Histamine impairs midbrain dopaminergic development in vivo by activating histamine type 1 receptors
© Escobedo-Avila et al. 2014
Received: 6 June 2014
Accepted: 30 July 2014
Published: 12 August 2014
Histamine (HA) regulates the sleep-wake cycle, synaptic plasticity and memory in adult mammals. Dopaminergic specification in the embryonic ventral midbrain (VM) coincides with increased HA brain levels. To study the effect of HA receptor stimulation on dopamine neuron generation, we administered HA to dopamine progenitors, both in vitro and in vivo.
Cultured embryonic day 12 (E12) VM neural stem/progenitor cells expressed transcripts for HA receptors H1R, H2R and H3R. These undifferentiated progenitors increased intracellular calcium upon HA addition. In HA-treated cultures, dopamine neurons significantly decreased after activation of H1R. We performed intrauterine injections in the developing VM to investigate HA effects in vivo. HA administration to E12 rat embryos notably reduced VM Tyrosine Hydroxylase (TH) staining 2 days later, without affecting GABA neurons in the midbrain, or serotonin neurons in the mid-hindbrain boundary. qRT-PCR and Western blot analyses confirmed that several markers important for the generation and maintenance of dopaminergic lineage such as TH, Lmx1a and Lmx1b were significantly diminished. To identify the cell type susceptible to HA action, we injected embryos of different developmental stages, and found that neural progenitors (E10 and E12) were responsive, whereas differentiated dopaminergic neurons (E14 and E16) were not susceptible to HA actions. Proliferation was significantly diminished, whereas neuronal death was not increased in the VM after HA administration. We injected H1R or H2R antagonists to identify the receptor responsible for the detrimental effect of HA on dopaminergic lineage and found that activation of H1R was required.
These results reveal a novel action of HA affecting dopaminergic lineage during VM development.
Histamine (HA) is a neurotransmitter that participates in the sleep cycle, motor activity, synaptic plasticity and memory –. The adult mammalian brain contains somas of HA-producing neurons in the hypothalamic tuberomamillary nucleus, and its projections reach most of the CNS ,,. HA exerts its actions through the activation of four G-protein-coupled receptors: H1R-H4R. H1R, H2R and H3R are widely distributed throughout the CNS –, but the presence of H4R in the brain remains unclear –.
During embryonic development, HA is present at higher concentrations than those observed in the adult brain –. HA increases its content at embryonic days (E) 14–16, and then decreases perinatally ,–. Histidine decarboxylase (HDC), which synthetizes HA, and the mRNAs for H1R, H2R and H3R – are also expressed at these developmental stages ,,. The transitory expression of HDC in the midbrain, together with elevated HA levels, and the expression of HRs, coincides with periods of dopaminergic specification , suggesting that HA might influence dopamine (DA) neuron generation in the ventral midbrain (VM). In rats, differentiated dopaminergic neurons are first identified at E12 by expression of Tyrosine Hydroxylase (TH), the rate-limiting enzyme for DA synthesis. The peak of DA neurogenesis is at E13. By E16, all DA cells are post-mitotic and their axons are en route to reach forebrain structures such as the striatum and the cerebral cortex ,.
On cortical neural stem/progenitor cell (NSPC) cultures, the effect of activating H1 and H2 receptors has been reported . HA increases cell proliferation by H2R activation and has a neuronal-differentiating action mediated by H1R stimulation, probably by rising prospero 1 and neurogenin 1 expression  and favoring differentiation to FOXP2-positive neurons . Furthermore, HA stimulates neuronal differentiation of adult subventricular zone NSPC . The HA concentrations required to observe these effects are 100 μM to 1 mM.
Elucidating the developmental pathways that control neuronal specification in the VM is of great relevance to increase our knowledge in differentiation of dopaminergic cells. In this work, we aimed to study the effect of HA on dopaminergic development. We found that HA is detrimental to dopaminergic differentiation of NSPC in vitro, and demonstrate that HA administration in vivo at early developmental stages decreases dopaminergic induction in the VM through H1R stimulation. This study establishes the inhibitory relationship of HA to DA neuron generation during development, and provides a novel mechanism for the future treatment of Parkinson’s disease.
