Abstract
The generation and specification of pyramidal neuron subpopulations during development relies on a complex network of transcription factors. The CB1 cannabinoid receptor is the major molecular target of endocannabinoids and marijuana active compounds. This receptor has been shown to influence neural progenitor proliferation and axonal growth, but its involvement in neuronal differentiation and the functional impact in the adulthood caused by altering its signaling during brain development are not known. Here we show that the CB1 receptor, by preventing Satb2 (special AT-rich binding protein 2)-mediated repression, increased Ctip2 (COUP-TF interacting protein 2) promoter activity, and Ctip2-positive neuron generation. Unbalanced neurogenic fate determination found in complete CB1−/− mice and in glutamatergic neuron-specific Nex–CB1−/− mice induced overt alterations in corticospinal motor neuron generation and subcerebral connectivity, thereby resulting in an impairment of skilled motor function in adult mice. Likewise, genetic deletion of CB1 receptors in Thy1–YFP–H mice elicited alterations in corticospinal tract development. Altogether, these data demonstrate that the CB1 receptor contributes to the generation of deep-layer cortical neurons by coupling endocannabinoid signals from the neurogenic niche to the intrinsic proneurogenic Ctip2/Satb2 axis, thus influencing appropriate subcerebral projection neuron specification and corticospinal motor function in the adulthood.
Introduction
The development of the cerebral cortex involves the sequential formation and specification of the excitatory neuron populations that constitute the definitive six-layered cortical structure (Molyneaux et al., 2007; Fishell and Hanashima, 2008). Among the cell intrinsic mechanisms involved in corticogenesis, a complex proneurogenic transcription factor program is responsible for the correct establishment of neuronal identity within an area or layer (Guillemot et al., 2006; Molyneaux et al., 2007). These factors coordinate cell-cycle exit, neuronal migration, and the specific gene expression program that dictates neuronal identity of upper and deep cortical neurons and axonal connectivity. Neurons projecting to subcortical areas are primarily located in early-generated deep layers 5 and 6, whereas callosal projecting neurons are abundant in upper layers 2 through 4 (Sur and Rubenstein, 2005; Molyneaux et al., 2007). Specification of subcortical (i.e., corticothalamic and subcerebral) projection neurons relies on the combinatorial action of a transcriptional regulation network composed of different factors, such as special AT-rich sequence-binding protein 2 (Satb2), chicken ovoalbumin upstream promoter transcription factor I (COUP-TFI), and its interacting protein 2 (Ctip2/Bcl11b), a zinc finger transcriptional repressor (Arlotta et al., 2005; Alcamo et al., 2008; Tomassy et al., 2010). Forebrain embryonic zinc finger-like protein 2 (Fezf2) and its downstream regulator COUP-TF interacting protein 2 (Ctip2) are sufficient to induce ectopic specification of subcerebral projection neurons, and, in particular, of corticospinal motor neurons (CSMNs), which reside in layer 5b and extend their axons to the spinal cord (Molyneaux et al., 2005; Chen et al., 2008). In contrast, upper-layer specification factor Satb2 directly binds to matrix attachment regions (MARs) of the Ctip2 promoter and prevents Ctip2 expression, thus favoring the neuronal fate of callosal projection (Alcamo et al., 2008).
In addition to these endogenous determinants, an array of extracellular cues from the neurogenic niche is necessary for the precise coordination of cortical development. The endocannabinoid (eCB) system, via the cannabinoid CB1 receptor, has been shown to exert a regulatory role in corticogenesis (Aguado et al., 2005; Berghuis et al., 2007; Morozov et al., 2009). Specifically, CB1 receptor inactivation leads to defective ventricular/subventricular zone (VZ/SVZ) progenitor cell proliferation and axonal guidance alterations (Aguado et al., 2005; Mulder et al., 2008), thus impairing long-range corticothalamic connectivity (Mulder et al., 2008; Wu et al., 2010). However, the molecular mechanism of CB1 receptor action in neuronal specification has not yet been investigated. Likewise, the impact on cortical development and the subsequent alterations in adult brain function as a consequence of altered prenatal CB1 receptor function on intake of cannabinoid receptor agonists or antagonists by pregnant women remains primarily elusive (Galve-Roperh et al., 2009; Jutras-Aswad et al., 2009; Schneider, 2009). Hence, in the present study, we investigated the regulatory role of the CB1 receptor in the transcription factor program that controls pyramidal neurogenesis and laminar differentiation, as well as its potential additional impact for adulthood neural functions. Our findings reveal that CB1 receptor signaling, by modulating the Ctip2/Satb2 transcriptional regulatory code in differentiating neurons, controls neuronal projection fate differentiation, thereby tuning subsequent CSMN development and function.
Materials and Methods
Materials.
The following materials were kindly donated: anti-CB1 receptor antibody (K. Mackie, Indiana University, Bloomington, IN), FAAH−/− mice (B. Cravatt, Scripps Institute, San Diego, CA), pCAG–DsRed (M. Nieto, National Center of Biotechnology, Madrid, Spain), pfosluc constructs with the MAR sequences A2–A5 of the Ctip2 promoter and pMSCV–Satb2 expression vector (R. Grosschedl, Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany), and HU-210 [(6aR,10aR)-3-(1,1′-dimethylheptyl)-6a,7,10,10a-tetrahydro-1-hydroxy-6,6-dimethyl-6H-dibenzo[b,d]pyran-9-methanol] (R. Mechoulam, Hebrew University, Jerusalem, Israel). Rat monoclonal anti-5-bromo-2′-deoxyuridine (BrdU), rabbit polyclonal anti-Ctip2, anti-T-box brain 1 (Tbr1), anti-Tbr2, and mouse monoclonal anti-Satb2 antibodies were from Abcam. Rabbit polyclonal anti-CB1 antibody (Frontier Institute, Hokkaido, Japan), mouse monoclonal anti-CRE (Covance), mouse monoclonal anti-GFP (Invitrogen), and chicken polyclonal anti-GFP (Millipore) antibodies were also used.
