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Animal and clinical studies show that gestational exposure to nicotine increases the propensity of offspring to consume nicotine, but the precise mechanism mediating this behavioral phenomenon is unclear. The present study in Sprague Dawley rats examined the possibility that the orexigenic peptide systems, enkephalin (ENK) and orexin (OX), which are stimulated by nicotine in adult animals and promote consummatory behavior, may be similarly responsive to nicotine's stimulatory effect in utero while having long-term behavioral consequences. The results demonstrated that nicotine exposure during gestation at low doses (0.75 or 1.5 mg/kg/d) significantly increased mRNA levels and density of neurons that express ENK in the hypothalamic paraventricular nucleus and central nucleus of the amygdala, OX, and another orexigenic peptide, melanin-concentrating hormone, in the perifornical lateral hypothalamus in preweanling offspring. These effects persisted in the absence of nicotine, at least until puberty. Colabeling of the cell proliferation marker BrdU with the neuronal marker NeuN and peptides revealed a marked stimulatory effect of prenatal nicotine on neurogenesis, but not gliogenesis, and also on the number of newly generated neurons expressing ENK, OX, or melanin-concentrating hormone. During adolescence, offspring also exhibited significant behavioral changes, increased consumption of nicotine and other substances of abuse, ethanol and a fat-rich diet, with no changes in chow and water intake or body weight. These findings reveal a marked sensitivity during gestation of the orexigenic peptide neurons to low nicotine doses that may increase the offspring's propensity to overconsume substances of abuse during adolescence.

Glucoprivation or hypoglycemia induces a range of counterregulatory responses, including glucose mobilization, reduced glucose utilization, and de novo glucose synthesis. These responses are mediated in part by the sympathetic nervous system. The aim of this study was to determine the chemical codes of sympathetic preganglionic neurons (SPN) activated by glucoprivation, induced by 2-deoxy-D-glucose (2DG). SPN controlling the adrenal glands and celiac ganglia, which ultimately can innervate the liver and pancreas, were targeted together with the superior cervical ganglia (control). 23.9% ± 1.3% of SPN in the T4-T11 region contained c-Fos immunoreactivity following 2DG; 70.3% ± 1.8% of SPN innervating the adrenal glands and 37.4% ± 3% of SPN innervating celiac ganglia were activated. 14.8% ± 3.5% of SPN (C8-T3) innervating superior cervical ganglia were activated. In the C8-T3 region 55% ± 10% of SPN activated contained PPCART, with only 12% ± 3% expressing PPE mRNA, whereas, in the T4-T11 region, 78% ± 4% contained PPE, with only 6.0% ± 0.6% expressing PPCART mRNA. Thus CART is not involved in glucose mobilization. Two chemically distinct populations of SPN (PPE⁺ 57.4% ± 5%, PPE⁻ ∼40%) were identified to regulate adrenaline release in response to glucoprivation. Multiple chemically distinct SPN populations innervating a specific target could suggest their graded recruitment. The two distinct populations of SPN (PPE⁺ 67.6% ± 9%, PPE⁻ ∼30%) projecting to celiac ganglia activated by glucoprivation could direct pancreatic and hepatic or other counterregulatory responses. Nearly all SPN that expressed PPE mRNA and projected to the adrenal glands or celiac ganglia were activated, suggesting a role for the inhibitory peptide enkephalin in responses evoked by glucoprivation., (Copyright © 2013 Wiley Periodicals, Inc., A Wiley Company.)

Despite their widespread use in opioid maintenance treatment and pain management, little is known about the intracellular effectors of methadone and buprenorphine and the transcriptional responses they induce. We therefore studied the acute effects of these two opioids in rats, comparing our observations with those for the reference molecule, morphine. We determined the analgesic ED50 of the three molecules in the tail flick test, to ensure that transcriptional effects were compared between doses of equivalent analgesic effect. We analysed changes in gene expression over time in three cerebral structures involved in several opioid behaviours-the dorsal striatum, thalamus and nucleus accumbens-by real-time quantitative PCR. We analysed the expression of genes encoding proteins of the endogenous opioid system in parallel with that of Fos, a marker of neuronal activation. The acute transcriptional effects of methadone resembled those of morphine more closely than did those of buprenorphine, in terms of kinetics and intensities. Our results provide the first evidence that these two drugs widely used in pain management and opioid maintenance treatment can disturb the regulation of endogenous opioid system genes and induce molecular outcomes different from those observed with morphine., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: Housing animals in an enriched environment (EE) enhances behavioral function. However, the mechanism underlying this EE-mediated functional improvement and the resultant changes in gene expression have yet to be elucidated., Objectives: We attempted to investigate the underlying mechanisms associated with long-term exposure to an EE by evaluating gene expression patterns., Methods: We housed 6-week-old CD-1 (ICR) mice in standard cages or an EE comprising a running wheel, novel objects, and social interaction for 2 months. Motor and cognitive performances were evaluated using the rotarod test and passive avoidance test, and gene expression profile was investigated in the cerebral hemispheres using microarray and gene set enrichment analysis (GSEA)., Results: In behavioral assessment, an EE significantly enhanced rotarod performance and short-term working memory. Microarray analysis revealed that genes associated with neuronal activity were significantly altered by an EE. GSEA showed that genes involved in synaptic transmission and postsynaptic signal transduction were globally upregulated, whereas those associated with reuptake by presynaptic neurotransmitter transporters were downregulated. In particular, both microarray and GSEA demonstrated that EE exposure increased opioid signaling, acetylcholine release cycle, and postsynaptic neurotransmitter receptors but decreased Na+ / Cl- -dependent neurotransmitter transporters, including dopamine transporter Slc6a3 in the brain. Western blotting confirmed that SLC6A3, DARPP32 (PPP1R1B), and P2RY12 were largely altered in a region-specific manner., Conclusion: An EE enhanced motor and cognitive function through the alteration of synaptic activity-regulating genes, improving the efficient use of neurotransmitters and synaptic plasticity by the upregulation of genes associated with postsynaptic receptor activity and downregulation of presynaptic reuptake by neurotransmitter transporters.

We have recently identified missense mutations in prodynorphin (PDYN), the precursor to dynorphin opioid peptides, as the cause for spinocerebellar ataxia (SCA23) in Dutch ataxia cases. We report a screen of PDYN for mutations in 371 cerebellar ataxia cases, which had a positive family history; most are of French origin. Sequencing revealed three novel putative missense mutations and one heterozygous two-base pair deletion in four independent SCA patients. These variants were absent in 400 matched controls and are located in the highly conserved dynorphin domain. To resolve the pathogenicity of the heterozygous variants, we assessed the peptide production of the mutant PDYN proteins. Two missense mutations raised dynorphin peptide levels, the two-base pair deletion terminated dynorphin synthesis, and one missense mutation did not affect PDYN processing. Given the outcome of our functional analysis, we may have identified at least two novel PDYN mutations in a French and a Moroccan SCA patient. Our data corroborates recent work that also showed that PDYN mutations only account for a small percentage (~0.1 %) of European SCA cases.

The chronic use of nicotine, the main psychoactive ingredient of tobacco smoking, alters diverse physiological processes and consequently generates physical dependence. To understand the impact of chronic nicotine on neuropeptides, which are potential molecules associated with dependence, we conducted qualitative and quantitative neuropeptidomics on the rat dorsal striatum, an important brain region implicated in the preoccupation/craving phase of drug dependence. We used extensive LC-FT-MS/MS analyses for neuropeptide identification and LC-FT-MS in conjunction with stable isotope addition for relative quantification. The treatment with chronic nicotine for 3 months led to moderate changes in the levels of endogenous dorsal striatum peptides. Five enkephalin opioid peptides were up-regulated, although no change was observed for dynorphin peptides. Specially, nicotine altered levels of nine non-opioid peptides derived from precursors, including somatostatin and cerebellin, which potentially modulate neurotransmitter release and energy metabolism. This broad but selective impact on the multiple peptidergic systems suggests that apart from the opioid peptides, several other peptidergic systems are involved in the preoccupation/craving phase of drug dependence. Our finding permits future evaluation of the neurochemical circuits modulated by chronic nicotine exposure and provides a number of novel molecules that could serve as potential therapeutic targets for treating drug dependence.

