Down-regulation of fatty acid binding protein 7 (Fabp7) is a hallmark of the postpartum brain
Fatty acid binding protein 7 (Fabp7) is a versatile protein that is linked to glial differentiation and proliferation, neurogenesis, and multiple mental health disorders. Recent microarray studies identified a robust decrease in Fabp7 expression in key brain regions of the postpartum rodents. Given its diverse functions, Fabp7 could play a critical role in sculpting the maternal brain and promoting the maternal phenotype. The present study aimed at investigating the expression profile of Fabp7 across the postpartum CNS. Quantitative real-time PCR (qPCR) analysis showed that Fabp7 mRNA was consistently down-regulated across the postpartum brain. Of the 9 maternal care-related regions tested, seven exhibited significant decreases in Fabp7 in postpartum (relative to virgin) females, including medial prefrontal cortex (mPFC), nucleus accumbens (NA), lateral septum (LS), bed nucleus of stria terminalis dorsal (BnSTd), paraventricular nucleus (PVN), lateral hypothalamus (LH), and basolateral and central amygdala (BLA/CeA). For both ventral tegmental area (VTA) and medial preoptic area (MPOA) levels of Fabp7 were lower in mothers, but levels of changes did not reach significance. Confocal microscopy revealed that protein expression of Fabp7 in the LS paralleled mRNA findings. Specifically, the caudal LS exhibited a significant reduction in Fabp7 immunoreactivity, while decreases in medial LS were just above significance. Double fluorescent immunolabeling confirmed the astrocytic phenotype of Fabp7-expressing cells. Collectively, this research demonstrates a broad and marked reduction in Fabp7 expression in the postpartum brain, suggesting that down-regulation of Fabp7 may serve as a hallmark of the postpartum brain and contribute to the maternal phenotype.
1. Introduction<!>2.1. Animals<!>2.2. Tissue collection and slicing<!>2.3. Western blotting<!>2.4. cDNA preparation and quantitative real-time PCR (qPCR)<!>2.5. Fluorescent immunohistochemistry with tyramide signal amplification (TSA)<!>2.6. Relative quantitation of Fabp7immunoreactivity<!>2.7. Statistical analysis<!>3.1. Validation of Fabp7 antibody specificity<!>3.2. Levels of Fabp7 mRNA were reliably decreased across the postpartum brain<!>3.3. Immunoreactivity of Fabp7 was significantly decreased in the postpartum LS<!>4. Discussion<!>4.1. Fabp7 is linked to neural differentiation, and may mediate specific astro-neuronal plasticity of the postpartum brain<!>4.2. Alterations in astrocytic morphology and gene expression in the postpartum brain<!>4.3. Regulation of Fabp7 expression in the CNS<!>4.4. Possible roles of Fabp7 down-regulation in the postpartum brain<!>5. Conclusions
<p>Postpartum female rodents exhibit a wide array of behaviors that promote the survival and development of their offspring. The manifestation of these maternal behaviors has been associated with alterations in circulating hormones (Bridges, 1984; Numan and Insel, 2003), and neurosignaling pathways, such as oxytocin, enkephalin, and GABA (Gammie, 2005; Insel, 1990; Petraglia et al., 1985; Qureshi et al., 1987; Young et al., 1997). However, it is likely that other unidentified genes are contributing to the reconstruction of the maternal brain that regulates postpartum behavioral phenotype. Recent microarray studies have identified hundreds of genes that display altered expression in postpartum females compared to virgin controls. However, the extent and functional significance of these alterations in gene expression have yet to be clarified.</p><p>Microarray studies from our laboratory and others have identified that one gene, fatty acid binding protein 7 (Fabp7), also referred to as brain lipid binding protein (BLBP), has altered expression in several regions of postpartum brain. Fabp7 expression was found to be decreased in arcuate nucleus of postpartum rats (Xiao et al., 2005), and in mouse hypothalamus (Gammie et al., 2005). These initial findings have been complemented by recent microarray studies in the medial prefrontal cortex (mPFC) (Eisinger et al., 2014), medial preoptic area (MPOA) (Driessen et al., 2014a), nucleus accumbens (NA) (Zhao et al., 2014), and the septum of two different strains of mice (Eisinger et al., 2013b; Zhao et al., 2012b). Combined, these studies indicate that there is overall a consistent down-regulation of Fabp7 in postpartum females, though verification of microarray findings were only conducted for the septum microarrays (Eisinger et al., 2013b; Zhao et al., 2012b).