Acute insult of ammonia leads to calcium-dependent glutamate release from cultured astrocytes, an effect of pH

Hyperammonemia is a key factor in the pathogenesis of hepatic encephalopathy (HE) as well as other metabolic encephalopathies, such as those associated with inherited disorders of urea cycle enzymes and in Reye's syndrome. Acute HE results in increased brain ammonia (up to 5 mM), astrocytic swelling, and altered glutamatergic function. In the present study, using fluorescence imaging techniques, acute exposure (10 min) of ammonia (NH4+/NH3) to cultured astrocytes resulted in a concentration-dependent, transient increase in [Ca2+]i. This calcium transient was due to release from intracellular calcium stores, since the response was thapsigargin-sensitive and was still observed in calcium-free buffer. Using an enzyme-linked fluorescence assay, glutamate release was measured indirectly via the production of NADH (a naturally fluorescent product when excited with UV light). NH4+/NH3 (5 mM) stimulated a calcium-dependent glutamate release from cultured astrocytes, which was inhibited after preincubation with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester but unaffected after preincubation with glutamate transport inhibitors dihydrokainate and DL-threo-beta-benzyloxyaspartate. NH4+/NH3 (5 mM) also induced a transient intracellular alkaline shift. To investigate whether the effects of NH4+/NH3 were mediated by an increase in pH(i), we applied trimethylamine (TMA+/TMA) as another weak base. TMA+/TMA (5 mM) induced a similar transient increase in both pH(i) and [Ca2+]i (mobilization from intracellular calcium stores) and resulted in calcium-dependent release of glutamate. These results indicate that an acute exposure to ammonia, resulting in cytosolic alkalinization, leads to calcium-dependent glutamate release from astrocytes. A deregulation of glutamate release from astrocytes by ammonia could contribute to glutamate dysfunction consistently observed in acute HE

Properties of Doublecortin-(DCX)-Expressing Cells in the Piriform Cortex Compared to the Neurogenic Dentate Gyrus of Adult Mice

The piriform cortex receives input from the olfactory bulb and (via the entorhinal cortex) sends efferents to the hippocampus, thereby connecting the two canonical neurogenic regions of the adult rodent brain. Doublecortin (DCX) is a cytoskeleton-associated protein that is expressed transiently in the course of adult neurogenesis. Interestingly, the adult piriform cortex, which is usually considered non-neurogenic (even though some reports exist that state otherwise), also contains an abundant population of DCX-positive cells. We asked how similar these cells would be to DCX-positive cells in the course of adult hippocampal neurogenesis. Using BAC-generated transgenic mice that express GFP under the DCX promoter, we studied DCX-expression and electrophysiological properties of DCX-positive cells in the mouse piriform cortex in comparison with the dentate gyrus. While one class of cells in the piriform cortex indeed showed features similar to newly generated immature granule neurons, the majority of DCX cells in the piriform cortex was mature and revealed large Na+ currents and multiple action potentials. Furthermore, when proliferative activity was assessed, we found that all DCX-expressing cells in the piriform cortex were strictly postmitotic, suggesting that no DCX-positive “neuroblasts” exist here as they do in the dentate gyrus. We conclude that DCX in the piriform cortex marks a unique population of postmitotic neurons with a subpopulation that retains immature characteristics associated with synaptic plasticity. DCX is thus, per se, no marker of neurogenesis but might be associated more broadly with plasticity

Incorporation of N-propanoylneuraminic acid leads to calcium oscillations in oligodendrocytes upon the application of GABA

