AMPA Receptor Regulation at the mRNA and Protein Level in Rat Primary Cortical Cultures

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.

AMPA Receptors Are Involved in Store-Operated Calcium Entry and Interact with STIM Proteins in Rat Primary Cortical Neurons

“…Cells that responded with rapid, high [Ca 2+ ] i rise were identified as neurons and only these cells were analyzed in the experiments (Figures 1, 2). Approximately 60% of cells responded to KCl, which is in line with previous studies (Orlandi et al, 2011). Cells that responded with a delay or did not respond at all were assumed to be non-neuronal glial cells, most likely astrocytes.…”

“…This likely reflects the amount and type of AMPARs in both types of cells (Figure 3B; Verkhratsky and Steinhäuser, 2000). Although glial cells possess functional AMPARs (Pizzo et al, 2001; Lalo et al, 2006), the glial population does not contribute significantly to the total amount of AMPARs when analyzed by Western blot and real-time polymerase chain reaction (Orlandi et al, 2011). The expression of all four AMPA subunits was detected in rat cortical glia, but the GluA2 subunit was most abundant (Holzwarth et al, 1994).…”

Maturation of AMPAR Composition and the GABAAR Reversal Potential in hPSC-Derived Cortical Neurons

“…The principal AMPAR composition in native cortical preparations has been described to develop from an immature GluA1/ GluA4-containing to an adult GluA1/GluA2-containing AMPAR complex (Monyer et al, 1991;Talos et al, 2006aTalos et al, , 2006bOrlandi et al, 2011). Our data are consistent with an upregulation of the GluA2 subunit from weeks 2 to 5, however, the mRNA data for hECNs also demonstrate an equivalent upregulation of the GluA4 subunit.…”

“…An increase in the proportion of AMPARs containing GluA2(R) subunits was confirmed in hECNs by assessing the polyamine sensitivity of AMPAR-mediated current to 1-naphthyl acetyl spermine (NASPM), a selective antagonist of GluA2(R)-lacking AMPARs (Koike et al, 1997). At week 2, NASPM (3 M) strongly inhibited (77 Ϯ 5%) AMPA-evoked steady-state whole-cell currents ( Fig.…”

“…AMPARs are tetrameric assemblies of different combinations of GluA1, GluA2, GluA3, or GluA4 subunits. AMPAR composition in rodent and human excitatory cortical neurons is developmentally regulated; GluA1/GluA4-containing stoichiometry predominates in immature neurons, whereas mature neurons have an increased expression of GluA1 and GluA2 subunits (Monyer et al, 1991;Talos et al, 2006aTalos et al, , 2006bOrlandi et al, 2011). We assessed relative AMPAR subunit mRNA expression levels at 2 and 5 weeks after aNPC plate-down by qRT-PCR (Fig.…”