Effect of chronic L-dopa or melatonin treatments after dopamine deafferentation in rats: dyskinesia, motor performance, and cytological analysis

Abstract

The present study examines the ability of melatonin to protect striatal dopaminergic loss induced by 6-OHDA in a rat model of Parkinson's disease, comparing the results with L-DOPA-treated rats. The drugs were administered orally daily for a month, their therapeutic or dyskinetic effects were assessed by means of abnormal involuntary movements (AIMs) and stepping ability. At the cellular level, the response was evaluated using tyrosine hydroxylase immunoreactivity and striatal ultrastructural changes to compare between L-DOPA-induced AIMs and Melatonin-treated rats. Our findings demonstrated that chronic oral administration of Melatonin improved the alterations caused by the neurotoxin 6-OHDA. Melatonin-treated animals perform better in the motor tasks and had no dyskinetic alterations compared to L-DOPA-treated group. At the cellular level, we found that Melatonin-treated rats showed more TH-positive neurons and their striatal ultrastructure was well preserved. Thus, Melatonin is a useful treatment to delay the cellular and behavioral alterations observed in Parkinson's disease.

Figure 1

Synaptic ending (B) showing the two axes measured, establishing a synaptic contact with a dendritic spine (S).

Figure 2

Mean latencies to cross two narrow beams (6 and 12 mm) (±SEM) before and after 6-OHDA lesion and during treatments. Note that from the beginning of LD treatment the rats improve their motor behavior until 21 days and afterwards showed a significant increase in the time to transverse the beam compared to controls. In contrast, the time to cross the beam of melatonin-treated rats until day 21th was similar to those animals with lesion and no treatment; afterwards the time was reduced drastically resembling the values of the control group ( ^∗ P < .001 versus control group; ^• P < .001 LD-treated group versus melatonin-treated group; ANOVA test).

Figure 3

The three groups, 6-OHDA, 6-OHDA + LD, and 6-OHDA + melatonin confer certain susceptibility to dyskinesia during the course of the experiment, but the overall AIM severity is most pronounced in rats with 6-OHDA + LD treatment. (a) Time course of AIM development during the chronic LD and melatonin treatments period. Values give total (locomotive + axial + orolingual + limb AIMs) integrated AIM scores per testing session as group means ± SEM. (b) Time course of total AIM scores/monitoring period after a single treatment of LD or melatonin (treatment day 30).

Figure 4

Sequences of video recordings from rats affected by locomotive (a), axial (b) orolingual (c), and forelimb AIMs (d). Locomotive AIMs (a) comprise circular movement towards the contralateral side to the lesion. Only locomotive movements involving all four limbs are rated under this AIM category. The sequence in (b) shows a torsion movement of the neck and upper trunk towards the contralateral side to the lesion. Body torsion is maximally severe (>90°), causing the rat to lose equilibrium. Orolingual AIMs (c) include opening and closing of the jaws and tongue protrusion towards the side contralateral to the lesion (arrow). A black circle in (d) highlights purposeless up and down translocation of the Parkinsonian (right) forelimb.

Figure 5

Integrated AIM scores were generated separately for locomotive (a), orolingual (b), axial (c), and forelimb (d) AIMs using data from day 30 of chronic treatments. Note that animals treated with melatonin did not develop locomotive AIMs and axial rotation ( ^∗ P < 0.005 LD-treated group versus 6-OHDA-lesioned and melatonin-treated groups).

Figure 6

TH-immunoreactive cell counts from the SNc. The data are presented as the mean ± SEM. A statistically significant decrease in TH-immunoreactive cells was detected in both ipsilateral (I) and contralateral (C) SNc in the three experimental groups ( ^∗ P < 0.05 versus control group; ^• P < 0.05 between melatonin and 6-OHDA and LD-treated groups; ANOVA test).

Figure 7

Representative TH-immunostained from coronal sections containing the SNc of control, 6-OHDA-lesioned, 6-OHDA-lesioned + LD and 6-OHDA-lesioned + melatonin-treated rats. Note the profound cell loss in the ipsilateral SNc in the three experimental groups, being more evident in the 6-OHDA and LD treated ones; also, the contralateral SNc of melatonin-treated rats lost fewer neurons than the other two experimental groups (magnification 4×).

Figure 8

Synaptic ending mean diameter in ipsilateral (I) and contralateral (C) striata after stereotactic surgery and treatments, major and minor axes ( ^∗ P < 0.05 versus control group; ^• P < 0.05 between melatonin and 6-OHDA and LD-treated groups; ANOVA test).

Figure 9

Electron micrographs from control group rat striatum neuropile (A); 6-OHDA-lesioned ipsilateral striatum (B); 6-OHDA + LD treated rat ipsilateral striatum (C); 6-OHDA + melatonin-treated rat ipsilateral striatum (D). (A) In control group, the mean size of the synaptic buttons (b) was 700 × 696 nm, and the predominant Postsynaptic target was the dendritic spines (s); it can be observed that the neuropile is well preserved. (B) This image shows a swollen synaptic button (b) establishing a synaptic contact with a dendritic spine (s), altered mitochondria (arrowhead), and some vacuoles (*) within neuropile. (C) This image demonstrates three edematous presynaptic endings (b) of the LD-treated ipsilateral striatum establishing three synaptic contacts, one with a dendritic spine with dilated spine apparatus (sa), and two with a dendrite (d). Note the altered mitochondria (arrow heads) and neuropile vacuoles (*). (D) An increase in the presence of perforated synaptic contacts was notorious in striata of the three experimental groups (arrow). Note that the neuropile of the melatonin-treated group is well preserved, similar to control group neuropile. (b) Synaptic bouton, (sa) spine apparatus, (s) dendritic spine. Bar 0.2 μm.

Figure 10

This graph shows the average number of synaptic boutons that established synaptic contact with dendritic spine in the ipsilateral and contralateral striata of the four analyzed groups (*P < 0.005 versus control group and melatonin-treated group; ANOVA test).

Figure 11

This graph shows the average of the total number of perforated synapses in the ipsilateral and contralateral striata of the four analyzed groups (*P < 0.005 versus control group and melatonin-treated group; ANOVA test).