Supplementary Information

Involvement of Cholinergic System in Hyperactivity in Dopamine-Deficient Mice

Yoko Hagino, Shinya Kasai, Masayo Fujita, Susumu Setogawa, Hiroshi Yamaura, Dai Yanagihara, Makoto Hashimoto, Kazuto Kobayashi, Herbert Y Meltzer, Kazutaka Ikeda*

*Corresponding author. E-mail:

Figure S1. Locomotor activity in wildtype mice with daily L-DOPA injections. Locomotor activity in wildtype mice with (n = 7) of without (n = 6) daily L-DOPA injections.

Figure S2. Catecholamine biosynthetic pathway and catecholamine presence in wildtype, TH-KO, DD, and DD with L-DOPA mice. Dopamine (DA) and norepinephrine (NE) are sequentially synthesized from tyrosine by three enzymes, tyrosine hydroxylase (TH), aromatic amino acid decarboxylase (AADC), and dopamine β-hydroxylase (DBH). TH-KO mice which resulted in deficiency in both DA and NE, died at a late stage of embryonic development or shortly after birth. DD mice have extremely low levels of DA and died by 4 weeks of age. DD mice can be rescued by daily treatment with L-DOPA.

Figure S3. Microdialysis analysis of wildtype and DD mice. (a) L-DOPA-induced elevation of DAex levels in DD mice 24 h after the last L-DOPA injection. Time course of DAex (percentage of baseline DAex) before and after the intraperitoneal injection of saline or L-DOPA (50 mg/kg). The arrows indicate the drug injection time. Each point represents the mean ± SEM. (b) L-DOPA-induced elevation of DAex levels in DD mice 24 h after the last L-DOPA injection. Time course of DAex (fmol/10 μl/10 min) before and after the intraperitoneal injection of saline or L-DOPA (50 mg/kg) (DD mice, inset). The arrows indicate the drug injection time. Each point represents the mean ± SEM.

Figure S4. Locomotor activity in wildtype and DD mice in the home cage. Before locomotor activity was measured, the mice were placed in their home cages (30 ´ 20 ´ 14 cm) for 1 day for acclimatization and then placed in the Supermex. Black bars represent the dark cycle. The arrows indicate the L-DOPA (50 mg/kg) injection time (n = 6).

Figure S5. Kinematic parameters during locomotion. (a) Comparison of joint angles during the step cycle period in wildtype mice (blue) and DD mice (red). Averaged angular excursions of the hip, knee, and ankle joints are plotted. The cycle period was normalized and is expressed as a percentage. Vertical blue and red lines in each panel correspond to the stance-swing phase transition in wildtype and DD mice, respectively. The SEM is plotted on either side of the mean. (b) Iliac crest heights in the step cycle during locomotion in wildtype mice (blue) and DD mice (red). The data are expressed as mean ± SEM. (c) Greater trochanter heights in the step cycle during locomotion in wildtype mice (blue) and DD mice (red) mice. The data are expressed as mean ± SEM.

Figure S6. Temporal parameters during locomotion. (a) Scatterplot of stride length vs. walking speed in wildtype mice (N = 8, n = 5-24) and dopamine-deficient mice (N = 9, n = 5-28). N indicates the number of mice, and n is the number of steps. (b) Temporal parameters in wildtype and DD mice during locomotion. (A) Stance phase duration. (B) Swing phase duration. (C) Step cycle duration. (D) Duty rate. The data are expressed as mean ± SEM.

Figure S7. Effect of ziprasidone on hyperactivity in DD mice. Locomotor response to ziprasidone (3 mg/kg) in wildtype mice (n = 5) and DD mice (n = 4). The arrow indicates the drug infection time.

Movie S1. This movie shows an example of locomotor activity and cataleptic behavior in wildtype (left) and DD (right) mice.

Supplementary Methods

Hindlimb movement analyses. Movement analyses were limited to the sagittal plane parallel to the direction of walking. Custom-designed image analysis software (DIPP-Motion 2D, DITECT, Tokyo, Japan) was used to extract the two-dimensional coordinates of the various joint markers and reconstruct a stick-diagram representation of the right hindlimb. Because of skin slippage above the knee joint during walking, the actual knee position was corrected by triangulation from the position of the hip and ankle joints using the measured lengths of the femur and tibia. In this study, we defined a step cycle as always having a phase in which the mouse supported its body weight with both hindlimbs (bisupport phase). If no bisupport phase was found in the observed cycle, then we excluded the data from the analysis because the mouse was unable to walk. We analyzed the temporal parameters, swing phase duration, stance phase duration, step cycle period, and walking speed, which was calculated as the horizontal velocity of the iliac crest. The step cycle can be divided into the swing phase and stance phase. The swing phase is defined as starting at the moment when the animal lifts its foot from the floor and ending when the foot comes back into contact with the floor. The stance phase is defined as starting when the foot touches the floor and ending when the foot lifts from the floor. The duty rate of a hindlimb is the time fraction of the step cycle duration for which that limb is in the stance phase. To analyze the angular excursions of the hip, knee, and ankle during a cycle period, the step cycle duration was normalized, and cubic-spline interpolation was applied to the original data on the joint angles of the hip, knee, and ankle to obtain 100 samples per step cycle regardless of their duration using MATLAB computer software (MathWorks, Natick, MA, USA).

Immunohistochemistry. The paraffin sections were autoclaved in 0.01 M citrate buffer (pH 6.0) to retrieve antigen. The paraffin sections were then immersed in 0.3% hydrogen peroxide in methanol and incubated in 5% normal goat or rabbit serum for blocking. The sections were incubated with primary antibodies overnight at 4°C, followed by detection with biotinylated secondary antibodies and the avidin-biotin complex kit (Vector Laboratories, Burlingame, CA, USA). A positive reaction was detected using diaminobenzidine tetrahydrochloride that contained 0.001% hydrogen peroxide.

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