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Cerebrospinal fluid (CSF) production occurs at a rate of 500 ml per day in the adult human. Conventional osmotic forces do not suffice to support such production rate and the molecular mechanisms underlying this fluid production remain elusive. Using ex vivo choroid plexus live imaging and isotope flux in combination with in vivo CSF production determination in mice, we identify a key component in the CSF production machinery. The Na+/K+/2Cl- cotransporter (NKCC1) expressed in the luminal membrane of choroid plexus contributes approximately half of the CSF production, via its unusual outward transport direction and its unique ability to directly couple water transport to ion translocation. We thereby establish the concept of cotransport of water as a missing link in the search for molecular pathways sustaining CSF production and redefine the current model of this pivotal physiological process. Our results provide a rational pharmacological target for pathologies involving disturbed brain fluid dynamics.
Neuronal activity causes substantial Na+ transients in fine cellular processes such as dendrites and spines. The physiological consequences of such Na+ transients are still largely unknown. High-resolution Na+ imaging is pivotal to study these questions, and, up to now, two-photon imaging with the fluorescent Na+ indicator sodium-binding benzofuran isophthalate (SBFI) has been the primary method of choice. Recently, a new Na+ indicator dye, CoroNa Green (CoroNa), that has its absorbance maximum at 492 nm, has become available. In the present study, we have compared the properties of SBFI with those of CoroNa by performing Na+ measurements in neurons of hippocampal slices. We show that CoroNa is suitable for measurement of Na+ transients using non-confocal wide-field imaging with a CCD camera. However, substantial transmembrane dye leakage and lower Na+ sensitivity are clearly disadvantages when compared to SBFI. We also tested CoroNa for its suitability for high-resolution imaging of Na+ transients using a confocal laser scanning system. We demonstrate that CoroNa, in contrast to SBFI, can be employed for confocal imaging using a conventional argon laser and report the first Na+ measurements in dendrites using this dye. In conclusion, CoroNa may prove to be a valuable tool for confocal Na+ imaging in fine cellular processes.
Inhalation of (nano)particles may lead to pulmonary inflammation. However, the precise mechanisms of particle uptake and generation of inflammatory mediators by alveolar macrophages (AM) are still poorly understood. The aim of this study was to investigate the interactions between particles and AM and their associated pro-inflammatory effects in relation to particle size and physico-chemical properties.NR8383 rat lung AM were treated with ultrafine (uf), fine (f) TiO2 or fine crystalline silica (DQ12 quartz). Physico-chemical particle properties were investigated by transmission electron microscopy, elemental analysis and thermogravimetry. Aggregation and agglomeration tendency of the particles were determined in assay-specific suspensions by means of dynamic light scattering.All three particle types were rapidly taken up by AM. DQ12 and ufTiO2 , but not fTiO2 , caused increased extracellular reactive oxygen species (ROS), heme oxygenase 1 (HO-1) mRNA expression and tumor necrosis factor (TNF)-α release. Inducible nitric oxide synthase (iNOS) mRNA expression was increased most strongly by ufTiO2 , while DQ12 exclusively triggered interleukin (IL) 1β release. However, oscillations of intracellular calcium concentration and increased intracellular ROS were observed with all three samples. Uptake inhibition experiments with cytochalasin D, chlorpromazine and a Fcγ receptor II (FcγRII) antibody revealed that the endocytosis of fTiO2 by the macrophages involves actin-dependent phagocytosis and macropinocytosis as well as clathrin-coated pit formation, whereas the uptake of ufTiO2 was dominated by FcγIIR. The uptake of DQ12 was found to be significantly reduced by all three inhibitors. Our findings suggest that the contrasting AM responses to fTiO2 , ufTiO2 and DQ12 relate to differences in the involvement of specific uptake mechanisms.