Animals.
Experimental designs and procedures were approved by the Complutense University Animal Research Committee in accordance with Directive 86/609/EU of the European Commission. All efforts were made to minimize the number of animals and their suffering throughout the experiments. Mice were maintained in standard conditions, keeping littermates grouped in breeding cages, at a constant temperature (20 ± 2°C) on a 12 h light/dark cycle with food and water ad libitum. The generation and genotyping of CB1−/−, CB1f/f,Nex–Cre, CB1f/f,Dlx5/6-Cre, and FAAH−/− mice and their respective littermate controls have been reported previously and was performed accordingly (Cravatt et al., 2001; Marsicano et al., 2003; Monory et al., 2007; Massa et al., 2010). Thy1–eYFP (line H) mice were obtained from The Jackson Laboratory [B6.Cg–Tg(Thy1–YFP–H)2Jrs/J] and crossed with CB1−/− mice. The heterozygous F1 generation was crossed again with CB1−/− mice. Mouse tissues of either sex were obtained during timed mating as assessed by vaginal plug.
Immunofluorescence and confocal microscopy.
Coronal brain slices (10 μm) were processed as described previously (Mulder et al., 2008), and layers were identified by their discrete cell densities as visualized by Hoechst 33528 (Sigma) and β-III-tubulin counterstaining. After blockade with 5% goat serum, brain sections were incubated overnight at 4°C with the indicated primary antibodies. Confocal fluorescence images were obtained in a blinded manner by an independent observer, and all quantifications were obtained from a minimum of six sections from 1-in-10 series per mice. Immunofluorescence of cortical sections was performed along the rostral-to-caudal axis, and the quantifications were performed in the mediolateral area of rostromedial sections that correspond to the motor/somatosensory cortex. Pyramidal layer specification was determined at embryonic day 16.5 (E16.5), postnatal day 2 (P2), and P8 in a 50-μm-wide cortical column divided into 10 equally sized bins, from the ventricular surface to the marginal zone. At least two independent cortical columns were analyzed per section, and results were averaged (see Figs. 1, 2, 6, 8). Positive cells for the corresponding markers were quantified and referred to the total cell number in the bin identified by Hoechst 33528.
Protein kinase Cγ immunohistochemistry.
Protein kinase Cγ (PKCγ) immunohistochemistry was performed in 100-μm-thick sagittal sections. After permeabilization, quenching of endogenous peroxidase activities and blockade with goat serum free-floating sections were incubated with rabbit anti-PKCγ antibody (Santa Cruz Biotechnology) and processed according to the instructions of the manufacturer with 1:500 goat biotinylated anti-rabbit IgG and with avidin-biotinylated peroxidase complex (ABC Standard Vectastain ABC kit; Vector Laboratories). Stained sections were mounted, and bright-field images were captured from an Olympus BX51 upright microscope using an Olympus DP70 CCD camera.
In situ hybridization.
Coronal paraffin sections (10 μm) of E12.5, E13.5, E14.5, and E16.5 heads were obtained, deparaffinized, and processed for in situ hybridization as described previously (Monory et al., 2007). Clim1 (National Center for Biotechnology Information reference sequence NM_ 010698.3) riboprobe for in situ hybridization was amplified with the following primers: forward, ACCCTCATTCCCCGTTATT; and reverse, TGGCTCTCCTACCACCATC.
Real-time quantitative PCR.
RNA was isolated using RNeasy Plus kit (Qiagen). cDNA was obtained with Transcriptor (Roche). Real-time quantitative PCR assays were performed using the FastStart master mix with Rox (Roche), and probes were obtained from the Universal Probe Library Set (Roche). Amplifications were run in a 7900 HT-Fast Real-Time PCR System (Applied Biosystems). Each value was adjusted by using 18S RNA and β-actin levels as reference.
Ex vivo and in utero electroporation.
Ex vivo electroporation experiments were performed at E13.5 as described previously (Mulder et al., 2008) with pCAG–GFP and pCAG–CB1–GFP expression vectors, or pGFP-V-RS (Origene) shControl and shCB1 and dissociated cells were cultured as described below. In addition, CB1f/f cortices were electroporated with pCAG–GFP or pCAG–Cre–GFP (Addgene). In addition, in utero electroporations were performed at E14.5 or E13.5 with pCAG–CB1–GFP or DsRed (respectively) and analyzed at E16.5 (see Figs. 1I,J, 7A–D). In utero electroporation experiments with pCAG–CRE–GFP and pCAG-GFP control plasmids were also performed from E12.5 to P0 in CB1f/f mice (see Fig. 4D,E).
Pyramidal and organotypical cortical cell cultures.
Cortical neural progenitors were cultured from dissected cortices isolated at E13.5. Cells were mechanically dissociated and plated in polylysine- and laminin-coated dishes after ex utero electroporation and dissection of the cortical electroporated area (fast green positive). The neuronal phenotype was assessed after 7 d in vitro by quantification of Ctip2 and Satb2 expression in GFP-positive (GFP+) cells from ≥10 randomly selected view fields/coverslip after Hoechst 33528 cell counterstaining in genetically manipulated cells. In addition, pharmacological regulation experiments were performed in wild-type (WT) brain slices.
