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Role of lateral hypothalamic orexin neurons in reward processingand addictionGary Aston-Jones*, Rachel J. Smith, David E. Moorman, and Kimberlei A. RichardsonDepartment of Neurosciences, Medical University of South Carolina, Basic Science Building 403,173 Ashley Ave, MSC 510, Charleston, SC, 29425-5100SummaryOrexins (also known as hypocretins) are recently discovered neuropeptides made exclusively inhypothalamic neurons that have been shown to be important in narcolepsy/cataplexy and arousal.Here, we conducted behavioral, anatomical and neurophysiological studies that show that a subsetof these cells, located specifically in lateral hypothalamus (LH), are involved in reward processingand addictive behaviors. We found that Fos expression in LH orexin neurons varied in proportion topreference for morphine, cocaine or food. This relationship obtained both in drug naïve rats and inanimals during protracted morphine withdrawal, when drug preference was elevated but foodpreference was decreased. Recent studies showed that LH orexin neurons that project to ventraltegmental area (VTA) have greater Fos induction in association with elevated morphine preferenceduring protracted withdrawal than non-VTA-projecting orexin neurons, indicating that the VTA isan important site of action for orexin's role in reward processing. In addition, we found thatstimulation of LH orexin neurons, or microinjection of orexin into VTA, reinstated an extinguishedmorphine preference. Most recently, using a self-administration paradigm we discovered that theOx1 receptor antagonist SB-334867 (SB) blocks cocaine-seeking induced by discrete or contextualcues, but not by a priming injection of cocaine. Neurophysiological studies revealed that locallyapplied orexin often augmented responses of VTA dopamine (DA) neurons to activation of the medialprefrontal cortex (mPFC), consistent with the view that orexin facilitates activation of VTA DAneurons by stimulus-reward associations. We also recently showed that orexin in VTA is necessaryfor learning a morphine place preference. These findings are consistent with results from othersshowing that orexin facilitates glutamate-mediated responses, and is necessary for glutamate-dependent long-term potentiation, in VTA DA neurons. We surmise from these studies that LH orexinneurons play an important role in reward processing and addiction, and that LH orexin cells are animportant input to VTA for behavioral effects associated with reward-paired stimuli.IntroductionOrexin behavioral neurobiologyOrexin A and orexin B (also known as hypocretin 1 and hypocretin 2) are recently discoveredpeptides that are produced from a prepro-orexin molecule made solely in hypothalamicneurons. Since their nearly simultaneous discovery by de Lecea et al and Sakurai et al in 1998(de Lecea et al., 1998; Sakurai et al., 1998), considerable work has been done to characterizethis new peptide neurotransmitter system. Sakurai et al. (1998) also characterized 2 receptors*author for correspondence: Gary Aston-Jones, email@example.com Phone: 843-792-6092 Fax: 843-792-4423.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customerswe are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resultingproof before it is published in its final citable form. Please note that during the production process errors may be discovered which couldaffect the content, and all legal disclaimers that apply to the journal pertain.NIH Public AccessAuthor ManuscriptNeuropharmacology. Author manuscript; available in PMC 2010 January 1.Published in final edited form as:Neuropharmacology. 2009 ; 56(Suppl 1): 112–121. doi:10.1016/j.neuropharm.2008.06.060.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript
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for the orexin system, termed Ox1 and Ox2. The Ox1 receptor binds orexin A with 30 nMaffinity but has much lower affinity for orexin B, whereas the Ox2 receptor binds both orexinpeptides with similar high affinity. The orexin neurons give rise to a highly divergent systemof fiber projections that spans the entire neuraxis, including innervation in the cerebral cortex,hippocampus, thalamus, midbrain, and spinal cord (Peyron et al., 1998; Sutcliffe and de Lecea,2002). Likewise, the two orexin receptors are widely distributed in the CNS, but are regionallyselective (Lu et al., 2000; Marcus et al., 2001; Trivedi et al., 1998).Great interest was focused on this system in the year after its discovery, when again two groupsvirtually simultaneously reported that dysfunction in the orexin system is strongly associatedwith narcoleptic symptoms in animals (Chemelli et al., 1999; Lin et al., 1999). Subsequentwork in humans verified that narcoleptic patients (particularly those with cataplexy) have littleorexin in their CSF, and lack most or all orexin neurons (Nishino, 2007; Nishino et al.,2000). With these compelling findings, the prevailing view of orexin function focused onarousal and maintenance of the waking state. Supporting this view were findings that majortargets of orexin projections are classic brain arousal nuclei such as the locus coeruleus (Peyronet al., 1998; Sutcliffe and de Lecea, 2002), and that orexin application typically stronglyactivates these cells (Brown et al., 2001; Eriksson et al., 2001; Horvath et al., 1999; Ivanovand Aston-Jones, 2000; Korotkova et al., 2003).However, a potential role for orexins in reward processing was evident from one of the firstpublications of their discovery. Sakurai and colleagues reported that administration of orexinA or orexin B into the lateral ventricle produced feeding in rats, which prompted them to namethe new peptides "orexins", meaning appetite stimulants (Sakurai et al., 1998). The first reportof a possible role for orexins in addiction appeared in 2003, with findings that orexin neuronsplay a role in opiate withdrawal (Georgescu et al., 2003). These and other findings promptedus to examine a possible role for this novel neuropeptide system in reward processing and drugabuse. As reviewed below, our studies along with those from others now indicate that orexinsplay a prominent role in conditioned responses to stimuli associated with food and drugrewards. This reward-associated function of the orexin system may be separate from its rolein maintenance of the waking state, and mediated by a separate population of (laterally located)orexin neurons.Orexins and reward processingOrexin neurons are activated by reward-associated stimuliOur report in 2005 was the first to demonstrate that orexin neurons in lateral hypothalamus(LH) play an active role in reward processing and drug abuse (Harris et al., 2005). We used aconditioned place preference (CPP) paradigm to measure Fos activation in orexin neuronsassociated with preference expressed for drug or natural rewards. We found that ratsconditioned with morphine, cocaine or food in a CPP paradigm exhibited substantiallyincreased Fos staining in LH orexin neurons on the drug-and food-free CPP test session (Fig.1, Table 1). Notably, this Fos induction in LH orexin neurons was in close proportion to thedegree of preference that animals exhibited during the CPP test day (r = 0.72 to 0.90, p<.01).Moreover, this behavioral correspondence with Fos induction was selective for orexin neuronsin the lateral part of the orexin cell field (in LH), and was not found for orexin neurons outsideLH (e.g., perifornical, PeF, or dorsomedial hypothalamus, DMH), nor for non-orexin neuronswithin LH (Table 1). Note also that LH orexin neurons have a lower baseline (constituitive)level of Fos expression, and show a larger increase in Fos with the CPP test, than PeF or DMHorexin neurons.These findings prompted us to examine whether activation of LH orexin neurons plays a rolein driving the associated preference. We tested this using systemic administration of the Ox1Aston-Jones et al.Page 2Neuropharmacology. Author manuscript; available in PMC 2010 January 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript
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receptor antagonist, SB-334867 (SB). Results indicated that, indeed, this antagonist givenbefore the test session significantly attenuated expression of a morphine CPP (Harris et al.,2005).Orexin neurotransmission in VTA drives reinstatement of drug-preferenceWe reasoned that the above findings might indicate a role for conditioned activation of orexinneurons in driving reinstatement of extinguished drug-seeking. To test this, we conditionedrats for morphine CPP, and then extinguished their morphine preference by repeatedlyexposing them to the CPP environment without drug reward. After achieving extinction ofpreference (which typically required 1–3 weeks), we microinjected the Y4 receptor agonist ratpancreatic polypeptide (rPP) into LH to stimulate orexin neurons. We used rPP becauseCampbell and colleagues (Campbell et al., 2003) showed previously that this compoundpreferentially stimulates Fos induction in orexin neurons when injected into LH. We foundthat rPP microinjected into LH produced a robust reinstatement of preference for the previous(extinguished) morphine-paired side (Fig. 2). This effect was specific to LH, as injectionsdorsal to LH, or medial to LH among non-LH orexin neurons, were not effective in reinstatingpreference (Harris et al., 2005). This reinstatement with LH rPP was also specific for orexinneurotransmission, as it was completely blocked by systemic pretreatment with SB.The orexin system projects widely throughout the CNS, so there are many possible targetswhere orexin might act to produce this reinstatement of preference. One site that seemed likelywas the ventral tegmental area (VTA), where dopamine (DA) neurons that play a critical rolein reward and reinforcement mechanisms are located. We tested the VTA as a site of orexinaction in reinstatement of preference, and found that microinjections of orexin directly intoVTA robustly elicited morphine preference in animals that had previously been extinguished(Harris et al., 2005). Together with the preceding results, these findings provide strong evidencethat orexin projections from LH to VTA play an important role in expression of drug preference,and may also be involved in relapse of drug-seeking following extinction.Orexin neurotransmission is necessary for cue-induced, but not for cocaine-induced,reinstatement of cocaine-seeking in self-administering ratsWe recently conducted a set of experiments using the self-administration paradigm todetermine whether orexin is involved in stimulus-cocaine interactions and relapse of cocaine-seeking (Smith et al., 2007). Rats self-administered intravenous cocaine (0.2 mg/infusion) inthe presence of discrete tone and light cues during daily 2-hour sessions. Animals were thengiven extinction trials, in which lever presses no longer produced cocaine infusions or cues.After animals extinguished lever responding, we elicited reinstatement of cocaine-seeking viacue presentation or a cocaine prime (10 mg/kg, ip). We found that systemic administration of20 – 30 mg/kg SB significantly attenuated cue-induced reinstatement of cocaine-seeking, ascompared to reinstatement following vehicle injection in the same animals (Fig. 3). In a within-subjects design, SB or vehicle was also administered prior to a late extinction trial whenresponding had tapered off to an average of less than 10 presses per 2-hour session. SB had noeffect on lever responding in this extinction trial, indicating that the reinstatement effect wasnot due to general effects of the antagonist on locomotion or arousal.In contrast to the effectiveness in blocking cue-induced reinstatement of cocaine-seeking, 10or 30 mg/kg SB did not attenuate reinstatement of responding induced by a priming injectionof cocaine. Animals similarly reinstated lever-pressing after a cocaine prime whether pretreatedwith vehicle or the antagonist. These results suggest that antagonism of the Ox1 receptor doesnot block the memory of stimulus properties of cocaine, but acts more selectively to blockcertain aspects of drug-seeking.Aston-Jones et al.Page 3Neuropharmacology. Author manuscript; available in PMC 2010 January 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript
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To investigate mechanisms underlying this selectivity, we administered SB during other stagesof drug-taking and drug-seeking in the cocaine self-administration paradigm. Administrationof 30 mg/kg SB during established self-administration produced no significant effects on thenumber of lever presses or drug infusions as compared to self-administration the day beforeand after, when no pretreatment was given. However, we saw a large effect when SB was givenprior to the first day of extinction (Fig. 3). Typically, animals engage in a particularly highamount of drug-seeking during the first extinction session despite being unrewarded by drugsor cues; this is taken to represent contextually-driven drug-seeking. Pretreatment with 30 mg/kg SB significantly attenuated this early extinction effect, as compared to animals that werepretreated with vehicle on that day. During the subsequent extinction days, when nopretreatment with SB was given, the animals that received the antagonist on Extinction Day 1showed a burst of extinction responding on Day 2, and then showed a tapered gradual extinctionof responding across days, similar to animals that initially received vehicle. Thus, the effect ofSB on extinction responding was limited to the day on which it was administered. Thesefindings have a marked similarity to the reinstatement results; that is, drug-seeking elicited byexternal triggers, such as cues or context associated with cocaine, was vulnerable to orexinreceptor antagonism, whereas drug-seeking elicited by a cocaine prime, or direct self-administration of cocaine, was not susceptible to SB.Taken together, these self-administration findings have led us to hypothesize that SB onlyaffects behaviors that rely on increased activation of VTA DA cells, including cue- or context-elicited drug-seeking. SB alone does not alter the spontaneous activity of DA neurons, but doesreduce excitatory effects on DA neurons, such as those caused by orexin and antipsychotics(Borgland et al., 2006; Massi et al., 2007; Rasmussen et al., 2007). Acutely, cocaine increasesDA release via direct actions on DA terminals, and does not require increased activation ofDA cells in the VTA. As discussed in more detail below, we propose that a primary mechanismof action for orexin to augment drug-seeking behavior is to increase glutamate responsivenessof VTA DA neurons (consistent with previous physiological results; Borgland et al., 2006).Orexin neurons are functionally dichotomousAs reviewed above, orexin neurons appear to be involved both in arousal and in reward. Wefound that the reward functions are associated primarily with orexin cells in LH, whereas othersprovided evidence that arousal-related processes were more associated with orexin neurons inthe DMH and PeF. We reviewed and integrated such information in a recent article, andconcluded that orexin neurons are functionally dichotomous (Harris and Aston-Jones,2006a). In this view, orexin neurons of DMH and PeF activate with stress and arousal, whereasthose in LH activate with reward-related stimuli. For example, Estabrooke et al (Estabrookeet al., 2001) reported that LH, but not PeF or DMH, orexin neurons are Fos-activated duringwaking compared to sleep. Fadel et al. (Fadel et al., 2002) found that neuroleptics that causeweight gain preferentially activate LH, rather than more medial, orexin neurons, consistentwith our data for LH involvement in reward processes. In addition, morphine withdrawalactivates Fos in DMH and PeF but not in LH orexin neurons (Sharf et al., 2008), whereaschronic ethanol consumption increased the area of orexin mRNA expression in LH, but not inmore medial hypothalamic areas (Lawrence et al., 2006). We found that footshock stimulationinduced Fos in PeF and DMH, but not in LH, orexin neurons (Harris et al., 2005). These andother results also lead us to propose that the role of orexin reported in stress-inducedreinstatement of cocaine-seeking by Boutrel et al (Boutrel et al., 2005) involves activation ofnoradrenergic and CRF neurons by stress-responsive orexin neurons in DMH and PeF, but notby LH orexin cells (Harris and Aston-Jones, 2006a).The dichotomy of orexin function implies that orexin neurons differ in their input-outputconnections according to reward vs arousal roles. There is already evidence along these lines:Aston-Jones et al.Page 4Neuropharmacology. Author manuscript; available in PMC 2010 January 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript
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Fadel and colleagues found that VTA- and mPFC-projecting orexin cells originatepreferentially from the LH (Fadel et al., 2002; Fadel and Deutch, 2002). Yoshida and colleaguesreported that PeF/DMH orexin neurons are innervated by other hypothalamic regions involvedin homeostatic and arousal-related drive states, whereas LH orexin neurons are preferentiallytargeted by brainstem areas involved in autonomic and visceral processing, and by reward-related areas such as the VTA and NAc shell (Yoshida et al., 2006). We are conducting studiesto further examine this possibility (Richardson et al., 2007; Sartor and Aston-Jones, 2008);some are briefly reviewed below.Altered hedonic processing during protracted withdrawal: possible role of LH orexinneuronsWe previously reported that chronic exposure to cocaine or morphine, followed by protractedforced abstinence, resulted in dramatically altered preferences for drug and natural rewards.Thus, preference for morphine or cocaine increased, and for food or novelty decreased, at 2 or5 weeks following protracted forced abstinence from chronic morphine or cocaine (Aston-Jones and Harris, 2004; Harris et al., 2001; Harris and Aston-Jones, 2001). This increasedpreference for drug, and decreased interest in natural rewards, is similar to reports by addicts(Jaffe, 1990) and could be a source of difficulty in maintaining prolonged abstinence fromdrugs after previous chronic exposure.We examined the neural substrates of this altered hedonic processing during protractedwithdrawal by examining Fos expression on the preference test day. We hypothesized thatbrain areas with altered Fos that mirrored altered preferences for drug and natural rewardswould be good candidates for structures that might underlie the altered hedonic processingduring protracted withdrawal. Thus, we sought brain areas where Fos expression was higherthan normal when withdrawn animals were tested for drug preference, and where Fosexpression was lower than normal when withdrawn animals were tested for food or noveltypreference. Our studies to date revealed 3 such areas: the basolateral amygdala, nucleusaccumbens shell, and LH (Aston-Jones and Harris, 2004). Notably, the LH region thatcontained altered Fos expression in this study coincided with the area that contains orexinneurons. Subsequent analysis of LH sections with Fos co-stained for orexin revealed that,indeed, Fos activation in orexin neurons in LH was higher than normal for animals in protractedmorphine withdrawal tested for drug preference, but lower than normal for animals inprotracted morphine withdrawal tested for food preference – ie, the Fos expression in LH orexinneurons closely mirrored the altered preferences that resulted from protracted drug withdrawal(Fig. 4). This result is consistent with our previous finding that Fos expression in LH orexinneurons correlates closely with preferences for morphine, cocaine or food reward (Harris etal., 2005), and indicates that LH orexin neurons may play a significant role in the alteredhedonic processing that occurs during protracted drug withdrawal. Other of our recent studiesindicate that neural systems in addition to LH orexin neurons may also be involved in thisaltered hedonic processing during protracted withdrawal as well (Harris et al., 2007a; Harrisand Aston-Jones, 2007).Topography of orexin projections to reward-related brain areasThe functional dichotomy reviewed above also implies that subpopulations of orexin neuronsdiffer in their projection targets, so that some are involved in arousal whereas others are moreconcerned with reward and reinforcement. We have begun to address this issue using tract-tracing to retrogradely label orexin neurons from various targets, combined with Fos to identifyorexin neurons that are activated by a CPP preference test in previously-naïve or morphine-dependent animals (Richardson et al., 2007). Rats received a unilateral injection of theretrograde tracer wheat germ agglutinin-colloidal gold (WGA-Au) in VTA. Animals weremade morphine-dependent for 2 weeks by subcutaneously implanting two morphine pellets 7Aston-Jones et al.Page 5Neuropharmacology. Author manuscript; available in PMC 2010 January 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript
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days after WGA-Au microinjections; other rats were given placebo pellets. CPP conditioningbegan two weeks after pellet removal; conditioning and testing were identical to our previouslypublished reports for morphine (Byrne et al., 2003). Results showed that dependent animalsexhibited an enhanced preference for the morphine-associated environment on the CPP testday compared to placebo-pelleted rats, as in our previous studies (Harris and Aston-Jones,2003c). A higher percentage of LH orexin neurons that exhibit Fos were retrogradely labeledfrom VTA in dependent than in non-dependent animals (26.4 ± 4.0% versus 11.4 ± 5.4%,p<0.05). The number of Fos-activated LH orexin neurons that project to VTA was significantlycorrelated with the intensity of conditioned preference in dependent animals (R=0.743,p<0.05). In addition, a greater percentage of retrogradely labeled orexin neurons were Fos-activated in dependent than in non-dependent animals (27.2 ± 4.0% versus 14.9 ± 5.0%, p=.06, n = 6 per group); this difference was not found for non-retrogradely-labeled neurons. Inaddition, the number of VTA-projecting orexin neurons that were Fos-activated wassignificantly correlated with the intensity of conditioned preference in dependent animals(R=0.740, n=6/group, p<0.05).Thus, as preference scores increased in dependent rats, thepercentage of Fos-activated, VTA-projecting orexin neurons increased. There was no suchcorrelation with preference found for Fos+ LH orexin neurons that were not retrogradelylabeled from VTA, nor was this correlation significant in non-dependent animals.This study extends past studies by demonstrating that Fos activation in VTA-projecting LHorexin neurons correlates with the intensity of reward during protracted withdrawal, andsupports the view that the VTA is an important site of action of LH orexin neurons in rewardprocessing and drug abuse. This study also confirms our previous finding that prior morphineexposure enhances the preference for drug-associated environments (Harris and Aston-Jones,2003a), and that animals that undergo prolonged forced abstinence have a higher degree ofactivation in LH orexin neurons that project to the VTA than control animals. Studies areunderway to determine if this phenomenon is exclusive to LH orexin neurons or if enhancedactivation of PeF or DMH orexin neurons that project to the VTA is also observed.Additionally, it will be determined if the activation of LH orexin neurons that project to otherareas less directly linked with reward (e.g., locus coeruleus, tuberomammillary nucleus) is alsoenhanced after morphine preference testing.Orexin modulates glutamate responses in VTA DA neuronsGlutamate transmission in VTA is important for several aspects of drug abuse. Cocaine causesrelease of glutamate in VTA (Kalivas and Duffy, 1995), and glutamate inputs to VTA arecritical for reinstatement of extinguished drug-seeking behavior, including that evoked bycocaine or conditioned cues (Bossert et al., 2004; Sun et al., 2005; Vorel et al., 2001). Severalstudies have demonstrated that a single injection of cocaine induces long-term potentiation(LTP) in VTA DA neurons that is dependent upon NMDA receptor activation (Borgland et al.,2004; Borgland et al., 2006; Thomas and Malenka, 2003; Ungless et al., 2001). Consistent withthese results, we showed that glutamate transmission and plasticity in VTA is important forlearning a cocaine CPP, and for learning or expressing a morphine CPP (Harris and Aston-Jones, 2003b; Harris et al., 2004). A recent study extended the LTP results by showing thatrepeated cocaine injections produced larger LTP in DA neurons than single injections (Liu etal., 2005), suggesting that the stronger abuse potential with repeated cocaine experience mayresult in part from plasticity in these cells.Recent work also indicates that orexin interacts in an important manner with glutamate functionin VTA DA neurons, and that this interaction may be significant for drug abuse behavior.Borgland et al (2006) demonstrated that cocaine-induced plasticity in VTA DA neurons(described above) depends critically upon orexin inputs. They found that the Ox1 antagonistSB, when co-administered with cocaine, blocked the glutamate-dependent LTP in midbrainAston-Jones et al.Page 6Neuropharmacology. Author manuscript; available in PMC 2010 January 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript
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DA neurons. That study also showed that orexin administration to midbrain slices produced alate-phase glutamate-dependent LTP in DA neurons, and that this was due primarily to theinsertion of new NMDA receptors in the synapse which then facilitated an AMPA receptor-mediated LTP. Finally, they showed that SB injected either systemically or directly into theVTA blocked the development of locomotor sensitization following repeated cocaineinjections. These findings provide important new information indicating that orexin in VTAplays an important role in synaptic plasticity of DA neurons. As described below, additionalwork by others and our group indicates that this plasticity is important behaviorally as well.Orexin modulates responses of VTA DA neurons to PFC activationAs described above, glutamate inputs and plasticity in VTA are critical for learning stimulus-drug associations (Harris and Aston-Jones, 2003b; Harris et al., 2004). Also, orexin increasesresponses and plasticity of VTA DA neurons evoked by glutamate inputs (Borgland et al.,2006). However, these previous studies did not specify which glutamate inputs are modulatedby orexin. Other studies showed that glutamate inputs to VTA originate from several sources,including PFC, bed nucleus of the stria terminalis (BNST), subthalamic nucleus andpedunculopontine tegmental nucleus (Carr and Sesack, 2000; Geisler and Zahm, 2005; Georgesand Aston-Jones, 2002b; Kita and Kitai, 1987; Rinvik and Ottersen, 1993; Sesack and Pickel,1992). We reasoned that inputs representing stimulus information that becomes associated withdrug administration might provide glutamate inputs to VTA that would be modulated by orexin.Several lines of evidence indicate that mPFC inputs to VTA are important to the function ofthe DA system, including its role in reward and drug abuse. The mPFC provides directglutamate innervation of DA and GABA neurons in VTA (Carr and Sesack, 2000; Sesack etal., 2003; Sesack et al., 1989; Sesack and Pickel, 1992), and regulates the release of DA innucleus accumbens (Karreman and Moghaddam, 1996; Taber et al., 1995; You et al., 1998),which plays a central role in drug abuse (Kalivas and McFarland, 2003; Kalivas and Volkow,2005; Koob, 1999; Wolf et al., 2004). The mPFC is a critical region for goal-directed behaviorsand impulse control. It receives highly processed information from several brain areas, and isinvolved in complex cognitive and behavioral processes (Bubser and Schmidt, 1990; Granonet al., 1994; Kolb, 1984; Muir et al., 1996; Watanabe, 1996). In addition, the mPFC has beenshown to be important in drug abuse and, specifically, in reinstatement of cocaine-seekingbehavior (Kalivas and McFarland, 2003; McFarland et al., 2004; McFarland and Kalivas,2001). In humans, imaging studies have revealed decreased metabolic activity in mPFC duringdrug withdrawal (Goldstein and Volkow, 2002) and large increases in activity in the mPFCfollowing exposure to drug cues (Childress et al., 1999; Grant et al., 1996; Maas et al., 1998).Therefore, mPFC afferents to VTA are a potential source of VTA glutamate that is involvedin reinstatement, learning and expression of drug-seeking behavior. Because our resultsindicate that orexin neurons are stimulated by cues that reinstate extinguished drug-seeking(Harris et al., 2005), we anticipate that orexin will be released onto VTA DA neurons at aboutthe same time that mPFC inputs representing stimulus-response information are arriving atthese cells. We hypothesize that the conditioned orexin input from LH would augmentresponses to glutamate (NMDA receptor-mediated) inputs from mPFC in a manner similar tothat described by Borgland et al. (Borgland et al., 2006), serving to increase responding ofVTA DA neurons to mPFC inputs.We tested the influence of orexin on prefrontal projections to the VTA by recording the activityof DA neurons in isoflurane-anesthetized rats (Moorman and Aston-Jones, 2007). Responseswere evoked from DA neurons by stimulating the prelimbic/infralimbic areas of the mPFC.Through the use of a second pipette glued to the recording pipette (Akaoka and Aston-Jones,1991; Georges and Aston-Jones, 2002a), we delivered orexin A directly to the site of therecorded neuron. Using different combinations of microstimulation and orexin delivery duringAston-Jones et al. Page 7Neuropharmacology. Author manuscript; available in PMC 2010 January 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript
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recording, we were able to ascertain the influence of orexin on mPFC-evoked responses in DAneurons.Stimulation of the prelimbic/infralimbic regions in mPFC in the absence of orexin evokedshort-latency (<50 ms) responses in 40% of putative DA neurons. Approximately 22% of DAneurons exhibited short-latency evoked responses within the range of monosynapticprojections (<25 ms (Thierry et al., 1983)). Orexin A (1.4 μM, 60 nl) applied directly torecorded neurons (in the absence of mPFC stimulation) produced strong increases in firing rate(in 58% neurons) and bursting (in 28% neurons) (Moorman and Aston-Jones, 2007), consistentwith more global applications of orexin in slice (Korotkova et al., 2003) and in vivo (Muschampet al., 2007).Our main interest was to test whether orexin application would facilitate mPFC-evokedresponses in DA neurons, possibly indicating a circuit substrate for orexin-induced facilitationof glutamate responses, or plasticity, in VTA necessary for stimulus-drug conditioning. Wetested the influence of mPFC stimulation on DA neurons when given before, during orfollowing local orexin application. Results showed that for most cells mPFC-evoked responsesfollowing orexin application were enhanced. An example of such an orexin-enhanced mPFC-evoked response is shown in Fig. 5. In contrast, we observed an equivalent number of neuronsexhibiting enhanced and diminished mPFC-evoked responses (45% each) when tested duringorexin application. We speculate that the difference between effects of orexin on responses tomPFC stimulation during vs after orexin application may be due to different time courses oforexin effects on DA vs GABA neurons in VTA. That is, Borgland et al. (2006) found thatorexin produced a long-lasting potentiation of glutamate-mediated responses in DA neurons,but others found that orexin produced only a transient increase in glutamate responses inpresumed GABA neurons (which may in turn inhibit DA neurons) (Korotkova et al., 2003).Thus, effects of orexin on DA neurons may outlast those on inhibitory GABA interneurons,which could result in the effects observed here.Orexin likely facilitates additional excitatory inputs to VTA also. Several other areas send (insome cases relatively strong) glutamatergic projections to the VTA, including the LH (Geisleret al., 2007). Indeed, it has been shown that orexin neurons co-express orexin and glutamate(Rosin et al., 2003); the co-release of these transmitters could produce enhanced excitatorydrive from the LH itself. The influence of orexin on other glutamatergic inputs remains to betested. However, we speculate that complex stimulus information is relayed to VTA frommPFC, and therefore that orexin-facilitated mPFC drive on dopamine neurons is particularlyrelevant for learning stimulus-drug associations and for stimulus-induced reinstatement ofextinguished drug-seeking. This may underlie the observations that activation of both themPFC and the LH orexin system are critical in learning stimulus-drug associations and inreinstatement of drug-seeking.LH orexin neurons are critical for learning morphine preferenceA recent study (Narita et al., 2006) showed that microinjection of SB into VTA duringconditioning reduced acquisition of a morphine CPP, confirming that orexin in VTA is criticalfor learning this stimulus-drug relationship, just as glutamate is (reviewed above). Thesestudies suggest a critical behavioral role of the glutamate-dependent plasticity in DA neuronsthat is regulated by orexin inputs. We examined this issue using lesion methods and examinedspecifically the role of LH orexin projections to the VTA in learning stimulus-drugrelationships (Harris et al., 2007b). We made bilateral neurotoxic lesions of LH neurons, orwe made a neurotoxic lesion of LH neurons unilaterally, and then injected the orexin antagonistSB into the contralateral VTA just preceding each of 3 morphine CPP conditioning trials.Results were similar with both manipulations, and are illustrated for the contralateraldisconnection procedure in Fig. 6. These manipulations both prevented learning the CPP, asAston-Jones et al.Page 8Neuropharmacology. Author manuscript; available in PMC 2010 January 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript
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evidenced by no preference expressed on the subsequent CPP test day. Normal preference forthe morphine-paired side appeared in animals in which either a neurotoxic lesion was outsideof LH, or the contralateral microinjection of SB was outside of VTA. These results areconsistent with those of other recent studies (Narita et al., 2006), and indicate that LH orexinis not only involved in conditioned reward processes, but also in plasticity in the VTAassociated with learning stimulus-drug relationships.To test whether orexin is also necessary for the acquisition of cocaine-associated cues duringself-administration, we used a Pavlovian conditioned-cues paradigm previously described bySee and colleagues (See, 2005). Although acquisition of self-administration typically occursacross several sessions, the Pavlovian conditioned-cues paradigm has the advantage ofconfining cue acquisition to a single session. Animals were trained to self-administer cocaine(0.2 mg/infusion) in the absence of cues. After five days of stable responding, animals wereexposed to a single Pavlovian conditioning session, in which no levers were extended and theanimals received passive infusions of cocaine paired with discrete tone and light cues. Thenumber of infusions was based on the self-administration for each animal in previous sessions.The Pavlovian session was followed by five more days of self-administration without cues.Following extinction of lever pressing in the absence of cocaine, drug-seeking was robustlyreinstated by the cocaine-paired cues. Administration of 30 mg/kg SB prior to the Pavlovianacquisition trial had no effect on subsequent expression of cue-elicited reinstatement of leverresponding. That is, animals pretreated with SB or vehicle showed similar reinstatementresponding, indicating that orexin is not necessary for acquiring cocaine-cue associations inthis paradigm. In contrast, 30 mg/kg SB in these same animals significantly reduced theexpression of conditioned-cue-elicited drug-seeking when administered prior to the subsequentreinstatement trial (Smith and Aston-Jones, unpublished). This corresponds to our resultsdescribed above showing that cue-induced reinstatement of cocaine-seeking requires orexintransmission.Together, these experiments indicate that orexin is necessary for learning morphine-stimulusassociations and preference, but not for learning the cocaine-stimulus relationships requiredfor cue-evoked reinstatement of cocaine-seeking behavior. There are several proceduraldifferences between these two learning paradigms that might underlie these different resultsfor SB administration, including CPP vs self-administration. However, we note that theseresults are consistent with the finding that SB is ineffective on drug-seeking behaviors thatinclude cocaine administration. Thus, one possibility, discussed in more detail below, is thatorexin may be involved in morphine, but not in cocaine, conditioning because DA releasefollowing morphine requires VTA activation, whereas DA release following cocaine does not.Discussion and hypothesisOrexin is involved in cue- but not cocaine-induced relapse: an hypothesisOne interesting outcome of our recent studies with self-administration of cocaine is that SBdid not block reinstatement of cocaine-seeking induced by cocaine itself, nor did this compoundsignificantly affect established cocaine self-administration. This contrasts sharply with theability of SB to block cocaine-seeking induced by cues, and to attenuate responding on the firstday of extinction. What might explain this difference in SB effects on stimulus-elicited vscocaine-elicited reinstatement of cocaine-seeking and maintenance of cocaine self-administration? We hypothesize that this is because the VTA is a major site of action for orexinin reward processes. Previous work established that DA release is an essential neural substratefor many types of motivated behavior (Wise, 2004). Orexin potentiates glutamate-mediatedresponses of VTA DA neurons, and it is possible that this orexin potentiation of responsivenessis a critical element in sufficient DA release to drive motivated behavior (eg., cocaine-seeking)for drug-associated stimuli. In contrast, cocaine itself elicits DA release without alteringAston-Jones et al.Page 9Neuropharmacology. Author manuscript; available in PMC 2010 January 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript
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impulse activity of VTA DA neurons, so that effects of cocaine on behavior (such as cocaine-induced reinstatement or cocaine self-administration) do not require orexin transmission. Thishypothesis is illustrated in Fig. 7, and is consistent with other previous results showing that SBcan block or attenuate (i) ethanol self-administration, or cue-induced reinstatement or ethanol-seeking (Lawrence et al., 2006), (ii) expression of a morphine CPP (Harris et al., 2005), and(iii) stress-induced reinstatement of cocaine-seeking (Boutrel et al., 2005). In all of these cases,the drug or stimulus causes DA release by activating VTA DA neurons, and we hypothesizethat therefore they require orexin transmission. This view is also consistent with the findingthat intra-VTA SB attenuates development of locomotor sensitization to cocaine (Borgland etal., 2006), a process that requires plasticity in VTA DA neurons (Kalivas and Alesdatter,1993; Sorg and Ulibarri, 1995).This hypothesis predicts that agents that act via glutamate release in VTA to motivate behaviorwould require orexin release in VTA. Studies examining the effects of local administration ofSB in VTA on reinstatement behavior are underway to test this possibility. We also predictthat SB should attenuate opiate self-administration, or opiate-induced drug-seeking behavior.Corresponding studies are also underway to test these ideas.This view indicates that the VTA is an important site of action for orexin in reward-associatedfunctions. LH orexin neurons are more prominently involved in reward processes than are moremedial orexin neurons in PeF and DMH (Harris and Aston-Jones, 2006b). Also, we expect thatLH orexin neurons that project to VTA may be more strongly activated by reward-relatedstimuli than non-VTA-projecting LH or PeF/DMH orexin neurons. Our preliminary resultsshowing that VTA-projecting orexin neurons show elevated Fos in proportion to preferencefor a drug-associated environment, compared to non-VTA-projecting orexin neurons(described above), is consistent with this hypothesis. Additional work tracing inputs andoutputs of orexin neurons is underway to further define the topography of connections forreward-associated orexin neurons.Although most of our analysis of the orexin system to date has centered on drug reward, it isnotable that these neurons are also Fos-activated by CSs associated with food reward. Resultsfrom Petrovich et al [Petrovich, 2002 #4291] show that amygdala inputs to hypothalamusregulate cue-induced feeding in sated animals. We speculate that orexin neurons may beinvolved in this circuit, and that the amygdala stimulates orexin neurons in response to foodCSs, which in turn may play a role in conditioned overeating and obesity. Further studies areneeded to evaluate this possibility.Orexin and reward: a role in drug-seeking and relapseThe results reviewed above indicate that LH orexin neurons are stimulated in proportion todrug or food preference, that stimulation of LH orexin neurons drives reinstatement of anextinguished preference for morphine, and that orexin neurotransmission is needed forstimulus-induced (but not for cocaine-induced) reinstatement of extinguished cocaine-seeking.We propose that these results indicate a role for orexin systems in drug-seeking and addiction.Interestingly, human narcoleptics, who have few or no orexin neurons, rarely exhibit stimulantabuse and seeking despite the fact that they are treated for years with stimulants (Sakurai,2007). Together, these findings indicate that LH orexin neurons may be stimulated by cuesassociated with drug acquisition and exposure, and that these neurons may be part of circuitrythat is critically involved in drug abuse, and specifically in stimulus-induced drug relapse.Moreover, we propose that VTA is a critical region for orexin actions in reward-seekingbehaviors. These findings are clinically significant, and indicate that development ofcompounds that specifically target LH orexin neurons, or orexin receptor actions in VTA DAneurons, may provide novel treatments for addiction and relapse. In particular, we propose thatan orexin antagonist would reduce the propensity to relapse to drug-taking behavior, and wouldAston-Jones et al.Page 10Neuropharmacology. Author manuscript; available in PMC 2010 January 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript
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help maintain abstinence in addicts re-exposed to previous drug cues. Additional studies in thisvein will advance our understanding of the role of orexin in addiction and lead to possible noveltherapeutic targets.AcknowledgementsThis work was supported by PHS grants R37 DA06214, R01 DA017289, P50 DA015369, F31 DA019733, and T32AA007474.ReferencesAkaoka H, Aston-Jones G. Opiate withdrawal-induced hyperactivity of locus coeruleus neurons issubstantially mediated by augmented excitatory amino acid input. J Neurosci 1991;11:3830–3839.