VGluT2 immunochemistry identifies thalamocortical terminals in layer 4 of adult and developing visual cortex

Abstract

A vesicular glutamate transporter, VGluT2, has been suggested to be the transporter utilized in the thalamocortical pathway. We examined the reliability of this marker in identifying and discriminating thalamic terminals in adult and developing ferret visual cortex. We studied brain sections stained for the transporter protein and/or anterogradely filled thalamocortical or intracortical axons, by using light, confocal, and electron microscopy. Under light microscopy, VGluT2 immunoreactivity (ir) in adult animals [past postnatal day (P)90] and in neonatal animals as early as P27 formed a dense band in layer 4 and appeared as scattered puncta in layers 6 and 1. Confocal dual-labeling analyses of P46 and adult striate cortices indicated that VGluT2 was present in thalamocortical axons, suggesting that thalamic projections utilize this transporter during postnatal development as well as adulthood. In contrast, extracellularly filled intracortical axons failed to colocalize with VGluT2-ir, suggesting that no significant terminal population originating in cortex contained VGluT2 in layer 4. Electron microscopic analysis revealed that, in adult layer 4, VGluT2-ir was present in large terminals, forming asymmetric synapses. Similar to anterogradely labeled thalamocortical terminals, VGluT2-ir synaptic terminals were different from their unlabeled counterparts in terms of terminal area (0.6 vs. 0.3 microm), synaptic length (486 vs. 353 nm), and preference for synapsing on spines (77% vs. 59%). Moreover, no significant differences were found between VGluT2-ir and anterogradely labeled thalamocortical terminals. Comparable similarities were also demonstrated at P46. These results indicate that thalamocortical terminals in layer 4 of visual cortex utilize VGluT2 and suggest that this marker can be used to identify thalamic axons specifically in adult and developing animals.

Gene Expression Identifies Distinct Ascending Glutamatergic Pathways to Frequency-Organized Auditory Cortex in the Rat Brain

“…This expression pattern is consistent with a VGLUT2 protein-dense terminal field found within thalamic recipient layers of sensory cortex (Kaneko et al, 2002;Nahmani and Erisir, 2005;Graziano et al, 2008;Hackett and de la Mothe, 2009). However, recent studies find that type 1 glutamate transporter (VGLUT1) mRNA is coexpressed with VGLUT2 mRNA in a subset of MGBv neurons in rodents (Barroso-Chinea et al, 2007;.…”

“…2D,G). This resembles that seen in other auditory and sensory cortices (Nahmani and Erisir, 2005;Graziano et al, 2008;Hackett and de la Mothe, 2009) and suggests that VGLUT2 protein expression alone does not readily distinguish the thalamocortical pathways to frequency-organized fields, A1 versus cSRAF.…”

Synapses, spines, zinc and pathology of Alzheimer's disease.

“…Using this method, we calculated that 19.63 ± 1.6% of the TdTomato-labeled axons contain vGLUT2. This is comparable to previous studies of geniculocortical terminals using these methods (Nahmani and Erisir, 2005 …”

“…The labeled geniculocortical axons formed arbors that are oriented parallel to the cortical surface, similar to the orientation of the dendrites of spiny stellate cells (Muly and Fitzpatrick, 1992 We also confirmed geniculocortical terminals in the tree shrew contain vGLUT2, as has been found in the ferret (Nahmani and Erisir, 2005) and mouse (Coleman et al, 2010). Not all geniculocortical axons were double-labeled with the vGLUT2 antibody, but this can be attributed to limitations in antibody penetration as well as the fact that vGLUT2 is limited to regions of the axons that contain synaptic vesicles.…”

Signal processing in the tectothalamic pathway.

“…1987;Ding and Casagrande 1998;Latawiec et al 2000;Erisir and Dreusicke 2005;Nahmani and Erisir 2005;Anderson et al 2009). …”