Midbrain NSPC cultures are multipotent and express histaminergic receptors
HA increases intracellular calcium in VM NSPC
HA increases apoptotic cell death of cultured undifferentiated midbrain NSPC without affecting cell proliferation
HA decreases the number of dopaminergic neurons in differentiated VM NSPC
In vivo experiments
Intrauterine administration of HA does not modify the morphology of the VM
HA injection decreases dopaminergic induction in VM
Injection of HA does not alter GABA or serotonin neurons
HA interferes with dopaminergic development only when administered at early stages
HA decreases cell proliferation without altering cell death
The effect of HA on dopaminergic differentiation is mediated by H1R
In this study we report that: i) midbrain NSPC express HA receptors; ii) HA stimulus increases intracellular calcium in proliferative VM NSPC; iii) one millimolar HA promotes apoptotic death of cultured VM NSPC by activating H1R; iv) Ten μM HA increases neuronal differentiation, but 1 mM HA decreases the number of differentiated dopaminergic neurons, in a H1R-dependent fashion, on VM NSPC cultures; v) HA injection at E12 decreases dopaminergic induction in VM without altering GABA or serotonin neurons in vivo; vi) HA interferes with dopaminergic development only when administered at early developmental stages; vii) HA impaired cell proliferation in the VM in vivo and viii) the effect of HA on dopaminergic differentiation is mediated by activation of H1R.
We found that H1 and H2 receptors are present at both mRNA and protein levels in proliferating and differentiated cultured VM NSPC. We showed that upon HA stimulation, undifferentiated cultures of VM NSPC significantly increased the cytoplasmic Ca2+ concentration. This data strongly suggest the presence of functional H1R coupled to the canonical calcium response in VM NSPC, consistent with the calcium responses evoked by 100 μM HA in cortical NSPC, which is blocked by chlorpheniramine , supporting the notion that H1R activation in undifferentiated NSPC occurs in both the forebrain and midbrain. It is well established that intracellular Ca2+ regulates neurogenesis as well as neurite/nerve growth and axonal pathfinding ,,. In cortical NSPC, H1R was responsible for the increased neuronal differentiation caused by HA. We explored the effect of HA on proliferation, cell death and differentiation of cultured midbrain NSPC. No significant differences on cell proliferation were found in HA-treated VM NSPC, when compared to control conditions. Regarding cell death, we observed an increasing effect of HA on apoptosis with 1 mM HA, which was clearly prevented by chlorpheniramine. We have previously reported that HA, through H1R activation, increases differentiation of E14 cortical NSPC to neurons that are positive for FOXP2, a marker of deep cortical layers ,. In contrast, in this study we found that in E12 VM NSPC, 10 μM HA increased neurogenesis and 1 mM HA affected DA neuron generation. These results highlight that the effects of HA vary depending on the studied brain region and the concentration of HA used. In VM NSPC, the decrease in TH + cells was mediated by H1R.
Previous studies have shown the expression patterns of HA-immunoreactive neurons and HRs at different stages of development . HA-immunoreactive neurons are first detected on E13 in the regions of mesencephalon and metencephalon, this expression pattern remains until E17 and gradually decreases towards birth . The expression patterns of HRs were reported at later stages of development (from E14 onwards) by in situ hybridization ,. We now show by RT-PCR and Western blot the presence of H1 and H2 receptors in E12 cultured VM NSPC.