</p><p>Fabp7 is primarily expressed in neural progenitor cells and astrocytes (Kurtz et al., 1994), and has long been involved in glial differentiation and proliferation, neurogenesis and multiple behavioral processes. For example, Fabp7 knockout (KO) mice displayed aberrant dendritic morphology and decreased spine density (Ebrahimi et al., 2016), increased anxiety and fear memory compared to wild-type controls (Owada et al., 2006). Several lines of research have also indicated the involvement of Fabp7 in maternal phenotype. Inhibition or deletion of cannabinoid receptor 1 (Cnr1) disrupts maternal behaviors (Schechter et al., 2012, 2013), and Fabp7 binds to anandamide, an endocannabinoid (Berger et al., 2012; Kaczocha et al., 2009). This suggests that Fabp7 may modulate maternal behavior via acting on endocannabinoid signaling pathway, although this effect has not been tested. Moreover, bioinformatics studies have indicated that alterations in genes associated with development and mental health disorders may contribute to the maternal phenotype by promoting neural remodeling and sociability in the maternal female (Driessen et al., 2014a; Eisinger et al., 2013a, 2013b, 2014). Correspondingly, Fabp7 has been linked to both CNS development and mental health disorders, including schizophrenia, autism, bipolar disorder, and depression (Ayalew et al., 2012; Iwayama et al., 2010; Kumar et al., 2011; Uriguen et al., 2008; Watanabe et al., 2007; Ziats and Rennert, 2011). The association of altered Fabp7 with CNS development and mental health disorders suggests the involvement of Fabp7 in maternal behavioral phenotypes. Postpartum depression in human studies is correlated with decreased levels of free fatty acids (Liu et al., 2010; Storch and Corsico, 2008). Increased free fatty acids or a high fat diet can reduce postpartum depression in rats (Feng et al., 1994; Owada et al., 1996), indicating that Fabp7 levels in mothers could contribute to a healthy maternal phenotype.</p><p>The purpose of the present study was to investigate in depth the alterations in Fabp7 expression in broad functional brain regions of postpartum females compared to virgin controls. Using quantitative real-time PCR (qPCR), we validated previous microarray findings and extended our investigation to include other brain regions associated with maternal behaviors. Further, we utilized fluorescent immunohistochemistry to examine the subregional distribution of Fabp7 in the lateral septum (LS) and confirm qPCR findings at the protein level.</p><!><p>Nulliparous age-matched outbred hsd:ICR female mice (~70 days of age; Harlan, Madison, WI) were used for all experiments. One half of the females remained housed together (two mice per cage) while the remaining females were pair-housed with breeder hsd:ICR males (Harlan, Madison, WI). After two weeks of cohousing, all females were individually housed. This housing strategy minimized the isolation-induced stress (Brown and Grunberg, 1995; Palanza et al., 2001), and provided all females with a similar social environment. All mice were housed in polypropylene cages with nestlets and ad libitum access to water and breeder chow (Harlan, Madison, WI). On postpartum day 1, litters were culled to standardize litter size to 11 pups. Animals were housed in a 12:12 light/dark cycle with lights on at 6:00h CST. All experimental procedures were in compliance with the guidelines of the National Institutes of Health Guide and Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committee of the University of Wisconsin.</p><p>For verifying the specificity of Fabp7 antibody, C57BL/6 wild-type (WT) and Fabp7 knockout (KO) male mice of same genetic background (Owada et al., 2006) were used. The mice were 19 months old and maintained under a 12:12 light/dark cycle (lights on at 8:00 AM) with ad libitum access to standard food and water. All experimental protocols were performed according to the Guidelines for Animal Experimentation of the Tohoku University Graduate School of Medicine under the laws and notification requirements of the Japanese Government.