Association of Cocaine- and Amphetamine-Regulated Transcript-Immunoreactive Elements with Thyrotropin-Releasing Hormone-Synthesizing Neurons in the Hypothalamic Paraventricular Nucleus and Its Role in the Regulation of the Hypothalamic–Pituitary–Thyroid Axis during Fasting

Electron micrographs showing synaptic associations (arrows) between CART-containing axon terminals and neuronal perikarya containing pro-TRH-IR (A) or pro-TRH- and CART-IR (B) in the medial parvocellular subdivision of the PVN. The pro-TRH-IR is labeled with highly electron-dense silver granules, whereas the CART-IR is recognized by the presence of the electron-dense DAB. A, Medium-power magnification view of a CART-IR asymmetric synapse on the perikarya of a pro-TRH neuron, shown in greater detail in theinset. A dendrite co-containing CART and pro-TRH is also apparent in the image. B, High-power magnification of an axosomatic synapse between a CART-IR axon terminal and a neuronal perikarya co-containing CART and pro-TRH. Scale bars: A, 1 μm; B and inset, 0.4 μm.

Double immunolabeling for CART- and α-MSH-IR in the arcuate nucleus and commissural part of the nucleus of the solitary tract (NTS). A and B, andC and D are the same tissue section. In the arcuate nucleus (A, B), most of the CART-IR neurons (A) co-contain α-MSH-IR (B) in its perikarya. Only occasional single-labeled neurons are visible in the nucleus. In contrast, no colocalization between CART (C) and α-MSH (D) is seen in photomicrographs taken from the identical field of the commissural part of NTS. III, Third ventricle;CC, central canal. Scale bars (shown inB): A, B, 100 μm; (shown in D): C, D, 50 μm.

Colocalization of CART and pro-TRH-containing neuronal elements in the rat hypothalamus. A–C, Low-power micrographs show pro-TRH mRNA- (labeled with AMCA, but pseudocolored red for improved illustration of the CART/pro-TRH colocalization) and CART-containing (green) perikarya in the PVN. Neurons co-containing CART and pro-TRH appear yellow. In the anterior parvocellular subdivision (A), only occasional double-labeled neurons (yellow) can be found, whereas most of the pro-TRH mRNA-containing neurons in the periventricular and medial parvocellular subdivisions co-contain CART-IR (B, C). D, Low-power micrographs of the anterior parvocellular subdivision of the PVN again illustrate that the majority of pro-TRH-IR neurons (green) in the subdivision form a separate and distinct population from CART-IR neurons (red), but as seen in E, these singly labeled TRH neurons do not accumulate Fluoro-Gold from the blood stream. In contrast, the majority of pro-TRH-IR neurons in the periventricular and medial parvocellular subdivisions co-contain CART-IR (yellow cells inF) and accumulate Fluoro-Gold from the blood stream (G). In the external zone of the median eminence (ME) (H) and in the OVLT (I), nearly all pro-TRH-IR axons co-contain CART-IR, as indicated by theyellow color in these regions resulting from color mixing (green fluorescence = pro-TRH-IR;red fluorescence = CART-IR). High-power photomicrograph (J) shows that pro-TRH axon terminals innervating pro-TRH neurons in the PVN do not contain CART-IR. III, Third ventricle. Scale bars (shown inG): A–G, 100 μm;H, 50 μm; I, 100 μm;J, 10 μm.

Dark-field illumination micrographs of pro-TRH mRNA in the medial and periventricular parvocellular subdivisions of the hypothalamic PVN in fed (A) and fasted (B) animals and fasted animals receiving an intracerebroventricular infusion of CART at a dose of 0.5 μg (C) every 6 hr for 64 hr. Note the reduction in the accumulation of silver grains over the PVN in fasted animals compared with the fed controls. Fasted animals receiving CART every 6 hr, however, show a marked increase in pro-TRH mRNA, similar to that of fed control animals. III, Third ventricle. Scale bar, 100 μm.

Computerized image analysis of pro-TRH mRNA content in the PVN of fed and fasted animals and fasted animals receiving an intracerebroventricular infusion of CART at a dose of 0.5 μg. *p < 0.05 compared with fasted animals.