Gene promoter activity assays.
The neural stem cell line HiB5 was used after transient transfection with 0.75 μg of the A4- or A3–MAR–pfosluc reporter of the Ctip2 promoter, 0.5 μg of pCAG–CB1–GFP, 0.25 μg of pMSCV–Satb2, and 0.02 μg of renilla-derived luciferase as internal transfection control with Lipofectamine 2000. Transcriptional promoter-driven luciferase activity was performed using the Dual-Luciferase Reporter Promega assay system and renilla-derived luciferase activity as internal transfection control in a Lumat LB9507 luminometer (Berthold Technologies). Control experiments were performed with an excess of Satb2 expression that was able to efficiently block the Ctip2-reporter activity (data not shown).
Behavioral analyses.
CB1−/− and Nex–CB1−/− mice and their respective wild-type littermates (WT and CB1f/f) were analyzed at 8 weeks of age with paired mean age among groups. Animals were always tested during the same light phase and acclimatized to the testing room for at least 30 min. All tests were video recorded for subsequent analysis and double-blind quantification. Mice were food deprived the night before the testing day (mean body weight at the end of the test becoming 92.6 ± 2.2% of the initial body weight). Skilled motor test and patch removal tasks were performed according to established protocols (Tomassy et al., 2010). Briefly, a Plexiglas reaching box (20 cm long × 8 cm wide × 20 cm high), with a 1-cm-wide vertical slit in the front side of the box was used. Animals had to reach the palatable food pellet (20 mg dustless precision sucrose-flavored food pellets (Bio Serv) from a shelf (4 cm wide × 8 cm long) in front of the vertical slit. Mice were habituated to the sucrose-flavored pellets for 3 consecutive days before the tests, which consisted of three phases: habituation, unskilled reaching, and skilled reaching. In the habituation phase (two sessions), mice were placed in the testing cage and 10 pellets were scattered on the floor. The session finished when mice had eaten all the pellets or 10 min had passed. For the unskilled reaching test, food pellets were placed one by one on the shelf within the mouse's tongue-reaching distance. The test finished when 10 pellets were eaten or 4 min had passed. In the skilled reaching test, food pellets were placed one by one on the shelf 1.5 cm away from the slit, so that mice had to use their forelimbs to reach them. Pellet grasping and retrieval was scored as a success, and pellet displacement without retrieval was scored as an error. The test finished within 6 min. Results are represented as percentage of success [(total successes/total trials) × 100]. The absence of phenotypic alterations in CB1−/− and Nex–CB1−/− mice in the unskilled task was used as a control that CB1 receptor ablation per se does not interfere with the test by influencing factors different from corticospinal function. Complementary analysis of motor impairment in CB1−/− mice was conducted by using the staircase reaching test (Campden Instruments). This test allows measurement of coordinated paw reaching in rodents. The system is formed by two stairs with eight steps each on which two rewarding food pellets can be placed. The test consisted of three phases: training, unskilled reaching, and skilled reaching. Habituation to sucrose-flavored food pellets was performed for 3 consecutive days, and then mice were habituated to the apparatus for 2 d by placing some pellets along the central corridor and stairs. On 5 consecutive training days, both stairs were filled with two pellets per stair, and mice were challenged to reach pellets that had been placed exclusively in the stairs for 10 min. In two additional test sessions, animals were challenged to reach pellets placed in the five lowest steps, which need the use of a paw to be reached (note that the three upper steps can be reached with the tongue and thus are not useful to assess paw skilled reaching ability). Finally, the patch removal test was conducted to measure skilled motor performance by assessing the ability of the mouse to remove a piece of adhesive patch placed in each hindpaw as described previously (Tomassy et al., 2010). Adhesive patches were placed in each hindpaw of the mouse, and the animal was placed in a normal housing cage and video recorded. The test finished when both patches had been removed or 4 min had passed. The time of latency for the first nose contact with the patch was determined, thus providing a general assessment of the sensorial status. Skilled patch removal ability was measured by calculating the mean number of contacts until each adhesive patch had been removed. Results shown correspond to the average of two tests. Additional characterization of general motor activity, exploration, and coordination was performed in ActiTrack and RotaRod devices as described previously (Blazquez et al., 2011).
DiI labeling.
Labeling of corticospinal tracts with the lipophilic carbocyanine dye DiI (1,1′-diotadecyl-3,3,3′,3′-tetramethylindocarbocyanine; Invitrogen) was performed in the brains of Nex–CB1−/− and CB1f/f mice at P2. A single crystal of DiI was picked up on a fire-polished tip of a broken glass micropipette and inserted into the presumptive motor cortex. The DiI-containing brains were incubated in PBS/0.1% sodium azide at 50°C in the dark for 4 weeks. Brains embedded in 3% agar were sectioned at 100 μm in the sagittal plane, counterstained with DAPI, and mounted with PBS. Axon projections were visualized using a Carl Zeiss AxioImager M1 with 5× magnification and 0.16 numerical aperture Zeiss objectives. Confocal images were obtained using a Carl Zeiss 510 system with 10×/0.45 numerical aperture (air) objective lens.
Data analyses and statistics.
Results shown represent the means ± SEM, and the number of experiments is indicated in every case. Statistical analysis was performed by one- or two-way ANOVA, as appropriate. A post hoc analysis was made by the Student–Neuman–Keuls test.
