Amyloid β-peptide directly induces spontaneous calcium transients, delayed intercellular calcium waves and gliosis in rat cortical astrocytes

The contribution of astrocytes to the pathophysiology of AD (Alzheimer's disease) and the molecular and signalling mechanisms that potentially underlie them are still very poorly understood. However, there is mounting evidence that calcium dysregulation in astrocytes may be playing a key role. Intercellular calcium waves in astrocyte networks in vitro can be mechanically induced after Aβ (amyloid β-peptide) treatment, and spontaneously forming intercellular calcium waves have recently been shown in vivo in an APP (amyloid precursor protein)/PS1 (presenilin 1) Alzheimer's transgenic mouse model. However, spontaneous intercellular calcium transients and waves have not been observed in vitro in isolated astrocyte cultures in response to direct Aβ stimulation in the absence of potentially confounding signalling from other cell types. Here, we show that Aβ alone at relatively low concentrations is directly able to induce intracellular calcium transients and spontaneous intercellular calcium waves in isolated astrocytes in purified cultures, raising the possibility of a potential direct effect of Aβ exposure on astrocytes in vivo in the Alzheimer's brain. Waves did not occur immediately after Aβ treatment, but were delayed by many minutes before spontaneously forming, suggesting that intracellular signalling mechanisms required sufficient time to activate before intercellular effects at the network level become evident. Furthermore, the dynamics of intercellular calcium waves were heterogeneous, with distinct radial or longitudinal propagation orientations. Lastly, we also show that changes in the expression levels of the intermediate filament proteins GFAP (glial fibrillary acidic protein) and S100B are affected by Aβ-induced calcium changes differently, with GFAP being more dependent on calcium levels than S100B

Cortical Layer 1 and Layer 2/3 Astrocytes Exhibit Distinct Calcium Dynamics In Vivo

Cumulative evidence supports bidirectional interactions between astrocytes and neurons, suggesting glial involvement of neuronal information processing in the brain. Cytosolic calcium (Ca2+) concentration is important for astrocytes as Ca2+ surges co-occur with gliotransmission and neurotransmitter reception. Cerebral cortex is organized in layers which are characterized by distinct cytoarchitecture. We asked if astrocyte-dominant layer 1 (L1) of the somatosensory cortex was different from layer 2/3 (L2/3) in spontaneous astrocytic Ca2+ activity and if it was influenced by background neural activity. Using a two-photon laser scanning microscope, we compared spontaneous Ca2+ activity of astrocytic somata and processes in L1 and L2/3 of anesthetized mature rat somatosensory cortex. We also assessed the contribution of background neural activity to the spontaneous astrocytic Ca2+ dynamics by investigating two distinct EEG states (“synchronized” vs. “de-synchronized” states). We found that astrocytes in L1 had nearly twice higher Ca2+ activity than L2/3. Furthermore, Ca2+ fluctuations of processes within an astrocyte were independent in L1 while those in L2/3 were synchronous. Pharmacological blockades of metabotropic receptors for glutamate, ATP, and acetylcholine, as well as suppression of action potentials did not have a significant effect on the spontaneous somatic Ca2+ activity. These results suggest that spontaneous astrocytic Ca2+ surges occurred in large part intrinsically, rather than neural activity-driven. Our findings propose a new functional segregation of layer 1 and 2/3 that is defined by autonomous astrocytic activity

COX-2, CB2 and P2X7-immunoreactivities are increased in activated microglial cells/macrophages of multiple sclerosis and amyotrophic lateral sclerosis spinal cord

