The block of P2Y1R counteracts the synaptic failure induced by severe OGD in the DG.

<p>AD was recorded as a negative d.c. shift in response to 9-min OGD in control (<b>a</b>, n = 26). MRS2179 (10 µM) completely prevented the appearance of AD in 23 out of 28 slices during 9-min OGD (<b>b</b>). MRS2179 (10 µM) had no effect in 5 out of 28 slices after 9-min OGD (<b>c</b>, n = 6), in which AD was recorded as a negative d.c. shift. <b>d</b>. example traces taken at the time-points indicated on the graph, immediately before (1), 10 min after the beginning of MRS2179 application (2), 9 minutes after OGD+MRS2179 (3) or 50 min after washout in oxygenated aCSF (4). Inset: example trace taken from the same hippocampal slice 24 hours from the end of OGD applied in the presence of 10 µM MRS2179. In each graph, traces are averages of 3 consecutive responses. Scale bars: 10 ms, 0.5 mV. <b>f</b>. The graph shows the time-course of the effect caused by 9-min OGD on fEPSP amplitude (mean±SEM) in untreated OGD slices (n = 26) and in OGD slices treated with 10 µM MRS2179, in which AD was absent (n = 23) or present (n = 5). fEPSPs amplitude is expressed as percent of the respective pre-ischemic baseline. Open bar: time of drug application.</p

The P2X7R antagonist, BBG, prevents the synaptic failure induced by 9-min OGD in the DG.

<p>AD was recorded as a negative d.c. shift in response to 9-min OGD in control conditions (<b>a</b>, n = 21). BBG (10 µM) completely prevented the appearance of AD in 22 out of 28 slices during 9-min OGD (<b>b</b>). BBG (10 µM) had no effect in 6 out of 28 slices after 9-min OGD (<b>c</b>, n = 6), in which AD was recorded as a negative d.c. shift. <b>d</b>. example traces taken at the time-points indicated on the graph, immediately before (1), 10 min after the beginning of BBG application (2), 9 minutes after OGD+BBG (3) or 50 min after washout in oxygenated aCSF (4). Inset: example trace taken from the same hippocampal slice 24 hours from the end of OGD applied in the presence of 10 µM BBG. In each graph, traces are averages of 3 consecutive responses. Scale bars: 10 ms, 0.5 mV. <b>f</b>. The graph shows the time-course of the effect caused by 9-min OGD on fEPSP amplitude (mean ± SEM) in untreated OGD slices (n = 21) and in OGD slices treated with 10 µM BBG, in which AD was absent (n = 22) or present (n = 6). fEPSPs amplitude is expressed as percent of the respective pre-ischemic baseline. Open bar: time of drug application.</p

Temporal profile of cell proliferation in the SGZ of DG.

<p>Quantification of BrdU⁺ cells in the SGZ of the DG at 3, 6 and 24 hours after the end of OGD. Each column shows the total number of BrdU⁺ cells in the SGZ. Bars represent the mean±SEM. In parentheses is the number of slices investigated. *<i>P</i><0.05 and **<i>P</i><0.01 <i>vs</i> control, One-way ANOVA followed by Newman–Keuls post hoc test.</p

The Selective Antagonism of P2X₇ and P2Y₁ Receptors Prevents Synaptic Failure and Affects Cell Proliferation Induced by Oxygen and Glucose Deprivation in Rat Dentate Gyrus

