Stroke induces the recruitment of neuronal precursors from the subventricular zone (SVZ) into the ischemic striatum. In injured areas, de-routed neuroblasts use blood vessels as a physical scaffold to their migration, in a process that resembles the constitutive migration seen in the rostral migratory stream (RMS). The molecular mechanism underlying injury-induced vasculature-mediated migration of neuroblasts in the post-stroke striatum remains, however, elusive. Using adult mice we now demonstrate that endothelial cells in the ischemic striatum produce brain-derived neurotrophic factor (BDNF), a neurotrophin that promotes the vasculature-mediated migration of neuronal precursors in the RMS, and that recruited neuroblasts maintain expression of p75NTR, a low-affinity receptor for BDNF. Reactive astrocytes, which are widespread throughout the damaged area, ensheath blood vessels and express TrkB, a high-affinity receptor for BDNF. Despite the absence of BDNF mRNA, we observed strong BDNF immunolabeling in astrocytes, suggesting that these glial cells trap extracellular BDNF. Importantly, this pattern of expression is reminiscent of the adult RMS, where TrkB-expressing astrocytes bind and sequester vasculature-derived BDNF, leading to the entry of migrating cells into the stationary phase. Real-time imaging of cell migration in acute brain slices revealed a direct role for BDNF in promoting the migration of neuroblasts to ischemic areas. We also demonstrated that cells migrating in the ischemic striatum display higher exploratory behavior and longer stationary periods than cells migrating in the RMS. Our findings suggest that the mechanisms involved in the injury-induced vasculature-mediated migration of neuroblasts recapitulate, at least partially, those observed during constitutive migration in the RMS.

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.

Emerging evidence indicates that neuronal activity-evoked changes in sodium concentration in astrocytes Na a represent a special form of excitability, which is tightly linked to all other major ions in the astrocyte and extracellular space, as well as to bioenergetics, neurotransmitter uptake, and neurovascular coupling. Recently, one of us reported that Na a transients in the neocortex have a significantly higher amplitude than those in the hippocampus. Based on the extensive data from that study, here we develop a detailed biophysical model to further understand the origin of this heterogeneity and how it affects bioenergetics in the astrocytes. In addition to closely fitting the observed experimental Na a changes under different conditions, our model shows that the heterogeneity in Na a signaling leads to substantial differences in the dynamics of astrocytic Ca2+ signals in the two brain regions, and leaves cortical astrocytes more susceptible to Na+ and Ca2+ overload under metabolic stress. The model also predicts that activity-evoked Na a transients result in significantly larger ATP consumption in cortical astrocytes than in the hippocampus. The difference in ATP consumption is mainly due to the different expression levels of NMDA receptors in the two regions. We confirm predictions from our model experimentally by fluorescence-based measurement of glutamate-induced changes in ATP levels in neocortical and hippocampal astrocytes in the absence and presence of the NMDA receptor's antagonist (2R)-amino-5-phosphonovaleric acid.

Cell transplantation is a promising approach for the reconstruction of neuronal circuits after brain damage. Transplanted neurons integrate with remarkable specificity into circuitries of the mouse cerebral cortex affected by neuronal ablation. However, it remains unclear how neurons perform in a local environment undergoing reactive gliosis, inflammation, macrophage infiltration, and scar formation, as in traumatic brain injury (TBI). To elucidate this, we transplanted cells from the embryonic mouse cerebral cortex into TBI-injured, inflamed-only, or intact cortex of adult mice. Brain-wide quantitative monosynaptic rabies virus (RABV) tracing unraveled graft inputs from correct regions across the brain in all conditions, with pronounced quantitative differences: scarce in intact and inflamed brain versus exuberant after TBI. In the latter, the initial overshoot is followed by pruning, with only a few input neurons persisting at 3 months. Proteomic profiling identifies candidate molecules for regulation of the synaptic yield, a pivotal parameter to tailor for functional restoration of neuronal circuits.

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.

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.