Synthetic κ-opioid receptor (KOR) agonists induce dysphoric and pro-depressive effects and variations in the KOR (OPRK1) and prodynorphin (PDYN) genes have been shown to be associated with alcohol dependence. We genotyped 23 single nucleotide polymorphisms (SNPs) in the PDYN and OPRK1 genes in 816 alcohol-dependent subjects and investigated their association with: (1) negative craving measured by a subscale of the Inventory of Drug Taking Situations; (2) a self-reported history of depression; (3) the intensity of depressive symptoms measured by the Beck Depression Inventory-II. In addition, 13 of the 23 PDYN and OPRK1 SNPs, which were previously genotyped in a set of 1248 controls, were used to evaluate association with alcohol dependence. SNP and haplotype tests of association were performed. Analysis of a haplotype spanning the PDYN gene (rs6045784, rs910080, rs2235751, rs2281285) revealed significant association with alcohol dependence (p = 0.00079) and with negative craving (p = 0.0499). A candidate haplotype containing the PDYN rs2281285-rs1997794 SNPs that was previously associated with alcohol dependence was also associated with negative craving (p = 0.024) and alcohol dependence (p = 0.0008) in this study. A trend for association between depression severity and PDYN variation was detected. No associations of OPRK1 gene variation with alcohol dependence or other studied phenotypes were found. These findings support the hypothesis that sequence variation in the PDYN gene contributes to both alcohol dependence and the induction of negative craving in alcohol-dependent subjects.

Objective: To explore the central mechanism of moxibustion on analgesic effect., Methods: Male Wistar rats were screened by pain threshold value before making model, and 48 rats whose pain threshold was (250 +/- 25) g were selected. Twelve male Wistar rats were randomly selected as a normal group. For the rest rats the rheumatoid arthritis (RA) model was duplicated by raising in a windy, cold and wet environment combined with injection of Freund's complete adjuvant (FCA), and then they were randomly divided into a model group, a moxibustion group and a moxa volatile oil group, 12 rats in each group. The moxibustion and the moxa volatile oil igroup were treated with moxibustion and moxa volatile oil at "Shenshu"(BL 23) and "Zusanli"(ST 36), respectively, for 15 days. No interventions were added on the model group and the normal group. The pain threshold in Iinjured foot and the expression of hypothalamic POMC mRNA and PDYN mRNA in rats were observed., Results: Compared with the normal group, the pain threshold and the expression of hypothalamic POMC mRNA and PDYN mRNA in the model group were increased (all P < 0.01). Compared with the model group, the pain threshold and the expression of hypothalamic POMC mRNA and PDYN mRNA in the moxibustion group were increased significantly (all P < 0.01), but no statistically significance in the moxa volatile oil group (P > 0.05). Compared with the moxa volatile oil group, the above-mentioned observative indices in moxibustion group were all increased significantly (all P < 0.01)., Conclusion: Moxibustion has obvious analgesic effect and its mechanism may be related to the increasing expression of hypothalamic POMC and PDYN mRNA through the warming effect of moxibustion.

Parkinson's disease (PD) is an asymmetric neurodegenerative disorder, and secondary adaptive mechanisms of the less-affected side could potentially compensate for parkinsonian symptoms. Here, we analyzed gene expression changes on the healthy side of a unilateral PD rat model and correlated these changes with locomotor velocity, which is known to be decreased in PD. Four weeks after a unilateral 6-hydroxydopamine lesion, the spontaneous locomotor velocity of rats was recorded just prior to brain extraction. We then analyzed the gene expression levels of markers of the direct (dynorphin and D1-class dopamine receptors) and indirect (enkephalin and D2-class dopamine receptors) pathways in the contralateral healthy striatum by in situ hybridization histochemistry. In addition, we analyzed the expression of several striatal and cortical glutamatergic markers, as well as nigral tyrosine hydroxylase (TH) and nigral dopamine transporter (DAT). We found a significant positive correlation between the mRNA expression levels of contralateral D1-class dopamine receptors and the mean locomotor velocity, at 4 weeks after surgery in parkinsonian rats but not in controls. Moreover, we observed a significant increase in the level of dynorphin mRNA in the lateral part of the contralateral striatum of parkinsonian rats compared to the controls. In contrast, no contralateral changes were observed in the striatal indirect pathway. We also did not find any significant contralateral modifications of TH, DAT or glutamatergic markers in PD animals, indicating that changes in direct pathway genes are not due to nigrostriatal dopaminergic or corticostriatal glutamatergic innervation. In conclusion, our results suggest a role of the healthy striatal direct pathway in counteracting dopaminergic denervation effects on motor symptoms., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

Many studies have utilized the Inbred Long Sleep and Inbred Short Sleep mouse strains to model the genetic influence on initial sensitivity to ethanol. The mechanisms underlying this divergent phenotype are still not completely understood. In this study, we attempt to identify genes that are differentially expressed between these two strains and to identify baseline networks of co-expressed genes, which may provide insight regarding their phenotypic differences. We examined the whole brain and striatal transcriptomes of both strains, using next generation RNA sequencing techniques. Many genes were differentially expressed between strains, including several in chromosomal regions previously shown to influence initial sensitivity to ethanol. These results are in concordance with a similar sample of striatal transcriptomes measured using microarrays. In addition to the higher dynamic range, RNA-Seq is not hindered by high background noise or polymorphisms in probesets as with microarray technology, and we are able to analyze exome sequence of abundant genes. Furthermore, utilizing Weighted Gene Co-expression Network Analysis, we identified several modules of co-expressed genes corresponding to strain differences. Several candidate genes were identified, including protein phosphatase 1 regulatory unit 1b (Ppp1r1b), prodynorphin (Pdyn), proenkephalin (Penk), ras association (RalGDS/AF-6) domain family member 2 (Rassf2), myosin 1d (Myo1d) and transthyretin (Ttr). In addition, we propose a role for potassium channel activity as well as map kinase signaling in the observed phenotypic differences between the two strains., (© 2013 Blackwell Publishing Ltd and International Behavioural and Neural Genetics Society.)

Spinocerebellar ataxias (SCA) are a genetically heterogeneous group of neurodegenerative diseases characterised by progressive cerebellar ataxia, dysarthria and oculomotor abnormalities. Recently the prodynorphin (PDYN) gene was identified as the cause of SCA23 in four Dutch families displaying progressive gait and limb ataxia. In this study we aimed to assess the frequency of PDYN gene defects and extend the phenotype of SCA23 patients in a UK ataxia series and also in patients from Greece, Egypt and India. We sequenced the coding and flanking intronic regions of the PDYN gene in a total of 852 ataxia patients, of which 356 were sporadic with no family history, 320 had a positive family history, and 176 probands had a positive family history and at least one family member had also been investigated. We also analysed 190 patients with multiple-system atrophy with cerebellar features (MSA-C), a phenocopy of SCA23. We identified a novel putative pathogenic heterozygous missense variant in the PDYN gene in an early onset SCA patient with an unknown family history. This variant was not present in 570 matched British controls. This is the first study to screen for SCA23 in UK patients and confirms that PDYN mutations are a very rare cause of spinocerebellar ataxia, accounting for ~ 0.1 % of ataxia cases but perhaps with a higher frequency in pure cerebellar ataxia. Given the rarity of PDYN mutations, front-line diagnostic evaluation of UK familial and early onset pure spinocerebellar ataxia patients should focus on other known ataxia genes.

Several studies demonstrated the role of the endogenous opioid system in the development of susceptibility to alcohol dependence. Recently, we reported that binge intragastric administration of ethanol induces selective alterations of pronociceptin and prodynorphin gene expression in the rat amygdala complex depending on the days of exposures and on the development of tolerance and dependence. The aim of the present study was to investigate the potential epigenetic mechanisms leading to these alcohol-induced changes in gene expression. Specific histone modifications and DNA methylation at opioid peptide precursor promoters were analyzed by chromatin immunoprecipitation and real-time methylation-specific PCR, respectively. We found a linkage between gene expression alterations and epigenetic modulation at pronociceptin and prodynorphin promoters following alcohol treatment. In animals treated for 1 day, we observed a reversed correlation, with a decrease of histone 3 lysine 27 trimethylation (repressive mark) and an increase of histone 3 lysine 9 acetylation (activating mark), associated with both gene expression up-regulation. In rats treated with alcohol for up to 5 days, we found an increase in histone 3 lysine 9 acetylation in the pronociceptin promoter providing further evidence of the already proposed possible role for histone deacetylases for addiction treatment. No significant alterations in DNA methylation and histone 3 lysine 4 trimethylation following different alcohol exposures were present, suggesting the selectivity of epigenetic effects induced by alcohol. These data demonstrate that ethanol induces selective epigenetic changes, thus better defining the role of opioid peptides in the ethanol-induced effects in the amygdala complex.

