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Food addiction is linked to obesity and eating disorders and is characterized by a loss of behavioral control and compulsive food intake. Here, using a food addiction mouse model, we report that the lack of cannabinoid type-1 receptor in dorsal telencephalic glutamatergic neurons prevents the development of food addiction-like behavior, which is associated with enhanced synaptic excitatory transmission in the medial prefrontal cortex (mPFC) and in the nucleus accumbens (NAc). In contrast, chemogenetic inhibition of neuronal activity in the mPFC-NAc pathway induces compulsive food seeking. Transcriptomic analysis and genetic manipulation identified that increased dopamine D2 receptor expression in the mPFC-NAc pathway promotes the addiction-like phenotype. Our study unravels a new neurobiological mechanism underlying resilience and vulnerability to the development of food addiction, which could pave the way towards novel and efficient interventions for this disorder., This work was supported by the Spanish Ministerio de Economía y Competitividad-MINECO (#SAF2017-84060-R-AEI/FEDER-UE), the Spanish Instituto de Salud Carlos III, RETICS-RTA (#RD12/0028/0023), the Generalitat de Catalunya, AGAUR (#2017 SGR-669), ICREA-Acadèmia (#2015) and the Spanish Ministerio de Sanidad, Servicios Sociales e Igualdad, Plan Nacional Sobre Drogas (#PNSD-2017I068) to R.M., Fundació La Marató-TV3 (#2016/20-30) to E.M-G., the German Research Foundation (#CRC1193 “Neurobiology of Resilience”, TP A05 and B04) to B.L. and S.G., and the Boehringer Ingelheim Foundation to B.L., S.G. and I.R.A. The work of M.N.A was supported by the Emergent AI Center funded by the Carl-Zeiss-Stiftung. NARSAD Young Investigator Award (#22434), MINECO Ramón y Cajal (#RYC- 2014-15784) and (#SAF2016-76565-R) and Fondo Europeo de Desarrollo Regional (FEDER) to R.A. DIUE Generalitat de Catalunya (#2017 SGR 595), MINECO (#SAF2016-79956-R), EU (#Era Net Neuron PCIN-2013-060), Fundació La Marató-TV3 (#2016/20-31), CRG acknowledges support of the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) to the EMBL partnership, the Center of Excellence Severo Ochoa (#SEV-2012-0208), the CERCA Programme/Generalitat de Catalunya, the CIBER of Rare Diseases of the ISCIII to M.D. MINECO Ramón y Cajal (#RYC-2016-20414), AGAUR (#2017 SGR 926), (#RTI2018-094887-B-I00) and Fondo Europeo de Desarrollo Regional (FEDER) to M.N.

The generation and specification of pyramidal neuron subpopulations during development relies on a complex network of transcription factors. The CB1cannabinoid 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 CB1receptor, 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 completeCB1−/−mice and in glutamatergic neuron-specificNex–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 CB1receptors inThy1–YFP–Hmice elicited alterations in corticospinal tract development. Altogether, these data demonstrate that the CB1receptor 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.

Journal Article; Exposure to unbalanced diets during pre-gestational and gestational periods may result in long-term alterations in metabolism and behavior. The contribution of the endocannabinoid system to these long-term adaptive responses is unknown. In the present study, we investigated the impact of female rat exposure to a hypercaloric-hypoproteic palatable diet during pre-gestational, gestational and lactational periods on the development of male offspring. In addition, the hypothalamic and hippocampal endocannabinoid contents at birth and the behavioral performance in adulthood were investigated. Exposure to a palatable diet resulted in low weight offspring who exhibited low hypothalamic contents of arachidonic acid and the two major endocannabinoids (anandamide and 2-arachidonoylglycerol) at birth. Palmitoylethanolamide, but not oleoylethanolamide, also decreased. Additionally, pups from palatable diet-fed dams displayed lower levels of anandamide and palmitoylethanolamide in the hippocampus. The low-weight male offspring, born from palatable diet exposed mothers, gained less weight during lactation and although they recovered weight during the post-weaning period, they developed abdominal adiposity in adulthood. These animals exhibited anxiety-like behavior in the elevated plus-maze and open field test and a low preference for a chocolate diet in a food preference test, indicating that maternal exposure to a hypercaloric diet induces long-term behavioral alterations in male offspring. These results suggest that maternal diet alterations in the function of the endogenous cannabinoid system can mediate the observed phenotype of the offspring, since both hypothalamic and hippocampal endocannabinoids regulate feeding, metabolic adaptions to caloric diets, learning, memory, and emotions. Yes

The annexin A5 gene ( Anxa5 ) was recently found to be expressed in the developing and adult vascular system as well as the skeletal system. In this paper, the expression of an Anxa5 - lacZ fusion gene was used to define the onset of expression in the vasculature and to characterize these Anxa5-lacZ -expressing vasculature-associated cells. After blastocyst implantation, Anxa5 - lacZ -positive cells were first detected in extra-embryonic tissues and in angioblast progenitors forming the primary vascular plexus. Later, expression is highly restricted to perivascular cells in most blood vessels resembling pericytes or vascular smooth muscle cells. Viable Anxa5 - lacZ + perivascular cells were isolated from embryos as well as adult brain meninges by specific staining with fluorescent X-gal substrates and cell-sorting. These purified lacZ + cells specifically express known markers of pericytes, but also markers characteristic for stem cell populations. In vitro and in vivo differentiation experiments show that this cell pool expresses early markers of chondrogenesis, is capable of forming a calcified matrix and differentiates into adipocytes. Hence, Anxa5 expression in perivascular cells from mouse defines a novel population of cells with a distinct developmental potential.