Genetically encoded nanosensors such as the FRET-based adenosine triphosphate (ATP) sensor ATeam enable the measurement of changes in ATP levels inside cells, promoting our understanding of metabolic interactions between astrocytes and neurons. The sensors are usually well characterized in vitro but display altered properties when expressed inside cells, precluding a meaningful conversion of changes in FRET ratios into changes in intracellular ATP concentrations ([ATP]) on the basis of their in vitro properties. Here, we present an experimental strategy for the intracellular calibration of two different variants of ATeam in organotypic tissue slice culture of the mouse brain. After cell-type-specific expression of the sensors in astrocytes or neurons, slices were first perfused with a saline containing the saponin β-escin to permeabilize plasma membranes for ATP. Next, cells were depleted of ATP by perfusion with ATP-free saline containing metabolic inhibitors. Finally, ATP was re-added at defined concentrations and resulting changes in the FRET ratio recorded. When employing this protocol, ATeam1.03 expressed in astrocytes reliably responds to changes in [ATP], exhibiting an apparent K D of 9.4 mM. The high-affinity sensor ATeam1.03YEMK displayed a significantly lower intracellular K D of 2.7 mM. On the basis of these calibrations, we found that induction of a recurrent neuronal network activity resulted in an initial transient increase in astrocytic [ATP] by ~0.12 mM as detected by ATeam1.03YEMK, a result confirmed using ATeam1.03. In neurons, in contrast, [ATP] immediately started to decline upon initiation of a network activity, amounting to a decrease by an average of 0.29 mM after 2 min. Taken together, our results demonstrate that ATeam1.03YEMK and ATeam1.03 display a significant increase in their apparent K D when expressed inside cells as compared with in vitro. Moreover, they show that both ATeam variants enable the quantitative detection of changes of astrocytic and neuronal [ATP] in the physiological range. ATeam1.03YEMK, however, seems preferable because its K D is close to baseline ATP levels. Finally, our data support the idea that synchronized neuronal activity initially stimulates the generation of ATP in astrocytes, presumably through increased glycolysis, whereas ATP levels in neurons decline.
Astrocytes and oligodendrocytes in different brain regions form panglial networks and the topography of such networks can correlate with neuronal topography and function. Astrocyte-oligodendrocyte networks in the lateral superior olive (LSO)-an auditory brainstem nucleus-were found to be anisotropic with a preferred orientation orthogonally to the tonotopic axis. We hypothesized that such a specialization might be present in other tonotopically organized brainstem nuclei, too. Thus, we analyzed gap junctional coupling in the center of the inferior colliculus (IC)-another nucleus of the auditory brainstem that exhibits tonotopic organization. In acute brainstem slices obtained from mice, IC networks were traced employing whole-cell patch-clamp recordings of single sulforhodamine (SR) 101-identified astrocytes and concomitant intracellular loading of the gap junction-permeable tracer neurobiotin. The majority of dye-coupled networks exhibited an oval topography, which was preferentially oriented orthogonal to the tonotopic axis. Astrocyte processes showed preferentially the same orientation indicating a correlation between astrocyte and network topography. In addition to SR101-positive astrocytes, IC networks contained oligodendrocytes. Using Na+ imaging, we analyzed the capability of IC networks to redistribute small ions. Na+ bi-directionally diffused between SR101-positive astrocytes and SR101-negative cells-presumably oligodendrocytes-showing the functionality of IC networks. Taken together, our results demonstrate that IC astrocytes and IC oligodendrocytes form functional anisotropic panglial networks that are preferentially oriented orthogonal to the tonotopic axis. Thus, our data indicate that the topographic specialization of glial networks seen in IC and LSO might be a general feature of tonotopically organized auditory brainstem nuclei.
In the vertebrate olfactory bulb (OB), axonless granule cells (GC) mediate self- and lateral inhibitory interactions between mitral/tufted cells via reciprocal dendrodendritic synapses. Locally triggered release of GABA from the large reciprocal GC spines occurs on both fast and slow time scales, possibly enabling parallel processing during olfactory perception. Here we investigate local mechanisms for asynchronous spine output. To reveal the temporal and spatial characteristics of postsynaptic ion transients, we imaged spine and adjacent dendrite Ca2 +- and Na+-signals with minimal exogenous buffering by the respective fluorescent indicator dyes upon two-photon uncaging of DNI-glutamate in OB slices from juvenile rats. Both postsynaptic fluorescence signals decayed slowly, with average half durations in the spine head of t1 / 2_Δ[Ca2 +]i ∼500 ms and t1 / 2_Δ[Na+]i ∼1,000 ms. We also analyzed the kinetics of already existing data of postsynaptic spine Ca2 +-signals in response to glomerular stimulation in OB slices from adult mice, either WT or animals with partial GC glutamate receptor deletions (NMDAR: GluN1 subunit; AMPAR: GluA2 subunit). In a large subset of spines the fluorescence signal had a protracted rise time (average time to peak ∼400 ms, range 20 to >1,000 ms). This slow rise was independent of Ca2 + entry via NMDARs, since similarly slow signals occurred in ΔGluN1 GCs. Additional Ca2 + entry in ΔGluA2 GCs (with AMPARs rendered Ca2 +-permeable), however, resulted in larger ΔF/Fs that rose yet more slowly. Thus GC spines appear to dispose of several local mechanisms to promote asynchronous GABA release, which are reflected in the time course of mitral/tufted cell recurrent inhibition.
