Figure 1.
Illustration of the injection sites in dopaminergic lesions in the rat brain.
(A) Anatomical locations of the two injection sites in the Paxinos Rat Atlas (circles). (B) Actual tissue slice demonstrating the positions of the two injection sites (blue colour). (C) Illustration of the main projection areas of the ventral tegmental area (VTA) and lateral substantia nigra (SN). (D) Projection areas of the locus coeruleus.
Table 1.
Details of primary antibodies.
Figure 2.
Successful dopaminergic lesions do not cause changes in the COMT protein levels.
A normalized optical density (OD) analysis of (A) tyrosine hydroxylase (TH) or (B) COMT positive neurons in the substantia nigra (SN) and striatum (Stri) after a unilateral 6-OHDA (8 µg) lesion of the SN, and TH and COMT positive neurons in the prefrontal cortex (PFC) after 6-OHDA lesion of the VTA. At least 3 separate stainings were done, and mean ± SEM are shown. Significant decrease in TH OD was seen in all areas analyzed but none in COMT OD. ***, P<0.001; **, P<0.01 vs. control side (Student t-test).
Table 2.
Dopamine levels after 6-OHDA-lesions and noradrenaline levels after DSP-4 lesions.
Figure 3.
A–C. Selective dopaminergic lesions do not affect COMT protein expression and activity.
6-OHDA (8 µg) was unilaterally infused either to the lateral substantia nigra (SN) or ventral tegmental area (VTA). COMT protein expressions were unaltered in any of the dopaminergic projection areas in the SN (A) or VTA (B) in agreement with unchanged enzyme activity (C). In dopaminergic lesion studies, animals received unilateral infusions of 6-OHDA and therefore the results are expressed as percentage of the intact side set as 100. Mean ± SEM, n = 4 in the SN control group, otherwise n = 5.
Figure 4.
A–X. COMT protein neither colocalized with tyrosine hydroxylase (TH) positive neurons nor decreased after destruction of dopamine neurons.
The effect of dopaminergic lesion on the COMT immunoreactivity. The 6-OHDA lesion of the substantia nigra (SN) and ventral tegmental area (VTA) were made as described and COMT immunoreactivity analysed in the projection areas. In the intact SN, COMT (red color) was not colocalized with dopaminergic neurons (tyrosine hydroxylase, TH; green color; A–D), and lesion in the SN did not affect COMT immunoreactivity (E–H) although TH-immunoreactivity disappeared (E). The situation was similar in the striatum (Stri; I–L versus M–P). Moreover, in the intact prefrontal cortex (PFC), the projection area of the VTA, COMT did not colocalize with dopaminergic nerves (Q–T). Moreover, the lesion in the VTA did not effect on COMT on the PFC (U–W). Nuclei are visualized by DAPI (blue color). Scale bars are 10 µm in all pictures. Three separate stainings were made and representative examples are shown. Quantification of ODs was done in Fig.2.
Figure 5.
A–B. Selective noradrenergic lesions do not change COMT protein expression and activity.
COMT protein expression (A) and activity (B) were unaltered in any of the noradrenergic projection areas. To destroy noradrenergic neurons, animals were injected i.p. with DSP-4 (50 mg/kg) and control animals received i.p. injections of saline (vehicle). The results are expressed as percentage of the control animals set as 100. Mean ± SEM, n = 5.
Figure 6.
A–G. Lack of the effect of noradrenergic lesion on COMT immunoreactivity.
DSP-4 lesion of noradrenergic neurons did not affect COMT-immunoreactivity (red color) in noradrenergic projection areas, the prefrontal cortex (PFC; A–B), striatum (Stri; C–D) and hippocampus CA1 (Hc CA1; E–F). Three separate stainings were done, and the COMT optical densities are quantified in Fig. 6G. Nuclei are visualized by DAPI (blue color). Scale bars are 10 µm in all pictures.
Figure 7.
A–T. COMT protein is colocalized with a GABAergic (glutamate decarboxylase, GAD65/67; green) but less or none with a cholinergic marker (choline acetyltransferase, ChAT; green).
In the striatum (Stri), COMT (red) was found to be colocalized with GABAergic neurons and fibres (A–D; white arrows and yellow color depicting colocalization), while no colocalization with cholinergic nerves was seen (E–H). Moreover, COMT was not colocalized with cholinergic neurons in the medial septum (MS; I–L). In the cortex, COMT was also colocalized with GABAergic interneurons (M–P; white arrows, yellow color) and a weak colocalization with some cholinergic nerves was seen (Q–T). Nuclei are visualized by DAPI (blue color). Scale bars are 10 µm in all pictures. Three separate stainings were done and representative examples are shown.