions in the extracellular space. The network of astrocytes connected via gap junctions allows a wider or more confined distribution of these ions depending on the open probability of the gap junctions. K+ clearance relies on the K+ uptake by astrocytes and the subsequent diffusion of K+ through the astrocyte network. When astrocytes become uncoupled, K+ clearance becomes hindered. Accumulation of extracellular K+ leads to hyperexcitability of neurons. Here, using acute hippocampal slices from mice, we uncovered that brief periods of epileptiform activity result in gap junction uncoupling. In slices that experienced short-term epileptiform activity, extracellular K+ transients in response to glutamate became prolonged. Na+ imaging with a fluorescent indicator indicated that intercellular diffusion of small cations in the astrocytic syncytium via gap junctions became rapidly restricted after epileptiform activity. Using a transgenic mouse with astrocyte-specific expression of a pH sensor (Lck-E2GFP), we confirmed that astrocytes react to epileptiform activity with intracellular alkalization. Application of Na+/HCO3- cotransporter blocker led to the suppression of intracellular alkalization of astrocytes and to the prevention of astrocyte uncoupling and hyperactivity intensification both in vitro and in vivo Therefore, the inhibition of astrocyte alkalization could become a promising therapeutic strategy for countering epilepsy development.SIGNIFICANCE STATEMENT We aimed to understand the mechanisms underlying the plastic change of forebrain circuits associated with the intensification of epilepsy. Here, we demonstrate that first-time exposure to only brief periods of epileptiform activity results in acute disturbance of the intercellular astrocyte network formed by gap junctions in hippocampal tissue slices from mice. Moreover, rapid clearance of K+ from the extracellular space was impaired. Epileptiform activity activated inward Na+/HCO3- cotransport in astrocytes by cell depolarization, resulting in their alkalization. Our data suggest that alkaline pH shifts in astrocytes lead to gap junction uncoupling, hampering K+ clearance, and thereby to exacerbation of epilepsy. Pharmacological intervention could become a promising new strategy to dampen neuronal hyperexcitability and epileptogenesis.

Ion dynamics at the energy-deprived tripartite synapse.

The anatomical and functional organization of neurons and astrocytes at 'tripartite synapses' is essential for reliable neurotransmission, which critically depends on ATP. In low energy conditions, synaptic transmission fails, accompanied by a breakdown of ion gradients, changes in membrane potentials and cell swelling. The resulting cellular damage and cell death are causal to the often devastating consequences of an ischemic stroke. The severity of ischemic damage depends on the age and the brain region in which a stroke occurs, but the reasons for this differential vulnerability are far from understood. In the present study, we address this question by developing a comprehensive biophysical model of a glutamatergic synapse to identify key determinants of synaptic failure during energy deprivation. Our model is based on fundamental biophysical principles, includes dynamics of the most relevant ions, i.e., Na+, K+, Ca2+, Cl- and glutamate, and is calibrated with experimental data. It confirms the critical role of the Na+/K+-ATPase in maintaining ion gradients, membrane potentials and cell volumes. Our simulations demonstrate that the system exhibits two stable states, one physiological and one pathological. During energy deprivation, the physiological state may disappear, forcing a transit to the pathological state, which can be reverted when blocking voltage-gated Na+ and K+ channels. Our model predicts that the transition to the pathological state is favoured if the extracellular space fraction is small. A reduction in the extracellular space volume fraction, as, e.g. observed with ageing, will thus promote the brain's susceptibility to ischemic damage. Our work provides new insights into the brain's ability to recover from energy deprivation, with translational relevance for diagnosis and treatment of ischemic strokes.

Changes in Astroglial K+ upon Brief Periods of Energy Deprivation in the Mouse Neocortex.

