Rab18在哺乳時期母鼠的成年神經元新生中所扮演的角色

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(1)國立臺灣師範大學生命科學系碩士論文. Rab18 在哺乳時期母鼠的成年神經元新生中所扮演的角色 The Role of Rab18 in Adult Neurogenesis of Lactating Female Mice. 研究生：周雅文 Ya-Wen Chou 指導教授：王慈蔚博士 Dr. Tsu-Wei Wang. 中華民國 104 年八月.

(2) Content. Chinese abstract ........................................................................... 2 English abstract ............................................................................ 4 Introduction .................................................................................. 6 Materials and methods ............................................................... 12 Results ........................................................................................ 17 Discussion .................................................................................. 30 Figures ........................................................................................ 35 References .................................................................................. 54. 1.

(3) 摘要. 在成年哺乳類大腦中，神經元新生持續發生於嗅球與海馬迴的齒狀迴。嗅球神經元新生的功能在於氣味的辨認與產生育幼行為；而齒狀迴神經元新生的功能則是空間學習以及抗憂鬱。Rab18 是 Ras 相關的小 GTP 水解酶 Rab 家族中的一員。有趣的是，Rab18 基因剃除母鼠的育幼行為是受損的，而成年神經元新生被阻斷的母鼠也無法執行育幼行為，讓我們懷疑 Rab18 與成年神經元新生有關。文獻指出在神經內分泌細胞中，Rab18 負調節多巴胺的分泌；而多巴胺已知會抑制成年神經元新生及泌乳激素釋放。根據這些發現，我們假設在哺乳時期， Rab18 會透過負調節多巴胺的釋放，使泌乳激素上升，進而調節成年神經元新生，使母鼠能夠執行育幼行為。我們發現在哺乳時期的 Rab18 缺失的母鼠成年神經元新生降低，並且在兩個神經幹細胞存在的微環境，即側腦室下區和齒狀迴顆粒細胞下區中，神經幹細胞及神經母細胞的密度和增生也比野生型母鼠低。此外，我們也發現在產後第一天， Rab18 剃除母鼠血清中的泌乳激素濃度較低，而中腦的多巴胺則較高。以上研究顯示在哺乳時期，Rab18 可能會透過負調節多巴胺的分泌，使得由泌乳激素居中調節的成年神經元新生上升。然而，當對 Rab18 剃除母鼠皮下注射泌乳激素後，儘管牠們的成年神經元新 2.

(4) 生回復到和野生型相當的程度，牠們的育幼行為仍然是失常的，顯示由 Rab18 在哺乳時期透過泌乳激素所調節的成年神經元新生並非誘發育幼行為的唯一因素。. 關鍵字：Rab18、成年神經元新生、多巴胺、哺乳時期、育幼行為、泌乳激素. 3.

(5) Abstract. In the adult mammalian brain, neurogenesis continuously occurs in the olfactory bulb (OB) and the dentate gyrus (DG) of the hippocampus. The function of adult neurogenesis in the OB and DG is odor discrimination/maternal behaviors and spatial learning/anti-depression, respectively. Rab18 is a member of Rab proteins belongings to Ras-related small GTPase superfamily. Interestingly, Rab18 null mice have impaired maternal behaviors, which are also observed in female mice when their adult neurogenesis is blocked. A previous study shows that Rab18 negatively regulates dopamine secretion in neuroendocrine cells and dopamine is shown to decrease adult neurogenesis and prolactin. Based on these findings, we hypothesize that Rab18 governs prolactin level during lactation by negatively regulating dopamine secretory pathway, which in turn regulates adult neurogenesis and maternal behaviors. We found that Rab18-/- female mice had impaired adult neurogenesis postpartum. Progenitor cells and proliferating cells in the neural stem cell niches of OB and DG, the subventricular zone (SVZ) and the subgranular zone (SGZ), in Rab18-/- female mice were decreased postpartum. Compared to control female mice, lower serum prolactin level and higher midbrain dopamine level in Rab18-/- female mice on postpartum day 1 (PD1) were also found. Moreover, exogenous prolactin rescued adult neurogenesis, but not maternal behavior defects in postpartum Rab18-/- female mice. Our results suggest that prolactin. 4.

(6) mediates Rab18-modulated adult neurogenesis, which alone is not sufficient to induce maternal behaviors postpartum.. Keywords: Rab18, adult neurogenesis, dopamine, lactation, maternal behaviors, prolactin. 5.

(7) Introduction Adult neurogenesis Neurogenesis occurs in the olfactory bulb (OB) and dentate dyrus (DG) throughout adult life in the mammalian brain. Many newborn neurons are produced by neural stem cells in the subventricular zone (SVZ), a layer of cells lining the lateral ventricle (LV), and the subgranular zone (SGZ), the innermost cell layer of DG, in the adult mammal brain. Adult neurogenesis is regulated by physiological and pathological conditions, such as pregnancy, exercise and brain injuries [1,2]. Many teams have attempted to study the contribution of adult generated neurons by altering the level of adult neurogenesis and their data reveal that impairments of adult neurogenesis results in abnormality in behaviors, such as olfactory and cognitive defects [3-13].. The SVZ-OB pathway Rodents highly rely on olfaction to survive. The olfactory system includes the main olfactory system (MOS) and the accessory olfactory system (AOS) [11,14], which are responsible for the detection of odors and pheromones, respectively. For odor detection, odorant molecules are detected by olfactory sensory neurons (OSNs) in the olfactory epithelium (MOE). These OSNs relay odorant signals to mitral and tufted cells in the main olfactory bulb (MOB), which further send these signals to the olfactory cortex (OC) for odor perception. For pheromone detection, OSNs in the vomeronasal organ (VNO) detect pheromones and send signals to the accessory olfactory bulb (AOB), which further transmit 6.

(8) signals to the amygdala, hypothalamus to initiate innate behaviors. The AOB and MOB are the destination of newborn neurons derived from neural stem cells in the SVZ, suggesting that newborn neurons may take part in olfaction-related functions. The steps and functions of adult neurogenesis in the SVZ-OB pathway have been investigated. In the SVZ, neural stem cells proliferate slowly and produce transient amplifying cells, which further give rise to neuroblasts. Neuroblasts migrate to the OB along the rostral migratory stream (RMS) and differentiate into neurons. The overwhelming majority of these neurons are GABAergic interneurons in granule cell layer (GCL) and periglomerular layer (PGL) [1,2]. Newborn neurons in the OB form dendrodendritic synapses on mitral and tufted cells and top-down projections from the OC [14]. To study the role of newborn neurons in the OB, adult neurogenesis in the OB is blocked by local SVZ irradiation [6,11], by treatment of antimitotic drugs [6,8,15] or by genetic ablation [4,9,13] and find that adult OB neurogenesis is required for olfactory discrimination, olfaction-dependent learning and initiation of innate behaviors, including maternal behaviors.. The SGZ-DG pathway The steps and functions of SGZ-DG pathway adult neurogenesis have been investigated. In the SGZ, quiescent neural stem cells produce progenitor cells, which give rise to neuroblasts. Neuroblasts differentiate into neurons, which extend their dendrites to the molecular layer of the DG and project their axons to the CA3 region of the hippocampus to be 7.

(9) incorporated into the hippocampal circuit. Newborn neurons in the DG have higher structural plasticity than preexisting neurons. During critical period, newborn neurons receive functional inputs and compete with preexisting ones for their survival [1,2]. Previous studies have shown that voluntary wheel running and enriched environments rescue the age-dependent decline of adult neurogenesis, which may further improve the performances of paradigms related to cognition and spatial learning [16]. Moreover, antidepressants alleviate emotional illness by promoting the survival of newborn DG neurons [17]. By using genetic ablation, adult DG neurogenesis has been found that it buffers stress responses and depression [10]. Taken together, adult hippocampal neurogenesis is essential for cognition, spatial learning and modulation of mood.. Maternal behaviors Maternal behaviors including nursing and protecting newborns play a key role in the reproduction in almost all mammals. During pregnancy, neurological transformations in the central and peripheral nervous systems of female mammals occur to prepare for lactation and decreasing aversion to infants. Previously, increased adult neurogenesis in the OB on gestation day 7 (GD7) and postpartum day 7 (PD7) are found. In that research, they also find that prolactin mediates pregnancy-induced adult neurogenesis [18]. Prolactin is a hormone produced and released by lactotroph cells in the anterior pituitary and it is elevated during pregnancy and lactation [19,20]. Secretion of prolactin ensures implantation of embryos by maintaining the corpus luteum [20]. 8.

