Laminar Distribution of Subsets of GABAergic Axon Terminals in Human Prefrontal Cortex

Abstract

In human prefrontal cortex (PFC), ~85% of γ-aminobutyric acid (GABA)-expressing neurons can be subdivided into non-overlapping groups by the presence of calbindin (CB), calretinin (CR) or parvalbumin (PV). Substantial research has focused on the differences in the laminar locations of the cells bodies of these neurons, with limited attention to the distribution of their axon terminals, their sites of action. We previously reported that in non-human primates subtypes of these cells are distinguishable by differences in terminal protein levels of the GABA synthesizing enzymes glutamic acid decarboxylase 65 (GAD65) and GAD67. Here we used multi-label fluorescence microscopy in human PFC to assess: (1) the laminar distributions of axon terminals containing CB, CR, or PV; and (2) the relative protein levels of GAD65, GAD67 and vesicular GABA transporter (vGAT) in CB, CR and PV terminals. The densities of the different CB, CR and PV terminal subpopulations differed across layers of the PFC. PV terminals comprised two subsets based on the presence of only GAD67 (GAD67+) or both GADs (GAD65/GAD67+), whereas CB and CR terminals comprised three subsets (GAD65+, GAD67+, or GAD65/GAD67+). The densities of the different CB, CR and PV GAD terminal subpopulations also differed across layers. Finally, within each of the three calcium-binding protein subpopulations intra-terminal protein levels of GAD and vGAT differed by GAD subpopulation. These findings are discussed in the context of the laminar distributions of CB, CR and PV cell bodies and the synaptic targets of their axons.

Keywords: GAD65; GAD67; glutamic acid decarboxylase; human PFC; vGAT.

Figure 1

The proportion of GABAergic calbindin+ (CB+), calretinin+ (CR+) and parvalbumin+ (PV+) terminals differs across cortical layers. (A,B) Bar graphs showing the relative proportions of vesicular GABA transporter (vGAT) terminals in prefrontal cortex (PFC) that were CB+, CR+ or PV+. Error bars = SEM.

Figure 2

Subtypes of CB, CR and PV neurons are distinguishable by the expression of GAD65 mRNA in human PFC.(A,B) Single plane image of a PFC tissue section labeled for CB, GAD65 and GAD67 mRNAs. (A5) and (B5) are merged images of (A1–A3) and (B1–B3), respectively. Lipofuscin autofluorescence is shown in (A4,B4). (C,D) Single plane image of a PFC tissue section labeled for CR, GAD65 and GAD67 mRNAs. (C5) and (D5) are merged images of (C1–C3) and (D1–D3), respectively. Lipofuscin autofluorescence is shown in (C4,D4). (E,F) Single plane image of a PFC tissue section labeled for PV, GAD65 and GAD67 mRNAs.(E5) and (F5) are merged images of (E1–E3) and (F1–F3), respectively. Lipofuscin autofluorescence is shown in (E4,F4). In all images, the outline of the nucleus, which was visualized using DAPI, is shown. Scale bars = 5 μm.

Figure 3

The proportion of three CB/GAD+ terminal subpopulations differs across cortical layers. (A) Projection image (6 z-planes separated by 0.25 μm) of a human PFC tissue section immunolabeled for CB, vGAT (not shown), GAD65 and GAD67. Gray scale images (A1–A3) are single channel images of the multichannel image (A4) and are representative of the immunohistochemistry labeling. Scale bar = 5 μm. (B,C) Bar graphs showing the relative proportions of vGAT/CB+ terminals in PFC that were GAD65+, GAD67+, or GAD65/GAD67+. Error bars = SEM.

Figure 4

The proportion of three CR/GAD+ terminal subpopulations differs across cortical layers. (A) Projection image (7 z-planes separated by 0.25 μm) of a human PFC tissue section immunolabeled for CR, vGAT (not shown), GAD65 and GAD67. Gray scale images (A1–A3) are single channel images of the multichannel image (A4) and are representative of the immunohistochemistry labeling. Scale bar = 5 μm. (B,C) Bar graphs showing the relative proportions of vGAT/CR+ terminals in PFC that were GAD65+, GAD67+, or GAD65/GAD67+. Error bars = SEM.

Figure 5

The proportion of two PV/GAD+ terminal subpopulations differs across cortical layers. (A) Projection image (5 z-planes separated by 0.25 μm) of a human PFC tissue section immunolabeled for PV, vGAT (not shown), GAD65 and GAD67. Gray scale images (A1–A3) are single channel images of the multichannel image (A4) and are representative of the immunohistochemistry labeling. Scale bar = 5 μm. (B,C) Bar graphs showing the relative proportions of vGAT/PV+ terminals in PFC that were GAD67+ or GAD65/GAD67+. Error bars = SEM.

Figure 6

Summary. Bar graph showing the percentage of all γ-aminobutyric acid (GABA) neurons (black bars) and terminals assessed (gray bars) that is CB+, CR+, or PV+ in PFC total gray matter. Table showing the distribution of soma and terminals for each of the three GABA neuron subtypes across the six cortical layers. The soma data for CB and CR were taken from previously published findings in human PFC (Daviss and Lewis, 1995).