Undifferentiated embryonic stem cells express ionotropic glutamate receptor mRNAs.

Ionotropic glutamate receptors (iGluRs) do not only mediate the majority of excitatory neurotransmission in the vertebrate CNS, but also modulate pre- and postnatal neurogenesis. Most of the studies on the developmental role of iGluRs are performed on neural progenitors and neural stem cells (NSCs). We took a step back in our study by examining the role of iGluRs in the earliest possible cell type, embryonic stem cells (ESCs), by looking at the mRNA expression of the major iGluR subfamilies in undifferentiated mouse ESCs. For that, we used two distinct murine ES cell lines, 46C ESCs and J1 ESCs. Regarding 46C ESCs, we found transcripts of kainate receptors (KARs) (GluK2 to GluK5), AMPA receptors (AMPARs) (GluA1, GluA3, and GluA4), and NMDA receptors (NMDARs) (GluN1, and GluN2A to GluN2D). Analysis of 46C-derived cells of later developmental stages, namely neuroepithelial precursor cells (NEPs) and NSCs, revealed that the mRNA expression of KARs is significantly upregulated in NEPs and, subsequently, downregulated in NSCs. However, we could not detect any protein expression of any of the KAR subunits present on the mRNA level either in ESCs, NEPs, or NSCs. Regarding AMPARs and NMDARs, GluN2A is weakly expressed at the protein level only in NSCs. Matching our findings for iGluRs, all three cell types were found to weakly express pre- and postsynaptic markers of glutamatergic synapses only at the mRNA level. Finally, we performed patch-clamp recordings of 46C ESCs and could not detect any current upon iGluR agonist application. Similar to 46C ESCs, J1 ESCs express KARs (GluK2 to GluK5), AMPARs (GluA3), and NMDARs (GluN1, and GluN2A to GluN2D) at the mRNA level, but these transcripts are not translated into receptor proteins either. Thus, we conclude that ESCs do not contain functional iGluRs, although they do express an almost complete set of iGluR subunit mRNAs.

Plasticity in D1-like receptor expression is associated with different components of cognitive processes.

Dopamine D1-like receptors consist of D1 (D1A) and D5 (D1B) receptors and play a key role in working memory. However, their possibly differential contribution to working memory is unclear. We combined a working memory training protocol with a stepwise increase of cognitive subcomponents and real-time RT-PCR analysis of dopamine receptor expression in pigeons to identify molecular changes that accompany training of isolated cognitive subfunctions. In birds, the D1-like receptor family is extended and consists of the D1A, D1B, and D1D receptors. Our data show that D1B receptor plasticity follows a training that includes active mental maintenance of information, whereas D1A and D1D receptor plasticity in addition accompanies learning of stimulus-response associations. Plasticity of D1-like receptors plays no role for processes like response selection and stimulus discrimination. None of the tasks altered D2 receptor expression. Our study shows that different cognitive components of working memory training have distinguishable effects on D1-like receptor expression.

Functional excitatory GABAA receptors precede ionotropic glutamate receptors in radial glia-like neural stem cells.

The involvement of neurotransmission in neuronal development is a generally accepted concept. Nevertheless, the precise regulation of neurotransmitter receptor expression is still unclear. To investigate the expression profiles of the most important ionotropic neurotransmitter receptors, namely GABA(A) receptors (GABA(A)Rs), NMDA receptors (NMDARs), and AMPA receptors (AMPARs), quantitative RT-PCR, immunoblot analysis and patch clamp studies were performed in in vitro-generated neural stem cells (NSCs). This clearly defined cell line is closely related to radial glia cells, the stem cells in the neonate brain. We found functional GABA(A)Rs of the subunit composition alpha2, beta3, and gamma1 to be expressed. Unexpectedly, functional ionotropic glutamate receptors were absent. However, NSCs expressed the NMDAR subunits NR2A and NR3A, and the AMPAR subunit GluR4 at the protein level, and GluR3 at the mRNA level. The overexpression of functional NMDARs in NSCs led to an increased mRNA level of AMPAR subunits, indicating a role in synaptogenesis. Early neuronal markers remained unchanged. These data extend our knowledge about ionotropic neurotransmitter receptor expression during neuronal development and will aid further investigations on activity-dependent neurogenesis.

Quantitative analysis of cotransfection efficiencies in studies of ionotropic glutamate receptor complexes.

Transient transfection of cultured mammalian cells is widely employed in the study of ionotropic glutamate receptors. Heteromeric expression is usually achieved by simultaneous transfection of various combinations of glutamate receptor subunit-encoding cDNAs. This approach is based on an "all-or-none" assumption, rarely verified experimentally, that any given cell expresses all subunits present during transfection. A similar assumption implicitly is made when cotransfection of a cDNA encoding a fluorescent marker protein is applied to distinguish transfected from untransfected cells. A further frequent assumption alleges that the ratio between cDNAs used in cotransfection experiments directs the assembly of receptor complexes in heterologous expression systems. To check the validity of these assumptions for ionotropic glutamate receptors as model transmembrane receptors, we generated fluorescently labeled receptor subunits and introduced them into HEK-293 cells by the calcium phosphate method. Analyzing the expression of multiple fusion proteins by confocal microscopy, we evaluated the coexpression efficiencies for various glutamate receptor cDNA combinations, cDNA amounts, and cDNA ratios. Several factors were found to influence the individual, cumulative, and cotransfection efficiencies, including the cDNA ratio, the nature of the expressed protein, and the specific combination of cotransfected cDNAs. After simultaneous transfection with equal amounts of several cDNAs, we demonstrate the consistent generation of several distinct populations of cells that express different receptor subunit combinations. The evidence we present suggests that cotransfected cells should always be independently tested for the expression of all target subunits before picking cells for the analysis of specific heteromeric receptor assemblies.