Based on studies demonstrating a role for BDNF in regulating cell proliferation in the adult hippocampus, as well as the expression of BDNF and TrKB receptors in the neonatal hippocampus of rats (Solum and Handa, 2001 ), we hypothesized that estradiol modulates proliferation in the developing hippocampus by regulating BDNF expression. We report here a study in which BDNF transcript and peptide levels were quantified in hippocampal subregions of neonatal male and female rats following estradiol treatment. A baseline sex difference in BDNF gene expression was observed that mirrors the sex difference in hippocampal cell genesis. However, the effects of estradiol on BDNF differed among subregions of the hippocampus, and BDNF peptide levels did not change in parallel with BDNF gene expression. Together, these observations indicate region‐specific roles for BDNF in sculpting the developing hippocampus in males vs. females.

The downstream cellular mechanisms that mediate the effects of estradiol on neurogenesis in the neonatal hippocampus are largely unknown, although a likely candidate is brain‐derived neurotrophin (BDNF). Many of the effects of estradiol in the hippocampus are mirrored by BDNF or have a demonstrated requirement for activation of the cognate BDNF receptor, TrKB (Scharfman and MacLusky, 2006 ). Numerous studies indicate a role for BDNF in regulating neurogenesis. For example, administration of BDNF to adult hippocampus increases cell proliferation (Scharfman et al., 2005 ), while knockdown of BDNF or conditional deletion of TrKB reduces proliferation (Lee and Son, 2009 ; Taliaz et al., 2010 ). BDNF also negatively impacts cell survival via p75 NTR receptor signaling (Catts et al., 2008 ).

Previous studies in our laboratory have characterized a robust sex difference in cell genesis in the neonatal hippocampus of rats that is modulated by gonadal steroids (Zhang et al., 2008 ; Bowers et al., 2010 ; Waddell et al., 2013 ). During the first postnatal week, males have twice as many proliferating cells in the dentate gyrus and CA1 regions of the hippocampus compared with females, and this difference is not due to altered rates of cell death. Although estradiol administration to neonatal females increases the number of proliferating cells in dentate and CA1 to levels seen in males, exogenous estradiol does not increase cell genesis in males. Conversely, disruption of estrogen signaling in neonatal males decreases cell proliferation, but has no effect in females (Bowers et al., 2010 ). The effects of estradiol on developmental cell genesis in the hippocampus are rapid, occurring within hours, and yet also persistent, as indicated by the number of proliferating cells that survive and differentiate (Zhang et al., 2008 ; Bowers et al., 2010 ). Up to 80% of new cells in the neonatal hippocampus of males or estradiol‐treated females will differentiate into neurons, whereas only 40% will do so in untreated females (Bowers et al., 2010 ). Thus, estradiol promotes both proliferation and differentiation of new neurons and thereby has an important role in organizing the hippocampal circuitry in a sex‐specific manner.

The hippocampus is critically involved in contextual learning and stress responding, and as such plays a vital role in both complex and innate behaviors (Sweatt, 2004 ; Fanselow and Dong, 2010 ). Aspects of learning and stress responding differ in males and females across the life span (Weinstock, 2007 ; Schoenfeld and Gould, 2012 ; Mahmoud et al., 2016 ), and disorders in which hippocampal dysfunction is implicated, such as depression, anxiety, or schizophrenia, differ between the sexes in terms of prevalence and/or presentation (McLean et al., 2011 ; Schoenfeld and Cameron, 2015 ). While evidence suggests that many of these differences are secondary to the developmental organizational effects of gonadal steroids, the mechanisms by which this would occur are not known.

Quantitative PCR (qPCR) and ELISA data from untreated animals were analyzed by two‐factor analysis of variance (ANOVA) with sex and region as independent variables, followed by pairwise post hoc comparisons using Bonferroni correction. qPCR data from animals treated with estradiol and tamoxifen were analyzed using two‐factor ANOVA with sex and treatment as independent factors and Bonferroni post hoc comparisons. ELISA data from animals treated with estradiol and tamoxifen were analyzed using the Kruskal–Wallis test of ranks. Western immunoblotting data from treated and untreated animals were analyzed using single‐factor ANOVA across groups for each hippocampal region with Tukey multiple‐comparisons posttest. All data were first analyzed using the Kolmogorov–Smirnov test for normalcy and the Bartlett test for homogeneity of variances. Significance was set at P < 0.05 for all analyses.