(10) Interestingly, infusion of prolactin promotes maternal behaviors, whereas injection of a dopamine D2 receptor agonist bromocriptine, which inhibits release of endogenous prolactin, delays maternal behaviors in adult female rats [21], suggesting that dopamine-governed prolactin release plays a key role in maternal behaviors. Moreover, male pheromones initiate prolactin-induced neurogenesis, which advances maternal behaviors in adult female mice [22]. During early pregnancy, prolactin-induced adult OB neurogenesis is required for maternal behaviors and has anxiolytic effects [6,20]. Taken together, these studies reveal that prolactin-induced adult OB neurogenesis is required for maternal behaviors.. Rab proteins and Rab18 Rab proteins belong to Ras-related superfamily of small GTPase and are known to take part in many cellular processes, such as endocytosis, exocytosis, intracellular signaling, differentiation and development [23,24]. Rab proteins cycle between inactive GDP-bound form and active GTP-bound form. Rab guanine nucleotide exchange factors (Rab GEFs), Rab GTPase activating proteins (Rab GAPs) and Rab Guanosine dissociation inhibitors (Rab GDIs) regulate the activity of Rab protein at the right time and the right place. In human, mutations of RAB genes, its upstream regulators or downstream effectors have been found in different types of cancer, endocrine diseases and genetic disorders [24], suggesting that RAB proteins have diverse functions.. 9.

(11) Rab18, a member of Rab proteins, has been reported to participate in lipolysis and lipogenesis [23], be required for the maintenance of the structure of endoplasmic reticulum (ER) [25], play a key role in eye development [26,27] and regulate Ca2+-mediated exocytosis of dopamine in neuroendocrine cell lines [24,28]. Moreover, overexpression of Rab18 inhibits vesicle trafficking in PC12 cells and pituitary AtT 20 cells [24,28]. Taken together, Rab18 has distinct roles in different cell types. In the recent study by Bem et al., loss-of-function mutations of RAB18 result in Warberg Micro Syndrome (WARMS), a severe recessive genetic disorder. The clinical features in WARMS are characterized as microcephaly, brachycephaly, microphthalmia, microcornea, low anterior hairline, large protruding pinnae and downturned mouth corners, progressive optic nerve atrophy, severe spastic quadriplegia with contractures and universal developmental retardation [23]. With the collaboration with C-J Hong laboratory, they find a null mouse line with deleted promoter, axon 1 and partial intron 1 in Rab18 from ENU-mutagenized screening [27]. Rab18 null mice develop neurological features that are associated with WARMS, such as progressive hind limb spasticity and smaller heads. Interestingly, Rab18 null female mice have impaired maternal behaviors, such as neonaticide and infanaticide, which can be rescued by expressing Rab18 under the control of ubiquitously expressing polymerase II promoter (C-J Hong, personal communication). Since adult neurogenesis is required for maternal behaviors, it is possible that Rab18 regulates maternal behaviors by mediating adult neurogenesis.. 10.

(12) Previously, we demonstrate that Rab18-/- virgin female mice have lower adult neurogenesis in the OB and DG (unpublished data). Since Rab18 negatively regulates the secretion of dopamine in neuroendocrine cell lines [28], it is possible that Rab18 also regulates dopamine level in vivo. Indeed, we also find that dopamine is higher in the midbrain of Rab18-/- virgin mice (data not shown). Dopamine inhibits the production and release of prolactin [19-21] and dopamine also inhibits cell proliferation in the SVZ [29,30] and SGZ [30,31]. Based on these findings, we hypothesize that Rab18 governs prolactin level during lactation by negatively regulating dopamine secretion, which in turn regulates adult neurogenesis and maternal behaviors. In the present study, we found that Rab18-/- female mice had impaired adult neurogenesis postpartum. Progenitor cells and proliferating cells in the SVZ and SGZ of Rab18-/- female mice were also decreased. Compared to control female mice, lower serum prolactin level and higher midbrain dopamine level in Rab18-/- female mice on PD1 were also found. Adult neurogenesis of lactating Rab18-/- mice was increased by exogenous prolactin treatments. However, after prolactin treatments, Rab18-/- mice still had impaired maternal behaviors. Taken together, these data suggest that prolactin mediates Rab18-modulated adult neurogenesis, which is not sufficient to induce maternal behaviors.. 11.

(13) Materials and methods. Experimental animals Rab18+/- mice were kindly provided by Dr. C-J Hong laboratory, National Yang-Ming University [27] and were bred in the animal facility at Department of Life Science, National Taiwan Normal University. These mice were from the C57BL6 background. Mice were housed in the IVC room (bioLASCO, Taiwan Co.) under a 12-h light, 12-h dark cycle (lights on at 7:00 AM), with ad libitum access to food and water. Mice were weaned at 4-week-old, and genotyped by PCR. 10-week-old WT and Rab18-/- female mice were paired with adult WT male mice and were considered pregnant by checking the vaginal plug half day later.. BrdU administration WT and Rab18-/- female mice received intraperitoneal injections of 5-bromo-2-deoxyuridine (BrdU; 100 mg/kg body weight in saline; SIGMA-ALDRICH Co.) on PD7 twice, six hours apart. All mice were sacrificed two weeks later.. Perfusion and tissue preparation Mice were deeply anaesthetized by an intraperitoneal injection with Avertin solution (0.025 g of 2,2,2-tribromomethylalcohol and 0.025 ml of 2-methyl-2-butanol in 0.975 ml water; 17 ml/kg body weight) and perfused transcardially with 0.9% saline, followed by 4% paraformaldehyde (PFA) in 1× phosphate buffer saline (PBS), pH=7.4. 12.

(14) Brains were removed from the skull, post-fixed overnight in 4% PFA and then dehydrated in 20% sucrose in 1× PBS with 0.02% sodium azide for 24 hours at 4°C. Dehydrated brains were frozen by dry ice and stored in the -80°C refrigerator. Frozen brains were cut coronally with a microtome (Leica SM 2010R). Serial sections (40 μm thick) were collected in multi-well plate (Becton Dickinson Labware, USA). Sections were stored at 4°C in 1× PBS with 0.02% sodium azide before used.. Immunofluorescence To examine adult neurogenesis, BrdU and NeuN double labeling was performed. Six serial OB or DG sections (160 μm apart) were selected and washed with 1× Tris-buffered saline (TBS) for three times. Sections were then denatured with 2N hydrochloride for 30 minutes at 37°C and neutralized with 0.1M sodium borate for 10 minutes at room temperature. After rinsed in 1× TBS for three times, tissues were incubated in goat serum-containing blocking buffer at room temperature for at least one hour. Then, tissues were incubated in rat anti-BrdU (1:500; ACCURATE CHEMICAL & SCIENTEFIC Co.) and mouse anti-NeuN (1:1000; Millipore Co.) for 16-18 hours at 4°C. After washed in 1× TBS for three times, sections were incubated in goat anti-rat 488 (1:500; Abcam Co.) 16 and goat anti-mouse 550 (1:500; Abcam Co.) secondary antibodies for two hours at room temperatures. Finally, they were rinsed, mounted on slide, and sealed with anti-fade reagent (Invitrogen) and stored at 4°C. To examine cell proliferation, progenitor cells (SVZ and SGZ) and dopaminergic neurons (OB and SN), Six serial sections were labeled with, 13.

(15) rabbit anti-Ki67 (1:250; Novocastra Laboratones Ltd, UK), rabbit anti-Sox2 (1:1000; Millipore Co.), guinea pig anti-DCX (1:5000; Millipore Co.) or rabbit anti-TH (1:1000; Millipore Co.) primary antibody. Immunofluorescence protocol was similar to the above without denaturing and neutralization steps. After incubated in primary antibodies, sections were washed again and incubated in secondary antibodies, including goat anti-rabbit 488 and donkey anti-guinea pig 549 (1:500; Jackson Immuno-Research Laboratory, USA). Finally, they were incubated in 1× PBS with 4, 6-diamidino-2-phenylindole (DAPI; 1:10000 in 1× PBS) for 30 minutes, followed by being washed, coverslipped and stored at 4°C.. Enzyme-linked immunosorbent assay (ELISA) Blood samples from female mice were collected on PD1, PD4, and PD7. Besides, blood samples from virgin female mice were collected to examine whether serum prolactin concentration increases postpartum. All blood samples stood for at least one hour for blood coagulation and were centrifuged at 4000 rpm for 15 minutes. Plasma was obtained and stored at -80°C until use. To measure the serum prolactin concentrations, DuoSet ELISA Development Kit (R&D system) was used according to the manufacturer’s instructions. All of our results have been normalized to serum total protein levels. Serum total protein concentrations were measured by Bio-Rad Protein Assay (BIO-RAD Laboratories) according to the manufacturer’s instructions.. 14.

(16) High-performance liquid chromatography (HPLC) Mice were sacrificed and the OB and the midbrain were isolated. Tissues were homogenized and incubated in 0.1N ice-cold perchloric acid (3-30 μl/mg of tissue) for dialyzing neurotransmitter monoamines and metabolites. 20 minutes later, samples were centrifuged at 10000 rpm for 10 minutes at 4°C. Supernatants were collected and stored at -80°C until use. To detect dopamine in the OB and midbrain, HPLC was used. Our system was HPLC-EGC system, which contained a C-18 HPLC column (5μm, 4.6 × 250 mm, Alltima), a model 1100 series pump (Agilent) and an electrochemical detector. The composition of mobile phase was 2.1 g/L Heptane-1-sulfonic acid, 0.1 g/L EDTA, 3 ml/L pro analysi, 3.5 ml/L Triethylamine, 170 ml/L acetonitrile and distilled water. The pH of mobile phase was adjusted to 2.7.. Confocal microscopy and cell counting All brain sections were photographed by a confocal microscope (Leica TCS SP2 Confocal Spectral Microscope Imaging System). The number of cells counted per experiment was performed in 2-μm-thick confocal sections. All scale bars of these photos were 40 μm excluding that of photos from substantia nigra (SN) (160 μm).. Exogenous prolactin administration To increase prolactin early postpartum, female mice received subcutaneous injections of mouse recombinant prolactin (0.8 μg/mL in 0.9% saline, 100 μL/day; R&D system) each day from gestation day 18 15.

(17) (GD18) to PD2.. Pup retrieval test and pup survival rate To test whether female mice have maternal behaviors, pup retrieval test was performed. Our protocols were according to the previous study with modifications [9]. Female mice were separated from their home cages for 15 minutes. Before female mice were re-introduced, their pups were scattered. To exclude the number effect, 4 pups were used in each group. Latencies to retrieve all pups within 5 minutes were recorded. If female mice do not retrieve all pups, the results are indicated as 300 seconds. Proportions of survival pups after PD1 were quantified as previously described [9]. Proportions of survival pups were calculated by pup number on PD1/pup number on PD0 × 100%. Proportions of survival pups on PD21 were calculated by pup number on PD21/pup number on PD0 × 100%.. Statistical analysis Comparison between two groups was analyzed by two-tailed Student’s t-test. Multiple comparisons were analyzed by one-way or two-way ANOVA followed by Tukey’s post-hoc analysis. Comparison of latency to retrieve all pups between genotypes and treatments was analyzed by nonparametric method followed by Mann-Whitney U test. Statistical significance was indicated as *: p<0.05, **: p<0.01.. 16.

(18) Results Rab18 is required for adult neurogenesis in the AOB, MOB, and DG in lactating mice Previously, we found that Rab18-/- virgin female mice had impaired adult neurogenesis (data not shown). Given that adult neurogenesis is required for maternal behaviors [9] and it is increased on PD7 [18], it is important to study whether adult neurogenesis in Rab18-/- mice is also impaired at this time. To study the role of Rab18 in lactating female mice, mice were injected with BrdU, a thymidine analog, to label newborn cells in Rab18-/female mice and their WT littermates on PD7 (Fig. 1A). Since a newborn cell takes about two weeks to migrate to the OB and differentiate into a mature neuron, mice were sacrificed two weeks after BrdU injections. We found that newborn cells in Rab18 null mice were decreased in the MOB (WT: 6.0 ± 0.5 × 104 cells/mm3; Rab18-/-: 4.2 ± 0.3 × 104 cells/mm3; p=0.0201; Fig. 1G, H and I) and a trend decreased in the DG (WT: 14.4 ± 1.8 × 103 cells/mm3; Rab18-/-: 8.6 ± 2.3 × 103 cells/mm3; p= 0.0589; Fig. 1L, M and N), but not in the AOB (WT: 3.8 ± 0.8 × 104 cells/mm3; Rab18-/-: 2.3 ± 0.7 × 104 cells/mm3, p=0.10; Fig. 1B, C and D). Newborn neurons in the AOB (WT: 2.7 ± 0.4 × 104 cells/mm3; Rab18-/-: 1.6 ± 0.3 × 104 cells/mm3; p=0.0271; Fig. 1B, C and E), MOB (WT: 4.7 ± 0.5 × 104 cells/mm3; Rab18-/-: 2.9 ± 0.3 × 104 cells/mm3; p=0.0134; Fig. 1G, H and J) and DG (WT: 10.8 ± 1.3 × 103 cells/mm3; Rab18-/-: 6.0 ± 1.7 × 103 cells/mm3; p=0.0362; Fig. 1L, M and O) were also decreased in Rab18-/female mice. There was no difference in neuronal differentiation between Rab18-/- mice and WT littermates in the AOB (WT: 76.6 ± 7.0 %; 17.

(19) Rab18-/-: 71.2 ± 2.8 % ; p=0.46; Fig. 1B, C and F), MOB (WT: 78.8 ± 1.8 %; Rab18-/-: 69.9 ± 5.0 %; p=0.11; Fig. 1G, H and K) and DG (WT: 76.1 ± 4.8 %; Rab18-/-: 67.0 ± 3.9 %; p=0.14; Fig. 1L, M and P). These results suggest that Rab18 is required for OB and DG adult neurogenesis in lactating mice.. Rab18 is necessary for cell proliferation in the SVZ and SGZ in lactating mice Rab18-/- female mice have impaired adult neurogenesis postpartum, which may be due to decreased cell proliferation in two neural stem cell niches, the SVZ and SGZ. To test this possibility, we labeled proliferating cells with Ki67 antibody, a cell cycle marker, in Rab18-/- female mice on PD21. We found that proliferating cell number was deceased in the SVZ (WT: 16.5 ± 1.4 × 104 cells/mm3; Rab18-/-: 10.7 ± 1.1 × 104 cells/mm3; p=0.0083; Fig. 2A-C) and SGZ (WT: 2.2 ± 0.2 × 104 cells/mm3; Rab18-/-: 1.6 ± 0.2 × 104 cells/mm3; p=0.0469; Fig. 2D-F). These results suggest Rab18 is necessary for cell proliferation in the SVZ and SGZ in lactating mice.. Rab18 is essential for the maintenance of progenitor cells in the SVZ and SGZ in lactating mice Our data show that adult neurogenesis and cell proliferation are decreased in Rab18-/- mice postpartum. Reduced adult neurogenesis and cell proliferation may be resulted from fewer progenitor cells, such as neural stem cells and neuroblasts. To study this, we labeled neural stem cells 18.

(20) with Sox2 antibody. Our data revealed that there were fewer neural stem cells in the SVZ (WT: 4.0 ± 0.2 × 105 cells/mm3; Rab18-/-: 3.0 ± 0.2 × 105 cells/mm3; p=0.0055; Fig. 3A-C) and SGZ (WT: 11.5 ± 0.8 × 104 cells/mm3; Rab18-/-: 7.6 ± 0.8 × 104 cells/mm3; p=0.0063; Fig. 3D-F) in Rab18-/- female mice on PD21. Our results suggest that Rab18 plays a key role in the maintenance of neural stem cells in the SVZ and SGZ in lactating mice. Neuroblasts are also progenitor cells in the SVZ and the SGZ. Next we labeled neuroblasts with Doublecortin antibody in Rab18-/- female postpartum. Quantification of neuroblasts revealed that there were fewer neuroblasts in the SVZ (WT: 38.1 ± 5.0 × 104 cells/mm3; Rab18-/-: 21.1 ± 3.1 × 104 cells/mm3; p=0.0157; Fig. 4A-C) and SGZ (WT: 9.9 ± 0.5 × 104 cells/mm3; Rab18-/-: 7.4 ± 0.6 × 104 cells/mm3; p=0.0087; Fig. 4D-F) in Rab18-/- female mice. These results indicate that Rab18 is essential for the production of neuroblasts in the SVZ and SGZ in lactating mice. Taken together, Rab18 is required for the maintenance of progenitor cells in the SVZ and SGZ in lactating mice.. Rab18 positively and negatively regulate serum prolactin concentration and midbrain dopamine concentration postpartum, respectively Rab18 is required for adult neurogenesis in the OB and DG, cell proliferation and production of progenitor cells in the SVZ and SGZ in lactating mice. However, the mechanism is still unclear. Previously, Larsen and her colleague find that prolactin is increased during early 19.

(21) pregnancy and mediates pregnancy-induced adult OB neurogenesis and is required for maternal behaviors [6,18]. Serum prolactin concentration is also increased postpartum when female mice lactate [20], and infusion of prolactin promotes maternal behaviors in female rat [21]. It is possible that Rab18 regulates serum prolactin concentration in postpartum female mice. Moreover, dopamine inhibits the release and the mRNA expression of prolactin [19] and is negatively regulated by Rab18 in a neuroendocrine cell line [28]. Previously, we found that Rab18-/- virgin female mice had higher dopamine concentration in the midbrain (data not shown). Based on these findings, we hypothesize that Rab18 may positively regulate prolactin by decreasing dopamine. To investigate this, we measured serum prolactin concentration in WT and Rab18-/- female mice by ELISA on PD1, PD4 and PD7 and age-matched virgin female mice (Fig. 5A). Compared to aged-match virgin female mice, there was a significant increase in serum prolactin concentration on PD1 (WT Virgin: 2.7 ± 0.8 ng/mg, PD1: 6.4 ± 0.9 ng/mg; p=0.0390; Fig. 5B), but not in PD4 (PD4: 3.4 ± 0.5 ng/mg; p=1; Fig. 5B) and PD7 WT mice (PD7: 2.1 ± 0.6 ng/mg, p=1; Fig. 5B). WT female mice had significantly lower serum prolactin concentration on PD4 and PD7 than that of PD1 (PD1-PD4: p=0.0350; PD1-PD7: p=0.0020; Fig. 5B), but there was no difference between PD4 and PD7 (p=0.81, Fig. 5B). We also found that Rab18-/mice had significantly lower serum prolactin concentration on PD1 than that of WT littermates (Rab18-/-; PD1: 2.8 ± 0.9 ng/mg; p=0.0010; Fig. 5B), but there was no difference in serum prolactin level of Rab18-/female mice among PD1, PD4 and PD7 (Rab18-/-; PD4: 2.3 ± 0.8 ng/mg; 20.

(22) PD7: 3.6 ± 0.8 ng/mg; PD1-PD4: p=0.94; PD1-PD7: p=0.83; Fig. 5B). Our results suggest that Rab18 is required for the increase of serum prolactin level on PD1 in lactating mice. Since we found that Rab18-/- female mice had lower serum prolactin concentration on PD1, it suggests that Rab18-/- female mice may have higher dopamine level on PD1. To test this possibility, we measured the dopamine concentration in the OB and the midbrain, which are two areas with dopaminergic neurons, in WT and Rab18-/- female mice on PD1 by HPLC (Fig. 6A). We found that there was no difference in dopamine concentration in the OB between WT and Rab18-/- female mice (WT= 0.7 ± 0.2 pmol/mg; Rab18-/-: 1.1 ± 0.4 pmol/mg; p=0.28; Fig. 6B). Interestingly, there was significantly higher dopamine concentration in the midbrain in Rab18-/- female mice (WT= 1.5 ± 0.4 pmol/mg; Rab18-/-: 4.2 ± 1.0 pmol/mg; p=0.0253; Fig. 6C). Our data suggest that Rab18 inhibits dopamine release in the midbrain in lactating mice postpartum. Together, our results suggest that Rab18 positively and negatively regulate serum prolactin concentration and midbrain dopamine concentration postpartum, respectively.. Rab18-/- mice have more dopaminergic neurons in the substantia nigra Rab18-/- mice have higher dopamine concentration postpartum, suggesting that Rab18 negatively regulate dopamine release. Alternatively, Rab18 may also regulate the number of dopaminergic neurons. To examine this possibility, we labeled dopaminergic neurons 21.

(23) with tyrosine hydroxylase (TH) antibody. We found that Rab18-/- female mice had more dopaminergic neurons in the substantia nigra (SN) (WT: 3.6 ± 0.7 × 104 cells/mm3; Rab18-/-: 6.0 ± 0.3 × 104 cells/mm3; p=0.0417; Fig. 7D-F), a part of the midbrain, but not in the glomerular layer (GL) of the OB (WT: 6.4 ± 0.5 × 104 cells/mm3; Rab18-/-: 5.9 ± 0.3 × 104 cells/mm3; p=0.70; Fig. 7A-C). Therefore, our data suggest that Rab18 negatively regulate dopaminergic neuron number, which may also contribute to the dopamine level in the midbrain.. Prolactin treatments rescue adult neurogenesis in the MOB and DG but not in AOB in lactating Rab18-/- mice Since Rab18 deficient mice have impaired adult neurogenesis and lower prolactin level, it is possible that Rab18-modulated adult neurogenesis is mediated by prolactin. To investigate this possibility, Rab18-/- mice received consecutive subcutaneous injections of prolactin or vehicle since GD18 to PD2 and BrdU on PD7, and their adult neurogenesis was examined (Fig. 8A). After vehicle treatments, there was a trend decrease in newborn cells in AOB in Rab18-/- mice (WT: 3.6 ± 0.6 × 104 cells/mm3; Rab18-/-: 2.1 × 104 cells/mm3; p=0.0760; Fig. 8B, C and F). There was no difference in newborn neurons in the AOB in Rab18-/- mice after vehicle treatments (WT: 2.5 ± 0.5 × 104 cells/mm3; Rab18-/-: 1.6 × 104 cells/mm3; p=0.14; Fig. 8B, C and G). There was no difference in neuronal differentiation in the AOB after vehicle treatments (WT: 68.0 ± 3.9%; Rab18-/-: 71.4%; p=0.63; Fig. 8B, C and H). There was a trend increase in newborn cells (Prolactin: 3.7 ± 0.6 × 104 cells/mm3; p=0.0800; Fig. 8C, E 22.

