Neurotoxic effects of DSP-4 on the central noradrenergic system in male zebra finches

Abstract

When administered systemically, the noradrenergic neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) appears to target the noradrenergic innervation originating in the locus coeruleus causing long-term decrements in noradrenergic function. In songbirds, DSP-4-treatment decreased female-directed singing by males and copulation solicitation responses of females to male songs. However, DSP-4 treatment in songbirds did not lower measures of NE function in the brain to the same extent as it does in mammals. The current study had two goals: determining if two DSP-4 treatments 10 days apart would cause significant decrements in noradrenergic function in male zebra finches and determining if, as in other species, the noradrenergic innervation of midbrain and cortical areas would be profoundly affected while hypothalamic areas were spared. Dopamine-beta-hydroxylase immunoreactivity (DBH-ir) was quantified in thirteen brain regions (five vocal control nuclei, one auditory nucleus, two hypothalamic nuclei, and five additional areas that demonstrated high DBH labeling in controls). Within 20 days, DSP-4 treatment profoundly reduced the number of DBH-ir cells in both the locus coeruleus and ventral subcoeruleus. Unlike a previous study, DBH labeling delineated four out of five vocal control nuclei and an auditory nucleus. As expected, DSP-4 treatment significantly decreased DBH labeling in all areas examined in the mesencephalon and telencephalon without significantly affecting DBH-ir in hypothalamic areas. This double treatment regime appears to be much more effective in decreasing noradrenergic function in songbirds than the single treatment typically used.

Figure 1

Mean (± SEM) estimated DBH-ir cells in locus coeruleus (LoC), ventral subcoeruleus (SCv), dorsal LoC, and ventral LoC in saline-treated (SAL) and DSP-4-treated males. Significant decrease in cell numbers compared to saline group, **p<0.01, ***p<0.0001.

Figure 2

Mean area (± SEM) covered by DBH-ir labeling per coronal section of A. areas outside the vocal control system and B. vocal control and auditory nuclei. Mean (± SEM) percent of each brain area covered by DBH-ir labeling per coronal section in C. areas outside the vocal control system and D. in vocal control and auditory nuclei. Note broken Y axes in A and D. Scale is not linear in A. Significant change compared to saline-treated males, *p<0.05, **p<0.01, ***p<0.001. See text for abbreviations.

Figure 3

Photomicrographs of DBH ir in areas outside of the vocal control system in male zebra finch brains. In each pair, the brain on the left is from a saline treated male, that on the right from a DSP-4 treated male. A) LoC, B) VTA, C) S, D) Cb, E) POA, F) PVN. See text for abbreviations.

Figure 4

Photomicrographs of DBH-ir labeling in vocal control and auditory nuclei in male zebra finch brains. In each pair, the brain on the left is from a saline-treated male, that on the right from a DSP-4-treated male. A) Uva, B) lMAN & X, C) HVC, D) RA, E) TeO & MLd. See text for abbreviations. Note that in the saline-treated controls, the DBH-ir labeling inside the vocal control nuclei is denser than that in surrounding tissue, except in Area X. There are two issues here. First, there are more aggregations of dense punctate labeling and fibers within the vocal control nuclei compared to surrounding tissue. Second, the background of the VCN is darker than that of the surrounding tissue. This is caused by smaller, lighter puncta of DBH ir, making the vocal control areas in the DSP-4-treated birds darker than the surrounding tissue. DSP-4 treatment significantly lowered, but did not eliminate DBH-ir. The VCN in DSP-4-treated birds stand out because they still have more DBH-ir than the surround. For the most part, tissue from DSP-4-treated birds lacks the dense fibers and puncta seen in controls. This can be seen in higher magnification in Figure 5. The arrows in HVC and the asterisks in lMAN in this Figure correspond to those in these areas in Figure 5.

Figure 5

Higher magnification of DBH-ir labeling in the sections of HVC and lMAN shown in Figure 4. In each pair, the brain on the left is from a saline-treated male, that on the right from a DSP-4-treated male. The arrows in HVC and the asterisks in lMAN in this Figure correspond to those in these areas in Figure 4. Note how the pattern and density of DBH-ir labeling over lMAN in the control bird is different from that outside the nucleus. This is particularly clear in comparison to the area below lMAN. If you refer back to Figure 4, you can see that the arrow in HVC in the control bird points to an area of dense labeling on the ventral border of the nucleus. It is clear at higher magnification that no labeling below the arrow matches the aggregations of high density DBH-ir labeling seen within HVC.