Histamine impairs midbrain dopaminergic development in vivo by activating histamine type 1 receptors

NSPC have the capacity to self-renew, and the potential to differentiate into neurons, astrocytes and oligodendrocytes. To characterize the expression of NSPC markers, as well as their capacity to differentiate to neuronal and glial populations, we cultured VM NSPC isolated from E12 rat embryos. We maintained these cells in proliferation during 4 days in the presence of the mitogen Fibroblast growth factor (FGF)-2, and then induced differentiation for 6 days after removal of FGF-2. We found that a very high proportion of these cells express Sox2, Vimentin and Nestin, which are markers widely used to identify undifferentiated NSPC (Figure 1A). After removing FGF-2 from cultures, cells readily differentiate into neurons (MAP2- and β-III Tubulin-positive), astrocytes (Glial Fibrillary Acidic Protein, GFAP-positive) and oligodendrocytes (O4-positive) (Figure 1B), confirming that our cultures are indeed NSPC.

Previous work has demonstrated that mRNAs for histaminergic receptors are present in rodent embryos from E14 onwards [18],[20],[21], but no information has been reported regarding the expression of these receptors at earlier stages of embryogenesis neither in the VM tissue, nor in midbrain NSPC in vitro. Therefore, using primers verified to amplify HA receptors [27], we examined whether these receptors were present in cultured VM NSPC, before and after differentiation. We found by RT-PCR that proliferative and differentiated VM NSPC cultures, isolated from E12 rat embryos, express transcripts of H₁R, H₂R, and three isoforms reported for the H₃R, which are generated by alternative splicing [19],[21],[31],[32] (Figure 2A). The VM tissue without culture also had mRNAs for H₁, H₂ and H₃ receptors (data not shown). We also performed Western blot analyses and found that the corresponding proteins for H₁R and H₂R were present in cultured NSPC (Figure 2B) and in VM tissue (data not shown). Even when the level of mRNA for H₁ and H₂ receptors was low, a single band was clearly detected, showing that H₁R and H₂R proteins are present in E12 cultured VM NSPC.

Calcium (Ca²⁺) is an important regulator of various neuronal functions. During the formation of CNS, Ca²⁺ signaling is involved in the regulation of processes such as neural induction and differentiation of neural progenitors into neurons and glia, in a defined spatiotemporal pattern [33],[34]. Since the activation of H₁R leads to the accumulation of Ca²⁺ as a second messenger, we tested if undifferentiated VM NSPC were responsive to HA stimulation by elevating intracellular Ca²⁺ concentration ([Ca²⁺]_i). To address this hypothesis, we loaded cultured Nestin + VM NSPC with the ratiometric calcium probe Fura-2 AM, and measured the fluorescence changes resulting from alternating excitation of Fura-2 between 340 nm and 380 nm (R = F₃₄₀/F₃₈₀). We found a significant 3-fold increase in [Ca²⁺]_i after the addition of 10 μM HA to VM NSPC, compared to the basal levels observed in control conditions (Figure 3), indicating that undifferentiated VM NSPC are responsive to HA. These results are consistent with our previous work on cerebrocortical NSPC [29], where 100 μM HA also caused calcium increases in VM cells through H₁R activation.

In order to examine the effects of HA on VM NSPC proliferation and apoptotic cell death, we treated cultures with increasing concentrations of HA (from 1 μM to 1 mM) in the presence of FGF-2 for 4 days. We used BrdU incorporation assays to analyze proliferation and the TUNEL assay to analyze apoptotic cell death. We calculated the percentage of BrdU- and TUNEL-positive cells relative to the total number of cells in control and HA-treated cultures and found that HA did not affect BrdU incorporation (Figure 4A and B). In contrast, 1 mM HA significantly increased the proportion of TUNEL-positive cells and this effect was prevented by co-incubation with the H₁R antagonist chlorpheniramine (Figure 4C and D).