Malfunction of astrocytic K+ regulation contributes to the breakdown of extracellular K+ homeostasis during ischemia and spreading depolarization events. Studying astroglial K+ changes is, however, hampered by a lack of suitable techniques. Here, we combined results from fluorescence imaging, ion-selective microelectrodes, and patch-clamp recordings in murine neocortical slices with the calculation of astrocytic [K+]. Brief chemical ischemia caused a reversible ATP reduction and a transient depolarization of astrocytes. Moreover, astrocytic [Na+] increased by 24 mM and extracellular [Na+] decreased. Extracellular [K+] increased, followed by an undershoot during recovery. Feeding these data into the Goldman-Hodgkin-Katz equation revealed a baseline astroglial [K+] of 146 mM, an initial K+ loss by 43 mM upon chemical ischemia, and a transient K+ overshoot of 16 mM during recovery. It also disclosed a biphasic mismatch in astrocytic Na+/K+ balance, which was initially ameliorated, but later aggravated by accompanying changes in pH and bicarbonate, respectively. Altogether, our study predicts a loss of K+ from astrocytes upon chemical ischemia followed by a net gain. The overshooting K+ uptake will promote low extracellular K+ during recovery, likely exerting a neuroprotective effect. The resulting late cation/anion imbalance requires additional efflux of cations and/or influx of anions, the latter eventually driving delayed astrocyte swelling.

Cell-Type Dependent Regulation of the Electrogenic Na+/HCO3- Cotransporter 1 (NBCe1) by Hypoxia and Acidosis in Glioblastoma.

Glioblastoma multiforme (GBM) is the most common and malignant brain tumour. It is characterised by transcriptionally distinct cell populations. In tumour cells, physiological pH gradients between the intracellular and extracellular compartments are reversed, compared to non-cancer cells. Intracellular pH in tumour cells is alkaline, whereas extracellular pH is acidic. Consequently, the function and/or expression of pH regulating transporters might be altered. Here, we investigated protein expression and regulation of the electrogenic sodium/bicarbonate cotransporter 1 (NBCe1) in mesenchymal (MES)-like hypoxia-dependent and -independent cells, as well as in astrocyte-like glioblastoma cells following chemical hypoxia, acidosis and elucidated putative underlying molecular pathways. Immunoblotting, immunocytochemistry, and intracellular pH recording with the H+-sensitive dye 2',7'-bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein were applied. The results show NBCe1 protein abundance and active NBCe1 transport. Hypoxia upregulated NBCe1 protein and activity in MES-like hypoxia-dependent GBM cells. This effect was positively correlated with HIF-1α protein levels, was mediated by TGF-ß signalling, and was prevented by extracellular acidosis. In MES-like hypoxia-independent GBM cells, acidosis (but not hypoxia) regulated NBCe1 activity in an HIF-1α-independent manner. These results demonstrate a cell-specific adaptation of NBCe1 expression and activity to the microenvironment challenge of hypoxia and acidosis that depends on their transcriptional signature in GBM.

On the origin of ultraslow spontaneous Na+ fluctuations in neurons of the neonatal forebrain.

Spontaneous neuronal and astrocytic activity in the neonate forebrain is believed to drive the maturation of individual cells and their integration into complex brain-region-specific networks. The previously reported forms include bursts of electrical activity and oscillations in intracellular Ca2+ concentration. Here, we use ratiometric Na+ imaging to demonstrate spontaneous fluctuations in the intracellular Na+ concentration of CA1 pyramidal neurons and astrocytes in tissue slices obtained from the hippocampus of mice at postnatal days 2-4 (P2-4). These occur at very low frequency (∼2/h), can last minutes with amplitudes up to several millimolar, and mostly disappear after the first postnatal week. To further investigate their mechanisms, we model a network consisting of pyramidal neurons and interneurons. Experimentally observed Na+ fluctuations are mimicked when GABAergic inhibition in the simulated network is made depolarizing. Both our experiments and computational model show that blocking voltage-gated Na+ channels or GABAergic signaling significantly diminish the neuronal Na+ fluctuations. On the other hand, blocking a variety of other ion channels, receptors, or transporters including glutamatergic pathways does not have significant effects. Our model also shows that the amplitude and duration of Na+ fluctuations decrease as we increase the strength of glial K+ uptake. Furthermore, neurons with smaller somatic volumes exhibit fluctuations with higher frequency and amplitude. As opposed to this, larger extracellular to intracellular volume ratio observed in neonatal brain exerts a dampening effect. Finally, our model predicts that these periods of spontaneous Na+ influx leave neonatal neuronal networks more vulnerable to seizure-like states when compared with mature brain.NEW & NOTEWORTHY Spontaneous activity in the neonate forebrain plays a key role in cell maturation and brain development. We report spontaneous, ultraslow, asynchronous fluctuations in the intracellular Na+ concentration of neurons and astrocytes. We show that this activity is not correlated with the previously reported synchronous neuronal population bursting or Ca2+ oscillations, both of which occur at much faster timescales. Furthermore, extracellular K+ concentration remains nearly constant. The spontaneous Na+ fluctuations disappear after the first postnatal week.

