Edited by: João O. Malva, University of Coimbra, Portugal
Reviewed by: Juan Andrés De Carlos, Consejo Superior de Investigaciones Científicas (CSIC), Spain; Sara Xapelli, Universidade de Lisboa, Portugal
*Correspondence: Anayansi Molina-Hernández
This article was submitted to Neurogenesis, a section of the journal Frontiers in Neuroscience
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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
Maternal diabetes is a risk factor that increases the incidence of neural tube defects (NTDs) by at least 11/1000 in humans (estimated from: Soler et al.,
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
Fu et al. (
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,
Additionally, extrinsic and intrinsic factors participate in the chronological programming of NSC proliferation, migration, differentiation, and survival, which lead to the final CNS cytoarchitecture.
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.,
The messenger RNA (mRNA) of the histamine receptors 1 (H1R) and 2 (H2R) are expressed in the cortical neuroepithelium at E14, while the H3R appears in cerebral cortex until E19 (Kinnunen et al.,
HA, as other neurotransmitters, (GABA, serotonin, acetylcholine and glutamate) are important extrinsic factors affecting NSC differentiation
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 H1R 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.
Wistar rats (250–300 g) from (INB-UNAM) were maintained in our animal facilities, house dindividually 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.
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 H1R 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 H1R antagonist chlorpheniramine (Sigma–Aldrich; Naranjo and de Naranjo,
To determine the HA and H1R 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.
The glucose level was measured immediately after embryo decapitation and then heads were promptly placed in cold Kreb's solution (100 mM NaCl, 2 mM KCl, 0.6 mM KH2PO4, 12 mM NaHCO3, 7 mM glucose, 0.1% phenol red, 0.3% bovine serum albumin, and 3% magnesium sulfate; pH 7.4 and 4°C) for dorsal telencephalon or ventral mesencephalon/rhombencephalon dissection using a stereoscopic microscope (Olympus SZX16, Shinjuku, Tokyo, Japan). Finally, tissue was washed with phosphate-buffered saline (PBS; pH 7.4 and 4°C).
For immunohistochemistry, E14 embryos were fixed by immersion in Bouin's solution (15:15:1 saturated picric aqueous solution:formalin 40%:glacial acetic acid) followed by immersion in 15 and 30% sucrose gradients for 24 h each. After fixation, the embryos were frozen in isopentane (Sigma–Aldrich) and 10 μm coronal slices using a cryostat (Leica CM1850 UV, Wetzlar, Germany) were recovered.
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® 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,
Immunofluorescence procedures were conducted using standard protocols (Molina-Hernandez and Velasco,
qRT-PCR was performed to analyze the temporal expression of H1R 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 H1R, HDC, neurogenic factors (Prox1 and Ngn1), and neuron markers (βIII-Tub and MAP2). The dorsal cortical neuroepithelium (for H1R, 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 retro-transcription 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® 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 (H1R) or 400 ng (other mRNAs) of cDNA, 20 pmol of forward (F) and reverse (R) primers, and the commercial KAPA™ SYBER FAST® 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 (H1R 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−ΔΔCT, where CT is the cycle threshold (Livak and Schmittgen,
The primer sequences and product size were as follows: H1R (292bp), F:5′-CTTCTACCTCCCCACTTTGCT-3′ and R: 5′-TTCCCTTTCCCCCTCTTG-3′; Prox1 (384 bp), F: 5′-TGTTCTTTTACACCCGTTACCC-3′ and R: 5′-CACTATCCAGCTTGCAGATGAC-3′; Ngn1 (131 bp), F: 5′-AGCCCGGCCAGCGATACAGA-3′ and R: 5′-GGACCACCCGGGCCATAGGT-3′; βIII-Tub (102bp), F: 5′-GCCAAGTTCTGGGAGGCTCATC-3′ and R: 5′-GTAGTAGACACTGTAGCGTTCCA-3′; MAP2 (132 bp), F: 5′-GAG AAG GAG GCC CAA CAC AA-3′ and R: 5′-TCTTCGAGGCTTCTTCCAGTG-3′; and GAPDH (189 bp), F: 5′-GGA CCT CAT GGC CTA CAT GG-3′ and R: 5′-CCCCTCCTGTTGTTATGGGG-3′.
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 H1R 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.
Protein extracts from a pool of dorsal telencephalon (E12-E14, whole litter and E16-E20, 4 embryos) or ventral mesencephalon/rhombencephalon (E12–E16, whole litter) were obtained after lysis in buffer containing 2.5 mM Tris-HCl pH7.5, IGEPAL 1%, 100 mM NaCl, and protease inhibitors (AMRESCO, Solon, OH, USA). Total protein was determined by the Bradford method (Bradford,
Denaturalizing protein electrophoresis in 10 or 8% (for MAP2) acrylamide gel was performed with 80 μg (H1R and MAP2) or 40 μg (HDC and βIII-Tub) of total protein using a MiniProtean II system (Bio-Rad, Hercules, CA, USA). Proteins were transferred to nitrocellulose membranes (Amersham TM Hybond TM-ECL, Buckinghamshire, UK) using the Trans-Blot® semi-dry transfer cell system (Bio-Rad), as described previously (Villanueva,
GAPDH or actin were used as the internal controls. Because a non-specific band was observed in the H1R immunoblots and to ensure H1R detection, a cell line that highly expresses H1R, the U373MG glioblastoma cells were transfected with H1R siRNA-Smart-pool or scramble sequences (Accell, GE Healthcare) were used as negative or positive controls, respectively (
A modified method was used to measure the HDC activity (Keeling et al.,
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.