Results
The CB1 cannabinoid receptor regulates cortical layer neuron specification
We initially determined the impact of CB1 receptor inactivation on cortical development by analyzing cortical thickness of CB1−/− and WT littermates. CB1−/− mice showed enlarged ventricles at P2, in agreement with the described alterations induced by in utero CB1 receptor pharmacological blockade (Mulder et al., 2008). A reduction of total cortical thickness was evident at P2 in CB1−/− mice (total cortical thickness, 527 ± 26 μm in WT mice vs 475 ± 15 μm in CB1−/− mice; p < 0.05), which was attributable to deep-layer but not upper-layer thickness reduction (upper- and deep-layer cortical thickness, 174 ± 26 and 352 ± 28 μm in WT mice vs 172 ± 5 and 302 ± 20 μm in CB1−/− mice; p < 0.05 for deep-layer cortical thickness; n = 6 for each group). These results support a significant role of the CB1 receptor in controlling progenitor population size and raise the question of whether CB1 receptor signaling exerts a selective function in the differentiation of the neuronal populations of the different cortical layers. Because Tbr1 is an early expressed T-box transcription factor that promotes deep-layer specification and corticothalamic neuron projections (Hevner et al., 2001; Han et al., 2011), we first analyzed postmitotic Tbr1+ neuroblasts along the developing cortex in CB1 receptor-deficient mice. Quantification of Tbr1+ cells in equal-sized bins at E16.5 showed that these postmitotic cells accumulated abnormally in deep bins of the cortical plate of CB1−/− mice compared with WT littermates (Fig. 1A, middle column, B). Moreover, the distribution of neurons that express the upper-layer neuronal marker Satb2 was also affected (Fig. 1A, right column), and, in particular, Satb2+ cells were expanded in the lower bins 2–3 (Fig. 1C). Double marker analysis further showed that CB1 receptor deletion increased the number of cells that coexpress Tbr1 and Satb2 (Fig. 1D,E). Satb2 is a well-known repressor of Ctip2, a selective layer 5b marker of subcerebral projection neurons, and this repressive action of Satb2 promotes callosal projection-neuron identity (Alcamo et al., 2008). We sought to investigate the impact of CB1 deletion in the development of Satb2+ and Ctip2+ neuronal cell populations. Satb2+ cells in CB1−/− cortices at E16.5 were intermingled among Ctip2+ cells, and an increased number of cells coexpressing both markers were evident during CB1 receptor inactivation (Fig. 1D,F). Satb2+ cell number in the total cortical column was significantly increased (Fig. 1G), and consequently Ctip2+ cells were reduced in CB1−/− mice compared with WT littermates (Fig. 1H).
Considering the altered expression of neuronal specification markers induced by genetic ablation of CB1, we performed gene expression analysis of selected transcription factors involved in the regulation of corticogenesis in E14.5 cortical extracts. CB1 receptor inactivation impaired the expression of determinants known to be involved in fate specification and development of deep- and upper-layer cortical neurons. Thus, CB1 receptor deficiency decreased the expression of the deep-layer neuronal markers Fezf2 and Ctip2 (relative mRNA levels, 0.55 ± 0.07 vs 1.00 ± 0.15 in WT cortices and 0.47 ± 0.06 vs 1.00 ± 0.13 in WT cortices; p < 0.05 and p < 0.01, respectively; n = 4 for each group) (Arlotta et al., 2005; Chen et al., 2008) and the corticofugal neuronal marker Sox5 (relative mRNA levels, 0.50 ± 0.05 vs 1.00 ± 0.11 in WT cortices; p < 0.05) (Lai et al., 2008). Conversely, levels of the upper-layer transcription factor Satb2 were upregulated in CB1−/− cortices (relative mRNA levels, 2.65 ± 0.15 vs 1.00 ± 0.23 in WT cortices; p < 0.05). To validate the role of the CB1 receptor in the correct expression of upper/deep-layer neuronal specification markers, we performed a gain-of-function strategy aimed at rescuing CB1 receptor expression in a CB1−/− background by in utero electroporation of a pCAG–CB1–GFP expression vector (Fig. 1I). Importantly, reexpression of the CB1 receptor reduced the expansion of Satb2+ cells that occurred in deep bins of CB1−/− cortices and thus reduced the number of GFP+Satb2+ cells (Fig. 1I, arrows, J).
The CB1 cannabinoid receptor controls deep-layer neuron specification through the regulation of Ctip2–Satb2 balance
We further evaluated the role of the CB1 receptor in the regulation of deep-layer neuronal specification by analyzing the changes in the Ctip2–Satb2 axis in cortical organotypic cultures of E13.5 WT mice. Treatment with HU-210, a CB1 receptor synthetic agonist, increased the number of Ctip2-only positive cells but decreased the number of Satb2+ cells (Fig. 2A–C, arrows and arrowheads, respectively). The induction of neuronal differentiation to Ctip2+ cells by HU-210 and its inhibitory effect on Satb2+ cell differentiation were prevented by the CB1 receptor-selective antagonist SR141716 [N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole carboxamide]. Because Satb2 negatively regulates Ctip2 expression by binding to MAR sequences at the Ctip2 promoter (Alcamo et al., 2008; Britanova et al., 2008), we performed luciferase reporter assays for different MAR regions of the Ctip2 promoter in the HiB5 neural stem cell line. HiB5 cells were transiently transfected with luciferase constructs under the control of the Satb2-binding sites A4 or A3 of the Ctip2 promoter together with expression vectors for CB1 and Satb2. HU-210 treatment increased A4–fosluc activity (Fig. 2D), and this effect was prevented by CB1 receptor blockade with SR141716. Likewise, HU-210 increased A3–fosluc activity (data not shown). Thus, CB1 receptor activity prevents the repressive effect of Satb2 on the Ctip2 promoter, thereby increasing its activity.
