preproenkephalin and Disease-Models--Animal

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.

Topics: Adaptation, Biological; Animals; Behavior, Addictive; Disease Models, Animal; Drug Discovery; Dynorphins; Enkephalins; Genetic Predisposition to Disease; Humans; Illicit Drugs; Narcotic Antagonists; Polymorphism, Genetic; Protein Precursors; Receptors, Opioid, kappa; Recurrence

Topics: Animals; Calcium-Binding Proteins; Disease Models, Animal; Enkephalins; Kv Channel-Interacting Proteins; Mice; Mice, Knockout; Nociceptors; Pain; Physical Stimulation; Protein Precursors; Receptors, Opioid; Repressor Proteins; Transcription, Genetic

Topics: Animals; Disease Models, Animal; Dopamine; Dystonia; