Dopamine in Motor Cortex Is Necessary for Skill Learning and Synaptic Plasticity methods
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Results
The surgical procedure to perform intracortical injections caused a transient impairment of motor function as assessed using an accelerated rotarod test. The deficit between sham-lesioned (vehicle, n = 4) and 6-OHDA-treated rats (n = 4) was not different (, group × time interaction: p = 0.61) suggesting that the impairment of motor function resulted from the surgical procedure and not from the drug itself. Rotarod performance was lower at 3 hr, 6 hr and 9 hr post-injection (post hoc tests: p<0.05, power >0.75) with a statistical trend of reduced performance at 24 hr (p = 0.069) as compared with baseline. Performance recovered to pre-injection levels within 48 hr (, post-hoc difference to baseline: p = 1.00; overall effect of time F(6,30) = 3.44, p = 0.011, power 0.88). To guarantee full recovery a...
Materials and Methods
All experiments were performed in adult male Long-Evans rats (8-10 weeks, 250-350 g) raised in our animal facility. Animals were housed individually in a 12/12-hr light/dark cycle (light on: 3am, off: 3pm). All procedures were conducted according to national and international guidelines and were approved by the Animal Care Committee of the State of Baden-Württemberg, Germany. Chemicals and antibodies were purchased from Sigma-Aldrich Chemie GmbH, Munich, Germany, unless noted otherwise.
Materials and Methods
Training sessions were performed at the beginning of the dark phase. Animals were food-restricted for 24 hr before the first pre-training session (see below). During training animals were kept slightly over their initial weight (332.1±29.4 g) by providing 50 mg/kg of standard lab diet after each training session. Water was given ad libitum.
Materials and Methods
Motor skill training was performed as previously described. The training cage was a 15 × 40 cm chamber (height 30 cm) with a vertical window (1 cm wide, 5 cm high beginning 2 cm above floor) in the front wall and a small light sensor in the rear wall (7 cm above ground). Animals were first pre-trained for five days learning to open the motorized sliding door that covered the front window, by nose-poking the sensor in the rear. Opening the window gave access to one food pellet (45 mg, Bio-serve, Frenchtown, NJ, USA) located on a small horizontal board outside of the cage. During pre-training pellets were retrieved by tongue. Upon retrieval the pellet was automatically replaced by a pellet dispenser. Pre-training was followed by 6-15-days of motor skill training that was initiated by removing the board and placing the pellet on a vertical post 1.5 cm away from the window. In t...
Surgical Procedures
All surgical procedures were performed under ketamine (70 mg/kg, i.p.) and xylazine anesthesia (5 mg/kg, i.p.) with the rats fixed in a stereotactic frame (Stoelting Co., Wood Dale, IL, USA). Additional ketamine doses were administered if necessary. Body temperature was controlled using a heating pad. Buprenorphin (0.01 mg/kg, i.p.) was given after surgery for pain relief. All permanent implants were anchored onto the skull with two screws (2 mm diameter) placed in the frontal and occipital skull. Bone flaps were replaced and fixated using bone cement (FlowLine, Heraus Kulzer, Dormagen, Germany).
Surgical Procedures
The forelimb representation was identified in each animal for optimal placement of injection needles (6-OHDA and levodopa) and cannula implantation (repeated antagonist injections). The brain was exposed by craniotomy leaving the dura intact (coordinates with respect to Bregma: 4 mm posterior to 5 mm anterior, 5 mm to 1 mm lateral). M1 somatotopy was mapped using a thin-film microelectrode array (Multichannel Systems, Reutlingen, Germany) as previously described. The array was placed onto the dura over the frontoparietal cortex. Biphasic stimuli (100 stimuli at 300 Hz, 1-5 mA constant current, 10 ms stimulus interval) were applied to the 64 contacts of the electrode array in a random sequence. Evoked limb twitches were visually identified. The forelimb area was typically 2 to 3.5 mm lateral and 1.5 to 2.5 mm anterior to Bregma. All drug injections were performed in a depth of 1...
