Immunohistochemical amplification of mCherry fusion protein is necessary for proper visualization.

Fluorescent reporter proteins are a powerful tool being increasingly integrated into biological experiments. Their utility spans techniques such as live-cell imaging, validating transgene expression, and studying cell-type specific anatomy. As these reporters become more widely used, it is necessary to fully understand their benefits and limitations. One such recently developed red fluorescent protein, mCherry, has been well utilized due to its stability, brightness, and pH resistance. In the course of an experiment using the fluorescent reporter protein mCherry fused to a G-protein coupled receptor (mCherry fusion protein), our lab discovered a notable inability for the fusion protein to faithfully produce fluorescent signal representative of its expression in fixed tissue. Here, we demonstrate the importance of immunohistochemical amplification in tissue injected with various adeno-associated viruses (AAVs), containing mCherry fusion protein as a reporter. Our findings demonstrate that antibody amplification consistently provides a stronger signal when mCherry fusion protein is used as a reporter protein.

Pubertal development of estradiol-induced hypothalamic progesterone synthesis.

In females, a hallmark of puberty is the luteinizing hormone (LH) surge that triggers ovulation. Puberty initiates estrogen positive feedback onto hypothalamic circuits, which underlie the stimulation of gonadotropin releasing hormone (GnRH) neurons. In reproductively mature female rodents, both estradiol (E2) and progesterone (P4) signaling are necessary to stimulate the surge release of GnRH and LH. Estradiol membrane-initiated signaling facilitates progesterone (neuroP) synthesis in hypothalamic astrocytes, which act on E2-induced progesterone receptors (PGR) to stimulate kisspeptin release, thereby activating GnRH release. How the brain changes during puberty to allow estrogen positive feedback remains unknown. In the current study, we hypothesized that a critical step in estrogen positive feedback was the ability for estradiol-induced neuroP synthesis. To test this idea, hypothalamic neuroP levels were measured in groups of prepubertal, pubertal and young adult female Long Evans rats. Steroids were measured with liquid chromatography tandem mass spectrometry (LC-MS/MS). Hypothalamic neuroP increases from pre-puberty to young adulthood in both gonad-intact females and ovariectomized rats treated with E2. The pubertal development of hypothalamic E2-facilitated progesterone synthesis appears to be one of the neural switches facilitating reproductive maturation.

Single neuron morphology in vivo with confined primed conversion.

Unraveling the structural organization of neurons can provide fundamental insights into brain function. However, visualizing neurite morphology in vivo remains difficult due to the high density and complexity of neural packing in the nervous system. Detailed analysis of neural morphology requires distinction of closely neighboring, highly intricate cellular structures such as neurites with high contrast. Green-to-red photoconvertible fluorescent proteins have become powerful tools to optically highlight molecular and cellular structures for developmental and cell biological studies. Yet, selective labeling of single cells of interest in vivo has been precluded due to inefficient photoconversion when using high intensity, pulsed, near-infrared laser sources that are commonly applied for achieving axially confined two-photon (2P) fluorescence excitation. Here we describe a novel optical mechanism, "confined primed conversion," which employs continuous dual-wave illumination to achieve confined green-to-red photoconversion of single cells in live zebrafish embryos. Confined primed conversion exhibits wide applicability and this chapter specifically elaborates on employing this imaging modality to analyze neural morphology of optically targeted single neurons in the developing zebrafish brain.

Hypothalamic galanin-like peptide rescues the onset of puberty in food-restricted weanling rats.

Galanin-like peptide (GALP) is a known mediator of metabolism and reproduction; however, the role that GALP plays in the onset of puberty is unknown. First, we tested the hypothesis that central GALP administration could rescue puberty in food-restricted weanling rats. GALP treatment in food-restricted rats of both sexes rescued the timing of the onset of puberty to that seen in ad lib. fed controls. Second, we tested whether GALP translation knocked-down in ad lib. fed, prepubertal rats would alter the timing of puberty. Knock-down females, but not males, showed a significant (P < 0.01) delay in the onset of puberty compared to controls. Third, we sought evidence that the role of GALP in pubertal onset is mediated by the kisspeptin system. In situ hybridisation analyses showed a significant (P < 0.01) reduction in Kiss1 mRNA within the hypothalamic arcuate nucleus in food-restricted rats compared to ad lib. fed controls and this reduction was prevented with i.c.v. GALP administration. Furthermore, analyses of Fos-immunoreactivity (-IR) after i.c.v. GALP treatment did not elicit Fos-IR within any kisspeptin neurones, nor are GALP and kisspeptin peptides or mRNA colocalised. These data demonstrate that hypothalamic GALP infusion maintained the onset of puberty in food-restricted weanling rats, although probably not via direct innervation of kisspeptin neurones.