Astrocytes of the early postnatal brain.

In the rodent forebrain, the majority of astrocytes are generated during the early postnatal phase. Following differentiation, astrocytes undergo maturation which accompanies the development of the neuronal network. Neonate astrocytes exhibit a distinct morphology and domain size which differs to their mature counterparts. Moreover, many of the plasma membrane proteins prototypical for fully developed astrocytes are only expressed at low levels at neonatal stages. These include connexins and Kir4.1, which define the low membrane resistance and highly negative membrane potential of mature astrocytes. Newborn astrocytes moreover express only low amounts of GLT-1, a glutamate transporter critical later in development. Furthermore, they show specific differences in the properties and spatio-temporal pattern of intracellular calcium signals, resulting from differences in their repertoire of receptors and signalling pathways. Therefore, roles fulfilled by mature astrocytes, including ion and transmitter homeostasis, are underdeveloped in the young brain. Similarly, astrocytic ion signalling in response to neuronal activity, a process central to neuron-glia interaction, differs between the neonate and mature brain. This review describes the unique functional properties of astrocytes in the first weeks after birth and compares them to later stages of development. We conclude that with an immature neuronal network and wider extracellular space, astrocytic support might not be as demanding and critical compared to the mature brain. The delayed differentiation and maturation of astrocytes in the first postnatal weeks might thus reflect a reduced need for active, energy-consuming regulation of the extracellular space and a less tight control of glial feedback onto synaptic transmission.

Dysregulation of Astrocyte Ion Homeostasis and Its Relevance for Stroke-Induced Brain Damage.

Ischemic stroke is a leading cause of mortality and chronic disability. Either recovery or progression towards irreversible failure of neurons and astrocytes occurs within minutes to days, depending on remaining perfusion levels. Initial damage arises from energy depletion resulting in a failure to maintain homeostasis and ion gradients between extra- and intracellular spaces. Astrocytes play a key role in these processes and are thus central players in the dynamics towards recovery or progression of stroke-induced brain damage. Here, we present a synopsis of the pivotal functions of astrocytes at the tripartite synapse, which form the basis of physiological brain functioning. We summarize the evidence of astrocytic failure and its consequences under ischemic conditions. Special emphasis is put on the homeostasis and stroke-induced dysregulation of the major monovalent ions, namely Na+, K+, H+, and Cl-, and their involvement in maintenance of cellular volume and generation of cerebral edema.

Heterogeneity of Activity-Induced Sodium Transients between Astrocytes of the Mouse Hippocampus and Neocortex: Mechanisms and Consequences.

