Homer1 Scaffold Proteins Govern Ca2+ Dynamics in Normal and Reactive Astrocytes.

In astrocytes, the intracellular calcium (Ca2+) signaling mediated by activation of metabotropic glutamate receptor 5 (mGlu5) is crucially involved in the modulation of many aspects of brain physiology, including gliotransmission. Here, we find that the mGlu5-mediated Ca2+ signaling leading to release of glutamate is governed by mGlu5 interaction with Homer1 scaffolding proteins. We show that the long splice variants Homer1b/c are expressed in astrocytic processes, where they cluster with mGlu5 at sites displaying intense local Ca2+ activity. We show that the structural and functional significance of the Homer1b/c-mGlu5 interaction is to relocate endoplasmic reticulum (ER) to the proximity of the plasma membrane and to optimize Ca2+ signaling and glutamate release. We also show that in reactive astrocytes the short dominant-negative splice variant Homer1a is upregulated. Homer1a, by precluding the mGlu5-ER interaction decreases the intensity of Ca2+ signaling thus limiting the intensity and the duration of glutamate release by astrocytes. Hindering upregulation of Homer1a with a local injection of short interfering RNA in vivo restores mGlu5-mediated Ca2+ signaling and glutamate release and sensitizes astrocytes to apoptosis. We propose that Homer1a may represent one of the cellular mechanisms by which inflammatory astrocytic reactions are beneficial for limiting brain injury.

Synaptic Integration of Adult-Born Hippocampal Neurons Is Locally Controlled by Astrocytes.

Adult neurogenesis is regulated by the neurogenic niche, through mechanisms that remain poorly defined. Here, we investigated whether niche-constituting astrocytes influence the maturation of adult-born hippocampal neurons using two independent transgenic approaches to block vesicular release from astrocytes. In these models, adult-born neurons but not mature neurons showed reduced glutamatergic synaptic input and dendritic spine density that was accompanied with lower functional integration and cell survival. By taking advantage of the mosaic expression of transgenes in astrocytes, we found that spine density was reduced exclusively in segments intersecting blocked astrocytes, revealing an extrinsic, local control of spine formation. Defects in NMDA receptor (NMDAR)-mediated synaptic transmission and dendrite maturation were partially restored by exogenous D-serine, whose extracellular level was decreased in transgenic models. Together, these results reveal a critical role for adult astrocytes in local dendritic spine maturation, which is necessary for the NMDAR-dependent functional integration of newborn neurons.

Pannexin-1-mediated ATP release from area CA3 drives mGlu5-dependent neuronal oscillations.

The activation of Group I metabotropic glutamate receptors (GI mGluRs) in the hippocampus results in the appearance of persistent bursts of synchronised neuronal activity. In response to other stimuli, such activity is known to cause the release of the purines ATP and its neuroactive metabolite, adenosine. We have thus investigated the potential release and role of the purines during GI mGluR-induced oscillations in rat hippocampal areas CA3 and CA1 using pharmacological techniques and microelectrode biosensors for ATP and adenosine. The GI mGluR agonist DHPG induced both persistent oscillations in neuronal activity and the release of adenosine in areas CA1 and CA3. In contrast, the DHPG-induced release of ATP was only observed in area CA3. Whilst adenosine acting at adenosine A1 receptors suppressed DHPG-induced burst activity, the activation of mGlu5 and P2Y1 ATP receptors were necessary for the induction of DHPG-induced oscillations. Selective inhibition of pannexin-1 hemichannels with a low concentration of carbenoxolone (10 µM) or probenecid (1 mM) did not affect adenosine release in area CA3, but prevented both ATP release in area CA3 and DHPG-induced bursting. These data reveal key aspects of GI mGluR-dependent neuronal activity that are subject to bidirectional regulation by ATP and adenosine in the initiation and pacing of burst firing, respectively, and which have implications for the role of GI mGluRs in seizure activity and neurodevelopmental disorders.

G-protein coupled receptor-evoked glutamate exocytosis from astrocytes: role of prostaglandins.

Astrocytes are highly secretory cells, participating in rapid brain communication by releasing glutamate. Recent evidences have suggested that this process is largely mediated by Ca(2+)-dependent regulated exocytosis of VGLUT-positive vesicles. Here by taking advantage of VGLUT1-pHluorin and TIRF illumination, we characterized mechanisms of glutamate exocytosis evoked by endogenous transmitters (glutamate and ATP), which are known to stimulate Ca(2+) elevations in astrocytes. At first we characterized the VGLUT1-pHluorin expressing vesicles and found that VGLUT1-positive vesicles were a specific population of small synaptic-like microvesicles containing glutamate but which do not express VGLUT2. Endogenous mediators evoked a burst of exocytosis through activation of G-protein coupled receptors. Subsequent glutamate exocytosis was reduced by about 80% upon pharmacological blockade of the prostaglandin-forming enzyme, cyclooxygenase. On the other hand, receptor stimulation was accompanied by extracellular release of prostaglandin E2 (PGE2). Interestingly, administration of exogenous PGE2 produced per se rapid, store-dependent burst exocytosis of glutamatergic vesicles in astrocytes. Finally, when PGE2-neutralizing antibody was added to cell medium, transmitter-evoked exocytosis was again significantly reduced (by about 50%). Overall these data indicate that cyclooxygenase products are responsible for a major component of glutamate exocytosis in astrocytes and that large part of such component is sustained by autocrine/paracrine action of PGE2.