Protein (12 μg) from tissue homogenates was resolved on 10% to 20% gradient polyacrylamide gels (NuPage, LifeTechnologies) under reducing and denaturing conditions, and transferred to polyvinylidene difluoride membrane. Membranes were blocked with Odyssey Blocking Buffer (LiCor Biosciences) diluted 1:1 in Tris‐buffered saline (TBS; pH 7.4) prior to incubation overnight at 4 °C in Blocking Buffer diluted 1:1 in TBS‐Tween (0.05%) containing primary antibodies against BDNF (rabbit IgG against the middle portion of human BDNF, Aviva Systems Biology, Cat# ARP41970_P050; RRID:AB_10644597; diluted 1:800) and GAPDH (mouse monoclonal, Abcam, Cat# ab9484; RRID:AB_307274; diluted 1:5,000). Membranes were washed three times in TBS‐Tween (0.05%) and incubated in Blocking Buffer diluted 1:1 in TBS‐Tween/0.02% SDS containing secondary antibodies (IRDye©800 goat‐anti‐rabbit and IRDye©680 goat‐anti‐mouse IgG, LiCor Biosciences; both diluted 1:5000). After washing, membranes were imaged on an Odyssey CLx Infrared Imaging System (LiCor). Fluorescence intensity for each BDNF band was normalized to fluorescence intensity of the corresponding GAPDH band to obtain an integrated fluorescence value.

Frozen tissues were homogenized in lysis buffer containing protease and phosphatase inhibitors, as described above. Total free BDNF propeptide was measured using the BDNF E max Immunoassay System (Promega), according to the manufacturer's protocol. Tissue homogenates were diluted 1:5 in PBS (pH 7.4), acid treated at pH 3.5 for 15 min by the addition of 2N HCl, and neutralized with NaOH prior to dilution in Block & Sample Buffer. According to the manufacturer, this enzyme‐linked immunosorbent assay (ELISA) has a sensitivity of 15.6 pg/ml and exhibits less than 3% cross‐reactivity to other neurotrophins.

Total RNA was isolated from tissue homogenates (untreated animals) or frozen tissues (animals treated in estradiol experiments) using a modified phenol extraction method, according to the manufacturer's instructions (TriReagent RT, MRC Inc.). cDNA from hippocampal subregions was transcribed from 1 μg total RNA using a High‐Capacity cDNA Reverse Transcription Kit and random hexamers (LifeTechnologies). cDNA products were diluted 1:3, and 5 μl was used for fluorescence‐based, real‐time polymerase chain reaction (PCR) in a reaction mixture containing SYBR© Green PCR Master Mix (LifeTechnologies) and 250 nM primers. PCR reactions were performed for 40 cycles on a ViiA°7 Real‐Time PCR System (ABI). PCR primers for BDNF expression targeted the coding region of BDNF exon IX and were designed to quantify all transcripts. BDNF transcripts were normalized to Gapdh as a proxy for total cellular RNA content, and expression among groups was calculated using the ΔΔC t method (Pfaffl, 2001 ). Data were expressed as mean fold expression relative to females ± standard error of mean fold expression. Mean C t values for Gapdh differed by less than 0.2 C t between males and females for all three hippocampal regions. Primer sequences were as follows:

To dissect individual subregions of the hippocampal formation, brains were bisected sagittally in ice‐cold phosphate‐buffered saline (PBS; pH 7.4), and thalamic tissue was removed to expose the ventricular surface of the hippocampus. The entire dentate gyrus was removed as a discrete structure by inserting a fine‐gauge needle along the length of the hippocampal fissure. Fiber projections along the dentate axis proximal to CA3 were removed with fine forceps. Ammon's horn was dissected from the neocortical tissue, and meninges were removed using fine forceps. CA1 and CA3 areas were separated along the length of the hippocampus. Tissues from untreated animals were homogenized in lysis buffer consisting of 10 mM Tris‐HCl pH 7.6, 150 mM NaCl, 1% Nonidet P‐40, 1% sodium deoxycholate, and containing protease and phosphatase inhibitors (Sigma). A portion of the homogenate was frozen for subsequent analysis of BDNF peptide content. The remaining homogenate was used for isolation of total RNA. In this way, both protein and total RNA were obtained from the same cohort of untreated animals. Tissues were collected from seven animals of each sex at PN4 and PN15. For pups receiving drug treatments, estradiol benzoate (Sigma) or tamoxifen (Sigma) was administered at 100 μg in 0.1 ml sesame oil subcutaneously on PN0 and PN1. An equivalent volume of sesame oil was similarly administered as vehicle control. This dose of estradiol and tamoxifen is necessary to overcome circulating steroid hormone–binding globulins present in the neonatal rat, and has previously been shown to elicit sexual differentiation of the rodent brain, including sex‐specific effects in the developing hippocampus (Mong et al., 1999 ; Amateau et al., 2004 ; Bowers et al., 2010 ). Hippocampal subregions of treated animals were collected on PN4 and frozen for analyses of gene or protein expression ( n = 5–7 per group).