Activity-related sodium transients induced by glutamate uptake represent a special form of astrocyte excitability. Astrocytes of the neocortex, as opposed to the hippocampus proper, also express ionotropic glutamate receptors, which might provide additional sodium influx. We compared glutamate-related sodium transients in astrocytes and neurons in slices of the neocortex and hippocampus of juvenile mice of both sexes, using widefield and multiphoton imaging. Stimulation of glutamatergic afferents or glutamate application induced sodium transients that were twice as large in neocortical as in hippocampal astrocytes, despite similar neuronal responses. Astrocyte sodium transients were reduced by ∼50% upon blocking NMDA receptors in the neocortex, but not hippocampus. Neocortical, but not hippocampal, astrocytes exhibited marked sodium increases in response to NMDA. These key differences in sodium signaling were also observed in neonates and in adults. NMDA application evoked local calcium transients in processes of neocortical astrocytes, which were dampened upon blocking sodium/calcium exchange (NCX) with KB-R7943 or SEA0400. Mathematical computation based on our data predict that NMDA-induced sodium increases drive the NCX into reverse mode, resulting in calcium influx. Together, our study reveals a considerable regional heterogeneity in astrocyte sodium transients, which persists throughout postnatal development. Neocortical astrocytes respond with much larger sodium elevations to glutamatergic activity than hippocampal astrocytes. Moreover, neocortical astrocytes experience NMDA-receptor-mediated sodium influx, which hippocampal astrocytes lack, and which drives calcium import through reverse NCX. This pathway thereby links sodium to calcium signaling and represents a new mechanism for the generation of local calcium influx in neocortical astrocytes.SIGNIFICANCE STATEMENT Astrocyte calcium signals play a central role in neuron-glia interaction. Moreover, activity-related sodium transients may represent a new form of astrocyte excitability. Here we show that activation of NMDA receptors results in prominent sodium transients in neocortical, but not hippocampal, astrocytes in the mouse brain. NMDA receptor activation is accompanied by local calcium signaling in processes of neocortical astrocytes, which is augmented by sodium-driven reversal of the sodium/calcium exchanger. Our data demonstrate a significant regional heterogeneity in the magnitude and mechanisms of astrocyte sodium transients. They also suggest a close interrelation between NMDA-receptor-mediated sodium influx and calcium signaling through the reversal of sodium/calcium exchanger, thereby establishing a new pathway for the generation of local calcium signaling in astrocyte processes.

Ionic signalling in astroglia beyond calcium.

Astrocytes are homeostatic and protective cells of the central nervous system. Astroglial homeostatic responses are tightly coordinated with neuronal activity. Astrocytes maintain neuronal excitability through regulation of extracellular ion concentrations, as well as assisting and modulating synaptic transmission by uptake and catabolism of major neurotransmitters. Moreover, they support neuronal metabolism and detoxify ammonium and reactive oxygen species. Astroglial homeostatic actions are initiated and controlled by intercellular signalling of ions, including Ca2+ , Na+ , Cl- , H+ and possibly K+ . This review summarises current knowledge on ionic signals mediated by the major monovalent ions, which occur in microdomains, as global events, or as propagating intercellular waves and thereby represent the substrate for astroglial excitability.

Bmal1-deficiency affects glial synaptic coverage of the hippocampal mossy fiber synapse and the actin cytoskeleton in astrocytes.

Bmal1 is an essential component of the molecular clockwork, which drives circadian rhythms in cell function. In Bmal1-deficient (Bmal1-/-) mice, chronodisruption is associated with cognitive deficits and progressive brain pathology including astrocytosis indicated by increased expression of glial fibrillary acidic protein (GFAP). However, relatively little is known about the impact of Bmal1-deficiency on astrocyte morphology prior to astrocytosis. Therefore, in this study we analysed astrocyte morphology in young (6-8 weeks old) adult Bmal1-/- mice. At this age, overall GFAP immunoreactivity was not increased in Bmal1-deficient mice. At the ultrastructural level, we found a decrease in the volume fraction of the fine astrocytic processes that cover the hippocampal mossy fiber synapse, suggesting an impairment of perisynaptic processes and their contribution to neurotransmission. For further analyses of actin cytoskeleton, which is essential for distal process formation, we used cultured Bmal1-/- astrocytes. Bmal1-/- astrocytes showed an impaired formation of actin stress fibers. Moreover, Bmal1-/- astrocytes showed reduced levels of the actin-binding protein cortactin (CTTN). Cttn promoter region contains an E-Box like element and chromatin immunoprecipitation revealed that Cttn is a potential Bmal1 target gene. In addition, the level of GTP-bound (active) Rho-GTPase (Rho-GTP) was reduced in Bmal1-/- astrocytes. In summary, our data demonstrate that Bmal1-deficiency affects morphology of the fine astrocyte processes prior to strong upregulation of GFAP, presumably because of impaired Cttn expression and reduced Rho-GTP activation. These morphological changes might result in altered synaptic function and, thereby, relate to cognitive deficits in chronodisruption.

