7 research outputs found
Activation of Type 1 Cannabinoid Receptor (CB1R) promotes neurogenesis in murine subventricular zone cell cultures
The endocannabinoid system has been implicated in the modulation of adult neurogenesis. Here, we describe the effect of type 1 cannabinoid receptor (CB1R) activation on self-renewal, proliferation and neuronal differentiation in mouse neonatal subventricular zone (SVZ) stem/progenitor cell cultures. Expression of CB1R was detected in SVZ-derived immature cells (Nestin-positive), neurons and astrocytes. Stimulation of the CB1R by (R)-(+)-Methanandamide (R-m-AEA) increased self-renewal of SVZ cells, as assessed by counting the number of secondary neurospheres and the number of Sox2+/+ cell pairs, an effect blocked by Notch pathway inhibition. Moreover, R-m-AEA treatment for 48 h, increased proliferation as assessed by BrdU incorporation assay, an effect mediated by activation of MAPK-ERK and AKT pathways. Surprisingly, stimulation of CB1R by R-m-AEA also promoted neuronal differentiation (without affecting glial differentiation), at 7 days, as shown by counting the number of NeuN-positive neurons in the cultures. Moreover, by monitoring intracellular calcium concentrations ([Ca2+](i)) in single cells following KCl and histamine stimuli, a method that allows the functional evaluation of neuronal differentiation, we observed an increase in neuronal-like cells. This proneurogenic effect was blocked when SVZ cells were co-incubated with R-m-AEA and the CB1R antagonist AM 251, for 7 days, thus indicating that this effect involves CB1R activation. In accordance with an effect on neuronal differentiation and maturation, R-m-AEA also increased neurite growth, as evaluated by quantifying and measuring the number of MAP2-positive processes. Taken together, these results demonstrate that CB1R activation induces proliferation, self-renewal and neuronal differentiation from mouse neonatal SVZ cell cultures.Fundacao para a Ciencia e a Tecnologia - Portugal [POCTI/SAU-NEU/68465/2006, PTDC/SAU-NEU/104415/2008, PTDC/SAU-NEU/101783/2008, POCTI/SAU-NEU/110838/2009]; Fundacao Calouste Gulbenkian [96542]; Fundacao para a Ciencia e Tecnologiainfo:eu-repo/semantics/publishedVersio
Primary antibodies used for immunocytochemistry.
<p>Primary antibodies used for immunocytochemistry.</p
SVZ cells express CB1R.
<p><b>A:</b> Detection of CB1R by Western blotting in SVZ. Lane 1 corresponds to SVZ proliferating cells, lane 2 to SVZ extract from adult C57Bl6 mice and lane 3 to the negative control (total proteins from CB1R-KO mice). <b>B–F:</b> Representative confocal digital images depicting CB1R immunoreactivity in SVZ cells after 7 days of differentiation [CB1R (in red); nestin (in green), GFAP (in green), PSA-NCAM (in green), DCX (in green), βIII tubulin (in green), MAP2 (in green) and Hoechst 33342 (used to visualize cell nuclei, in blue)]. c1, e1 and f1 are magnifications of squares in C, E and F, respectively. Scale bars = 20 µm. SVZ: subventricular zone; GFAP: Glial fibrillary acidic protein; PSA-NCAM: Polysialylated neural cell adhesion; βIII tubulin: Neuron-specific class III beta-tubulin; MAP2: Microtubule-associated protein 2; CB1R: CB1 receptor.</p
(R)-(+)-Methanandamide induces the differentiation of GABAergic neurons and neuritogenesis.
<p><b>A:</b> Schematic representation of the protocol. <b>B:</b> Bar graph depicts the numbers of either VGAT- or TH/βIII tubulin-positive cells, expressed as percentage of total cells. The data are expressed as percentage ± SEM. N = 3. *<i>P</i><0.05 using unpaired Student’s t test for comparison with control. <b>C:</b> Schematic representation of the protocol used for studying neuritogenesis. <b>D:</b> Representative confocal digital images of the GFP (green), MAP2 (red), Hoechst staining (blue), in control cultures and in cultures exposed to R-m-AEA. Scale bar = 20 µm. <b>E:</b> Bar graphs depict (from left to right): total length (µm), number of primary and number of secondary ramifications of MAP2 neurites per cell. N = 3. **P<0.01 using unpaired student’s t test for comparison with control. MAP2: Microtubule-associated protein 2; TH: tyrosine hydroxylase; βIII tubulin: Neuron-specific class III beta-tubulin; VGAT: vesicular GABA transporter.</p
(R)-(+)-Methanandamide does not induce glial differentiation in SVZ cultures through CB1R activation.
<p><b>A:</b> Protocol used for studying glial differentiation. <b>B:</b> Western blot analysis of GFAP and Olig2 protein levels in SVZ. Data are expressed as mean ± SEM. N = 4. <b>C:</b> Bar graph depicts the number of GFAP and Olig2-positive cells, expressed as the percentage of total cells <i>per</i> culture. Data are expressed as mean ± SEM. N = 3. <b>D:</b> Representative fluorescent digital images of GFAP-positive cells (green), Olig2-positive cells (red) and Hoechst staining (blue nuclei). Scale bar = 50 µm.</p
(R)-(+)-Methanandamide promotes the expression of the proneurogenic genes <i>Ngn1</i>.
<p><b>A:</b> Scheme of the protocol. <b>B:</b> Bar graph depicts the fold increase of H3K36m3 recruitment in the promoter region of <i>Ngn1</i> gene quantified by qChIP analysis. <b>C</b>: Bar graph depicts the fold increase of mRNA expression for Ngn1 protein evaluated by qRT-PCR analysis. Data are expressed as mean ± SEM. N = 4–7. *P<0.05, using Dunnett’s test for comparison with control (set to 1). H3K36m3: Histone H3 trimethylated on lysine 36; Ngn1: Neurogenin 1; qChIP: quantitative chromatin immunoprecipitation; qRT-PCR: quantitative real time polymerase chain reaction.</p
(R)-(+)-Methanandamide promotes self-renewal.
<p><b>A:</b> Experimental protocol. <b>B:</b> Bar graphs represent the number of primary and secondary neurospheres. Data are expressed as mean ± SEM. N = 6. *P<0.05, **P<0.01 and ***<i>P</i><0.001 using Dunnett’s multiple comparison test, for comparison with control; <sup>###</sup>P<0.001 using Dunnett’s multiple comparison test, for comparison with R-m-AEA. <b>C:</b> Protocol used for studying cell-fate. <b>D:</b> Confocal digital images of cell pairs obtained following (a) the symmetrical division of a SVZ cell into two Sox2+ cells (Sox2+/+), (b) the asymmetrical division into a Sox2+ and a Sox2- progenitor (Sox2+/−) and (c) the symmetrical terminal division into two Sox2- progenitors (Sox2−/−). Scale bars 20 µm. <b>E:</b> Bar graph illustrates the number of each type of cell divisions counted. Data are expressed as the percentage of total cell pairs and are represented as the mean ± SEM. N = 5. *P<0.05 and ***P<0.001 using Bonferroni’s multiple comparison test, for comparison with the respective controls; <sup>###</sup>P<0.001 using Bonferroni’s multiple comparison test, for comparison with the respective R-m-AEA. SOX2: sex determining region Y-box 2.</p