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Evidence quotes (6)
The surgical procedure to perform intracortical injections caused a transient impairment of motor function as assessed using an accelerated rotarod test. The deficit between sham-lesioned (vehicle, n = 4) and 6-OHDA-treated rats (n = 4) was not different (, group × time interaction: p = 0.61) suggesting that the impairment of motor function resulted from the surgical procedure and not from the drug itself. Rotarod performance was lower at 3 hr, 6 hr and 9 hr post-injection (post hoc tests: p<0.05, power >0.75) with a statistical trend of reduced performance at 24 hr (p = 0.069) as compared with baseline. Performance recovered to pre-injection levels within 48 hr (, post-hoc difference to baseline: p = 1.00; overall effect of time F(6,30) = 3.44, p = 0.011, power 0.88). To guarantee full recovery a 3-day post-injection period was allowed following surgery before training was continued.
All experiments were performed in adult male Long-Evans rats (8-10 weeks, 250-350 g) raised in our animal facility. Animals were housed individually in a 12/12-hr light/dark cycle (light on: 3am, off: 3pm). All procedures were conducted according to national and international guidelines and were approved by the Animal Care Committee of the State of Baden-Württemberg, Germany. Chemicals and antibodies were purchased from Sigma-Aldrich Chemie GmbH, Munich, Germany, unless noted otherwise.
Training sessions were performed at the beginning of the dark phase. Animals were food-restricted for 24 hr before the first pre-training session (see below). During training animals were kept slightly over their initial weight (332.1±29.4 g) by providing 50 mg/kg of standard lab diet after each training session. Water was given ad libitum.
Motor skill training was performed as previously described. The training cage was a 15 × 40 cm chamber (height 30 cm) with a vertical window (1 cm wide, 5 cm high beginning 2 cm above floor) in the front wall and a small light sensor in the rear wall (7 cm above ground). Animals were first pre-trained for five days learning to open the motorized sliding door that covered the front window, by nose-poking the sensor in the rear. Opening the window gave access to one food pellet (45 mg, Bio-serve, Frenchtown, NJ, USA) located on a small horizontal board outside of the cage. During pre-training pellets were retrieved by tongue. Upon retrieval the pellet was automatically replaced by a pellet dispenser. Pre-training was followed by 6-15-days of motor skill training that was initiated by removing the board and placing the pellet on a vertical post 1.5 cm away from the window. In this position pellets were only retrievable by using the forelimb. The first skill training session was to determine forelimb preference and consisted of 50 door openings ( = trials). Determination of preference was necessary before surgical instrumentation of the hemisphere contralateral...
All surgical procedures were performed under ketamine (70 mg/kg, i.p.) and xylazine anesthesia (5 mg/kg, i.p.) with the rats fixed in a stereotactic frame (Stoelting Co., Wood Dale, IL, USA). Additional ketamine doses were administered if necessary. Body temperature was controlled using a heating pad. Buprenorphin (0.01 mg/kg, i.p.) was given after surgery for pain relief. All permanent implants were anchored onto the skull with two screws (2 mm diameter) placed in the frontal and occipital skull. Bone flaps were replaced and fixated using bone cement (FlowLine, Heraus Kulzer, Dormagen, Germany).
The forelimb representation was identified in each animal for optimal placement of injection needles (6-OHDA and levodopa) and cannula implantation (repeated antagonist injections). The brain was exposed by craniotomy leaving the dura intact (coordinates with respect to Bregma: 4 mm posterior to 5 mm anterior, 5 mm to 1 mm lateral). M1 somatotopy was mapped using a thin-film microelectrode array (Multichannel Systems, Reutlingen, Germany) as previously described. The array was placed onto the dura over the frontoparietal cortex. Biphasic stimuli (100 stimuli at 300 Hz, 1-5 mA constant current, 10 ms stimulus interval) were applied to the 64 contacts of the electrode array in a random sequence. Evoked limb twitches were visually identified. The forelimb area was typically 2 to 3.5 mm lateral and 1.5 to 2.5 mm anterior to Bregma. All drug injections were performed in a depth of 1 mm below the dura.
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