Two classes of GABAergic neurons in the inferior colliculus

Abstract

The inferior colliculus (IC) is unique, having both glutamatergic and GABAergic projections ascending to the thalamus. Although subpopulations of GABAergic neurons in the IC have been proposed, criteria to distinguish them have been elusive and specific types have not been associated with specific neural circuits. Recently, the largest IC neurons were found to be recipients of somatic terminals containing vesicular glutamate transporter 2 (VGLUT2). Here, we show with electron microscopy that VGLUT2-positive (VGLUT2(+)) axonal terminals make axosomatic synapses on IC neurons. These terminals contain only VGLUT2 even though others in the IC have VGLUT1 or both VGLUT1 and 2. We demonstrate that there are two types of GABAergic neurons: larger neurons with VGLUT2(+) axosomatic endings and smaller neurons without such endings. Both types are present in all subdivisions of the IC, but larger GABAergic neurons with VGLUT2(+) axosomatic terminals are most prevalent in the central nucleus. The GABAergic tectothalamic neurons consist almost entirely of the larger cells surrounded by VGLUT2(+) axosomatic endings. Thus, two types of GABAergic neurons in the IC are defined by different synaptic organization and neuronal connections. Larger tectothalamic GABAergic neurons are covered with glutamatergic axosomatic synapses that could allow them to fire rapidly and overcome a slow membrane time constant; their axons may be the largest in the brachium of the IC. Thus, large GABAergic neurons could deliver IPSPs to the medial geniculate body before EPSPs from glutamatergic IC neurons firing simultaneously.

Figure 1.

Electron micrograph of large IC neuron with dense VGLUT2⁺ axosomatic endings. a, VGLUT2⁺ terminals contain an electron-dense reaction product and surround the cell body of a large IC neuron which has highly folded (asterisk) nucleus and stacked rough endoplasmic reticulum (arrowhead). The box shows the location of panel b. b, Higher-magnification view of synaptic terminals (T) that form asymmetric synapses (arrows). Scale bars: a, 5 μm; b, 1 μm.

Figure 2.

Confocal, deconvoluted micrographs showing three populations of glutamatergic terminals in the IC. a–d, VGLUT2⁺ terminals (green) contact a neuronal cell body labeled with MAP2 (blue) and its proximal dendrites. VGLUT1⁺ terminals (red) and terminals that are both VGLUT1⁺ and VGLUT2⁺ (arrowheads) are not associated with somata. e, Scatter plot of ICC terminals showing mean fluorescence of VGLUT1 and VGLUT2 immunoreactivities. Blue dots indicate terminals which were not apposed to cell bodies or primary dendrites, while red dots indicate terminals encircling cell bodies and primary dendrites. Terminals from one animal are illustrated. f, Percentage of terminals immunopositive for only VGLUT2, only VGLUT1, or both. Blue bars indicate proportions of terminals which were not apposed to cell bodies or primary dendrites, while red bars indicate proportions of terminals encircling cell bodies and primary dendrites. Scale bar, 10 μm.

Figure 3.

Confocal images that show two types of neurons immunopositive for GAD67 (red). a, d, g, Immunopositive cells with dense VGLUT2⁺ axosomatic endings (green) are indicated with arrows. b, e, h, GAD67⁺ cells without VGLUT2⁺ terminals (white arrowheads). c, f, g, GAD67-negative cells (black arrowheads) also lack VGLUT2⁺ axosomatic endings. These three types were seen in all IC subdivisions (a–c, ICC; d–f, DC; g and h, LC). Scale bar: (in h′′′) a–h′′′, 10 μm.

Figure 4.

