Extracellular Molecular Markers and Soma Size of Inhibitory Neurons: Evidence for Four Subtypes of GABAergic Cells in the Inferior Colliculus

Abstract

Inhibition plays an important role in shaping responses to stimuli throughout the CNS, including in the inferior colliculus (IC), a major hub in both ascending and descending auditory pathways. Subdividing GABAergic cells has furthered the understanding of inhibition in many brain areas, most notably in the cerebral cortex. Here, we seek the same understanding of subcortical inhibitory cell types by combining staining for two types of extracellular markers--perineuronal nets (PNs) and perisomatic rings of terminals expressing vesicular glutamate transporter 2 (VGLUT2)--to subdivide IC GABAergic cells in adult guinea pigs. We found four distinct groups of GABAergic cells in the IC: (1) those with both a PN and a VGLUT2 ring; (2) those with only a PN; (3) those with only a VGLUT2 ring; and (4) those with neither marker. In addition, these four GABAergic subtypes differ in their soma size and distribution among IC subdivisions. Functionally, the presence or absence of VGLUT2 rings indicates differences in inputs, whereas the presence or absence of PNs indicates different potential for plasticity and temporal processing. We conclude that these markers distinguish four GABAergic subtypes that almost certainly serve different roles in the processing of auditory stimuli within the IC.

Significance statement: GABAergic inhibition plays a critical role throughout the brain. Identification of subclasses of GABAergic cells (up to 15 in the cerebral cortex) has furthered the understanding of GABAergic roles in circuit modulation. Inhibition is also prominent in the inferior colliculus, a subcortical hub in auditory pathways. Here, we use two extracellular markers to identify four distinct groups of GABAergic cells. Perineuronal nets and perisomatic rings of glutamatergic boutons are present in many subcortical areas and often are associated with inhibitory cells, but they have rarely been used to identify inhibitory subtypes. Our results further the understanding of inhibition in the inferior colliculus and suggest that these extracellular molecular markers may provide a key to distinguishing inhibitory subtypes in many subcortical areas.

Keywords: GABA; VGLUT2; auditory; inhibition; perineuronal net; plasticity.

Figure 1.

GAD⁺ and GAD⁻ neurons are present throughout the IC. A, Photographs of GAD⁺ neurons (arrows) and GAD⁻ neurons (arrowheads) in the ICc (left column) and the IClc (right column). Scale bar, 20 μm. B, A schematic of a representative section showing the three main subdivisions of the IC, as well as the three layers of the IClc. Aq, Cerebral aqueduct; I, II, and III, layers of the IClc; NLL, nuclei of the lateral lemniscus. C, Bar graph showing the percentage of NeuN⁺ neurons that are GAD⁺ in each IC subdivision and overall. The percentage is highest in IClc layer II and lowest in the ICd. Error bars indicate 1 SD. Total sample size, 28,607 neurons. Sample sizes for subdivisions: ICc, 10,514; ICd, 14,023; IClc I, 406; IClc II, 1480; IClc III, 2184.

Figure 2.

Neurons of different profile areas differ in their distribution within the IC. A, Composite plot with all NeuN⁺ neurons from eight transverse sections through the IC. Soma locations were normalized to overlay sections (see Materials and Methods). Dorsal is up, and medial is left. Subdivision outlines from one representative section are shown for reference. On the right is a legend showing the soma size categories represented by each marker color. Red markers indicate the smallest somatic profiles (<75 μm²), and purple markers indicate the largest somatic profiles (>275 μm²), with intermediate colors indicating groups with a range of 50 μm². I, II, and III, Layers of the IClc; NLL, nuclei of the lateral lemniscus. B, Distribution of all NeuN⁺ neurons (left column), GAD⁺ neurons (center column), or GAD⁻ neurons (right column), separated by soma size categories: the smallest neurons are represented at the top and the largest at the bottom. The background in row 3 was colored gray to enhance visibility of the yellow markers.

Figure 3.

GAD⁺ neurons are larger, on average, than GAD⁻ neurons. A, Box and whisker plot showing the range and median of soma profile area for GAD⁺ (magenta) and GAD⁻ (yellow) neurons. The box indicates the interquartile range (and thus contains the central 50% of the values), a bold horizontal line in the box indicates the median, and the 95% confidence intervals are indicated by the whiskers. Circles beyond the whiskers indicate outliers. Both GAD⁺ and GAD⁻ neurons represent a range of sizes, but GAD⁺ neurons are significantly larger, on average, than GAD⁻ neurons. ***p < 0.001. Note that the y-axis is scaled logarithmically. Sample sizes: 7575 GAD⁺ neurons and 21,032 GAD⁻ neurons. B, Plot showing the percentage of neurons that are GAD⁺ as a function of soma area. Areas were binned in 10 μm² increments. The percentage of neurons that are GAD⁺ increases with increasing soma size, and the two parameters are significantly correlated.

Figure 4.