Altered Gap Junction Network Topography in Mouse Models for Human Hereditary Deafness.

Anisotropic gap junctional coupling is a distinct feature of astrocytes in many brain regions. In the lateral superior olive (LSO), astrocytic networks are anisotropic and oriented orthogonally to the tonotopic axis. In CaV1.3 knock-out (KO) and otoferlin KO mice, where auditory brainstem nuclei are deprived from spontaneous cochlea-driven neuronal activity, neuronal circuitry is disturbed. So far it was unknown if this disturbance is also accompanied by an impaired topography of LSO astrocyte networks. To answer this question, we immunohistochemically analyzed the expression of astrocytic connexin (Cx) 43 and Cx30 in auditory brainstem nuclei. Furthermore, we loaded LSO astrocytes with the gap junction-permeable tracer neurobiotin and assessed the network shape and orientation. We found a strong elevation of Cx30 immunoreactivity in the LSO of CaV1.3 KO mice, while Cx43 levels were only slightly increased. In otoferlin KO mice, LSO showed a slight increase in Cx43 as well, whereas Cx30 levels were unchanged. The total number of tracer-coupled cells was unaltered and most networks were anisotropic in both KO strains. In contrast to the WTs, however, LSO networks were predominantly oriented parallel to the tonotopic axis and not orthogonal to it. Taken together, our data demonstrate that spontaneous cochlea-driven neuronal activity is not required per se for the formation of anisotropic LSO astrocyte networks. However, neuronal activity is required to establish the proper orientation of networks. Proper formation of LSO astrocyte networks thus necessitates neuronal input from the periphery, indicating a critical role of neuron-glia interaction during early postnatal development in the auditory brainstem.

Relation between activity-induced intracellular sodium transients and ATP dynamics in mouse hippocampal neurons.

KEY POINTS: Employing quantitative Na+ -imaging and Förster resonance energy transfer-based imaging with ATeam1.03YEMK (ATeam), we studied the relation between activity-induced Na+ influx and intracellular ATP in CA1 pyramidal neurons of the mouse hippocampus. Calibration of ATeam in situ enabled a quantitative estimate of changes in intracellular ATP concentrations. Different paradigms of stimulation that induced global Na+ influx into the entire neuron resulted in decreases in [ATP] in the range of 0.1-0.6 mm in somata and dendrites, while Na+ influx that was locally restricted to parts of dendrites did not evoke a detectable change in dendritic [ATP]. Our data suggest that global Na+ transients require global cellular activation of the Na+ /K+ -ATPase resulting in a consumption of ATP that transiently overrides its production. For recovery from locally restricted Na+ influx, ATP production as well as fast intracellular diffusion of ATP and Na+ might prevent a local drop in [ATP]. ABSTRACT: Excitatory neuronal activity results in the influx of Na+ through voltage- and ligand-gated channels. Recovery from accompanying increases in intracellular Na+ concentrations ([Na+ ]i ) is mainly mediated by the Na+ /K+ -ATPase (NKA) and is one of the major energy-consuming processes in the brain. Here, we analysed the relation between different patterns of activity-induced [Na+ ]i signalling and ATP in mouse hippocampal CA1 pyramidal neurons by Na+ imaging with sodium-binding benzofurane isophthalate (SBFI) and employing the genetically encoded nanosensor ATeam1.03YEMK (ATeam). In situ calibrations demonstrated a sigmoidal dependence of the ATeam Förster resonance energy transfer ratio on the intracellular ATP concentration ([ATP]i ) with an apparent KD of 2.6 mm, indicating its suitability for [ATP]i measurement. Induction of recurrent network activity resulted in global [Na+ ]i oscillations with amplitudes of ∼10 mm, encompassing somata and dendrites. These were accompanied by a steady decline in [ATP]i by 0.3-0.4 mm in both compartments. Global [Na+ ]i transients, induced by afferent fibre stimulation or bath application of glutamate, caused delayed, transient decreases in [ATP]i as well. Brief focal glutamate application that evoked transient local Na+ influx into a dendrite, however, did not result in a measurable reduction in [ATP]i . Our results suggest that ATP consumption by the NKA following global [Na+ ]i transients temporarily overrides its availability, causing a decrease in [ATP]i . Locally restricted Na+ transients, however, do not result in detectable changes in local [ATP]i , suggesting that ATP production, together with rapid intracellular diffusion of both ATP and Na+ from and to unstimulated neighbouring regions, counteracts a local energy shortage under these conditions.

