The Roles of BDNF, pCREB and Wnt3a in the Latent Period Preceding Activation of Progenitor Cell Mitosis in The Adult Dentate Gyrus by Fluoxetine

The formation of new neurons continues into adult life in the dentate gyrus of the rat hippocampus, as in many other species. Neurogenesis itself turns out to be highly labile, and is regulated by a number of factors. One of these is the serotoninergic system: treatment with drugs (such as the SSRI fluoxetine) markedly stimulates mitosis in the progenitor cells of the dentate gyrus. But this process has one remarkable feature: it takes at least 14 days of continuous treatment to be effective. This is despite the fact that the pharmacological action of fluoxetine occurs within an hour or so of first administration. This paper explores the role of BDNF in this process, using the effect of a Trk antagonist (K252a) on the labelling of progenitor cells with the mitosis marker Ki67 and the associated expression of pCREB and Wnt3a. These experiments show that (i) Fluoxetine increased Ki67 counts, as well as pCREB and Wnt3a expression in the dentate gyrus. The action of fluoxetine on the progenitor cells and on pCREB (but not Wnt3a) depends upon Trk receptor activation, since it was prevented by icv infusion of K252a. (ii) These receptors are required for both the first 7 days of fluoxetine action, during which no apparent change in progenitor mitosis occurs, as well as the second 7 days. Increased pCREB was always associated with progenitor cell mitosis, but Wnt3a expression may be necessary but not sufficient for increased progenitor cell proliferation. These results shed new light on the action of fluoxetine on neurogenesis in the adult dentate gyrus, and have both clinical and experimental interest

DLK1/PREF1 regulates nutrient metabolism and protects from steatosis.

Nonalcoholic fatty liver disease (NAFLD) is associated with insulin resistance and obesity, as well as progressive liver dysfunction. Recent animal studies have underscored the importance of hepatic growth hormone (GH) signaling in the development of NAFLD. The imprinted Delta-like homolog 1 (Dlk1)/preadipocyte factor 1 (Pref1) gene encodes a complex protein producing both circulating and membrane-tethered isoforms whose expression dosage is functionally important because even modest elevation during embryogenesis causes lethality. DLK1 is up-regulated during embryogenesis, during suckling, and in the mother during pregnancy. We investigated the normal role for elevated DLK1 dosage by overexpressing Dlk1 from endogenous control elements. This increased DLK1 dosage caused improved glucose tolerance with no primary defect in adipose tissue expansion even under extreme metabolic stress. Rather, Dlk1 overexpression caused reduced fat stores, pituitary insulin-like growth factor 1 (IGF1) resistance, and a defect in feedback regulation of GH. Increased circulatory GH culminated in a switch in whole body fuel metabolism and a reduction in hepatic steatosis. We propose that the function of DLK1 is to shift the metabolic mode of the organism toward peripheral lipid oxidation and away from lipid storage, thus mediating important physiological adaptations associated with early life and with implications for metabolic disease resistance.This is the final published version. It is available online from PNAS here: http://www.pnas.org/content/111/45/16088.abstract

Novel control by the CA3 region of the hippocampus on neurogenesis in the dentate gyrus of the adult rat.

The dentate gyrus is a site of continued neurogenesis in the adult brain. The CA3 region of the hippocampus is the major projection area from the dentate gyrus. CA3 sends reciprocal projections back to the dentate gyrus. Does this imply that CA3 exerts some control over neurogenesis? We studied the effects of lesions of CA3 on neurogenesis in the dentate gyrus, and on the ability of fluoxetine to stimulate mitotic activity in the progenitor cells. Unilateral ibotenic-acid generated lesions were made in CA3. Four days later there was no change on the number of either BrdU or Ki67-positive progenitor cells in the dentate gyrus. However, after 15 or 28 days, there was a marked reduction in surviving BrdU-labelled cells on the lesioned side (but no change in Ki-67+ cells). pCREB or Wnt3a did not co-localise with Ki-67 but with NeuN, a marker of mature neurons. Lesions had no effect on the basal expression of either pCREB or Wnt3a. Subcutaneous fluoxetine (10 mg/kg/day) for 14 days increased the number of Ki67+ cells as expected on the control (non-lesioned) side but not on that with a CA3 lesion. Nevertheless, the expected increase in BDNF, pCREB and Wnt3a still occurred on the lesioned side following fluoxetine treatment. Fluoxetine has been reported to decrease the number of "mature" calbindin-positive cells in the dentate gyrus; we found this still occurred on the side of a CA3 lesion. We then showed that the expression GAP-43 was reduced in the dentate gyrus on the lesioned side, confirming the existence of a synaptic connection between CA3 and the dentate gyrus. These results show that CA3 has a hitherto unsuspected role in regulating neurogenesis in the dentate gyrus of the adult rat

