Effects of oxygen and glucose deprivation on synaptic transmission and neural progenitor cells maturation in rat dentate gyrus: role of A2A adenosine receptors

G Maraula1, C Traini1, T Mello2, F Pedata1, AM Pugliese1. 1University of Florence, Department of Preclinical and Clinical Pharmacology 50139, Italy, 2University of Florence, Gastroenterology Unit, Department of Clinical Pathophysiology 50139, Italy

The release of substantial amounts of adenosine (Latini and Pedata, J Neurochem. 79:463-484, 2001), that acts in the brain activating four receptor subtypes, A1, A2A, A2B and A3, is one of the early events occurring during ischemia, both in vivo or in vitro.

The role of adenosine A2A receptor activation during oxygen and glucose deprivation (OGD) was investigated using extracellular recordings of field excitatory post-synaptic potentials (fEPSPs) from the dentate gyrus of slices acutely prepared from male Wistar rats (150-200g body weight) (Pugliese et al., Br J Pharmacol., 147:524-532, 2006). Nine-min OGD elicited an irreversible loss of fEPSP (recorded up to 24 hours from the end of OGD) and was invariably followed by the appearance of anoxic depolarization (AD), an unambiguous sign of neuronal damage. The application of the selective adenosine A2A receptor antagonist 4-(2-[7-Amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM241385, 100 nM, n=21) did not modify fEPSP outcome during OGD, but prevented or delayed AD appearance (from 7.6 ± 0.3 min, n=11 in the absence to 9.9 ± 0.6 min, n=9 in the presence of the A2A antagonist, P<0.002, unpaired Student’s t-test) and allowed a significant recovery of neurotransmission after 9-min OGD (96.5 ± 5.0%, n=12, in comparison to 3.4 ± 2.4%, n=35, found in untreated OGD slices, P<0.001, unpaired Student’s t-test).

In related experiments, we investigated whether 9-min OGD modified cell proliferation and maturation in the subgranular zone (SGZ) of dentate gyrus by performing an immunohistochemistry analysis using 5-Bromo-2’-deoxyuridine (BrdU), a DNA replication marker and Doublecortin (DCX), an immature neuronal marker, respectively. Two intraperitonealy injections of BrdU (50mg/kg in 0.9% NaCl) were given for three consecutive days (6 h apart). After 24 hours from last injection, slices were prepared as previously described.

The number of BrdU positive (BrdU+) cells, 24 hours from the end of 9-min OGD, was similar to control, untreated slices (30.4 ± 3.6, n = 10, after OGD in comparison to 29.5 ± 3.3 found in control, n =10). Similarly, in OGD slices treated with ZM241385 (100 nM) the number of BrdU+ cells did not change in comparison to both untreated OGD or control slices.

DCX-immunoreactive neuroblasts (DCX+) were clearly observed in the dentate gyrus of control slices. Their cell bodies were located in the SGZ and their processes appeared very short and without tertiary dendrites. Conversely, 24 hours from the end of OGD, the DCX+ neuroblasts showed a well-developed arborisation of tertiary dendrites. We may conclude that, in the early phase of in vitro ischemia, OGD facilitates the maturation toward a neuronal phenotype without affecting cell proliferation in the SGZ. ZM241385, when applied during OGD, does not modify the effect on cell maturation induced by the ischemic insult.

Data indicate that the selective antagonism of adenosine A2A receptors by ZM241385 prevents the depression of synaptic activity and delays AD appearance induced by a severe OGD in the rat dentate gyrus, thus providing neuroprotection in this pathological condition.

Supported by the Italian Ministry of Health and “Ente Cassa di Risparmio of Florence” (F. Pedata).