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.