Preprodynorphin mRNA Levels Research Articles

Механизмы контроля потребления алкоголя: экспрессия генов ноцицептин/каппа-опиоидной системы в мозге крыс

Каппа-опиоидная и ноцицептиновая системы играют важную роль в модуляции функций дофаминергической системы «награды», однако их роль в механизмах формирования аддиктивного поведения до конца не ясна. Цель исследования: сравнить уровни мРНК каппа-опиоидного (OPRK1) и ноцицептинового (OPRL1) рецепторов, а также их эндогенных лигандов - препродинорфина (PDYN) и препроноцицептина (PDYN) в мозге животных с различным уровнем предпочтения алкоголя. Методы: потребление алкоголя у половозрелых крыс-самцов Wistar регистрировали с 60-го по 85-й дни жизни в модели «свободный выбор» между водой и 10% раствором этанола. Относительный уровень мРНК в структурах мозга определяли на 86-й день жизни методом ПЦР в реальном времени. Результаты: Экспрессия мРНК PDYN и PNOC в стриатуме, и OPRK1 и OPRL1 в миндалине была достоверно выше у «отвергающих», т.е. контролирующих потребление алкоголя на постоянно низком уровне животных, по сравнению с «предпочитающими», увеличившими потребление алкоголя в течение эксперимента. Заключение: более высокий уровень экспрессии генов эндогенных опиоидов PDYN и PNOC и их рецепторов в исследованных областях мозга может обусловливать меньшую гедоническую привлекательность алкоголя, играя превентивную роль и снижая риск злоупотребления алкоголем Kappa-opioidergic and nociceptinergic systems both inhibit the mesolimbic reward circuitry but their role in mechanisms of addictive behavior still is not well understood. Objective of the study was to compare expression of kappa opioid and nociceptin receptor genes (OPRK1 and OPRL1, respectively) and their endogenous ligands, preprodynorphin (PDYN) and prepronociceptin (PNOC), in the mesolimbic areas of rats with high and low voluntary alcohol consumption. Methods. Individually housed male Wistar rats were given a 25-day-long two-bottle (10% ethanol/water) free choice trial. mRNA levels were measured using the real-time polymerase chain reaction (qPCR). Results. Alcohol preferring rats progressively increased their ethanol consumption over the course of experiment while non-preferring, low-drinking animals controlled their alcohol intake near the baseline level. Alcohol non-preferring rats were identified to have significantly higher levels of PDYN and PNOC mRNA in the striatum and OPRK1 and OPRL1 mRNA in the amygdala compared to ethanol preferring animals. Conclusion. We suggested that higher levels of the “anti-reward opioid system” gene expression in mesolimbic structures may blunt the hedonic response to alcohol and thus preclude the increase in alcohol preference and uncontrolled excessive alcohol intake.

P-44 * INVOLVEMENT OF MU- AND DELTA-OPIOID RECEPTOR FUNCTION IN THE REWARDING EFFECT OF PENTAZOCINE

Most opioid receptor agonists have abuse potential, and the rewarding effects of opioids can be reduced in the presence of pain. Pentazocine has been used clinically for the treatment of pain. However, little information is available regarding whether the pentazocine-induced rewarding effects can be suppressed under pain. Therefore, the present study was performed to investigate the effects of pain on the acquisition of the rewarding effects of pentazocine, and to examine the mechanism of the rewarding effects of pentazocine using the conditioned place preference paradigm. Pentazocine produced significant rewarding effects. Even though the rewarding effects induced by pentazocine were significantly suppressed under pain induced by formalin, accompanied by increase of preprodynorphin mRNA levels in the nucleus accumbens, a high dose of pentazocine produced significant rewarding effects under pain. In the normal condition, pentazocine-induced rewarding effects were blocked by a low dose of naloxone, whereas the rewarding effects induced by high doses of pentazocine under pain were suppressed by naltrindole (a delta-opioid receptor antagonist). These findings suggest that the rewarding effects of pentazocine is mediated by activation of mu- as well as delta-opioid receptors. We also found that neural adaptations can reduce the abuse potential of pentazocine under pain.

