The Histamine H1 Receptor Participates in the Increased Dorsal Telencephalic Neurogenesis in Embryos from Diabetic Rats

Increased neuron telencephalic differentiation during deep cortical layer formation has been reported in embryos from diabetic mice. Transitory histaminergic neurons within the mesencephalon/rhombencephalon are responsible for fetal histamine synthesis during development, fibers from this system arrives to the frontal and parietal cortex at embryo day (E) 15. Histamine is a neurogenic factor for cortical neural stem cells in vitro through H1 receptor (H1R) which is highly expressed during corticogenesis in rats and mice. Furthermore, in utero administration of an H1R antagonist, chlorpheniramine, decreases the neuron markers microtubuline associated protein 2 (MAP2) and forkhead box protein 2. Interestingly, in the diabetic mouse model of diabetes induced with streptozotocin, an increase in fetal neurogenesis in terms of MAP2 expression in the telencephalon is reported at E11.5. Because of the reported effects on cortical neuron differentiation of maternal diabetes in one hand and of histamine in the other, here the participation of histamine and H1R on the increased dorsal telencephalic neurogenesis was explored. First, the increased neurogenesis in the dorsal telencephalon at E14 in diabetic rats was corroborated by immunohistochemistry and Western blot. Then, changes during corticogenesis in the level of histamine was analyzed by ELISA and in H1R expression by qRT-PCR and Western blot and, finally, we tested H1R participation in the increased dorsal telencephalic neurogenesis by the systemic administration of chlorpheniramine. Our results showed a significant increase of histamine at E14 and in the expression of the receptor at E12. The administration of chlorpheniramine to diabetic rats at E12 prevented the increased expression of βIII-tubulin and MAP2 mRNAs (neuron markers) and partially reverted the increased level of MAP2 protein at E14, concluding that H1R have an important role in the increased neurogenesis within the dorsal telencephalon of embryos from diabetic rats. This study opens new perspective on the participation of HA and H1R receptor in early corticogenesis in health and disease.

