Fluorogold fg

Mice were anesthetized with chloral hydrate and placed in a stereotactic apparatus. A 1 μl volume of 4% Fluoro-Gold (FG, Invitrogen) suspension was injected into the superior colliculi as previously described^{51 (link), 52} . Seven days were allotted for the retrograde transport of FG to label RGCs. After the experiment, a retinal wholemount was prepared, and FG positive RGCs were identified under a confocal fluorescence microscope (LSM710, Carl Zeiss).

Above cathetered rats were anesthetized with 3.5% chloral hydrate (10 ml/kg, i.p.), and underwent laminectomy at vertebral level T9/10 to expose the spinal cord. Before transection of the spinal cord, retrograde neuronal tracer Fluorogold (FG, 2% in PBS, Invitrogen, 2 μl per each side in four points) was injected into the left and the right dorsal column to label pyramidal cells in the sensorimotor cortex layer V and VI. And then the dorsal half of the spinal cord was cut with a pair of previously marked microscissors to sever the dorsal corticospinal tract (CST) at a depth of 1.8 mm from the dorsal surface, and a 25 gauge needle was used to plough the lesion site to assure complete transection of the CST. Muscles and skin were closed in layers, and animals were placed in a temperature-controlled chamber until thermoregulation was re-established. Histological examination had revealed that these lesions severed all dorsal CST fibers in the dorsal funiculus as well as in the lateral CST and extended past the central canal in all animals. Manual voiding of the bladder was performed twice per day until reflex bladder emptying was reestablished.

Five days after OVX and E2 treatment, rats (n = 9) were stereotaxically implanted with a stainless-steel guide cannula (23-gage; Plastics One, Roanoke, VA) in the MRF with the tip end at 11.4 mm posterior and 9.0 mm ventral to the bregma and 0.7 lateral to the midline, according to the brain atlas (Paxinos and Watson, 2006 ). FluoroGold (FG; Invitrogen, Carlsbad, CA) was dissolved in saline at 2% and unilaterally injected into the MRF at a rate of 0.25 μl/min for 2 min using a microsyringe pump through an internal cannula (26 gage). This procedure was performed under anesthesia with pentobarbital (13 mg/ml Somnopentyl, 0.15ml/100g body weight). Two days after FG injection, some rats (n = 6) received sexual stimulation and others (n = 3) were used as non-stimulated controls. Brains were processed for FG and cFos double-IHC. After cFos-ir was detected as described above, free-floating sections were sequentially incubated with 0.3% H₂O₂ in PBS for 15 min, 2% NGS in PBS for 1 h, and primary rabbit antiserum against FG (1:20,000; Invitrogen) for 24h at RT. FG-ir neurons were visualized with a streptavidin-biotin kit, followed by DAB as a chromogen.