Subthalamotomy-induced changes in dopamine receptors in parkinsonian monkeys

Subthalamotomy allows a reduction of doses of l-DOPA in dyskinetic patients while its antiparkinsonian benefits are preserved. However, the mechanisms of the potentiation of this response to medication remain to be elucidated. Hence, dopamine D1 and D2 receptors as well as the dopamine transporter were investigated using receptor binding autoradiography. D1 and D2 receptors as well as preproenkephalin and preprodynorphin mRNA levels were measured by in situ hybridization. Four dyskinetic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) parkinsonian monkeys that underwent unilateral subthalamotomy were compared to four controls, four saline-treated and four l-DOPA-treated MPTP monkeys. Dopamine, its metabolites and its transporter were extensively and similarly decreased in all parkinsonian monkeys. D1 receptor specific binding was decreased in the striatum of all MPTP monkeys. The l-DOPA-induced decrease in D1 receptor specific binding was reversed in the striatum ipsilateral to subthalamotomy. D1 receptor mRNA levels followed a similar pattern. D2 receptor specific binding and mRNA levels remained unchanged in all groups. Striatal preproenkephalin mRNA levels were overall increased in MPTP monkeys; the STN-lesioned parkinsonian group had significantly lower values than the saline-treated and l-DOPA-treated parkinsonian monkeys in the dorsolateral putamen. Striatal preprodynorphin mRNA levels remained unchanged in MPTP monkeys compared to controls whereas it increased in all monkeys treated with l-DOPA compared to controls; subthalamotomy induced a decrease in the dorsolateral putamen ipsilateral to surgery. The improved motor response to l-DOPA after subthalamotomy in the parkinsonian monkeys investigated may be associated with an increased synthesis and expression of D1 receptors ipsilateral to STN lesion of the direct pathway.

The DPP-IV inhibitor linagliptin and GLP-1 induce synergistic effects on body weight loss and appetite suppression in the diet-induced obese rat

Linagliptin is a dipeptidyl peptidase (DPP)-IV inhibitor approved for the treatment of type 2 diabetes. DPP-IV inhibitors are considered weight neutral, suggesting that elevation of endogenous incretin levels is not sufficient to promote weight loss per se. Here we evaluated the effect of linagliptin in combination with subcutaneous treatment of GLP-1(7-36) on body weight regulation in diet-induced obese (DIO) rats. Linagliptin administered perorally (1.5mg/kg, b.i.d.), but not subcutaneously (0.5mg/kg, b.i.d.), evoked a very modest body weight loss (2.2%) after 28 days of treatment. GLP-1 (0.5mg/kg, s.c.) treatment alone induced a body weight loss of 4.1%. In contrast, combined linagliptin (1.5mg/kg, p.o., or 0.5mg/kg, s.c.) and GLP-1 (0.5mg/kg) treatment evoked a marked anorectic response with both routes of linagliptin administration being equally effective on final body weight loss (7.5–8.0%). In comparison, liraglutide monotherapy (0.2mg/kg, s.c., b.i.d.) reduced body weight by 10.1%. Interestingly, the weight lowering effect of combined linagliptin and GLP-1 treatment was associated with a marked increase in chow preference, being more pronounced as compared to liraglutide treatment. In addition, linagliptin and GLP-1 co-treatment, but not liraglutide, specifically increased prepro-dynorphin mRNA levels in the caudate–putamen, an effect not obtained with administration of the compounds individually. In conclusion, co-treatment with linagliptin and GLP-1 synergistically reduces body weight in obese rats. The anti-obesity effect was caused by appetite suppression with a concomitant change in diet preference, which may potentially be associated with increased dynorphin activity in forebrain regions involved in reward anticipation and habit learning.

Involvement of μ‐ and δ‐opioid receptor function in the rewarding effect of (±)‐pentazocine