[PubMed: 1683899]Aston-Jones G, Harris GC. Brain substrates for increased drug seeking during protracted withdrawal.Neuropharmacology 2004;47S1:167–179. [PubMed: 15464135]Borgland SL, Malenka RC, Bonci A. Acute and chronic cocaine-Induced potentiation of synaptic strengthin the ventral tegmental area: Electrophysiological and behavioral correlates in individual rats. JNeurosci 2004;24:7482–7490. [PubMed: 15329395]Borgland SL, Taha SA, Sarti F, Fields HL, Bonci A. Orexin A in the VTA is critical for the induction ofsynaptic plasticity and behavioral sensitization to cocaine. Neuron 2006;49:589–601. [PubMed:16476667]Bossert JM, Liu SY, Lu L, Shaham Y. A role of ventral tegmental area glutamate in contextual cue-induced relapse to heroin seeking. J Neurosci 2004;24:10726–10730. [PubMed: 15564590]Boutrel B, Kenny PJ, Specio SE, Martin-Fardon R, Markou A, Koob GF, de Lecea L. Role for hypocretinin mediating stress-induced reinstatement of cocaine-seeking behavior. PNAS 2005;102:19168–19173. [PubMed: 16357203]Brown RE, Sergeeva O, Eriksson KS, Haas HL. Orexin A excites serotonergic neurons in the dorsal raphenucleus of the rat. Neuropharmacology 2001;40:457–459. [PubMed: 11166339]Bubser M, Schmidt WJ. 6-Hydroxydopamine lesion of the rat prefrontal cortex increases locomotoractivity, impairs acquisition of delayed alternation tasks, but does not affect uninterrupted tasks in theradial maze. Behav Brain Res 1990;37:157–168. [PubMed: 2108704]Byrne R, Harris GC, Aston-Jones G. Glutamate input to the ventral tegmental area is necessary for bothlearning and expression of morphine place preference. Soc Neurosci Abstr 2003;29:110.111.Campbell RE, Smith MS, Allen SE, Grayson BE, Ffrench-Mullen JM, Grove KL. Orexin neurons expressa functional pancreatic polypeptide Y4 receptor. J Neurosci 2003;23:1487–1497. [PubMed:12598637]Carr DB, Sesack SR. Projections from the rat prefrontal cortex to the ventral tegmental area: targetspecificity in the synaptic associations with mesoaccumbens and mesocortical neurons. J Neurosci2000;20:3864–3873. [PubMed: 10804226]Chemelli RM, Willie JT, Sinton CM, Elmquist JK, Scammell T, Lee C, Richardson JA, Williams SC,Xiong Y, Kisanuki Y, Fitch TE, Nakazato M, Hammer RE, Saper CB, Yanagisawa M. Narcolepsyin orexin knockout mice: molecular genetics of sleep regulation. Cell 1999;98:437–451. [PubMed:10481909]Childress AR, Mozley PD, McElgin W, Fitzgerald J, Reivich M, O'Brien CP. Limbic activation duringcue-induced cocaine craving. Am J Psychiatry 1999;156:11–18. [PubMed: 9892292]de Lecea L, Kilduff TS, Peyron C, Gao X, Foye PE, Danielson PE, Fukuhara C, Battenberg EL, GautvikVT, Bartlett FS 2nd, Frankel WN, van den Pol AN, Bloom FE, Gautvik KM, Sutcliffe JG. Thehypocretins: hypothalamus-specific peptides with neuroexcitatory activity. Proc Natl Acad Sci U SA 1998;95:322–327. [PubMed: 9419374]Eriksson KS, Sergeeva O, Brown RE, Haas HL. Orexin/hypocretin excites the histaminergic neurons ofthe tuberomammillary nucleus. J Neurosci 2001;21:9273–9279. [PubMed: 11717361]Aston-Jones et al. Page 11Neuropharmacology. Author manuscript; available in PMC 2010 January 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript
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Sesack SR, Pickel VM. Prefrontal cortical efferents in the rat synapse on unlabeled neuronal targets ofcatecholamine terminals in the nucleus accumbens septi and on dopamine neurons in the ventraltegmental area. J Comp Neurol 1992;320:145–160. [PubMed: 1377716]Sharf R, Sarhan M, DiLeone RJ. Orexin Mediates the Expression of Precipitated Morphine Withdrawaland Concurrent Activation of the Nucleus Accumbens Shell. Biol Psychiatry. 200810.1016/j.biopsych.2008.03.006Smith R, See R, Aston-Jones G. The orexin-1 receptor antagonist SB-334867 blocks cue inducedreinstatement of cocaine-seeking. Neuroscience Meeting Planner Online. 2007Program No. 916.1Sorg BA, Ulibarri C. Application of a protein synthesis inhibitor into the ventral tegmental area, but notthe nucleus accumbens, prevents behavioral sensitization to cocaine. Synapse 1995;20:217–224.[PubMed: 7570353]Sun W, Akins CK, Mattingly AE, Rebec GV. Ionotropic Glutamate Receptors in the Ventral TegmentalArea Regulate Cocaine-Seeking Behavior in Rats. Neuropsychopharmacology 2005;30:2073–2081.[PubMed: 15841101]Sutcliffe JG, de Lecea L. The hypocretins: setting the arousal threshold. Nat Rev Neurosci 2002;3:339–349. [PubMed: 11988773]Swanson, LW. Brain maps: structure of the rat brain. Elsevier; Amsterdam: 1992.Taber MT, Das S, Fibiger HC. Cortical regulation of subcortical dopamine release: mediation via theventral tegmental area. J Neurochem 1995;65:1407–1410. [PubMed: 7643120]Thierry AM, Chevalier G, Ferron A, Glowinski J. Diencephalic and mesencephalic efferents of the medialprefrontal cortex in the rat: electrophysiological evidence for the existence of branched axons. ExpBrain Res 1983;50:275–282. [PubMed: 6641860]Thomas MJ, Malenka RC. Synaptic plasticity in the mesolimbic dopamine system. Philos Trans R SocLond B Biol Sci 2003;358:815–819. [PubMed: 12740128]Trivedi P, Yu H, MacNeil DJ, Van der Ploeg LH, Guan XM. Distribution of orexin receptor mRNA inthe rat brain. FEBS Lett 1998;438:71–75. [PubMed: 9821961]Ungless MA, Whistler JL, Malenka RC, Bonci A. Single cocaine exposure in vivo induces long-termpotentiation in dopamine neurons. Nature 2001;411:583–587. [PubMed: 11385572]Vorel S, Liu X, Hayes R, Spector J, Gardner E. Relapse to cocaine-seeking after hippocampal theta burststimulation. Science 2001;292:1175–1178. [PubMed: 11349151]Watanabe M. Reward expectancy in primate prefrontal neurons. Nature 1996;382:629–632. [PubMed:8757133]Wise RA. Dopamine, learning and motivation. Nat Rev Neurosci 2004;5:483–494. [PubMed: 15152198]Wolf ME, Sun X, Mangiavacchi S, Chao SZ. Psychomotor stimulants and neuronal plasticity.Neuropharmacology 2004;47:61–79. [PubMed: 15464126]Yoshida K, McCormack S, Espana RA, Crocker A, Scammell TE. Afferents to the orexin neurons of therat brain. J Comp Neurol 2006;494:845–861. [PubMed: 16374809]You ZB, Tzschentke TM, Brodin E, Wise RA. Electrical stimulation of the prefrontal cortex increasescholecystokinin, glutamate, and dopamine release in the nucleus accumbens: an in vivo microdialysisstudy in freely moving rats. J Neurosci 1998;18:6492–6500. [PubMed: 9698337]Aston-Jones et al.Page 15Neuropharmacology. Author manuscript; available in PMC 2010 January 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript
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June 12, 2012
That dog is hilarious. That whole video is pretty comical.
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That dog is hilarious. That whole video is pretty comical.
The dog totally makes this video, he seems unimpressed.
Freaking most ridiculous I have ever seen!
Omg the dog was killing me, the whole video was pretty hilarious
Lol that dogs looking at him like he can do a waaaaaay better job
http://www.youporn.com/watch/341602/big-woman-shows-interviewer-all-angles/?from=partner http://fr.youporn.com/watch/259340/sexy-semen-swallowers-swallow-semen/?from=partner

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Dec. 22, 2013
FORT COLLINS, Colo. -- Overcoming a rough first half, the Colorado State women's basketball team took the game over in the finals minutes, defeating Weber State, 59-54. The Rams are now 8-3 this season, which is the team's best start since 2001-02, while the Wildcats fall to 4-5. This is the most non-conference wins for CSU since 2009-10.
The Rams struggled to find their shot in the first half, shooting 32.0 percent from the field, but they were able to keep Weber State in check, behind 24-22 at the midway point.
"We've won about every way imaginable," Head Coach Ryun Williams praised. "We've won close games, we've won shooting the ball extremely well. We've won with different kids stepping up at different times. The thing that I'm the most proud of, with all the turnovers and all the bad decsions we made today, is that we still found a way to win. That's what we have to do. You're not going to win brilliant basketball, beautiful basketball, 30 nights out of the year."
With 27 seconds left in the game, Colorado State led 57-54. Senior Sam Martin pulled down a crucial offensive rebound and drew a foul on the put-back. Martin made both of the free-throw shots, sealing the Rams victory.
Junior Gritt Ryder led the Rams with 25 points, a career-high, scoring 15 points in the second half. She added six rebounds, three steals, and two assists.
"She had a great game today and I think that's because she stepped up when we needed her too and helped our team out," Martin praised.
Colorado State was unable to keep hold of the ball in the first frame, turning the ball over 12 times, while Weber State had just five. The Rams only gave it away four times in the second stanza.
Martin scored 10 points for CSU, and was one rebound short of a double-double with nine rebounds. She recorded four blocks, a career-high. Sophomore Caitlin Duffy added eight rebounds, seven points and two assists. Freshman Ellen Nystrom, who missed playing time because of foul trouble, put up six points, three rebounds and one assist. Senior Alicia Nichols tallied four points and six rebounds.
Sophomore Emilie Hesseldal made her first appearance for the Rams today after being ineligible due to NCAA transfer rules.
"We're not satisfied with eight wins, we want it to be 11-0," explained Ryder. "That's a strong thing about our team, 8-3 is good, but we want to get better and improve."
Colorado State takes a break for the holidays and begins the new year with Mountain West action at San Diego State (2-6) on Jan. 2. Tipoff at Viejas Arena is scheduled for 7 p.m. MT.