Increased neuron telencephalic differentiation during deep cortical layer formation has been reported in embryos from diabetic mice. Transitory histaminergic neurons within the mesencephalon/rhombencephalon are responsible for fetal histamine synthesis during development, fibers from this system arrives to the frontal and parietal cortex at embryo day (E) 15. Histamine is a neurogenic factor for cortical neural stem cells in vitro through H 1 receptor (H 1 R) which is highly expressed during corticogenesis in rats and mice. Furthermore, in utero administration of an H 1 R antagonist, chlorpheniramine, decreases the neuron markers microtubuline associated protein 2 (MAP2) and forkhead box protein 2. Interestingly, in the diabetic mouse model of diabetes induced with streptozotocin, an increase in fetal neurogenesis in terms of MAP2 expression in the telencephalon is reported at E11.5. Because of the reported effects on cortical neuron differentiation of maternal diabetes in one hand and of histamine in the other, here the participation of histamine and H 1 R on the increased dorsal telencephalic neurogenesis was explored. First, the increased neurogenesis in the dorsal telencephalon at E14 in diabetic rats was corroborated by immunohistochemistry and Western blot. Then, changes during corticogenesis in the level of histamine was analyzed by ELISA and in H 1 R expression by qRT-PCR and Western blot and, finally, we tested H 1 R participation in the increased dorsal telencephalic neurogenesis by the systemic administration of chlorpheniramine. Our results showed a significant increase of histamine at E14 and in the expression of the receptor at E12. The administration of chlorpheniramine to diabetic rats at E12 prevented the increased expression of βIII-tubulin and MAP2 mRNAs (neuron markers) and partially reverted the increased level of MAP2 protein at E14, concluding that H 1 R have an important role in the increased neurogenesis within the dorsal telencephalon of embryos from diabetic rats. This study opens new perspective on the participation of HA and H 1 R receptor in early corticogenesis in health and disease.
Maternal diabetes may affect neural stem cells (NSC) proliferation, migration, differentiation, and survival. These effects can, in turn, lead to cytoarchitectonic defects that affect neural development and, consequently, to the impairment of diverse CNS functions. The type and extent of these disturbances will depend on which anatomic structure is affected and the time window in which the insult occurs during neural tube development. Indeed, several studies have shown that high glucose levels lead to abnormal NSC death, proliferation, and cell-fate choice both in vivo and in vitro (Liao et al., 2004;Fu et al., 2006;Chatzigeorgiou et al., 2009;Pavlinkova et al., 2009;Xu et al., 2013). Fu et al. (2006) reported increases in both neuron and glial differentiation in embryos from diabetic mice within the ventral and dorsal telencephalon at embryo day (E) 11.5 and in NSCs treated with a high glucose concentration in vitro. These changes were attributed to the increased expression of the neurogenic factors neurogenin1 (Ngn1), Ngn2, and Achaete-Scute family basic helix-loop-helix (bHLH) transcription factor 1 (Mash1). These authors also reported enhanced expression of Sonic Hedgehog, which may promote neurogenesis through decreased expression of Hes family bHLH transcription factor 1 (Hes1) and Hes5. In contrast, Xu et al. (2013) reported delayed and decreased neurogenesis during neural tube development (E8) in the same model.
During cortical development in mice, it is estimated that the birth of deep cortical layer neurons begins at E10.5 and ends at E14.5 (Angevine and Sidman, 1961;Gaspard et al., 2008Gaspard et al., , 2009Sansom and Livesey, 2009), while in the rat the neurogenesis starts at E12 (deep cortical layer birth; Valverde et al., 1989;Bayer and Altman, 1991).
In rat, histamine (HA) is one of the first neurotransmitters to be detected in CNS, with the highest concentration at E14 and E16 (Vanhala et al., 1994), coinciding with the peak of neuron differentiation in the cerebral cortex (Gaspard et al., 2008(Gaspard et al., , 2009Sansom and Livesey, 2009). Fibers from the transient histaminergic neurons in the mesencephalon can be observed through the ventral tegmental area, within the medial forebrain bundle and the optic tract, reaching the frontal and the parietal cortex at E15, earlier than other monoaminergic systems (Specht et al., 1981;Lidov and Molliver, 1982;Auvinen and Panula, 1988;Reiner et al., 1988;Vanhala et al., 1994).
Given the emerging knowledge on the role of HA in neuron differentiation and the effect of hyperglycemia on neurogenesis, here we investigated whether the levels of HA and/or the expression of the H 1 R increases in embryos from diabetic rats during early corticogenesis and if these play a role in the increased neurogenesis in the dorsal telencephalon at E14.

MATERIALS AND METHODS
Wistar rats (250-300 g) from (INB-UNAM) were maintained in our animal facilities, housed individually and maintained in standard conditions (12:12 h light/dark cycle, 21 ± 2 • C and 40% relative humidity) with free access to food and water were used. A vaginal smear was performed to confirm the presence of spermatozoids the morning after mating, and this time point was defined as E0.5. All experiments followed both the National Institutes of Health (NIH, USA) "Guide for the Care and Use of Laboratory Animals (NIH Publication No. 80-23, revised 1978)" and the "Norma Official Mexicana para la Producción Cuidado y Uso de Animales de Laboratorio" (NOM-062- ZOO-1999). The accepted protocol number received from the institutional research, biosecurity and ethic committees was 3230-21202-01-2015.

Diabetes Induction and Antagonist Treatment
At day 5 of pregnancy, pregnant rats received a single intraperitoneal injection of either a buffered citrate solution (vehicle; pH 7.4) for control rats or streptozotocin (STZ; Sigma-Aldrich, St. Louis, MO, USA; body weight: 50 mg/kg) for experimental rats (diabetic rats). From 24 h after vehicle or STZ injection until sacrifice the glucose level was measured daily using a drop of blood taken from the tail vein and a glucometer (ACCU-Chek Performa, Roche Diagnostics, Basel, Switzerland). Rats with glucose levels above 200 mg/dl were included in the diabetic group, while animals with <200 mg/dl levels were discarded.
To explore the possible role of H 1 R in the increased neuron differentiation in embryos from diabetic rats, we performed a series of experiments, where injectable water (vehicle; Laboratorios PiSA S. A. de C. V., GDL. Jal. MEX) or 5 mg/kg of the H 1 R antagonist chlorpheniramine (Sigma-Aldrich; Naranjo and de Naranjo, 1968) was intraperitoneally administrated to control and diabetic pregnant rats, at E12, and evaluated at E14.