We previously reported the effects of herpes simplex virus (HSV) vector-mediated enkephalin on bladder overactivity and pain. In this study, we evaluated the effects of vHPPE (E1G6-ENK), a newly engineered replication-deficient HSV vector encoding human preproenkephalin (hPPE). vHPPE or control vector was injected into the bladder wall of female rats 2 weeks prior to the following studies. A reverse-transcription PCR study showed high hPPE transgene levels in L6 dorsal root ganglia innervating the bladder in the vHPPE group. The number of freezing behaviors, which is a nociceptive reaction associated with bladder pain, was also significantly lower in the vHPPE group compared with the control group. The number of L6 spinal cord c-fos-positive cells and the urinary interleukin (IL)-1β and IL-6 levels after resiniferatoxin (RTx) administration into the bladder of the vHPPE group were significantly lower compared with those of the control vector-injected group. In continuous cystometry, the vHPPE group showed a smaller reduction in intercontraction interval after RTx administration into the bladder. This antinociceptive effect was antagonized by naloxone hydrochloride. Thus, the HSV vector vHPPE encoding hPPE demonstrated physiological improvement in visceral pain induced by bladder irritation. Gene therapy may represent a potentially useful treatment modality for bladder hypersensitive disorders such as bladder pain syndrome/interstitial cystitis.

Background: Huntington's disease (HD) is a neurological disorder caused by mutations in the huntingtin (HTT) gene, the product of which leads to selective and progressive neuronal cell death in the striatum and cortex. Transcriptional dysregulation has emerged as a core pathologic feature in the CNS of human and animal models of HD. It is still unclear whether perturbations in gene expression are a consequence of the disease or importantly, contribute to the pathogenesis of HD., Objective: To examine if transcriptional dysregulation can be ameliorated with antisense oligonucleotides that reduce levels of mutant Htt and provide therapeutic benefit in the YAC128 mouse model of HD., Methods: Quantitative real-time PCR analysis was used to evaluate dysregulation of a subset of striatal genes in the YAC128 mouse model. Transcripts were then evaluated following ICV delivery of antisense oligonucleotides (ASO). Rota rod and Porsolt swim tests were used to evaluate phenotypic deficits in these mice following ASO treatment., Results: Transcriptional dysregulation was detected in the YAC128 mouse model and appears to progress with age. ICV delivery of ASOs directed against mutant Htt resulted in reduction in mutant Htt levels and amelioration in behavioral deficits in the YAC128 mouse model. These improvements were correlated with improvements in the levels of several dysregulated striatal transcripts., Conclusions: The role of transcriptional dysregulation in the pathogenesis of Huntington's disease is not well understood, however, a wealth of evidence now strongly suggests that changes in transcriptional signatures are a prominent feature in the brains of both HD patients and animal models of the disease. Our study is the first to show that a therapeutic agent capable of improving an HD disease phenotype is concomitantly correlated with normalization of a subset of dysregulated striatal transcripts. Our data suggests that correction of these disease-altered transcripts may underlie, at least in part, the therapeutic efficacy shown associated with ASO-mediated correction of HD phenotypes and may provide a novel set of early biomarkers for evaluating future therapeutic concepts for HD.

Approximately 500,000 infants are born prematurely each year in the United States. These infants typically require an extensive stay in the neonatal intensive care unit (NICU), where they experience on average 14 painful and invasive procedures each day. These procedures, including repeated heel lance, insertion of intravenous lines, and respiratory and gastric suctioning, typically result in an inflammatory response, inducing pain and stress in the newborn. Remarkably, the majority of these procedures are performed in the complete absence of pre- or post-emptive analgesics. Recent clinical studies report that former NICU patients have increased thresholds for pain and stress later in life as compared with term-born infants. However, to date, the mechanisms whereby early-life inflammation alters later-life response to stress and pain are not known. The present studies were conducted to determine if neonatal injury impairs adult responses to anxiety- and stress-provoking stimuli. As we have previously reported that early-life pain results in a significant increase in opioid peptide expression within the midbrain periaqueductal gray, the role of endogenous opioids in our behavioral studies was also examined. Male and female rats received an intraplantar injection of the inflammatory agent carrageenan (1%) on the day of birth. In adulthood, animals were assessed for changes in response to anxiety- and stress-provoking stimuli using the open field and forced swim tests, respectively. Injury-induced changes in sucrose preference and stress-induced analgesia were also assessed. As adults, neonatally injured animals displayed a blunted response to both anxiety- and stress-provoking stimuli, as indicated by significantly more time spent in the inner area of the open field and a 2-fold increase in latency to immobility in the forced swim test as compared to controls. No change in sucrose preference was observed. Using in situ hybridization and immunohistochemistry, we observed a 2-fold increase in enkephalin mRNA and protein expression, respectively, in stress-related brain regions including the central amygdala and lateral septum. Administration of the opioid receptor antagonist naloxone reversed the attenuated responses to forced swim stress and stress-induced analgesia, suggesting the changes in stress-related behavior were opioid-dependent. Together, these data contribute to mounting evidence that neonatal injury in the absence of analgesics has adverse effects that are both long-term and polysystemic., (Copyright © 2013 S. Karger AG, Basel.)

Background: In Parkinson's disease (PD), bradykinesia, or slowness of movement, only appears after a large striatal dopamine depletion. Compensatory mechanisms probably play a role in this delayed appearance of symptoms., Objective: Our hypothesis is that the striatal direct and indirect pathways participate in these compensatory mechanisms., Methods: We used the unilateral 6-hydroxydopamine (6-OHDA) rat model of PD and control animals. Four weeks after the lesion, the spontaneous locomotor activity of the rats was measured and then the animals were killed and their brain extracted. We quantified the mRNA expression of markers of the striatal direct and indirect pathways as well as the nigral expression of dopamine transporter (DAT) and tyrosine hydroxylase (TH) mRNA. We also carried out an immunohistochemistry for the striatal TH protein expression., Results: As expected, the unilateral 6-OHDA rats presented a tendency to an ipsilateral head turning and a low locomotor velocity. In 6-OHDA rats only, we observed a significant and positive correlation between locomotor velocity and both D1-class dopamine receptor (D1R) (direct pathway) and enkephalin (ENK) (indirect pathway) mRNA in the lesioned striatum, as well as between D1R and ENK mRNA., Conclusions: Our results demonstrate a strong relationship between both direct and indirect pathways and spontaneous locomotor activity in the parkinsonian rat model. We suggest a synergy between both pathways which could play a role in compensatory mechanisms and may contribute to the delayed appearance of bradykinesia in PD.

Aims: The aim of this study was to determine if genetic variation in the pain-modulating gene DREAM and its pathway genes influence susceptibility to reporting musculoskeletal pain in the population., Methods: Pairwise tag single nucleotide polymorphisms (SNPs) in DREAM, PDYN and OPRK1 were genotyped in a UK population-based discovery cohort in whom pain was assessed using blank body manikins at three time points. Depression and anxiety symptoms were assessed at the first time point. Zero-inflated negative binomial regression was used to test for association between SNPs and the maximum number of pain sites reported (0-29) across the three time points. Significantly associated SNPs (p < 0.05) were subsequently genotyped for validation in a cohort of European men with pain assessed at two time points., Results: Thirty-five SNPs were genotyped in 1055 subjects, of whom 83% reported pain, in the discovery cohort. SNPs in each gene were associated with the maximum number of pain sites reported, were independent of symptoms of anxiety and depression and had a significant cumulative effect (p = 7.0 × 10(-5) ). Significantly associated SNPs were successfully genotyped in 1733 men, 76% of whom reported pain, in the validation cohort, but did not show significant association with the number of pain sites., Conclusions: Genetic variation in the DREAM pathway genes was associated with the extent of pain reporting in a population-based cohort. These findings were not replicated in a single independent cohort; however, given the potential of this pathway as a therapeutic target, further investigation in additional cohorts is warranted., (© 2012 European Federation of International Association for the Study of Pain Chapters.)