Most opioid receptor agonists have abuse potential, and the rewarding effects of opioids can be reduced in the presence of pain. While each of the enantiomers of pentazocine has a differential pharmacologic profile, (±)-pentazocine has been used clinically for the treatment of pain. However, little information is available regarding which components of pentazocine are associated with its rewarding effects, and whether the (±)-pentazocine-induced rewarding effects can be suppressed under pain. Therefore, the present study was performed to investigate the effects of pain on the acquisition of the rewarding effects of (±)-pentazocine, and to examine the mechanism of the rewarding effects of (±)-pentazocine using the conditioned place preference paradigm. (±)-Pentazocine and (-)-pentazocine, but not (+)-pentazocine, produced significant rewarding effects. Even though the rewarding effects induced by (±)-pentazocine were significantly suppressed under pain induced by formalin, accompanied by increase of preprodynorphin mRNA levels in the nucleus accumbens, a high dose of (±)-pentazocine produced significant rewarding effects under pain. In the normal condition, (±)-pentazocine-induced rewarding effects were blocked by a low dose of naloxone, whereas the rewarding effects induced by high doses of pentazocine under pain were suppressed by naltrindole (a δ-opioid receptor antagonist). Interestingly, (±)-pentazocine did not significantly affect dopamine levels in the nucleus accumbens. These findings suggest that the rewarding effects of (-)-pentazocine may contribute to the abuse potential of (±)-pentazocine through μ- as well as δ-opioid receptors, without robust activation of the mesolimbic dopaminergic system. We also found that neural adaptations can reduce the abuse potential of (±)-pentazocine under pain.

Effects of voluntary alcohol drinking on corticotropin-releasing factor and preprodynorphin mRNA levels in the central amygdala of Sardinian alcohol-preferring rats

The stress-response corticotropin-releasing factor (CRF) and dynorphin systems are critically involved in alcohol drinking and “anxiety”-related behaviors. Selectively bred Sardinian alcohol-preferring (sP) rats display high inherent “anxiety”-related behaviors, in comparison with their alcohol-nonpreferring counterpart (sNP rats). The present study was undertaken to investigate: (1) if there were genetically determined differences in basal gene expression levels of CRF, CRF-R1, preprodynorphin (ppDyn) and kappa opioid receptor (KOP-r) between sP and sNP rats; specifically, mRNA levels of the above genes were measured in the central amygdala (CeA), hypothalamus and other stress responsive and mesolimbic regions of alcohol-naive sP and sNP rats; and (2) if the above mRNA levels were altered by voluntary alcohol drinking in sP rats exposed to the standard, homecage 2-bottle “alcohol vs. water” choice regimen 24h/day for 17 days. Higher basal CRF mRNA levels were found only in CeA of alcohol-naive sP rats, compared with sNP rats; these levels were decreased after alcohol consumption. In contrast, ppDyn mRNA levels in CeA of sP rats were increased by alcohol consumption, but with no basal difference from sNP rats. Although higher basal ppDyn mRNA levels were found in hypothalamus of sP rats, compared with sNP rats, there was no alteration after alcohol drinking in sP rats. No difference for the above mRNA levels was observed in other regions, including nucleus accumbens shell or core, caudate-putamen, ventral tegmental area and medial/basolateral amygdala, between the two rat lines before or after alcohol consumption. Our results demonstrate the existence of genetically determined high basal CRF mRNA levels in CeA of sP rats. Alcohol consumption decreased CeA CRF mRNA levels with parallel increases in CeA ppDyn mRNA levels.

A functional haplotype implicated in vulnerability to develop cocaine dependence is associated with reduced PDYN expression in human brain.

Dynorphin peptides and the kappa-opioid receptor are important in the rewarding properties of cocaine, heroin, and alcohol. We tested polymorphisms of the prodynorphin gene (PDYN) for association with cocaine dependence and cocaine/alcohol codependence. We genotyped six single nucleotide polymorphisms (SNPs), located in the promoter region, exon 4 coding, and 3' untranslated region, in 106 Caucasians and 204 African Americans who were cocaine dependent, cocaine/alcohol codependent, or controls. In Caucasians, we found point-wise significant associations of 3'UTR SNPs (rs910080, rs910079, and rs2235749) with cocaine dependence and cocaine/alcohol codependence. These SNPs are in high linkage disequilibrium, comprising a haplotype block. The haplotype CCT was significantly experiment-wise associated with cocaine dependence and with combined cocaine dependence and cocaine/alcohol codependence (false discovery rate, q=0.04 and 0.03, respectively). We investigated allele-specific gene expression of PDYN, using SNP rs910079 as a reporter, in postmortem human brains from eight heterozygous subjects, using SNaPshot assay. There was significantly lower expression for C allele (rs910079), with ratios ranging from 0.48 to 0.78, indicating lower expression of the CCT haplotype of PDYN in both the caudate and nucleus accumbens. Analysis of total PDYN expression in 43 postmortem brains also showed significantly lower levels of preprodynorphin mRNA in subjects having the risk CCT haplotype. This study provides evidence that a 3'UTR PDYN haplotype, implicated in vulnerability to develop cocaine addiction and/or cocaine/alcohol codependence, is related to lower mRNA expression of the PDYN gene in human dorsal and ventral striatum.

