Different properties of P2X(7) receptor in hippocampal and cortical astrocytes.
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ABSTRACT:
SUBMITTER: Bianco F
PROVIDER: S-EPMC2686827 | biostudies-other | 2009 Jun
REPOSITORIES: biostudies-other
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Project description:ATP plays an important role in signal transduction between neuronal and glial circuits and within glial networks. Here we describe currents activated by ATP in astrocytes acutely isolated from cortical brain slices by non-enzymatic mechanical dissociation. Brain slices were prepared from transgenic mice that express enhanced green fluorescent protein under the control of the human glial fibrillary acidic protein promoter. Astrocytes were studied by whole-cell voltage clamp. Exogenous ATP evoked inward currents in 75 of 81 astrocytes. In the majority ( approximately 65%) of cells, ATP-induced responses comprising a fast and delayed component; in the remaining subpopulation of astrocytes, ATP triggered a smoother response with rapid peak and slowly decaying plateau phase. The fast component of the response was sensitive to low concentrations of ATP (with EC(50) of approximately 40 nm). All ATP-induced currents were blocked by pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS); they were insensitive to ivermectin. Quantitative real-time PCR demonstrated strong expression of P2X(1) and P2X(5) receptor subunits and some expression of P2X(2) subunit mRNAs. The main properties of the ATP-induced response in cortical astrocytes (high sensitivity to ATP, biphasic kinetics, and sensitivity to PPADS) were very similar to those reported for P2X(1/5) heteromeric receptors studied previously in heterologous expression systems.
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Project description:BackgroundSchema, a concept from cognitive psychology used to explain how new information is integrated with previous experience, is a framework of acquired knowledge within associative network structures as biological correlate, which allows new relevant information to be quickly assimilated by parallel cortical encoding in the hippocampus (HPC) and cortex. Previous work demonstrated that myelin generation in the anterior cingulate cortex (ACC) plays a critical role for dynamic paired association (PA) learning and consolidation, while astrocytes in ACC play a vital role in cognitive decision-making. However, circuit components and mechanism involving HPC-anterior cingulate cortex (ACC) during schema formation remain uncertain. Moreover, the correlation between HPC-ACC circuit and HPC astrocytic activity is unclear.ResultsUtilizing a paired association (PA) behavioral paradigm, we dynamically recorded calcium signals of CA1-ACC projection neurons and ACC neurons during schema formation. Depending on the characteristics of the calcium signals, three distinct stages of schema establishment process were identified. The recruitment of CA1-ACC network was investigated in each stage under CA1 astrocytes Gi pathway chemogenetic activation. Results showed that CA1-ACC projecting neurons excitation gradually decreased along with schema development, while ACC neurons revealed an excitation peak in the middle stage. CA1 astrocytic Gi pathway activation will disrupt memory schema development by reducing CA1-ACC projection neuron recruitment in the initial stage and prevent both CA1-ACC projection neurons and ACC neuron excitation in the middle stage. CA1 astrocytes Gi markedly suppress new PA assimilation into the established memory schema.ConclusionsThese results not only reveal the dynamic feature of CA1-ACC network during schema establishment, but also suggest CA1 astrocyte contribution in different stages of schema establishment.
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Project description:The known seven mammalian receptor subunits (P2X1-7) form cationic channels gated by ATP. Three subunits compose a receptor channel. Each subunit is a polypeptide consisting of two transmembrane regions (TM1 and TM2), intracellular N- and C-termini, and a bulky extracellular loop. Crystallization allowed the identification of the 3D structure and gating cycle of P2X receptors. The agonist-binding pocket is located at the intersection of two neighbouring subunits. In addition to the mammalian P2X receptors, their primitive ligand-gated counterparts with little structural similarity have also been cloned. Selective agonists for P2X receptor subtypes are not available, but medicinal chemistry supplied a range of subtype-selective antagonists, as well as positive and negative allosteric modulators. Knockout mice and selective antagonists helped to identify pathological functions due to defective P2X receptors, such as male infertility (P2X1), hearing loss (P2X2), pain/cough (P2X3), neuropathic pain (P2X4), inflammatory bone loss (P2X5), and faulty immune reactions (P2X7).