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text: Robin Thicke - Give It 2 U ft. Kendrick Lamar by Free Britney at July 3, 2013 6:20 am. Comments Robin Thicke and Kendrick Lamar want to "Give It 2 U." It sounds dirty because it is. Share Tweet Email Edit Delete Follow Robin Thicke? Facebook Disqus Star: Robin Thicke Tag: Music Related Videos: Robin Thicke Videos, Music Videos Related Post: Robin Thicke "Give It 2 U" Single: Even More Inappropriate Than "Blurred Lines"! Uploaded by: Free Britney Uploaded: July 03, 2013 Duration: 230 seconds


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Beware Republicans in Greek clothing
In less than a month, before John Boehner leaves his job as Speaker of the House of Representatives, Congress must once again consider raising the debt ceiling.
In less than a month, before John Boehner leaves his job as Speaker of the House of Representatives, Congress must once again consider raising the debt ceiling.Shares
Every Republican and Democratic candidate in the 2016 presidential race is unfit for the White House.Shares
Puritans and the Press; a response to Oregon; Trump, evangelicals, Reagan; animal affirmationShares
Based on his writings, I have been admittedly critical of Pope Francis I. My principal observation is that he does not understand democratic capitalism as we practice it in the United States. His preconceptions of society and economics lead him inexorably to the conclusion that society's solutions to the macro problems of the world lie with big government. This view conflicts with the "bottom-up" democracy held dear by Americans.Shares
Russian state media has been working overtime since the start of the Russian intervention in the Syrian civil war.Shares
Kim Davis' hair and Donald Trump's prayer; Chick-fil-A in New York City; Roman Catholics and evangelicalsShares
For the better part of the last two years, the experts, pundits and Washington insiders have proclaimed that Hillary Rodham Clinton was the inevitable 2016 presidential nominee of the Democratic Party.Shares
Tim Constantine reports on Dr. Ben Carson's comments on gun control, Hillary Clinton taking on Donald Trump, and Alec Baldwin calling outHillary Clinton and Donald Trump as warmongers.Shares
OK, watch out! Incoming! I'm going to let them have it, full bore. Having just spent a month in the former Soviet Union, specifically Russia and Ukraine, I'm going to say some things that many Americans won't want to hear. But these things need to be said. As the old saying goes, truth is the best medicine.Shares
Jonathan Edwards (1703-1758) is considered one of America's first and most important philosophers and theologians. Here are the 10 best books by and about Edwards.Shares
We all know someone who has been or is suffering from addiction -- which has even been referred to by some as our national plague and the most ignored health crisis in our country.Shares
After completing a grueling debt renegotiation with its international creditors, Ukraine still has a large mountain to climb solving the problem of the $3 billion bond with Russia.Shares
Newsvesting is a term you are going to hear a lot about.Shares
Tim Constantine reports on good news for the Bernie Sanders campaign, and more good news for Fiat.Shares
From the newspaper archives. "A little extreme for my taste," 1974 congressional candidate Bill Clinton said, weighing in on the recent fad of "streaking."Shares
One might have been forgiven for thinking that the negative effects of President Obama's nuclear deal with Iran would take some time to begin manifesting themselves in the Middle East.Shares
Ben Carson's faith; Evangelicals and Israel; GOP failures in pro-life PR; virgin birth IVF; Kim Davis and FrancisShares
Pro-Russian rebel forces and the Ukrainian government have signed an agreement to withdraw weapons under 100mm caliber from the contact line in East Ukraine. I have met with and spoken with Ukrainian military sources here in Mariupol and can confirm this is beginning to happen. This development effectively ends the armed portion of the conflict in East Ukraine between Kyiv and pro-Russian separatists for the time being.Shares
In air defense terms, it's called anti-access/area denial, or A2/AD in Pentagon speak.Shares
One of the places where jihad - the effort to achieve our submission to the Islamic supremacist doctrine of shariah - is playing out in America is in our courts.Shares


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What Cosmetic Procedure is Done the Most in New England? Answer May Be Surprising
What do you think is the most performed procedure in New England when it comes to plastic surgery? Hint: It's not a breast augmentation.
What do you think is the most performed procedure in New England when it comes to plastic surgery? Hint: It's not a breast augmentation.
It has been a month since my hernia repair and for the most part I'm back to normal. It's still a little uncomfortable to bend over and pick something up off of the floor and sudden body movements are also a little taxing on the midsection but everything seems to be getting better.
Greg Michaels is a little concerned about his upcoming hernia surgery, which I've actually had done. I did the best I could do to ease his fears. I wrote a little song (that sounds a tad like something else!)
On Tuesday, over my vacation, I spent it at St. Mary's Hospital with my wife waiting for her during surgery. She was ready to go in but I was freaking even though it wasn't me. When the doctor told me it would take two to two and half hours, I knew I could leave and I did, but only for about an hour. I grabbed lunch and took my 6th grade son out of school early because as it worked out, she would have been out of surgery around the same time as my son was out of school.
As I sit here at Maine General, Thayer Unit in Waterville, I can't wait to see dad. My sister, Bonnie and Marie Anne are here and dad made it through surgery well.


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updated 11/26/2012 AT 3:00 PM ET
•originally published 11/26/2012 AT 4:00 PM ET
Kim Zolciak is calling it quits on the Real Housewives of Atlanta, but the drama is far from over.
In a teaser for the Dec. 2 episode, a pregnant Zolciak argues with her fellow Housewives about going on a vacation with them before declaring, "I'm done with this whole thing." She walks out on them and shoves a camera out of her way in a huff.
Bravo has confirmed to PEOPLE that Zolciak is off Real Housewives of Atlanta after the Dec. 2 episode.
Meanwhile, Zolciak has said on Twitter that she is leaving the Real Housewives for a spinoff. Earlier this year, she starred in her own Bravo spinoff show called Don't Be Tardy for the Wedding, and Bravo has also confirmed to PEOPLE that a second season, currently titled Don't Be Tardy…, has been picked up.
So is Zolciak's seemingly-abrupt exit by choice? Housewives costar NeNe Leakes is fueling the (gossip) fire with her assertion that Zolciak was fired. And now the former friends have taken to Twitter to battle it out.


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Nice to meet you here at our free ringtones website, where it's possible to download epic ringtones for your ringer. Today we have a ringtone of "Baby I'm Yours feat. Irfane (" by Breakbot as well as 386 other ringtones by Breakbot. You can listen to all 4 Breakbot – "Baby I'm Yours feat. Irfane (" ringtone variations available here and download it for free to your PC. You can also select other 4 albums produced by Breakbot for all time. "Baby I'm Yours feat. Irfane (" is referred to Electro, and you may listen to a huge amount of Electro collaborations absolutely for free. And finally, we have collected lyrics for this track and you can view it here as well. So feel free to browse, listen, download ringtones at Audiko.net. Make your phone sound cool!
Breakbot Baby I'm Yours feat. Irfane ( Ringtone
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Hey Adventurers!
Better Off Bowling is starting up their summer league this month and I thought I'd pass some info along to our group! They've given us a discount code so registration is only $63 - use code MEETUP104. Since this sounds like fun, I've created a team for our group called Bowling For Beer! - here's the link:
and you can get info on the season: http://www.betteroffbowling.com/philly
Also, there are some other Adventure peeps who are making their own teams with friends, so feel free to do that as well. It doesn't matter if you're a horrible bowler or a fanstastic one as this league is mainly about fun and meeting people, so that's right up our alley. Anyone who's interested in their meetup group can check it out here:
This is the info on their meetup below! We get registration at only $63 with the promo code, but the meetup ends soon!! Bowling begins June 26th!!
Better Off Bowling, Philadelphia's Biggest Social Bowling League!
Hundreds of fun young professionals (20's and 30's), co-ed and non-competitive, and at only $63 for the entire season, incredibly affordable!
- 6 weeks of coed social bowling (1-3 games each week) at Lucky Strike!
- Bowling on June 26, July 3, July 10, July 17, July 24, July 31
- Hundreds of fun bowlers with an awesome after-bowling bar scene!
- Bowling shoe rentals and league Management
- Drink specials, after-bowling bar, and End-of-Season Party!
Registration is only $63 with promo code: MEETUP104
Join as a team, small group, or individual!
Check out pics from the league: Bowling Pics!!
The league is filling up, so sign up soon!!