Increased brain ammonium (NH4(+)/NH3) plays a central role in the manifestation of hepatic encephalopathy (HE), a complex syndrome associated with neurological and psychiatric alterations, which is primarily a disorder of astrocytes. Here, we analysed the influence of NH4(+)/NH3 on the calcium concentration of astrocytes in situ and studied the underlying mechanisms of NH4(+)/NH3-evoked calcium changes, employing fluorescence imaging with Fura-2 in acute tissue slices derived from different regions of the mouse brain. In the hippocampal stratum radiatum, perfusion with 5 mM NH4(+)/NH3 for 30 minutes caused a transient calcium increase in about 40% of astrocytes lasting about 10 minutes. Furthermore, the vast majority of astrocytes (∼ 90%) experienced a persistent calcium increase by ∼ 50 nM. This persistent increase was already evoked at concentrations of 1-2 mM NH4(+)/NH3, developed within 10-20 minutes and was maintained as long as the NH4(+)/NH3 was present. Qualitatively similar changes were observed in astrocytes of different neocortical regions as well as in cerebellar Bergmann glia. Inhibition of glutamine synthetase resulted in significantly larger calcium increases in response to NH4(+)/NH3, indicating that glutamine accumulation was not a primary cause. Calcium increases were not mimicked by changes in intracellular pH. Pharmacological inhibition of voltage-gated sodium channels, sodium-potassium-chloride-cotransporters (NKCC), the reverse mode of sodium/calcium exchange (NCX), AMPA- or mGluR5-receptors did not dampen NH4(+)/NH3-induced calcium increases. They were, however, significantly reduced by inhibition of NMDA receptors and depletion of intracellular calcium stores. Taken together, our measurements show that sustained exposure to NH4(+)/NH3 causes a sustained increase in intracellular calcium in astrocytes in situ, which is partly dependent on NMDA receptor activation and on release of calcium from intracellular stores. Our study furthermore suggests that dysbalance of astrocyte calcium homeostasis under hyperammonemic conditions is a widespread phenomenon, which might contribute to the disturbance of neurotransmission during HE.
Astrocytes are in close contact to excitatory synapses and express transporters which mediate the sodium-dependent uptake of glutamate. In cultured astrocytes, selective activation of glutamate transport results in sodium elevations which stimulate Na(+)/K(+)-ATPase and glucose uptake, indicating that synaptic release of glutamate might couple excitatory neuronal activity to glial sodium homeostasis and metabolism. Here, we analysed intracellular sodium transients evoked by synaptic stimulation in acute mouse hippocampal slices using quantitative sodium imaging with the sodium-sensitive fluorescent indicator dye SBFI (sodium-binding benzofuran isophthalate). We found that short bursts of Schaffer collateral stimulation evoke sodium transients in the millimolar range in both CA1 pyramidal neurons and in SR101-positive astrocytes of the stratum radiatum. At low stimulation intensities, glial sodium transients were confined to one to two primary branches and adjacent fine processes and only weakly invaded the soma. Increasing the number of activated afferent fibres by increasing the stimulation intensity elicited global sodium transients detectable in the processes as well as the somata of astrocytes. Pharmacological analysis revealed that neuronal sodium signals were mainly attributable to sodium influx through ionotropic glutamate receptors. Activation of ionotropic receptors also contributed to glial sodium transients, while TBOA-sensitive glutamate transport was the major pathway responsible for sodium influx into astrocytes. Our results thus establish that glutamatergic synaptic transmission in the hippocampus results in sodium transients in astrocytes that are mainly mediated by activation of glutamate transport. They support the proposed link between excitatory synaptic activity, glutamate uptake and sodium signals in astrocytes of the hippocampus.
The identification of factors that compromise neurogenesis is aimed at improving stem cell-based approaches in the field of regenerative medicine. Interferon gamma (IFNγ) is a main pro-inflammatory cytokine and up-regulated during several neurological diseases. IFNγ is generally thought to beneficially enhance neurogenesis from fetal or adult neural stem/precursor cells (NSPCs).
Astrocytes express the sodium-dependent glutamate transporters GLAST and GLT-1, which are critical to maintain low extracellular glutamate concentrations. Here, we analyzed changes in their expression and function following a mechanical lesion in the CA1 area of organotypic hippocampal slices. 6-7 days after lesion, a glial scar had formed along the injury site, containing strongly activated astrocytes with increased GFAP and S100 β immunoreactivity, enlarged somata, and reduced capability for uptake of SR101. Astrocytes in the scar's periphery were swollen as well, but showed only moderate upregulation of GFAP and S100 β and efficiently took up SR101. In the scar, clusters of GLT-1 and GLAST immunoreactivity colocalized with GFAP-positive fibers. Apart from these, GLT-1 immunoreactivity declined with increasing distance from the scar, whereas GLAST expression appeared largely uniform. Sodium imaging in reactive astrocytes indicated that glutamate uptake was strongly reduced in the scar but maintained in the periphery. Our results thus show that moderately reactive astrocytes in the lesion periphery maintain overall glutamate transporter expression and function. Strongly reactive astrocytes in the scar, however, display clusters of GLAST and GLT-1 immunoreactivity together with reduced glutamate transport activity. This reduction might contribute to increased extracellular glutamate concentrations and promote excitotoxic cell damage at the lesion site.
