MCT8 and D3 immunoreactivities in axon varicosities of the rat parvocellular hypophysiotropic neurons.

<p>Confocal images were subjected to deconvolution. Boxed areas are enlarged in <b>B</b> and <b>C</b>, respectively. The immunofluorescent signal for MCT8 (red) is distributed as small dots throughout the external zone of median eminence and appears (arrowheads) on the surface of gonadotropin-releasing hormone (GnRH, <b>C</b>), thyrotropin-releasing hormone (TRH, <b>D</b>), corticotropin-releasing hormone (<b>E</b>), growth hormone-releasing hormone (GHRH, <b>F</b>) and somatostatin (SST, E) immunofluorescent axon varicosities (green). (<b>H,I</b>) Representative images of triple immunofluorescent labelings demonstrate D3 (blue), MCT8 (red) and a hypophyseotroph hormone (green) in the axons of the median eminence. MCT8-immunoreactive puncta (arrowheads) appear on the surface of the following categories of axon varicosities; single-labeled for D3 (<b>Ha</b>), single-labeled for GnRH (<b>Hb</b>), and double labeled for D3 and GnRH (<b>Hc</b>) or CRH (<b>I</b>). Scale bars: A: 10 µm, B: 5 µm, C-I: 2 µm.</p

D3-immunoreactivity in the infundibular stalk of the human hypothalamus.

<p>(<b>A</b>) D3 immunoreactive fibers (arrowheads) are present in the human infundibular stalk (IS); these fibers are shown in medium (<b>B</b>) and high (inset in A) power micrographs. opt: tractus opticus, VMH: ventromedial hypothalamic nucleus, III: third ventricle Scale bars: 500 µm in A, 50 µm in B, 10 µm in inset.</p

Schematic illustration of axonal uptake and regulation of T3 in the mediobasal hypothalamus.

<p>We suggest that T3 generated by D2 is released from tanycyte processes and taken up by MCT8 into axons of hypophysiotropic neurons. T3 concentrations are subjected to local regulation by D3-containing axon varicosities (At1), but absent in D3-negative axons (At2). T3 could be subjected to retrograde transport to reach the soma and nucleus of hypophysiotropic neurons and/or could act locally by affecting mitochondrial function and local thermogenesis. At, axon terminal; Tc, tanycyte; Pc, portal capillary.</p

D3 expression in processes of mouse immortalized GT 1-7 GnRH neurons <i>in vitro.</i>

<p>(<b>A</b>) GT 1-7 cells endogenously express D3 mRNA. –RT: minus reverse transcriptase control; + and -: positive and negative controls, respectively (<b>B</b>) Schematic illustration of Bimolecular Fluorescence Complementation used to study D3 homodimerization. YFP(N) and YFP(C) stand for YFP(1-158aa) and YFP(159–238aa), respectively. (<b>C</b>) GT 1-7 cells were transfected with a plasmid encoding the fusion protein D3-YFP(full-length). (<b>D</b>) Co-expression of YFP(1-158)-D3 and YFP-(159–238)-D3 fusion proteins results in fluorescence complementation and demonstrates D3 homodimers in the axon-like processes of GT1-7 cells. Arrows indicate D3 along the processes. Scale bars: 10 uM. (<b>E</b>). Axonal D3 activity is increased by T3-treatment in the median eminence of male Wistar rats. Mean±SEM (n = 9) *P<0.05 by t-test.</p

Ultrastructure of D3 immunoreactive elements in the rat mediobasal hypothalamus.

<p>(<b>A</b>) D3 immunoreactivity, identified by silver grain deposits, appear primarily in axon varicosities containing dense core vesicles of 80–120 nm diameter in the upper external zone of the median eminence, characteristic of axons of parvocellular (PC) neurons. No or a few silver grains could be observed in association with organelles of magnocellular neurons (MC) or tanycytes (Tc), respectively. (<b>B</b>) D3-positive axons exhibiting various degrees of labeling are mixed with non-labeled fibers (asterisks) in the external zone of the median eminence. (<b>C</b>) Although silver grains occasionally appear in association with the plasma membrane (black arrowheads) and with small clear vesicles (white arrowheads) of the axon varicosities, the majority are not labeled (asterisks). (<b>D</b>) In contrast, the dense core vesicles accumulate most the reaction product (black arrowheads), as visible at high power magnification in the vicinity of the capillaries of the external zone of the median eminence. Unlabeled small clear vesicles are indicated with asterisk. Tc, tanycyte; Scale bars: 1 µm in A–B, 250 nm in C, 100 nm in inset on C, 500 nm in D.</p

MCT8 immunoreactivity in the rodent mediobasal hypothalamus.

<p>Low magnification photograph illustrates the presence of MCT8-immunoreactivity associated with tanycytes (<b>A</b>). In the median eminence, strong MCT8-immunoreactivity is observed in tanycyte processes (<b>B, arrows</b>). In addition to occurring in tanycyte processes (arrows), MCT8-immunoreactivity is also observed in small dot like structures reminiscent of axon varicosities (arrow heads) (<b>C</b>). MCT8 immunoreactivity in the mediobasal hypothalamus of wild-type (<b>D</b>) and MCT8-KO mice (<b>E</b>). III, third ventricle; Scale bars: 200 µm in A, 100 µm in B, 20 µm in C, 50 µm in D,E.</p

Dual-immunofluorescence images illustrate the overlapping distribution of fibers immunoreactive for D3 (green fluorochrome) and GnRH (A) or TRH (D) (red color) in the median eminence.

<p>Sites of overlap (yellow color) occur along the axonal pathway in the median eminence (<b>B,E</b>). High power confocal images demonstrate dual-labeled axon varicosities (yellow color in composite images) immunoreactive for D3 and GnRH (<b>C</b>) or D3 and TRH (<b>F</b>). The D3 immunoreactivity appears as yellow patches (arrowheads) within the axon varicosities. Single channels are also shown in <b>C’, C”</b> and <b>F’, F”</b>, respectively. High power dual-immunofluorescent images are also shown for fibers labeled for D3 (green fluorochrome) and CRH (<b>G</b>), GHRH (<b>H</b>) or somatostatin (<b>I</b>) (red color). These D3-immunoreactive sites (arrowheads) correspond to axon varicosities (<b>G</b>, <b>H</b>). Somatostatin (SS)- immunoreactive axon varicosities show virtually no signal for D3. Scale bars: 20 µm in A, 5 µm in B, 5 µm in C, 10 µm in D,E, 5 µm in F–I.</p

Quantification of D3 distribution in parvocellular axon terminals in the rat median eminence.

<p>(n = 3; *P<0.01 TRH <i>vs.</i> GnRH, GHRH, CRH and somatostatin (SST) by ANOVA followed by Newman-Keuls post-test.)</p

Diameter of immunoreactive clusters in the outer zone of the rat hypothalamic median eminence stained for D3, GnRH or Rab3 detected by N-STORM superresolution microscopy.

<p>**P<0.001; *P<0.05 by ANOVA followed by Newman-Keuls post test. Data are shown as Mean ± SEM (N = 500).</p

D3 detection by Western Blot in the rat and human hypothalamus.

<p>Note that the human D3 protein runs slightly faster compared to rat D3, in agreement with the calculated ∼3 kDa size difference between the two proteins.</p