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Project description:Remote memories depend on coordinated activity in the hippocampus and frontal cortices, but the timeline of these interactions is debated. Astrocytes sense and modify neuronal activity, but their role in remote memory is scarcely explored. We expressed the Gi-coupled designer receptor hM4Di in CA1 astrocytes and discovered that astrocytic manipulation during learning specifically impaired remote, but not recent, memory recall and decreased activity in the anterior cingulate cortex (ACC) during retrieval. We revealed massive recruitment of ACC-projecting CA1 neurons during memory acquisition, which was accompanied by the activation of ACC neurons. Astrocytic Gi activation disrupted CA3 to CA1 communication in vivo and reduced the downstream response in the ACC. In behaving mice, it induced a projection-specific inhibition of CA1-to-ACC neurons during learning, which consequently prevented ACC recruitment. Finally, direct inhibition of CA1-to-ACC-projecting neurons spared recent and impaired remote memory. Our findings suggest that remote memory acquisition involves projection-specific functions of astrocytes in regulating CA1-to-ACC neuronal communication.
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Project description:Zn²(+) is an essential ion that is stored in and co-released from glutamatergic synapses and it modulates neurotransmitter receptors involved in long-term potentiation (LTP). However, the mechanism(s) underlying Zn²(+) -induced modulation of LTP remain(s) unclear. As the purinergic P2X receptors are relevant targets for Zn²(+) action, we have studied their role in LTP modulation by Zn²(+) in the CA1 region of rat hippocampal slices. Induction of LTP in the presence of Zn²(+) revealed a biphasic effect - 5-50 ?m enhanced LTP induction, whereas 100-300 ?m Zn²(+) inhibited LTP. The involvement of a purinergic mechanism is supported by the fact that application of the P2X receptor antagonists 2',3'-O-(2,4,6-trinitrophenyl) ATP (TNP-ATP) and periodate-oxidized ATP fully abolished the facilitatory effect of Zn²(+) . Notably, application of the P2X? receptor-specific antagonist Brilliant Blue G did not modify the Zn²(+) -dependent facilitation of LTP. Exogenous ATP also produced a biphasic effect - 0.1-1 ?m ATP facilitated LTP, whereas 5-10 ?m inhibited LTP. The facilitatory effect of ATP was abolished by the application of TNP-ATP and was modified in the presence of 5 ?m Zn²(+) , suggesting that P2X receptors are involved in LTP induction and that Zn²(+) leads to an increase in the affinity of P2X receptors for ATP. The latter confirms our previous results from heterologous expression systems. Collectively, our results indicate that Zn²(+) at low concentrations enhances LTP by modulating P2X receptors. Although it is not yet clear which purinergic receptor subtype(s) is responsible for these effects on LTP, the data presented here suggest that P2X? but not P2X? is involved.
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Project description:Astrocytic contributions to neuroinflammation are widely implicated in disease, but they remain incompletely explored. We assess medial prefrontal cortex (PFC) and visual cortex (VCX) astrocyte and whole-tissue gene expression changes in mice following peripherally induced neuroinflammation triggered by a systemic bacterial endotoxin, lipopolysaccharide, which produces sickness-related behaviors, including anhedonia. Neuroinflammation-mediated behavioral changes and astrocyte-specific gene expression alterations peak when anhedonia is greatest and then reverse to normal. Notably, region-specific molecular identities of PFC and VCX astrocytes are largely maintained during reactivity changes. Gene pathway analyses reveal alterations of diverse cell signaling pathways, including changes in cell-cell interactions of multiple cell types that may underlie the central effects of neuroinflammation. Certain astrocyte molecular signatures accompanying neuroinflammation are shared with changes reported in Alzheimer's disease and mouse models. However, we find no evidence of altered neuronal survival or function in the PFC even when neuroinflammation-induced astrocyte reactivity and behavioral changes are significant.
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Project description:Normal nervous system function is critically dependent on the balance of water and ions in the extracellular space (ECS). Pathological reduction in brain interstitial osmolarity results in osmotically-driven flux of water into cells, causing cellular edema which reduces the ECS and increases neuronal excitability and risk of seizures. Astrocytes are widely considered to be particularly susceptible to cellular edema due to selective expression of the water channel aquaporin-4 (AQP4). The apparent resistance of pyramidal neurons to osmotic swelling has been attributed to lack of functional water channels. In this study we report rapid volume changes in CA1 pyramidal cells in hypoosmolar ACSF (hACSF) that are equivalent to volume changes in astrocytes across a variety of conditions. Astrocyte and neuronal swelling was significant within 1 min of exposure to 17 or 40% hACSF, was rapidly reversible upon return to normosmolar ACSF, and repeatable upon re-exposure to hACSF. Neuronal swelling was not an artifact of patch clamp, occurred deep in tissue, was similar at physiological vs. room temperature, and occurred in both juvenile and adult hippocampal slices. Neuronal swelling was neither inhibited by TTX, nor by antagonists of NMDA or AMPA receptors, suggesting that it was not occurring as a result of excitotoxicity. Surprisingly, genetic deletion of AQP4 did not inhibit, but rather augmented, astrocyte swelling in severe hypoosmolar conditions. Taken together, our results indicate that neurons are not osmoresistant as previously reported, and that osmotic swelling is driven by an AQP4-independent mechanism.