Recently, we established that recurrent activity evokes network sodium oscillations in neurons and astrocytes in hippocampal tissue slices. Interestingly, metabolic integrity of astrocytes was essential for the neurons' capacity to maintain low sodium and to recover from sodium loads, indicating an intimate metabolic coupling between the 2 cell types. Here, we studied if lactate can support neuronal sodium homeostasis during impaired energy metabolism by analyzing whether glucose removal, pharmacological inhibition of glycolysis and/or addition of lactate affect cellular sodium regulation. Furthermore, we studied the effect of lactate on sodium regulation during recurrent network activity and upon inhibition of the glial Krebs cycle by sodium-fluoroacetate. Our results indicate that lactate is preferentially used by neurons. They demonstrate that lactate supports neuronal sodium homeostasis and rescues the effects of glial poisoning by sodium-fluoroacetate. Altogether, they are in line with the proposed transfer of lactate from astrocytes to neurons, the so-called astrocyte-neuron-lactate shuttle.
One hallmark of adult neurogenesis is its adaptability to environmental influences. Here, we uncovered the epithelial sodium channel (ENaC) as a key regulator of adult neurogenesis as its deletion in neural stem cells (NSCs) and their progeny in the murine subependymal zone (SEZ) strongly impairs their proliferation and neurogenic output in the olfactory bulb. Importantly, alteration of fluid flow promotes proliferation of SEZ cells in an ENaC-dependent manner, eliciting sodium and calcium signals that regulate proliferation via calcium-release-activated channels and phosphorylation of ERK. Flow-induced calcium signals are restricted to NSCs in contact with the ventricular fluid, thereby providing a highly specific mechanism to regulate NSC behavior at this special interface with the cerebrospinal fluid. Thus, ENaC plays a central role in regulating adult neurogenesis, and among multiple modes of ENaC function, flow-induced changes in sodium signals are critical for NSC biology.
Dendrodendritic synaptic interactions are a hallmark of neuronal processing in the vertebrate olfactory bulb. Many classes of olfactory bulb neurons including the principal mitral cells (MCs) and the axonless granule cells (GCs) dispose of highly efficient propagation of action potentials (AP) within their dendrites, from where they can release transmitter onto each other. So far, backpropagation in GC dendrites has been investigated indirectly via Ca2+ imaging. Here, we used two-photon Na+ imaging to directly report opening of voltage-gated sodium channels due to AP propagation in both cell types. To this end, neurons in acute slices from juvenile rat bulbs were filled with 1 mM SBFI via whole-cell patch-clamp. Calibration of SBFI signals revealed that a change in fluorescence ΔF/F by 10% corresponded to a Δ[Na+]i of ∼22 mM. We then imaged proximal axon segments of MCs during somatically evoked APs (sAP). While single sAPs were detectable in ∼50% of axons, trains of 20 sAPs at 50 Hz always resulted in substantial ΔF/F of ∼15% (∼33 mM Δ[Na+]i). ΔF/F was significantly larger for 80 Hz vs. 50 Hz trains, and decayed with half-durations τ1/2 ∼0.6 s for both frequencies. In MC lateral dendrites, AP trains yielded small ΔF/F of ∼3% (∼7 mM Δ[Na+]i). In GC apical dendrites and adjacent spines, single sAPs were not detectable. Trains resulted in an average dendritic ΔF/F of 7% (16 mM Δ[Na+]i) with τ1/2 ∼1 s, similar for 50 and 80 Hz. Na+ transients were indistinguishable between large GC spines and their adjacent dendrites. Cell-wise analysis revealed two classes of GCs with the first showing a decrease in ΔF/F along the dendrite with distance from the soma and the second an increase. These classes clustered with morphological parameters. Simulations of Δ[Na+]i replicated these behaviors via negative and positive gradients in Na+ current density, assuming faithful AP backpropagation. Such specializations of dendritic excitability might confer specific temporal processing capabilities to bulbar principal cell-GC subnetworks. In conclusion, we show that Na+ imaging provides a valuable tool for characterizing AP invasion of MC axons and GC dendrites and spines.
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.
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