</p><!><p>On postpartum day 6 or 7 between 9:00h and 12:00h CST, brains were removed from lactating and age-matched virgin females. Estrous states for virgin females were determined using a vaginal lavage (Marcondes et al., 2002), and only diestrus naive females were used in subsequent experiments (Zhao and Gammie, 2015). For the qPCR analysis, females were lightly anaesthetized with isoflurane before brains were removed and flash frozen in methylbutane on dry ice. Brains were stored at −80°C until sliced to a 200 μm thickness on a cryostat (Leica, CM1850, Bannockburn, IL, USA). Sections were mounted on gelatin coated glass slides and regions of interest were collected using the Brain Punch Set (Stoelting, Wood Dale, IL, USA) under a dissection microscope. The bregma coordinates for the collected brain regions are as follows according to The Allen Mouse Brain Atlas (reference atlas version 1, 2008): mPFC (1.98mm-1.54mm), NA (1.70mm to 0.98mm), LS (1.10mm to 0.14mm), BnSTd (0.26mm to 0.02mm), MPOA (0.26mm to −0.10mm), PVN (−0.58mm to −0.94mm), LH (−0.70mm to −1.06mm), BLA/CeA (−0.82mm to −1.70mm), and VTA (−2.92mm to −3.80mm). These brain regions were selected because they are linked to maternal behavior, other socially motivated behaviors and mood state (D'Anna and Gammie, 2009; Febo et al., 2010; Fleming et al., 1980; Insel and Harbaugh, 1989; Li and Fleming, 2003; Numan and Numan, 1996; Numan et al., 2005; Zhao and Li, 2010). For fluorescent immunohistochemistry, females were lightly anaesthetized with isoflurane, injected with 0.15 mL sodium pentobarbital, then transcardially perfused with approximately 50 mL saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Brains were postfixed overnight in 4% paraformaldehyde, then submerged in 30% sucrose in 0.1 M phosphate buffer for two days before being stored at −80°C. Brains were sliced to a 30 μm thickness on a cryostat, then stored at −20°C in anti-freeze cryoprotectant until labeling.</p><p>Preparation of brain sections from C57BL/6 wild-type (WT) and Fabp7 knockout (KO) mice for immunofluorescent labeling was made as described previously (Sharifi et al., 2011, 2013). Briefly, Fabp7 KO and WT mice brains were perfusion-fixed in 4% paraformaldehyde, postfixed overnight at 4°C and cryoprotected in graded concentrations of sucrose. Coronal sections (30 μm) containing the LS were sliced, and stored in anti-freeze cryoprotectant until staining.</p><!><p>Western blot analysis was performed to characterize the specificity of Fabp7 antibodies according to our previously published protocol (Zhao et al., 2012a; Zhao and Gammie, 2015). Briefly, brain tissue from a postpartum female was homogenized with ice-cold RIPA buffer containing protease and phosphatase inhibitors. Following tissue homogenization, samples were centrifuged and the supernatant fraction was collected. Protein concentration was determined using BCA Protein Assay (Pierce Chemical Co.; Rockford, IL). Twenty μg of total protein was loaded and gel electrophoresis was run using a 4-20% Mini-PROTEAN TGX precast gel (Bio-Rad; Hercules, CA) and transferred to a PVDF membrane. Membrane was blocked for 1 hr in 0.1 M TBST containing 5% nonfat dry milk and incubated with the primary antibody rabbit anti-Fabp7 (Abcam ab32423, Cambridge, MA, USA; diluted 1:1000) in 5% nonfat dry milk overnight at 4°C. Following washes, the membrane was incubated in an HRP-linked secondary antibody (goat anti-rabbit IgG, diluted 1:5000; Cell Signaling Technology, Beverly, MA) for 1 hr at RT. Immunoreactive bands were detected using a chemiluminescence kit (Amersham ECL Prime Western Blotting detection Reagent, GE Healthcare UK Ltd.; Buckinghamshire, UK), and the images were captured using C-Digit Blot Scanner (LI-COR Biosciences; Lincoln, NE).</p><!