An automated group-housed oral fentanyl self-administration method in mice.

RATIONALE AND OBJECTIVES: Social factors play a critical role in human drug addiction, and humans often consume drugs together with their peers. In contrast, in traditional animal models of addiction, rodents consume or self-administer the drug in their homecage or operant self-administration chambers while isolated from their peers. Here, we describe HOMECAGE ("Home-cage Observation and Measurement for Experimental Control and Analysis in a Group-housed Environment"), a translationally relevant method for studying oral opioid self-administration in mice. This setting reduces experimental confounds introduced by social isolation or interaction with the experimenter. METHODS: We have developed HOMECAGE, a method in which mice are group-housed and individually monitored for their consumption of a drug vs. a reference liquid. RESULTS: Mice in HOMECAGE preserve naturalistic aspects of behavior, including social interactions and circadian activity. The mice showed a preference for fentanyl and escalated their fentanyl intake over time. Mice preferred to consume fentanyl in bouts during the dark cycle. Mice entrained to the reinforcement schedule of the task, optimizing their pokes to obtain fentanyl rewards, and maintained responding for fentanyl under a progressive ratio schedule. HOMECAGE also enabled the detection of cage-specific and individual-specific behavior patterns and allowed the identification of differences in fentanyl consumption between co-housed control and experimental mice. CONCLUSIONS: HOMECAGE serves as a valuable procedure for translationally relevant studies on oral opioid intake under conditions that more closely mimic the human condition. The method enables naturalistic investigation of factors contributing to opioid addiction-related behaviors and can be used to identify novel treatments.

Claustral neurons projecting to frontal cortex restrict opioid consumption.

The synthetic opioid fentanyl is a major contributor to the current opioid addiction crisis. We report that claustral neurons projecting to the frontal cortex limit oral fentanyl self-administration in mice. We found that fentanyl transcriptionally activates frontal-projecting claustrum neurons. These neurons also exhibit a unique suppression of Ca2+ activity upon initiation of bouts of fentanyl consumption. Optogenetic stimulation of frontal-projecting claustral neurons, intervening in this suppression, decreased bouts of fentanyl consumption. In contrast, constitutive inhibition of frontal-projecting claustral neurons in the context of a novel, group-housed self-administration procedure increased fentanyl bout consumption. This same manipulation also sensitized conditioned-place preference for fentanyl and enhanced the representation of fentanyl experience in the frontal cortex. Together, our results indicate that claustrum neurons exert inhibitory control over frontal cortical neurons to restrict oral fentanyl intake. Upregulation of activity in the claustro-frontal projection may be a promising strategy for reducing human opioid addiction.

Protease-activated receptor-1 (PAR1) acts via a novel Galpha13-dishevelled axis to stabilize beta-catenin levels.

We have previously shown a novel link between hPar-1 (human protease-activated receptor-1) and beta-catenin stabilization. Although it is well recognized that Wnt signaling leads to beta-catenin accumulation, the role of PAR1 in the process is unknown. We provide here evidence that PAR1 induces beta-catenin stabilization independent of Wnt, Fz (Frizzled), and the co-receptor LRP5/6 (low density lipoprotein-related protein 5/6) and identify selective mediators of the PAR1-beta-catenin axis. Immunohistological analyses of hPar1-transgenic (TG) mouse mammary tissues show the expression of both Galpha(12) and Galpha(13) compared with age-matched control counterparts. However, only Galpha(13) was found to be actively involved in PAR1-induced beta-catenin stabilization. Indeed, a dominant negative form of Galpha(13) inhibited both PAR1-induced Matrigel invasion and Lef/Tcf (lymphoid enhancer factor/T cell factor) transcription activity. PAR1-Galpha(13) association is followed by the recruitment of DVL (Dishevelled), an upstream Wnt signaling protein via the DIX domain. Small interfering RNA-Dvl silencing leads to a reduction in PAR1-induced Matrigel invasion, inhibition of Lef/Tcf transcription activity, and decreased beta-catenin accumulation. It is of note that PAR1 also promotes the binding of beta-arrestin-2 to DVL, suggesting a role for beta-arrestin-2 in PAR1-induced DVL phosphorylation dynamics. Although infection of small interfering RNA-LRP5/6 or the use of the Wnt antagonists, SFRP2 (soluble Frizzled-related protein 2) or SFRP5 potently reduced Wnt3A-mediated beta-catenin accumulation, no effect was observed on PAR1-induced beta-catenin stabilization. Collectively, our data show that PAR1 mediates beta-catenin stabilization independent of Wnt. We propose here a novel cascade of PAR1-induced Galpha(13)-DVL axis in cancer and beta-catenin stabilization.