The endogenous opioid system (EOS) plays a critical role in addictive processes. Molecular dysregulations in this system may be specific for different stages of addiction cycle and neurocircuitries involved and therefore may differentially contribute to the initiation and maintenance of addiction. Here we evaluated whether the EOS is altered in brain areas involved in cognitive control of addiction including the dorsolateral prefrontal cortex (dl-PFC), orbitofrontal cortex (OFC) and hippocampus in human alcohol-dependent subjects. Levels of EOS mRNAs were measured by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and levels of dynorphins by radioimmunoassay (RIA) in post-mortem specimens obtained from 14 alcoholics and 14 controls. Prodynorphin mRNA and dynorphins in dl-PFC, κ-opioid receptor mRNA in OFC and dynorphins in hippocampus were up-regulated in alcoholics. No significant changes in expression of proenkephalin, and µ- and δ-opioid receptors were evident; pro-opiomelanocortin mRNA levels were below the detection limit. Activation of the κ-opioid receptor by up-regulated dynorphins in alcoholics may underlie in part neurocognitive dysfunctions relevant for addiction and disrupted inhibitory control., (© 2011 The Authors, Addiction Biology © 2011 Society for the Study of Addiction.)

The rostral ventrolateral medulla (RVLM) contains neurons critical for cardiovascular, respiratory, metabolic, and motor control. The activity of these neurons is controlled by inputs from multiple identified brain regions; however, the neurochemistry of these inputs is largely unknown. Gamma-aminobutyric acid (GABA) and enkephalin tonically inhibit neurons within the RVLM. The aim of this study was to identify all brain regions that provide GABAergic or enkephalinergic input to the rat RVLM. Neurons immunoreactive for cholera toxin B (CTB-ir), retrogradely transported from the RVLM, were assessed for expression of glutamic acid decarboxylase (GAD67) or preproenkephalin (PPE) mRNA using in situ hybridization. GAD67 mRNA was expressed in CTB-ir neurons in the following regions: the nucleus of the solitary tract (NTS, 6% of CTB-ir neurons), area postrema (AP, 8%), caudal ventrolateral medulla (17%), midline raphe (40%), ventrolateral periaqueductal gray (VLPAG, 15%), lateral hypothalamic area (LHA, 25%), central nucleus of the amygdala (CeA, 77%), sublenticular extended amygdala (SLEA, 86%), interstitial nucleus of the posterior limb of the anterior commissure (IPAC, 56%), bed nucleus of the stria terminals (BNST, 59%), and medial preoptic area (MPA, 53%). PPE mRNA was expressed in CTB-ir neurons in the following regions: the NTS (14% of CTB-ir neurons), midline raphe (26%), LHA (22%), zona incerta (ZI, 15%), CeA (5%), paraventricular nucleus (PVN, 13%), SLEA (66%), and MPA (26%). Thus, limited brain regions contribute GABAergic and/or enkephalinergic input to the RVLM. Multiple neurochemically distinct pathways originate from these brain regions projecting to the RVLM., (Copyright © 2012 Wiley Periodicals, Inc.)

The discovery of biomarkers able to predict biological age of individuals is a crucial goal in aging research. Recently, researchers' attention has turn toward epigenetic markers of aging. Using the Illumina Infinium HumanMethylation450 BeadChip on whole blood DNA from a small cohort of 64 subjects of different ages, we identified 3 regions, the CpG islands of ELOVL2, FHL2, and PENK genes, whose methylation level strongly correlates with age. These results were confirmed by the Sequenom's EpiTYPER assay on a larger cohort of 501 subjects from 9 to 99 years, including 7 cord blood samples. Among the 3 genes, ELOVL2 shows a progressive increase in methylation that begins since the very first stage of life (Spearman's correlation coefficient = 0.92) and appears to be a very promising biomarker of aging., (© 2012 The Authors Aging Cell © 2012 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland.)

Background: Marijuana use by teenagers often predates the use of harder drugs, but the neurobiological underpinnings of such vulnerability are unknown. Animal studies suggest enhanced heroin self-administration (SA) and dysregulation of the endogenous opioid system in the nucleus accumbens shell (NAcsh) of adults following adolescent Δ(9)-tetrahydrocannabinol (THC) exposure. However, a causal link between proenkephalin (Penk) expression and vulnerability to heroin has yet to be established., Methods: To investigate the functional significance of NAcsh Penk tone, selective viral-mediated knockdown and overexpression of Penk was performed, followed by analysis of subsequent heroin SA behavior. To determine whether adolescent THC exposure was associated with chromatin alteration, we analyzed levels of histone H3 methylation in the NAcsh via chromatin immunoprecipitation at five sites flanking the Penk gene transcription start site., Results: Here we show that regulation of the Penk opioid neuropeptide gene in NAcsh directly regulates heroin SA behavior. Selective viral-mediated knockdown of Penk in striatopallidal neurons attenuates heroin SA in adolescent THC-exposed rats, whereas Penk overexpression potentiates heroin SA in THC-naïve rats. Furthermore, we report that adolescent THC exposure mediates Penk upregulation through reduction of histone H3 lysine 9 (H3K9) methylation in the NAcsh, thereby disrupting the normal developmental pattern of H3K9 methylation., Conclusions: These data establish a direct association between THC-induced NAcsh Penk upregulation and heroin SA and indicate that epigenetic dysregulation of Penk underlies the long-term effects of THC., (Copyright © 2012 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

Post-traumatic stress disorder (PTSD) is an anxiety disorder that develops in predisposed individuals following a terrifying event. Studies on isogenic animal populations might explain susceptibility to PTSD by revealing associations between the molecular and behavioural consequences of traumatic stress. Our study employed four inbred mouse strains to search for differences in post-stress response to a 1.5-mA electric foot shock. One day to 6 weeks after the foot shock anxiety, depression and addiction-like phenotypes were assessed. In addition, expression levels of selected stress-related genes were analysed in hippocampus and amygdala. C57BL/6J mice exhibited up-regulation in the expression of Tsc22d3, Nfkbia, Plat and Crhr1 genes in both brain regions. These alterations were associated with an increase of sensitized fear and depressive-like behaviour over time. Traumatic stress induced expression of Tsc22d3, Nfkbia, Plat and Fkbp5 genes and developed social withdrawal in DBA/2J mice. In 129P3/J strain, exposure to stress produced the up-regulation of Tsc22d3 and Nfkbia genes and enhanced sensitivity to the rewarding properties of morphine. Whereas, SWR/J mice displayed increase only in Pdyn expression in the amygdala and had the lowest conditioned fear. Our results reveal a complex genetic background of phenotypic variation in response to stress and indicate the SWR/J strain as a valuable model of stress resistance. We found potential links between the alterations in expression of Tsc22d3, Nfkbia and Pdyn, and different aspects of susceptibility to stress., (© 2012 The Authors. Genes, Brain and Behavior © 2012 Blackwell Publishing Ltd and International Behavioural and Neural Genetics Society.)

The spinocerebellar ataxias (SCAs) are a clinically and genetically heterogeneous group of neurodegenerative diseases. In 2010, four missense mutations in the prodynorphin (PDYN) gene were found in two families and two sporadic cases of SCA type 23 (SCA23) from the Netherlands. In addition, one missense mutation in PDYN was also found in one sporadic SCA23 case in America in 2012. To date, there have been no reports of PDYN gene mutations in mainland China. To investigate the frequency of SCA23 among the Chinese Han population, we performed polymerase chain reaction (PCR) and DNA direct sequencing of the PDYN gene in 305 unrelated ataxia patients. Although no SCA23 mutation was identified, one novel single nucleotide polymorphism (c.255G>A, p.Lys85Lys) in exon 4 of the PDYN gene was found. This suggests that SCA23 is a rare form of dominant ataxia in the Chinese Han population., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Gene transfer to target delivery of neurotrophic factors to the primary sensory afferent for treatment of polyneuropathy, or of inhibitory neurotransmitters for relief of chronic pain, offers the possibility of a highly selective targeted release of bioactive molecules within the nervous system. Preclinical studies with non-replicating herpes simplex virus (HSV)-based vectors injected into the skin to transduce neurons in the dorsal root ganglion have demonstrated efficacy in reducing-pain related behaviors in animal models of inflammatory pain, neuropathic pain, and pain caused by cancer, and in preventing progression of sensory neuropathy caused by toxins, chemotherapeutic drugs or resulting from diabetes. Successful completion of the first phase 1 clinical trial of HSV-mediated gene transfer in patients with intractable pain from cancer has set the stage for further clinical trials of this approach., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Addictions to cocaine or heroin/prescription opioids [short-acting μ-opioid receptor (MOPr) agonists] involve relapsing cycles, with experimentation/escalating use, withdrawal/abstinence, and relapse/re-escalation. κ-Opioid receptors (KOPr; encoded by OPRK1), and their endogenous agonists, the dynorphins (encoded by PDYN), have counter-modulatory effects on reward caused by cocaine or MOPr agonist exposure, and exhibit plasticity in addictive-like states. KOPr/dynorphin activation is implicated in depression/anxiety, often comorbid with addictions. In this opinion article we propose that particular stages of the addiction cycle are differentially affected by KOPr/dynorphin systems. Vulnerability and resilience can be due to pre-existing (e.g., genetic) factors, or epigenetic modifications of the OPRK1 or PDYN genes during the addiction cycle. Pharmacotherapeutic approaches limiting changes in KOPr/dynorphin tone, especially with KOPr partial agonists, may hold potential for the treatment of specific drug addictions and psychiatric comorbidity., (Copyright © 2012. Published by Elsevier Ltd.)

Stem cell therapy is one of the most promising treatments in neuroregenerative medicine. Considering the role of the endogenous opioid system in controlling the pathophysiology of neurological disorders and behavioral aberrations, current studies have focused on enkephalins as a part of the opioid system. Due to high capability of unrestricted somatic stem cells (USSCs) and human mesenchymal stem cells (hMSCs) for cell therapy and transplantation; here, we examined their enkephalinergic differentiation potential through Ikaros-related pathways in order to develop in vitro models to help drug screening and stem cell therapy for the opioid-related disorders. The authenticity of the stem cells was verified by differentiation experiments along with flow cytometry for surface markers. Later, we confirmed their neurogenic differentiation with semiquantitative and quantitative transcriptional and translational evaluations of the enkephalinergic-related genes such as proenkephalin, CREBZF, Ikaros, and prodynorphin. Our findings supported the enkephalinergic differentiation of these stem cells. Noteworthy, USSCs showed higher potential for differentiating into enkephalinergic neurons under Ikaros activation than hMSCs, which makes them appropriate for neurological therapeutic applications. In conclusion, this study suggests a powerful in vitro model for neurogenesis that may help clarification of enkephalinergic differentiation and related signaling networks along with neural drug screening. Such investigations may be beneficial to ameliorate the neural-related therapeutic approaches.

The dorsal striatum, which contains the dorsolateral striatum (DLS) and dorsomedial striatum (DMS), integrates the acquisition and implementation of instrumental learning in cooperation with the nucleus accumbens (NAc). The dorsal striatum regulates the basal ganglia circuitry through direct and indirect pathways. The mechanism by which these pathways mediate the learning processes of instrumental actions remains unclear. We investigated how the striatal indirect (striatopallidal) pathway arising from the DLS contributes to the performance of conditional discrimination. Immunotoxin targeting of the striatal neuronal type containing dopamine D(2) receptor in the DLS of transgenic rats resulted in selective, efficient elimination of the striatopallidal pathway. This elimination impaired the accuracy of response selection in a two-choice reaction time task dependent on different auditory stimuli. The impaired response selection was elicited early in the test sessions and was gradually restored as the sessions continued. The restoration from the deficits in auditory discrimination was prevented by excitotoxic lesion of the NAc but not by that of the DMS. In addition, lesion of the DLS mimicked the behavioral consequence of the striatopallidal removal at the early stage of test sessions of discriminative performance. Our results demonstrate that the DLS-derived striatopallidal pathway plays an essential role in the execution of conditional discrimination, showing its contribution to the control of selection accuracy of learned motor responses. The results also suggest the presence of a mechanism that compensates for the learning deficits during the repetitive sessions, at least partly, demanding accumbal function.

Antagonism of the kappa opioid receptor (KOR) has been reported to have anti-depressant-like properties. The dynorphin/KOR system is a crucial neurochemical substrate underlying the pathologies of addictive diseases, affective disorders and other disease states. However, the molecular underpinnings and neuroanatomical localization of the dysregulation of this system have not yet been fully elucidated. Utilizing the Porsolt Forced Swim Test (FST), an acute stressor commonly used as in rodent models measuring antidepressant efficacy, male Sprague-Dawley rats were subject to forced swimming for 15 min, treated 1h with vehicle or norbinaltorphimine (nor-BNI) (5 or 10mg/kg), and then 1 day later subject to FST for 5 min. In accordance with previous findings, nor-BNI dose dependently increased climbing time and reduced immobility. In comparison to control animals not exposed to FST, we observed a significant elevation in prodynorphin (pDyn) mRNA levels following FST using real-time optical polymerase chain reaction (PCR) in the caudate putamen but not in the nucleus accumbens, hypothalamus, amygdala, frontal cortex, or hippocampus. nor-BNI treatment did not affect pDyn mRNA levels in comparison to animals that received vehicle. The corresponding brain regions from the opposite hemisphere were analyzed for underlying chromatin modifications of the prodynorphin gene promoter region using chromatin immunoprecipitation with antibodies against specifically methylated histones H3K27Me2, H3K27Me3, H3K4Me2, and H3K4Me3, as well as CREB-1 and MeCP2. Significant alterations in proteins bound to DNA in the Cre-3, Cre-4, and Sp1 regions of the prodynorphin promoter were found in the caudate putamen of the FST saline-treated animals compared to control animals, with no changes observed in the hippocampus. Epigenetic changes resulting in elevated dynorphin levels specifically in the caudate putamen may in part underlie the enduring effects of stress., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