><p>Total RNA was extracted from regions of interest using the Aurum Total RNA Fatty and Fibrous tissue kit (Bio-Rad, Hercules, CA), with two minor alterations to the manufacturer's protocol as previously described (Driessen et al., 2014a, 2014b). RNA integrity was assessed for randomly selected samples using the Agilent RNA 6000 Nano Chips and Pico Chips with the Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA). RNA concentrations for each sample were determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE). 100 ng of RNA from each sample was used for reverse transcription with the SuperScript III First Strand Synthesis for RT-qPCR kit (Invitrogen, Carlsbad, CA) in an Eppendorf MasterCycler Personal PCR machine (Eppendorf, Hamburg, Germany). The concentration of cDNA was measured and diluted before amplification in a StepOnePlus Real Time PCR machine (Applied Biosystem, Foster City, CA) or a Bio-Rad CFX96 Touch Real Time System (Bio-Rad, Hercules, CA). All reactions were run in triplicate with SsoFast EvaGreen Supermix (Bio-Rad, Hercules, CA), and primers specific to the genes of interest (Table 1). The following thermal profile was used for all reactions: an initial melting step of 95°C for 30 seconds, followed by 40 cycles of a 5 second 95°C melting step, a 57-60°C annealing step for 20 seconds (Table 1), and a 72°C elongation step for 20 seconds. Each sample was run in triplicate and the average Cq value of triplicate qPCR reactions was used for data analysis. The gene expression was analyzed using a relative expression software tool REST 2009, which calculates relative expression of target gene transcripts in comparison to reference gene transcripts based on an efficiency corrected mathematical model. It calculates the relative expression ratio on the basis of the PCR efficiency (E) and crossing point deviation (ΔCP) of the investigated transcripts and on a newly developed randomization test macro (Pfaffl, 2001; Pfaffl et al., 2002). The relative expression of Fabp7 was assessed in postpartum versus virgin females using two of the following stable reference genes (Ywhaz, Ppia, Ywhah, or Sdha) for each region tested except for the PVN, where only Sdha was used due to the failure to find the second stable reference gene. Multiple reference genes were used because initial analysis found that no single reference gene is stably expressed across all of the regions tested between virgin and postpartum females. Reference genes that were significantly different between the two groups were excluded from data analysis. The number of animals used for the qPCR assay was as follows: virgin, N = 12; postpartum, N = 12.</p><!><p>To verify the specificity of Fabp7 antibody and understand whether the decrease in Fabp7 mRNA reflects a reduction in protein level, we examined Fabp7 immunoreactivity in the LS. We chose LS as a representative target brain region because LS is a core brain region involved in maternal behaviors (D'Anna and Gammie, 2009; Fleischer and Slotnick, 1978; Scotti et al., 2011) and other socially motivated behaviors (Bredewold et al., 2015; Liu et al., 2001), and the connectivity and chemoarchitecture of the major LS subdivisions are well characterized (Risold and Swanson, 1997a, 1997b; Sheehan et al., 2004). Additionally, our previous work has confirmed the significant downregulation of Fabp7 mRNA in the LS (Eisinger et al., 2013b; Zhao et al., 2012b), while its protein level was not measured. Fluorescent immunolabeling was carried out at room temperature unless otherwise noted. Sections were washed 5x 5 min in TBS (0.15M NaCl and 0.1 M Tris-HCl, pH 7.5) then incubated in 3% H2O2 in TBS for 30 minutes to inhibit peroxidase activity. Following 3x 10 min washes in TBST (0.05% Tween-20 in TBS), sections were incubated in blocking buffer (2% Roche Applied Sciences blocking reagent in TBS) for 1 hr. Sections were then incubated overnight at 4°C with primary antibodies in blocking buffer, using rabbit anti-Fabp7 (Abcam ab32423, Cambridge, MA, USA; diluted 1:1000). Sections were washed 3x 10 min with TBST, then incubated in anti-rabbit HRP-conjugated secondary antibodies (Cell Signaling #7074, Danvers, MA, USA; diluted 1:100). Following 3x 10 min washes in TBST, sections were incubated for 10 min in a Cy3-conjugated Tyramide (TSA™ Plus Cyanine 3 kit, Perkin Elmer, Waltham, MA, USA) in a 1:50 dilution with 1x Amplification Diluent. After washing 3x 10 min in TBST, sections were mounted onto slides and coverslipped using DePex (Serva, Heidelberg, Germany), and stored in the dark at 4°C. For controls, removal of the primary antibodies, secondary antibodies, or Cy3-conjugated tyramide resulted in no immunoreactivity (data not shown).