High-frequency stimulation of the subthalamic nucleus potentiates L-DOPA-induced neurochemical changes in the striatum in a rat model of Parkinson's disease.

This study examined the cellular changes produced in the striatum by chronic L-DOPA treatment and prolonged subthalamic nucleus high-frequency stimulation (STN-HFS) applied separately, successively, or in association, in the 6-hydroxydopamine-lesioned rat model of Parkinson's disease (PD). Only animals showing severe L-DOPA-induced dyskinesias (LIDs) were included, and STN-HFS was applied for 5 d at an intensity efficient for alleviating akinesia without inducing dyskinesias. L-DOPA treatment alone induced FosB/deltaFosB immunoreactivity, exacerbated the postlesional increase in preproenkephalin, reversed the decrease in preprotachykinin, and markedly increased mRNA levels of preprodynorphin and of the glial glutamate transporter GLT1, which were respectively decreased and unaffected by the dopamine lesion. STN-HFS did not affect per se the postlesion changes in any of these markers. However, when applied in association with L-DOPA treatment, it potentiated the positive modulation exerted by L-DOPA on all of the markers examined and tended to exacerbate LIDs. After 5 d of L-DOPA withdrawal, the only persisting drug-induced responses were an elevation in preprodynorphin mRNA levels and in the number of FosB/deltaFosB-immunoreactive neurons. Selective additional increases in these two markers were measured when STN-HFS was applied subsequently to L-DOPA treatment. These data provide the first evidence that STN-HFS exacerbates the responsiveness of striatal cells to L-DOPA medication and suggest that STN-HFS acts specifically through an L-DOPA-modulated signal transduction pathway associated with LIDs in the striatum. They point to striatal cells as a primary site for the complex interactions between these two therapeutic approaches in PD and argue against a direct anti-dyskinetic action of STN-HFS.

MK-801 attenuates cocaine induction of c-fos and preprodynorphin mRNA levels in Fischer rats

This study shows that single (15 mg/kg) or binge (3×15 mg/kg) cocaine administration increases c-fos mRNA levels, but only binge cocaine administration increases preprodynorphin mRNA levels in the caudate/putamen of Fischer rats. This increase in both c-fos and preprodynorphin mRNA levels is attenuated by the noncompetitive NMDA antagonist MK-801 (0.25 mg/kg), suggesting a preferential role for NMDA receptor co-activation in cellular events leading to cocaine-induced behaviors.

Effects of cocaine on c-fos and preprodynorphin mRNA levels in intact and ovariectomized Fischer rats

Psychostimulants such as cocaine have been shown to regulate c-fos and opioid gene expression in male rats. However, little information is available on cocaine effects in female rats or how the ovarian hormones, estrogen and progesterone, modulate these effects. In this study we used quantitative solution hybridization assays to measure c-fos and preprodynorphin (PDYN) mRNA levels after cocaine administration in the caudate/putamen of intact male and female rats or ovariectomized (OVX) female rats that were pretreated with vehicle, estrogen and/or progesterone. The c-fos mRNA levels were increased in intact male and female rats after 30min or 3h of one single cocaine injection and after 14 days of single daily cocaine injections. The c-fos mRNA levels were also increased after 30min of a single cocaine injection in OVX female rats that were treated with vehicle, estrogen and/or progesterone. The PDYN mRNA levels did not change after 30min, 3h or 14 days in intact male or female rats. However, PDYN mRNA levels were increased in the caudate/putamen of OVX female rats pretreated with vehicle or a combination of estrogen and progesterone but not in OVX female rats that were pretreated with either estrogen or progesterone alone. Our data suggest hormonal regulation of cocaine effects on PDYN mRNA levels which may modulate cocaine-induced behaviors in female rats.