All procedures involving experimental animals were approved by the Institutional Animal Care and Use Committee of the University of Maryland School of Medicine. Sprague‐Dawley rat pups were obtained from dams mated in the University of Maryland School of Medicine Animal Care Facility and housed in a 12‐hr light/dark cycle. The day of birth was designated postnatal day 0 (PN0). On postnatal day 4 (PN4), subregions of the hippocampal formation were dissected, frozen on dry ice, and stored at −80 °C until subsequent processing. For each hippocampal subregion, tissue was combined from both hemispheres of the brain of an individual pup.

As shown in Figures 3 and 4 , BDNF peptide levels did not differ in vehicle‐treated males and females on PN4. Western immunoblotting was used to test for baseline sex differences in BDNF peptide content in hippocampal subregions during the first postnatal week using the same cohort of untreated PN4 animals used to demonstrate a sex difference in BDNF gene expression. As with the hippocampal homogenates from animals treated with estradiol and tamoxifen, a single band corresponding to the 28‐KDa BDNF propeptide was detected. Two‐factor ANOVA confirmed no effect of sex on BDNF propeptide content among the three hippocampal subregions at postnatal day 4 ( P = 0.9091), nor were there regional differences in propeptide content ( P = 0.9389; Fig. 5 A). No differences in BDNF propeptide content were seen between males and females (two‐factor ANOVA, P = 0.5086) or among the three hippocampal subregions (two‐factor ANOVA, P = 0.0551) from untreated animals at postnatal day 15 (Fig. 5 B). To confirm the results from Western immunoblotting, BDNF peptide content in dentate and CA1 was additionally assessed by ELISA in the same samples. Two‐factor ANOVA confirmed there was no effect of sex on BDNF peptide content in either region ( P = 0.7120; Fig. 6 ). A significant main effect of hippocampal subregion on BDNF peptide levels was indicated (F[1,26] = 58.45, n = 7, P < 0.0001). BDNF peptide was higher in the dentate of both sexes, compared with CA1, recapitulating the relative regional abundances for BDNF transcript (Fig. 1 A).

To examine the effect of estradiol on BDNF peptide levels, a third cohort of animals was treated on PN0 and PN1 with estradiol benzoate (female pups only), tamoxifen (males only), or vehicle. Because hippocampal cell genesis is not altered by estrogen treatment in males or tamoxifen treatment in females, these two groups were not included in this cohort in an effort to limit the number of experimental animals. BDNF peptide levels were determined in hippocampal subregions collected on PN4 using a commercially available ELISA, and reported as picograms of BDNF peptide per milligram of total protein (Fig. 3 ). A Bartlett test indicated inhomogeneity of variance among treatment groups within the dentate, so means were compared within the three hippocampal subregions using nonparametric ANOVA. No differences among groups within the dentate ( P = 0.0508; Fig. 3 A), CA1 ( P = 0.1463, Fig. 3 B), or CA3 ( P = 0.1598; Fig. 3 C) regions were indicated using the Kruskal–Wallis test by ranks.

Regional effects of estradiol and tamoxifen on BDNF gene expression in postnatal hippocampus, as determined by real‐time qPCR at PN4. Transcript levels were normalized to Gapdh and expressed relative to vehicle‐treated females within each region. A : In dentate, estradiol decreases BDNF transcripts in both sexes, while tamoxifen decreases BDNF transcripts in males only (ANOVA, * P < 0.05, ** P < 0.01 compared with same‐sex vehicle control). B : In CA1, estradiol increases BDNF gene expression in both sexes, while tamoxifen has no effect (ANOVA, *** P < 0.0001, compared with same‐sex vehicle control). BDNF expression was higher in vehicle‐treated males compared with vehicle‐treated females in both regions (ANOVA, # P < 0.05). n = 5–6 animals per treatment group for each sex.

