Ebastine br Discussion Our results indicate

Discussion Our results indicate that marked changes in the relative distribution of AMPA receptors in microsomal and synaptic fractions occur during the postnatal period, and that these changes are correlated with changes in different populations of AMPA binding sites. At early postnatal ages (PND 2 to PND 7), the relative abundance of AMPA receptor subunits GluR1 and GluR2/3 in microsomal fractions vs. synaptosomal fractions is higher than in adults. It is only after PND 14 that the adult pattern for the distribution of subunits between these 2 membrane fractions is established. Furthermore, the results clearly indicate that the levels of AMPA receptor subunits present in the microsomal fractions are higher at early postnatal ages than in adults. This overexpression of GluR subunits during the postnatal period is reminiscent of the overexpression of the mRNA for these subunits reported by us as well as by others 7, 22, 26. Indeed, there was a very significant correlation between the average values for GluR1-3 mRNA, and the average values for GluR1-3 subunits in P3 fractions (not shown). Furthermore, our results concerning the changes in GluR subunits in P2 fractions are very similar to those reported by Hall and Bahr [8], and indicate a gradual increase in GluR subunits in the P2 fractions; these authors also showed that the changes in GluR subunits in crude mitochondrial fractions (which correspond to our P2 fractions) were correlated with the changes in low affinity, but not in high affinity binding for -AMPA. They concluded that the relative increase in low affinity over the high-affinity binding sites during development could be due to a shift in subunit composition of the receptors, or to the gradual association of the receptors with a factor or group of factors related to postsynaptic density and conferring low affinity to the AMPA receptors. Our results suggest a different interpretation. In particular, we observed very significant correlations between the changes in high-affinity binding and the levels of receptor subunits present in P3 fractions. In addition, we also previously reported a higher proportion of high affinity binding sites for -AMPA in the microsomal fraction at early postnatal ages than in adults [25]. Furthermore, we recently showed that anti-GluR2/3 Ebastine label 2 distinct states of the receptors in Western blots, especially in P3 fractions, a band with an apparent Mr of 108 kDa and another one with a Mr of 103.5 kDa. We presented evidence that the lower molecular weight band represented a partially glycosylated species of the subunits and we proposed that it corresponded to an immature form of AMPA receptors present in endoplasmic reticulum/early Golgi. Taken together, these findings strongly suggest the existence of 2 distinct populations of AMPA receptors with different structures and affinities for -AMPA and with a different ontogeny. One population of receptors would be constituted of partially glycosylated subunits, exhibit high affinity for -AMPA and would be localized predominantly in endoplasmic reticulum/early Golgi. Furthermore, these receptors would be more abundant at early postnatal ages, and would represent precursors for the other population of receptors. The latter would be constituted of fully glycosylated subunits, would exhibit low affinity for -AMPA and would be predominantly located in synaptic membranes. Finally, this population would increase relatively to the other one during the developmental period. This would fit well with the notion that receptors would be overexpressed and form an intracellular pool of pro-receptors at early developmental stages, and would become gradually inserted into postsynaptic structures as synaptogenesis takes place. Analysis of synaptic responses throughout development in rat hippocampus has revealed a developmental decrease in the relative number of `silent synapses', i.e., of synapses with functional NMDA but not AMPA receptors [6], with PND 2 exhibiting approximately 82% silent synapses and PND 6 just under 30% of silent synapses. Our data do indicate a dramatic increase in the expression of AMPA receptor subunits in P3 fractions between PND 2 and PND 7, which could possibly be related to the marked increase in active synapses during this period of time. They also indicate that while AMPA/GluR receptors may be inactive at PND 2, a significant number appear to be present in synaptic plasma membranes at this age (about 25–50% of adult levels; Fig. 2A,C). However, it is possible that subcellular fractionation of rat brain changes with development such that more non-synaptic AMPA/GluR receptors are isolated in the P2 fraction at early developmental stages. In particular, GluR2/3 immunoreactivity exhibited separable molecular weight banding at early time points in P2 fractions, suggesting more contamination of synaptic membranes by microsomal membranes at these ages. We proposed elsewhere that insertion of receptors from microsomal fractions into synaptic membranes could participate in the phenomenon of long-term potentiation of synaptic transmission [24]. If this is true, the relative difficulty in eliciting LTP in hippocampus before PND 10-12 [3]would not be due to the lack of intracellular receptors, but in steps involved in the mobilization of these receptors, or in the stabilization of newly inserted receptors. In addition, our data would fit well with the notion that early developmental period is associated with greater synaptic plasticity of excitatory transmission 15, 29, inasmuch as overproduction of precursor receptors is a critical determinant of such plasticity.