Abstract
Quantitative in situ hybridization histochemistry was used to determine the age-related changes in tyrosine hydroxylase (TH) mRNA and cholecystokinin (CCK) mRNA in the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc) of the rat. Coronal sections (10 μm) were cut in a cryostat through the VTA and SNc of brains from 3 month and 33 month old Sprague-Dawley rats and immediately adjacent sections hybridized with 35S-labelled 45-mer oligonucleotide probes specific for either the rat TH or CCK genes. The mRNA levels of each gene were estimated by computerised densitometric analysis of the signal on X-ray film autoradiograms and estimation of the number of mRNA expressing cells as well as the density of expression per cell (grain density) was made from high resolution emulsion autoradiograms. Analysis of the TH mRNA on X-ray film autoradiograms indicated that the levels averaged 25% lower in the SNc ( P < 0.01) and 18% lower in the VTA ( P < 0.05) of the old rats. However, analysis of the emulsion autoradiograms showed that this reduction in TH mRNA in the VTA and SNc in the old rats was not due to a loss of TH mRNA expressing cells but due to a reduction in the hybridization signal per expressing cell. Analysis of the CCK mRNA signal on X-ray film autoradiograms showed that the levels averaged 9 and 15% higher (but non - significant) in the VTA and SNc respectively of the old rats while analysis of the emulsion autoradiograms showed no increase in the number of expressing cells but an averaged 20% (though not significant) increase in the hybridization signal per CCK mRNA expressing cell in the VTA and SNc of the old rats. The results demonstrate that in the aged rat there is no significant loss of dopamine (DA)-containing neurones in the VTA and SNc but there is a clear decrease in the synthetic activity of the neurones and this may be responsible for the neurochemical and behavioural deficits associated with the ageing process. Since in the rat CCK is extensively colocalized with DA in neurones in both the VTA and SNc, the trend of increased activity of the CCK gene (with a concomitant decrease in TH gene activity) suggests a complex regulation of colocalized neurotransmitters possibly via autofeedback mechanisms at the transcriptional level.
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