Embryonic Tissue Processing
To determine the HA and H 1 R levels, we sacrificed pregnant rats by decapitation at E12, E14, E16, E18, and E20. The embryos were extracted, and the total and reabsorbed embryos were registered.

HA Level Measurement Using Enzyme-Linked Immunosorbent Assay (ELISA)
We used the ELISA technique (sensitivity: 0.2 ng/ml; 100% HA specificity) to determine the HA level following the protocol recommended by the supplier (ALPCO R immunoassays, Salem, NH, USA). Tissues from the dorsal telencephalon (E12-E20), placenta (E14) and serum of pregnant rat (E14; 50 µl) were used.
The embryo or placenta tissue was homogenized in 100 µl of cold PBS (pH 7.4; POLYTRON PT 2100 Homogenizer, Kinematica, Switzerland) and centrifuged at 10,500 × g for 5 min. Then 50 µl of the supernatant (or serum) was used to determine the HA concentration.
The absorbance values were measured at 495 nm in a multiple detection system (GLOMAX, Promega, Madison, WI, USA). The HA concentration was determined using a reference curve constructed using 0, 0.5, 1.5, 5, 15, and 50 ng/ml HA, and the results were corrected by the amount of protein per sample and expressed as molarity. The protein concentration was determined using the Bradford method (Bradford, 1976).

Quantitative Reverse-Transcription Polymerase Chain Reaction (qRT-PCR)
qRT-PCR was performed to analyze the temporal expression of H 1 R and histidine decarboxylase (HDC; EC 4.1.1.22, enzyme responsible for convert histidine to histamine) as the relative expression to E12 and differences between groups in terms of the relative expression to the control of H 1 R, HDC, neurogenic factors (Prox1 and Ngn1), and neuron markers (βIII-Tub and MAP2). The dorsal cortical neuroepithelium (for H 1 R, Prox1, Ngn1, βIII-Tub, and Map2) or mesencephalon/rhombencephalon neuroepithelia (for HDC) were obtained and immediately stored at −80 • C until use.
Total RNA from E12-E14 (4 epithelia per experiment) and E16-E18 (2 epithelia per experiment) cortical neuroepithelia were isolated using TRIZOL reagent (Invitrogen). The RNA integrity was determined by visualizing 18S and 28S ribosomal RNA stained with ethidium bromide (0.2 mg/ml) in 2% agarose gel. One microgram of RNA was used for the retrotranscription reaction with 0.5 µg of oligo-dT, 1 mM dNTPs, 0.2 mM dithiothreitol (DTT), 1U of RNase inhibitor, and 15U of Super Script R III (Invitrogen). The reaction was incubated at 25 • C for 5 min and then at 50 • C for 1 h, and the reaction was stopped at 70 • C for 15 min. Dynamic ranges were measured for each gene before the qPCR analysis to determine the fluorescence threshold and reaction efficiency.
PCR analysis was performed using 800 ng (H 1 R) or 400 ng (other mRNAs) of cDNA, 20 pmol of forward (F) and reverse (R) primers, and the commercial KAPA TM SYBER FAST R qPCR mix (KAPA Biosystems, Wilmington, MA, USA) with a Rotor-Gene 6000 thermocycler (Qiagen, Germantown, MD, USA). The amplification conditions were as follows: 95 • C for 10 min, followed by 35 cycles at 56 • C (H 1 R and glyceraldehyde 3-phosphate dehydrogenase [GAPDH]) or 62 • C (Prox1, Ngn1, βIII-Tub, and Map2) for 15 s, and a final amplification at 72 • C for 30 s. The relative fold changes (relative expression) were determined using the mathematical algorithm 2 − CT , where CT is the cycle threshold (Livak and Schmittgen, 2001). GAPDH was used as the internal control to obtain CT values per sample. After PCR was performed, denaturation curves were calculated to confirm the amplification of a single product (Kubista et al., 2006).
Adult rat cerebral cortex or U373MG (CVCL_2219) cell cDNA was used as the positive control, and U373MG was transfected with a rat Hrh1 siRNA-Smart-pool siRNA (Accell, GE Healthcare, Chicago, IL, USA) as a negative control for H 1 R amplification. Endpoint PCR and electrophoresis in 2% agarose gel were performed to verify the size of the products with GelRed (Biotium, Inc., Fremont, CA, USA). Finally, fragments were purified for sequencing.
GAPDH or actin were used as the internal controls. Because a non-specific band was observed in the H 1 R immunoblots and to ensure H 1 R detection, a cell line that highly expresses H 1 R, the U373MG glioblastoma cells were transfected with H 1 R siRNA-Smart-pool or scramble sequences (Accell, GE Healthcare) were used as negative or positive controls, respectively ( Figure 5B).