CREB-binding protein (CBP) is an important transcriptional cofactor for various intracellular signaling pathways, including Ca(2+)- and cAMP-mediated gene activation. The loss of one CBP allele causes the human Rubinstein-Taybi syndrome, which is characterized by mental retardation and other severe developmental defects. Deletion of both CBP alleles in the mouse leads to early embryonic lethality. To address the function of CBP in late embryogenesis and in adult physiology, a floxed CBP allele (CBP(fl)) was generated. Using the Cre/loxP recombination system, CBP function was disrupted in principal forebrain neurons by breeding with a transgenic CaMKIIalpha-Cre mouse line to obtain CBP(fl/fl;CaMKIIalphaCre) mice. These mice contain CBP(stop523) alleles specifically in principal forebrain neurons, presumably resulting in the production of a truncated CBP protein unable to interact with a number of transcription factors, including phosphorylated CREB.

Acquisition and storage of aversive memories is one of the basic principles of central nervous systems throughout the animal kingdom. In the absence of reinforcement, the resulting behavioural response will gradually diminish to be finally extinct. Despite the importance of extinction, its cellular mechanisms are largely unknown. The cannabinoid receptor 1 (CB1) and endocannabinoids are present in memory-related brain areas and modulate memory. Here we show that the endogenous cannabinoid system has a central function in extinction of aversive memories. CB1-deficient mice showed strongly impaired short-term and long-term extinction in auditory fear-conditioning tests, with unaffected memory acquisition and consolidation. Treatment of wild-type mice with the CB1 antagonist SR141716A mimicked the phenotype of CB1-deficient mice, revealing that CB1 is required at the moment of memory extinction. Consistently, tone presentation during extinction trials resulted in elevated levels of endocannabinoids in the basolateral amygdala complex, a region known to control extinction of aversive memories. In the basolateral amygdala, endocannabinoids and CB1 were crucially involved in long-term depression of GABA (gamma-aminobutyric acid)-mediated inhibitory currents. We propose that endocannabinoids facilitate extinction of aversive memories through their selective inhibitory effects on local inhibitory networks in the amygdala.

Annexin A5 (annexin V, anchorin CII) represents the prototype member of the large annexin family, characterized by its ability to interact with phospholipids in a calcium-dependent manner and to form calcium-specific ion channels. Despite intense biochemical analysis, the in vivo expression and function of this annexin during mouse development, still remains unclear. Immunohistochemistry, in situ hybridization and reporter gene expression were used to define expression of annexin A5 during early mouse development. First, annexin A5 expression is associated with the developing vascular system. Later, expression is detected within the notochord and found in parallel to the differentiation of cartilage and bone. Therefore, expression of the Anxa5 gene may represent a novel marker characterizing cell lineages involved in the development of the vascular as well as the skeletal system.

Pax1 expression in vertebrate limb buds is confined to cells in a discrete anterior proximal domain (Timmons et al. [1994] Development 120:2773-2785; Ebensperger et al. [1995] Anat. Embryol. 191:297-310). In dorsoventral patterning of Drosophila, expression of pox meso, an insect gene with high sequence similarity to Pax1, is repressed by decapentaplegic (dpp) in dorsal mesoderm and, thus, is restricted to a discrete ventral domain (Staehling-Hampton et al. [1994] Nature 372:783-786). In the chick wing, cells expressing a vertebrate homolog of dpp, bone morphogenetic protein 4 (Bmp4), abut the Pax1 domain, suggesting a similar relationship between homologous genes in both vertebrates and invertebrates. Here, we show that two BMPs (BMP4, and BMP2, also highly related to dpp) can repress Pax1 in the developing chick wing. Chick wing bud cells expressing Pax1 give rise to the shoulder girdle. Cells in an equivalent position in the mouse forelimb also express Pax1, and Pax1 mutant mice display shoulder girdle defects. Similarly in chick embryos, girdle defects are produced by treatments with signalling molecules that lead to expression of BMPs, which subsequently reduce Pax1 expression in the limb bud. Recently, BMP4 has been shown to inhibit Pax1 expression in the developing trunk (Monsoro-Burq et al. [1996] Development 122:3607-3616) and Pax9 expression in developing teeth (Neubuser et al. [1997] Cell 90:247-255). Thus, a property of BMPs appears to be to regulate pox meso homologs negatively and, thus, limit their expression domains.