</p><p>The specificity of Fabp7 antibodies was further validated in the present study using double fluorescent immunohistochemistry with anti-Fabp7 antibodies in combination with Gfap antibodies (Gfap Millipore, MAB360, 1:1000 dilution), or the neuronal marker Rbfox2 (NeuN2), which is related to and highly colocalized with NeuN (Kim et al., 2009; Kuroyanagi, 2009; McKee et al., 2005) (NeuN2 Millipore, MAB377, Bilerica, MA, USA 1:1000 dilution). For double fluorescent immunohistochemistry, sections were incubated in a mixture of rabbit anti-Fabp7 and mouse anti-NeuN2 or rabbit anti-Fabp7 and mouse anti-Gfap antibodies overnight at 4°C. Following incubation with anti-rabbit HRP-conjugated secondary antibodies and Cy3-conjugated Tyramide as described previously (Zhao et al., 2013; Zhao and Gammie, 2015), sections were washed 3x 10 min in TBST, then incubated in 3% H2O2 for 30 minutes. Sections were then incubated in anti-mouse HRP-conjugated secondary antibodies (Cell Signaling #7076, diluted 1:100) in TBST for 1 hr before 3x10 min washes in TBST. Sections were then incubated for 30 min in Alexa Fluor 488-conjugated tyramide (Molecular Probes, Eugene, OR, USA) in a 1:100 TSA stock solution with 0.0015% H2O2 in amplification buffer. After 3x 10 min washes in TBST, sections were mounted onto slides and coverslipped. As controls, quenching of HRP activity, omission of the primary antibodies or HRP-conjugated secondary antibodies completely eliminated immunoreactivity.</p><!><p>The confocal imaging was performed at the Newcomb Imaging Center, Department of Botany, University of Wisconsin-Madison using a laser scanning confocal microscope (Zeiss 510 Meta) with acquisition software. For quantitative analysis of Fabp7 immunolabeling in virgin and postpartum females, mean density of Fabp7 immunoreactivity was bilaterally measured in one representative section per mouse (Zhao et al., 2008) that is anatomically well-matched across animals using 40x magnification photomicrographs within a 230.3 × 230.3 μm unit area in three subregions of the LS (LSD, LSI, and LSV) across rostral, medial, and caudal LS (Fig. 1). NIH ImageJ software was used for determining the mean density of immunolabeling in virgin and postpartum females, using a uniform threshold and subtracting out the background staining for each individual section. The values of mean density from five mice of each group were averaged (N = 5 for virgins; N = 5 for postpartum). All microscopy work and data analysis was conducted by an investigator blind to the experimental conditions.</p><!><p>Statistical analysis was performed using the freely available R platform. T-tests, or in the case of non-normality, Kruskal-Wallis tests, were conducted for all comparisons, and outliers were removed as determined by the Dixon's Q Test. Overall level of statistical significance was considered at p < 0.05.</p><!><p>To verify the specificity of Fabp7 antibody, we performed the following three experiments. First, Western blotting with anti-Fabp7 antibodies resulted in one band at approximately 15 kDa (Fig. 2), which is the predicted size and consistent with previous findings (Haunerland and Spener, 2004). Second, we examined Fabp7 immunoreactivity in the Fabp7 knockout mouse. We observed strong staining of astrocytic cells in the LS of WT mouse and absence of specific immunoreactivity in the LS of a Fabp7 KO mouse (Fig. 3). However, Fabp7 is one of three total Fabps (i.e., Fabp7, Fabp3 and Fabp5) expressed in the brain, all with similar molecular weights and sequence similarity (Liu et al., 2010; Owada et al., 1996). To further confirm the specificity of anti-Fabp7 antibodies, double fluorescent immunohistochemistry was conducted with the astrocytic marker Gfap, and the neuronal marker Rbfox2 (here referred to as NeuN2). Since Fabp7 is expressed primarily in astrocytes, while Fabp3 is found in neurons, and Fabp5 is localized in a mixture of neurons and glial cells (Owada et al., 1996), it is expected that the specific anti-Fabp7 antibodies would highly colabel astrocytes. Double fluorescent immunolabeling found that no Fabp7 was colocalized with NeuN2 (Fig. 4d-f), but largely coexpressed with Gfap (Fig. 4a-c), indicating that the anti-Fabp7 antibodies are specific to Fabp7. Further, the cells labeled with Fabp7 appear to have an astrocytic morphology (Fig. 4a,d), consistent with previous findings (Owada et al., 2006).