Ultrasound guided-injection is a valuable technique that allows visualization and manipulation of embryos at specific developmental stages –. We did not find morphological differences between the vehicle- and HA-injected embryos, assessed by hematoxylin-eosin staining and the VM thickness, which allowed us to confidently proceed to analyze the effect of HA on midbrain development. We corroborated, by co-injections with Cell tracker, that HA reached all cells in the VM. When we evaluated neuronal differentiation by immunostaining, qRT-PCR and immunoblot, HA did not modify β-III Tubulin expression, suggesting that HA is not precluding neuronal differentiation. However, consistent with the data in VM NSPC cultures, when the limiting enzyme for DA synthesis, TH, was evaluated by immunohistochemistry, qRT-PCR and Western blot, we found significant decreases after HA application at E12, pointing out to a detrimental effect on dopaminergic neurons in vivo. These results suggest that HA might be affecting either DA specification and/or differentiation. To study if DA specification was being affected, we analyzed the expression of the Lim homeodomain genes Lmx1a and Lmx1b, which are specific markers for midbrain dopaminergic precursors. Lmx1a is an early marker of dividing mesencephalic precursors in the floor plate ventricular cells at early stages of development, instructing their differentiation into the midbrain dopaminergic phenotype, while Lmx1b is essential for the generation of properly differentiated midbrain dopaminergic neurons and its regional specification ,. HA significantly decreased the expression levels of both markers. Pitx3, a protein important for maintenance of dopaminergic identity, also decreased in HA-injected embryos, relative to vehicle-treated animals. To further support that HA is acting on DA precursors, E10 and E12 embryos were injected either with vehicle or HA. We demonstrated that the impairing effect of HA over the dopaminergic lineage is present in both developmental stages. In sharp contrast, DA neurons were unaffected when HA was applied at E14 or E16, when most cells already differentiated to dopaminergic neurons. These results strongly support that detrimental action of HA is on VM dopaminergic neural precursors rather than early differentiated neurons.
We found that HA did not modify BrdU incorporation in cultured VM NSPC, but significantly decreased the number of ventricular pHH3-positive cells in vivo. This different effect of HA on proliferation might be explained by the fact that BrdU was added to cultures for 3 h, and is incorporated by cells in the S phase of cell cycle, whereas pHH3 is present in cells undergoing mitoses. An additional factor to consider is that 100 μM HA caused a significant increase of BrdU incorporation in cultured cortical NSPC in the presence of daily-added 10 ng/ml FGF-2 , the same conditions used here for VM NSPC. A potentiation of thymidine incorporation has also been reported for tracheal smooth muscle human cells incubated with 10 ng/ml Epidermal Growth Factor and 10 μM HA, compared to the growth factor-only condition; this increased proliferation was associated to activation of Akt with participation of Gq/11 receptors . It is possible that in cultured VM cells there are subpopulations that differentially respond to HA: some might decrease (i.e. dopaminergic precursors), while others (non-dopaminergic cells) could increase BrdU incorporation, resulting in no overall changes after HA addition. This possibility is supported by the fact that HA caused a significant decrease in the number of differentiated dopaminergic neurons both in vitro and in vivo.
We also analyzed the differentiation of other neuronal phenotypes present in midbrain-surrounding regions. Both serotoninergic and dopaminergic neurons arise from the ventral-most neuroepithelial progenitors, although at different rostro-caudal levels . GABAergic cells are present in the substantia nigra pars reticulata and to some extent within the substantia nigra pars compacta and also in the ventral tegmental area . Here, we demonstrate that the HA effect on midbrain development was selective for dopaminergic phenotype without affecting the generation of GABA neurons. In line with these findings, serotonin neurons, which are formed in the hindbrain very close to the midbrain-hindbrain boundary, were unaffected by HA.
In order to identify the cellular mechanism by which HA was exerting its action on decreasing TH + dopaminergic neurons, we co-injected HA with H1R or H2R antagonists. We found that only chlorpheniramine, a selective H1R antagonist, completely prevented the impairing action of HA over DA neurons. Our data support the hypothesis that HA action is on precursor dopaminergic cells by activation of H1R. We suggest that HA might be acting as a detrimental signal during early stages of DA specification. As we showed in vitro, incubation with HA leads to the elevation of Ca2+ in Nestin-positive VM NSPC, which might suggest that the mechanistic basis of HA effect over NSPC population affecting dopaminergic differentiation/specification involves intracellular Ca2+. Interestingly, Ca2+ signaling appears to control NSPC proliferation, regulating the switch from proliferation to neuronal differentiation . For instance, in human NSPC, mobilization of IP3-dependent Ca2+ stores lengthens the cell cycle and increases the number of intermediate neural progenitors . In this model, Ca2+ controls the duration of cell cycle by increasing the level of p53 protein, a known regulator of the cyclin-dependent kinase inhibitor p21 . We have previously shown in cortical NSPC cultures the effect of HA on cell division patterns, evaluating asymmetric and symmetric divisions . In the developing VM, proliferating cells are present in close to the ventricular zone ; we show that the number of mitotic cells is decreased by HA injection in vivo, and this could be a contributing mechanism for the decreased number of dopaminergic neurons.