AbstractSialylation of glycoproteins and glycolipids plays an important role during development, regeneration and pathogenesis. It has been shown that unnatural sialylation within glial cell cultures can have distinct effects on their proliferation and antigenic profiles. These cultures metabolize N-propanoylmannosamine (N-propanoylneuraminic acid precursor=P-NAP), a synthetic non-physiological precursor of neuraminic acid, resulting in the expression of N-propanoylneuraminic acid in glycoconjugates of their cell membranes [Schmidt, C., Stehling, P., Schnitzer, J., Reutter, W. and Horstkorte, R. (1998) J. Biol. Chem. 273, 19146–19152]. To determine whether these biochemically engineered sialic acids influence calcium concentrations in cells of the oligodendrocyte lineage, mixed glial cultures of oligodendrocytes growing on top of an astrocyte monolayer were exposed to glutamate, histamine, adrenaline, γ-aminobutyric acid (GABA), high potassium (high K+) and ATP. Calcium responses in P-NAP-treated oligodendrocytes were determined by confocal microscopy with the calcium indicator fluo-3 AM, and compared with control cultures. We showed that P-NAP differentially modulated the calcium responses of individual oligodendrocytes when GABA was applied. GABA induced calcium oscillations with up to four spikes per min in 60% of oligodendrocytes when treated with P-NAP

De fabrica systematis nervosi evertebratorum

Hermann von Helmholtz, der Namensgeber der Helmholtz-Gemeinschaft, ist den meisten als Physiker bekannt und wurde auch als der "Reichskanzler der Physik" bezeichnet. In Vergessenheit geraten ist die Tatsache, dass er Medizin studiert hat und als Mediziner seine wissenschaftliche Laufbahn begann. 1842 schrieb er als 21-Jähriger seine Promotion über ein neurowissenschaftliches Thema. In dieser Zeit publizierte Christian Ehrenberg, Professor an der Berliner Universität, das erste Bild einer Nervenzelle. Gleichzeitig wurde auch die Zelltheorie von Jacob Henle und Matthias Schleiden in Berlin formuliert. Sie besagt, dass alle Gewebe einschließlich des Gehirns aus Zellen bestehen. Hermann Helmholtz stellte sich in seiner Doktorarbeit die Frage, wie das Nervensystem von wirbellosen Tieren aufgebaut ist. Sind die Prinzipien ähnlich oder ganz anders als bei Wirbeltieren und dem Menschen? Betreut von dem bekanntesten Physiologen und Anatomen dieser Zeit, Johannes Müller, untersuchte er das Nervensystem von Blutegel, Hausspinne, Schmetterling, Regenwurm, Flusskrebs oder Teichmuschel. Er kam zu der Erkenntnis, dass sich die Nervensysteme dieser Wirbellosen, oder auch Invertebraten genannt, nicht grundsätzlich von den Nervensystemen der Wirbeltiere inklusive des Menschen unterscheiden. Alle Grundelemente wie Zellen und Fortsätze sind identisch, so dass der Aufbau von Nervensystemen nach einem in der Tierwelt einheitlichen Plan erfolgt. Diese neurowissenschaftlichen Erkenntnisse von Helmholtz sind in der "Neuroscience Community" in Vergessenheit geraten, denn er schrieb seine Doktorarbeit in Latein. Sie wurde bisher nie in eine andere Sprache übersetzt. Helmut Kettenmann, Neurowissenschaftler am Max-Delbrück Centrum für Molekulare Medizin in der Helmholtz-Gemeinschaft, hat nun zusammen mit der Altphilologin Julia Heideklang von der Humboldt-Universität und Joachim Pflüger, Invertebraten-Neurobiologe an der Freien Universität, diese Dissertation sowohl ins Englische als auch ins Deutsche übersetzt. Die Doktorarbeit wird ausführlich eingeführt und kommentiert und zeigt , dass Hermann Helmholtz schon als 21-Jähriger noch heute gültige Erkenntnisse auf neurowissenschaftlichem Gebiet formulierte, die seine herausragenden, teils visionären Fähigkeiten schon früh unter Beweis stellten

