Fig. 1.

Identification of Rostral Preoptic Area (rPO)-Projecting A2 Noradrenergic Neurons by Dual Cytoplasmic Retrobead Epifluorescence and Tyrosine Hydroxylase (TH)-Immunoreactivity (-ir) and Their Sub-Categorization According to Presence versus Absence of Nuclear Glucokinase Regulatory Protein (nGKRP) Immunolabeling. Ovariectomized (OVX) rats were implanted with a subcutaneous (sc) estradiol (E)-filled silastic capsule [30 ug E/mL (E-30) versus 300 ug E/mL (E-300)], then injected into the rPO with Retrobead retrograde tracer prior to 18 h food deprivation (FD) or uninterrupted feeding (full-fed, i.e., FF controls). Hindbrain A2 neurons were identified in situ by tyrosine hydroxylase (TH)-immunoreactivity (-ir) (Panel I); TH-ir-positive cells exhibiting red cytoplasmic tracer epifluorescence (Panel II) were individually laser-catapult-microdissected for single-cell multiplex qPCR following sub-categorization based upon the presence or absence of nGCKR-ir (Panel III). The encircled area in Panels I, II, and III contains two representative cytoplasmic TH-ir- and Retrobead epifluorescence-positive hindbrain A2 neurons (one indicated by a yellow arrow, the other by a red arrow) that exhibit immunostaining for nGKRP-ir. Panel IV presents a merged image of cytoplasmic and nuclear staining patterns. The inset to each Panel, upper right-hand corner, shows an encircled TH-ir- and Retrobead epifluorescence-positive neuron that lacks nGKRP-ir.