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  • br Experimental procedures br Results br Discussion Although


    Experimental procedures
    Discussion Although the presence and localization of the GlyR subunits have been determined in the adult retina, the proportion of each GlyR subunit has not been assessed in the adult or in the developing retina. In the present study, we carried out the absolute quantification of GlyR mRNA by qPCR, as well as the protein expression of the α1-4 and βGlyR subunits during the postnatal development of the rat retina. During rat development, although retina cells genesis begins around embryonic day 10 (E10), 50% of cells are born around P1 and retinogenesis is complete near P12 (Rapaport et al., 2004). Glycinergic amacrine cells are born primarily in the postnatal period (P1P7) (Fletcher and Kalloniatis, 1997). The first synapses in the IPL appear around P11, probably from amacrine cell processes, although not completely differentiated synapses have been reported around P7 (Weidman and Kuwabara, 1968). After eye opening (P12–13), decrease in the rate of synapses formation has been noted (Horsburgh and Sefton, 1987). The stratification and maturation of ganglion cell dendrites and spines occur at the first postnatal month Yamasaki and Ramoa, 1993, Landi et al., 2007); also, the acuity and contrast sensitivity improve between P30 and P45 (Praputpittaya and Wililak, 2003). Our results showed that transcript levels of the GlyR subunits α1, α3, α4, and β, increased gradually during the postnatal development of the retina, while α2 levels were constantly high in the immature retina showing a considerable increase in the adult; those for the α4 were relatively low at all stages studied. Also, protein expression of α1, α4, and α3, increased progressively during retina development, but protein expression for α2 was relatively high through all development stages to the adult. These data agree with the low expression of the α1 and α3 subunits found at early stages of the central nervous system (CNS) development, as well as with the late onset of both α subunits during CNS development (Malosio et al., 1991, Jonsson et al., 2012). These data also agree with the relatively low α4 immunoreactivity that has been detected at the IPL in the adult mouse retina (Heinze et al., 2007, Wässle et al., 2009). But the high expression of α2 at all time points seems to contradict other studies where α2 is highly expressed in fetal spinal cord and Hexamethonium Bromide regions and its expression decreases in late stages of development (Malosio et al., 1991, Becker et al., 1988, Aroeira et al., 2011). Our results also appear to be in accordance with those observed at early postnatal rat retina, where the presence of the α2 subunit was found as a diffuse staining in the neuroblastic layer, as well as with immunohistochemical studies which found the expression of this subunit in the adult rat and mice retina (Greferath et al., 1994, Haverkamp et al., 2004). The α1 subunit exhibited a relatively high proportion of expression in the immature (20%) and adult (40%) retina, which is in agreement with previous immunohistochemical studies that have extensively demonstrated strong expression of GlyRα1 in the adult retina (Sassoè-Pognetto et al., 1994, Sassoè-Pognetto and Wässle, 1997, Greferath et al., 1994). Our results indicate that similar to the high expression of α1 in spinal cord and brain, the α1 subunit is also highly expressed in the adult retina. In this respect, bipolar cells and AII amacrine have fast inhibitory post-synaptic currents (IPCs), which agrees with the fast kinetics observed for the α1 GlyRs (Majudar et al, 2007); moreover, All amacrine is known to release glycine and hyperpolarize OFF cone bipolar cells. Furthermore, α1 mediates predominant glycinergic synaptic input to A-type ganglion cells, which transmit light signals with high temporal resolution (Majudar et al, 2007). In our study we found the α2 subunit as the predominant GlyR subunit in both the developing and mature rat retina (Fig. 2, Fig. 3). The high proportion of expression of the α2 subunit observed in the adult retina agrees with its even distribution across the IPL (Haverkamp et al., 2004, Heinze et al., 2007). GlyR α2 has been shown to be expressed by bipolar cell axon terminals and amacrine cells, as well as ganglion cell dendrites (Haverkamp et al., 2004, Jusuf et al., 2005). Despite that, mice lacking the GlyR α2 (Glra2 -/-) did not show gross morphological changes (Nobles et al., 2012, Zhang et al., 2015). The high expression of GlyR α2 is most likely related to its role in retinal development. At an early postnatal stage of the rat, immunohistochemical studies found the presence of α2 as a diffuse staining in the neuroblastic retina layer and it was later restricted to the IPL. Therefore, our results may support the role of the α2 GlyR subunit as a modulator of retina differentiation (Young and Cepko, 2004). The high proportion of the α2 subunit we found at early postnatal ages may indicate the existence of homomeric GlyR α2 in retina, as has been suggested in cerebellar synapses at P7-10 (Avila et al., 2013).