The process of ethanol anticipation is a particularly important phenomenon that can determine subsequent drug-taking behavior. Recent studies suggest that systems within the medial prefrontal cortex (mPFC), during anticipation, may contribute to the goal-directed seeking of ethanol. The current investigation examined the possibility that the opioid peptide enkephalin (ENK), known to mediate some of the reinforcing properties of ethanol, may function in the mPFC during the anticipation of ethanol access. Using a limited access (3 h/d) paradigm for 10 days with 20% ethanol, Sprague-Dawley rats were first identified either as low drinkers (LD, <1.0 g/kg/3 h) or as high drinkers (HD, >2.0 g/kg/3 h) that exhibited a long-term phenotype of high ethanol consumption and a significant ethanol deprivation effect. During the anticipation period immediately preceding daily ethanol access, the HD rats compared to LD or Control animals with ad libitum ethanol access exhibited increased anticipatory behaviors, including greater exploratory behavior in a novel open field as revealed by significantly more time spent in the rearing position (+53-65%, p < 0.05) and increased number of rears made (+33-44%, p < 0.05) and greater novelty-seeking behavior in a hole-board apparatus revealed by an increase in total (+50-52%, p < 0.05) and novel nose pokes (+45-48%, p < 0.05). In the HD rats, analysis of the mPFC using real-time quantitative PCR showed significantly greater mRNA levels of ENK (p < 0.05) and the mu-opioid receptor (MOR) (p < 0.05), but not delta-opioid receptor (DOR), and this increase in ENK expression was found, using in situ hybridization, to occur specifically in the prelimbic (PrL) subregion of the mPFC. When injected into the PrL during the anticipation period, a MOR agonist but not DOR agonist significantly increased consumption of 20% ethanol (p < 0.05). These findings support the role of ENK, acting through MOR within the PrL to promote the anticipation and excessive consumption of ethanol., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Unlabelled: Relapse is a serious problem for the effective treatment of cocaine addiction., Rationale: Examining cocaine re-exposure-induced behavioral and neurobiological alterations following chronic escalating-dose binge cocaine administration and withdrawal may provide insight into the neurobiological basis of cocaine relapse., Objectives: Our goal was to determine how exposure to chronic escalating-dose cocaine affects development of subsequent cocaine-induced conditioned place preference (CPP) and changes in endogenous opioid systems., Methods: Mice were injected with either escalating-dose binge cocaine (15-30 mg/kg/injection × 3/day) or saline for 14-days and conditioned with 15 mg/kg of cocaine or saline (once per day for 10-days), starting either 1 or 14-days after the last day of binge injections., Results: Mice exposed to chronic escalating cocaine did not develop CPP to cocaine when conditioning commenced on the first day of withdrawal (CPP test on day 10 of withdrawal). By contrast, mice did develop CPP to cocaine when conditioning started on the 14th day of withdrawal (CPP test on day 24 of withdrawal). Furthermore, preproenkephalin (Penk) mRNA levels in caudate putamen were significantly higher in mice that received 14-day withdrawal from escalating-dose binge cocaine before the CPP procedure (tested 24 days post-binge) than those that received 1-day withdrawal (tested 10 days post-binge)., Conclusions: The rewarding effect of cocaine was blunted in early withdrawal from chronic escalating exposure, but recovered in more prolonged withdrawal. Time-dependent elevations in Penk mRNA levels may be part of the underlying mechanisms of this effect., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Analyses of prodynorphin and proenkephalin cDNAs cloned from the central nervous system of the shark, Heterodontus portusjacksoni, provided additional evidence that these two opioid precursor-coding genes were most likely directly derived from a common ancestral gene. The two cDNAs could be aligned by inserting only seven gaps. The prodynorphin cDNA encodes five opioid sequences which could be aligned to opioid positions B through F in the proenkephalin cDNA. The sequence identity within the opioid positions was 59% at the amino acid level. Shark α-neo-endorphin, dynorphin A, and dynorphin B have amino acid motifs in common with shark met-enkephalin-8, and shark proenkephalin opioid positions E and F, respectively, which have not been observed in other gnathostome prodynorphin and proenkephalin precursor sequences. Shark prodynorphin encodes both kappa (α-neo-endorphin, dynorphin A, and dynorphin B) and delta (met-enkephalin and leu-enkephalin) opioid sequences. Mixed function prodynorphin precursors (encoding both enkephalins and dynorphins) are also found in representatives of the teleost fishes, lungfishes, and amphibians. It appears that only mammals evolved a prodynorphin precursor that exclusively encodes kappa opioid agonists (dynorphins)., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Genetic factors are believed to account for 30-50% of the risk for cocaine and heroin addiction. Dynorphin peptides, derived from the prodynorphin (PDYN) precursor, bind to opioid receptors, preferentially the kappa-opioid receptor, and may mediate the aversive effects of drugs of abuse. Dynorphin peptides produce place aversion in animals and produce dysphoria in humans. Cocaine and heroin have both been shown to increase expression of PDYN in brain regions relevant for drug reward and use. Polymorphisms in PDYN are therefore hypothesized to increase risk for addiction to drugs of abuse. In this study, 3 polymorphisms in PDYN (rs1022563, rs910080 and rs1997794) were genotyped in opioid-addicted [248 African Americans (AAs) and 1040 European Americans (EAs)], cocaine-addicted (1248 AAs and 336 EAs) and control individuals (674 AAs and 656 EAs). Sex-specific analyses were also performed as a previous study identified PDYN polymorphisms to be more significantly associated with female opioid addicts. We found rs1022563 to be significantly associated with opioid addiction in EAs [P = 0.03, odds ratio (OR) = 1.31; false discovery rate (FDR) corrected q-value]; however, when we performed female-specific association analyses, the OR increased from 1.31 to 1.51. Increased ORs were observed for rs910080 and rs199774 in female opioid addicts also in EAs. No statistically significant associations were observed with cocaine or opioid addiction in AAs. These data show that polymorphisms in PDYN are associated with opioid addiction in EAs and provide further evidence that these risk variants may be more relevant in females., (© 2012 The Authors. Genes, Brain and Behavior © 2012 Blackwell Publishing Ltd and International Behavioural and Neural Genetics Society.)

Proteases are required for processing precursors into active neuropeptides that function as neurotransmitters for cell-cell communication. This study demonstrates the novel function of human cathepsin V protease for producing the neuropeptides enkephalin and neuropeptide Y (NPY). Cathepsin V is a human-specific cysteine protease gene. Findings here show that expression of cathepsin V in neuroendocrine PC12 cells and human neuronal SK-N-MC cells results in production of (Met)enkephalin from proenkephalin. Gene silencing of cathepsin V by siRNA in human SK-N-MC cells results in reduction of (Met)enkephalin by more than 80%, illustrating the prominent role of cathepsin V for neuropeptide production. In vitro processing of proenkephalin by cathepsin V occurs at dibasic residue sites to generate enkephalin-containing peptides and an ∼24-kDa intermediate present in human brain. Cathepsin V is present in human brain cortex and hippocampus where enkephalin and NPY are produced and is present in purified human neuropeptide secretory vesicles. Colocalization of cathepsin V with enkephalin and NPY in secretory vesicles of human neuroblastoma cells was illustrated by confocal microscopy. Furthermore, expression of cathepsin V with proNPY results in NPY production. These findings indicate the unique function of human cathepsin V for producing enkephalin and NPY neuropeptides required for neurotransmission in health and neurological diseases.

We investigated the antinociceptive effect of microinjection of HSV-1 amplicon vector-mediated human proenkephalin (hPPE) into the ventral periaqueductal grey (PAG) on neuropathic pain in rats. Male Sprague-Dawley rats with chronic constriction injury (CCI)-induced neuropathic pain were microinjected into the ventral PAG with normal saline (NS), pHSVIRES-lacZ (SHZ), or HSV-1 amplicon vector pHSVIRES-hPPE-lacZ (SHPZ), respectively. Pain thresholds in the SHPZ-treated rats were significantly higher at day 3, then reached peak at day 14 and lasted until day 35 after PAG administration, and these effects were reversed by naloxone. In contrast, NS or SHZ-treated rats did not significantly affect pain thresholds. These results demonstrated that microinjection of HSV-1 amplicon vector-mediated hPPE into the ventral PAG attenuates neuropathic pain in rats.

Tissue injury and/or opioids induce plastic changes in the spinal cord resulting in pain hypersensitivity; the contribution of the dorsal root ganglia (DRG) is poorly understood. We evaluated DRG phenotypic changes induced by surgery and/or remifentanil in a mice model of postoperative pain using as neuronal markers ERK1/2 and c-Fos; prodynorphin mRNA and dynorphin levels were also determined. We hypothesized that a correlation between nociception and DRG reactivity would occur. Surgery and/or remifentanil induced mechanical hypersensitivity, correlated with ERK1/2 phosphorylation and c-Fos expression in the DRG; changes were greater in the remifentanil + incision group and still present on day 14 (p < 0.01 vs. control). Intrathecal PD98059 (ERK1/2 inhibitor) partially reversed the mechanical hypersensitivity (44%, p < 0.05) observed in the remifentanil + incision group. In this group, significant increases in prodynorphin mRNA (at 2, 7, and 14 days, p < 0.01) roughly coincided with increases in dynorphin (days 2 and 14, p < 0.001) in the DRG. Remifentanil or incision (alone) also induced an up-regulation in prodynorphin mRNA expression on days 7 and 14 (p < 0.01, p < 0.05, respectively), partially correlating with dynorphin levels. On day 21, all molecular changes returned to control levels in all experimental conditions, concurring with the complete recovery of nociceptive thresholds. Surgery and/or remifentanil induce up-regulation of c-Fos and pERK in the DRG, approximately correlating with nociceptive behavior, also associated with an increased expression of prodynorphin/dynorphin. These changes support the role of the DRG in the development and maintenance of pain hypersensitivity after surgery. The findings could contribute to the development of new therapeutic agents focused on peripheral targets.