To analyze whether HA has an effect on the differentiation of VM NSPC, cultures were treated for 6 days in the absence of FGF-2. Then, we evaluated by immunocytochemistry the expression of the neuronal marker β-III Tubulin. We found that 10 μM HA significantly increased the percentage of β-III Tubulin -positive cells from 21% to 37%, compared to control (Figure 5A and B). Additionally, we analyzed whether HA could have a role on dopaminergic differentiation by detecting the expression of the rate limiting enzyme for dopamine synthesis, TH, normalized by the number of neurons positive to β-III Tubulin. Figure panels 5C and D show that 1 mM HA significantly reduced the proportion of dopaminergic neurons when compared to controls. To establish if the decrease on TH-positive neurons was mediated by H₁R activation, we treated cultures with chlorpheniramine, alone or in combination with 1 mM HA. We observed a significant recovery in the number of TH + cells when HA plus chlorpheniramine were added, compared to HA alone (Figure 5E and F).

Multipotent NSPC were isolated from E12-E12.5 embryos obtained from time-pregnant Wistar rats as described [27]. For simplicity, we only use E12 to designate these embryos and the same criterion applies for other embryonic stages. VM was dissected in Krebs solution then mechanically dissociated. Cells were recovered by centrifugation. Isolated cells were cultured on plastic ware treated with 15 μg/ml poly-L-ornithine (Sigma) and 1 μg/mL human Fibronectin (Invitrogen) in N2 medium (DMEM/F12 1:1, supplemented with 25 μg/L of human insulin, 30 nM sodium selenite, 100 μM putrescine, 20 nM progesterone and 100 mg/L of apotransferrin) containing 10 ng/mL of basic fibroblast growth factor (FGF-2; Peprotech). Cultures were maintained at 37°C in humidified 5% CO₂/95% air atmosphere. These cultures were considered as passage (P) 0. When cultures reached 80% confluence, cells were detached with 0.1 mM EDTA in phosphate buffered saline (PBS) and re-plated to P1. Cells were maintained under proliferation conditions for 4 days in control (N2 medium + 10 ng/ml FGF-2) and experimental conditions that vary concentrations of HA (from 1 μM to 1 mM) with or without HA receptor antagonists. Differentiation was promoted by removal of FGF-2 during 6 additional days in control and experimental conditions. HA antagonists for H₁R (1 μM chlorpheniramine, Sigma), H₂R (30 μM cimetidine, Sigma) were added daily to HA-treated cells during proliferation and differentiation stages. For immunocytochemistry analysis, BrdU and TUNEL assays, cells were seeded at 1.5×10⁴/well in 24-well plates (Corning). For RNA extraction, cells were seeded at 3×10⁵/well in 6-well plates (Corning). HA dihydrochloride was used without neutralization.

Total RNA was isolated from proliferating cultures, differentiated cultures, and midbrain tissue from ultrasound injected and control embryos using TRIZOL reagent (Invitrogen). One μg of RNA was reverse transcribed using dNTPs, 100 U of Superscript III Reverse Transcriptase, 50 μM of random hexamers and 40 U of Recombinant RNase Inhibitor (Invitrogen). PCR reactions were performed using 100 ng of cDNA, 2 U of recombinant Taq DNA polymerase (Invitrogen), 20 pmol of each oligonucleotide, 500 μM dNTPs (Sigma) and 1.5 mM MgCl_2. PCR products were analyzed by electrophoresis to determine their molecular weights after ethidium bromide staining. The sequences of the PCR products were verified and correspond to the indicated HA receptor. Reactions with RNA in the absence of retrotranscription were included as negative controls. RNA extracted from adult rat cerebral cortex was used as a positive control for HA receptors. The forward (F) and reverse (R) primers listed in Table 1 were used.

These primers have been used previously by our group [27] and after retrotranscription and amplification, PCR products were extracted from the gel and sequenced, confirming that these sequences correspond to H₁, H₂ and the 3 isoforms of H₃ receptors.