Mean Optical Density (pixels) for pCREB or Wnt3a after either saline or ibotenic acid lesions for 14 days (Experiment 2) or 28 days (Experiment 3) and following treatment with subcutaneous saline or fluoxetine-containing pumps for 14 (Experiment 4), 3 animals/group and 3 sections/animal.

<p>Mean Optical Density (pixels) for pCREB or Wnt3a after either saline or ibotenic acid lesions for 14 days (Experiment 2) or 28 days (Experiment 3) and following treatment with subcutaneous saline or fluoxetine-containing pumps for 14 (Experiment 4), 3 animals/group and 3 sections/animal.</p

The effect of CA3 lesions on (A) the survival of BrdU cells and (B) number of Ki-67+ progenitor cells in the dentate gyrus after 14 days treatment with either fluoxetine (10 mg/kg/day) or saline subcutaneously (osmotic minipump).

<p>Values are means ± SEM., **p<0.01 compared to control. (C) Immunofluorescent staining of BDNF after the two treatments. Values are mean optical density of BDNF ±SEM. **p<0.001. Bar represents a distance of 50 µm.</p

The effect of CA3 lesion on (A) the newly formed neurons staining for BrdU and (B) the number of Ki-67-positive progenitor cells in the dentate gyrus.

<p>Mean number per section after 28 days, compared to controls (saline infusion). Values are means ± SEM. Values are mean ±SEM. **p<0.01, p:ns compared to control. (C) double-stained cells with BrdU and NeuN. Bar represents a distance of 200 µm and 100 µm.</p

(A) Cresyl violet staining of the CA3 lesion following ibotenic acid infusions.

<p>(B) The effect of CA3 lesion on the newly formed neurons BrdU and the mitosis rates Ki-67 (C) of progenitor cells in the dentate gyrus. Mean number per section after 14 days, compared to controls (saline infusion). Values are mean ±SEM. <i>**</i>p<0.01, compared to control. (D) Immunofluorescent Co-localisation of NeuN stained in green, pCREB (left) and Wnt3a stained in red (right) in saline treated animals. Double labelling for NeuN/pCREB and NeuN/Wnt3a appears orange, Ki67 staining is green. Bar represents a distance of 100 µm and 50 µm.</p

Effect of treatment with K252a icv for either the first or second 7 days of a 14 day fluoxetine (10 mg/kg/day) treatment.

<p>A: Ki67-labelled cell counts; B: Wnt3a expression in the dentate gyrus. Values as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013652#pone-0013652-g001" target="_blank">Figure 1</a>. Bar represents 100 µm.</p

Summary of the effects of fluoxetine (flx), and K252a on the expression of Ki67, pCREB and Wnt3a in the dentate gyrus of the adult male rat.

<p>0 =  no increase compared to control; 1 =  increased compared to controls.</p

The effect of either 7 or 14 days treatment with fluoxetine (10 mg/kg/day).

<p>A: Ki67 cell counts, B: BDNF mRNA expression, C: pCREB D: Wnt3a. E,F: <i>in situ hybridization</i> images of BDNF mRNA. Values as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013652#pone-0013652-g001" target="_blank">Figure 1</a>. Bar represents 100 µm. Values are mean ±SEM.**<i>p</i><0.001 compared to control. Representative sections are shown from fluoxetine-treated animals only, since there is virtually no expression in controls.</p