GAD⁺ neurons surrounded by VGLUT2 rings are larger, on average, than those lacking VGLUT2 rings. A, Photographs of GAD⁺ neurons of a range of sizes surrounded by (arrows) or lacking (arrowheads) dense rings of VGLUT2⁺ terminals. Scale bar, 10 μm. B, A composite plot showing all GAD⁺ neurons from eight IC sections, similar to Figure 2, but with colored markers representing only GAD⁺ neurons surrounded by dense rings of VGLUT2⁺ terminals. Gray markers represent GAD⁺ neurons lacking VGLUT2 rings. Note that many GAD⁺ neurons with VGLUT2 rings are in the largest size category, with somatic profile areas >275 μm² (purple markers). C, Box and whisker plot showing the range and median of soma areas for GAD⁺ neurons without (magenta) and with (cyan) dense perisomatic rings of VGLUT2⁺ terminals. GAD⁺ neurons with VGLUT2 rings are larger on average than GAD⁺ neurons lacking VGLUT2 rings. ***p < 0.001.

Figure 5.

GAD⁺ neurons surrounded by PNs are larger, on average, than those lacking PNs. A, Photographs of GAD⁺ neurons of a range of sizes surrounded by (arrows) or lacking (arrowheads) PNs. Scale bar, 10 μm. B, A composite plot showing all GAD⁺ neurons from eight IC sections, similar to Figure 2, but with colored markers representing only GAD⁺ neurons surrounded by PNs. Smaller gray markers represent GAD⁺ neurons lacking PNs. Note that many GAD⁺ neurons with PNs are in the largest size category, with somatic profile areas >275 μm² (purple markers). C, Box and whisker plot showing the range and median of soma areas for GAD⁺ neurons without (magenta) and with (green) PNs. GAD⁺ neurons with PNs are larger on average than GAD⁺ neurons lacking PNs. ***p < 0.001.

Figure 6.

GAD⁺ neurons can be divided into four distinct subtypes. A–D, Photographs of four subtypes of GAD⁺ neurons in the IC. Each image row shows a single field that was imaged to reveal four markers. The first column shows an overlay of images stained for GAD and PNs; the second column shows an overlay of images stained for NeuN and VGLUT2; the third column shows an overlay of all four images. Some neurons have both a PN and a ring of VGLUT2⁺ terminals (GAD–PN–VGLUT2 ring; arrows in A–C), some neurons only have a PN (GAD–PN; open arrowheads in B and C), some neurons only have a ring of VGLUT2⁺ terminals (GAD–VGLUT2 ring; double arrow in D), and some neurons lack both a PN and a ring of VGLUT2⁺ terminals (GAD-only; arrowheads in A and C). The complete lack of PN staining in D is attributable to a local lack of PNs rather than a staining issue, because other photographs (not shown here) of PN-surrounded cells were collected from the same section, and a small amount of WFA labeling of the extracellular matrix is present in the background. Scale bar, 20 μm. E, Bar graph showing the proportion of each GAD subtype in each IC subdivision. GAD⁺ neurons lacking both a PN and a ring of VGLUT2⁺ terminals (light blue) are the majority in each subdivision but are a smaller majority in ICc. Overall and in each subdivision, most GAD⁺ neurons with a PN (light and dark green bars) lack a VGLUT2 ring (light green bars).

Figure 7.

Four subtypes of GAD⁺ neurons differ in distribution. A–D, Composite plots showing all GAD⁺ neurons from eight IC sections, with each GAD subtype shown in a separate panel. For each, the composite plot shows all neurons of that subtype, with subdivision outlines from one representative case for reference. GAD-only neurons (A) are denser in IClc, whereas GAD–PN and GAD–PN–VGLUT2 ring neurons (C, D) cluster in the ventromedial part of central IC. There is no definitive clustering of GAD–VGLUT2 ring neurons (B). To the right of each composite plot, a bar graph shows the normalized average density (cells per square millimeter divided by the maximum) for each GAD subtype across IC subdivisions.

Figure 8.

Four subtypes of GAD⁺ neurons differ in soma profile area. A, Box and whisker plot showing the soma profile area range and median for GAD⁻ neurons (yellow), as well as each GAD subtype. GAD-only neurons (light blue) are significantly smaller than GAD⁺ neurons in the other groups, whereas GAD–PN–VGLUT2 ring neurons (dark green) are significantly larger than GAD⁺ neurons in other groups. GAD⁻ neurons are significantly smaller than all four groups of GAD⁺ neurons. ***p < 0.001. B, Frequency distributions of soma profile area for each of the GAD⁺ subtypes, as well as GAD⁻ neurons. Vertical red lines indicate the soma size classification scheme proposed in this study for IC GAD⁺ neurons (small, <105 μm²; medium, 105–318 μm²; large, >318 μm²).

Figure 9.

Average soma diameter of neuron types in this study compared with IC cell size classifications from previous studies. The top shows histograms of average soma diameters for each GAD⁺ subtype and for GAD⁻ neurons from the present study. Vertical red lines indicate the soma size classification scheme proposed in this study (small, <12 μm; medium, 12–22 μm; and large, >22 μm). In the bottom, each row indicates a previous study that used soma diameter to classify IC cells. Filled boxes indicate finite ranges, and arrows indicate unbounded classification windows (i.e., < or >).