To directly assess the role of the CB1 receptor in deep-layer neuronal differentiation, we electroporated E13.5 mouse cortices, when CSMN generation is maximal (Tomassy et al., 2010), with a GFP+ vector, and the CB1 receptor was knocked down or overexpressed with shCB1 and pCAG–CB1, respectively. Cortical cultures were obtained by dissociation and allowed to differentiate for 7 d in vitro. Efficient manipulation of CB1 receptor expression under these conditions was evaluated by CB1 and GFP immunofluorescence (Fig. 3A,E). In addition, we validated the effect of shCB1 by Western blot analysis (Fig. 3B) and real-time PCR. shCB1 reduced CB1 protein and mRNA levels when compared with shControl (relative CB1 protein levels in shControl and shCB1, 1.00 ± 0.13 and 0.59 ± 0.15, p < 0.01; relative CB1 mRNA levels in shControl and shCB1, 1.00 ± 0.23 and 0.46 ± 0.17, p < 0.01). Immunofluorescence characterization of neuronal populations among the electroporated GFP+ population after differentiation revealed that CB1 receptor downregulation decreased the generation of Ctip2-only-positive cells (Fig. 3C,D), whereas CB1 receptor overexpression increased Ctip2+ cells (Fig. 3F,G). In addition, CB1 ablation by ex utero electroporation of pregnant E13.5 CB1f/f brains was conducted with a Cre recombinase expression vector (Fig. 4A). Using this strategy to achieve acute CB1 receptor loss of function, we also observed reduced Ctip2+ differentiation of cortical cells when compared with GFP+ cells (Fig. 4B, arrows, C). To further validate the results derived from ex utero CB1 receptor manipulation, we performed in vivo in utero electroporation with the pCAG–Cre–GFP and pCAG–GFP plasmids, and pups were analyzed at P0 (Fig. 4D). Noteworthy, in utero CB1 receptor ablation decreased Ctip2+ neuronal differentiation when compared with control CB1f/f cortices (Fig. 4E). These results support that CB1 receptor activity is required for the appropriate expression of the deep-layer neuronal determinant Ctip2.
The CB1 cannabinoid receptor independently regulates progenitor proliferation and neuronal differentiation
Because CB1 receptor loss of function is ensued by alterations in neuronal specification during cortical development (present study) and the CB1 receptor is also known to be expressed in progenitor cells, on which it drives VZ/SVZ progenitor proliferation in a cell-autonomous manner (Aguado et al., 2005; Mulder et al., 2008), a plausible hypothesis would be that CB1-receptor-mediated regulation of neuronal specification was a direct consequence of its role on progenitor generation. To investigate the putative relationship between changes in cortical progenitor proliferation and neuronal specification, we next compared complete CB1−/− mutant mice and glutamatergic-specific Nex–CB1−/− mutant mice. In the latter mutants, CB1 is selectively deleted in glutamatergic neurons of the dorsal telencephalon (Monory et al., 2006). Cre recombinase expression under the control of the Nex regulatory sequences was described to selectively target neurons of the developing cortex rather than cortical progenitors (Wu et al., 2005). However, the impact of conditional Nex–CB1−/− deletion during cortical development is unknown. Complete CB1−/− mice showed reduced progenitor proliferation in the developing cortex (Aguado et al., 2005), whereas no differences in progenitor cell proliferation were evident between CB1f/f and Nex–CB1−/− mice (Fig. 5A,B). Immunofluorescence analysis showed that Cre expression at E14.5 occurred in postmitotic areas of the developing cortex beyond the basal edge of the SVZ, as identified by the expression pattern of its marker Tbr2 (Fig. 5C). In situ hybridization experiments at different stages demonstrated that Nex–CB1−/− mice preserved CB1 expression at early proliferative stages in VZ/SVZ cells and only postmitotic neuroblasts lost their expression (Fig. 5D). In agreement with the notion that the CB1 receptor regulates neuronal differentiation independently of its actions on the progenitor cell pool, the observed increase in Satb2+ cells in CB1−/− was not attributable to its aberrant expression in apical or basal progenitor cells (data not shown). These findings demonstrated that the combined use of conditional Nex–CB1−/− and complete CB1−/− mice allows discriminating between CB1 receptor actions in VZ/SVZ neural progenitor populations and CB1-receptor-mediated regulation of differentiating postmitotic neuronal cells.
Deficient development of deep-layer Ctip2+ neurons in the absence of the CB1 cannabinoid receptor
To avoid the possible confounding interactions with impaired progenitor proliferation that occur in CB1−/− mice, we therefore turned to investigate the development of deep-layer Ctip2+ neurons in Nex–CB1−/− mice. The number of Ctip2+ cells at P2 along the cortical plate was reduced in Nex–CB1−/− mice compared with their WT littermates (Fig. 6A). Of importance, this effect was still evident at P8 (Fig. 6D). The direct regulatory action of CB1 receptor in the specification of the pyramidal lineage was confirmed by analyzing Dlx5/6–CB1−/− mice, which lack CB1 in GABAergic neurons of the forebrain (Monory et al., 2006). CB1 receptor deletion in the GABAergic lineage did not interfere with the generation of Ctip2+ cells (Fig. 6B). Substantiating a putative regulatory action of the eCB tone in cortical layer specification, mice deficient in fatty acid amide hydrolase (FAAH), a major eCB-degrading enzyme, showed an increased number of Ctip2+ cells and, therefore, an opposite phenotype to Nex–CB1−/− mice (Fig. 6C). In addition, in situ hybridization for Clim1 (Ldb2), a transcription factor that labels subcerebral projection neurons of layer 5 (Azim et al., 2009), revealed a severe reduction of Clim1+ neurons in Nex–CB1−/− mice (Fig. 6E). Furthermore, Western blot analysis of nuclear extracts from Nex–CB1−/− cortices showed reduced Ctip2 protein levels relative to Satb2 expression, whereas the opposite was observed in FAAH−/− cortical extracts (Fig. 6F).
