Altered nucleolar morphology in substantia nigra dopamine neurons following 6-hydroxydopamine lesion in rats

Abstract

The nucleolus, the site of ribosomal ribonucleic acid (rRNA) transcription and assembly, is an important player in the cellular response to stress. Altered nucleolar function and morphology, including decreased nucleolar volume, has been observed in Parkinson's disease; thus the nucleolus represents a potential indicator of neurodegeneration in the disease. This study determined the effects of a partial unilateral intrastriatal 6-hydroxydopamine (6-OHDA) lesion, which models the dopaminergic loss found in Parkinson's disease, on the nucleoli of dopaminergic cells in the substantia nigra pars compacta (SNpc). Adult male Long-Evans rats underwent unilateral intrastriatal infusion of 6-OHDA (12.5μg). Lesions were verified by amphetamine-stimulated rotation 7 days later, and rats were euthanized 14 days after infusion. Coronal sections (50μm) were stained for tyrosine hydroxylase-silver nucleolar (TH-AgNOR) stain using MultiBrain Technology (NeuroScience Associates), which resulted in clearly defined nucleoli and neuronal outlines. Stereological methods were used to compare dopaminergic morphology between lesioned and intact hemispheres in each rat. In cells exhibiting a definable nucleolus, nucleolar volume was decreased by 16% on the ipsilateral side. The ipsilateral SNpc also exhibited an 18% decrease in SNpc planimetric volume, a 46% decrease in total TH-positive neuron number, and an 11% decrease in neuronal body volume (all P<0.05 by paired t-test). These findings suggest that the 6-OHDA lesion alters nucleolar morphology and that these changes are similar to those occurring in Parkinson's disease.

Fig. 1. High-power and low-power micrographs of TH-AgNOR stained sections of contralateral and ipsilateral substantia nigra

4X images of TH-AgNOR-stained sections of contralateral and ipsilateral SN qualitatively show depletion of the lesioned dopaminergic regions (1A and B). 100X images show the outlines of the contralateral and ipsilateral neurons and the AgNOR-stained nucleoli (1C and D). Representative sections from the same rat are shown. Abbreviations: SNL- substantia nigra lateralis, SNpc- substantia nigra pars compacta, VTA- ventral tegmental area

Fig. 2. The double nucleator technique

Markers are placed on the nucleolus center and where the nucleator rays intersect with the borders of the nucleolus and the neuronal body outline.

Fig. 3. Effects of unilateral intrastriatal 6-OHDA lesion on TH-AgNOR-stained planimetric volume (A), neuron number (B) and neuron volume (C) in the SNpc

All rats showed decreased SNpc planimetric volume between the ipsilateral and contralateral hemispheres. The mean within-subject decrease was 18.3 ± 2.2% (P<0.05 by paired t-test, n=11). All rats showed decreased neuronal number between the ipsilateral and contralateral SNpc. The mean within-subject decrease was 45.7% ± 5.1% (P<0.05 by paired t-test, n=11). All rats showed decreased neuronal volume between the ipsilateral and contralateral SNpc. The mean within-subject decrease was 11.2 ± 3.1% (P<0.05 by paired t-test, n=11).

Fig. 4. Effects of unilateral intrastriatal 6-OHDA lesion on TH-AgNOR-stained nucleolar volume in the SNpc

All rats showed decreased nucleolar volume between the ipsilateral and contralateral SNpc (4A). The mean within-subject decrease was 16.4 ± 2.4% (P<0.05 by paired t-test, n=11). The ratio of nucleolar volume to neuronal volume in the ipsilateral SNpc was smaller in only 6 of the 11 rats the contralateral SNpc and there was no significant difference between sides (4B).