Anatomical characterization of subcortical descending projections to the inferior colliculus in mouse

Abstract

Descending projections from the thalamus and related structures to the midbrain are evolutionarily highly conserved. However, the basic organization of this auditory thalamotectal pathway has not yet been characterized. The purpose of this study was to obtain a better understanding of the anatomical and neurochemical features of this pathway. Analysis of the distributions of retrogradely labeled cells after focal injections of retrograde tracer into the inferior colliculus (IC) of the mouse revealed that most of the subcortical descending projections originated in the brachium of the IC and the paralaminar portions of the auditory thalamus. In addition, the vast majority of thalamotectal cells were found to be negative for the calcium-binding proteins calbindin, parvalbumin, or calretinin. Using two different strains of GAD-GFP mice, as well as immunostaining for GABA, we found that a subset of neurons in the brachium of the IC is GABAergic, suggesting that part of this descending pathway is inhibitory. Finally, dual retrograde injections into the IC and amygdala plus corpus striatum as well into the IC and auditory cortex did not reveal any double labeling. These data suggest that the thalamocollicular pathway comprises a unique population of thalamic neurons that do not contain typical calcium-binding proteins and do not project to other paralaminar thalamic forebrain targets, and that a previously undescribed descending GABAergic pathway emanates from the brachium of the IC. J. Comp. Neurol. 525:885-900, 2017. © 2016 Wiley Periodicals, Inc.

Keywords: GABA; RRID: AB_2619710; RRID:AB_2278725; RRID:AB_476667; RRID:AB_476894; RRID:AB_477329; RRID:IMSR_JAX:007677; RRID:SCR_001775; RRID:SCR_003070; RRID:SCR_013286; RRID:SCR_014235; RRID:SCR_014432; colliculus; thalamocollicular; thalamotectal; thalamus.

Figure 1. Example of the distribution of cells projecting to the IC from thalamus and adjacent structures

A) Four images from the injection site after deposit of Fluorogold-soaked gelfoam was placed into the IC. Numbers in lower right corner correspond to the caudal-rostral location of the slice (0.0 = most caudal portion of the IC, 1.0 = most rostral portion of the IC). Backlabeled cells are seen in the BIC (B) as well as MGB and adjacent structures (C and D). Scale bars = 150 μm. D=dorsal, L=lateral.

Figure 2. Distribution of backlabeled cells after injection of tracer into LC

Top: Five representative sections from caudal to rostral illustrating the distribution of backlabeled cells from three different animals, color-coded by animal. Intra-MGB borders are not distinguished in the caudal most section of the MGB (see text for details). Bottom: Injection sites for three different animals. Dotted lines correspond to nuclear borders, when available either via nuclear or GAD staining. Shape and color in bottom left corner of each injection site photo correspond to the backlabeled cells projected onto the drawings above. In all cases, fluorogold was used to inject. Scale bar = 100μm.

Figure 3. Distribution of backlabeled cells after injection of tracer into DC

The figure is organized in an identical manner as Figure 2.

Figure 4. Distribution of backlabeled cells after injection of tracer into CNIC

The figure is organized in an identical manner as Figures 2 and 3, except that only two injection sites are shown.

Figure 5. Evaluation of double-labeling of thalamotectal cells with calcium binding proteins

A-B) Composite 20X photos (9 photos each, arranged 3 × 3) views of backlabeled cells after FG injection into the IC (A) and immunostain for calbindin (B). C-E) Zoomed 20X views showing backlabeled thalamotectal cells (C), calbindin positive cells (D) and an overlay image (E) with thalamotectal cells in green and calbindin cells in magenta, from the square shown in (A). F-J) Similar to A-E, but for calretinin. K-O) Similar to A-E, but for parvalbumin. Scale bar (A, B, F, G, K, L) = 250μm. Scale bar (C-E, H-J, M-O) = 20 μm. Inset in bottom left of (K) is the same injection site for all three sets of images.

Figure 6. Positive control for combining immunostaining with backlabeling of cells with fluorogold

A and B) Low-powered images of the MGB after immunostaining for calbindin (A) or placing fluorogold into the AC (B). Scale bar = 100 μm. C) Higher-power view from square shown in panel A showing calbindin-positive neurons. D) Higher-power view from same field in panel C showing backlabeled thalamocortical cells. E) Overlay image showing multiple double labeled cells (denoted with white arrows). Green = fluorogold labeled cells. Magenta = calbindin labeled cells. Scale bar for panels C-E = 30 μm.

Figure 7. GABAergic cells from the BIC project to the IC

A) Low-power image of backlabeled cells backlabeled after injection of FG into the IC (injection site in inset). Boxes correspond to BIC (upper box) and PP (lower box). Scale bar = 500 μm. B) Same section as (A), immunostained for GAD67+. C) 40X confocal image of backlabeled cells in the BIC. D) 40X confocal image of GAD+ tissue in the BIC. E) Overlay between (C) and (D). White arrows point to double-labeled cells. F) 40X confocal image of backlabeled cells in the PP. G) 40X confocal image of GAD+ tissue in the PP. H) Overlay between (F) and (G). No double-labeled cells are seen. Scale bar = 20 μm. I and J) Distributions of backlabeled cells in the posterior thalamus and BIC of two GAD-GFP mice injected with CTB in the IC (injection sites in insets). Backlabeled cells which were either GAD+ (black circles) or GAD- (red circles).

Figure 8. Immunostaining of GABAergic cells from the BIC that project to the IC

A) Backlabeled cells in the BIC after injection of FG into the IC (injection site in inset). B) GABA+ cells in the same section of the BIC. C) Overlay between (A) and (B). Arrows show double-labeled cells. Arrowheads point to backlabeled cells that are negative for GABA. Scale bar = 20 μm.

Figure 9. Thalamotectal cells do not project to the AC, amygdala or corpus striatum

A) Injection site of fluorogold into the AC. Backlabeled cells are seen in the MGB. B) Injection site of Retrobeads into the IC. C) High-powered image from the mMGB showing backlabeled thalamocortical cells. D) High-powered image from the same mMGB field showing backlabeled thalamotectal cells E) Overlay image from panels C and D showing absence of double-labeled cells. Scale bar for panels C-E = 25 μm. F) Injection site of Retrobeads into the IC, G) Large injection site of Retrobeads encompassing the amygdala (AMYG) and corpus striatum (CS). H) High-powered image from the mMGB showing backlabeled thalamotectal cells. I) High-powered image from the same mMGB field showing backlabeled thalamic cells projecting to the CS and/or the AMYG. J) Overlay image from panels H and I showing absence of double-labeled cells. Scale bar for panels H-J = 50 μm.

Figure 10. Hypothesized organizational scheme of descending projections to the IC

Neurons in the IC receive excitatory input from the AC and posterior thalamus (blue circles representing neurons) and GABAergic input from the BIC (black circles representing neurons). It is not yet known if IC-projecting neurons in the posterior thalamus and BIC receive direct input from corticothalamic axons (question marks) or if individual IC neurons receive both cortical and subcortical descending input. Although the diagram is focused on the LC for clarity, since both DC and LC receive substantial projections from AC and posterior thalamus, the hypothesized connectional scheme could also be applied to the DC.