<div><p>Purinergic P2X and P2Y receptors are broadly expressed on both neurons and glial cells in the central nervous system (CNS), including dentate gyrus (DG). The aim of this research was to determine the synaptic and proliferative response of the DG to severe oxygen and glucose deprivation (OGD) in acute rat hippocampal slices and to investigate the contribution of P2X₇ and P2Y₁ receptor antagonism to recovery of synaptic activity after OGD. Extracellular field excitatory post-synaptic potentials (fEPSPs) in granule cells of the DG were recorded from rat hippocampal slices. Nine-min OGD elicited an irreversible loss of fEPSP and was invariably followed by the appearance of anoxic depolarization (AD). Application of MRS2179 (selective antagonist of P2Y₁ receptor) and BBG (selective antagonist of P2X₇ receptor), before and during OGD, prevented AD appearance and allowed a significant recovery of neurotransmission after 9-min OGD. The effects of 9-min OGD on proliferation and maturation of cells localized in the subgranular zone (SGZ) of slices prepared from rats treated with 5-Bromo-2′-deoxyuridine (BrdU) were investigated. Slices were further incubated with an immature neuron marker, doublecortin (DCX). The number of BrdU⁺ cells in the SGZ was significantly decreased 6 hours after OGD. This effect was antagonized by BBG, but not by MRS2179. Twenty-four hours after 9-min OGD, the number of BrdU⁺ cells returned to control values and a significant increase of DCX immunofluorescence was observed. This phenomenon was still evident when BBG, but not MRS2179, was applied during OGD. Furthermore, the P2Y₁ antagonist reduced the number of BrdU⁺ cells at this time. The data demonstrate that P2X₇ and P2Y₁ activation contributes to early damage induced by OGD in the DG. At later stages after the insult, P2Y₁ receptors might play an additional and different role in promoting cell proliferation and maturation in the DG.</p></div

Data_Sheet_2_The Selective Antagonism of Adenosine A2B Receptors Reduces the Synaptic Failure and Neuronal Death Induced by Oxygen and Glucose Deprivation in Rat CA1 Hippocampus in Vitro.DOCX

<p>Ischemia is a multifactorial pathology characterized by different events evolving in time. Immediately after the ischemic insult, primary brain damage is due to the massive increase of extracellular glutamate. Adenosine in the brain increases dramatically during ischemia in concentrations able to stimulate all its receptors, A₁, A_2A, A_2B, and A₃. Although adenosine exerts clear neuroprotective effects through A₁ receptors during ischemia, the use of selective A₁ receptor agonists is hampered by their undesirable peripheral side effects. So far, no evidence is available on the involvement of adenosine A_2B receptors in cerebral ischemia. This study explored the role of adenosine A_2B receptors on synaptic and cellular responses during oxygen and glucose deprivation (OGD) in the CA1 region of rat hippocampus in vitro. We conducted extracellular recordings of CA1 field excitatory post-synaptic potentials (fEPSPs); the extent of damage on neurons and glia was assessed by immunohistochemistry. Seven min OGD induced anoxic depolarization (AD) in all hippocampal slices tested and completely abolished fEPSPs that did not recover after return to normoxic condition. Seven minutes OGD was applied in the presence of the selective adenosine A_2B receptor antagonists MRS1754 (500 nM) or PSB603 (50 nM), separately administered 15 min before, during and 5 min after OGD. Both antagonists were able to prevent or delay the appearance of AD and to modify synaptic responses after OGD, allowing significant recovery of neurotransmission. Adenosine A_2B receptor antagonism also counteracted the reduction of neuronal density in CA1 stratum pyramidale, decreased apoptosis at least up to 3 h after the end of OGD, and maintained activated mTOR levels similar to those of controls, thus sparing neurons from the degenerative effects caused by the simil-ischemic conditions. Astrocytes significantly proliferated in CA1 stratum radiatum already 3 h after the end of OGD, possibly due to increased glutamate release. A_2Breceptor antagonism significantly prevented astrocyte modifications. Both A_2B receptor antagonists did not protect CA1 neurons from the neurodegeneration induced by glutamate application, indicating that the antagonistic effect is upstream of glutamate release. The selective antagonists of the adenosine A_2B receptor subtype may thus represent a new class of neuroprotective drugs in ischemia.</p

AD development and synaptic failure in the DG of rat hippocampal slices after 9-min OGD.

