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After stroke, macrophages in the ischemic brain may be derived from either resident microglia or infiltrating monocytes. Using bone marrow (BM)-chimerism and dual-reporter transgenic fate mapping, we here set out to delimit the responses of either cell type to mild brain ischemia in a mouse model of 30 min transient middle cerebral artery occlusion (MCAo). A discriminatory analysis of gene expression at 7 days post-event yielded 472 transcripts predominantly or exclusively expressed in blood-derived macrophages as well as 970 transcripts for microglia. The differentially regulated genes were further collated with oligodendrocyte, astrocyte, and neuron transcriptomes, resulting in a dataset of microglia- and monocyte-specific genes in the ischemic brain. Functional categories significantly enriched in monocytes included migration, proliferation, and calcium signaling, indicative of strong activation. Whole-cell patch-clamp analysis further confirmed this highly activated state by demonstrating delayed outward K+ currents selectively in invading cells. Although both cell types displayed a mixture of known phenotypes pointing to the significance of 'intermediate states' in vivo, blood-derived macrophages were generally more skewed toward an M2 neuroprotective phenotype. Finally, we found that decreased engraftment of blood-borne cells in the ischemic brain of chimeras reconstituted with BM from Selplg-/- mice resulted in increased lesions at 7 days and worse post-stroke sensorimotor performance. In aggregate, our study establishes crucial differences in activation state between resident microglia and invading macrophages after stroke and identifies unique genomic signatures for either cell type.

Astrocytes form large networks, in which individual cells are connected via gap junctions. It is thought that this astroglial gap junction coupling contributes to the buffering of extracellular K+ increases. However, it is largely unknown how the control of extracellular K+ by astroglial gap junction coupling depends on the underlying activity patterns and on the magnitude of extracellular K+ increases. We explored this dependency in acute hippocampal slices (CA1, stratum radiatum) by direct K+ -sensitive microelectrode recordings and acute pharmacological inhibition of gap junctions. K+ transients evoked by synaptic and axonal activity were largely unaffected by acute astroglial uncoupling in slices obtained from young and adult rats. Iontophoretic K+ -application enabled us to generate K+ gradients with defined spatial properties and magnitude. By varying the K+ -iontophoresis position and protocol, we found that acute pharmacological uncoupling increases the amplitude of K+ transients once their initial amplitude exceeded ~10 mM. Our experiments demonstrate that the contribution of gap junction coupling to buffering of extracellular K+ gradients is limited to large and localized K+ increases.

Microglia, the central nervous system resident innate immune cells, cluster around Aβ plaques in Alzheimer's disease (AD). The activation phenotype of these plaque-associated microglial cells, and their differences to microglia distant to Aβ plaques, are incompletely understood. We used novel three-dimensional cell analysis software to comprehensively analyze the morphological properties of microglia in the TgCRND8 mouse model of AD in spatial relation to Aβ plaques. We found strong morphological changes exclusively in plaque-associated microglia, whereas plaque-distant microglia showed only minor changes. In addition, patch-clamp recordings of microglia in acute cerebral slices of TgCRND8 mice revealed increased K+ currents in plaque-associated but not plaque-distant microglia. Within the subgroup of plaque-associated microglia, two different current profiles were detected. One subset of cells displayed only increased inward currents, while a second subset showed both increased inward and outward currents, implicating that the plaque microenvironment differentially impacts microglial ion channel expression. Using pharmacological channel blockers, multiplex single-cell PCR analysis and RNA fluorescence in situ hybridization, we identified Kir and Kv channel types contributing to the in- and outward K+ conductance in plaque-associated microglia. In summary, we have identified a previously unrecognized level of morphological and electrophysiological heterogeneity of microglia in relation to amyloid plaques, suggesting that microglia may display multiple activation states in AD.

Substance P (SP) is implicated in stress regulation and affective and anxiety-related behavior. Particularly high expression has been found in the main output region of the amygdala complex, the central amygdala (CE). Here we investigated the cellular mechanisms of SP in CE in vitro, taking advantage of glutamic acid decarboxylase-green fluorescent protein (GAD67-GFP) knockin mice that yield a reliable labeling of GABAergic neurons, which comprise 95% of the neuronal population in the lateral section of CE (CEl). In GFP-positive neurons within CEl, SP caused a membrane depolarization and increase in input resistance, associated with an increase in action potential firing frequency. Under voltage-clamp conditions, the SP-specific membrane current reversed at −101.5 ± 2.8 mV and displayed inwardly rectifying properties indicative of a membrane K+ conductance. Moreover, SP responses were blocked by the neurokinin type 1 receptor (NK1R) antagonist L-822429 and mimicked by the NK1R agonist [Sar9,Met(O2)11]-SP. Immunofluorescence staining confirmed localization of NK1R in GFP-positive neurons in CEl, predominantly in PKCδ-negative neurons (80%) and in few PKCδ-positive neurons (17%). Differences in SP responses were not observed between the major types of CEl neurons (late firing, regular spiking, low-threshold bursting). In addition, SP increased the frequency and amplitude of GABAergic synaptic events in CEl neurons depending on upstream spike activity. These data indicate a NK1R-mediated increase in excitability and GABAergic activity in CEl neurons, which seems to mostly involve the PKCδ-negative subpopulation. This influence can be assumed to increase reciprocal interactions between CElon and CEloff pathways, thereby boosting the medial CE (CEm) output pathway and contributing to the anxiogenic-like action of SP in the amygdala.