Enzymatic Activity
A modified method was used to measure the HDC activity (Keeling et al., 1984;Barnes and Hough, 2002;Shoji et al., 2006). Five hundred milligrams of mesencephalon/rhombencephalon tissue (where fetal histaminergic transitory neurons reside) was obtained from E14 and E16, or from placenta at E14. The tissues were homogenized in 2.5 ml of cold PBS (0.02 M, pH 6.2) containing 20 µM pyridoxal phosphate, 200 µM DTT, and 25 mg phosphorylated cellulose (pH 7.5). The homogenate was then centrifuged at 10,000 × g at 4 • C for 15 min, and the supernatant was diluted 1:1 with the enzyme reaction solution (0.1 M PBS pH 6.7, 50 mM pyridoxal phosphate, 100 mM aminoguanidine, 2 mM of L-histidine as a substrate, and 30 µM of histamine N-methyltransferase inhibitor SKF 91488). The mixture was incubated at 37 • C for 5 min, and the reaction was terminated by the addition of 0.4 ml of perchloric acid (2.8 M).
The synthesized HA was separated by ion-exchange chromatography using a phosphorylated cellulose support. HA was eluted with perchloric acid (2.8 M) and quantified by ELISA.

Experimental Design and Statistical Analysis
Sample size was estimated for independent samples and studies analyzed by t-test with the power and sample size program (Casagrande et al., 1978;Dupont and Plummer, 1990) using the following parameters: power = 0.9, α = 0.05, δ = 1, σ = 0.7 and m = 3. A minimum of four experiments per group were included.
Unpaired t-test was performed for comparison between control and diabetic. For temporal analysis and comparisons between control, chlorfeniramine, diabetic, and diabetic+chlorpheniramine groups ANOVA followed the uncorrected Fisher's LSD multiple comparison test were performed. Statistical analysis and graphic creation were performed in GraphPad Prism version 6.0 (GraphPad Software, Inc., La Jolla, CA, USA).

RESULTS
The glucose levels in control embryos were lower than those in pregnant rats, and "normal" glycemic was not reached until E18, in contrast, diabetic embryos showed significant increased blood glucose levels in all days evaluated with respect diabetic pregnant rats. As expected, glycemic changes in embryos from the diabetic group presented significantly higher glucose levels than the control group embryos during all stages of development, exhibiting values >200 mg/dl starting at E14 ( Table 1).

Embryos from Diabetic Rats Presented Increased Neuron Differentiation
Increased dorsal telencephalic neurogenesis in diabetic rat embryos was evidenced by immunohistochemistry and Western blot analyses. In control embryos, MAP2 immunocytochemistry was localized in the cortical plate, and βIII-Tub was observed in the cortical plate and subplate (Figures 1A-G). In addition to a disorganized epithelium making it difficult to distinguish the layers at the time of development evaluated, the distributions of both neuron markers were altered in the diabetic group: MAP2 was observed throughout the neuroepithelium showing an intense mark in what is suggested to be the marginal zone and cortical plate, whereas βIII-Tub exhibited an apical distribution in which its suggested to be the marginal zone (Figures 1H-M). Western blot analysis revealed that embryos from diabetic rats presented significantly increased MAP2c (70 kDa; Figures 1N,O). A high-molecular weight band corresponding to MAP2a/b (>260 kDa; Figure 1N) not observed in control embryos was barely observed in the diabetic. After overexposing the autoradiography film in the area which the high-molecular weight band was observed, this was clearly observed in the diabetic group (Figure 1N'). No significant changes were obtained for βIII-Tub (Figures 1P,Q).

