Optogenetic Manipulation of RTN Neurons
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Corresponding Organization :
Other organizations : University of Virginia
Protocol cited in 6 other protocols
Variable analysis
- Injection of virus (PRSx8-hChR2 (H134R)-mCherry or PRSx8-Allato-EGFP)
- Antidromic field potentials elicited by stimulating the mandibular branch of the facial nerve
- Sprague-Dawley rats, males, weight: 219–282 gm
- Anesthesia with a mixture of ketamine (75mg/kg), xylazine (5mg/kg) and acepromazine (1mg/kg) administered i.m.
- Standard aseptic surgical methods
- Antibiotic ampicillin (100mg/kg, i.m.) and analgesic ketorolac (0.6mg/kg, i.p.) administered post-surgery
- Lentivirus delivered into the RTN by controlled pressure injection (60 PSI, 3–8 ms pulses) using glass pipettes (25μm external tip diameter, 6–12Ω resistance)
- Injections made unilaterally at 2 or 3 different rostro-caudally aligned sites separated by 200 or 300μm for a total volume of 400 nl
- Animals maintained for no less than 3 weeks before physiological experiments
- Injection of anti-dopamine-β-hydroxylase conjugated to saporin (antiDβH-sap) bilaterally (4 sites total, 100nl per site) into the region of the lateral horn of the second thoracic segment (1.0–1.2mm lateral of midline, 1mm below lateral sulcus) to destroy the C1 neurons that project to the spinal cord
- No observable behavioral or respiratory effects from the surgical procedures and virus injections, and the rats gained weight normally
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