Motor activity tests
To control for any effects of doxycycline on motor activity and motor deficits following 6-OHDA+L-DOPA treatment, the forepaw adjusting steps test and the open field test were performed. Vehicle or doxycycline was injected 30 minutes before L-DOPA.
Forepaw adjusting steps test: The stepping test has been used as a measure of forelimb akinesia, demonstrating sensitivity to dopamine loss as well as the reversal of the deficit by dopamine replacement therapy (Chang et al., 1999; Olsson et al., 1995). Briefly, the experimenter restrained the rat, so that the hind limbs were lifted, and one front paw was free for weight-bearing. The rats were dragged laterally across a table (90 cm/10s), and a trained rater blind to the condition counted the steps. Three trials were conducted for each forepaw and included both forward and backward stepping. Each forepaw was alternated, with the forepaw that was contralateral to the lesion tested first. Baseline stepping measurements were obtained before initiation of the study (pre-test), and 60 min after L-DOPA treatment on days 1, 7, and 14. The results are presented as a percentage of the number of forehand adjusting steps taken with the intact paw relative to the total number of adjusted steps.
Open field test: The rats’ spontaneous activity in the open field was tested 60 min after L-DOPA or vehicle administration. Each rat was individually placed in the center of the arena and allowed to explore the open field for 5 min. The behavior recognition system (ANY-maze 4.9, Stoelting USA) was used to calculate the distance traveled and time of immobility.