Analysis of GAD67⁺ IC cells based on cell diameter and density of VGLUT2⁺ axosomatic endings and comparison to subjective classification. a–c, Scatter plots of GAD67⁺ cells (blue circles) in subdivisions of the IC and kernel density estimates of populations. Kernel density estimates are visualized by both color depth (white and red indicate high and low probability density, respectively) and contour plots. d, Best fit of bivariate Gaussian distribution to the kernel density estimate from the ICC with two bivariate normal distributions calculated by the least-squares method. The distribution with lower terminal density (blue contours) shows no covariance (ρ = 0) between diameter and density, while the distribution with higher terminal density (red contours) shows covariance between these parameters (ρ = 0.49). Points at which both distributions have the same probability density aligned with a slope y = −0.0071x + 0.41. e, Scatter plot of the ICC GAD67⁺ cells (circles, 92 cells) is divided by slope (y) into two populations. Neurons were classified subjectively, as VGLUT2-axosomatic positive (red, 61 cells) or VGLUT2-axosomatic negative (dark blue, 23 cells). A few cells were miscategorized and represent false-positive (cyan, 2 cells) and false-negative (orange, 6 cells) choices. See also Table 1. f, Scatter plot of all GAD67⁺ cells in all IC subdivisions classified by our subjective analysis and separated by slope (y) used in d and e. VGLUT2-axosomatic positive (140 cells; red circles) and VGLUT2-axosomatic negative (98 cells; blue circles). Errors were 21 false-positive and 20 false-negative cells. See also Table 2. g–j, Histograms of perimeter show the distribution of GAD67⁺ cells with or without VGLUT2⁺ axosomatic endings (red and blue bars, respectively) classified objectively in the ICC (g), DC (h), LC (i), and entire IC (j). In all cases, these two cell types make two distinct but overlapping populations of GAD67⁺ neurons.

Figure 5.

Distribution and size of GAD67⁺ IC neurons. a, Composite image of whole IC section immunostained for VGLUT2 (green) and GAD67 (red) and counterstained with Neurotrace fluorescent Nissl dye (blue) reconstructed from 78 confocal images using a ×40 lens (NA 1.3). GAD67⁺ cells with VGLUT2⁺ endings were distributed throughout the IC. The ICC and GABA modules (Chernock et al., 2004) are prominent at low magnification as red areas since there is less dense VGLUT2 immunoreactivity in these locations. b, Drawing of the section in a showing the subdivisions of the IC and location of GAD67-immunopositive cells with and without VGLUT2⁺ axosomatic endings (red and blue dots, respectively). GAD67-negative cells with VGLUT2⁺ axosomatic endings were rare (green dots). c, Percentage of GAD67⁺ cells with VGLUT2⁺ axosomatic endings (red bars), GAD67⁺ cells without axosomatic endings (blue bars), and GAD67-negative cells with axosomatic endings (green bars) in subdivisions of the IC. n = 3. d, Mean diameters of GAD67⁺ somata with VGLUT2⁺ axosomatic endings (red bars), and without such endings (blue bars), in subdivisions of IC. In b–d, n = 3; error bars in b and c indicate SEM. See also Tables S1 and S2. Scale bar, 500 μm.

Figure 6.

Identification of IC GAD67 neurons that project to the MGB. a, One neuron with VGLUT2⁺ axosomatic endings (white arrow). b, Large GAD67⁺ with axosomatic endings (white arrow) and a second GAD67⁺ neuron (black arrow) without axosomatic endings. c, After an FG injection into the medial geniculate body, retrogradely labeled FG-positive cells were found in the IC (white arrows and arrowheads). d, The large GAD67⁺ cells with VGLUT2⁺ axosomatic endings were labeled with FG (white arrow). GAD67⁺ neurons without VGLUT2⁺ axosomatic endings were not labeled with FG (black arrows). The majority of FG-positive cells are negative for GAD67 (white arrowheads). Green, VGLUT2; red, GAD67; blue, FG. Scale bar: (in d) a–d, 50 μm.

Figure 7.

Three types of neurons in the IC. Large GABAergic tectothalamic cells (1) are encircled by VGLUT2⁺ endings. Small GABAergic neurons lack axosomatic glutamate inputs (2). Non-GABAergic neurons (3) that presumably use glutamate as a transmitter express VGLUT2 (Herzog et al., 2001). There are two kinds of tectothalamic cells; inhibitory and excitatory, and the vast majority of inhibitory tectothalamic neurons are large GABAergic cells with VGLUT2⁺ axosomatic endings. All cell types receive inhibitory axosomatic inputs (i.e., GABA and glycine) (Merchán et al., 2005). Note that distribution of terminals on dendrites is speculative.