FRET-based imaging of intracellular ATP in organotypic brain slices.

Active neurons require a substantial amount of adenosine triphosphate (ATP) to re-establish ion gradients degraded by ion flux across their plasma membranes. Despite this fact, neurons, in contrast to astrocytes, do not contain any significant stores of energy substrates. Recent work has provided evidence for a neuro-metabolic coupling between both cell types, in which increased glycolysis and lactate production in astrocytes support neuronal metabolism. Here, we established the cell type-specific expression of the Förster resonance energy transfer (FRET) based nanosensor ATeam1.03YEMK ("Ateam") for dynamic measurement of changes in intracellular ATP levels in organotypic brain tissue slices. To this end, adeno-associated viral vectors coding for Ateam, driven by either the synapsin- or glial fibrillary acidic protein (GFAP) promoter were employed for specific transduction of neurons or astrocytes, respectively. Chemical ischemia, induced by perfusion of tissue slices with metabolic inhibitors of cellular glycolysis and mitochondrial respiration, resulted in a rapid decrease in the cellular Ateam signal to a new, low level, indicating nominal depletion of intracellular ATP. Increasing the extracellular potassium concentration to 8 mM, thereby mimicking the release of potassium from active neurons, did not alter ATP levels in neurons. It, however, caused in an increase in ATP levels in astrocytes, a result which was confirmed in acutely isolated tissue slices. In summary, our results demonstrate that organotypic cultured slices are a reliable tool for FRET-based dynamic imaging of ATP in neurons and astrocytes. They moreover provide evidence for an increased ATP synthesis in astrocytes, but not neurons, during periods of elevated extracellular potassium concentrations.

Reverse NCX Attenuates Cellular Sodium Loading in Metabolically Compromised Cortex.

In core regions of ischemic stroke, disruption of blood flow causes breakdown of ionic gradients and, ultimately, calcium overload and cell death. In the surrounding penumbra, cells may recover upon reperfusion, but recovery is hampered by additional metabolic demands imposed by peri-infarct depolarizations (PIDs). There is evidence that sodium influx drives PIDs, but no data exist on PID-related sodium accumulations in vivo. Here, we found that PIDs in mouse neocortex are associated with propagating sodium elevations in neurons and astrocytes. Similar transient sodium elevations were induced in acute tissue slices by brief chemical ischemia. Blocking NMDA-receptors dampened sodium and accompanying calcium loads of neurons in tissue slices, while inhibiting glutamate transport diminished sodium influx into astrocytes, but amplified neuronal sodium loads. In both cell types, inhibition of sodium/calcium exchange (NCX) increased sodium transients. Blocking NCX also significantly reduced calcium transients, a result confirmed in vivo. Our study provides the first quantitative data on sodium elevations in peri-infarct regions in vivo. They suggest that sodium influx drives reversal of NCX, triggering a massive secondary calcium elevation while promoting export of sodium. Reported neuroprotective effects of NCX activity in stroke models might thus be related to its dampening of ischemia-induced sodium loading.

CD73-derived adenosine and tenascin-C control cytokine production by epicardium-derived cells formed after myocardial infarction.

Epicardium-derived cells (EPDCs) play a fundamental role in embryonic cardiac development and are reactivated in the adult heart in response to myocardial infarction (MI). In this study, EPDCs from post-MI rat hearts highly expressed the ectoenzyme CD73 and secreted the profibrotic matricellular protein tenascin-C (TNC). CD73 on EPDCs extensively generated adenosine from both extracellular ATP and NAD. This in turn stimulated the release of additional nucleotides from a Brefeldin A-sensitive intracellular pool via adenosine-A2BR signaling, forming a positive-feedback loop. A2BR activation, in addition, strongly promoted the release of major regulatory cytokines, such as IL-6, IL-11, and VEGF. TNC was found to stimulate EPDC migration and, together with ATP-P2X7R signaling, to activate inflammasomes in EPDCs via TLR4. Our results demonstrate that EPDCs are an important source of various proinflammatory factors in the post-MI heart controlled by purinergic and TNC signaling.-Hesse, J., Leberling, S., Boden, E., Friebe, D., Schmidt, T., Ding, Z., Dieterich, P., Deussen, A., Roderigo, C., Rose, C. R., Floss, D. M., Scheller, J., Schrader, J. CD73-derived adenosine and tenascin-C control cytokine production by epicardium-derived cells formed after myocardial infarction.

