Inhibitory Projections from the Inferior Colliculus to the Medial Geniculate body Originate from Four Subtypes of GABAergic Cells

Abstract

GABAergic cells constitute 20-40% of the cells that project from the inferior colliculus [(IC) a midbrain auditory hub] to the medial geniculate body [(MG) the main auditory nucleus of the thalamus]. Four subtypes of GABAergic IC cells have been identified based on their association with perineuronal nets (PNs) and dense rings of axosomatic terminals expressing vesicular glutamate transporter 2 (VGLUT2 rings). These subtypes differ in their soma size and distribution within the IC. Based on previous work emphasizing large GABAergic cells as the origin of GABAergic IC-MG projections, we hypothesized that GABAergic IC cells surrounded by PNs and VGLUT2 rings, which tend to have larger somas, were more likely to project to the MG than smaller cells lacking these extracellular markers. Here, we injected retrograde tract tracers into the MG of guinea pigs of either sex and analyzed retrogradely labeled GABAergic cells in the ipsilateral IC for soma size and association with PNs and/or VGLUT2 rings. We found a range of GABAergic soma sizes present within the IC-MG pathway, which were reflective of the full range of GABAergic soma sizes present within the IC. Further, we found that all four subtypes of GABAergic IC cells participate in the IC-MG pathway, and that GABAergic cells lacking PNs and VGLUT2 rings were more prevalent within the pathway than would be expected based on their overall prevalence in the IC. These results may provide an anatomical substrate for the multiple roles of inhibition in the IC-MG pathway, which have emerged in electrophysiological studies.

Keywords: GABA; VGLUT2; perineuronal net; soma size; tectothalamic; thalamus.

Figure 1.

Tracer deposits included lemniscal and non-lemniscal MG subdivisions. Depictions of 4 of the 9 retrograde tracer deposits. A, B, Tracer deposits centered in non-lemniscal regions of the MG. C, D, Tracer deposits largely in lemniscal MGv. A, Photograph showing a single rostrocaudal level of the RB deposit in case GP733. B, Series of tracings showing the rostrocaudal extent of the FB deposit in case GP767. C, Photograph showing a single rostrocaudal level of the CTB deposit in case GP745. D, Series of tracings showing the rostrocaudal extent of the GB deposit in case GP744. Variations like these in tracer deposits ensured that all portions of the IC–MG projection were included in the study. Transverse sections. Scale bars, 1 mm. CTB, Cholera toxin B; D, dorsal; M, medial; MGm, medial subdivision of the MG; MGsg, suprageniculate subdivision of the MG; MGv, ventral subdivision of the MG; RB, red RetroBeads.

Figure 2.

Retrograde tracers label small, medium and large GABAergic IC cells. Photographs of cells (arrows) labeled with retrograde tracer (first column, red, Retro) that were also GAD+ (second column, green) and NeuN+ (third column, yellow). Note that all retrograde tracers are pseudo-colored red for simplicity. The first row shows examples of small GAD+ IC–MG cells (soma profile area <105 µm²), the second and third row show examples of medium GAD+ IC–MG cells (soma profile area between 105 and 318 µm²), and the bottom row shows an example of a large GAD+ IC–MG cell (soma profile area >318 µm²). Scale bar, 20 µm.

Figure 3.

GABAergic retrogradely labeled IC cells exhibit a wide range of soma sizes. Bar graphs showing the proportions of IC GAD+ cells overall (white bars) or the subset that were labeled by retrograde transport from the MG (gray bars). Each population is broken into small, medium, and large soma size categories. The total sample was made up of 6511 GAD+ IC cells, of which 258 were retrogradely labeled. Of these, 3114/100 were in ICc (A), 2081/72 were in ICd (B), and 1316/86 were in IClc (C). Medium cells form the majority of GAD+ cells and GAD+ IC–MG cells in each subdivision. The proportions of GABAergic soma sizes within the IC–MG pathway are reflective of the proportions of GABAergic soma sizes of the overall GABAergic population within the IC. Error bars represent SEM.

Figure 4.

Four subtypes of GABAergic neurons participate in the IC–MG projection. Photographs of neurons (NeuN stain in the first column, yellow) labeled with retrograde tracer (first column, Retro, red) that are also GAD+ (second column, green). In the first two rows (GAD-only), the indicated cells (white arrows) lack both a PN (third column, cyan) and a VGLUT2 ring (fourth column, magenta). In the third and fourth rows (GAD-PN), the indicated cells lack VGLUT2 rings but are surrounded by PNs. In the fifth row (GAD-VGLUT2 ring), the indicated cell lacks a PN but is surrounded by a VGLUT2 ring. In the sixth and seventh rows (GAD-PN-VGLUT2 ring), the indicated cells are surrounded by both PNs and VGLUT2 rings. Note that all tracers have been pseudo-colored red for simplicity. Scale bar, 20 µm.

Figure 5.

The four subtypes of GABAergic neurons in the IC–MG projection differ in soma size and spatial distribution. A, Box and whisker plot showing the range and median soma profile areas of non-GAD (yellow), GAD-only (light blue), GAD-PN (light green), GAD-VGLUT2 ring (dark blue), and GAD-PN-VGLUT2 ring (dark green) IC–MG cells. On average, GAD-only cell somas are smaller than GAD-PN cell somas, and GAD-PN-VGLUT2 ring cells have the largest somas. Note that the y-axis of the box plot is logarithmically scaled. Sample sizes: non-GAD = 4024 cells; GAD-only = 170 cells; GAD-VGLUT2 ring = 3 cells; GAD-PN = 63 cells; GAD-PN-VGLUT2 ring = 22 cells. Three asterisks indicate a significant difference in soma size between two groups at the p = 0.001 level. B, Composite plot of all the GAD+ IC–MG projecting cells in this study (cells from two sections each from 6 cases). Light blue markers indicate GAD-only cells, dark blue X’s indicate GAD-VGLUT2 ring cells, light green markers indicate GAD-PN cells, and dark green markers indicate GAD-PN-VGLUT2 ring cells. GAD-only cells are relatively more dense along the lateral and dorsal edges of the IC, whereas GAD+ cells surrounded by PNs and/or VGLUT2 rings are relatively more dense in deeper regions of the IC (ICc and IClc layer III). ICc- central nucleus of the IC; ICd- dorsal cortex of the IC, IClc I, II, and III, Layers I, II, and III of the lateral cortex of the IC.

Figure 6.

Retrogradely-labeled IC cells comprise four GABAergic subtypes. Bar graphs showing the proportions of IC GAD+ cells overall (solid bars) or the subset that were labeled by retrograde transport from the MG (striped bars). The cells were classified as GAD-only (light blue), GAD-PN (light green), GAD-VGLUT2 ring (dark blue), or GAD-PN-VGLUT2 ring (dark green) in each of the three main IC subdivisions. The total sample was made up of 6511 GAD+ IC cells, of which 258 were retrogradely labeled. Of these, 3114/100 were in ICc (A), 2081/72 were in ICd (B), and 1316/86 were in IClc (C). In each IC subdivision, the proportions of GAD subtypes within the IC–MG pathway were significantly different from the proportions of GAD subtypes within the IC overall, with GAD-Only cells overrepresented in the IC–MG pathway. Error bars represent SEM.

Figure 7.

A range of soma sizes and subtypes of cells makes up the GABAergic portion of the IC–MG projection. Black arrows indicate the GABAergic IC–MG projection, with the weight of each arrow reflecting the proportion of the pathway that is made up of each GABAergic subtype. Green circles represent small, medium, and large GABAergic IC cells; subtypes are distinguished by the presence of perineuronal nets (cyan) and/or perisomatic rings of VGLUT2 immunopositive boutons (magenta).