(24) and F), but not in newborn neurons (Prolactin: 2.6 ± 0.1 × 104 cells/mm3; p=0.12; Fig. 8C, E and G) in the AOB in Rab18-/- mice after prolactin treatments. Moreover, there was no difference in neuronal differentiation rate in the AOB in Rab18-/- mice after prolactin treatments (Prolactin: 73.0 ± 8.1%; p=0.74; Fig. 8C, E and H). There was no difference in newborn cells (Prolactin: 4.0 ± 0.3 × 104 cells/mm3; p=0.58; Fig. 8B, D and F) and newborn neurons (Prolactin: 3.0 ± 0.2 × 104 cells/mm3; p=0.42; Fig. 8B, D and G) in the AOB in WT mice after prolactin treatments. There was no difference in neuronal differentiation rate in the AOB between two treatments in WT mice (Prolactin: 73.6 ± 3.6%; p=0.39; Fig. 8B, D and H). These results indicate that prolactin treatments may rescue the production of newborn cells in the AOB in Rab18-/- mice. Numbers of newborn cells and newborn neurons in the MOB in WT and Rab18-/- mice after prolactin treatments were also examined. After vehicle treatments, there was a significant decrease in newborn cells (WT: 5.7 ± 0.4 × 104 cells/mm3; Rab18-/-: 4.2 × 104 cells/mm3; p=0.0440; Fig. 9A, B and E) and newborn neurons (WT: 4.5 ± 0.3 × 104 cells/mm3; Rab18-/-: 3.4 × 104 cells/mm3; p=0.0390; Fig. 9A, B and F) in the MOB in Rab18-/- mice. There was no difference in neuronal differentiation in the MOB in Rab18-/- mice (WT: 79.1 ± 2.8%; Rab18-/-: 80.5%; p=0.65; Fig. 9A, B and G). There was a significant increase in newborn cells (Prolactin: 6.7 ± 0.6 × 104 cells/mm3; p=0.005 Fig. 9B, D and E) and newborn neurons (Prolactin: 5.5 ± 0.4 × 104 cells/mm3; p=0.002; Fig. 9B, D and F) in the MOB in Rab18-/- mice after prolactin treatments. Also, 23.

(25) there was no difference in the neuronal differentiation in the MOB in Rab18-/- mice after prolactin treatments (Prolactin: 81.8 ± 1.3%; p=0.68; Fig. 9B, D and G). There was a trend increase in newborn cells (Prolactin: 6.9 ± 0.0 × 104 cells/mm3; p= 0.0560; Fig. 9A, C and E) and a significant increase in newborn neurons (Prolactin: 5.6 ± 0.1 × 104 cells/mm3; p=0.0200; Fig. 9A, C and F) in the MOB in WT mice after prolactin treatments. But there was no difference in neuronal differentiation rate in the MOB between treatments in WT mice (Prolactin: 81.84 ± 1.12%; p=0.33; Fig. 9A, C and G). These results suggest that prolactin treatments rescue adult neurogenesis in the MOB in Rab18-/- mice. The number of newborn cells and newborn neurons in the DG in WT and Rab18-/- mice after prolactin treatments were also examined. After vehicle treatments, there was no difference in newborn cells (WT: 11.4 ± 2.0 × 103 cells/mm3; Rab18-/-: 6.5 × 103 cells/mm3; p=0.10; Fig. 10A, B and E), newborn neurons (WT: 8.3 ± 1.2 × 103 cells/mm3; Rab18-/-: 4.9 × 103 cells/mm3; p=0.10; Fig. 10A, B and F) and neuronal differentiation (WT: 73.7 ± 4.8%; Rab18-/-: 73.3%; p=0.95; Fig. 10A, B and G) in the DG in Rab18-/- mice. There was a trend increase in newborn cells (Prolactin: 12.9 ± 1.8 × 103 cells/mm3; p=0.0540; Fig. 10B, D and E) and a significant increase in newborn neurons (Prolactin: 10.3 ± 1.6 × 103 cells/mm3; p=0.0250; Fig. 10B, D and F) in the DG in Rab18-/- mice after prolactin treatments. There was no difference in neuronal differentiation in the DG in Rab18-/- mice after prolactin treatments (Prolactin: 79.3 ± 1.5%; p=0.35; Fig. 10B, D and G). There was no difference in newborn cells (Prolactin: 9.9 ± 2.1 × 103 cells/mm3; p=0.54; Fig. 10A, C and E) 24.

(26) and newborn neurons (Prolactin: 8.0 ± 1.3 × 103 cells/mm3; p=0.86; Fig. 10A, C and F) in the DG in WT mice after prolactin treatments. Also, there was no difference in neuronal differentiation rate in the DG between two treatments in WT mice (Prolactin: 81.71% ± 4.94%; p=0.15; Fig. 10A, C and G). Our data indicate that prolactin treatments rescue adult neurogenesis in the DG in Rab18-/- mice. Taken together, our data reveal that prolactin treatments rescue adult neurogenesis in the MOB and DG, but not in the AOB in the Rab18-/mice. Moreover, it suggests that prolactin mediates Rab18-dependent adult neurogenesis.. Prolactin treatments rescue cell proliferation in the SVZ, but not in the SGZ in lactating Rab18-/- mice Since Rab18 is required for cell proliferation in the SVZ and SGZ, and prolactin treatments rescue adult neurogenesis in Rab18-/- mice postpartum, it is important to investigate whether Rab18-modulated adult neurogenesis in lactating mice is mediated by prolactin-induced cell proliferation. To investigate this, proliferating cells in the SVZ and SGZ in lactating mice were detected by Ki67 antibody. After vehicle treatments, there were fewer proliferating cells in the SVZ in Rab18-/mice than that in WT mice (WT: 14.5 ± 1.3 × 104 cells/mm3; Rab18-/-: 8.9 ± 0.8 × 104 cells/mm3; p=0.0150; Fig. 11A, B and E). There was no difference in proliferating cells in the SGZ between two genotypes after vehicle treatments (WT: 2.0 ± 0.3 × 104 cells/mm3; Rab18-/-: 1.9 ± 0.3 × 104 cells/mm3; p=0.75; Fig. 11F, G and J). After prolactin treatments, 25.

(27) there was a significant increase in proliferating cells in the SVZ (Prolactin: 14.2 ± 1.4 × 104 cells/mm3; p=0.0270; Fig.11B, D and E), but not in SGZ (Prolactin: 2.4 ± 0.4 × 104 cells/mm3; p=0.23; Fig.11G, I and J) in Rab18-/- mice. There was no difference in proliferating cells in the SVZ in WT mice after prolactin treatments (Prolactin: 18.8 × 104 cells/mm3; p=0.29; Fig.11A, C and E). Also, there was no difference in proliferating cell number in the SGZ between treatments in WT mice (Prolactin: 2.0 ± 0.3 × 104 cells/mm3; p=0.99; Fig.11F, H and J). Our data suggest that prolactin treatments rescue cell proliferation in Rab18-/- mice in the SVZ, but not in the SGZ.. Prolactin treatments rescue the maintenance of neural stem cells in the SGZ, but not in the SVZ in Rab18-/- mice Since Rab18 is required for the maintenance of progenitor cells and prolactin treatments rescue cell proliferation in Rab18-/- mice in the SVZ, it is important to study which cell population is increased after prolactin treatments in Rab18-/- mice. To study this, neural stem cells in the SVZ and SGZ were detected by Sox2 antibody. There was a significant decrease in neural stem cells in the SVZ in Rab18-/- mice after vehicle treatment (WT: 35.7± 1.9 × 104 cells/mm3; Rab18-/- : 27.9 ± 1.3 × 104 cells/mm3; p=0.035; Fig. 12A, B and E). In Rab18-/- mice, there was no difference in neural stem cells in the SVZ after prolactin treatments (Prolactin: 33.9 ± 4.6 × 104 cells/mm3; p=0.11; Fig. 12B, D and E). There was no difference in neural stem cells in the SVZ in WT mice after prolactin treatments (Prolactin: 39.2 ± 3.4 × 104 cells/mm3; p=0.31; Fig. 26.