</p><!><p>Fabp7 mRNA was found to be significantly down-regulated in postpartum females in seven of the nine brain regions tested (Fig. 5). Those regions include: mPFC (p < 0.001, fold change = 0.48), NA (p < 0.001, fold change = 0.67), LS (p < 0.001, fold change = 0.62), bed nucleus of the stria terminalis dorsal (BnSTd) (p = 0.015, fold change = 0.76), paraventricular nucleus of the hypothalamus (PVN) (p = 0.019, fold change = 0.71), lateral hypothalamus (LH) (p < 0.001, fold change = 0.60), and basolateral and central amygdala BLA/CeA (p < 0.001, fold change = 0.74). Results for the ventral tegmental area (VTA) and medial preoptic area (MPOA) did not reach the statistical significance, but levels of Fabp7 mRNA still displayed a trend towards a decrease in maternal females compared to virgins (VTA p = 0.066, fold change = 0.73; MPOA p = 0.18, fold change = 0.84). Combined, these results indicate an overall decrease in Fabp7 mRNA across the postpartum brain compared to virgin controls.</p><!><p>Three subdivisions of the LS, the dorsal LS (LSD), intermediate LS (LSI), and ventral LS (LSV), were examined for Fabp7 protein expression across three rostrocaudal extent of LS (rostral, medial, and caudal LS) (Fig. 1). In parallel with the significant down-regulation of mRNA, Fabp7 proteins were also decreased within the LS of postpartum brain. Specifically, significant or near significant down-regulation in Fabp7 protein between virgin and maternal females appeared to occur toward the medial (medial LS: LSD p = 0.063; LSI p = 0.03; LSV p = 0.056) and caudal (caudal LS: LSD p = 0.002; LSI p = 0.006; LSV p = 0.007) (Figs. 6,7) portion of the LS, with no significant differences found in any of the three subregions of the rostral LS (rostral LS: LSD p = 0.12; LSI p = 0.644; LSV p = 0.425) (Fig. 7). Further, quantitative analysis showed that expression changes in Fabp7 protein across the entire LS were heterogenously distributed, with large scale alterations in the caudal portion of LS (Figs. 6,7).</p><!><p>This study confirmed, and extended upon, previous findings demonstrating that expression of Fabp7 mRNA and protein was widely and markedly decreased in brains of postpartum females compared to virgins. A substantial decrease in Fabp7 mRNA was found in 7 of the 9 brain regions tested, with expression levels dropping approximately 25% to 50% in postpartum females. The two brain regions that were not significantly decreased, VTA and MPOA, still showed a downward trend in expression (fold change = 0.73 and 0.84, respectively), indicating that there is an overall decrease in Fabp7 mRNA in brain regions linked to maternal behavior and postpartum mood state. Furthermore, in parallel with reduced mRNA expression, Fabp7 protein was also identified to be decreased in subregions of medial and caudal LS, reflecting the functional roles for reduced Fabp7 in the postpartum females.</p><!><p>Fabp7 expression has been associated with specific stages of neurogenesis in several reports (Arai et al., 2005; Feng et al., 1994; Owada et al., 2006). Reductions in Fabp7 via either small interfering RNAs or knockout mice result in decreased proliferation of neural stem and progenitor cells but promote cellular differentiation toward a neuronal phenotype, with no changes in Gfap labeled cells, indicating that lack of Fabp7 influences the ratio of the production of immature neurons, but not astrocytes in vivo (Arai et al., 2005; Matsumata et al., 2012). Additionally, Fabp7 has a high binding affinity for docosahexaenoic acid (DHA) (Xu et al., 1996), and DHA has previously been linked to CNS development (Uauy et al., 2001) and cognitive function (Morley and Banks, 2010). Thus, Fabp7 may participate in CNS development and cognition via a DHA-mediated mechanism.