<p><b>a</b>. Upper panel: fEPSP taken from a typical experiment at the time-points indicated on the graph in a control hippocampal slice (Ctr) continuously superfused with oxygenated aCSF. Lower panel: example traces taken at the time-points indicated on the graph recorded from a hippocampal slice before (4), at the end of 9-min OGD (5) or 50 min after washout in oxygenated aCSF (6). <b>b</b>. The graph shows the time-course of the fEPSP amplitude, expressed as percent of pre-ischemic baseline in the DG (mean±SEM) in a group of control slices (Ctr, n = 7) or in a group of slices subjected to 9-min OGD (OGD, n = 34). Note that, while a stable fEPSP was recorded in control slices, the ischemic insult elicited a gradual reduction of fEPSPs amplitude, which completely disappears and does not recover even after prolonged washing in oxygenated aCSF. Inset: example traces taken from the same respective hippocampal slices shown in panel <b>a</b>, in control (Ctr, recorded 24 hours from slice preparation) or 24 hours from the end of OGD. In each graph, traces are averages of 3 consecutive responses. Scale bars: 10 ms, 0.5 mV. <b>c</b>. Anoxic depolarization (AD) was recorded as a negative d.c. shift in response to 9-min OGD. The d.c. shift was always recorded (n = 34) during 9-min OGD. <b>d</b>. Each column represents the mean±SEM of AD latency or AD amplitude recorded in the DG during 9-min OGD. AD latency was measured from the beginning of OGD insult.</p

Data_Sheet_1_The Selective Antagonism of Adenosine A2B Receptors Reduces the Synaptic Failure and Neuronal Death Induced by Oxygen and Glucose Deprivation in Rat CA1 Hippocampus in Vitro.DOCX

<p>Ischemia is a multifactorial pathology characterized by different events evolving in time. Immediately after the ischemic insult, primary brain damage is due to the massive increase of extracellular glutamate. Adenosine in the brain increases dramatically during ischemia in concentrations able to stimulate all its receptors, A₁, A_2A, A_2B, and A₃. Although adenosine exerts clear neuroprotective effects through A₁ receptors during ischemia, the use of selective A₁ receptor agonists is hampered by their undesirable peripheral side effects. So far, no evidence is available on the involvement of adenosine A_2B receptors in cerebral ischemia. This study explored the role of adenosine A_2B receptors on synaptic and cellular responses during oxygen and glucose deprivation (OGD) in the CA1 region of rat hippocampus in vitro. We conducted extracellular recordings of CA1 field excitatory post-synaptic potentials (fEPSPs); the extent of damage on neurons and glia was assessed by immunohistochemistry. Seven min OGD induced anoxic depolarization (AD) in all hippocampal slices tested and completely abolished fEPSPs that did not recover after return to normoxic condition. Seven minutes OGD was applied in the presence of the selective adenosine A_2B receptor antagonists MRS1754 (500 nM) or PSB603 (50 nM), separately administered 15 min before, during and 5 min after OGD. Both antagonists were able to prevent or delay the appearance of AD and to modify synaptic responses after OGD, allowing significant recovery of neurotransmission. Adenosine A_2B receptor antagonism also counteracted the reduction of neuronal density in CA1 stratum pyramidale, decreased apoptosis at least up to 3 h after the end of OGD, and maintained activated mTOR levels similar to those of controls, thus sparing neurons from the degenerative effects caused by the simil-ischemic conditions. Astrocytes significantly proliferated in CA1 stratum radiatum already 3 h after the end of OGD, possibly due to increased glutamate release. A_2Breceptor antagonism significantly prevented astrocyte modifications. Both A_2B receptor antagonists did not protect CA1 neurons from the neurodegeneration induced by glutamate application, indicating that the antagonistic effect is upstream of glutamate release. The selective antagonists of the adenosine A_2B receptor subtype may thus represent a new class of neuroprotective drugs in ischemia.</p

Image_1_The Selective Antagonism of Adenosine A2B Receptors Reduces the Synaptic Failure and Neuronal Death Induced by Oxygen and Glucose Deprivation in Rat CA1 Hippocampus in Vitro.TIF