Neural stem cells (NSCs) from the subventricular zone (SVZ) have been indicated as a source of new oligodendrocytes to use in regenerative medicine for myelin pathologies. Indeed, NSCs are multipotent cells that can self-renew and differentiate into all neural cell types of the central nervous system. In normal conditions, SVZ cells are poorly oligodendrogenic, nevertheless their oligodendrogenic potential is boosted following demyelination. Importantly, progressive restriction into the oligodendrocyte fate is specified by extrinsic and intrinsic factors, endocannabinoids being one of these factors. Although a role for endocannabinoids in oligodendrogenesis has already been foreseen, selective agonists and antagonists of cannabinoids receptors produce severe adverse side effects. Herein, we show that hemopressin (Hp), a modulator of CB1 receptors, increased oligodendroglial differentiation in SVZ neural stem/progenitor cell cultures derived from neonatal mice. The original results presented in this work suggest that Hp and derivates may be of potential interest for the development of future strategies to treat demyelinating diseases.

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.

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.

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.

Uncovering the physiological role of individual proteins that are part of the intricate process of cellular signaling is often a complex and challenging task. A straightforward strategy of studying a protein's function is by manipulating the expression rate of its gene. In recent years, the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9-based technology was established as a powerful gene-editing tool for generating sequence specific changes in proliferating cells. However, obtaining homogeneous populations of transgenic post-mitotic neurons by CRISPR/Cas9 turned out to be challenging. These constraints can be partially overcome by CRISPR interference (CRISPRi), which mediates the inhibition of gene expression by competing with the transcription machinery for promoter binding and, thus, transcription initiation. Notably, CRISPR/Cas is only one of several described approaches for the manipulation of gene expression. Here, we targeted neurons with recombinant Adeno-associated viruses to induce either CRISPRi or RNA interference (RNAi), a well-established method for impairing de novo protein biosynthesis by using cellular regulatory mechanisms that induce the degradation of pre-existing mRNA. We specifically targeted hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels, which are widely expressed in neuronal tissues and play essential physiological roles in maintaining biophysical characteristics in neurons. Both of the strategies reduced the expression levels of three HCN isoforms (HCN1, 2, and 4) with high specificity. Furthermore, detailed analysis revealed that the knock-down of just a single HCN isoform (HCN4) in hippocampal neurons did not affect basic electrical parameters of transduced neurons, whereas substantial changes emerged in HCN-current specific properties.

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.

Disturbances in the brain fluid balance can lead to life-threatening elevation in the intracranial pressure (ICP), which represents a vast clinical challenge. Nevertheless, the details underlying the molecular mechanisms governing cerebrospinal fluid (CSF) secretion are largely unresolved, thus preventing targeted and efficient pharmaceutical therapy of cerebral pathologies involving elevated ICP.

Elevated intracranial pressure (ICP) is observed in many neurological pathologies, e.g. hydrocephalus and stroke. This condition is routinely relieved with neurosurgical approaches, since effective and targeted pharmacological tools are still lacking. The carbonic anhydrase inhibitor, acetazolamide (AZE), may be employed to treat elevated ICP. However, its effectiveness is questioned, its location of action unresolved, and its tolerability low. Here, we determined the efficacy and mode of action of AZE in the rat .

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.

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.

Transplantation is a clinically relevant approach for brain repair, but much remains to be understood about influences of the disease environment on transplant connectivity. To explore the effect of amyloid pathology in Alzheimer's disease (AD) and aging, we examined graft connectivity using monosynaptic rabies virus tracing in APP/PS1 mice and in 16- to 18-month-old wild-type (WT) mice. Transplanted neurons differentiated within 4 weeks and integrated well into the host visual cortex, receiving input from the appropriate brain regions for this area. Unexpectedly, we found a prominent several-fold increase in local inputs, in both amyloid-loaded and aged environments. State-of-the-art deep proteome analysis using mass spectrometry highlights complement system activation as a common denominator of environments promoting excessive local input connectivity. These data therefore reveal the key role of the host pathology in shaping the input connectome, calling for caution in extrapolating results from one pathological condition to another.

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.

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).