PAR1 plays a role in epithelial malignancies: transcriptional regulation and novel signaling pathway.

Protease-activated receptor 1 (PAR(1)) is the first and prototype member of an established PAR family comprising four members. The role of PAR(1) in tumor biology has been established, and is characterized by a consistent direct correlation between overexpression of its levels and epithelial tumor aggressiveness. We have found that high expression of the human Par(1) (hPar(1)) gene in epithelial tumors is controlled largely at the transcriptional level. This led us to assign Egr-1, a transcription activator, as an inducer of hPar(1), and p53, a tumor suppressor gene, as an inhibitor, both acting to achieve fine tuning of hPar(1) in prostate carcinoma. High PAR(1) levels maintain prosurvival signals in tumor cells while silencing or ablation of the gene induce apoptosis. Studies of our hPar(1) transgenic mice, which overexpress hPar(1) in the mammary glands, revealed a novel PAR(1)-induced ß-catenin stabilization function. The components connecting PAR(1) to ß-catenin stabilization have been determined, assigning at first G(α)(13) as a selective immediate component. The PAR(1)-G(α) (13) axis recruits disheveled (DVL), an upstream signaling partner of the canonical Wnt signaling pathway. Silencing of DVL by siRNA-DVL potently abrogates PAR(1)-induced ß-catenin stabilization, demonstrating its critical role in the process. We, thus, propose that transcriptional regulation of hPar(1) gene over expression in epithelia malignancies initiates a novel signaling pathway, directly connecting to ß-catenin stabilization, a core event in both tumorigenesis and developmental processes.

Egr2 induction in spiny projection neurons of the ventrolateral striatum contributes to cocaine place preference in mice.

Drug addiction develops due to brain-wide plasticity within neuronal ensembles, mediated by dynamic gene expression. Though the most common approach to identify such ensembles relies on immediate early gene expression, little is known of how the activity of these genes is linked to modified behavior observed following repeated drug exposure. To address this gap, we present a broad-to-specific approach, beginning with a comprehensive investigation of brain-wide cocaine-driven gene expression, through the description of dynamic spatial patterns of gene induction in subregions of the striatum, and finally address functionality of region-specific gene induction in the development of cocaine preference. Our findings reveal differential cell-type specific dynamic transcriptional recruitment patterns within two subdomains of the dorsal striatum following repeated cocaine exposure. Furthermore, we demonstrate that induction of the IEG Egr2 in the ventrolateral striatum, as well as the cells within which it is expressed, are required for the development of cocaine seeking.

The human brain is ever changing, constantly rewiring itself in response to new experiences, knowledge or information from the environment. Addictive drugs such as cocaine can hijack the genetic mechanisms responsible for this plasticity, creating dangerous, obsessive drug-seeking and consuming behaviors. Cocaine-induced plasticity is difficult to apprehend, however, as brain regions or even cell populations can react differently to the compound. For instance, sub-regions in the striatum ­ the brain area that responds to rewards and helps to plan movement ­ show distinct responses during progressive exposure to cocaine. And while researchers know that the drug immediately changes how neurons switch certain genes on and off, it is still unclear how these genetic modifications later affect behavior. Mukherjee, Gonzales et al. explored these questions at different scales, first focusing on how progressive cocaine exposure changed the way various gene programs were activated across the entire brain. This revealed that programs in the striatum were the most affected by the drug. Examining this region more closely showed that cocaine switches on genes in specific 'spiny projection' neuron populations, depending on where these cells are located and the drug history of the mouse. Finally, Mukherjee, Gonzales et al. used genetically modified mice to piece together cocaine exposure, genetic changes and modifications in behavior. These experiments revealed that the drive to seek cocaine depended on activation of the Egr2 gene in populations of spiny projection neurons in a specific sub-region of the striatum. The gene, which codes for a protein that regulates how genes are switched on and off, was itself strongly activated by cocaine intake. Cocaine addiction can have devastating consequences for individuals. Grasping how this drug alters the brain could pave the way for new treatments, while also providing information on the basic mechanisms underlying brain plasticity.