Increased HA Level in the Embryonic Dorsal Telencephalon from Diabetic Rats at E14
To explore the roles of HA and H 1 R in increased neuron differentiation, we first determined the levels of HA and H 1 R in the cerebral cortex neuroepithelium of embryos from both diabetic and control rats.
The temporal patterns of the HA levels during corticogenesis revealed a significant increase in the HA concentration at E14 in both groups. Comparing the groups revealed that HA level in the diabetic group was 3.5 times higher at E14 than in the control group (Figure 2).
Since we presume that the main source of fetal HA was the transient histaminergic neurons (Vanhala et al., 1994), we measured HDC mRNA, its protein levels (at E12, E14, and E16) and activity (E14 and E16) in ventral mesencephalon/rhombencephalon fetal tissue where fetal HDC expression and HA positive neurons reside.
Differences in the glucose levels between pregnant rats and its litters, and between embryos from control and diabetic pregnant rats were evaluated. ***p < 0.001 vs. maternal. a p < 0.001 and b p < 0.01 vs. Ctl embryo.
Frontiers in Neuroscience | www.frontiersin.org The results showed significantly lower levels of HDC mRNA in embryos from diabetic rats at E12 (3.8 times) and E14 (2.1 times; Figure 3A). The decreased expression of HDC observed in embryos from the diabetic group was consistent with a decrease in the level of its protein at E14 (1.8 times), however, a significant increase was obtained at E12 with respect the control group (Figures 3B,C). No amplification was detected for HDC at E14 in dorsal telencephalon tissue (data not shown).
Our results revealed a significant decrease at E14 and an increase at E16 in the diabetic group embryos relative to the control embryos ( Figure 3D). As a result of the amount of tissue required to assess the HDC activity, this was not determined at E12.
Because the increased level of telencephalon HA at E14 in the diabetic group was not explained by the fetal HDC mRNA, protein level or the HDC activity, other sources, such as the mother or placenta, likely provide HA to the embryos. The HA concentrations in the diabetic maternal serum was lower with respect to the control (Figure 4A). When the levels of HA and HDC activity were evaluated in the placenta, we found FIGURE 2 | Fetal histamine levels during corticogenesis. The graph shows the histamine (HA) concentrations (nM) in the control and diabetic groups in telencephalic (E12-E20) tissue. Data are the mean (S.E.M.) from 5 to 8 experiments per triplicate (n is shown in bars). For changes between different embryo days within the same group, one-way ANOVA was performed [Control,F (4,27) = 3.1, P = 0.03, and R 2 = 0.3; Diabetic, F (4, 27) = 3.1, P = 0.03, and R 2 = 0.3] followed by Fisher's LSD test, P = 0.01 in the control for E14 vs. E12 and DF = 26, and in the diabetic P = 0.01 for E14 vs. E12 and DF = 26. For comparison between groups, unpaired t-test was used and significant P-value is shown in the graph. E = embryo day.
that the HA concentration in the diabetic group was 2.3 times higher than in control placentas ( Figure 4B). Although there was a tendency to increase, no significant difference in the HDC activity was observed between groups in placenta HDC activity ( Figure 4C).

