Layer-Specific Vesicular Glutamate Transporter 1 Immunofluorescence Levels Delineate All Layers of the Human Hippocampus Including the Stratum lucidum

Abstract

The hippocampal formation consists of the Ammon’s horn (cornu Ammonis with its regions CA1-4), dentate gyrus, subiculum, and the entorhinal cortex. The rough extension of the regions CA1-3 is typically defined based on the density and size of the pyramidal neurons without clear-cut boundaries. Here, we propose the vesicular glutamate transporter 1 (VGLUT1) as a molecular marker for the CA3 region. This is based on its strong labeling of the stratum lucidum (SL) in fluorescently stained human hippocampus sections. VGLUT1 puncta of the intense SL band co-localize with synaptoporin (SPO), a protein enriched in mossy fibers (MFs). Owing to its specific intensity profile throughout all hippocampal layers, VGLUT1 could be implemented as a pendant to Nissl-staining in fluorescent approaches with the additional demarcation of the SL. Furthermore, by high-resolution confocal microscopy, we detected VGLUT2 in the human hippocampus, thus reconciling two previous studies. Finally, by VGLUT1/SPO co-staining, we provide evidence for the existence of infrapyramidal MFs in the human hippocampus and we show that SPO expression is not restricted to MF synapses as demonstrated for rodent tissue.

Expression profiles of the autism-related SHANK proteins in the human brain

“…In a frontal cortex section from a male individual (case 2, [ 32 ] and Table 1 ), SHANK2-positive puncta were found in the soma of pyramidal neurons and in the neuropil, while the nuclei were devoid of SHANK2 IF (Fig. 1 a, 1 PN and 2).…”

Combined DiI and Antibody Labeling Reveals Complex Dysgenesis of Hippocampal Dendritic Spines in a Mouse Model of Fragile X Syndrome

“…The 2-step protocol can be applied to visualize presynaptic sites expressing different subsets of proteins, thus distinguishing individual synapses based on both morphological and molecular identity and allowing one to access the complexity and heterogeneity of synapses in the brain. Using this method we were successfully able to visualize a subset of dendritic spines with different microanatomy—presence vs. absence of a SA labeled with anti-Synpo—adjacent to presynaptic terminals expressing either Vesicular Glutamate Transporter 2 (VGluT2; Figure 2 a,b; Figure S2 ) or Synaptoporin (Synpr; Figure 2 a,b; Figure S3 ) enriched in hippocampal mossy fibers [ 40 ].…”

Combined DiI and antibody labeling reveals complex dysgenesis of hippocampal spine synapses in a mouse model of Fragile X Syndrome

“…Using this method we were successfully able to visualize a subset of dendritic spines with different microanatomy – presence vs . absence of a SA labeled with anti-Synaptopodin – adjacent to presynaptic terminals expressing either Vesicular Glutamate Transporter 2 (VGluT2; Figure 2a,b) or Synaptoporin (Synpr; Figure 2a,b) enriched in hippocampal mossy fibers [40].…”

“…The 2-step protocol can be applied to visualize presynaptic sites expressing different subsets of proteins, thus distinguishing individual synapses based on both morphological and molecular identity and allowing one to access the complexity and heterogeneity of synapses in the brain. Using this method we were successfully able to visualize a subset of dendritic spines with different microanatomy -presence vs. absence of a SA labeled with anti-Synaptopodin -adjacent to presynaptic terminals expressing either Vesicular Glutamate Transporter 2 (VGluT2; Figure 2a,b) or Synaptoporin (Synpr; Figure 2a,b) enriched in hippocampal mossy fibers [40].…”