(28) 12A, C and E). After vehicle treatments, there was no difference in neural stem cells in the SGZ in Rab18-/- mice than that in WT mice (WT: 10.2 ± 0.5 × 104 cells/mm3; Rab18-/-: 9.3 ± 0.5 × 104 cells/mm3; p=0.15; Fig. 12F, G and J). There was a significant increase in neural stem cells in the SGZ in Rab18-/- mice after prolactin treatments (Prolactin: 11.3 ± 0.3 × 104 cells/mm3; p=0.0040; Fig. 12G, H and J). There was no difference in neural stem cell number in the SGZ between two treatments in WT mice (Prolactin: 10.6 ± 0.5 × 104 cells/mm3; p=0.29; Fig. 12F, H and J). These results indicate that prolactin treatments rescue neural stem cells in the SGZ, but not in the SVZ in Rab18-/- mice.. Prolactin treatments rescue the production of neuroblasts in the SVZ, but not in the SGZ in Rab18-/- mice After vehicle treatments, there was a significant decrease in neuroblasts in the SVZ (WT: 36.9 ± 4.4 × 104 cells/mm3; Rab18-/-: 17.5 ± 1.3 × 104 cells/mm3; p=0.0100; Fig. 13A, B and E), but not in the SGZ (WT: 9.4 ± 0.8 × 104 cells/mm3; Rab18-/-: 8.0 ± 2.0 × 104 cells/mm3; p=0.27; Fig. 13F, G and J) in Rab18-/- mice. There was a significant increase in neuroblasts in the SVZ after prolactin treatments in WT mice (Prolactin: 43.6 ± 2.4 × 104 cells/mm3; p=0.0300; Fig 13A, C and E) and Rab18-/- mice (Prolactin: 33.7 ± 4.1 × 104 cells/mm3; p=0.0080; Fig 13B, D and E). In the SGZ, there was no difference in neuroblast number between two treatments in WT mice (Prolactin: 8.6 ± 0.6 × 104 cells/mm3; p=0.53; Fig 13F, H, and J). However, there was a trend increase in neuroblasts in the SGZ in Rab18-/- mice after prolactin treatments (Prolactin: 10.5 ± 0.3 × 104 27.

(29) cells/mm3; p=0.0930; Fig 13G, I and J). Taken together, prolactin treatments rescue the production of neuroblasts in the SVZ, but not in SGZ in Rab18-/- mice.. Prolactin treatments do not rescue maternal behavior defects in Rab18-/- mice To test whether prolactin-induced adult neurogenesis rescues maternal behavioral defects in Ra18-/- mice, Rab18 null mice received six consecutive injections of exogenous prolactin from GD18 to PD2 (Fig. 14A). Maternal behaviors were assessed by pup retrieval test on PD0. Mother mice were separated from the home cage for 15 minutes. Before it was re-introduced, its own pups were scattered. We found that WT mice retrieved their own pups within 5 minutes after consecutive injections of prolactin or vehicle (Vehicle: median=89 seconds, Q1=73.25 seconds, Q3=148.5 seconds; Prolactin: median=106.5 seconds, Q1=92.25 seconds, Q3=128.5 seconds; p=0.56; Fig. 14B). Rab18-/female mice did not retrieve any pups after injections of vehicle or prolactin (Vehicle: median=300 seconds, Q1=300 seconds, Q3=300 seconds; Prolactin: median=300 seconds, Q1=300 seconds, Q3=300 seconds; p=1; Fig. 14B). The survival rate of pups on PD1 and PD21 were also recorded (Fig. 14A, C, D). Compared to WT mice, there was a significant decrease in pup survival rate on PD1 (genotype effect: F=17.997; p=0.0010; Fig.14C) and on PD21 (genotype effect: F=108.715; p<0.001; Fig.14D) in Rab18-/- mice, suggesting that Rab18-/- mice have impaired maternal behaviors. After prolactin treatments, there was no 28.

(30) difference in pup survival rate on PD1 in either WT (Vehicle: 72.6 ± 13.4%; Prolactin: 93.1 ± 4.8%; p=0.26; Fig. 14C) or Rab18-/- mice (Veh: 38.1 ± 48.1%; Prolactin: 20.0 ± 34.6%; p=0.35; Fig.14C), suggesting that prolactin do not rescue behavioral defects in Rab18-/- mice. However, there was a significant increase in the pup survival rate in WT mice after prolactin treatments on PD21 (Vehicle: 61.2 ± 11.0%; Prolactin: 82.3 ± 11.7%; p=0.045; Fig.14D). It suggests that prolactin enhances maternal behaviors in WT mice. No pups survived on PD21 after either vehicle or prolactin treatments in Rab18-/- mice (Vehicle: 0.00%; Prolactin: 0.00 ± 0.00%; p=1; Fig. 14D). Although some pups of Rab18-/- mice survived on PD1, they were not lactated by Rab18-/- mice and died a few hours to days later. Taken together, our results indicate that prolactin-induced adult neurogenesis is not sufficient to rescue maternal behavioral defects in Rab18-/- mice.. 29.

(31) Discussion. In the present study, we study whether Rab18-modulated adult neurogenesis is responsible for maternal behaviors. Our data reveal that Rab18 is required for adult neurogenesis in the OB and DG and the maintenance of proliferating cells and progenitor cells. Moreover, Rab18 regulates the secretion of dopamine, which decreases the concentration of serum prolactin early postpartum. Moreover, prolactin might rescue adult neurogenesis in the MOB and DG, but not maternal behaviors in Rab18-/mice postpartum. Our results suggest that Rab18 may govern prolactin level during lactation by negatively regulating dopamine secretion, which in turn regulates adult neurogenesis (Fig. 15).. Prolactin may induce adult neurogenesis in the MOB by activating proliferation of neuroblasts in the SVZ Here we find that Rab18 modulates adult neurogenesis by increasing the release of prolactin. We also find that Rab18-/- mice have more neuroblasts, but not neural stem cells in the SVZ after prolactin treatments. It is possible that prolactin induces adult neurogenesis by activating the proliferation of neuroblasts. Previous study shows that prolactin treatments increase adult neurogenesis in the MOB by promoting cell proliferation in the SVZ [18]. Our data are consistent to their experiments. In that study, they also find that prolactin receptors express in the dorsolateral SVZ [18], suggesting that prolactin directly activate cells in the SVZ or indirectly through a paracrine manner. 30.

(32) Although it is unclear which cell population in the SVZ express prolactin receptors, its downstream factor, erk5, has been shown to be expressed by 70% of Dcx+ neuroblasts [32]. To investigate the expression pattern of prolactin receptors, we can immunostain prolactin receptor antibody and cell population-specific marker antibody, such as Sox2, Dcx, NeuN, GFAP (astrocyte marker) and Olig2 (oligodendroprogenior cell marker).. Prolactin may induce adult neurogenesis in the DG by promoting cell proliferation in progenitors or rescuing dopamine-suppressed cell proliferation in the SGZ We also find that adult neurogenesis in the DG in Rab18-/- mice increases after prolactin treatments, suggesting that exogenous prolactin induces adult neurogenesis in the DG in Rab18-/- mice. These data are contradictory to previous experiments from Shingo and his colleagues. They find that exogenous prolactin do not promote adult neurogenesis in the DG [18]. We suspect that the serum concentration of prolactin in Rab18-/- mice is lower and this is caused by continuing higher dopamine concentration in the midbrain in Rab18-/- mice. Also, dopaminergic neurons in the midbrain project to the hippocampus and dopamine has been shown to decrease cell proliferation in the SGZ[31]. Although which cell population expresses prolactin receptor has not been found, prolactin receptors are expressed in the hippocampus[33]. In our experiments, we also find that neural stem cells and neuroblasts and their cell proliferation in the SGZ in Rab18-/- mice are increased after prolactin treatments. Taken together, these data suggest that prolactin may induce 31.