Sample size was estimated for independent samples and studies analyzed by
Unpaired
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
Glycemic values in control and diabetic pregnant rats and its corresponding litter.
0 | 96.28 ± 1.06 (21) | 91.3 ± 1.16 (20) | |||
2 | 96.58 ± 2.14 (12) | 363.23 ± 27.66 (17) | |||
4 | 96.41 ± 3.16 (12) | 367.92 ± 21.16 (13) | |||
5 | 94.30 ± 1.88 (10) | 350 ± 30.53 (9) | |||
6 | 90.70 ± 2.16 (10) | 470.28 ± 24.95 (7) | |||
7 | 100 ± 1.08 (12) | 414.12 ± 12.56 (25) | 12 | 44.43 ± 0.7 |
65.56 ± 4.73 |
9 | 91 ± 0.77 (12) | 501.7 ± 11.24 (10) | 14 | 51.08 ± 3.85 |
366.6 ± 20.98 |
11 | 92 ± 4.9 (8) | 542.77 ± 9 (18) | 16 | 63.23 ± 3.04 |
310.32 ± 6.35 |
13 | 91.2 ± 1.25 (5) | 395 ± 1.62 (8) | 18 | 89.6 ± 0.7 (4) | 453.9 ± 17.95 |
15 | 90.66 ± 4.63 (3) | 550.83 ± 13.15 (10) | 20 | 91.11 ± 2 (3) | 432.25 ± 7.81 |
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
Diabetes during pregnancy increases neuron differentiation and maturation.
To explore the roles of HA and H1R in increased neuron differentiation, we first determined the levels of HA and H1R 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
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 (
Since we presume that the main source of fetal HA was the transient histaminergic neurons (Vanhala et al.,
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
HDC expression and activity in mesencephalic/rhombencephalic tissue.
Our results revealed a significant decrease at E14 and an increase at E16 in the diabetic group embryos relative to the control embryos (Figure
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
Other possible fetal sources of histamine at E14.
A nucleotide BLAST for the sequence obtained from the H1R PCR product showed 100% identity (nucleotides 594–885 from NM_017018.1;
The H1R 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
Temporal fetal H1R expression during corticogenesis in embryos from control and diabetic rats.
Comparing the groups' H1R 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
Differences in histamine type 1 receptor expression between embryos from control and diabetic rats.
BLAST for the sequences obtained from the PCR products showed 100% identity for Prox1 (nucleotides 2248–2631 from NM_001107201), Ngn1 (nucleotides 780–910 from NM_019207.1), βIII-Tub (nucleotides 101–202 from NM_139254), and MAP2 (nucleotides 433–564 from NM_013066.1;
We found a tendency to rise in the expression of Ngn1 (1.5 times;
Chlorpheniramine prevents the increased expression of neuronal factors at E14.
The effect of the H1R 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
Chlorpheniramine partially prevents increased MAP2 in the dorsal telencephalons of embryos from diabetic rats.
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.,
The effects of high glucose concentrations on neuron differentiation are controversial. Some authors have reported increased differentiation in mice at E11.5 (both
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.,
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.,
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,
As previously reported, HA acts as a neurogenic factor in cortical NSC via H1R 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,
Our results suggest that increased levels of HA at E14 and/or H1R 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.,
Although HA is highly increased at E14 under hyperglycemia, the increased expression of H1R 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,
Low levels of HA and the effect of chlorpheniramine administration at E12 in the diabetic group suggest constitutive activity of H1R and inverse agonism of chlorpheniramine. Constitutive activity has been described for H1R in allergies (Nijmeijer et al.,
In addition to the important role of H1R 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 H1R 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 H1R receptor in early corticogenesis in health and disease.
All authors participated in the development of this research and drafting of the manuscript. KS and LM, performed the experiments and participated in the analysis of results. GG-L participate in immunohistochemistry experiments and analysis. WP animal acquisition and manipulation. ND, WP, and MDN-O, conception contributions, critical revision of the manuscript and experimental supervision. AM-H funding, experimental design, experimental and data supervision, data analysis and final approval of the manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We acknowledge the funding given from INPer and CONACyT (CB-2015-254847) to AM-H and CONACyT. IPN fellowships to SKH (508696). We also thank Dr. José Antonio Arias-Montaño for donating SKF 91488 and U373MG for positive control. We thank Francisco Camacho (INB-UNAM) for assisting with animal care. Additionally, we thank Advancing Research Worldwide and Michelle Therese Connolly for the language editing and correction.
histamine H1 receptor
histamine
microtubule associated protein 2
forkhead box protein 2
BetaIII-tubulin
neurogenin
neural tube defects
neural stem cells
central nervous system
embryo day
Achaete-Scute family basic helix-loop-helix (bHLH) transcription factor 1
Hes family bHLH transcription factor
Prospero 1
glyceraldehyde 3-phosphate dehydrogenase
forward
reverse
histidine decarboxylase
glucose transporter 3
bone morphogenetic protein 4
nuclear factor (erythroid-derived 2)-like 2
nuclear receptor binding SET domain protein 1
paired box 3
hypoxia inducible factor 1 alpha subunit.