A new approach to trace the transport routes of macronutrients in plants at the level of cells and tissues and to measure their elemental distributions was developed for investigating the dynamics and structure-function relationships of transport processes. Stem samples from Phaseolus vulgaris were used as a test system. Shock freezing and cryo-preparation were combined in a cryogenic chain with cryo-time-of-flight secondary ion mass spectrometry (cryo-ToF-SIMS) for element and isotope-specific imaging. Cryo-scanning electron microscopy (cryo-SEM) was integrated into the cryogenic workflow to assess the quality of structural preservation. We evaluated the capability of these techniques to monitor transport pathways and processes in xylem and associated tissues using supplementary sodium (Na) and tracers for potassium (K), rubidium (Rb), and 41K added to the transpiration stream. Cryo-ToF-SIMS imaging produced detailed mappings of water, K, calcium, magnesium, the K tracers, and Na without quantification. Lateral resolutions ranged from 10 μm in survey mappings and at high mass resolution to approximately 1 μm in high lateral resolution imaging in reduced areas and at lower mass resolution. The tracers Rb and 41K, as well as Na, were imaged with high sensitivity in xylem vessels and surrounding tissues. The isotope signature of the stable isotope tracer was utilized for relative quantification of the 41K tracer as a fraction of total K at the single pixel level. Cryo-SEM confirmed that tissue structures had been preserved with subcellular detail throughout all procedures. Overlays of cryo-ToF-SIMS images onto the corresponding SEM images allowed detailed correlation of nutrient images with subcellular structures.

Neuronal damage shortly after onset or after brief episodes of cerebral ischemia has remained difficult to assess with clinical and preclinical imaging techniques as well as with microscopical methods. We here show, in rodent models of middle cerebral artery occlusion (MCAO), that neuronal damage in acute focal cerebral ischemia can be mapped with single-cell resolution using thallium autometallography (TlAMG), a histochemical technique for the detection of the K+-probe thallium (Tl+) in the brain. We intravenously injected rats and mice with thallium diethyldithiocarbamate (TlDDC), a lipophilic chelate complex that releases Tl+ after crossing the blood–brain barrier. We found, within the territories of the affected arteries, areas of markedly reduced neuronal Tl+ uptake in all animals at all time points studied ranging from 15 minutes to 24 hours after MCAO. In large lesions at early time points, areas with neuronal and astrocytic Tl+ uptake below thresholds of detection were surrounded by putative penumbral zones with preserved but diminished Tl+ uptake. At 24 hours, the areas of reduced Tl+uptake matched with areas delineated by established markers of neuronal damage. The results suggest the use of 201TlDDC for preclinical and clinical single-photon emission computed tomography (SPECT) imaging of hyperacute alterations in brain K+ metabolism and prediction of tissue viability in cerebral ischemia.

The potassium (K(+)) analogue thallium (Tl(+)) can be used as a tracer for mapping neuronal activity. However, because of the poor blood-brain barrier (BBB) K(+) -permeability, only minute amounts of Tl(+) enter the brain after systemic injection of Tl(+) -salts like thallium acetate (TlAc). We have recently shown that it is possible to overcome this limitation by injecting animals with the lipophilic chelate complex thallium diethyldithiocarbamate (TlDDC), that crosses the BBB and releases Tl(+) prior to neuronal or glial uptake. TlDDC can thus be used for mapping CNS K(+) metabolism and neuronal activity. Here, we analyze Tl(+) -kinetics in the rodent brain both experimentally and using simple mathematical models. We systemically injected animals either with TlAc or with TlDDC. Using an autometallographic method we mapped the brain Tl(+) -distribution at various time points after injection. We show that the patterns and kinetics of Tl(+) -redistribution in the brain are essentially the same irrespective of whether animals have been injected with TlAc or TlDDC. Data from modeling and experiments indicate that transmembrane Tl(+) -fluxes in cells within the CNS in vivo equilibrate at similar rates as K(+) -fluxes in vitro. This equilibration is much faster than and largely independent of the equilibration of Tl(+) -fluxes across the BBB. The study provides further proof-of-concept for the use of TlDDC for mapping neuronal activity and CNS K(+) -metabolism. A theoretical guideline is given for the use of K(+) -analogues for imaging neuronal activity with general implications for the use of metal ions in neuroimaging.

Neuroenergetic models of synaptic transmission predicted that energy demand is highest for action potentials (APs) and postsynaptic ion fluxes, whereas the presynaptic contribution is rather small. Here, we addressed the question of energy consumption at Schaffer-collateral synapses. We monitored stimulus-induced changes in extracellular potassium, sodium, and calcium concentration while recording partial oxygen pressure (pO2) and NAD(P)H fluorescence. Blockade of postsynaptic receptors reduced ion fluxes as well as pO2 and NAD(P)H transients by ~50%. Additional blockade of transmitter release further reduced Na+, K+, and pO2 transients by ~30% without altering presynaptic APs, indicating considerable contribution of Ca2+-removal, transmitter and vesicle turnover to energy consumption.

Familial pseudohyperkalaemia (FP) is a symptomless, dominantly inherited red cell trait, which shows a 'passive leak' of K+ cations into the plasma upon storage of blood at room temperature (or below). There are no haematological abnormalities. The loss of K+ is due to a change in the temperature dependence of the leak. The Scottish case initially described, FP Edinburgh, maps to 16q23-qter. Here we studied a large kindred of Flemish descent with FP, termed FP Lille, which was phenotypically identical to the Edinburgh FP. In FP Lille, however, the responsible locus mapped to 2q35-36, with a Lod score of 8.46 for marker D2S1338. We infer that FP Edinburgh and FP Lille, although they are phenocopies of one another, stem from two distinct loci, FP1 (16q23-qter) and FP2 (2q35-36), respectively. This duality hints at the possibility that the protein mediating the leak might be a heterodimer. No mutation was found in three plausibly candidate genes: the KCNE4 gene, the TUBA1 gene and a predicted gene located in genomic contig NT\_005403.