To determine if estrogen signaling influences BDNF expression in the developing hippocampus, a second cohort of animals was treated systemically with estradiol benzoate, tamoxifen, or vehicle on the day of birth and 24 hr later, and BDNF transcripts were quantified in dentate and CA1 regions on PN4 via qPCR. Within the dentate, two‐factor ANOVA for sex and treatment indicated a significant main effect of treatment ( F [2,31] = 13.42, n = 5–6, P < 0.0001). Post hoc pairwise comparisons confirmed that BDNF expression was significantly increased by estradiol treatment in both males ( P < 0.01) and females ( P < 0.05) compared with vehicle controls (Fig. 2 A). Surprisingly, tamoxifen treatment also decreased BDNF expression in the dentate of males ( P < 0.05), to the same degree as estradiol, but had no effect in females ( P > 0.05). A significant interaction between sex and treatment in the dentate was indicated ( F [2,31] = 6.870, n = 5–6, P = 0.0040); however, a main effect of sex was not ( F [1,31] = 3.478, n = 5–6, P = 0.0735), most likely because mean values for estradiol and tamoxifen treatments did not differ between males and females ( P > 0.05). BDNF expression was significantly higher in vehicle‐treated males vs. females ( P < 0.01), recapitulating the sex difference observed in untreated animals.

Relative abundance of BDNF transcripts in the dentate gyrus, CA1, and CA3 regions of the hippocampal formation of Sprague‐Dawley rats at postnatal day 4 (PN4) and postnatal day (PN15), as determined by real‐time qPCR. Transcript levels are normalized to Gapdh transcripts and expressed as mean transcript abundance ± standard error of mean, relative to female dentate gyrus. A : On PN4, males have higher levels of BDNF transcripts in dentate gyrus and CA1 compared with females (ANOVA, ** P < 0.01, * P < 0.05). Regional differences in BDNF expression were also seen at PN4, where more BDNF transcripts were seen in dentate compared with CA1 or CA3 (ANOVA, # P < 0.01, males only compared with male dentate; @ P < 0.0001, both sexes, compared with dentate. B : No sex differences in BDNF transcripts seen at PN15. Greatest regional expression in BDNF was seen in dentate gyrus (ANOVA, # P < 0.01, @ P < 0.0001, compared with dentate). n = 7 animals per sex.

DISCUSSION

In this study, we examined BDNF expression in the dentate gyrus, CA1, and CA3 regions of the hippocampal formation in male and female rats during the first postnatal week. We used a qPCR assay targeted to the exon IX coding sequence of the BDNF gene to quantify total levels of all BDNF transcripts, and found a baseline sex difference in BDNF gene expression. Males had roughly 50% more BDNF transcripts in the dentate gyrus and CA1, but not CA3, regions of the hippocampus during the first postnatal week. This sex difference was not detected at postnatal day 15. Intriguingly, the sex difference in BDNF transcripts on postnatal day 4 mirrored cell proliferation in these regions of the hippocampus, where males produce roughly twice as many new cells in the dentate and CA1 at this time (Zhang et al., 2008; Bowers et al., 2010; Lima et al., 2014). However, the relationship between BDNF and cell genesis in the neonatal hippocampus is likely to be complex and region‐specific, as estradiol positively regulates cell proliferation in both the dentate and CA1 (Bowers et al., 2010; Zhang et al., 2008) but had opposite effects on BDNF expression between these two regions in the present study (Fig. 2).

In contrast to long‐standing interest in the role of BDNF in hippocampal plasticity and neurogenesis in adults, particularly with regard to effects of estrogen, far fewer studies have examined BDNF expression in the developing hippocampus. Damborsky and Winzer‐Serhan (2012) reported that neonatal male rats have higher hippocampal BDNF gene expression in dentate, CA1, and CA3 subregions at PN5, but not PN8. This is largely consistent with our observations with the exception of CA3, where we did not detect sex differences. This discrepancy may be due to methodological differences, as Damborsky and Winzer‐Serhan used in situ hybridization autoradiography to determine relative BDNF transcript levels, and the present study measured transcripts via qPCR in dissected subregions of the hippocampus.