Altered Expression of H 1 R during Corticogenesis in Embryos from Diabetic Rats
A nucleotide BLAST for the sequence obtained from the H 1 R PCR product showed 100% identity (nucleotides 594-885 from NM_017018.1; https://blast.ncbi.nlm.nih.gov).
The H 1 R mRNA temporal analysis revealed significant changes relative to E12, with the lowest levels at E14 (2.7 times) and E20 (7.7 times) and the highest at E16 (1.9 times). In contrast, in the diabetic group, the highest expression of the receptor was obtained at E12, and this value was statistically different from those recorded for embryos at other ages ( Figure 5A). The H 1 R protein levels were significantly decreased at E14 (1.4 times), E16 (1.9 times), E18 (1.6 times), and E20 (2.4 times) compared with E12 in the control group. Interestingly, no temporal changes in the receptor protein levels were observed in the diabetic group (Figures 5C,D).
Comparing the groups' H 1 R mRNA levels showed that embryos from diabetic rats presented significant increases at E12 (2 times) and E20 (2.9 times), and significantly lower levels at E16 (5.5 times) and E18 (1.3 times) relative to the control group ( Figure 6A). However, in the telencephalic tissue from diabetic dams, the protein levels increased significantly at E12 (1.6 times) and E16 (1.7 times) relative to the control group (Figures 6B,C).
MAP2 in the diabetic group and notably reduced the expression of Ngn1 (31.3 less) and βIII-Tub (34.5 less; Figure 7). No significant changes were obtained for Prox1 ( Figure 7A). The effect of the H 1 R antagonist on MAP2 was corroborated by immunohistochemistry and Western blot analysis in tissue samples obtained from the dorsal telencephalons of embryos from diabetic rats (Figure 8). As shown previously, the distributions of both neuron markers were altered in the diabetic group: MAP2 extended down from the marginal zone, and βIII-Tub exhibited a basal distribution corresponding to the marginal zone (Figures 8E,F). Diabetic rats treated with chlorpheniramine showed the characteristic cortical plate mark for MAP2 and βIII-Tub, but for βIII-Tub an inspected mark in the ventricular zone (Figures 8G,H). Furthermore, Western blot analysis demonstrated that chlorpheniramine partially prevented the increase in the MAP2 level in the diabetic group without affecting the protein level of βIII-Tub (Figures 8I-L).