</p><p>Microarray studies of postpartum mPFC, LS, and MPOA identified a significant number of genes related to CNS development, including groups linked to neuronal differentiation and cell migration (Driessen et al., 2014a; Eisinger et al., 2013b, 2014). This supports previous findings demonstrating altered neurogenesis during pregnancy and lactation (Levy et al., 2011; Pawluski et al., 2009) and suggests that the maternal brain may be undergoing a new stage of adult CNS development compared to virgin females. Decreased Fabp7 may play an important role in this development and differentiation, but future studies would be required to address this possibility.</p><!><p>Consistent with most previous studies showing decreased Fabp7 mRNA in the postpartum brain, our immunohistochemical assay found decreases in Fabp7 protein expression. Overall, there was a significant decrease in Fabp7 protein expression in postpartum mice, with large scale significant changes in caudal LS (Figs. 6,7). These striking findings are important as the decreased Fabp7 protein expression in the LS reflects transcriptional down-regulation and/or post-transcriptional stabilization of its mRNA, and may contribute to the formation of maternal brain and the behavioral phenotypes. Current nomenclature divides the LS into three individual subregions according to structural and functional characteristics: rostral LS, intermediate LS, and ventral LS. The rostral LS is involved in reproductive and aggressive behaviors, while the caudal LS is associated with reward and motivated behaviors (Albert and Walsh, 1982; Mongeau et al., 2003; Sheehan et al., 2004). Ventral LS is linked to stress coping strategies and this function may be consistent throughout the rostral to caudal extent of LS (Sheehan et al., 2004). Each subregion has specific efferent projections, including several regions that are linked to maternal behaviors, such as the BnSTd, MPOA, LH, PVN, amygdala, and VTA (Risold and Swanson, 1997a; Sheehan et al., 2004). The caudal LS specifically is densely interconnected with the LH to promote appetitive and motivated responses to reward stimuli (Sheehan et al., 2004), but whether Fabp7 contributes to this pathway is unknown.</p><p>The cells fluorescently immunolabeled via anti-Fabp7 antibodies have a very similar morphology to that previously seen in astrocytes (Fig. 4a,d). In addition, double fluorescent immunolabeling showed that a large number of Fabp7-expressing cells were colocalized with Glial fibrillary acidic protein (Gfap) (an astrocytic marker), but not the NeuN2 (a neuronal marker), consistent with the concept that Fabp7 is localized in the glial cells. Astrocytes have been implicated in a variety of physiological processes. For instance, astrocytes contribute to the formation of the tripartite synapse due to dense interconnection with neuronal processes and cell bodies (Bushong et al., 2002; Halassa et al., 2007; Halassa and Haydon, 2010; Meshul et al., 1987). They also express neurotransmitter transporters, such as glutamate (Anderson and Swanson, 2000) and GABA (Schousboe, 2000), and metabolize glutamate to glutamine via glutamine synthetase (Hertz and Zielke, 2004) before release onto glutamate and GABAergic terminals. The release of glutamate from astrocytes via gliotransmission can also contribute to neural signaling by activating neuronal receptors (Halassa and Haydon, 2010). Astrocytes also mediate brain energy homeostasis and metabolism by the uptake and transportation of glucose, and decreased glucose availability can inhibit synaptic activity (Anderson and Swanson, 2000; Halassa and Haydon, 2010; Hertz and Zielke, 2004; Magistretti, 2006; Schousboe, 2000). Because Fabp7 is expressed in astrocytes, an important but unanswered question is whether Fabp7 is involved in these physiological processes.