Hypocretin (orexin) facilitates reward by attenuating the antireward effects of its cotransmitter dynorphin in ventral tegmental area.

Hypocretin (orexin) and dynorphin are neuropeptides with opposing actions on motivated behavior. Orexin is implicated in states of arousal and reward, whereas dynorphin is implicated in depressive-like states. We show that, despite their opposing actions, these peptides are packaged in the same synaptic vesicles within the hypothalamus. Disruption of orexin function blunts the rewarding effects of lateral hypothalamic (LH) stimulation, eliminates cocaine-induced impulsivity, and reduces cocaine self-administration. Concomitant disruption of dynorphin function reverses these behavioral changes. We also show that orexin and dynorphin have opposing actions on excitability of ventral tegmental area (VTA) dopamine neurons, a prominent target of orexin-containing neurons, and that intra-VTA orexin antagonism causes decreases in cocaine self-administration and LH self-stimulation that are reversed by dynorphin antagonism. Our findings identify a unique cellular process by which orexin can occlude the reward threshold-elevating effects of coreleased dynorphin and thereby act in a permissive fashion to facilitate reward.

Molecular genetic analysis of the hepatocyte growth factor/MET signaling pathway in pediatric medulloblastoma.

The hepatocyte growth factor (HGF)/MET pathway plays a critical role in the development of the nervous system and has been implicated in medulloblastoma pathogenesis. Recent studies have shown a role for gene amplification of activators of this pathway, as well as silencing of its inhibitors in medulloblastoma pathogenesis. We analyzed exon array data from a cohort of 103 primary medulloblastomas to show that HGF/MET pathway elements are dysregulated in tumors compared to normal cerebellum. To determine if mutation of HGF/MET pathway genes is a mechanism for pathway dysregulation, we conducted a mutational analysis by exon resequencing of three key components of this pathway, including serine protease inhibitor Kunitz-type 1 (SPINT1), serine protease inhibitor Kunitz-type 2 (SPINT2), and MET, in 32 primary human medulloblastoma specimens. From this analysis, we identified multiple coding synonymous and nonsynonymous single nucleotide polymorphisms in these genes among the 32 tumor samples. Interestingly, we also discovered two unreported sequence variants in SPINT1 and SPINT2 in two tumors that resulted in Arginine to Histidine amino acid substitutions at codons 418 and 233, respectively. However, conservation assessment and functional assays of these two variants indicate that they involve nonconserved residues and that they do not affect the function of SPINT1 and SPINT2 as tumor suppressor genes. In conclusion, our data suggest that mutation alone plays a minor role in causing aberrancies of the HGF/MET pathway in medulloblastoma in comparison with other malignancies such as breast, hepatocellular, renal, and lung carcinomas.

Pain-related depression of the mesolimbic dopamine system in rats: expression, blockade by analgesics, and role of endogenous κ-opioids.

Pain is often associated with depression of behavior and mood, and relief of pain-related depression is a common goal of treatment. This study tested the hypothesis that pain-related behavioral depression is mediated by activation of endogenous κ-opioid systems and subsequent depression of mesolimbic dopamine release. Adult male Sprague-Dawley rats were implanted with electrodes targeting the medial forebrain bundle (for behavior studies of intracranial self-stimulation (ICSS)) or with cannulae for microdialysis measures of nucleus accumbens dopamine (NAc DA). Changes in ICSS and NAc DA were examined after treatment with a visceral noxious stimulus (intraperitoneal injection of dilute lactic acid) or an exogenous κ-agonist (U69593). Additional studies examined the sensitivity of acid and U69593 effects to blockade by two analgesics (the nonsteroidal antiinflammatory drug ketoprofen and the µ-opioid agonist morphine) or by the κ-antagonist norbinaltorphimine (norBNI). The effects of acid were also examined on mRNA expression for prodynorphin (PDYN) and κ-opioid receptors (KORs) in mesocorticolimbic brain regions. Both acid and U69593 depressed ICSS and extracellular levels of NAc DA. Pain-related acid effects were blocked by ketoprofen and morphine but not by norBNI. The U69593 effects were blocked by norBNI but not by ketoprofen, and were only attenuated by morphine. Acid did not significantly alter PDYN or KOR in NAc, but it produced a delayed increase in PDYN in prefrontal cortex. These results support a key role for the mesolimbic DA system, but a more nuanced role for endogenous κ-opioid systems, in mediating acute pain-related behavioral depression in rats.

Ablation of kappa-opioid receptors from brain dopamine neurons has anxiolytic-like effects and enhances cocaine-induced plasticity.

