Functional NMDA receptor subtype 2B is expressed in astrocytes after ischemia in vivo and anoxia in vitro

Abstract

NMDA-type glutamate receptors play a critical role in neuronal synaptogenesis, plasticity, and excitotoxic death. Recent studies indicate that functional NMDA receptors are also expressed in certain glial populations in the normal brain. Using immunohistochemical methods, we detected the presence of the NMDA receptor 2B (NR2B) subunit of the NMDA receptor in neurons but not astrocytes in the CA1 and subicular regions of the rat hippocampus. However, after ischemia-induced neuronal death in these regions, double immunohistochemical labeling revealed that NR2B subunits colocalized with the astrocyte marker glial fibrillary acid protein and with NR1 subunits that are required for functional NMDA receptors. NR2B expression was first observed 3 d after ischemia and reached a peak at 28 d. At 56 d, only a few NR2B-expressing astrocytes were still present. In vitro, when postnatal hippocampal cultures were subjected to 5 min of anoxia, it resulted in NR2B expression on astrocytes in the glial feed layer. Imaging of intracellular calcium with postanoxic cultures and astrocytes isolated acutely from the ischemic hippocampus revealed a rise in intracellular [Ca2+] after stimulation with the specific agonist NMDA. The response could be blocked reversibly with the competitive antagonist 2-amino-5-phosphonovalerate and attenuated by the NR2B-selective antagonist ifenprodil. Control astrocytes were not responsive to NMDA but responded to glutamate. An understanding of the role of astrocytes that express functional NMDA receptors in response to ischemia may guide development of novel stroke therapies.

Fig. 1.

Western blots probed with the NR2B antibodies obtained from Dr. R. Huganir (A) andChemicon (B). Lanes were loaded with 25 and 50 μg of protein obtained from transiently transfected HEK cells expressing NR1/2A, NR1/2B, or NR1/2C. Both antibodies showed high specificity for NR2B at 180 kDa, although slight cross-reactivity with NR2A could be detected with the Chemicon antibody.

Fig. 2.

Immunohistochemistry of control rat brains showing the normal distributions of NR2B and NR1. A,B, Overview (scale bar, 100 μm) of the hippocampus and high-power magnification (scale bar, 50 μm) of the CA1 region.A, NR2B staining is present in the soma and dendrites of pyramidal cells throughout the hippocampus, as well as the soma and dendrites of dentate granule cells. B, NR1 is present in the soma and dendrites of pyramidal cells throughout the hippocampus, as well as the soma and dendrites of dentate granule cells. Glial cells from control brains showed no detectable staining with either of these antibodies.

Fig. 3.

Time course of glial NR2B expression after transient forebrain ischemia. A–C, Overview (scale bar, 100 μm) of the hippocampus and high-power magnification (scale bar, 50 μm) of the CA1 region. A, At 7 d after ischemia, the pyramidal cell layer (py) of the CA1 contains pyknotic neuronal cell somata, and the stratum radiatum (ra) is devoid of NR2B-positive dendrites or neurons. Numerous NR2B-positive glial cells are present within the CA1 and subicular regions but are notably absent from the CA3 region and dentate gyrus. B, By 28 d after ischemia, there is an increase in the number and intensity of staining of NR2B-positive glial cells throughout the CA1 and subicular regions, and little remains of pyknotic neuronal cell somata.C, By 42 d after ischemia, there is a marked decline in the number and intensity of staining of NR2B-positive glial cells in the CA1 and subicular regions.

Fig. 4.

Double labeling with glial cell markers demonstrating that, after ischemia in vivo, NR1 and NR2B are colocalized in astrocytes. A, Stratum radiatum of the CA1 region, 14 d after ischemia. Scale bar, 50 μm. GFAP and NR2B are colocalized within astrocytes. B, Stratum radiatum of the CA1 21 d after ischemia. Scale bar, 50 μm. GSA1B4 and NR2B are not colocalized, which indicates that the NR2B-immunoreactive glial cells are not microglia. C, Stratum radiatum of the CA1 28 d after ischemia. Scale bar, 50 μm. NR1 and NR2B are colocalized within astrocytes, which demonstrates that both subunits necessary for functional NMDARs are present in the same cells.

Fig. 5.

Double labeling with glial cell markers demonstrating that, after anoxia in vitro, NR1 and NR2B are colocalized in astrocytes. A, In a control postnatal hippocampal culture after 10 d in vitro, NR2B immunoreactivity is restricted to neuronal cells, and GFAP immunoreactivity is restricted to glial cells, with no overlap. Scale bar, 20 μm. B, In a postnatal hippocampal culture 3 d after anoxia, GFAP and NR2B are colocalized in the surviving astrocytes. Note that the NR2B immunoreactivity extends into distal glial processes that are not visible with staining for GFAP. Scale bar, 50 μm. C, In a postnatal hippocampal culture 3 d after anoxia, NR1 and NR2B are colocalized in astrocytes, which demonstrates that both subunits necessary for functional NMDARs are present in the same cells. Scale bar, 20 μm.

Fig. 6.

Functional NMDARs in astrocytes isolated acutely from the CA1 region of the hippocampus 20 d after ischemia.A, Phase-contrast image of acutely isolated astrocytes.B, Superfusion with 1 mm NMDA (solid bar) for 1 min resulted in a monophasic increase in [Ca²⁺]_I, represented as the ΔF/F of fluo-4 fluorescence after background correction. C, Concentration–response experiment showing the need for high concentrations of NMDA to elicit a calcium response. D, An astrocyte isolated acutely from a nonischemic hippocampus showed no response to 5 min of NMDA, whereas superfusion of glutamate (glu) resulted in an increase in [Ca²⁺]_i. E, Superfusion of 1 mm NMDA and 1 mm APV for 5 min elicited no change in [Ca²⁺]_i, whereas superfusion of 1 mm NMDA alone resulted in a monophasic increase in [Ca²⁺]_i. F, Superfusion with 3 μm ifenprodil alone (1) or 3 μm ifenprodil with 1 mm NMDA (2) elicited only an attenuated calcium response. After washing with HBSS (3), a calcium response could be elicited with 1 mm NMDA alone (4). The representative examples shown in B–F were typical of results found in two to three astrocytes.

Fig. 7.

NMDA-induced increase in [Ca²⁺]_i demonstrates the presence of functional NMDARs in postanoxic astrocytes in culture. The representative example of a postnatal hippocampal astrocyte, 3 d after anoxia, shows a polyphasic increase in [Ca²⁺]_i resulting from the superfusion of 1 mm NMDA for 10 min (indicated by the solid line). The dotted line indicates the superfusion of a medium containing a blocking mixture to eliminate the contribution of non-NMDA glutamate receptors (see Results). A similar result was found in a total of four astrocytes examined. [Ca²⁺]_i is represented as the ΔF/F of fluo-4 fluorescence after background correction.

Fig. 8.

A, GFAP staining of the ipsilateral and contralateral facial nucleus 14 d after unilateral resection of the facial nerve. Reactive astrocytes can only be detected in the ipsilateral nucleus. B, NR2B staining of the ipsilateral and contralateral facial nucleus of the same animal. No NR2B immunoreactivity can be found on neurons or glia.