In summary, the generation of BMP-7-deficient mice has provided additional evidence that this family of growth factors regulate many morphogenetic processes including but not limited to skeletal development. In particular our experiments demonstrate that BMP 7 acts as an early inducer of glomeruli formation, and that it is required for skeletal patterning and lens formation. Our results not only demonstrate that BMP 7 is involved in the differentiation of several organs during development, but also raise the hypothesis that mutations in the Bmp 7 gene itself or in the genetic pathway could be responsible for several human genetic diseases in which glomerulus formation is impaired.

CREB-binding protein (CBP) is an important transcriptional cofactor for various intracellular signaling pathways, including Ca(2+)- and cAMP-mediated gene activation. The loss of one CBP allele causes the human Rubinstein-Taybi syndrome, which is characterized by mental retardation and other severe developmental defects. Deletion of both CBP alleles in the mouse leads to early embryonic lethality. To address the function of CBP in late embryogenesis and in adult physiology, a floxed CBP allele (CBP(fl)) was generated. Using the Cre/loxP recombination system, CBP function was disrupted in principal forebrain neurons by breeding with a transgenic CaMKIIalpha-Cre mouse line to obtain CBP(fl/fl;CaMKIIalphaCre) mice. These mice contain CBP(stop523) alleles specifically in principal forebrain neurons, presumably resulting in the production of a truncated CBP protein unable to interact with a number of transcription factors, including phosphorylated CREB.

We examined the molecular progression of ectopic bone development upon application of recombinant human bone morphogenetic protein-2 (rhBMP2), using a commercial collagen type I carrier, in the hind quarter muscles of mice. We performed a gene expression study using mRNA in situ hybridisation to compare embryonic cartilage and bone formation with BMP2-induced ectopic bone formation. As bone growth can be induced postnatally or in adult animals, we examined the expression of molecules regulating embryonic bone development. We found that the mRNAs of the same molecules, such as Indian hedgehog (IHH), parathyroid hormone (PTH)/PTH-related peptide receptor (PPR) and BMPs, that regulate embryonic cartilage and bone development, are expressed during BMP-induced ectopic bone formation, suggesting parallels in the mechanisms controlling these processes. Our studies support by molecular means the previous findings in rats that BMP2-induced ectopic bone formation in mice undergoes bone development involving both modes, endochondral and intramembranous ossification, simultaneously at different sites of the implant.

Annexins are highly conserved proteins that are characterized by their ability to interact with phospholipids in a calcium-dependent manner. Although diverse functions have been ascribed to annexins based on in vitro analyses, their in vivo functions still remain unclear. The intensively studied annexin A5 has been identified by its effects on blood coagulation, and subsequently, its function as a calcium-specific ion channel was described. In vitro experiments and expression studies suggested a potential role of annexin A5 during calcification processes in vivo, especially in endochondral ossification. To gain insights into the relevance of annexin A5 in this process, we generated an annexin A5-deficient mouse mutant. Mice lacking annexin A5 are viable, are fertile, and reveal no significant alterations in the biochemical parameters characteristic for metabolic or functional defects. Neither the development of skeletal elements nor the in vitro calcification properties of isolated chondrocytes is significantly impaired by the absence of annexin A5. Therefore, annexin A5 is dispensable for the formation and maintenance of skeletal elements in the mouse and may possibly be pointing to a compensatory effect of other members from the annexin family due to their high functional and structural similarity.

Valproate (VPA) is one of several effective anti-epileptic and mood-stabilizing drugs, many of which are also potent teratogens in humans and several other mammalian species. Variable teratogenicity among inbred strains of laboratory mice suggests that genetic factors influence susceptibility. While studying the genetic basis for VPA teratogenicity in mice, we discovered that parental factors influence fetal susceptibility to induced malformations. Detailed examination of these malformations revealed that many were homeotic transformations. To test whether VPA, like retinoic acid (RA), alters HOX expression, pluripotent human embryonal carcinoma cells were treated with VPA or RA and Hox expression assessed. Altered expression of specific Hox genes may thus account for the homeotic transformations and other malformations found in VPA-treated fetuses.

Expression of anchorin CII, a collagen-binding protein of the annexin family, was followed in the developing chick embryo using Northern and in situ hybridization and Western blotting. During chick somite development, anchorin CII mRNA was detected by Northern blotting as early as stage 11. At stage 24, anchorin mRNA accumulated in the anterior part of the somite sclerotome near the resegmentation line, as shown by in situ hybridization. The presence of anchorin CII protein during stages 11 to 20 was confirmed by Western blotting. In situ hybridization identified anchorin CII also in the otic vesicle adjacent to the site of contact with the statoacoustic ganglion and in the mandibular mesenchyme. The level of anchorin CII mRNA in differentiated hyaline cartilage, exemplified by sternal cartilage, was lower than that in differentiating somites or cultured chondrocytes. These findings are consistent with our notion that anchorin CII may be involved in cell-matrix interactions preceding chondrogenic differentiation events in the chick embryo. A significant level of anchorin CII mRNA and protein synthesis was also found in cultured myoblasts, but less than that in chondroblasts. This distribution pattern is different from that reported for a related protein, p34, or calpactin, the major protein substrate for tyrosine kinase phosphorylation in chick chondrocytes and fibroblasts. The results confirm suggestions from previous sequencing studies that anchorin CII and p34 are different proteins of the annexin/calpactin family.