The selective neurotoxin DSP-4 impairs the noradrenergic projections from the locus coeruleus to the inferior colliculus in rats

Abstract

The inferior colliculus (IC) and the locus coeruleus (LC) are two midbrain nuclei that integrate multimodal information and play a major role in novelty detection to elicit an orienting response. Despite the reciprocal connections between these two structures, the projection pattern and target areas of the LC within the subdivisions of the rat IC are still unknown. Here, we used tract-tracing approaches combined with immunohistochemistry, densitometry, and confocal microscopy (CM) analysis to describe a projection from the LC to the IC. Biotinylated dextran amine (BDA) injections into the LC showed that the LC-IC projection is mainly ipsilateral (90%) and reaches, to a major extent, the dorsal and lateral part of the IC and the intercollicular commissure. Additionally, some LC fibers extend into the central nucleus of the IC. The neurochemical nature of this projection is noradrenergic, given that tyrosine hydroxylase (TH) and dopamine beta hydroxylase (DBH) colocalize with the BDA-labeled fibers from the LC. To determine the total field of the LC innervations in the IC, we destroyed the LC neurons and fibers using a highly selective neurotoxin, DSP-4, and then studied the distribution and density of TH- and DBH-immunolabeled axons in the IC. In the DSP-4 treated animals, the number of axonal fibers immunolabeled for TH and DBH were deeply decreased throughout the entire rostrocaudal extent of the IC and its subdivisions compared to controls. Our densitometry results showed that the IC receives up to 97% of its noradrenergic innervations from the LC neurons and only 3% from non-coeruleus neurons. Our results also indicate that TH immunoreactivity in the IC was less impaired than the immunoreactivity for DBH after DSP-4 administration. This is consistent with the existence of an important dopaminergic projection from the substantia nigra to the IC. In conclusion, our study demonstrates and quantifies the noradrenergic projection from the LC to the IC and its subdivisions. The re-examination of the TH and DBH immunoreactivity after DSP-4 treatment provides insights into the source, extent, and topographic distribution of the LC efferent network in the IC, and hence, contributes to our understanding of the role of the noradrenaline (NA) system in auditory processing.

Keywords: biotinylated dextran amine; densitometry; dopamine beta hydroxylase deficiency; immunohistochemistry; neurotoxicity; noradrenergic efferents; noradrenergic modulation; tyrosine hydroxylase.

Figure 1

The target areas and projection pattern of the locus coeruleus (LC) fibers within the inferior colliculus (IC). The inset schemes represent coronal sections at the rostrocaudal level as a reference, and the black square represents the area photographed in high magnification. (A) A representative photomicrograph of a typical injection site (I.S.). Left LC received a BDA stereotaxic injection (black). (B) A photomicrograph of a typical labeled fiber in the IC from the LC. The arrow indicates a fiber and the arrowhead indicates a varicosity. (C–E) Confocal photomicrographs of BDA-labeled fibers in the IC. (C) The arrow indicates possible synaptic buttons. (D) The arrow indicates single fibers. (E) The arrow indicates a fiber and the arrowhead indicates possible synaptic buttons. (F) A schematic representation of the BDA-labeled fibers from the LC found in the IC and the internal anatomic subdivisions of the IC. Notice that there are ipsi- and contralateral labeled fibers. The interaural level for coronal placement is displayed in millimeters. See the provided list for abbreviations.

Figure 2

The colocalization of locus coeruleus fibers with tyrosine hydroxylase (TH) in the dorsal cortex of the inferior colliculus (DCIC). (A) A schematic representation of the coronal section in which the following confocal photomicrographs were taken. The black square depicts the area of the confocal images. (B) The confocal image shows TH-ir in DCIC (red). The arrow and arrowhead indicate representative TH-labeled fibers magnified in C and D, respectively. (E) The confocal image shows labeled fibers (green) after BDA injection in the LC. The arrow and arrowhead indicate representative BDA-labeled fibers in the DCIC. The high magnification images of those fibers are shown in the insets F and G. (H) The merged composition of the photomicrographs in B and E (yellow). The arrowhead and arrow indicate the colocalization of BDA and TH on the representative fibers that are magnified in I and J, respectively. The interaural level for coronal placement is displayed in millimeters. See the provided list for abbreviations.