Insanity as a Defense to the Civil Fraud Penalty

Most neurological diseases are associated with chronic inflammation initiated by the activation of microglia, which produce cytotoxic and inflammatory factors. Signal transducers and activators of transcription (STATs) are potent regulators of gene expression but contribution of particular STAT to inflammatory gene expression and STAT-dependent transcriptional networks underlying brain inflammation need to be identified. In the present study, we investigated the genomic distribution of Stat binding sites and the role of Stats in the gene expression in lipopolysaccharide (LPS)-activated primary microglial cultures. Integration of chromatin immunoprecipitation-promoter microarray data and transcriptome data revealed novel Stat-target genes including Jmjd3, Ccl5, Ezr, Ifih1, Irf7, Uba7, and Pim1. While knockdown of individual Stat had little effect on the expression of tested genes, knockdown of both Stat1 and Stat3 inhibited the expression of Jmjd3 and inflammatory genes. Transcriptional regulation of Jmjd3 by Stat1 and Stat3 is a novel mechanism crucial for launching inflammatory responses in microglia. The effects of Jmjd3 on inflammatory gene expression were independent of its H3K27me3 demethylase activity. Forced expression of constitutively activated Stat1 and Stat3 induced the expression of Jmjd3, inflammation-related genes, and the production of proinflammatory cytokines as potently as lipopolysacharide. Gene set enrichment and gene function analysis revealed categories linked to the inflammatory response in LPS and Stat1C + Stat3C groups. We defined upstream pathways that activate STATs in response to LPS and demonstrated contribution of Tlr4 and Il-6 and interferon-. signaling. Our findings define novel direct transcriptional targets of Stat1 and Stat3 and highlight their contribution to inflammatory gene expression

Intrathecal heat shock protein 60 mediates neurodegeneration and demyelination in the CNS through a TLR4- and MyD88-dependent pathway

Background Toll-like receptors (TLR) constitute a highly conserved class of receptors through which the innate immune system responds to both pathogen- and host-derived factors. Although TLRs are involved in a wide range of central nervous system (CNS) disorders including neurodegenerative diseases, the molecular events leading from CNS injury to activation of these innate immune receptors remain elusive. The stress protein heat shock protein 60 (HSP60) released from injured cells is considered an endogenous danger signal of the immune system. In this context, the main objective of the present study was to investigate the impact of extracellular HSP60 on the brain in vivo. Results We show here that HSP60 injected intrathecally causes neuronal and oligodendrocyte injury in the CNS in vivo through TLR4-dependent signaling. Intrathecal HSP60 results in neuronal cell death, axonal injury, loss of oligodendrocytes, and demyelination in the cerebral cortex of wild-type mice. In contrast both mice lacking TLR4 and the TLR adaptor molecule MyD88 are protected against deleterious effects induced by HSP60. In contrast to the exogenous TLR4 ligand, lipopolysaccharide, intrathecal HSP60 does not induce such a considerable inflammatory response in the brain. In the CNS, endogenous HSP60 is predominantly expressed in neurons and released during brain injury, since the cerebrospinal fluid (CSF) from animals of a mouse stroke model contains elevated levels of this stress protein compared to the CSF of sham- operated mice. Conclusions Our data show a direct toxic effect of HSP60 towards neurons and oligodendrocytes in the CNS. The fact that these harmful effects involve TLR4 and MyD88 confirms a molecular pathway mediated by the release of endogenous TLR ligands from injured CNS cells common to many forms of brain diseases that bi-directionally links CNS injury and activation of the innate immune system to neurodegeneration and demyelination in vivo

Bone morphogenetic protein-7 release from endogenous neural precursor cells suppresses the tumourigenicity of stem-like glioblastoma cells