One of the first studies to examine the ontogeny of BDNF expression in the developing hippocampus determined BDNF transcript and peptide levels in neonatal male rats in relation to gonadal steroids. Castration on the day of birth decreases BDNF gene expression for the first two postnatal weeks in CA1 and CA3, yet a single dose of estradiol on the day of birth in castrated animals restores BDNF transcript levels (Solum and Handa, 2002). This demonstrates that not only does estradiol upregulate BDNF expression in Ammon's horn of the developing hippocampus, but the duration of the effect of estradiol suggests that BDNF expression during the first two weeks can be primed by the perinatal surge in gonadal steroids. In the present study, we manipulated estradiol signaling in neonatal rats using a paradigm of systemic administration of estradiol benzoate or the estrogen receptor antagonist tamoxifen. The main source of estrogen in the neonatal brain of rodents is the aromatization of circulating testosterone in males (Naftolin et al.,1971, 1975), although de novo synthesis of estradiol does occur in various brain areas of both sexes (Amateau et al., 2004), and in particular the hippocampus (Mukai et al., 2006; Hojo et al., 2011). The developing brain is shielded from the effects of maternal estradiol by α‐fetoprotein present in the neonatal circulation (Bakker et al., 2006). The sequestering effects of this steroid‐binding globulin can be overcome with a relatively high dose of exogenous estradiol, and this has been titrated to achieve effects in females that mimic endogenous estradiol in males (Amateau et al., 2004). Using a masculinizing dose of estradiol, we observed an upregulation of BDNF transcripts in CA1 in male and female neonates, similar to what was seen in males by Solum and Handa (2002). Interestingly, in the dentate of both sexes we observed the opposite effect of estradiol, where BDNF gene expression was decreased. These opposing effects of estradiol between the two regions of the hippocampus may be explained by regional differences in estrogen receptor subtype and cellular localization. In rats, ERα mRNA and protein are upregulated in CA1 during the first two weeks postnatally, and immunocytochemistry localizes ERα to nuclei of pyramidal neurons (O'Keefe and Handa, 1990; Ivanova and Beyer, 2000; Solum and Handa, 2001). BDNF is expressed in pyramidal neurons of the CA1; its synthesis and secretion are promoted by estradiol in parallel with CREB activation (Zhou et al., 2005), and a functional estrogen response element is found in the BDNF gene (Sohrabji et al.,1995). Together, these findings suggest that the observed upregulation of BDNF gene expression in pyramidal neurons of neonatal CA1 occurs via classical genomic action of estrogen receptors. In the neonatal dentate gyrus, however, extranuclear ERβ is localized to the plasma membrane of immature granule neurons and also glia (Herrick et al., 2006), raising the possibility that estradiol may have nongenomic effects on BDNF expression from these cell types in the dentate.

Although exogenous estradiol elicited opposite effects on BDNF expression in dentate and CA1, these effects were nevertheless the same between neonatal males and females. In contrast, antagonizing endogenous estradiol signaling at ERα and ERβ with tamoxifen appeared to have a sex‐specific effect in the dentate gyrus, but not CA1. Tamoxifen had no effect on BDNF transcript levels in female dentate, but in males tamoxifen decreased BDNF gene expression to the same level as in females. Because the 4‐hydroxy metabolite of tamoxifen has significantly more affinity for the estrogen receptor than the drug itself (Fabian et al., 1981), the possibility is raised that the differential effects of tamoxifen in neonatal males and females may be due to sex differences in tamoxifen metabolism. However, the enzymes of the cytochrome P450 system that metabolize tamoxifen are present from birth in the rodent liver and, although low during the neonatal period, show a similar pattern of expression in males and females (Hart et al., 2009; Cui et al., 2012). In addition, effects of exogenous estradiol administration in the brains of female neonatal rodents are blocked by systemic administration of tamoxifen (Hilton et al., 2004; Gonzales et al., 2012). Together, these findings indicate that tamoxifen is able to antagonize estrogen receptors similarly in neonatal males and females, and the effects on BDNF expression in the dentate observed in this study are sex‐specific. At first glance, this suggests that the baseline sex difference in BDNF expression in dentate is promoted by estradiol. However, since estradiol and androgen content in the hippocampus at this age is equivalent in males and females (Amateau et al., 2004; Konkle and McCarthy, 2011), there must be sex‐specific mechanisms downstream of estradiol signaling that mediate this difference.