BACKGROUND: While multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS) are primarily inflammatory and degenerative disorders respectively, there is increasing evidence for shared cellular mechanisms that may affect disease progression, particularly glial responses. Cyclooxygenase 2 (COX-2) inhibition prolongs survival and cannabinoids ameliorate progression of clinical disease in animal models of ALS and MS respectively, but the mechanism is uncertain. Therefore, three key molecules known to be expressed in activated microglial cells/macrophages, COX-2, CB2 and P2X7, which plays a role in inflammatory cascades, were studied in MS and ALS post-mortem human spinal cord. METHODS: Frozen human post mortem spinal cord specimens, controls (n = 12), ALS (n = 9) and MS (n = 19), were available for study by immunocytochemistry and Western blotting, using specific antibodies to COX-2, CB2 and P2X7, and markers of microglial cells/macrophages (CD 68, ferritin). In addition, autoradiography for peripheral benzodiazepine binding sites was performed on some spinal cord sections using [3H] (R)-PK11195, a marker of activated microglial cells/macrophages. Results of immunostaining and Western blotting were quantified by computerized image and optical density analysis respectively. RESULTS: In control spinal cord, few small microglial cells/macrophages-like COX-2-immunoreactive cells, mostly bipolar with short processes, were scattered throughout the tissue, whilst MS and ALS specimens had significantly greater density of such cells with longer processes in affected regions, by image analysis. Inflammatory cell marker CD68-immunoreactivity, [3H] (R)-PK11195 autoradiography, and double-staining against ferritin confirmed increased production of COX-2 by activated microglial cells/macrophages. An expected 70-kDa band was seen by Western blotting which was significantly increased in MS spinal cord. There was good correlation between the COX-2 immunostaining and optical density of the COX-2 70-kDa band in the MS group (r = 0.89, P = 0.0011, n = 10). MS and ALS specimens also had significantly greater density of P2X7 and CB2-immunoreactive microglial cells/macrophages in affected regions. CONCLUSION: It is hypothesized that the known increase of lesion-associated extracellular ATP contributes via P2X7 activation to release IL-1 beta which in turn induces COX-2 and downstream pathogenic mediators. Selective CNS-penetrant COX-2 and P2X7 inhibitors and CB2 specific agonists deserve evaluation in the progression of MS and ALS

ATP signalling in epilepsy

This paper focuses on a role for ATP neurotransmission and gliotransmission in the pathophysiology of epileptic seizures. ATP along with gap junctions propagates the glial calcium wave, which is an extraneuronal signalling pathway in the central nervous system. Recently astrocyte intercellular calcium waves have been shown to underlie seizures, and conventional antiepileptic drugs have been shown to attenuate these calcium waves. Blocking ATP-mediated gliotransmission, therefore, represents a potential target for antiepileptic drugs. Furthermore, while knowledge of an antiepileptic role for adenosine is not new, a recent study showed that adenosine accumulates from the hydrolysis of accumulated ATP released by astrocytes and is believed to inhibit distant synapses by acting on adenosine receptors. Such a mechanism is consistent with a surround-inhibitory mechanism whose failure would predispose to seizures. Other potential roles for ATP signalling in the initiation and spread of epileptiform discharges may involve synaptic plasticity and coordination of synaptic networks. We conclude by making speculations about future developments

Pharmacometabolomics of Response to Sertraline and to Placebo in Major Depressive Disorder - Possible Role for Methoxyindole Pathway

10.1371/journal.pone.0068283PLoS ONE87-POLN

Nonlinear gap junctions enable long-distance propagation of pulsating calcium waves in astrocyte networks

A new paradigm has recently emerged in brain science whereby communications between glial cells and neuron-glia interactions should be considered together with neurons and their networks to understand higher brain functions. In particular, astrocytes, the main type of glial cells in the cortex, have been shown to communicate with neurons and with each other. They are thought to form a gap-junction-coupled syncytium supporting cell-cell communication via propagating Ca2+ waves. An identified mode of propagation is based on cytoplasm-to-cytoplasm transport of inositol trisphosphate (IP3) through gap junctions that locally trigger Ca2+ pulses via IP3-dependent Ca2+-induced Ca2+ release. It is, however, currently unknown whether this intracellular route is able to support the propagation of long-distance regenerative Ca2+ waves or is restricted to short-distance signaling. Furthermore, the influence of the intracellular signaling dynamics on intercellular propagation remains to be understood. In this work, we propose a model of the gap-junctional route for intercellular Ca2+ wave propagation in astrocytes showing that: (1) long-distance regenerative signaling requires nonlinear coupling in the gap junctions, and (2) even with nonlinear gap junctions, long-distance regenerative signaling is favored when the internal Ca2+ dynamics implements frequency modulation-encoding oscillations with pulsating dynamics, while amplitude modulation-encoding dynamics tends to restrict the propagation range. As a result, spatially heterogeneous molecular properties and/or weak couplings are shown to give rise to rich spatiotemporal dynamics that support complex propagation behaviors. These results shed new light on the mechanisms implicated in the propagation of Ca2+ waves across astrocytes and precise the conditions under which glial cells may participate in information processing in the brain.Comment: Article: 30 pages, 7 figures. Supplementary Material: 11 pages, 6 figure