(33) adult neurogenesis in the DG by promoting cell proliferation in progenitors or rescuing dopamine-suppressed cell proliferation in the SGZ.. Why Prolactin treatments rescue adult neurogenesis, but not maternal behaviors in Rab18-/- mice? Prolactin treatments increase adult neurogenesis in the MOB and DG, but they do not rescue maternal behavior defects in Rab18-/- mice, suggesting that Rab18-modulated adult neurogenesis postpartum, which is mediated by prolactin, is not sufficient to rescue maternal behaviors. However, there are four possibilities, which can explain why Rab18-/- mice still have maternal defects after prolactin treatments. Frist, there is also a peak of adult neurogenesis on GD7, which is required for maternal behaviors [18]. Newly-generated neurons are incorporated into the existing OB circuit and form synapses about 14 days after their birth [5]. It is possible that this population of newly generated neurons is responsible for maternal behaviors postpartum. Therefore, we might miss the appropriate time window to increase adult neurogenesis with prolactin injections. Therefore, we can inject exogenous prolactin to Rab18-/- mice during early pregnancy and examine their maternal behaviors postpartum. Second, Rab18-/- mice might not establish binding with their pups. Female mice form binding with their pups by olfaction [11]. Since Rab18-/- mice have progressively axonal degeneration in spinal sensory 32.

(34) neurons and optic nerves [27], it is possible that their olfactory nerves also degenerate. To test this hypothesis, we should examine the olfactory nerve and olfaction in Rab18-/- mice. If Rab18-/- mice have impaired olfaction, it may explain why increase of prolactin and adult neurogenesis in Rab18-/- mice cannot rescue maternal behaviors. Third, Rab18-/- mice have higher dopamine concentration, which has been suggested to be a cause of schizophrenia [34]. A previous study has shown that mothers with psychiatric disease and mood disorder have higher possibility of neonaticide [35]. Interestingly, the anxiety level of Rab18-/- mice appear to be higher, suggesting that Rab18-/- mice may have mood disorder-like symptoms. Therefore, increase of prolactin has no effect on lowering dopamine. To overcome this, Rab18-/- mice may receive dopamine receptor antagonist injections to block endogenous dopamine signaling. Moreover, dopamine inhibits adult neurogenesis through blocking cell proliferation in the SVZ and SGZ. We can also examine adult neurogenesis in Rab18-/- mice after dopamine antagonist treatments. If Rab18-/- mice have higher level of adult neurogenesis postpartum and their anxiety level is decreased after these treatments, they may perform maternal behaviors. Lastly, body odors from newborn pups are important for the formation of maternal bonding between mother and its own pups. However, which molecules in body odors from newborn pups initiate maternal behaviors is still unclear. Here we find that adult neurogenesis in the AOB is not rescued by prolactin treatments. Since AOB is responsible for pheromone detection, our results suggest pheromones 33.

(35) could be required for maternal bonding and rescuing adult MOB neurogenesis by prolactin, which is required for odor discrimination, is not enough to initiate maternal behaviors. In conclusion, this is the first study to demonstrate that Rab18 regulates adult neurogenesis during lactation. Rab18 modulates adult neurogenesis postpartum by inhibiting dopamine secretion and inducing prolactin secretion. In the future, we will try to block endogenous dopamine signaling postpartum. Because dopamine is the upstream regulator of prolactin, we suspect that these treatments can universally rescue Rab18-/- mice from maternal defects by increasing prolactin secretion and alleviating mood disorders.. 34.

(36) Figures. Figure 1. Rab18 is required for adult neurogenesis in the AOB, MOB and DG in lactating mice. To examine adult neurogenesis in Rab18-/mice postpartum, mice were injected with BrdU on PD7 and sacrificed on PD21 (A). Newborn cells were immunolabeled with anti-BrdU antibody (green) and mature neurons were labeled by anti-NeuN antibody (red). Newborn neurons were identified by double staining of BrdU and NeuN (arrowheads). We found that lactating Rab18-/- mice had a significant decrease in newborn cells in the MOB (G-I) and a trend decrease in the DG (L-N), but not in the AOB (B-D). Compared to WT, newborn neuron number was significantly decreased in lactating Rab18-/- mice in the AOB 35.

(37) (B, C and E), MOB (G, I and J) and DG (L, M and O). There was no difference in neuronal differentiation between WT and Rab18-/- female mice in the AOB (B, C and F), MOB (G, H and K) and DG (L, M and P). Data were from at least 5 animals per genotype and are indicated as means ± S.E. * compared to WT, p<0.05 (t-test); scale bar: 40 μm.. 36.

(38) Figure 2. Rab18 is necessary for cell proliferation in the SVZ and SGZ in lactating mice. Proliferating cells were labeled with Ki67 antibody (green) and nuclei were labeled with DAPI (blue). The proliferating cell number was significantly decreased in the SVZ (A-C) and SGZ (D-F) in Rab18-/- female mice postpartum. DG: dentate dyrus; H: hilus; LV: lateral ventricle; ST: striatum. Data were from at least 4 animals per genotype and indicated as means ± S.E. * compared to WT, p<0.05; **, p<0.01 (t-test); scale bar: 40 μm.. 37.

(39) Figure 3. Rab18 is essential for the maintenance of neural stem cells in the SVZ and SGZ in lactating mice. Neural stem cells were labeled with Sox2 antibody (green). Compared to WT littermates, Rab18-/- mice had significantly fewer neural stem cells in the SVZ (A-C) and SGZ (D-F) postpartum. DG: dentate dyrus; H: hilus; LV: lateral ventricle; ST: striatum. Data were from at least 4 animals per genotype and indicated as means ± S.E. ** compared to WT, p<0.01; scale bar: 40 μm.. 38.

(40) Figure 4. Rab18 is essential for the production of neuroblasts in the SVZ and SGZ in lactating mice. Neuroblasts were labeled with Doublecortin (DCX) antibody (red). Neuroblasts were significantly decreased in the SVZ (A-C) and SGZ (D-F) in Rab18-/- mice. DG: dentate dyrus; H: hilus; LV: lateral ventricle; ST: striatum. Data were from at least 5 animals per genotype and are indicated as means ± S.E. * compared to WT, p<0.05; **, p<0.01 (t-test); scale bar: 40 μm.. 39.

(41) Figure 5. Rab18 is required for the increase of prolactin on postpartum day 1. To test whether Rab18 regulates the prolactin level in lactating mice, serum prolactin concentrations of Rab18-/- mice were measured. Plasma samples were collected from virgin or PD1, PD4 and PD7 mice and serum prolactin was detected by ELISA (A). All of our results were normalized to serum total proteins. In WT mice, serum prolactin on PD1 was higher than that of virgin, PD4 and PD7 (B, white bars). On PD1, serum prolactin was significantly less in Rab18-/- mice (B, black bar) than that of WT littermates. Data were from at least 3 animals per genotype and indicated as means ± S.E. * compared to WT or compared to PD1, p<0.05; **, p<0.01; ***, p<0.001 (Two-way ANOVA followed by Tukey’s post-hoc analysis).. 40.

(42) Figure 6. Rab18 inhibits dopamine release in the midbrain in lactating mice. To measure the dopamine concentration in lactating mice, mice were sacrificed on PD1 (A). Dopamine concentrations in the OB and midbrain on PD1 were measured by HPLC. There was significantly higher dopamine concentration in the midbrain (C), but not in the OB (B) in Rab18-/- mice on PD1. Data were from at least 3 animals per genotype and indicated as means ± S.E. * compared to WT: p<0.05 (t-test).. 41.

(43) Figure 7. Rab18-/- mice have more dopaminergic neurons in the SN postpartum. Since the dopamine concentration is higher in Rab18-/- mice on PD1, we tested whether Rab18 regulated dopaminergic neuron numbers in the glomerular layer (GL) of the OB and substantia nigra (SN) of the midbrain by TH immunolabeling (green). DAPI+ nuclei were blue (A, B, D and E). There was no difference in dopaminergic neuron number in the GL between WT and Rab18-/- mice postpartum (A-C). Rab18-/mice had significantly more dopaminergic neurons in the SN postpartum (D-F). Data were from 3 animals per genotype and indicated as means ± S.E. * compared to WT: p<0.05 (t-test); scale bar: 40 μm.. 42.