Glioblastoma cells with stem-like properties control brain tumour growth and recurrence. Here, we show that endogenous neural precursor cells perform an anti-tumour response by specifically targeting stem-like brain tumour cells. In vitro, neural precursor cells predominantly express bone morphogenetic protein-7; bone morphogenetic protein-7 is constitutively released from neurospheres and induces canonical bone morphogenetic protein signalling in stem-like glioblastoma cells. Exposure of human and murine stem-like brain tumour cells to neurosphere-derived bone morphogenetic protein-7 induces tumour stem cell differentiation, attenuates stem-like marker expression and reduces self-renewal and the ability for tumour initiation. Neurosphere-derived or recombinant bone morphogenetic protein-7 reduces glioblastoma expansion from stem-like cells by down-regulating the transcription factor Olig2. In vivo, large numbers of bone morphogenetic protein-7-expressing neural precursors encircle brain tumours in young mice, induce canonical bone morphogenetic protein signalling in stem-like glioblastoma cells and can thereby attenuate tumour formation. This anti-tumour response is strongly reduced in older mice. Our results indicate that endogenous neural precursor cells protect the young brain from glioblastoma by releasing bone morphogenetic protein-7, which acts as a paracrine tumour suppressor that represses proliferation, self-renewal and tumour-initiation of stem-like glioblastoma cell

Decreased demand for olfactory periglomerular cells impacts on neural precursor cell viability in the rostral migratory stream

The subventricular zone (SVZ) provides a constant supply of new neurons to the olfactory bulb (OB). Different studies have investigated the role of olfactory sensory input to neural precursor cell (NPC) turnover in the SVZ but it was not addressed if a reduced demand specifically for periglomerular neurons impacts on NPC-traits in the rostral migratory stream (RMS). We here report that membrane type-1 matrix metalloproteinase (MT1-MMP) deficient mice have reduced complexity of the nasal turbinates, decreased sensory innervation of the OB, reduced numbers of olfactory glomeruli and reduced OB-size without alterations in SVZ neurogenesis. Large parts of the RMS were fully preserved in MT1-MMP-deficient mice, but we detected an increase in cell death-levels and a decrease in SVZ-derived neuroblasts in the distal RMS, as compared to controls. BrdU-tracking experiments showed that homing of NPCs specifically to the glomerular layer was reduced in MT1-MMP-deficient mice in contrast to controls while numbers of tracked cells remained equal in other OB-layers throughout all experimental groups. Altogether, our data show the demand for olfactory interneurons in the glomerular layer modulates cell turnover in the RMS, but has no impact on subventricular neurogenesis

The principal neurons of the medial nucleus of the trapezoid body and NG2+ glial cells receive coordinated excitatory synaptic input

Glial cell processes are part of the synaptic structure and sense spillover of transmitter, while some glial cells can even receive direct synaptic input. Here, we report that a defined type of glial cell in the medial nucleus of the trapezoid body (MNTB) receives excitatory glutamatergic synaptic input from the calyx of Held (CoH). This giant glutamatergic terminal forms an axosomatic synapse with a single principal neuron located in the MNTB. The NG2 glia, as postsynaptic principal neurons, establish synapse-like structures with the CoH terminal. In contrast to the principal neurons, which are known to receive excitatory as well as inhibitory inputs, the NG2 glia receive mostly, if not exclusively, α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid receptor–mediated evoked and spontaneous synaptic input. Simultaneous recordings from neurons and NG2 glia indicate that they partially receive synchronized spontaneous input. This shows that an NG2+ glial cell and a postsynaptic neuron share presynaptic terminals

Neuron–astrocyte interactions in the medial nucleus of the trapezoid body

The calyx of Held (CoH) synapse serves as a model system to analyze basic mechanisms of synaptic transmission. Astrocyte processes are part of the synaptic structure and contact both pre- and postsynaptic membranes. In the medial nucleus of the trapezoid body (MNTB), midline stimulation evoked a current response that was not mediated by glutamate receptors or glutamate uptake, despite the fact that astrocytes express functional receptors and transporters. However, astrocytes showed spontaneous Ca2+ responses and neuronal slow inward currents (nSICs) were recorded in the postsynaptic principal neurons (PPNs) of the MNTB. These currents were correlated with astrocytic Ca2+ activity because dialysis of astrocytes with BAPTA abolished nSICs. Moreover, the frequency of these currents was increased when Ca2+ responses in astrocytes were elicited. NMDA antagonists selectively blocked nSICs while D-serine degradation significantly reduced NMDA-mediated currents. In contrast to previous studies in the hippocampus, these NMDA-mediated currents were rarely synchronized