The effects of artificially administering HA during VM development are clear, but we did not directly assess the participation of endogenous HA on dopaminergic development for example by decreasing its levels by inhibiting Histidine decarboxylase activity; nonetheless, the injection of H1 or H2 receptor antagonists alone did not modify TH staining, suggesting that the endogenous HA levels are not sufficient to affect dopaminergic development. Interestingly, the participation of HA in the pathogenesis of Parkinson’s disease in adult organisms has been suggested by previous work. For example, there is an increase in brain HA content in post-mortem putamen and substantia nigra and HA blood levels are increased in parkinsonian patients . Also, the number of histaminergic fibers innervating the substantia nigra is increased in individuals suffering Parkinson’s disease relative to controls ,. In addition to these suggestive data, there is experimental evidence showing that HA can affect differentiated dopaminergic neurons in the adult brain. Direct infusion of HA in the rodent substantia nigra leads to a selective damage of DA neurons . One of the most widely used animal model for Parkinson’s disease is the intracerebral injection of the DA neurotoxin 6-hydroxydopamine in one hemisphere. When endogenous brain HA levels were increased or decreased, 6-hydroxydopamine-induced damage was potentiated or inhibited, respectively, assessed by TH immunostaining and by the rotational test after apomorphine administration. Supporting the participation of HA in the damage induced by this neurotoxin, the H1R antagonist pyrilamine, significantly, albeit transiently, decreased DA neuron degeneration; the H2R antagonist cimetidine, did not modify the 6-hydroxydopamine-induced damage .
Alternative mechanisms for the deleterious effects of HA on DA cells can include activation of microglial cells. In the adult substantia nigra injection of HA generates a microglial reaction, promoting dopaminergic cell death . The possibility that microglia might contribute to DA degeneration in our model needs to be tested. There is evidence that microglia are present at this developmental stages , and that activated microglial cells reduce the number of neural precursor cells by phagocytosis in the developing cerebral cortex .
Our findings reveal an unexpected role of HA receptor activation during embryogenesis in a specific neuronal phenotype. These effects are relevant for its significance as a possible therapeutic target in CNS pathologies that involve the interaction between both histamine and dopamine systems. In conclusion, our study contributes to the knowledge of factors participating on the generation and degeneration of DA neurons and reveals novel actions of HA during midbrain development, which might lead to design new and more specific protocols for the generation of dopaminergic neurons and future therapies for Parkinson’s disease.
Multipotent NSPC were isolated from E12-E12.5 embryos obtained from time-pregnant Wistar rats as described . 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.
RNA extraction, RT-PCR and Real-Time RT-PCR
Primer sequences for detection of histaminergic receptors by end-point RT-PCR
These primers have been used previously by our group  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.
Primer sequences for detection of transcripts by qRT-PCR
Cells were cultured and seeded at 3.5×104/well in 6-well plates (Corning) in control and experimental conditions, following standard protocols ,. 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 . 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.
BrdU incorporation assay
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 . 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 . 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 staining
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 ,.
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.
Intracellular Ca2+ imaging
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 . 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.
Ultrasound guided injections
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 , 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.
This work was supported by grants from Dirección General de Asuntos del Personal Académico, Universidad Nacional Autónoma de México (Papiit IN208713), Consejo Nacional de Ciencia y Tecnología (CONACyT 131281) to I.V., and by the Ministerio de Ciencia e Innovación de España (Grant BFU2010-21377) to J.A.D.C. Itzel Escobedo-Avila, Rodrigo López-González and Daniel Cortés received a graduate fellowship from CONACyT. Fernanda Vargas-Romero received an undergraduate fellowship from DGAPA (IN208713). We thank Drs. Rosa E. Navarro and Jesús Santa-Olalla for continuous discussion and advice. The technical assistance of the microscopy unit of Instituto de Fisiología Celular is appreciated. The Nestin monoclonal antibody developed by S. Hockfield was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242, USA. We thank Dr. Daniel Hoeppner for critical reading of the manuscript.
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