Homeostatic control of synaptic activity by endogenous adenosine is mediated by adenosine kinase.

Extracellular adenosine, a key regulator of neuronal excitability, is metabolized by astrocyte-based enzyme adenosine kinase (ADK). We hypothesized that ADK might be an upstream regulator of adenosine-based homeostatic brain functions by simultaneously affecting several downstream pathways. We therefore studied the relationship between ADK expression, levels of extracellular adenosine, synaptic transmission, intrinsic excitability, and brain-derived neurotrophic factor (BDNF)-dependent synaptic actions in transgenic mice underexpressing or overexpressing ADK. We demonstrate that ADK: 1) Critically influences the basal tone of adenosine, evaluated by microelectrode adenosine biosensors, and its release following stimulation; 2) determines the degree of tonic adenosine-dependent synaptic inhibition, which correlates with differential plasticity at hippocampal synapses with low release probability; 3) modulates the age-dependent effects of BDNF on hippocampal synaptic transmission, an action dependent upon co-activation of adenosine A2A receptors; and 4) influences GABAA receptor-mediated currents in CA3 pyramidal neurons. We conclude that ADK provides important upstream regulation of adenosine-based homeostatic function of the brain and that this mechanism is necessary and permissive to synaptic actions of adenosine acting on multiple pathways. These mechanistic studies support previous therapeutic studies and implicate ADK as a promising therapeutic target for upstream control of multiple neuronal signaling pathways crucial for a variety of neurological disorders.

Minor contribution of ATP P2 receptors to electrically-evoked electrographic seizure activity in hippocampal slices: Evidence from purine biosensors and P2 receptor agonists and antagonists.

While the position of adenosine as an endogenous anticonvulsant is well established, it is unclear to what extent its precursor, ATP, contributes to seizure activity via P2 receptors. In this study we have addressed this issue through the use of ATP biosensors and agonists and antagonists of ATP P2 receptors to detect the release and role of ATP, respectively, during electrically-evoked electrographic seizure-like events (eSLEs) in rat hippocampal slices. The broad-spectrum P2 receptor antagonists RB-2 and PPADS (10µM) caused a small ∼30% inhibition of eSLE duration, and a reduction in intensity. This inhibition of eSLEs was partially reproduced with the P2X(1,2/3,3) antagonist NF023 (10µM), but not the P2X(7) antagonist BBG (10µM). However, the P2X receptor agonist α,ß-meATP did not enhance eSLEs, but instead reduced their duration. Furthermore, we could discern no role for P2Y(1) receptors in electrically-evoked eSLEs: both the P2Y(1) antagonist MRS2179 (10µM) and the P2Y(1) receptor agonist 2-methylthioADP (10µM) were without effect on eSLEs. Consistent with a minor role for ATP P2 receptors on eSLEs we could detect no ATP release during eSLEs, although appreciable quantities of adenosine were detected, which had a pronounced inhibitory action on eSLEs via A(1) receptors. We conclude that the role of ATP P2 receptors in modulating electrographic seizure activity is limited, at least in models such as this one requiring electrical stimulation of afferent fibres. We further conclude that the presence and action of adenosine under these conditions may primarily reflect direct release of this purine.

The novel NTPDase inhibitor sodium polyoxotungstate (POM-1) inhibits ATP breakdown but also blocks central synaptic transmission, an action independent of NTPDase inhibition.

Understanding the mechanisms and properties of purinergic signalling would be greatly assisted by the discovery of subtype selective and potent inhibitors of the NTPDase enzymes, which metabolise nucleotides such as ATP and ADP in the extracellular space. Currently ARL 67156 is the best available NTPDase inhibitor, but its relatively poor efficacy means that negative results are difficult to interpret. POM-1 (sodium polyoxotungstate) is a novel NTPDase inhibitor, which has shown promising results with the inhibition of recombinant NTPDases 1, 2 and 3. We have tested the effectiveness and physiological effects of POM-1 with cerebellar and hippocampal slices. Using the malachite green phosphate assay, HPLC and biosensor measurements we have found that POM-1 is more effective at blocking ATP breakdown in cerebellar slices than ARL 67156. The site of inhibition is at the first step of the breakdown cascade (conversion of ATP to ADP) and the effects of POM-1 appear readily reversible. However, POM-1 has multiple effects on synaptic transmission. At the cerebellar parallel fibre-Purkinje cell (PF) synapse POM-1 produced a long lasting inhibition of transmission, which was preceded in a minority of synapses by a transient increase in PF excitatory postsynaptic potential (EPSP) amplitude (approximately 20%). This increase in PF EPSP amplitude appears to result from a reduction in the tonic activation of presynaptic A1 receptors, consistent with POM-1 preventing the breakdown of ATP to adenosine. The reduction in PF EPSP amplitude does not however appear to result from NTPDase inhibition as it persists when both adenosine and ATP (P2Y and P2X) receptors are blocked. An increase in paired pulse ratio and a reduction in presynaptic volley amplitude suggest that there is a presynaptic component of POM-1 action which reduces glutamate release. POM-1 produced similar inhibition at climbing fibre synapses and at hippocampal CA1 pyramidal synapses. Thus although POM-1 is more effective than ARL 67156 at blocking ATP breakdown its usefulness is limited by off-target actions on synaptic transmission.