For qRT-PCR [55], we used 100 ng of the synthesized cDNA, 2.5 U of recombinant Taq DNA polymerase (Invitrogen), 0.4 mM of each oligonucleotide and dNTPs, 1.5 mM MgCl₂ and 5 μM Syto 9 (Invitrogen). Quantitative analysis of cDNA amplification was performed using the Rotor Gene 6000 System (Corbett Life Science). Melting curves were performed in every reaction to ensure that only one product was present. GAPDH amplification was used as loading control for all reactions and every transcript value was normalized to GAPDH expression. The 2exp (−ΔΔCt) method was used to determine the relative expression of each gene compared to control condition. The primers listed in Table 2 were used.

Cells were cultured and seeded at 3.5×10⁴/well in 6-well plates (Corning) in control and experimental conditions, following standard protocols [27],[56]. Briefly, cells were homogenized in lysis buffer (25 mM Tris–HCl pH 7, 1% IGEPAL, 100 mM NaCl) supplemented with protease inhibitors (Complete mini, Roche). Proteins were obtained by centrifugation at 13 800 g at 4°C for 15 min, and quantified by Bradford assay (BioRad). Proteins (30 μg) were resolved on 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Amersham Bioscience). The membranes were blocked with 5% non-fat milk and incubated overnight with primary antibodies. Pre-stained markers (Invitrogen) were included for size determination. The following antibodies were used: rabbit anti-rat H₁R polyclonal antibody (1:1500, Santa Cruz Biotechnology); goat anti-rat H₂R polyclonal antibody (1:1500, Santa Cruz Biotechnology); rabbit anti-Lmx1a polyclonal antibody (1:100, Abcam); rabbit anti-Lmx1b polyclonal antibody (1:100, Abcam); rabbit polyclonal anti-tyrosine hydroxylase (TH, 1:1000; Pel-freez) and rabbit anti-Pitx3 polyclonal antibody (1:500, Zymed). Membranes were washed and incubated with corresponding horseradish peroxidase-coupled secondary antibodies (1:10 000, Santa Cruz Biotechnology) for 2 hours. Immunoreactive bands were detected using enhanced chemiluminescence method (Amersham Bioscience) and developed on photographic film (Kodak).

Cells were fixed with 4% paraformaldehyde in PBS, pH 7.4 at the end of proliferation and differentiation stages. Immunocytochemistry assays were performed following standard protocols [57]. Briefly, cells were permeabilized and blocked for 1 hour with 0.3% Triton X-100 and 10% normal goat serum (NGS) in PBS. Cells were incubated overnight at 4°C with the following primary antibodies diluted in PBS containing 10% NGS: rabbit polyclonal anti-β-III Tubulin (1:2000, Covance); rabbit polyclonal anti-glial fibrillary acidic protein (GFAP; 1:2000, DAKO); mouse monoclonal antibody anti-microtubule associated protein 2 (MAP2; 1:500, Chemicon); mouse monoclonal anti-Nestin (1:100, Developmental Studies Hybridoma Bank); rabbit anti-Nestin (1:1000, Covance); rabbit polyclonal anti-TH (1:1000; Pel-freez); rabbit polyclonal anti-Sox2 (1:200; Millipore). Appropriate Alexa-Fluor secondary antibodies were used diluted in PBS (1:1000; Molecular Probes) containing 10% NGS. Nuclei were stained with Hoechst 33258 (1 ng/mL; Sigma). Immunostainings were analyzed with an epifluorescence microscope (Nikon, Eclipse TE2000-U) and photographed with a Nikon digital camera (DMX1200 F). Negative controls were performed in the absence of primary antibodies and showed no unspecific staining.