Figure 3

The colocalization of locus coeruleus fibers with dopamine beta hydroxylase (DBH) in the dorsal cortex of the inferior colliculus (DCIC). (A) A schematic representation of the coronal section in which the following confocal photomicrographs were taken. The black square depicts the area of the confocal images. (B) A confocal image for DBH-ir (red). The arrows indicate a representative fiber in two parts, most likely due to the Z-axis frame, and the arrowhead shows another representative fiber that is magnified in C. (D) The confocal image shows labeled fibers (green) after BDA injection in the LC. The arrows indicate a representative fiber, and the arrowhead shows another representative fiber that is magnified in E. (F) The merged composition between B and D (yellow). The arrowhead indicates a merged fiber (yellow) that is magnified in G. The arrows show colocalization in another fiber (yellow). The interaural level for coronal placement is displayed in millimeters. See the provided list for abbreviations.

Figure 4

The distribution of immunolabeled fibers for tyrosine hydroxylase (TH) and dopamine beta hydroxylase (DBH) in the inferior colliculus (IC). (A) Schematic representation of the coronal section in which the following confocal photomicrographs were taken. The black square depicts the exact placement of the confocal images, which were taken near the commissure. (B) The confocal image for TH-ir (red). (C) The confocal image for DBH-ir (green). (D) The merged composition between B and C (yellow). (E) A magnification of the white square in D. The arrowhead indicates merged fibers (yellow). (F) An orthogonal view of a section of the confocal fibers. The arrowheads indicate merged fibers and their place in the Z-axis. (G) A three-dimensional view of the two channels. The arrowheads show the difference between the signals in the upper right part of the images where there are TH-ir fibers (red) but not DBH-ir, which leads to the conclusion that dopaminergic fibers (not noradrenergic) exist in the IC. The white square indicates the zone where the colocalization was found. There is a similar pattern of signal distribution. The interaural level for the coronal placement is displayed in millimeters. See the provided list for abbreviations.

Figure 5

The effects of DSP-4 administration in the locus coeruleus (LC) on immunohistochemistry (tyrosine hydroxylase and dopamine beta hydroxylase) compared to negative controls for the LC and the inferior colliculus (IC). (A) A photomicrograph of the locus coeruleus (LC) immunostained for tyrosine hydroxylase (TH) in a control animal. The black arrow represents a typical immunoreactive neuron, which is magnified in the inset. (B) A photomicrograph of the LC immunostained for dopamine-beta-hydroxylase (DBH) in a control animal. The black arrow represents a typical immunoreactive neuron, which is magnified in the inset. (C) A photomicrograph of the LC immunostained for TH in a DSP-4-treated animal. (D) A photomicrograph of the LC immunostained for DBH in a DSP-4-treated animal. Notice in this set of images how the immunoreactivity against the catecholaminergic enzymes is distinctly decreased compared to the controls. This reflects the immunoreactivity found in the IC in controls and treated animals. E,F,G, and H are images taken from the negative controls (no primary antibody added). The interaural level for the coronal placement is displayed in millimeters. See the provided list for abbreviations.

Figure 6

The effects of DSP-4 administration in the tyrosine hydroxylase-immunoreactivity (TH-ir) within the inferior colliculus (IC). A schematic representation of TH-ir in the IC from the caudal to the rostral part. (A) Drawings of the TH-ir in control animals. (B) Drawings of the TH-ir in DSP-4-treated animals. Notice how the immunoreactivity is markedly decreased in treated animals compared to controls. (C) The mean gray value of the distribution of TH-ir in control animals at different rostrocaudal levels and subdivisions. (D) The mean gray value of the distribution of TH-ir in DSP-4-treated animals at different rostrocaudal levels and subdivisions. (E) The percent reduction in TH-ir following DSP-4 treatment. The interaural level for the coronal placement is displayed in millimeters. The bars represent means ± SD of 24 stained sections. See the provided list for abbreviations.

Figure 7

The effects of DSP-4 administration on the dopamine beta hydroxylase-immunoreactivity (DBH-ir) in the inferior colliculus (IC). A schematic representation of DBH-ir in the IC from the caudal to the rostral part. (A) Drawings of the DBH-ir in control animals. (B) Drawings of the DBH-ir in DSP-4-treated animals. Notice how the immunoreactivity almost disappears in treated animals. (C) The mean gray value of the distribution of DBH-ir in control animals at different rostrocaudal levels and subdivisions. (D) The mean gray value of the distribution of DBH-ir in DSP-4-treated animals at different rostrocaudal levels and subdivisions. (E) The percent reduction in DBH-ir following DSP-4 treatment. The interaural level for the coronal placement is displayed in millimeters. The bars represent means ± SD of 24 stained sections. See the provided list for abbreviations.