Many studies indicate that thimet oligopeptidase (EC3.4.24.15; TOP) can be implicated in the metabolism of bioactive peptides, including dynorphin 1-8, α-neoendorphin, β-neoendorphin and GnRH. Furthermore, the higher levels of this peptidase are found in neuroendocrine tissue and testis. In the present study, we have evaluated the effect of acute cocaine administration in male rats on TOP specific activity and mRNA levels in prosencephalic brain areas related with the reward circuitry; ventral striatum, hippocampus, and frontal cortex. No significant differences on TOP specific activity were detected in the hippocampus and frontal cortex of cocaine treated animals compared to control vehicle group. However, a significant increase in activity was observed in the ventral striatum of cocaine treated-rats. The increase occurred in both, TOP specific activity and TOP relative mRNA amount determined by real time RT-PCR. As TOP can be implicated in the processing of many neuropeptides, and previous studies have shown that cocaine also alters the gene expression of proenkephalin and prodynorphin in the striatum, the present findings suggest that TOP changes in the brain could play important role in the balance of neuropeptide level correlated with cocaine effects., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The dynorphin/κ-opioid receptor system has been implicated in the pathogenesis and pathophysiology of several psychiatric disorders. In the present review, we present evidence indicating a key role for this system in modulating neurotransmission in brain circuits that subserve mood, motivation, and cognitive function. We overview the pharmacology, signaling, post-translational, post-transcriptional, transcriptional, epigenetic and cis regulation of the dynorphin/κ-opioid receptor system, and critically review functional neuroanatomical, neurochemical, and pharmacological evidence, suggesting that alterations in this system may contribute to affective disorders, drug addiction, and schizophrenia. We also overview the dynorphin/κ-opioid receptor system in the genetics of psychiatric disorders and discuss implications of the reviewed material for therapeutics development.

Background: Sporadic-onset ataxia is common in a tertiary care setting but a significant percentage remains unidentified despite extensive evaluation. Rare genetic ataxias, reported only in specific populations or families, may contribute to a percentage of sporadic ataxia., Methods: Patients with adult-onset sporadic ataxia, who tested negative for common genetic ataxias (SCA1, SCA2, SCA3, SCA6, SCA7, and/or Friedreich ataxia), were evaluated using a stratified screening approach for variants in 7 rare ataxia genes., Results: We screened patients for published mutations in SYNE1 (n = 80) and TGM6 (n = 118), copy number variations in LMNB1 (n = 40) and SETX (n = 11), sequence variants in SACS (n = 39) and PDYN (n = 119), and the pentanucleotide insertion of spinocerebellar ataxia type 31 (n = 101). Overall, we identified 1 patient with a LMNB1 duplication, 1 patient with a PDYN variant, and 1 compound SACS heterozygote, including a novel variant., Conclusions: The rare genetic ataxias examined here do not significantly contribute to sporadic cerebellar ataxia in our tertiary care population., (Copyright © 2012 Movement Disorder Society.)

The purpose of this study was to examine the role of the prodynorphin gene in alcohol sensitivity, preference and vulnerability to alcohol consumption. Handling-induced convulsion (HIC) associated to alcohol, alcohol-induced loss of righting reflex (LORR), hypothermic effects in response to acute ethanol challenge, blood ethanol levels (BELs), conditioned place preference, voluntary ethanol consumption and preference, tyrosine hydroxylase (TH), dopamine transporter (DAT) and proenkephalin (PENK) gene expression, and µ-, δ- and κ-opioid agonist-stimulated [S(35) ]- guanosine 5'-triphosphate-binding autoradiography were studied in prodynorphin knockout (PDYN KO) and wild-type (WT) mice. There were no differences in HIC, LORR or the decrease in body temperature in response to acute ethanol challenge between PDYN KO and WT mice. PDYN KO mice presented higher BEL, higher ethanol-conditioned place preference and more ethanol consumption and preference in a two-bottle choice paradigm than WT mice. These findings were associated with lower TH and higher DAT gene expression in the ventral tegmental area and substantia nigra, and with lower PENK gene expression in the caudate-putamen (CPu), accumbens core (AcbC) and accumbens shell (AcbSh) in PDYN KO. The functional activity of the µ-opioid receptor was lower in the CPu, AcbC, AcbSh and cingulate cortex (Cg) of PDYN KO mice. In contrast, δ- and κ-opioid receptor-binding autoradiographies were increased in the CPu and Cg (δ), and in the CPu, AcbC and Cg (κ) of PDYN KO. These results suggest that deletion of the PDYN gene increased vulnerability for ethanol consumption by altering, at least in part, PENK, TH and DAT gene expression, and µ-, δ- and κ-opioid receptor functional activity in brain areas closely related to ethanol reinforcement., (© 2011 The Authors, Addiction Biology © 2011 Society for the Study of Addiction.)

Effector CD4(+) T lymphocytes generated in response to antigens produce endogenous opioids. Thus, in addition to their critical role in host defenses against pathogens, effector CD4(+) T lymphocytes contribute to relieving inflammatory pain. In this study, we investigated mechanisms of opioid release by antigen-experienced effector CD4(+) T cells that leave draining lymph nodes and come back into the inflammatory site. Effector antigen-primed CD4(+) T lymphocytes generated in vitro were intravenously injected into nude mice previously immunized with either cognate or irrelevant antigens in complete Freund adjuvant (CFA). CFA-induced mechanical hyperalgesia was only reduced in mice immunized with cognate antigen. Thus, antinociceptive activity of effector CD4(+) T cells requires the presence of the antigen for which they are specific within the inflammatory site. Accordingly, analgesia was inhibited by neutralizing cognate T cell receptor-mediated interaction between effector CD4(+) T lymphocytes and antigen-presenting cells at the site of inflammation. Analgesia was observed by transferring effector CD4(+) T lymphocytes with Th1 or Th2 phenotype, suggesting that antinociceptive activity is a fundamental property of effector CD4(+) T lymphocytes irrespective of their effector functions. Based on the use of agonists and antagonists selective for each of the opioid receptor subclasses, we showed that analgesia induced by T cell-derived opioids is elicited via activation of δ-type opioid receptors in the periphery. Thus, the antinociceptive activity is a fundamental property associated with the effector phase of adaptive immunity, which is driven by recognition of the cognate antigen by effector CD4(+) T lymphocytes at the inflammatory site., (Copyright © 2011 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.)

The Val(108/158)Met polymorphism of the catechol-O-methyltransferase gene (COMT) is known to interact with the function of various neuroreceptor systems in the brain. We have recently shown by post-mortem receptor autoradiography that the number of mu-opioid (MOP) receptor binding sites depends on the number of COMT Met(108/158) alleles in distinct human brain regions. We now investigated COMT Val(108/158)Met related levels of the MOP receptor protein and its endogenous ligands met-enkephalin and beta-endorphin in the human frontal cortex, thalamus and basal ganglia. Semiquantitative immunostaining and in situ hybridization were applied in a cohort of 17 human brain tissues from healthy donors. MOP receptor protein levels paralleled previous ligand binding results with a significantly higher MOP receptor expression in the mediodorsal nucleus of the thalamus of COMT Met(108/158) allele carriers. Also met-enkephalin peptide levels correlated with the genotype in this structure, with the lowest expression in COMT Met(108/158) homozygous individuals. Beta-endorphin was not detectable in the cortex, basal ganglia or thalamus, and therefore is unlikely to contribute to changes of the MOP receptor system. These results confirm the impact of the COMT Val(108/158)Met polymorphism on the MOP receptor system and may support the hypothesis of an enkephalin related turnover of MOP receptors at least in some brain structures., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

By screening extracts of venom from the Asian scorpion Buthus martensii Karsch (BmK) for their abilities to activate opioid receptors, we have identified BmK-YA, an amidated peptide containing an enkephalin-like sequence. BmK-YA is encoded by a precursor that displays a signal sequence and contains four copies of BmK-YA sequences and four of His(4)-BmK-YA, all flanked by single amino acid residues. BmK-YA and His(4)-BmK-YA are amidated and thus fulfill the characteristics expected of bioactive peptides. BmK-YA can activate mammalian opioid receptors with selectivity for the δ subtype while His(4)-BmK-YA is inactive at opioid receptors. The discovery of BmK-YA suggests that scorpion venom may represent a novel source of bioactive molecules targeting G protein-coupled receptors (GPCRs) and reveal additional insights on the evolution of the opioid precursors.

