NMDA receptors are expressed in oligodendrocytes and activated in ischaemia

Abstract

Glutamate-mediated damage to oligodendrocytes contributes to mental or physical impairment in periventricular leukomalacia (pre- or perinatal white matter injury leading to cerebral palsy), spinal cord injury, multiple sclerosis and stroke. Unlike neurons, white matter oligodendrocytes reportedly lack NMDA (N-methyl-d-aspartate) receptors. It is believed that glutamate damages oligodendrocytes, especially their precursor cells, by acting on calcium-permeable AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)/kainate receptors alone or by reversing cystine-glutamate exchange and depriving cells of antioxidant protection. Here we show that precursor, immature and mature oligodendrocytes in the white matter of the cerebellum and corpus callosum exhibit NMDA-evoked currents, mediated by receptors that are blocked only weakly by Mg2+ and that may contain NR1, NR2C and NR3 NMDA receptor subunits. NMDA receptors are present in the myelinating processes of oligodendrocytes, where the small intracellular space could lead to a large rise in intracellular ion concentration in response to NMDA receptor activation. Simulating ischaemia led to development of an inward current in oligodendrocytes, which was partly mediated by NMDA receptors. These results point to NMDA receptors of unusual subunit composition as a potential therapeutic target for preventing white matter damage in a variety of diseases.

Figure 1. Glutamate-evoked current in oligodendrocytes

a-c Lucifer (green) and antibody (red) to (a) NG2, (b) O4, and (c) MBP (colocalization in overlay, yellow). Scale 20μm. d TTX blocks synaptic currents in precursor. e Membrane resistance (±s.e.m.), p<0.001 comparing precursors with immature or mature). f Glutamate (100μM) evoked current (p=0.11 and 0.14 comparing 33 mature with 22 immature or 22 precursors). g Effect of TTX (n=5) and TBOA (n=4). h Effect of 200μM D-AP5 (blocks NMDA responses by ~78%: Supplementary Material) and 25μM NBQX (blocks AMPA responses >99%) in 0mM Mg. i Current remaining in 2mM Mg²⁺ (n=7) in 50μM AP5 (p=0.02, blocks NMDA receptors by ~39%), 25μM NBQX (p=0.0003) and AP5+NBQX (p=0.0008), and in 0 Mg²⁺ (n=4-6) in 200μM AP5 (p=0.01), 25μM NBQX (p=0.16), AP5+NBQX (p=0.0003), 10μM MK-801 (p=0.002) and 1mM MCPG plus 200μM CPCCOEt (mGluR blockers, p=0.25). All 2mM Mg²⁺ (except h,i), −63mV, 24°C.

Figure 2. Oligodendrocyte NMDA receptors show weak Mg²⁺-block

a Response of mature corpus callosum oligodendrocyte to 60μM NMDA, 20μM AMPA, 30μM kainate (KA) and 100μM trans-ACPD (0mM Mg²⁺). b Peak current in a for NMDA (n=22 cells), AMPA (n=12), KA (n=10) and ACPD (n=5). c Mature Lucifer-filled oligodendrocyte in corpus callosum (scale 20μm). d Responses of mature cerebellar oligodendrocyte in 0mM Mg²⁺. e Current in d for NMDA (n=26), AMPA (n=23), KA (n=5) and ACPD (n=16). f NMDA-evoked current in cerebellar cells (p=0.059 and 0.042 comparing 79 mature with 19 immature or 26 precursors). g NMDA responses rundown linearly with time (dashed line) during repeated (1/10 mins) applications: top, specimen cell; bottom, normalised data (n=10). h Effect of D-AP5 (50μM). i Effect of TTX (1μM). j Effect of 50μM D-AP5 (n=16, p=10⁻¹⁸), 1μM TTX (n=5, p=0.64), 25μM NBQX (n=4, p=0.96), 5μM strychnine plus 20μM bicuculline (n=16, p=0.14), ifenprodil (10μM, n=3, p=0.18), pregnenolone sulphate (100μM, n=4, p=0.085), D-cycloserine (1mM, n=3, p=0.6) and D-serine (100μM, n=5, p=0.34) on NMDA-evoked current at −63mV. k NMDA response in 0mM, 2mM and 0mM Mg²⁺ again. l Fraction of NMDA-evoked current remaining in 2mM Mg²⁺, in 4 precursor, 6 immature and 9 mature cells (insignificantly different, p=0.51). Arrows: values, for NR1 with NR2C or 2D, 2A or 2B, or 2A and 3A. m Normalized I-V relation for NMDA-evoked current in two precursors in 0mM and three different precursors in 2mM Mg²⁺. Cerebellum except a-c; 0 Mg²⁺ (except k-m); 100μM glycine and 5μM strychnine (except D-cycloserine, D-serine in j); 60μM NMDA; 24°C, −63mV.

Figure 3. Oligodendrocyte NMDA receptors

a Cerebellar cortex labelled for NR2C (WM, white matter, GL granular layer, ML molecular layer). b Enlarged box in a. Arrows: oligodendrocyte process; arrow heads: oligodendrocyte soma. c Oligodendrocyte processes labelling with Chemicon NR1 antibody (see also Supplementary Fig. 6). d NR3 labelling. e Immunogold (black dots, arrowheads, Wenthold NR1 antibody) labelling cerebellar myelin. f Enlarged view of myelin. g Gold particle density over cerebellar myelin (17 sheaths), mossy fibre synapse (MF psd, n=20), parallel fibre-Purkinje synapse (PF psd, n=26), mitochondria in MF terminal (Mito, n=30), and axon cytoplasm (Axon, n=17). Scale bars: a 100μm, b-d 20μm, e-f 0.25μm.

Figure 4. Ischaemia activates NMDA receptors

a Ischaemia-evoked current in precursor (2mM Mg²⁺) blocked by NBQX (25μM) plus D-AP5 (50μM) Inset: ischaemia-induced synaptic-like currents. b Precursor response to ischaemia (0mM Mg²⁺), blocked by D-AP5, NBQX. c Precursor response (0mM Mg²⁺) showing transient increase in synaptic currents, and most inward current blocked by D-AP5. d Fractional block of inward current: in 2mM Mg²⁺ by D-AP5 plus NBQX in 7 precursors, and in 0mM Mg²⁺ by D-AP5 or NBQX in precursors (9 for D-AP5, 7 for NBQX) and mature cells (9 for D-AP5, 10 for NBQX). P=0.098 comparing D-AP5 in precursors and mature cells; P=0.019 comparing NBQX in precursors and mature cells. All cerebellum, −63mV, 33°C.