Abstract
Ionotropic glutamate α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors are the major mediators of fast synaptic neurotransmission. In this work, we used primary cortical cultures from rats as a model system to study AMPA receptor regulation during in vitro cell maturation and after synaptic activity modifications. The levels of AMPA receptor mRNA and protein, along with the alternative splicing and RNA editing of the AMPA receptor subunit (GluR1-4) mRNAs, were analyzed in immature (DIV5) and mature (DIV26) rat neuronal cultures. We observed an increase in the expression of all four AMPA receptor subunits during in vitro neuronal maturation. This finding might be due to the formation of new synapses between neurons during the development of a complex neuronal network. We also analyzed the effects of stimulation (KCl and glutamate) and inhibition (APV/TTX) on rat mature neuronal cultures (DIV26): stimulation with KCl led to an overall down-regulation of GluR1 and GluR3 AMPA receptor subunits and an up-regulation of the GluR2 subunit. Similarly, glutamate treatment induced a significant down-regulation of GluR1 together with an up-regulation of GluR2. In contrast, the chronic blockade of neuronal activity that resulted from APV/TTX treatment up-regulated GluR1 and GluR3 with a parallel down-regulation of GluR2 and GluR4. RNA editing at the R/G site increased during neuronal cell maturation for all AMPA receptors (from 8–39% at DIV5 to 28–67% at DIV26). Unexpectedly, all the treatments tested induced a marked reduction (ranging from −9% to −52%) of R/G editing levels in mature neurons, primarily for the mRNA flip variant.In summary, we showed that cultured rat cortical neurons are able to vary the stoichiometric ratios of the AMPA receptor subunits and to control post-transcriptional processes to adapt fast synaptic transmission under different environmental conditions.
Highlights
The ionotropic glutamate a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors are important mediators of fast synaptic neurotransmission in vivo and in cultured cortical neurons
We used an in vitro model of cortical neuron maturation to investigate the relationship between neuronal activity and the levels and post-transcriptional regulation of glutamate AMPA receptor subunits by performing a quantitative molecular analysis of the four AMPA receptor subunits (GluR1 and GluR2, and the less known GluR3 and GluR4) and a thorough analysis of post-transcriptional events such as flip/flop alternative splicing in association with RNA-editing
We observed that in vitro neuronal maturation led to increased expression of all four AMPA receptor subunits both at the mRNA and protein levels
Summary
The ionotropic glutamate a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors are important mediators of fast synaptic neurotransmission in vivo and in cultured cortical neurons. The number, composition and localization, as much as post-transcriptional and translational regulation, of AMPA receptors in neurons are critically important factors that determine the neuronal response to glutamate and play a significant role in synaptic plasticity [1,2]. Post-transcriptional modifications of AMPA receptor mRNAs such as RNA editing and alternative splicing are functionally relevant in the regulation of glutamatergic neurotransmission. They represent a sophisticated mechanism that allows to modify the receptor subunits in positions that are key in regulating the permeability of the channel, the kinetics of pore opening, the time of desensitization of the receptor, as well as the time of recovery from desensitization [16,17,18]
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