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Project description:We have cloned a P2X receptor (OtP2X) from the green algae Ostreococcus tauri. The 42-kDa receptor shares approximately 28% identity with human P2X receptors and 23% with the Dictyostelium P2X receptor. ATP application evoked flickery single channel openings in outside-out membrane patches from human embryonic kidney 293 cells expressing OtP2X. Whole-cell recordings showed concentration-dependent cation currents reversing close to zero mV; ATP gave a half-maximal current at 250 mum. alphabeta-Methylene-ATP evoked only small currents in comparison to ATP (EC(50) > 5 mm). 2',3'-O-(4-Benzoylbenzoyl)-ATP, betagamma-imido-ATP, ADP, and several other nucleotide triphosphates did not activate any current. The currents evoked by 300 mum ATP were not inhibited by 100 microm suramin, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid, 2',3'-O-(2,4,6-trinitrophenol)-ATP, or copper. Ion substitution experiments indicated permeabilities relative to sodium with the rank order calcium >choline >Tris >tetraethylammonium >N-methyl-D-glucosamine. However, OtP2X had a low relative calcium permeability (P(Ca)/P(Na) = 0.4) in comparison with other P2X receptors. This was due at least in part to the presence of an asparagine residue (Asn(353)) at a position in the second transmembrane domain in place of the aspartate that is completely conserved in all other P2X receptor subunits, because replacement of Asn(353) with aspartate increased calcium permeability by approximately 50%. The results indicate that the ability of ATP to gate cation permeation across membranes exists in cells that diverged in evolutionary terms from animals about 1 billion years ago.
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Project description:Background and purposeAstrocytes of the mouse neocortex express functional NMDA receptors, which are not blocked by Mg(2+) ions. However, the pharmacological profile of glial NMDA receptors and their subunit composition is far from complete.Experimental approachWe tested the sensitivity of NMDA receptor-mediated currents to the novel GluN2C/D subunit-selective antagonist UBP141 in mouse cortical astrocytes and neurons. We also examined the effect of memantine, an antagonist that has substantially different affinities for GluN2A/B and GluN2C/d-containing receptors in physiological concentrations of extracellular Mg(2+).Key resultsUBP141 had a strong inhibitory action on NMDA receptor-mediated transmembrane currents in the cortical layer II/III astrocytes with an IC(50) of 2.29 µM and a modest inhibitory action on NMDA-responses in the pyramidal neurons with IC(50) of 19.8 µM. Astroglial and neuronal NMDA receptors exhibited different sensitivities to memantine with IC(50) values of 2.19 and 10.8 µM, respectively. Consistent with pharmacological differences between astroglial and neuronal NMDA receptors, NMDA receptors in astrocytes showed lower Ca(2+) permeability than neuronal receptors with P(Ca) /P(Na) ratio of 3.4.Conclusions and implicationsThe biophysical and pharmacological properties of the astrocytic NMDA receptors strongly suggest that they have a tri-heteromeric structure composed of GluN1, GluN2C/D and GluN3 subunits. The substantial difference between astroglial and neuronal NMDA receptors in their sensitivity to UBP141 and memantine may enable selective modulation of astrocytic signalling that could be very helpful for elucidating the mechanisms of neuron-glia communications. Our results may also provide the basis for the development of novel therapeutic agents specifically targeting glial signalling.
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Project description:To assess the putative role of adenosine triphosphate (ATP) upon nitric oxide (NO) production in the hippocampus, we used as a model both rat hippocampal slices and isolated hippocampal neurons in culture, lacking glial cells. In hippocampal slices, additions of exogenous ATP or 2'(3')-O-(4-Benzoylbenzoyl) ATP (Bz-ATP) elicited concentration-dependent NO production, which increased linearly within the first 15 min and plateaued thereafter; agonist EC50 values were 50 and 15 µM, respectively. The NO increase evoked by ATP was antagonized in a concentration-dependent manner by Coomassie brilliant blue G (BBG) or by N(ω)-propyl-L-arginine, suggesting the involvement of P2X7Rs and neuronal NOS, respectively. The ATP induced NO production was independent of N-methyl-D-aspartic acid (NMDA) receptor activity as effects were not alleviated by DL-2-Amino-5-phosphonopentanoic acid (APV), but antagonized by BBG. In sum, exogenous ATP elicited NO production in hippocampal neurons independently of NMDA receptor activity.
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