Brain kappa-opioid receptors (KORs) are implicated in states of motivation and emotion. Activation of KORs negatively regulates mesolimbic dopamine (DA) neurons, and KOR agonists produce depressive-like behavioral effects. To further evaluate how KOR function affects behavior, we developed mutant mice in which exon 3 of the KOR gene (Oprk1) was flanked with Cre-lox recombination (loxP) sites. By breeding these mice with lines that express Cre-recombinase (Cre) in early embryogenesis (EIIa-Cre) or only in DA neurons (dopamine transporter (DAT)-Cre), we developed constitutive KOR knockouts (KOR(-/-)) and conditional knockouts that lack KORs in DA-containing neurons (DAT-KOR(lox/lox)). Autoradiography demonstrated complete ablation of KOR binding in the KOR(-/-) mutants, and reduced binding in the DAT-KOR(lox/lox) mutants. Quantitative reverse transcription PCR (qPCR) studies confirmed that KOR mRNA is undetectable in the constitutive mutants and reduced in the midbrain DA systems of the conditional mutants. Behavioral characterization demonstrated that these mutant lines do not differ from controls in metrics, including hearing, vision, weight, and locomotor activity. Whereas KOR(-/-) mice appeared normal in the open field and light/dark box tests, DAT-KOR(lox/lox) mice showed reduced anxiety-like behavior, an effect that is broadly consistent with previously reported effects of KOR antagonists. Sensitization to the locomotor-stimulating effects of cocaine appeared normal in KOR(-/-) mutants, but was exaggerated in DAT-KOR(lox/lox) mutants. Increased sensitivity to cocaine in the DAT-KOR(lox/lox) mutants is consistent with a role for KORs in negative regulation of DA function, whereas the lack of differences in the KOR(-/-) mutants suggests compensatory adaptations after constitutive receptor ablation. These mouse lines may be useful in future studies of KOR function.

Inhibition of the MET Receptor Tyrosine Kinase as a Novel Therapeutic Strategy in Medulloblastoma.

Medulloblastoma is the most common pediatric posterior fossa malignancy, with a 5-year overall survival of only 60% and many survivors experiencing treatment-related morbidity secondary to current therapeutic regimens. With an improved understanding of the molecular basis for this disease, the opportunity to develop novel treatments with more tolerable toxicity profiles that target key molecular pathways, now exists. Recently, the hepatocyte growth factor (HGF)/MET signaling pathway has been implicated in medulloblastoma pathogenesis. Several therapeutic strategies targeting this pathway exist, including small molecule inhibitor therapy against the MET receptor tyrosine kinase. We examined the in vitro efficacy of targeting the MET receptor using the highly specific small molecule inhibitor PHA665752 as a novel treatment strategy in medulloblastoma. MET inhibition using PHA665752 was effective at reducing the proliferative capacity of the D283, ONS76, and MED8A medulloblastoma cell lines as assessed by MTS assay. Furthermore, PHA665752 treatment reduced D283 and ONS76 cell motility and impaired the growth of D283 cells in soft agar. Pretreatment of D283, ONS76, and MED8A cells with PHA665752 blocked exogenous recombinant human HGF-induced up-regulation of the downstream RAS/mitogen-activated protein kinase signaling pathway in D283, ONS76 and MED8A cell lines. Similarly, PHA665752 prevented HGF-induced phosphatidylinositol 3-kinase/AKT signaling in ONS76 and MED8A cells. These results highlight the efficacy of targeting the MET receptor tyrosine kinase therapeutically in medulloblastoma and provide support for further preclinical testing of small molecule inhibitors targeting the MET receptor in medulloblastoma.

Genetics of medulloblastoma: clues for novel therapies.

Medulloblastoma is the most common malignant brain tumor in children. Current medulloblastoma therapy entails surgery, radiation and chemotherapy. The 5-year survival rate for patients ranges from 40 to 70%, with most survivors suffering from serious long-term treatment-related sequelae. Additional research on the molecular biology and genetics of medulloblastoma is needed to identify robust prognostic markers for disease-risk stratification, to improve current treatment regimes and to discover novel and more effective molecular-targeted therapies. Recent advances in molecular biology have led to the development of powerful tools for the study of medulloblastoma tumorigenesis, which have revealed new insights into the molecular underpinnings of this disease. Here we discuss the signaling pathway alterations implicated in medulloblastoma pathogenesis, the techniques used in molecular profiling of these tumors and recent molecular subclassification schemes. Particular emphasis is given to the identification of novel molecular targets for less toxic, patient-tailored therapeutic approaches.