The CB1 cannabinoid receptor regulates subcerebral projection neuron development
The alterations induced by CB1 receptor inactivation in the proneurogenic layer specification program prompted us to investigate its impact on axonal projection and connectivity. In line with previous observations (Mulder et al., 2008; Wu et al., 2010), aberrant corticofugal projections were found upon CB1 deletion. Immunofluorescence microscopy analysis of the L1 neural cell adhesion molecule revealed subcortical projection deficits in P2 CB1−/− and Nex–CB1−/− mice but not in Dlx5/6–CB1−/− mice (data not shown). The corticostriatal boundary of Nex–CB1−/− mice showed altered axonal trajectories, and, in the intermediate zone (IZ), disorganized and enlarged fascicles were evident (data not shown). These results, together with the involvement of the CB1 receptor in the differentiation of deep-layer Ctip2+ neurons, from where corticospinal projections arise (Molyneaux et al., 2007), prompted us to investigate the role of the CB1 receptor in subcerebral axonal projections. We performed in utero electroporation experiments with pCAG–DsRed in CB1−/− mice at E13.5 and analyzed DsRed+ projections at E16.5 (Fig. 7A). The corticospinal nature of DsRed-labeled axons was confirmed by the Ctip2+ immunoreactivity shown by DsRed+ somata (Fig. 7B). In WT embryos, labeled projecting axons expressed CB1 receptor (Fig. 7C) and showed straight trajectories, whereas profound alterations of navigating DsRed+ axons were observed at the IZ of CB1-deficient mice (Fig. 7D). To confirm more precisely the exact nature of these subcortical projection alterations, we performed immunohistochemical analysis of PKCγ, because this protein is present in corticospinal tracts. Similar to the aberrant pattern of L1 immunofluorescence, abnormal axonal trajectories of PKCγ-labeled tracts in the corticostriatal junction were evident at P8 in Nex–CB1−/− mice compared with WT littermates (Fig. 7E.1). Although no major alterations of the corticospinal tract in the posterior hindbrain of Nex–CB1−/− were observed, these animals had aberrant CSMN projections as axons traverse the pons (Fig. 7E.2,E.3), those projections reaching the spinal cord in a notably less organized manner (Fig. 7E.4).
To unequivocally ascribe a role for the CB1 receptor in the development of layer 5 subcerebral neurons, we took advantage of Thy1–YFP–H mice, in which the expression of the fluorescent protein under the control of the neuronal promoter of the Thy-1 gene (encoding Thy-1 membrane glycoprotein precursor) occurs selectively in layer 5 projection neurons, thus allowing the visualization of corticospinal tracts (Feng et al., 2000; Tomassy et al., 2010). We crossed CB1−/− and Thy1–YFP–H mice to study the specification and development of corticospinal neurons and axonal tracts in the absence of the CB1 receptor. At P21, a significant reduction in fluorescently labeled somata was evident in cortical layer 5 (Fig. 8A,B), whereas no changes were observed in the hippocampus (data not shown). CB1 receptor deletion induced an aberrant phenotype of subcerebral projection neurons similar to that observed by PKC γ immunohistochemistry (Fig. 7). Thus, misrouted axons were visible while traversing the internal capsule in the corticostriatal junction in CB1−/−:Thy1–YFP–H mice (Fig. 8C), axons that reached the pons were reduced, and the remaining ones showed fasciculation alterations (Fig. 8D,E). Finally, in agreement with a role of the CB1 receptor in subcerebral projection neuron development, anterograde DiI tracing from the motor cortex of Nex–CB1−/− mice confirmed the existence of misrouted fibers branching off the tracts and deviating from their normal path at the corticostriatal junction (Fig. 8F).
The CB1 cannabinoid receptor regulates CSMN function
The decreased Ctip2+ cell population and the disruption of subcerebral axonal projections observed in CB1−/− mice prompted us to investigate whether these alterations of CSMN projections result in defective cortical motor function. Evaluation of the skilled pellet-reaching task, which is dependent on CSMN-mediated connectivity (Tomassy et al., 2010), revealed that adult CB1−/− mice had a remarkable impairment in fine motor function (Fig. 9A). The total number of trials performed during the skilled task was not significantly different between the two groups of mice, and unskilled motor activity was not affected either (Fig. 9C,D), thus indicating the selectivity of the skilled motor function deficits. Additional support for this selectivity was provided by the observation that the general motor activity, including total distance traveled, resting time, and fast movements (ActiTrack test), as well as motor coordination (RotaRod test), did not differ between CB1−/− and WT mice (Fig. 10A). Moreover, and in concert with the neuroanatomical findings described above, the deficits in skilled motor activity observed in CB1−/− mice were recapitulated in Nex–CB1−/− mice (Fig. 9B).