KV7/KCNQ Channels Are Functionally Expressed in Oligodendrocyte Progenitor Cells

Background: KV7/KCNQ channels are widely expressed in neurons and they have multiple important functions, including control of excitability, spike afterpotentials, adaptation, and theta resonance. Mutations in KCNQ genes have been demonstrated to associate with human neurological pathologies. However, little is known about whether K V7/KCNQ channels are expressed in oligodendrocyte lineage cells (OLCs) and what their functions in OLCs. Methods and Findings: In this study, we characterized KV7/KCNQ channels expression in rat primary cultured OLCs by RT-PCR, immunostaining and electrophysiology. KCNQ2-5 mRNAs existed in all three developmental stages of rat primary cultured OLCs. K V7/KCNQ proteins were also detected in oligodendrocyte progenitor cells (OPCs, early developmental stages of OLCs) of rat primary cultures and cortex slices. Voltage-clamp recording revealed that the IM antagonist XE991 significantly reduced KV7/KCNQ channel current (IK(Q)) in OPCs but not in differentiated oligodendrocytes. In addition, inhibition of K V7/KCNQ channels promoted OPCs motility in vitro. Conclusions: These findings showed that K V7/KCNQ channels were functionally expressed in rat primary cultured OLCs an

Role of P2 purinergic receptors in synaptic transmission under normoxic and ischaemic conditions in the CA1 region of rat hippocampal slices

The role of ATP and its stable analogue ATPγS [adenosine-5′-o-(3-thio)triphosphate] was studied in rat hippocampal neurotransmission under normoxic conditions and during oxygen and glucose deprivation (OGD). Field excitatory postsynaptic potentials (fEPSPs) from the dendritic layer or population spikes (PSs) from the soma were extracellularly recorded in the CA1 area of the rat hippocampus. Exogenous application of ATP or ATPγS reduced fEPSP and PS amplitudes. In both cases the inhibitory effect was blocked by the selective A1 adenosine receptor antagonist DPCPX (8-cyclopentyl-1,3-dipropylxanthine) and was potentiated by different ecto-ATPase inhibitors: ARL 67156 (6-N,N-diethyl-D-β,γ-dibromomethylene), BGO 136 (1-hydroxynaphthalene-3,6-disulfonate) and PV4 [hexapotassium dihydrogen monotitanoundecatungstocobaltate(II) tridecahydrate, K6H2[TiW11CoO40]·13H2O]. ATPγS-mediated inhibition was reduced by the P2 antagonist suramin [8-(3-benzamido-4-methylbenzamido)naphthalene-1,3,5-trisulfonate] at the somatic level and by other P2 blockers, PPADS (pyridoxalphosphate-6-azophenyl-2′,4′-disulfonate) and MRS 2179 (2′-deoxy-N6-methyladenosine 3′,5′-bisphosphate), at the dendritic level. After removal of both P2 agonists, a persistent increase in evoked synaptic responses was recorded both at the dendritic and somatic levels. This effect was prevented in the presence of different P2 antagonists. A 7-min OGD induced tissue anoxic depolarization and was invariably followed by irreversible loss of fEPSP. PPADS, suramin, MRS2179 or BBG (brilliant blue G) significantly prevented the irreversible failure of neurotransmission induced by 7-min OGD. Furthermore, in the presence of these P2 antagonists, the development of anoxic depolarization was blocked or significantly delayed. Our results indicate that P2 receptors modulate CA1 synaptic transmission under normoxic conditions by eliciting both inhibitory and excitatory effects. In the same brain region, P2 receptor stimulation plays a deleterious role during a severe OGD insult