Cells treated with FGF-2 were incubated for 3 hours with 10 μM 5-bromo-2-deoxyuridine (BrdU, Roche), washed with medium and fixed with 4% paraformaldehyde in PBS 21 h later [27]. After fixation, cells were incubated with 1 N HCl for 30 min at 25°C, and neutralized by washing with 0.1 M borate buffer, pH 8.5. Preparations were blocked for 1 h with 10% NGS (Invitrogen) and 0.3% Triton X-100 (Sigma) in PBS. Cells were incubated overnight at 4°C with monoclonal rat anti-BrdU antibody (1:100; Accurate) followed by three washes with 1% bovine serum albumin in PBS. Alexa-Fluor 488 goat anti-rat IgG was added (1:1000, Invitrogen) in blocking solution for 1 h at RT and washed three times with PBS. Nuclei were stained with Hoechst 33258 (1 ng/mL; Sigma).

Proliferating and differentiated cells were fixed with 4% paraformaldehyde in PBS, and subsequently washed three times with PBS, pH = 7.4 [27]. For detection of apoptotic cells, the Terminal deoxytransferase-mediated deoxyuridine triphosphate nick-end labeling kit (TUNEL, InSitu Cell Death Detection Kit, Roche Diagnostics) was used to evaluate the effect of HA on cell death. Apoptotic cells were visualized by fluorescence microscopy.

Fluoro Jade was used to label and quantify degenerating neurons in the VM. The sections were mounted on slides and air-dried. The slides were dipped in absolute ethanol for 3 min followed by 1 min in 70% ethanol and washed in deionized water for 1 min. Tissue was then oxidized with a 0.06% KMnO₄ solution diluted in PBS for 15 min, followed by three rinses with deionized water. From this point onwards, the slides were protected from light. Tissue sections were stained with a 0.001% solution of Fluoro-Jade in 0.1% acetic acid for 30 min. Slides were rinsed with water and air-dried. The slides were cleared in xylene and coverslipped with DPX mounting medium [58],[59].

Cell counts from BrdU, immunocytochemistry, TUNEL, phosphorylated histone H3 (pHH3) and Fluoro Jade experiments were performed from digital pictures. Quantification of cells was performed by using the ImageJ software considering the total number of Hoechst-stained nuclei (total cells), in at least 10 random fields from a minimum of 3 independent experiments.

Proliferating VM NSPC in suspension were loaded with the Ca²⁺-sensitive fluorescence indicator Fura-2 AM (Invitrogen) at a final concentration of 5 μM at 37°C for 45 min [29]. Loaded cells were centrifuged and seeded at 1×10⁵ cells on 3.5 cm glass bottom dishes (MatTek Corp) treated with 15 mg/mL poly-L-ornithine (Sigma) and 1 μg/ml human Fibronectin (Invitrogen) in N2 medium. After 2 hours, attached cells were incubated in Hank’s solution (137 mM NaCl, 5 mM KCl, 0.3 mM NaH₂PO₄, 0.8 mM MgSO₄, 1 mM MgCl₂, 5 mM glucose, 2 mM glutamine, 10 mM Tris–HCl without CaCl₂; pH 7.4) at 37°C for 10 minutes. Subsequently, Hank’s solution was removed and new solution with 5 mM CaCl₂ was added. UV excitation was set to alternate between 340 and 380 nm and the emission was monitored at 510 nm in a Carl Zeiss microscope. Images and the emission ratio generated between both excitations wavelengths (R = E 340/E 380) were obtained and analyzed using AxioVision software.