Additional validation of the abnormal skilled motor function phenotype found in CB1−/− animals was obtained by using the staircase test, which also reflects fine motor activity and is useful to evaluate motor impairment after cortical lesions (Brooks and Dunnett, 2009). CB1−/− and Nex–CB1−/− mice had a lower performance than their respective WT littermates in their ability to grasp the most difficult food pellets (steps 4–8) (Fig. 9E–G). Moreover, we observed that retrieval of non-challenging pellets in the staircase test (steps 1–3) was not different among genotypes (Fig. 9H), thus confirming the selective impairment of fine motor activity. Finally, the patch-removal task, which also evaluates sensorimotor function, was also used. Nex–CB1−/− mice were significantly less efficient than their WT littermates in removing a piece of adhesive tape from their hindpaws, as demonstrated by the higher number of contacts required for patch removal (Fig. 10B, left). This decreased performance of Nex–CB1−/− mice in patch removal reflected a fine motor function impairment rather than an altered perception of the patch, as indicated by the quantification of the latency time for the first attempt to remove the patch and the total time spent removing the patch, which were not significantly different between both genotypes (Fig. 10B, middle and right).
Discussion
Recent studies have shown that the CB1 receptor regulates neural progenitor proliferation and axonal navigation, thus suggesting an instructive role of the eCB system in nervous system development (Harkany et al., 2007). The present study provides new insights into the developmental role of the CB1 receptor, revealing that it exerts a pivotal role in regulating the proneurogenic transcription factor code that controls cortical neuron differentiation. In particular, we show here that the CB1 receptor (1) tunes the differentiation balance of deep- and upper-layer cortical projection neurons, (2) is coupled to the regulation of the Ctip2–Satb2 transcriptional regulatory code, (3) plays a regulatory role in CSMNs development, and, as a consequence, (4) is required for the correct function of the mature CNS. Thus, cortical development is regulated by CB1 receptor signaling through a dual progenitor-dependent and progenitor-independent mechanism of action. Although the CB1 receptor present in neural progenitors promotes cortical progenitor self-renewal and VZ/SVZ proliferation in a cell-autonomous manner (Aguado et al., 2005; Mulder et al., 2008), the CB1 receptor expressed in radially migrating neuroblasts controls the intrinsic neurogenic transcriptional program involved in the appropriate balance of cortical layer differentiation (present study) by sensing the extracellular eCB neurogenic niche.
CB1 cannabinoid receptor-dependent regulation of the neurogenic niche during neocortical development
During early cortical development, the CB1 receptor is not expected to exert the classical neuromodulatory role of the eCB system in the mature adult brain, because synaptic maturation and activity is not yet completed. Therefore, during corticogenesis, CB1 receptor-mediated neural fate decisions occur in a cell-autonomous manner (Aguado et al., 2005, 2007). Later, CB1 receptor regulation of synaptic plasticity may turn a predominant mechanism of action, as exemplified at postnatal stages when the CB1 receptor regulates the whisker map development of the somatosensory cortex in a neuronal activity-dependent manner (Li et al., 2009).
Here, we demonstrate that the CB1 receptor allows differentiating neurons to transduce information from the surrounding neurogenic niche by modulating the intrinsic fate determinants involved in the developmental gene expression program of neuronal differentiation. It remains to be determined which signals control the production of eCBs released by neural progenitors and differentiating neurons (Aguado et al., 2005). Cortical progenitors express the enzyme involved in the synthesis of 2-arachidonoylglycerol (2AG) diacylglycerol lipase (Berghuis et al., 2007), which is downregulated during progenitor cell differentiation (Walker et al., 2010). In addition, the expression of the degradation enzymes FAAH and monoacylglycerol lipase (MAGL) during embryonic neuronal differentiation allows the delicate control of eCB levels (Aguado et al., 2005; Mulder et al., 2008; Keimpema et al., 2010). Regulation of 2AG availability by MAGL is responsible for the regulation of axonal growth by CB1 receptor signaling (Keimpema et al., 2010). 2AG produced in thalamocortical axons can activate the CB1 receptor present on growth cones of corticothalamic tracts, thus contributing to their correct integration and coordination (Wu et al., 2010). In addition, eCB depletion by the overexpression of the FAAH enzyme inhibits radial migration, suggesting a role of the endogenous cannabinoid tone in pyramidal neuron migration (Mulder et al., 2008). Overall, these evidences indicate that regulated expression and activity of the different signaling elements of the eCB system are part of the differentiation program of projection neurons.
CB1 cannabinoid receptor-dependent regulation of forebrain neurogenic transcription network drives CSMN differentiation
Analyses of CB1-deficient mice revealed that neuronal differentiation is altered, as shown by the delayed distribution of postmitotic Tbr1+ neuroblasts and fate decision changes mediated by the Ctip2/Satb2 code. Nex–Cre-driven deletion of CB1 allowed us to dissect the role of CB1 solely in neuronal differentiation because, in Nex–CB1−/− mice, cortical progenitors preserve CB1 receptor expression and cell proliferation is not affected. The prominent role of the gene expression program regulated by the Ctip2 pathway in subcerebral CSMN generation (Arlotta et al., 2005; Chen et al., 2008; Tomassy et al., 2010) and the ability of the CB1 receptor to control this transcriptional regulation axis reveal that the CB1 receptor constitutes a novel signaling platform involved in the appropriate corticofugal neuronal development and skilled motor function by tuning subcerebral-projecting versus callosal neuron-projecting differentiation.