<p>Ischemia is a multifactorial pathology characterized by different events evolving in time. Immediately after the ischemic insult, primary brain damage is due to the massive increase of extracellular glutamate. Adenosine in the brain increases dramatically during ischemia in concentrations able to stimulate all its receptors, A₁, A_2A, A_2B, and A₃. Although adenosine exerts clear neuroprotective effects through A₁ receptors during ischemia, the use of selective A₁ receptor agonists is hampered by their undesirable peripheral side effects. So far, no evidence is available on the involvement of adenosine A_2B receptors in cerebral ischemia. This study explored the role of adenosine A_2B receptors on synaptic and cellular responses during oxygen and glucose deprivation (OGD) in the CA1 region of rat hippocampus in vitro. We conducted extracellular recordings of CA1 field excitatory post-synaptic potentials (fEPSPs); the extent of damage on neurons and glia was assessed by immunohistochemistry. Seven min OGD induced anoxic depolarization (AD) in all hippocampal slices tested and completely abolished fEPSPs that did not recover after return to normoxic condition. Seven minutes OGD was applied in the presence of the selective adenosine A_2B receptor antagonists MRS1754 (500 nM) or PSB603 (50 nM), separately administered 15 min before, during and 5 min after OGD. Both antagonists were able to prevent or delay the appearance of AD and to modify synaptic responses after OGD, allowing significant recovery of neurotransmission. Adenosine A_2B receptor antagonism also counteracted the reduction of neuronal density in CA1 stratum pyramidale, decreased apoptosis at least up to 3 h after the end of OGD, and maintained activated mTOR levels similar to those of controls, thus sparing neurons from the degenerative effects caused by the simil-ischemic conditions. Astrocytes significantly proliferated in CA1 stratum radiatum already 3 h after the end of OGD, possibly due to increased glutamate release. A_2Breceptor antagonism significantly prevented astrocyte modifications. Both A_2B receptor antagonists did not protect CA1 neurons from the neurodegeneration induced by glutamate application, indicating that the antagonistic effect is upstream of glutamate release. The selective antagonists of the adenosine A_2B receptor subtype may thus represent a new class of neuroprotective drugs in ischemia.</p

Image_2_The Selective Antagonism of Adenosine A2B Receptors Reduces the Synaptic Failure and Neuronal Death Induced by Oxygen and Glucose Deprivation in Rat CA1 Hippocampus in Vitro.TIF

<p>Ischemia is a multifactorial pathology characterized by different events evolving in time. Immediately after the ischemic insult, primary brain damage is due to the massive increase of extracellular glutamate. Adenosine in the brain increases dramatically during ischemia in concentrations able to stimulate all its receptors, A₁, A_2A, A_2B, and A₃. Although adenosine exerts clear neuroprotective effects through A₁ receptors during ischemia, the use of selective A₁ receptor agonists is hampered by their undesirable peripheral side effects. So far, no evidence is available on the involvement of adenosine A_2B receptors in cerebral ischemia. This study explored the role of adenosine A_2B receptors on synaptic and cellular responses during oxygen and glucose deprivation (OGD) in the CA1 region of rat hippocampus in vitro. We conducted extracellular recordings of CA1 field excitatory post-synaptic potentials (fEPSPs); the extent of damage on neurons and glia was assessed by immunohistochemistry. Seven min OGD induced anoxic depolarization (AD) in all hippocampal slices tested and completely abolished fEPSPs that did not recover after return to normoxic condition. Seven minutes OGD was applied in the presence of the selective adenosine A_2B receptor antagonists MRS1754 (500 nM) or PSB603 (50 nM), separately administered 15 min before, during and 5 min after OGD. Both antagonists were able to prevent or delay the appearance of AD and to modify synaptic responses after OGD, allowing significant recovery of neurotransmission. Adenosine A_2B receptor antagonism also counteracted the reduction of neuronal density in CA1 stratum pyramidale, decreased apoptosis at least up to 3 h after the end of OGD, and maintained activated mTOR levels similar to those of controls, thus sparing neurons from the degenerative effects caused by the simil-ischemic conditions. Astrocytes significantly proliferated in CA1 stratum radiatum already 3 h after the end of OGD, possibly due to increased glutamate release. A_2Breceptor antagonism significantly prevented astrocyte modifications. Both A_2B receptor antagonists did not protect CA1 neurons from the neurodegeneration induced by glutamate application, indicating that the antagonistic effect is upstream of glutamate release. The selective antagonists of the adenosine A_2B receptor subtype may thus represent a new class of neuroprotective drugs in ischemia.</p