Three days were allowed before the experiments. Rats were implanted with a
stainless steel guide cannula (0.8 mm once daily) with sodium pentobarbital (60
mg/kg, i.p.), and then the guide cannula was secured to the skull. Stereotaxic
coordinates for the dorsolateral PAG (dl-PAG) were 7.6 mm posterior to the
bregma, 0.65 mm lateral to the midline, and 4.2 mm ventral to the brain
surface.
Following this, cannula was connected to an osmotic minipump (Alzet pump brain
infusion kit, DURECT Inc., Cupertino, CA) with polycarbonate tubing. The pumps
were placed subcutaneously between the scapulae and loaded with vehicle
(artificial cerebrospinal fluid) as control or each of PIC receptor antagonists,
namely IL-1Ra (IL-1β receptor antagonist) and SC144 (gp130 antagonist to block
IL-6R) and etanercept (ETAN; TNF-α receptor antagonist), respectively (Tocris
Co., Ellisville, MO). In a subgroup, muscimol, agonist of GABAa receptors was
loaded. The PIC receptor antagonists in 10 µM of concentration and muscimol in
100 µM of concentration were delivered at 0.25 μl per hour (Alzet Model 1003D/3
day delivery; DURECT Inc., Cupertino, CA). This intervention allowed animals to
receive continuous PAG infusion via the osmotic minipumps before the experiments
and brain tissues were taken out. Note that all drugs were dissolved in
artificial cerebrospinal fluid as a final concentration.
stainless steel guide cannula (0.8 mm once daily) with sodium pentobarbital (60
mg/kg, i.p.), and then the guide cannula was secured to the skull. Stereotaxic
coordinates for the dorsolateral PAG (dl-PAG) were 7.6 mm posterior to the
bregma, 0.65 mm lateral to the midline, and 4.2 mm ventral to the brain
surface.
Following this, cannula was connected to an osmotic minipump (Alzet pump brain
infusion kit, DURECT Inc., Cupertino, CA) with polycarbonate tubing. The pumps
were placed subcutaneously between the scapulae and loaded with vehicle
(artificial cerebrospinal fluid) as control or each of PIC receptor antagonists,
namely IL-1Ra (IL-1β receptor antagonist) and SC144 (gp130 antagonist to block
IL-6R) and etanercept (ETAN; TNF-α receptor antagonist), respectively (Tocris
Co., Ellisville, MO). In a subgroup, muscimol, agonist of GABAa receptors was
loaded. The PIC receptor antagonists in 10 µM of concentration and muscimol in
100 µM of concentration were delivered at 0.25 μl per hour (Alzet Model 1003D/3
day delivery; DURECT Inc., Cupertino, CA). This intervention allowed animals to
receive continuous PAG infusion via the osmotic minipumps before the experiments
and brain tissues were taken out. Note that all drugs were dissolved in
artificial cerebrospinal fluid as a final concentration.