Neuronal Adenosine A2A Receptors Are Critical Mediators of Neurodegeneration Triggered by Convulsions

A convulsive period triggers an early upregulation of A_2AR in glutamate synapses, likely involving a local translation of synaptic A_2AR mRNA, and a delayed upregulation of A_2AR in modified microglia cells in the hippocampus. A, The immuno-density of the microglia marker CD11b and of A_2AR were low in hippocampal sections from saline-treated mice, whereas they were increased and colocated circa 22 h after a convulsive period triggered by the subcutaneous injection of 35 mg/kg kainate (B; confocal images representative of n = 3 mice per group). C, Kainate-induced convulsions triggered an increase of mRNA levels of CD11b and Iba1, characteristic of reactive microglia, only after 12–24 h in mouse hippocampal extracts (n = 6 for each time point). D, In contrast, an increased A_2AR density was observed in synaptosomes (purified synapses) as soon as 2 h after kainate injection, whereas an A_2AR binding density was only upregulated in total membranes after 24 h in the hippocampus (n = 5 for each determination, except n = 6 at 24 h). E, F, Kainate-induced A_2AR upregulation was evident in glutamate synapses, as gauged by the kainate-induced increased A_2AR immunoreactivity in hippocampal synaptosomes immuno-positive for vesicular glutamate transporter type 1 (vGluT1; n = 6). G, A_2AR mRNA was identified in purified synaptosomes from the hippocampus (HIP), similarly to its presence in rat striatal extracts (STR) of the rat brain (n = 3). H, The synaptic localization of A_2AR mRNA was confirmed in a pure axonal preparation from hippocampal neurons cultured in microfluidic chambers, which ensures a physical segregation of axon terminals and cell bodies (n = 2). I, The contribution of the local translation of synaptic A_2AR mRNA for the kainate-induced upregulation of A_2AR was concluded by the ability of cycloheximide (n = 4) to prevent the increase of A_2AR binding density in synaptosomes exposed for 2 h to 5 μM kainate (n = 5). Data are mean ± SEM; *p < 0.05 between bars or versus control (saline, sal).

A convulsive period bolsters the function of synaptic A_2AR in glutamatergic synapses. A, B, A_2AR blockade with SCH58261 (50 nM) did not modified the depolarization-evoked release of glutamate (i.e., on augmentation of extracellular K⁺ as indicated by the horizontal insert line) from hippocampal synaptosomes from saline-treated mice (left panel, n = 8), but increased glutamate release from synaptosomes collected from convulsing rats 2 h after kainate administration (10 mg/kg, i.p.; right panel, n = 6). C, D, The amplitude of LTP, triggered by high-frequency stimulation (100 Hz for 1 s) of afferent Schaffer fibers, was larger in CA1 synapses from hippocampal slices collected 4 h after the administration of kainate (n = 6) than in saline-treated mice (n = 5). C, Pairs of superimposed fEPSP recorded 10 min before (black and red traces) and 60 min after the high-frequency train (dark or light blue traces) in slices from saline-treated mice (left pair) or 4 h after kainate administration (right pair). D, Time course of average fEPSPs before and after application of the high-frequency train (at time 0) in slices from control mice (black symbols), the aberrantly large LTP in slices collected 4 h after kainate administration (red symbols) and the ability of 50 nM SCH58261 to bring LTP amplitude back to control levels in these slices collected 4 h after kainate injection (orange symbols). This indicates that convulsions-induced aberrant synaptic plasticity is due to overfunctioning of A_2AR. Data are mean ± SEM; *p < 0.05 versus control (saline in B; WT – wild type in D); **p < 0.05 comparing kainate versus kainate+SCH58261 in D.

Neuronal A_2AR are critical to convert the initial convulsions-induced glutamate hyperfunctioning into a subsequent dysfunction and loss of glutamate synapses with the activation of calpains. A, B, Absence of alteration of the high-frequency train (100 Hz for 1 s, applied at time 0)-induced LTP of fEPSP recorded in the CA1 stratum radiatum on stimulation of afferent Schaffer fibers in hippocampal slices collected 4 h after the induction of a convulsive period on subcutaneous administration of 35 mg/kg kainate to mice with a genetic deletion of A_2AR selectively in forebrain neurons (fb-A_2AR-KO, n = 6). B, C, Likewise, fb-A_2AR-KO mice analyzed 24 h after the injection of kainate did not display neuronal damage (FluoroJade-C, FJ-C), microgliosis (CD11b immunoreactivity) or astrogliosis (GFAP immunoreactivity), which was present in “wild-type” littermates (n = 8–10 mice per group). The conversion from the initial convulsions-induced hyperfunctioning of glutamate synapses into neurodegeneration first involved a synaptic dysfunction, as shown (D) by the lower amplitude of LTP in hippocampal slices collected 12 h after the administration of kainate to trigger a convulsive period within the first 75 min (red symbols) compared to saline-treated mice (black symbols; n = 10). E, The kainate-induced reduction of LTP amplitude at 12 h is not present in fb-A_2AR-KO mice (n = 7). F, Summary of the time-dependent evolution of the impact of a kainate-induced convulsive period on hippocampal synaptic plasticity: LTP was first bolstered (at 4 h) and later depressed (at 12 h) in a manner strictly dependent on neuronal A_2AR (lack of alterations in fb-A_2AR-KO). G, H, The evolution from overexcitation to decreased synaptic plasticity caused by kainate-induced convulsions was associated with a loss of synaptic markers, namely, SNAP-25, syntaxin-I and vesicular glutamate transporters type 1 (vGluT1), which was seen 12 h after kainate administration (n = 4), but not after 4 h in wild-type mice (WT; open symbols, n = 4) or in fb-A_2AR-KO mice (filled symbols, n = 4). I–K, This putative synaptotoxicity likely involved the recruitment of calpains, which was strictly dependent on the presence of neuronal A_2AR: in fact, kainate triggered a decreased immunoreactivity of the calpain substrate, spectrin (I, J), and a parallel increase of the immunodensity of the calpain-derived spectrin breakdown degradation products (SBDP-145–150 kDa) in wild-type but not in fb-A_2AR-KO mice (I, K; n = 5 in each group). Data are mean ± SEM; *p < 0.05 between bars or between kainate and saline (SAL) or versus control (100%).