Naltrexone is not recommended during pregnancy. However, sustained-release naltrexone implant use in humans has resulted in cases of inadvertent foetal exposure. Here, we used clinically relevant dosing to examine the effects of maternally administered sustained-release naltrexone on the rat brain by examining offspring at birth and in adulthood. Maternal treatment (naltrexone or placebo implant) started before conception and ceased during gestation, birth or weaning. Morphometry was assessed in offspring at birth and adulthood. Adult offspring were evaluated for differences in locomotor behaviour (basal and morphine-induced, 10 mg/kg, s.c.) and opioid neurochemistry, propensity to self-administer morphine and cue-induced drug-seeking after abstinence. Blood analysis confirmed offspring exposure to naltrexone during gestation, birth and weaning. Naltrexone exposure increased litter size and reduced offspring birth-weight but did not alter brain morphometry. Compared to placebo, basal motor activity of naltrexone-exposed adult offspring was lower, yet they showed enhanced development of psychomotor sensitization to morphine. Developmental naltrexone exposure was associated with resistance to morphine-induced down-regulation of striatal preproenkephalin mRNA expression in adulthood. Adult offspring also exhibited greater operant responding for morphine and, in addition, cue-induced drug-seeking was enhanced. Collectively, these data show pronounced effects of developmental naltrexone exposure, some of which persist into adulthood, highlighting the need for follow up of humans that were exposed to naltrexone in utero.

The role of dynorphin/kappa opioid receptors in epilepsy and addiction are well accepted, but their function in emotional control is not yet fully understood. Data obtained from different strains of prodynorphin (Pdyn)- and kappa opioid receptor (KOP)-deficient mice do not provide a consistent picture of the functions of Dyn/KOP in anxiety, suggesting the influence of testing conditions and/or genetic background. Therefore, we investigated the behaviour and neurochemistry of male and female Pdyn KO mice on the balb/c and C57Bl/6N background. Consistent with our results obtained from male mice on the C57bl/6N background, we observed a less anxious phenotype in the elevated plus maze, open-field and light-dark test in male mice on the balb/c background. Female mice on the balb/c background also displayed less anxiety like behaviour; however these data reflect high trait anxiety and inter-individual differences. In contrast, female mice on the C57Bl/6N background displayed low trait anxiety and a paradigm-dependent reduction of anxiety. No differences were observed in the forced swim test, while balb/c Pdyn KO mice displayed prolonged immobility in the tail suspension test. In line with our previous results, we observed reduced CRH mRNA in the central amygdala in all groups of mice. In contrast, the recently observed CRH mRNA reduction in the hypothalamic paraventricular nucleus appears restricted to male, but not female mice. Our data support previous data suggesting a pronounced impact of endogenous prodynorphin-derived peptides on anxiety. Moreover, our data support the idea that the less anxious phenotype manifests only at elevated stress levels.

Chronic use of drugs of abuse profoundly alters stress-responsive system. Repeated exposure to morphine leads to accumulation of the transcription factor ΔFosB, particularly in brain areas associated with reward and stress. The persistent effects of ΔFosB on target genes may play an important role in the plasticity induced by drugs of abuse. Recent evidence suggests that stress-related hormones (e.g., glucocorticoids, GC) may induce adaptations in the brain stress system that is likely to involve alteration in gene expression and transcription factors. This study examined the role of GC in regulation of FosB/ΔFosB in both hypothalamic and extrahypothalamic brain stress systems during morphine dependence. For that, expression of FosB/ΔFosB was measured in control (sham-operated) and adrenalectomized (ADX) rats that were made opiate dependent after ten days of morphine treatment. In sham-operated rats, FosB/ΔFosB was induced after chronic morphine administration in all the brain stress areas investigated: nucleus accumbens(shell) (NAc), bed nucleus of the stria terminalis (BNST), central amygdala (CeA), hypothalamic paraventricular nucleus (PVN) and nucleus of the solitary tract noradrenergic cell group (NTS-A(2)). Adrenalectomy attenuated the increased production of FosB/ΔFosB observed after chronic morphine exposure in NAc, CeA, and NTS. Furthermore, ADX decreased expression of FosB/ΔFosB within CRH-positive neurons of the BNST, PVN and CeA. Similar results were obtained in NTS-A(2) TH-positive neurons and NAc pro-dynorphin-positive neurons. These data suggest that neuroadaptation (estimated as accumulation of FosB/ΔFosB) to opiates in brain areas associated with stress is modulated by GC, supporting the evidence of a link between brain stress hormones and addiction.

Background: Many young people experiment with cannabis, yet only a subgroup progress to dependence suggesting individual differences that could relate to factors such as genetics and behavioral traits. Dopamine receptor D2 (DRD2) and proenkephalin (PENK) genes have been implicated in animal studies with cannabis exposure. Whether polymorphisms of these genes are associated with cannabis dependence and related behavioral traits is unknown., Methodology/principal Findings: Healthy young adults (18-27 years) with cannabis dependence and without a dependence diagnosis were studied (N = 50/group) in relation to a priori-determined single nucleotide polymorphisms (SNPs) of the DRD2 and PENK genes. Negative affect, Impulsive Risk Taking and Neuroticism-Anxiety temperamental traits, positive and negative reward-learning performance and stop-signal reaction times were examined. The findings replicated the known association between the rs6277 DRD2 SNP and decisions associated with negative reinforcement outcomes. Moreover, PENK variants (rs2576573 and rs2609997) significantly related to Neuroticism and cannabis dependence. Cigarette smoking is common in cannabis users, but it was not associated to PENK SNPs as also validated in another cohort (N = 247 smokers, N = 312 non-smokers). Neuroticism mediated (15.3%-19.5%) the genetic risk to cannabis dependence and interacted with risk SNPs, resulting in a 9-fold increase risk for cannabis dependence. Molecular characterization of the postmortem human brain in a different population revealed an association between PENK SNPs and PENK mRNA expression in the central amygdala nucleus emphasizing the functional relevance of the SNPs in a brain region strongly linked to negative affect., Conclusions/significance: Overall, the findings suggest an important role for Neuroticism as an endophenotype linking PENK polymorphisms to cannabis-dependence vulnerability synergistically amplifying the apparent genetic risk.

Background and Aims: CpG islands of the promoter region of some genes are methylated in pancreatic cancer tissue and the detection of this methylation has been suggested to be useful in the diagnosis of various cancers. The aim of this study was to investigate whether the detection of methylated CpG islands in plasma can be used in the diagnosis of pancreatic cancer., Material and Methods: Plasma DNA was collected from patients with pancreatic cancer, chronic pancreatitis, and healthy controls. The methylation status of six genes, UCHL1, NPTX2, SARP2, ppENK, p16, and RASSF1A, was checked by methylation-specific PCR and was subsequently confirmed by direct sequencing after bisulfite treatment., Results: CpG island methylation was detectable in 13 of 16 patients (81.3%) with pancreatic cancer, 1 of 29 healthy controls (3.5%), and 8 of 13 patients with chronic pancreatitis (61.5%). The mean number of genes with CpG island methylation was 1.6±1.2 in pancreatic cancer, 0.04±0.19 in healthy controls, and 1.2±1.1 in chronic pancreatitis. Among six genes, p16 was more specifically methylated in pancreatic cancer compared with chronic pancreatitis (p=0.016). The methylation status was not correlated with smoking history, tumor size, or cancer stage., Conclusions: The detection of methylated genes in the plasma may have a role in differentiating between pancreatic cancers and healthy controls but not between pancreatic cancer and chronic pancreatitis.

Targeting supraspinal pain control centers by gene transfer is known to induce sustained analgesia. In this study, we evaluated the effects of injecting a Herpes Simplex Virus type 1 vector which expresses enkephalin (HSV-ENK vector) in the lateralmost part of the caudal ventrolateral medulla (VLMlat), a pain control center that exerts mainly descending inhibitory effects on pain modulation. Overexpression of enkephalin at the VLMlat reduced the number of flinches during the early and delayed phases of the formalin test and decreased c-fos expression in the spinal cord. These antinociceptive effects were detected at 2 and 10days after injection of HSV-ENK in the VLMlat and were completely reversed by local administration of naloxone. Virally driven-enkephalin was expressed from transduced neurons located in the VLMlat and, at lower extent, in the rostral ventromedial medulla. Our results show that HSV-mediated expression of enkephalin in the VLMlat induced antinociceptive effects, likely due to an enhancement of the opioidergic input to the VLMlat which accounted for descending inhibition of the nociceptive transmission at the spinal cord. This study also demonstrates the value of HSV-1 derived vectors to manipulate, in a sustained and directed manner, pain modulatory pathways in the brain, which is important in the study of supraspinal pain control circuits., (Copyright © 2011 European Federation of International Association for the Study of Pain Chapters. Published by Elsevier Ltd. All rights reserved.)