In CB1−/− mice, the transcriptional repressor Satb2 is deregulated and Satb2+ cells are expanded and occupy developing deep layers, in which they are normally absent (Alcamo et al., 2008; Britanova et al., 2008). Because of the inhibitory effect of Satb2 on Ctip2 expression (Alcamo et al., 2008; Britanova et al., 2008), CB1 receptor deletion led to a reduction of Ctip2+ cells generated in layer 5. These results suggest the existence of complementary changes in transcriptional regulators of CSMN specification during CB1 receptor loss. In CB1-deficient mice, reduced Ctip2 and Fezf2 expression was observed. Fezf2 acts upstream of Ctip2 and is necessary and sufficient to induce subcortical axonal projection neurons of deep cortical layers (Molyneaux et al., 2005; Chen et al., 2008). Upper-layer neurons develop normally with concomitant Fezf2 downregulation, whereas its overexpression induces the excessive generation of corticospinal projections from where callosal projections normally arise (Molyneaux et al., 2005). Of importance, Tbr1 and Fezf2 mutually repress each other, and this regulatory mechanism is essential for the development of the corticospinal tract (McKenna et al., 2011). Thus, alterations of the Tbr1–Fezf2 transcriptional balance may also contribute to the deficits in CSMN specification observed in the absence of CB1 receptor signaling. In addition, loss of CB1 receptor also affected Clim1, another member of the subcerebral specification transcriptional regulation program that allows distinction between callosal and subcerebral layer 5 projection neurons (Azim et al., 2009). Of note, our findings of unbalanced transcriptional regulation of projection neuron differentiation in vivo are supported by in vitro studies of CB1 receptor-induced regulation of transcriptional activity and neuronal differentiation. Considering our previous data supporting a role of the eCB system in radial migration during corticogenesis (Mulder et al., 2008) and its ability to regulate downstream signaling systems that coordinate cell migration and neural cell differentiation, such as the mammalian target of rapamycin signaling pathway (Puighermanal et al., 2009; Palazuelos et al., 2012), a plausible hypothesis to be tested in the future would be the potential relationship between CB1 receptor regulation of radial migration and neuronal differentiation.
Role of the CB1 cannabinoid receptor in CSMN development and function: pathophysiological implications
Elucidating the contribution of CB1 receptor expressed during brain development to the function of the future adult brain is a challenging task with important biomedical implications. In the present study, we provide evidence for the existence of functional impairments in the brains of adult CB1 receptor-deficient mice as a consequence of alterations in developmental neuronal differentiation. Adult CB1−/− mice show defective skilled motor function that ensues alterations of corticospinal tract development associated with the regulatory role of the CB1 receptor of Ctip2/Satb2 transcriptional activity. Skilled motor behavior relies on the establishment of appropriate corticospinal connectivity, and even relatively mild alterations of the corticospinal tract can critically impair fine motor skills (Tomassy et al., 2010).
In addition to the skilled motor phenotype described herein, an emerging scenario suggests that genetic alterations or polymorphisms of CB1 and eCB-synthesizing/degrading enzymes can contribute to changes, often subtle, in forebrain development, which might in turn contribute to the susceptibility to a variety of psychiatric disorders in the adult brain (Galve-Roperh et al., 2009; Fiskerstrand et al., 2010). Likewise, changes in prenatal CB1 receptor function may conceivably ensue during pregnancy during mother's consumption of cannabinoid receptor agonists or antagonists (Jutras-Aswad et al., 2009) or exposure to other xenobiotics that interact with the eCB system, such as ethanol or organophosphorous pesticides (Nomura et al., 2008). Human and animal model studies have shown that loss of function of neuronal specification determinants (e.g., Tbr1, Satb2, Ctip2) results in alterations of cortical development and neurogenesis, which produces in the adult brain severe consequences in behavioral processes, such as motor control, cognition, epileptogenesis, and sensorimotor integration (Tomassy et al., 2010; Saito et al., 2011). Deregulated gene expression levels of deep-neuronal specification factors are associated with temporal lobe epilepsy and a particular form of autism (Pasca et al., 2011; Rossini et al., 2011).
Defective genesis and maturation of glutamatergic projection neurons alter the excitation/inhibition neurochemical balance and influence the interneuron populations of the cortical plate (Sessa et al., 2010; Lodato et al., 2011), and these alterations could contribute to the etiology of a large variety of disorders, such as epilepsy and schizophrenia (Lewis and Sweet, 2009). The participation of the CB1 receptor in deep-layer neuronal differentiation and function reported here may provide a better understanding of the potential involvement of the eCB system in the neurodevelopmentally evoked susceptibility to motor neurodegenerative disorders (Blazquez et al., 2011), seizure occurrence (Marsicano et al., 2003), and psychiatric disorders (Jutras-Aswad et al., 2009).
Footnotes
This work was supported by Ministry of Science and Innovation Grants PLE2009-0117 (I.G-R.) Fundación Alicia Koplowitz (I.G.-R.), and SAF2009-08403 (M.G.), Community of Madrid–Complutense University of Madrid Grants S2011/BMD-2308, S2011/BMD-2336, and 950344 (M.G., I.G.-R.), National Institutes of Health Grants DA029381, HD065561, and NS048884 (H.-C.L.), and German Research Foundation Grant FOR926 (B.L.). J.D.-A. is supported by the Health Research Fund (PFIS), and T.A. and J.P. are supported by the Ministry of Science and Innovation. We are grateful to our collaborators that kindly contributed to this work by sharing reagents and molecular probes. We are also indebted to E. García-Taboada, E. Resel, M. Purrio, and A. Conrad for excellent experimental assistance and to A. González, A. García-Rodríguez, K. Mackie, O. Marín, and J. Ramírez-Franco for valuable comments.
- Correspondence should be addressed to Ismael Galve-Roperh, Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, 28040 Madrid, Spain. igr{at}quim.ucm.es