DISCUSSION
To the best of our knowledge, embryonic glucose levels have not been previously reported in diabetic and control rat embryos.
Here, we demonstrate that embryos from diabetic rats have significantly higher glucose levels than control embryos and that this phenomenon can be observed as early as E12. Nevertheless, embryo glycemia is lower than the glucose levels in pregnant rat serum at E12. The higher glucose levels observed in embryos from diabetic rats could be attributed to an increase in the expression of glucose transporter 3 (GLUT3) in the placentas of diabetic rats (Boileau et al., 1995).
The effects of high glucose concentrations on neuron differentiation are controversial. Some authors have reported increased differentiation in mice at E11.5 (both in vitro and in vivo) as a result of increased expression of the neurogenic factors Ngn1/2 and MAP2, decreased progenitor factors Hes1 and nestin, and changes in both the pattern and level of expression of Sonic Hedgehog and bone morphogenetic protein-4 (BMP4; Liao et al., 2004;Fu et al., 2006). In contrast, other groups have suggested that maternal diabetes promotes reduced neuron diferentiation related to the decreased expression of BMP4, nuclear factor (erythroid-derived 2)-like 2 (Nrf2), nuclear receptor binding SET domain protein 1 (Nsd1), paired box 3 (Pax3), hypoxia inducible factor 1 alpha subunit (Hif1a), cyclic adenosine monophosphate (cAMP) responsive element binding protein 1, and doublecortin (Liao et al., 2004;Pavlinkova et al., 2009;Salbaum and Kappen, 2010;Ejdesjo et al., 2011).
Discrepancies between these studies may be attributable to a variety of causes, including: timing during development, tissue origin within the neural tube, the presence (or absence) of NTDs in the embryos used, and erroneous inferences about mRNA levels and associated proteins, which depend on mRNA stability, protein half-life, and the action of translation regulators (de Sousa Abreu et al., 2009;Diaz et al., 2014). For example, we found that βIII-Tub was overexpressed in the diabetic group, even though we did not find any differences in the proteins associated with it.
Our findings support increased neuron differentiation in the dorsal telencephalon at the neurogenic peak (E14) in embryos from diabetic rats based on the increased levels of Ngn1, βIII-Tub, and MAP2 (determined via qRT-PCR) and the protein content (MAP2 via immunohistochemistry and Western blot analyses).
Although MAP2 is considered a mature neuron marker, high-and low-molecular weight isoforms are expressed differentially during the development of the CNS and adult tissue. Low-molecular weight isoforms (70 and 75 kDa, MAP2c/b, respectively) are highly expressed during embryo development and the early postnatal period (Garner et al., 1988;Riederer and Innocenti, 1992). In contrast, the two high-molecular weight isoforms are expressed throughout life (MAP2a) or predominantly in the adult brain (MAP2d; >270 kDa; Chung et al., 1996;Fujimori et al., 2002).
The presence of high-and increased low-molecular weight isoforms (as evidenced by the Western blots of diabetic rat embryo tissues) suggests that an early neuron maturation process may occur in addition to increased neuron differentiation. An alternative splicing process for this protein may also arise under our experimental conditions (Kalcheva and Shafit-Zagardo, = 218.2, P < 0.0001, and R 2 = 0.98], followed by Fisher's LDS multiple comparison test, and significant P values are shown in each graph. 1995), as observed in fetal cardiac pathogenesis under diabetic conditions (Verma et al., 2013).
As previously reported, HA acts as a neurogenic factor in cortical NSC via H 1 R activation, promoting MAP2 and FOXP2 phenotypes. This action also increases neuron commitment by increasing the levels of Ngn1 and Prox1 during NSC proliferation (Molina-Hernandez and Velasco, 2008;Rodriguez-Martinez et al., 2012). Furthermore, in utero treatment with chlorpheniramine (H 1 R antagonist/inverse agonist) at E12 promotes reduced βIII-Tub and FOXP2 immunoreactivity in the dorsal telencephalon at E14 (Molina-Hernandez et al., 2013).
Our results suggest that increased levels of HA at E14 and/or H 1 R expression at E12 may be related to alterations in neuron differentiation in the diabetic model. The placenta may be the main source of the increased HA observed in embryos from diabetic dams. Alternatively, this increased HA level could be attributed to reduced fetal HA catabolism; however, no changes in histamine catabolism have been reported in tissues from STZ diabetic rats with increased HA concentrations (Gill et al., 1990).
Although HA is highly increased at E14 under hyperglycemia, the increased expression of H 1 R at E12 may be responsible for the increased neuron differentiation at E14, because the neurogenic effect of HA depends on the activation of this receptor (Molina-Hernandez and Velasco, 2008;Molina-Hernandez et al., 2013). This hypothesis was supported by the systemic administration of chlorpheniramine, which prevented the increased expression of MAP2 and βIII-Tub mRNA and MAP2 protein level in dorsal telencephalic tissue from diabetic rats. Although, previous reports regarding the participation of H 1 R on fetal and adult NSC in neuron differentiation are convincing (Yasuda and Yasuda, 1999;Molina-Hernandez and Velasco, 2008;Bernardino et al., 2012;Rodriguez-Martinez et al., 2012;Molina-Hernandez et al., 2013), we cannot reject a muscarinic participation using chlorpheniramine (Yasuda and Yasuda, 1999) since M2 receptor also enhances neural proliferation and neuron differentiation in E14 NSC in vitro (Williams et al., 2004;Zhou et al., 2004). Nevertheless, muscarinic binding sites appear between E13 and E14 in rats (Schlumpf et al., 1991) and their expression before this moment have not been reported. These data suggest that the effect reported here of chlorpheniramine may be indeed by H 1 R antagonism.
Low levels of HA and the effect of chlorpheniramine administration at E12 in the diabetic group suggest constitutive activity of H 1 R and inverse agonism of chlorpheniramine. Constitutive activity has been described for H 1 R in allergies (Nijmeijer et al., 2010) and over-expressing heterologous systems (Bakker et al., 2000(Bakker et al., , 2001, indicating that these phenomena are receptor density dependent. In addition to the important role of H 1 R during CNS development, the possibility that the receptor exhibits constitutive activity in embryos from diabetic dams may have important consequences not only in the context of pregnancy and fetal CNS development but also regarding the development of other organs (i.e., the heart).
Here, we show changes in the fetal ontogeny of the histaminergic system in a maternal pathological condition that might be related with the increased neurogenesis in the dorsal telencephalon. The functional implication of an increased telencephalic neuron differentiation and/or neuron maturation is difficult to establish and, even more to relate it with the etiology of neurodevelopmental disorders, since several intrinsic, epigenetic, and environmental factors may influence neuronal development and potentially contribute to neurological and mental disorders. But it is possible that the increased telencephalic neurogenesis which is intimately related to cell proliferation and migration will have important consequences on laminar specification and neural circuit integration, aspects that will be further studied. Additionally, this is the first study revealing the expression of H 1 R and HDC as early as E12 and to demonstrate that HDC is active in the transitory fetal histaminergic system. This study opens up an important field of research regarding the participation of HA and H 1 R receptor in early corticogenesis in health and disease.