(44) Figure 8. Prolactin treatments may rescue adult neurogenesis in the AOB in lactating Rab18-/- mice. To investigate whether Rab18-modulated adult neurogenesis is mediated by prolactin postpartum, Rab18-/- mice received prolactin injections from GD18 to PD2 and BrdU on PD7 and adult neurogenesis was examined on PD21 (A). Newborn cells were detected by BrdU antibody (green) and mature neurons were detected by NeuN antibody (red). After vehicle treatments, there was a trend decrease in newborn cells in Rab18-/- mice (B, C and F). However, there was no difference in newborn neurons and neuronal differentiation in the AOB between two genotypes after vehicle treatments. In Rab18-/mice, there was a trend increase in newborn cells after prolactin 43.

(45) treatments (C, E and F). There was no difference in newborn neurons and neuronal differentiation in the AOB in Rab18-/- mice after prolactin treatments (C, E, G and H). In WT mice, there was no difference in newborn cells, newborn neurons and neuronal differentiation rate in the AOB between two treatments (B, D and F-H). Data are from at least 2 animals per genotype and treatment and indicated as means ± S.E. Data were counted by Two-way ANOVA and followed by Tukey’s post-hoc analysis; scale bar: 40 μm.. 44.

(46) Figure 9. Prolactin treatments rescue adult neurogenesis in the MOB in lactating Rab18-/- mice. Newborn cells were detected by BrdU antibody (green) and mature neurons were detected by NeuN antibody (red). After vehicle treatments, there was a significant decrease in newborn cells and newborn neurons, but not in neuronal differentiation in the MOB in Rab18-/- mice (A, B and E-G). In Rab18-/- mice, there was a significant increase in newborn cells and newborn neurons, but not in in neuronal differentiation rate in the MOB after prolactin treatments (B, D-G). In WT mice, there was a trend increase and a significant increase in newborn cells and newborn neurons, respectively, but not in neuronal differentiation rate in the MOB after prolactin treatments (A, C and E-G). Data are from at least 2 animals per genotype and treatment and indicated as means ± S.E. *: p<0.05; **: p<0.01 (Two-way ANOVA followed by Tukey’s post-hoc analysis); scale bar: 40 μm.. 45.

(47) Figure 10. Prolactin treatments may rescue adult neurogenesis in the DG in lactating Rab18-/- mice. Newborn cells were detected by BrdU antibody (green) and mature neurons were detected by NeuN antibody (red). After vehicle treatments, there was no difference in newborn cells, newborn neurons and neuronal differentiation between two genotypes (A, B and E-G). In Rab18-/- mice, there was a trend increase and significant increase in newborn cells and newborn neurons in the DG after prolactin treatments (B, D, E and F), respectively. There wasno difference in neuronal differentiation in the DG in Rab18-/- mice (B, D and G). In WT mice, there was no differences in newborn cells, newborn neurons and neuronal differentiation rate in the DG after prolactin treatments (A, C and E-G). Data are from at least 2 animals per genotype and treatment and indicated as means ± S.E. *: p<0.05 (Two-way ANOVA followed by Tukey’s post-hoc analysis); scale bar: 40 μm.. 46.

(48) Figure 11. Prolactin treatments may rescue cell proliferation in the SVZ, but not in the SGZ in lactating Rab18-/- mice. Proliferating cells were detected by Ki67 antibody. After vehicle treatments, there was a significant decrease in proliferating cells in the SVZ, but not in the SGZ in Rab18-/- mice (A, B, E-G and J). In Rab18-/- mice, there was a significant increase in proliferating cells in the SVZ (B, D and E), but not in the SGZ (G, I and J) after prolactin treatments.. In WT mice, there was no difference in proliferating cell number in the SVZ and SGZ between two treatments (A, C, E, F, H and J). DG: dentate dyrus; H: hilus; LV: lateral ventricle; ST: striatum. Data were from at least 2 animals per genotype and treatment and indicated as means ± S.E. *: p<0.05 (Two-way ANOVA followed by Tukey’s post-hoc analysis); scale bar: 40 μm. 47.

(49) Figure 12. Prolactin treatments rescue the maintenance of neural stem cells in the SGZ in lactating Rab18-/- mice. Neural stem cells were labeled by Sox2 antibody (green). After vehicle treatments, there was a significant decrease in neural stem cells in the SVZ, but not in the SGZ in Rab18-/- mice (A, B, E-G and J). In WT and Rab18-/- mice, there was no difference in neural stem cell number in the SVZ between two treatments (A- E). In the SGZ, there was no difference in neural stem cells in the SGZ between two treatments in WT mice (F, H and J). In Rab18-/- mice, neural stem cells in the SGZ were significantly increased after prolactin treatments (G, I and J). DG: dentate dyrus; H: hilus; LV: lateral ventricle; ST: striatum. Data are from at least 2 animals per genotype and treatment and indicated as means ± S.E. *: p<0.05 (Two-way ANOVA followed by Tukey’s post-hoc analysis); scale bar: 40 μm. 48.

(50) Figure 13. Prolactin treatments rescue the production of neuroblasts in the SVZ in Rab18-/- mice. Neuroblasts were detected by DCX antibody (red). After vehicle treatments, there was a significant decrease in neuroblasts in the SVZ in Rab18-/- mice (A, B and E). In Rab18-/- mice, there was a significant increase in neuroblasts in the SVZ after prolactin treatments (B, D and E). In WT mice, there was a signifcant increase in neuroblast numbers in the SVZ after prolactin treatments (A, C and E). There was no difference in neuroblast numbers in the SGZ in WT and Rab18-/- mice after vehicle treatments (F, G and J). In WT mice, there was also no difference in neuroblast numbers in the SGZ after prolactin treatments (F, H and J). However, there was a trend increase in neuroblasts in the SGZ in Rab18-/- mice after prolactin treatments (G, I and J). DG: dentate dyrus; H: hilus; LV: lateral ventricle; ST: striatum. 49.

(51) Data are from at least 3 animals per genotype and treatment and indicated as means ± S.E. *: p<0.05; **: p<0.01 (Two-way ANOVA followed by Tukey’s post-hoc analysis); scale bar: 40 μm.. 50.

(52) Figure 12: Prolactin treatments do not rescue maternal behavior impairment in Rab18-/- mice. To investigate whether prolactin treatments rescue maternal behavior defects in Rab18-/- mice, pup retrieval test was performed (A). The latency to retrieve all pups during 5 minutes was measured and was shown in boxplots (central line shows the median, upper bound of box shows the third quartile and lower bound of box shows the first quartile). With two treatments, WT female mice retrieved all their pups within 5 minutes, but Rab18-/- mice did not (Mann-Whitney U test) (B). Compared to WT mice, there was a significant decrease in the pup survival rate on PD1 and on PD21, suggesting that Rab18-/- mice have impaired maternal behaviors (C and D). Prolactin treatments did not rescue pup survival rate in Rab18-/- mice on PD1 and PD21 (C and D), suggesting that prolactin do not rescue maternal behaviors in Rab18-/- mice. Prolactin treatments significantly increased pup survival in WT mice on PD21 (D), indicating that prolactin 51.

(53) promotes maternal behaviors in WT mice. Data are from at least 3 animals per genotype and per treatment and indicated as means ± S.E. **: p<0.01; ***: p<0.001 (Two-way ANOVA followed by Tukey’s post-hoc analysis).. 52.

(54) Figure 13. A model of the regulatory mechanism of Rab18 for adult neurogenesis in lactating mice. Rab18 modulates adult neurogenesis by inhibiting the secretion of the midbrain dopamine, which decreases serum prolactin level. Prolactin promotes adult neurogenesis in the MOB. Prolactin also promotes adult neurogenesis in the DG. Adult OB/DG neurogenesis could be involved in maternal behaviors and anti-(postpartum) depression.. 53.

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