Suppression of serotonin hyperinnervation does not alter the dysregulatory influences of dopamine depletion on striatal neuropeptide gene expression in rodent neonates

Sixty days following neonatal dopamine depletion (>98%) with 6-hydroxydopamine, preprotachykinin and preprodynorphin mRNA levels were significantly reduced (67 and 78% of vehicle controls, respectively) in the anterior striatum as determined by in situ hybridization while preproenkephalin mRNA expression was elevated (133% of vehicle controls). Suppression of the serotonin hyperinnervation phenomenon in the dopamine-depleted rat with 5,7-dihydroxytryptamine yielded no significant alterations in reduced striatal preprotachykinin (66%) or preprodynorphin (64%) mRNA levels, while preproenkephalin mRNA expression remained significantly elevated (140%). These data suggest that striatal serotonin hyperinnervation does not contribute to the development of dysregulated striatal neuropeptide transmission in either direct or indirect striatal output pathways following neonatal dopamine depletion.

Serotonergic regulation of neuropeptide and glutamic acid decarboxylase mRNA levels in the rat striatum and globus pallidus: studies with fluoxetine and DOI

The serotonergic regulation of neuropeptide and glutamic acid decarboxylase (GAD) mRNA levels in the rat basal ganglia was investigated by determining the effects of chronic treatment with the serotonin uptake blocker fluoxetine and the serotonin 5-HT 2 agonist (±)-2,5-dimethoxy-4-iodoamphetamine hydrobromide (DOI). Fluoxetine (10 mg/kg) induced a reduction of preproenkephalin and GAD65 mRNA levels in the caudate-putamen and nucleus accumbens core and shell after 5 days of treatment. In addition, GAD65 mRNA levels were reduced in the globus pallidus. These changes appeared to be transient as they were not found after 14 days of fluoxetine treatment. DOI (7 mg/kg), administered for 9 days, induced a decrease of preprodynorphin mRNA levels in the caudate-putamen and the nucleus accumbens core and shell. No regional differentiation in the effects of fluoxetine and DOI was observed. Based on the present results, we propose that an increased 5-HT tone may reduce enkephalin and GABA mRNA levels in striatal regions and in the globus pallidus. Our results further show that preproenkephalin mRNA is not affected by chronic 5-HT 2 receptor stimulation, indicating that the fluoxetine-induced decrease in preproenkephalin mRNA levels involves other 5-HT receptors than the 5-HT 2 receptor. Preprodynorphin mRNA levels, on the other hand, were found to be reduced after chronic 5-HT 2 receptor stimulation. This observation, together with our previous finding that the 5-HT 2 antagonist ritanserin tends to increase preprodynorphin mRNA levels, suggests a 5-HT 2-mediated tonic inhibition of preprodynorphin mRNA levels.

Enhancement of dynorphin gene expression in spinal cord following experimental inflammation: stimulus specificity, behavioral parameters and opioid receptor binding

The stimulus specificity for enhancement of dynorphin gene expression in rat spinal cord was studied by combined measurements of the peptide dynorphin A 1–8 and preprodynorphin mRNA levels during peripheral inflammation induced by several agents. The density of kappa receptors, the putative receptor for dynorphin peptides, was examined using receptor binding with autoradiographic visualization. Mu and delta receptor classes were also studied. All inflammatory agents tested (carrageenan, phorbol ester, yeast and Freund's adjuvant) rapidly induced edema and thermal hyperalgesia. All agents also induced a rapid (within 8 h) elevation in dynorphin mRNA and, in comparison, a delayed (within 2 days) elevation of dynorphin A 1–8 peptide; peak peptide levels were reached at 4 days. No alteration of kappa, mu or delta receptor binding was observed at 4 h or 4 days post inflammation. The rapid development of thermal hyperalgesia and elevation of dynorphin mRNA and peptide content indicates that the involvement of dynorphin-containing neurons in nociceptive processing does not require a chronic abnormality and a dynamic picture of opioid modulation of sensory processing emerges. These data also demonstrate that activation of dynorphin biosynthesis in spinal cord is a feature common to hyperalgesia and peripheral inflammation and is not restricted to any one type of inflammatory agent. The lack of alteration in receptors suggests that the physiological effects of an increased biosynthesis are not accompanied by a concurrent down-regulation of opiate receptors.