Mammary gland tissue targeted overexpression of human protease-activated receptor 1 reveals a novel link to beta-catenin stabilization.

Protease-activated receptor 1 (PAR1) is emerging with distinct assignments in tumor biology. We show that tissue targeted overexpression of hPar1 in mice mammary glands results in precocious hyperplasia, characterized by a dense network of ductal side branching and accelerated proliferation. These glands exhibit increased levels of wnt-4 and wnt-7b and a striking beta-catenin stabilization. Nuclear localization of beta-catenin is observed in hPar1 transgenic mouse tissue sections but not in the wild-type, age-matched counterparts. PAR1 induces beta-catenin nuclear localization also in established epithelial tumor cell lines of intact beta-catenin system (transformed on the background of mismatch repair system; RKO cells). We propose hereby that PAR1-mediated beta-catenin stabilization is taking place primarily via the increase of Wnt expression. Enforced expression of a specific Wnt antagonist family member, secreted frizzled receptor protein 5 (SFRP5), efficiently inhibited PAR1-induced beta-catenin stabilization. Likewise, application of either SFRP2 or SFRP5 on epithelial tumor cells completely abrogated PAR1-induced beta-catenin nuclear accumulation. This takes place most likely via inhibition of Wnt signaling at the level of cell surface (forming a neutralizing complex of "Receptors-SFRP-Wnt"). Furthermore, depletion of hPar1 by small interfering RNA (siRNA) vectors markedly inhibited PAR1-induced Wnt-4. The striking stabilization of beta-catenin, inhibited by SFRPs on one hand and Wnt-4 silencing by hPar1 siRNA on the other hand, points to a novel role of hPar1 in Wnt-mediated beta-catenin stabilization. This link between PAR1 and beta-catenin may bear substantial implications both in developmental and tumor progression processes.

Salient experiences are represented by unique transcriptional signatures in the mouse brain.

It is well established that inducible transcription is essential for the consolidation of salient experiences into long-term memory. However, whether inducible transcription relays information about the identity and affective attributes of the experience being encoded, has not been explored. To this end, we analyzed transcription induced by a variety of rewarding and aversive experiences, across multiple brain regions. Our results describe the existence of robust transcriptional signatures uniquely representing distinct experiences, enabling near-perfect decoding of recent experiences. Furthermore, experiences with shared attributes display commonalities in their transcriptional signatures, exemplified in the representation of valence, habituation and reinforcement. This study introduces the concept of a neural transcriptional code, which represents the encoding of experiences in the mouse brain. This code is comprised of distinct transcriptional signatures that correlate to attributes of the experiences that are being committed to long-term memory.

Low-density lipoprotein receptor-related protein 6 is a novel coreceptor of protease-activated receptor-2 in the dynamics of cancer-associated ß-catenin stabilization.

Protease-activated receptor-2 (PAR2) plays a central role in cancer; however, the molecular machinery of PAR2-instigated tumors remains to be elucidated. We show that PAR2 is a potent inducer of ß-catenin stabilization, a core process in cancer biology, leading to its transcriptional activity. Novel association of low-density lipoprotein-related protein 6 (LRP6), a known coreceptor of Frizzleds (Fz), with PAR2 takes place following PAR2 activation. The association between PAR2 and LRP6 was demonstrated employing co-immunoprecipitation, bioluminescence resonance energy transfer (BRET), and confocal microscopy analysis. The association was further supported by ZDOCK protein-protein server. PAR2-LRP6 interaction promotes rapid phosphorylation of LRP6, which results in the recruitment of Axin. Confocal microscopy of PAR2-driven mammary gland tumors in vivo, as well as in vitro confirms the association between PAR2 and LRP6. Indeed, shRNA silencing of LRP6 potently inhibits PAR2-induced ß-catenin stabilization, demonstrating its critical role in the induced path. We have previously shown a novel link between protease-activated receptor-1 (PAR1) and ß-catenin stabilization, both in a transgenic (tg) mouse model with overexpression of human PAR1 (hPar1) in the mammary glands, and in cancer epithelial cell lines. Unlike in PAR1-Gα13 axis, both Gα12 and Gα13 are equally involved in PAR2-induced ß-catenin stabilization. Disheveled (DVL) is translocated to the cell nucleus through the DVL-PDZ domain. Collectively, our data demonstrate a novel PAR2-LRP6-Axin interaction as a key axis of PAR2-induced ß-catenin stabilization in cancer. This newly described axis enhances our understanding of cancer biology, and opens new avenues for future development of anti-cancer therapies.