Glutamate transporter-associated anion channels adjust intracellular chloride concentrations during glial maturation.

Astrocytic volume regulation and neurotransmitter uptake are critically dependent on the intracellular anion concentration, but little is known about the mechanisms controlling internal anion homeostasis in these cells. Here we used fluorescence lifetime imaging microscopy (FLIM) with the chloride-sensitive dye MQAE to measure intracellular chloride concentrations in murine Bergmann glial cells in acute cerebellar slices. We found Bergmann glial [Cl- ]int to be controlled by two opposing transport processes: chloride is actively accumulated by the Na+ -K+ -2Cl- cotransporter NKCC1, and chloride efflux through anion channels associated with excitatory amino acid transporters (EAATs) reduces [Cl- ]int to values that vary upon changes in expression levels or activity of these channels. EAATs transiently form anion-selective channels during glutamate transport, and thus represent a class of ligand-gated anion channels. Age-dependent upregulation of EAATs results in a developmental chloride switch from high internal chloride concentrations (51.6 ± 2.2 mM, mean ± 95% confidence interval) during early development to adult levels (35.3 ± 0.3 mM). Simultaneous blockade of EAAT1/GLAST and EAAT2/GLT-1 increased [Cl- ]int in adult glia to neonatal values. Moreover, EAAT activation by synaptic stimulations rapidly decreased [Cl- ]int . Other tested chloride channels or chloride transporters do not contribute to [Cl- ]int under our experimental conditions. Neither genetic removal of ClC-2 nor pharmacological block of K+ -Cl- cotransporter change resting Bergmann glial [Cl- ]int in acute cerebellar slices. We conclude that EAAT anion channels play an important and unexpected role in adjusting glial intracellular anion concentration during maturation and in response to cerebellar activity. GLIA 2017;65:388-400.

Rapid sodium signaling couples glutamate uptake to breakdown of ATP in perivascular astrocyte endfeet.

Perivascular endfeet of astrocytes are highly polarized compartments that ensheath blood vessels and contribute to the blood-brain barrier. They experience calcium transients with neuronal activity, a phenomenon involved in neurovascular coupling. Endfeet also mediate the uptake of glucose from the blood, a process stimulated in active brain regions. Here, we demonstrate in mouse hippocampal tissue slices that endfeet undergo sodium signaling upon stimulation of glutamatergic synaptic activity. Glutamate-induced endfeet sodium transients were diminished by TFB-TBOA, suggesting that they were generated by sodium-dependent glutamate uptake. With local agonist application, they could be restricted to endfeet and immunohistochemical analysis revealed prominent expression of glutamate transporters GLAST and GLT-1 localized towards the neuropil vs. the vascular side of endfeet. Endfeet sodium signals spread at an apparent maximum velocity of ∼120 µm/s and directly propagated from stimulated into neighboring endfeet; this spread was omitted in Cx30/Cx43 double-deficient mice. Sodium transients resulted in elevation of intracellular magnesium, indicating a decrease in intracellular ATP. In summary, our results establish that excitatory synaptic activity and stimulation of glutamate uptake in astrocytes trigger transient sodium increases in perivascular endfeet which rapidly spread through gap junctions into neighboring endfeet and cause a reduction of intracellular ATP. The newly discovered endfeet sodium signaling thereby represents a fast, long-lived and inter-cellularly acting indicator of synaptic activity at the blood-brain barrier, which likely constitutes an important component of neuro-metabolic coupling in the brain. GLIA 2017;65:293-308.