Optogenetic Activation of Indirect-Pathway MSNs
Objective: To directly activate basal ganglia circuitry using optogenetic control of direct- and indirect-pathway medium spiny projection neurons (MSNs) and measure effects on motor behavior including freezing, bradykinesia, and locomotor initiation
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Equipment2
Materials1
Not specified • Not specified • Not specified • Not specified
Software1
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Protocol Steps
Viral vector preparation and injection
Prepare Cre-dependent viral expression vectors containing channelrhodopsin-2 for injection into striatum
Note: Separate viral preparations for D1-Cre and D2-Cre transgenic mice to target direct- and indirect-pathway MSNs respectively
View evidence from paper
“Cre-dependent viral expression of channelrhodopsin-2 in the striatum of D1-Cre and D2-Cre BAC transgenic mice”
Stereotaxic injection of viral vectors
Inject Cre-dependent channelrhodopsin-2 viral vectors into the striatum of transgenic mice
Note: Bilateral injections to target both hemispheres for subsequent bilateral excitation
View evidence from paper
“Cre-dependent viral expression of channelrhodopsin-2 in the striatum of D1-Cre and D2-Cre BAC transgenic mice”
Expression period
Allow time for viral expression of channelrhodopsin-2 in target neurons
Note: Duration not specified in provided text
View evidence from paper
“Cre-dependent viral expression of channelrhodopsin-2 in the striatum”
Optogenetic stimulation of indirect-pathway MSNs
Deliver light stimulation to activate channelrhodopsin-2 expressing indirect-pathway MSNs bilaterally and measure resulting motor behavior
Note: Bilateral excitation used to elicit parkinsonian state
View evidence from paper
“Bilateral excitation of indirect-pathway MSNs elicited a parkinsonian state, distinguished by increased freezing, bradykinesia, and decreased locomotor initiations”
Behavioral assessment during indirect-pathway activation
Measure and record motor behaviors including freezing, bradykinesia, and locomotor initiation during optogenetic stimulation
Note: Parkinsonian phenotype characterized by increased freezing, bradykinesia, and decreased locomotor initiations
View evidence from paper
“Bilateral excitation of indirect-pathway MSNs elicited a parkinsonian state, distinguished by increased freezing, bradykinesia, and decreased locomotor initiations”
Optogenetic stimulation of direct-pathway MSNs
Deliver light stimulation to activate channelrhodopsin-2 expressing direct-pathway MSNs and measure resulting motor behavior
Note: Opposite effects to indirect-pathway activation
View evidence from paper
“activation of direct-pathway MSNs reduced freezing and increased locomotion”
Behavioral assessment during direct-pathway activation
Measure and record motor behaviors during optogenetic stimulation of direct-pathway MSNs
Note: Direct pathway activation produces reduced freezing and increased locomotion
View evidence from paper
“activation of direct-pathway MSNs reduced freezing and increased locomotion”
Testing in Parkinson's disease model
Perform optogenetic stimulation of direct-pathway MSNs in a mouse model of Parkinson's disease and assess rescue of motor deficits
Note: Direct pathway activation completely rescued deficits in freezing, bradykinesia, and locomotor initiation
View evidence from paper
“In a mouse model of Parkinson's disease, direct pathway activation completely rescued deficits in freezing, bradykinesia, and locomotor initiation”