One possible mechanism is the depolarizing action of GABA. In the adult brain, GABA is the main inhibitory neurotransmitter, but immature neurons are depolarized in response to GABA A receptor activation (Represa and Ben‐Ari, 2005). This results in numerous trophic effects in the developing brain, proximally mediated by an influx of Ca2+, which results in activation of the transcription factor CREB. Depolarizing GABA upregulates BDNF gene expression and peptide content in developing neurons in a CREB‐dependent manner (Berninger et al., 1995; Shieh et al., 1998; Obrietan et al., 2002). Sex differences in CREB content and the depolarizing actions of GABA are found in the neonatal hippocampus of rats. Males respond to GABA A receptor activation with a longer duration of calcium influx in a greater percentage of the neuronal population (Nuñez and McCarthy, 2007, 2009; Galanopoulou, 2008) and also have more activated CREB compared with females (Auger et al., 2001; Perrot‐Sinal et al., 2003). Moreover, both the depolarizing actions of GABA and activated CREB content are positively regulated by estradiol (Nugent et al., 2012; Nuñez et al., 2005; Nuñez and McCarthy, 2009). The baseline sex differences in depolarizing GABA and CREB suggest a mechanism through which greater BDNF expression is achieved in the neonatal hippocampus of males despite equivalent estradiol content between the sexes.

A surprising outcome of this study was the discordance between BDNF transcript and peptide levels. Although the ELISA captured relative differences between dentate and CA1 regions that mirrored the results from qPCR, it did not detect any differences in peptide content between males and females, or in response to estradiol, and this was confirmed with Western immunoblotting. BDNF is synthesized and secreted as a glycosylated precursor propeptide of approximately 28 KDa, which is proteolytically cleaved to yield a mature peptide of 14 KDa (Mowla et al., 2001). Both the ELISA and the anti‐BDNF antibody we used for Western immunoblotting are able to detect the 28‐KDa propeptide and the mature form of BDNF, and were intended to measure total translational output, although we only detected a band corresponding to the 28‐KDa propeptide in our Western immunoblots. The abundance of BDNF precursor peptide relative to the mature form is greatest in the hippocampus of neonatal and juvenile animals, due to developmentally regulated expression of tissue plasminogen activator, which is essential for zymogen activation of the protease which cleaves proBDNF to the mature peptide (Pang et al., 2004; Yang et al., 2009). The amount of mature BDNF found in the hippocampus of mice during the first 15 days postnatally is extremely low (Yang et al., 2014), and therefore our inability to quantify mature BDNF is likely due to insufficient sensitivity in our Western immunoblots. Previous studies have noted differences between BDNF transcript levels and peptide content. For example, Gibbs (1999) found that estradiol treatment of ovariectomized female rats increased BDNF transcripts but had no effect on peptide levels in the hippocampus. In the neonatal male rat, castration induces a significant increase in BDNF mature peptide, in opposition to transcripts (Solum and Handa, 2002). More recently, Hill et al (2014) found that adolescent stressed male rats that had experienced maternal deprivation have lower levels of hippocampal BDNF gene expression compared with unstressed animals, but higher levels of the mature BDNF peptide, while no changes are observed in the BDNF propeptide. Females undergoing maternal separation and adolescent stress also exhibit lower levels of mature BDNF peptide in the hippocampus, but no changes in propeptide or transcript levels. The BDNF propeptide and mature form can have opposing effects on several aspects of neuronal development, including cell proliferation (Hempstead, 2006). While BDNF increases cell genesis, as noted above, BDNF propeptide can promote apoptosis through preferential activation of p75NTR, rather than TrKB receptors, thereby decreasing cell genesis (Teng et al., 2005). We did not quantify mature BDNF in this study, but given the potential for sex‐specific regulation of proBDNF cleavage, and the opposing effects of proBDNF and mature BDNF on cell genesis, this is worth future study.

In summary, we have found sex‐ and region‐specific differences in the relationship between BDNF gene expression and estradiol in the neonatal hippocampus. Further studies that examine how this relationship is differentially mediated among the hippocampal subregions, as well as a more detailed analysis of the effects of sex and estradiol on BDNF propeptide processing, may identify BDNF as a key factor in sexual differentiation of the developing hippocampus.