Properties of N-methyl-D-aspartate (NMDA) receptor channels were studied using the patch-clamp technique in fluorescence-labelled control and axotomised motoneurons in thin spinal cord slices. Single-channel currents induced by NMDA in outside-out patches isolated from axotomised motoneurons and voltage clamped at -100 mV, exhibited six amplitude levels with a mean conductance of 14.9 +/- 1.9, 22.2 +/- 2.7, 35.6 +/- 4.4, 49.1 +/- 3.5, 59.6 +/- 3.5 and 69.0 +/- 2.9 pS. In contrast, the conductance of NMDA receptor channels, recorded under identical conditions in control motoneurons was characterised by only four levels corresponding to 20.1 +/- 2.5, 38.0 +/- 3.0, 58.6 +/- 3.4 and 71.5 +/- 2.6 pS. The time course of deactivation of NMDA receptor EPSCs in axotomised motoneurons voltage clamped at +40 mV was double exponential. The deactivation had a similar time course in control and axotomised motoneurons from 6-day-old animals; however, the deactivation became faster with increased time after injury. The fast and slow time constants in motoneurons 8 days after axotomy became three times faster than in controls. NMDA receptor-mediated responses were voltage dependent in the presence of extracellular Mg(2+). In axotomised motoneurons Boltzmann analysis of the relationship between the peak amplitude of NMDA receptor EPSCs or NMDA-induced responses and membrane potential suggested an apparent K(d) for Mg(2+) binding (at 0 mV) of 1.2 +/- 0.5 and 3.4 +/- 3.9 mM, respectively. Single-cell RT-PCR analysis of mRNA revealed that NR2A-D and NR3A subunit transcripts were expressed in axotomised motoneurons. The results of our experiments suggest that in addition to genotypic control of NMDA receptors in motoneurons, axotomy, an experimental model of neurodegeneration, alters functional properties of the receptors in motoneurons destined to die.

Institute of Physiology Academy of Sciences of the Czech Republic Vídenská 1083 142 20 Prague 4 Czech Republic

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