Abstract
Molecular cloning has revealed the existence of three distinct small conductance (SK1–3) Ca2+-activated K+ channels. Because SK channels underlie the afterhyperpolarization (AHP) that is critical for sculpturing phasic firing in hypothalamic neurons, we investigated the distribution of these channels in the female guinea pig. Both SK1 and SK3 cDNA fragments were cloned using PCR, and ribonuclease protection assay as well as in situ hybridization analysis illustrated that the SK3 channel was the predominant subtype expressed in the guinea pig hypothalamus. Combined in situ hybridization and fluorescence immunocytochemistry revealed that SK3 mRNA was expressed in GnRH, dopamine, and vasopressin neurons, and all of these neurons exhibited an AHP current. Moreover, SK3 mRNA was found in other brain areas, including the septum, bed nucleus, amygdala, thalamus, midbrain, and hippocampus. Using quantitative ribonuclease protection assay, the rank order of SK3 mRNA expression was septum ≥ midbrain > rostral thalamus ≥ rostral basal hypothalamus ≥ caudal thalamus ≥ preoptic area ≫ caudal basal hypothalamus ≥ hippocampus. Moreover, 17β-E2 treatment, which reduces plasma LH during the negative feedback phase, significantly increased SK3 mRNA levels in the rostral basal hypothalamus (P < 0.05; n = 6). Therefore, these findings suggest that estrogen increases the mRNA expression of SK3 channels, which may represent a mechanism by which estrogen regulates hypothalamic neuronal excitability during negative feedback.
Quantitation of TGF-?1 mRNA in porcine mesangial cells by comparative kinetic RT/PCR: Comparison with ribonuclease protection assay and in situ hybridization