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We studied the action potential–evoked autaptic N-methyl-d-aspartate receptor-mediated excitatory postsynaptic currents (NMDA-EPSCs) using solitary cerebellar neurons cultured in microislands from wild-type (+/+), NR2A subunit knockout (NR2A−/−), and NR2C subunit knockout (NR2C−/−) mice. The peak amplitude of autaptic NMDA-EPSCs increased for all genotypes between days in vitro 8 (DIV8) and DIV13. Compared with +/+ cells at DIV13, NR2A−/− cells had smaller and NR2C−/− cells had larger NMDA-EPSCs. The decay time of these currents were all unexpectedly fast, except in NR2A−/− neurons, and showed small but significant shortening with development. Comparison of quantal parameters during development indicated an increase in quantal content in all genotypes. The synaptic portion of NMDA receptors measured using MK-801 blockade was roughly 50% in all genotypes at DIV8, and this percentage became slightly larger in NR2A−/− and NR2C−/− neurons at DIV12. The NR2B-selective antagonists Conantokin G and CP101,606 differed in their blocking actions with development, suggesting the presence of both heterodimeric NR1/NR2B and heterotrimeric NR1/NR2A/NR2B receptors. The most striking result we obtained was the significant increase of NMDA-EPSC peak amplitude and charge transfer in NR2C−/− mice. This was mainly the result of an increase in quantal size as estimated from miniature NMDA-EPSCs. The expression of NR2C subunit containing receptors was supported by the decreased Mg2+ sensitivity of NMDA receptors at DIV13 in +/+ but not in NR2C−/− cells. Thus solitary cerebellar granule neurons provide a novel model to investigate the role of receptor subtypes in the developmental changes of synaptic NMDA receptors.

Deletion of the β3 subunit of the GABAAreceptor produces severe behavioral deficits and epilepsy. GABAAreceptor-mediated miniature inhibitory postsynaptic currents (mIPSCs) in cortical neurons in cultures from β3 −/− mice were significantly faster than those in β3 +/+ mice and were more prolonged by zolpidem. Surface staining revealed that the number of β2/3, α2, and α3 (but not of α1) subunit-expressing neurons and the intensity of subunit clusters were significantly reduced in β3 −/− mice. Transfection of β3 −/− neurons with β3 cDNA restored β2/3, α2, and α3 subunits immunostaining and slowed mIPSCs decay. We show that the deletion of the β3 subunit causes the loss of a subset of GABAAreceptors with α2 and α3 subunits while leaving a receptor population containing predominantly α1 subunit with fast spontaneous IPSC decay and increased zolpidem sensitivity.

Deletion of the beta3 subunit of the GABA(A) receptor produces severe behavioral deficits and epilepsy. GABA(A) receptor-mediated miniature inhibitory postsynaptic currents (mIPSCs) in cortical neurons in cultures from beta3 -/- mice were significantly faster than those in beta3 +/+ mice and were more prolonged by zolpidem. Surface staining revealed that the number of beta2/3, alpha2, and alpha3 (but not of alpha1) subunit-expressing neurons and the intensity of subunit clusters were significantly reduced in beta3 -/- mice. Transfection of beta3 -/- neurons with beta3 cDNA restored beta2/3, alpha2, and alpha3 subunits immunostaining and slowed mIPSCs decay. We show that the deletion of the beta3 subunit causes the loss of a subset of GABA(A) receptors with alpha2 and alpha3 subunits while leaving a receptor population containing predominantly alpha1 subunit with fast spontaneous IPSC decay and increased zolpidem sensitivity.