Abstract
NMDA receptor subunits change during development and their synaptic expression is modified rapidly after synaptic plasticity induction in hippocampal slices. However, there is scarce information on subunits expression after synaptic plasticity induction or memory acquisition, particularly in adults. GluN1, GluN2A and GluN2B NMDA receptor subunits were assessed by western blot in 1) adult rats that had explored an open field (OF) for 5 minutes, a time sufficient to induce habituation, 2) mature rat hippocampal neuron cultures depolarized by KCl and 3) hippocampal slices from adult rats where long term potentiation (LTP) was induced by theta-burst stimulation (TBS). GluN1 and GluN2A, though not GluN2B, were significantly higher 70 minutes –but not 30 minutes- after a 5 minutes session in an OF. GluN1 and GluN2A total immunofluorescence and puncta in neurites increased in cultures, as evaluated 70 minutes after KCl stimulation. Similar changes were found in hippocampal slices 70 minutes after LTP induction. To start to explore underlying mechanisms, hippocampal slices were treated either with cycloheximide (a translation inhibitor) or actinomycin D (a transcription inhibitor) during electrophysiological assays. It was corroborated that translation was necessary for LTP induction and expression. The rise in GluN1 depends on transcription and translation, while the increase in GluN2A appears to mainly depend on translation, though a contribution of some remaining transcriptional activity during actinomycin D treatment could not be rouled out. LTP effective induction was required for the subunits to increase. Although in the three models same subunits suffered modifications in the same direction, within an apparently similar temporal course, further investigation is required to reveal if they are related processes and to find out whether they are causally related with synaptic plasticity, learning and memory.
Highlights
NMDA (N-methyl-D-aspartate) receptors (NMDAR) are heterotetramers composed by two GluN1 obligatory subunits and two regulatory subunits: GluN2 (A–D) or GluN3 (A–B) [1]
Western Blot (WB) Each slice used in Field excitatory postsynaptic potentials (fEPSPs) recording assays, as well as both hippocampi from each animal exposed to the OF were separately homogenized in a Teflon glass potter (561599) in 100 mM NaCl, 0.2% Triton X-100, 1 mM EGTA, antiproteases cocktail (Sigma), 20 mM HEPES buffer; and incubated on ice 30 minutes to led to a complete lysis of the tissue
Grosshans et al [19] reported an enhanced GluN1 and GluN2A surface expression 15 and 30 minutes after long term potentiation (LTP) induction in CA1 mini-slices from adult rat; since the intracellular subunits levels concomitantly decreased, they proposed that GluN1 and GluN2A were recruited from available pools and suggested that this could represent a persistent postsynaptic modification since the change was present after 180 minutes
Summary
NMDA (N-methyl-D-aspartate) receptors (NMDAR) are heterotetramers composed by two GluN1 obligatory subunits and two regulatory subunits: GluN2 (A–D) or GluN3 (A–B) [1]. Most NMDAR contain GluN2 subunits [2], with GluN2A and GluN2B being the major regulatory subunits in the forebrain, in the hippocampus These two subunits have different pharmacological and biophysical properties [3] and are believed to play a determining role in synaptic plasticity. Their expression changes in the forebrain during postnatal development in rodents: GluN2B is first predominant and declines two weeks after birth [4,5]. NMDAR membrane expression changes immediately (seconds/minutes) after LTP induction by ‘‘tetanizing’’ high-frequency stimulation. In this work our goal was to investigate if the expression of the major NMDAR subunits undergoes changes driven by behavioral experience and synaptic plasticity in adult rats. We started to explore the putative mechanisms that would underlie those changes in hippocampal slices
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