</p><p>Changes in astrocyte morphology and gene expression have been reported in the maternal brain. Astrocyte morphology is altered in the supraoptic nucleus of postpartum females, which may contribute to the coupling of oxytocin release to stimulate milk let down (Hatton et al., 1987; Salm et al., 1985). Gfap is commonly used as a glial cell marker, and increased levels of Gfap have been found in the cingulate cortex area 2 of postpartum females (Salmaso et al., 2005, 2009). Further, glutamine synthetase, which is primarily expressed in astrocytes and necessary for the glutamate/GABA cycle between neurons and astrocytes, is up-regulated in the LS and MPOA of maternal females compared to virgins (Driessen et al., 2014a; Zhao et al., 2012b). While the emphasis in maternal behavior research has been placed on neuronal signaling, alterations in astrocyte morphology and gene expression changes in glial cells are also occurring during the transition into a maternal state, and future studies are necessary to fully understand whether and/or how Fabp7 down-regulation would impact alterations in neuronal signaling and neurocircuitry necessary for the construction of the maternal brain.</p><!><p>Several mechanisms have been identified that modulate transcription and translation of Fabp7. Pax6, a transcription factor which promotes neuronal differentiation during early CNS development, contributes to altered Fabp7 expression, with undetectable levels of Fabp7 in Pax6 knockout mice (Arai et al., 2005). Notch signaling has also been linked to Fabp7 expression, as activation of Notch signaling promotes Fabp7 expression (Gaiano et al., 2000), while reduction in Notch1 and Notch3 receptors inhibits the production of Fabp7 in radial glia (Anthony et al., 2005). While the present results demonstrate that Fabp7 is down-regulated in multiple brain regions in the postpartum female, the mechanisms of down-regulation are still not known. Previous microarray studies conducted in the mPFC, LS, and MPOA of maternal females have found significant expression changes in Pax6, Notch1, and Notch3 mRNA in distinct brain regions (Driessen et al., 2014a; Eisinger et al., 2013b, 2014). Therefore, it is possible that alterations in these genes may contribute to the down-regulation of Fabp7 in postpartum females. Additionally, it appears that Fabp7 down-regulation is dependent on the presence of offspring. When pups were removed from mothers after parturition, Fabp7 mRNA in the postpartum LS returned to levels seen in the virgin female (Eisinger et al., 2013b), suggesting that parturition alone is not sufficient to sustain a decreased expression of Fabp7. Pups provide olfactory and tactile stimulation which appear to be necessary for decreased Fabp7 expression, and future studies investigating Fabp7 in other brain regions in pup deprived maternal females, and virgins with pups, would provide further evidence of the link between Fabp7 expression and the presence of offspring.</p><!><p>Fabp7 is associated with autism, bipolar disorder, schizophrenia, anxiety and depression (Ayalew et al., 2012; Iwayama et al., 2010; Kumar et al., 2011; Owada et al., 2006; Uriguen et al., 2008; Watanabe et al., 2007; Ziats and Rennert, 2011). In the postpartum state, there is an increased risk for multiple disorders, including postpartum depression, postpartum psychosis, and postpartum bipolar disorder (Eberhard-Gran et al., 2002; Kendell et al., 1987; Stowe and Nemeroff, 1995). Given the link of Fabp7 to mental health disorders, it is possible that alterations in Fabp7 expression in mothers may provide a vulnerability factor for the emergence of a disorder. However, any suggested links are speculative at this point due to a lack of clinical and experimental evidence. Future studies investigating how Fabp7 modulates postpartum mood state are warranted.</p><!><p>In sum, this study demonstrated that Fabp7 was widely and significantly down-regulated across the postpartum brain compared to virgin females. This finding suggests that down-regulation of Fabp7 may serve as a hallmark of the postpartum brain. Furthermore, down-regulation of Fabp7 mRNA was in parallel with the decreased protein within the LS. Fabp7-expressing cells are primarily glial cells, but not neurons. Previous studies have identified a significant number of genes linked to CNS development in the postpartum brain, including Fabp7, suggesting that the postpartum brain may represent a new stage of development. Future studies investigating the involvement of Fabp7 in neural remodeling, synaptic plasticity, social and emotional reactivity in postpartum females are needed to fully understand its association with maternal phenotype.</p>