We used timed-pregnant Wistar rats with embryos at gestational ages E10, E12, E14 and E16 to perform ultrasound-guided injections. All animal procedures were approved by the Instituto de Fisiología Celular Animal Use and Care Committee and conformed to Mexican and NIH guidelines. All injections were performed by duplicate or triplicate in different embryos from the same dam, and repeated at least in 3 different pregnant rats. Control and experimental animals were included in the same pregnant rat, but to make sure that the different conditions do not mix, each group was evaluated in a single uterine horn. The results were reproducible when we analyzed embryos from different dams. To perform ultrasound guided injections [40], we used an MHF-1 Ultraview UltraSound System (E-Technologies) with a focal distance of 7 mm for imaging. Rats were deeply anesthetized in a chamber and then maintained with a facemask to administer inhaled sevoflurane (5% for induction and 1% for maintenance). We performed midline laparotomy, uterine horns were carefully exposed and the number of embryos was recorded. Intraventricular injections were performed through the uterine wall using pulled microcapillaries (borosilicate glass, Sutter Instruments). The glass needles were visualized in the ultrasound imaging system and the verification of the site of injection in the lateral ventricles was made during the procedure by observing the liquid going out of the needle. HA, chlorpheniramine (H₁R antagonist) or cimetidine (H₂R antagonist) were injected on the ventricular lumen at different doses in a final volume of 2 μl aided by an automatic injector (Quintessential injector, Stoelting). The injected amount was of HA dihydrochloride, which was used without neutralization. DMSO and Cell Tracker (Invitrogen) injections were performed as controls. Once the desired number of embryos was injected, the uterus was repositioned in its physiological site. The incisions of the abdominal muscle and skin were stitched with separate sutures. Finally we applied Buprenorphine as analgesic (0.1 mg/kg, Pisa Laboratories) and animals were monitored and left to recover. Two days after the injections, pregnant rats were euthanized by overdose of the anesthetic Sodium pentobarbital (Pfizer), and identified embryos were recovered for analysis. We performed the following number of successfully injected E12 embryos: vehicle: 48; HA: 12; chlorpheniramine: 9; cimetidine: 9; HA plus chlorpheniramine: 9; HA plus cimetidine: 9. The number of dams used was 18.

Embryos were removed and transferred to PBS. Subsequently, the embryos were fixed by immersion overnight in 4% PFA in PBS at 4°C. Thereafter, the tissues were cryoprotected overnight in 30% (v/v) sucrose, embedded in Tissue Freezing Medium (Tissue-Tek, Zakura Finetek) and frozen. Sagittal and coronal sections (20 μm) were obtained on a cryostat (Leica). For immunohistochemistry, slides were rehydrated in PBS. Tissue sections were permeabilized and blocked for 1 hour at RT with 0.3% Triton X-100 and 10% NGS in PBS. Slides were incubated overnight at 4°C with the following primary antibodies diluted in PBS containing 10% NGS: rabbit polyclonal anti-pHH3 (1:250, Cell Signaling), rabbit polyclonal anti-β-III Tubulin (1:1000, Babco Covance); mouse monoclonal anti-Nestin (1:100, Developmental Studies Hybridoma Bank); rabbit polyclonal anti-tyrosine hydroxylase (TH; 1:1000, Pel-freez); rabbit polyclonal anti-GAD65/67 (1:500, Millipore); rabbit polyclonal anti-serotonin (1:500, Sigma). Subsequently, the sections were washed three times for 5 minutes in PBS and incubated with the secondary antibodies Alexa-Fluor 488 anti-rabbit IgG and Alexa-Fluor 568 anti-mouse IgG (1:500; Molecular Probes) in PBS containing 10% NGS for 2 hours. Nuclei were stained with Hoechst 33258 (1 ng/mL). After three further washes in PBS, the sections were mounted in Aqua Poly/Mount (Polysciences, Inc.) Immunostainings were analyzed with an epifluorescence microscope (Nikon, Eclipse TE2000-U) and photographed with a Nikon digital camera (DMX1200 F). Some images were acquired with a FV1000 Olympus confocal microscope. Negative controls were performed in the absence of primary antibodies and showed no unspecific staining.

We performed 3–5 independent experiments, defined as duplicate or triplicate experiments from a different pregnant rat. All data are presented as mean ± standard error of mean (S.E.M). One-way ANOVA was performed for statistical analysis and multiple comparisons between treated and control groups were made using the post-hoc Student-Newman-Keuls test. Differences were considered statistically significant at P ≤ 0.05. Graphs were generated using GraphPad Prism software.