Changes of immunocytochemical localization of vesicular glutamate transporters in the rat visual system after the retinofugal denervation

Abstract

To clarify which vesicular glutamate transporter (VGluT) is used by excitatory axon terminals of the retinofugal system, we examined immunoreactivities and mRNA signals for VGluT1 and VGluT2 in the rat retina and compared immunoreactivities for VGluT1 and VGluT2 in the retinorecipient regions using double immunofluorescence method, anterograde tracing, and immunoelectron microscopy. Furthermore, the changes of VGluT1 and VGluT2 immunoreactivities were studied after eyeball enucleation. Intense immunoreactivity and mRNA signal for VGluT2, but not for VGluT1 immunoreactivity, were observed in most perikarya of ganglion cells in the retina. Immunoelectron microscopy revealed that VGluT1- and VGluT2-immunolabeled terminals made asymmetrical synapses, suggesting that they were excitatory synapses, and that VGluT1-immunolabeled terminals were smaller than VGluT2-labeled ones in many retinorecipient regions, such as the dorsal lateral geniculate nucleus (LGd) and superior colliculus (SC). Double immunofluorescence study further revealed that almost no VGluT2 immunoreactivity was colocalized with VGluT1 in the retinorecipient regions. After wheat germ agglutinin (WGA) injection into the eyeballs, WGA immunoreactivity was colocalized in the single axon terminals of LGd and SC with VGluT2 but not VGluT1 immunoreactivity. After unilateral enucleation, VGluT2 immunoreactivity in the LGd, SC, nucleus of the optic tract, and nuclei of the accessory optic tract in the contralateral side of the enucleated eye was clearly decreased. Although only a small change of VGluT2 immunoreactivity was observed in the contra- and ipsilateral suprachiasmatic nuclei, olivary pretectal nucleus, anterior pretectal nucleus, and posterior pretectal nucleus, moderate reduction of VGluT2 was found in these regions after bilateral enucleation. On the other hand, almost no change in VGluT1 immunoreactivity was found in the structures examined in the present enucleation study. Thus, the present results support the notion that the retinofugal pathways are glutamatergic, and indicate that VGluT2, but not VGluT1, is employed for accumulating glutamate into synaptic vesicles of retinofugal axons.

Comparison of the ontogeny of the vesicular glutamate transporter 3 (VGLUT3) with VGLUT1 and VGLUT2 in the rat retina

“…4). This observation is in agreement with previous reports from other groups studying VGLUT2 in rodent retina [13,25,41]. Very faint immunoreactivity in the somas was first observed in the GCL at P0 (Fig 4), the expression increased at P6 and remained strong through to the adult retina.…”

Input-Specific Intrasynaptic Arrangements of Ionotropic Glutamate Receptors and Their Impact on Postsynaptic Responses

“…The two distinct features correspond to the ultrastructural characteristics of RG and CG synapses, respectively (Erişir et al, 1997;Van Horn et al, 2000;Li et al, 2003). To confirm the identity of these synapses on the replica, we labeled for vGluT 2 and 1, which are selectively expressed in RG and CG fibers in the dLGN, respectively (see Materials and Methods) (Fremeau et al, 2001;Fujiyama et al, 2003). In accordance with their morphological features, the large presynaptic P-faces were always labeled for vGluT2 ( Fig.…”

“…The specificity of the NR1 antibody was confirmed by labeling of replicas obtained from forebrain-specific NR1 KO mice (Iwasato et al, 2000); synaptic NR1 immunoparticles in hippocampal pyramidal cells were mostly abolished in the KO region (data not shown). Antibodies against vesicular glutamate transporter (vGluT) 2 (Miyazaki et al, 2003) and vGluT1 (Kulik et al, 2002) raised in guinea pigs were used as markers of the RG and the CG fibers (Fremeau et al, 2001;Fujiyama et al, 2003), respectively, with an anti-guinea pig secondary antibody conjugated with 10 nm gold particles (BBI). Although vGluTs are vesicular proteins, they are often detected on presynaptic plasma membrane and thus have been used to identify the origin of presynaptic profiles (Hagiwara et al, 2005;Masugi-Tokita et al, 2007).…”

Vesicular Glutamate Transporter 1 Is Required for Photoreceptor Synaptic Signaling But Not For Intrinsic Visual Functions

“…Although VGLUT2 is considered the major isoform used in glutamatergic pathways from retina to brain (Fujiyama et al, 2003), we cannot conclude whether VGLUT1 neurons can mediate visual signaling in the thalamocortical pathways. Additional dual-recording experiments in retina and LGN or visual cortex would be needed to establish or rule out a role for VGLUT1.…”

“…Therefore, other VGLUT isoforms must be used by the ipRGCs to conduct glutamatergic signals from the retina to their target (suprachiasmatic and preoptic tectal nuclei) in the nonimage-forming visual brain areas. In the rat retina, VGLUT2 mRNA and protein have been localized to cell bodies in the ganglion cell layer (GCL) (Fujiyama et al, 2003), and retinal ganglion cells were found to be the source of VGLUT2-positive puncta in the LGN (Land et al, 2004). Although VGLUT3 immunoreactivity has been found in the somas of some cells in the GCL of the cat (Fyk-Kolodziej et al, 2004), in the mouse and rat retina, VGLUT3 immunostaining has only been found in a subset of amacrine cells with cell bodies in the inner nuclear layer (Haverkamp and Wässle, 2004;Johnson et al, 2004).…”