Email any questions or issues to: [masked]
Just want to remind everyone that registration for the coed social bowling league will be ending on Friday, June 21st! So if you haven't already registered but you're interested in doing so, just go to www.betteroffbowling.com/philly and sign up soon!
0 · June 14, 2013
Meetup has allowed me to meet people I wouldn't have met naturally - they're totally different than me.
— Allison, started Women's Adventure Travel
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Murrurundi NSW
sharing our local community with the world
Search local - Shop local - Stay local -
The top navigation bar below goes directly to areas on the Murrurundi Community Portal Website. The OzDirectory navigation bar lists local business and community groups.
116 Mayne Street Murrurundi NSW 2338
We are located behind the RSL Hall, enter through gates to the left of the hall.
Positions currently available on Wednesdays and Thursdays. We are also taking names for placements in 2016
Upper Hunter Shire CouncilLatest Media ReleasesHunter Shire Council's draft Delivery Program & Operational Plan (DPOP)The latest "Links Magazine" - September 2015_______________________________________
The Murrurundi Community Portal Website A proud initiative of the Murrurundi CommunityLeaders ForumNext Meeting - Monday 21st September 2015Murrurundi Library Rooms 5.00pm
__________________________________Our major sponsors are
Bettington RuralKing of the RangesMurrurundi & District Arts Council


text:
Trade and investment topics
Trade agreements
innovationXchange is DFAT's development innovation hub.
To help Australians avoid difficulties overseas, we maintain travel advisories for more than 170 destinations.
The Australian Passport Office and its agents are committed to providing a secure, efficient and responsive passport service for Australia.
The information you provide will help us contact you in an emergency.
On 11 September 2015, New Colombo Plan China alumni had the opportunity to share details about their overseas experiences during a roundtable discussi
Foreign Minister Bishop welcomes New Colombo Plan alumni from across Australia to Canberra for the inaugural New Colombo Plan Alumni Forum.
From 2016, 60 students from UWS Business School will participate in New Colombo Plan mobility projects in China, India, Singapore and Vietnam.
Minister Bishop and Minister Pyne MP, have announced details of the 461 mobility projects that will be funded in 2016 by the New Colombo Plan.
Ten Australian Catholic University nursing students undertook a clinical placement at hospitals in Taipei supported by New Colombo Plan mobility grant
Page 1 of 11
Subscribe to the New Colombo Plan newsletter for the latest news delivered to your inbox.


text:
Gorilla Nearly Destroys Zoo Glass, Terrifies Nearby Family
Watch a silver back gorilla randomly lose it and break zoo glass by running towards it.
Watch a silver back gorilla randomly lose it and break zoo glass by running towards it.
We always hear all the stories about troops and their heroic missions, but have you ever wondered what they eat out there in the harsh conditions overseas and during battles?
We all have our days, you know, when you miss the trashcan with that candy wrapper and just leave it there. That is normal lazy, right? These inventions take it beyond lazy.
Seems like everywhere you look, there is an animal video on the internet. This one will make your day. A compilation of animals trying to fit in small places seems to be hilariously satisfying.
Can we just sit back and swoon for a second? Justin and Garth on stage together! And what they sing is totally epic.
Having trouble with your bra ladies? You know, the usual problems that bug you throughout the day? Here are some much needed bra hacks every woman should know and use.
I know many guys will fight the argument that they have life way easier than women, but once you take a look at this video I think most guys would agree they have it pretty easy.
This video will for sure have you squeezing and loving your pet as soon as you see them.
The Arizona Diamondbacks unveiled the insanity of the Churro Dog today, their new star snack
This was no ordinary ride home.
These inventions are so outrageous you won't believe them. Who even thinks of these things?!
This looks like something out of a movie.


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The 2016 Punchestown Festival will take place from Tuesday 26th to Saturday 30th April.
Join us for five action packed days where the best of Irish and British racing talent will compete in 38 races including 12 Grade 1's.
We will have a full entertainment programme with live music & street theatre, hospitality packages, shopping village, best dressed lady competitions, children's entertainment and a buzzing social scene.
Tickets will be on sale from September 2015 . Make sure to sign up to our newsletter or follow us on Facebook and Twitter for early bird offers.
More InformationGo!
The Grade 3 Star Best For Racing Coverage Chase is the feature race supported by graded and listed jumping action. First race 2.15. Admission €12 and €8.
Book TicketsGo!
First race 2.05 ...Read More
LEADING owner Lloyd Williams has heaped praise on trainer Aidan O'Brien after making a rare public a...Read More


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who is jumping about among the dancers with a bunch of flowers in his hand? He has no partner but seems to be exercising his legs in sympathy with those who are really dancing. No! I was mistaken,--he has a partner, but the lady's pretty figure is so small that I could only see the top of her head, which is covered with scarlet verbenas and a profusion of roses; and I was under the illusion that the big man was going it alone with a magnificent bouquet in his grasp. Toney, do tell me, who is that man? He seems to be a great admirer of beauty, and has been flitting about among the ladies like a large bumble-bee in search of the sweetest and most delicious flowers."
"That is M. T. Pate, a distinguished lawyer, an eloquent orator, an able writer, a profound thinker, and the prince of lady-killers. He is possessed of a very original genius, and has recently written a remarkable pamphlet, in which is demonstrated the possibility as well as the immense importance of draining the Atlantic Ocean, and convertin
Cara Devon has always suffered curiosity and im... Read more
When the economic downturn ends Matty Cruzs co... Read more
A telekinetic teenager. A telepathic child. A p... Read more
A century after an apocalyptic war reduces all... Read more
Fall in love with the Kensington siblings in th... Read more
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Linksys WRT54G3G-UK Wireless-G Router for 3G/UMTS Broadband
£130 plus Vodafone connect card
Allows you to share your 3G network with colleagues
Large, even larger power pack, difficulties setting up, connection costs
For business travellers wanting to access the office anywhere in the UK most have switched to a 3G datacard. However if there is more than one of you on the road, you'll need a datacard for every man, women and child won't it?
Not according to Linksys. It's idea has been to create a networking device that allows you to not only connect to the internet using a Vodafone 3G datacard, but also to share that connection once connected.
There are two parts to the WRT54G3G. The first is that it works just as like anyother standard router, and can be plugged into your existing fixed line WAN (cable modem or DSL), sharing this connection with other four computers via ethernet (RJ45), or multiple computers via wireless (Wi-Fi).
The second is that plug in the Vodafone datacard and providing you are in a 3G or GPRS area you can not only connect to the internet, but now share that network wirelessly so other computers (ie your colleagues) can use that internet connection as well.
In practice and the router does what its says it does on the tin. Although only working with Vodafone's data card collection - either the current standard 3G offering or the new improved superfast HSDPA variant currently within the M25 - speeds are never too far behind regular broadband connections.
The newer of the cards offers a potential 1.4Mbps for downloads and 384kbps for uploads and Vodafone reckons it'll cover ninety per cent of the areas where 3G is currently in use by the end of 2006 so we would recommend checking Vodafone's web site before purchasing this product.
In terms of security, the unit ticks all the boxes and is compatible with WEP, WPA and WPA2. It comes with a fully configurable built-in DHCP server, VPN passthrough and a SPI firewall.
It also allows the user to configure port forward for specific applications and to define an IP address to be placed on the DMZ.
Setup is simple, although you will need a PC to do it. After that however if you've got Mac colleagues then they can join in with the fun too.
So what's the catch? Well the device although perfect as a travelling solution isn't very "travelable". The router, while flat is still fairly large and the Powerpack even larger. Then there is the set up. While incredibly simple, we still have teething problems getting the router to recognise our datacard a problem which judging by some comments on web forums isn't isolated.
The final worry is data. Not its protection, but its price. Unless you are on a decent package with Vodafone, more users means you are likely to speed though your allowance as fast as a group of kids with a box of doughnuts.
Grumbles aside, the WRT54G3G is a fantastic mobile networking solution that allows you to create a wireless network wherever you go as long as there is a 3G or GPRS signal.
It would have been nice to see a battery pack option and a smaller casing for ultimate portability however we love the fact that you can use this box to share your internet connection (and datacard) with your colleagues.
As an ad hoc network at a conference or trade show you'll be hard pushed to beat it as long as you can afford the connection fees.
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