Comprehensive analysis of transcription dynamics from brain samples following behavioral experience.

The encoding of experiences in the brain and the consolidation of long-term memories depend on gene transcription. Identifying the function of specific genes in encoding experience is one of the main objectives of molecular neuroscience. Furthermore, the functional association of defined genes with specific behaviors has implications for understanding the basis of neuropsychiatric disorders. Induction of robust transcription programs has been observed in the brains of mice following various behavioral manipulations. While some genetic elements are utilized recurrently following different behavioral manipulations and in different brain nuclei, transcriptional programs are overall unique to the inducing stimuli and the structure in which they are studied(1,2). In this publication, a protocol is described for robust and comprehensive transcriptional profiling from brain nuclei of mice in response to behavioral manipulation. The protocol is demonstrated in the context of analysis of gene expression dynamics in the nucleus accumbens following acute cocaine experience. Subsequent to a defined in vivo experience, the target neural tissue is dissected; followed by RNA purification, reverse transcription and utilization of microfluidic arrays for comprehensive qPCR analysis of multiple target genes. This protocol is geared towards comprehensive analysis (addressing 50-500 genes) of limiting quantities of starting material, such as small brain samples or even single cells. The protocol is most advantageous for parallel analysis of multiple samples (e.g. single cells, dynamic analysis following pharmaceutical, viral or behavioral perturbations). However, the protocol could also serve for the characterization and quality assurance of samples prior to whole-genome studies by microarrays or RNAseq, as well as validation of data obtained from whole-genome studies.

A new in vivo model of pantothenate kinase-associated neurodegeneration reveals a surprising role for transcriptional regulation in pathogenesis.

Pantothenate Kinase-Associated Neurodegeneration (PKAN) is a neurodegenerative disorder with a poorly understood molecular mechanism. It is caused by mutations in Pantothenate Kinase, the first enzyme in the Coenzyme A (CoA) biosynthetic pathway. Here, we developed a Drosophila model of PKAN (tim-fbl flies) that allows us to continuously monitor the modeled disease in the brain. In tim-fbl flies, downregulation of fumble, the Drosophila PanK homologue in the cells containing a circadian clock results in characteristic features of PKAN such as developmental lethality, hypersensitivity to oxidative stress, and diminished life span. Despite quasi-normal circadian transcriptional rhythms, tim-fbl flies display brain-specific aberrant circadian locomotor rhythms, and a unique transcriptional signature. Comparison with expression data from flies exposed to paraquat demonstrates that, as previously suggested, pathways others than oxidative stress are affected by PANK downregulation. Surprisingly we found a significant decrease in the expression of key components of the photoreceptor recycling pathways, which could lead to retinal degeneration, a hallmark of PKAN. Importantly, these defects are not accompanied by changes in structural components in eye genes suggesting that changes in gene expression in the eye precede and may cause the retinal degeneration. Indeed tim-fbl flies have diminished response to light transitions, and their altered day/night patterns of activity demonstrates defects in light perception. This suggest that retinal lesions are not solely due to oxidative stress and demonstrates a role for the transcriptional response to CoA deficiency underlying the defects observed in dPanK deficient flies. Moreover, in the present study we developed a new fly model that can be applied to other diseases and that allows the assessment of neurodegeneration in the brains of living flies.

Par genes: molecular probes to pathological assessment in breast cancer progression.

Taking the issue of tumor categorization a step forward and establish molecular imprints to accompany histopathological assessment is a challenging task. This is important since often patients with similar clinical and pathological tumors may respond differently to a given treatment. Protease-activated receptor-(1) (PAR(1)), a G protein-coupled receptor (GPCR), is the first member of the mammalian PAR family consisting of four genes. PAR(1) and PAR(2) play a central role in breast cancer. The release of N-terminal peptides during activation and the exposure of a cryptic internal ligand in PARs, endow these receptors with the opportunity to serve as a "mirror-image" index reflecting the level of cell surface PAR(1&2)-in body fluids. It is possible to use the levels of PAR-released peptide in patients and accordingly determine the choice of treatment. We have both identified PAR(1) C-tail as a scaffold site for the immobilization of signaling partners, and the critical minimal binding site. This binding region may be used for future therapeutic modalities in breast cancer, since abrogation of the binding inhibits PAR(1) induced breast cancer. Altogether, both PAR(1) and PAR(2) may serve as molecular probes for breast cancer diagnosis and valuable targets for therapy.

