Stable maintenance of glutamate receptors and other synaptic components in long‐term hippocampal slices

Abstract

Cultured hippocampal slices retain many in vivo features with regard to circuitry, synaptic plasticity, and pathological responsiveness, while remaining accessible to a variety of experimental manipulations. The present study used ligand binding, immunostaining, and in situ hybridization assays to determine the stability of AMPA- and NMDA-type glutamate receptors and other synaptic proteins in slice cultures obtained from 11 day postnatal rats and maintained in culture for at least 4 weeks. Binding of the glutamate receptor ligands [³H]AMPA and [³H]MK-801 exhibited a small and transient decrease immediately after slice preparation, but the binding levels recovered by culture day (CD) 5–10 and remained stable for at least 30 days in culture. Autoradiographic analyses with both ligands revealed labeling of dendritic fields similar to adult tissue. In addition, slices at CD 10–20 expressed a low to high affinity [³H]AMPA binding ratio that was comparable with that in the adult hippocampus (10:1). AMPA receptor subunits GluR1 and GluR2/3 and an NMDA receptor subunit (NMDAR1) exhibited similar postcutting decreases as that exhibited by the ligand binding levels, followed by stable recovery. The GluR4 AMPA receptor subunit was not evident during the first 10 CDs but slowly reached detectable levels thereafter in some slices. Immunocytochemistry and in situ hybridization techniques revealed adult-like labeling of subunit proteins in dendritic processes and their mRNAs in neuronal cell body layers. Long-term maintenance was evident for other synapse-related proteins, including synaptophysin, neural cell adhesion molecule isoforms (NCAMs), and an AMPA receptor related antigen (GR53), as well as for certain structural and cytoskeletal components (e. g., myelin basic protein, spectrin, microtubule-associated proteins). In summary, following an initial and brief depression, many synaptic components were expressed at steady-state levels in long-term hippocampal slices, thus allowing the use of such a culture system for investigations into mechanisms of brain synapses.