Immunochemical characterization of inhibitory mouse cortical neurons: three chemically distinct classes of inhibitory cells

Abstract

The cerebral cortex has diverse types of inhibitory neurons. In rat cortex, past research has shown that parvalbumin (PV), somatostatin (SOM), calretinin (CR), and cholecystokinin (CCK) label four distinct chemical classes of GABAergic interneurons. However, in contrast to rat cortex, previous studies indicate that there is significant colocalization of SOM and CR in mouse cortical inhibitory neurons. In the present study we further characterized immunochemical distinctions among mouse inhibitory cortical neurons by double immunochemical labeling with chemical markers. We found that, PV, SOM, and vasointenstinal peptide (VIP) reliably identify three nonoverlapping distinct subpopulations, as there was no overlap of immunoreactivity between PV and all the other chemical markers tested, and SOM and VIP did not show any overlap in labeled neurons in all the cortical areas. In comparison, there was significant overlap in combinations of other chemical markers. With some laminar and regional variations, the average overlap of SOM/CR (percentage of SOM+ cells expressing CR) and SOM/neuropeptide tyrosine (NPY) across all examined layers and cortical regions was 21.6% and 7.1%, respectively. The average overlap of VIP/CR, VIP/NPY, and CR/NPY was 34.2%, 9.5%, and 10%, respectively. We quantified and assessed the percentages of marker-positive GABAergic cells, and showed that the nonoverlapping subpopulations (i.e., PV+, SOM+ and VIP+ cells) accounted for about 60% of the GABAergic cell population. Taken together, our data reveal important chemical distinctions between mouse inhibitory cortical neurons and indicate that PV, SOM, and VIP can differentially label a majority of mouse inhibitory cortical neurons.

Fig. 1. Sub-populations of GABA immunopositive neurons in mouse cortex

As shown in A–C, almost all the cells immunopositive for parvalbumin (PV) were also positive for GABA. D–F, G–I and J–L show that a great majority of neurons expressing somatostatin (SOM), calretinin (CR), and neuropeptide tyrosine (NPY), respectively, were also immunopositive for GABA. The scale bar in C applied to A–I; and the scale bar in L applies to J–L.

Fig. 2. Co-localization of PV, SOM, CR, NPY and VIP with GFP-expressing GABAergic neurons in GAD-GFP transgenic mouse cortex

As shown in A–C, essentially all the cells immunopositive for parvalbumin (PV) were co-localized with GFP expressing GABAergic cells in the transgenic mouse in which basically all the GABAergic cells express GFP. The arrows in A–C point to the PV-immunopositive cells overlapping with GFP expression. Similarly, D–F, G–I, J–L and M–O show that nearly all the cells immunopositive for somatostatin (SOM), calretinin (CR), neuropeptide tyrosine (NPY) and vasoactive intestinal peptides (VIP), respectively, were also co-localized with GFP expressing GABAergic cells. The scale bar in O applies to all panels.

Fig. 3. No overlap between expressions of parvalbumin (PV) and the other chemical markers tested in mouse cortex

A–C, D–F, G–I and J–L show that PV immunopositive cells were not co-localized with somatostatin (SOM), calretinin (CR), neuropeptide tyrosine (NPY), or vasoactive intestinal peptide (VIP), respectively. White boxes denote the locations of PV+ cells. The sections were from mouse S1 cortex. Scale bar in C applies to all panels.

Fig. 4. Double immunochemical staining of mouse cortical sections with different combinations of somatostatin (SOM), calretinin (CR), neuropeptide tyrosine (NPY) and vasoactive intestinal peptide (VIP)

Some SOM+ neurons were immunopositive for CR (A–C) or NPY (D–F), but SOM+ neurons were not immunopositive for VIP (G–I). Some CR+ cells were immunopositive for VIP (J–L), and some NPY+ cells were also immunopositive for VIP (M–O). Arrows point to double-labeled cells. White boxes in panels G–I denote locations of SOM+ cells. The sections were from mouse S1 cortex. The scale bar in C applies to all panels.

Fig. 5. The cumulative histograms showing percentage laminar distributions of marker-specific GABAergic cells in mouse FC, S1 and V1

The labels on the horizontal axis indicate different markers. The histograms indicate the percentages of marker-positive cells with different colors and patterns coding different cortical layers.

Fig. 6

The histogram showing percentage compositions of marker-positive GABAergic cells across S1 cortical layers.