DVL as a scaffold protein capturing classical GPCRs.

The classical G-protein-coupled receptors (GPCRs) are characterized by their ability to interact with heterotrimeric G proteins upon activation and by structural features such as seven transmembrane spanning domains. Frizzleds (Fzs) are comparable seven transmembrane receptors (7 TMRs) that are activated via Wnts and play a critical role in embryogenesis, tissue hemostasis and oncogenicity. It remains controversial, however, whether they may be considered GPCRs. Hence, the ten members of Fzs constitute a distinct atypical family of seven-transmembrane receptors. Canonical Wnt/ß-catenin signaling leads to the core process of ß-catenin stabilization and, ultimately, to the translocation of ß-catenin to the nucleus where it acts as a co-transcription factor and induces Wnt target gene transcription. we have documented that activation by proteinase-activated receptor1 (PAR(1)), a classical 7TMR, recruits dishevelled (DvL), an upstream Wnt signaling protein, to mediate ß-catenin stabilization. DvL is selectively bound to activated G(α13) subunit, coupled to PAR(1) following activation. Formation of the PAR(1)-induced DvL-G(α13) axis is carried out independently of Wnt, Fz and the co-receptor LRP5/6 (low density lipoprotein-related protein 5/6) since neither siRNA-LRP5/6 co-receptors nor the presence of SFRPs; secreted Fz receptor proteins (Wnt antagonists) affect PAR(1)-induced ß-catenin stabilization. Similarly, PAR(1) induced placenta cytotrophoblast physiological invasion process was not affected by inhibiting Wnt, but was abrogated by siRNA-DvL. we propose that DvL serves as a central mediator protein that links classical GPCRs to ß-catenin stabilization in both pathological (tumor) and physiological (placenta) invasion processes.

Etk/Bmx regulates proteinase-activated-receptor1 (PAR1) in breast cancer invasion: signaling partners, hierarchy and physiological significance.

BACKGROUND: While protease-activated-receptor 1 (PAR(1)) plays a central role in tumor progression, little is known about the cell signaling involved. METHODOLOGY/PRINCIPAL FINDINGS: We show here the impact of PAR(1) cellular activities using both an orthotopic mouse mammary xenograft and a colorectal-liver metastasis model in vivo, with biochemical analyses in vitro. Large and highly vascularized tumors were generated by cells over-expressing wt hPar1, Y397Z hPar1, with persistent signaling, or Y381A hPar1 mutant constructs. In contrast, cells over-expressing the truncated form of hPar1, which lacks the cytoplasmic tail, developed small or no tumors, similar to cells expressing empty vector or control untreated cells. Antibody array membranes revealed essential hPar1 partners including Etk/Bmx and Shc. PAR(1) activation induces Etk/Bmx and Shc binding to the receptor C-tail to form a complex. Y/A mutations in the PAR(1) C-tail did not prevent Shc-PAR(1) association, but enhanced the number of liver metastases compared with the already increased metastases obtained with wt hPar1. We found that Etk/Bmx first binds via the PH domain to a region of seven residues, located between C378-S384 in PAR(1) C-tail, enabling subsequent Shc association. Importantly, expression of the hPar1-7A mutant form (substituted A, residues 378-384), which is incapable of binding Etk/Bmx, resulted in inhibition of invasion through Matrigel-coated membranes. Similarly, knocking down Etk/Bmx inhibited PAR(1)-induced MDA-MB-435 cell migration. In addition, intact spheroid morphogenesis of MCF10A cells is markedly disrupted by the ectopic expression of wt hPar1. In contrast, the forced expression of the hPar1-7A mutant results in normal ball-shaped spheroids. Thus, by preventing binding of Etk/Bmx to PAR(1) -C-tail, hPar1 oncogenic properties are abrogated. CONCLUSIONS/SIGNIFICANCE: This is the first demonstration that a cytoplasmic portion of the PAR(1) C-tail functions as a scaffold site. We identify here essential signaling partners, determine the hierarchy of binding and provide a platform for therapeutic vehicles via definition of the critical PAR(1)-associating region in the breast cancer signaling niche.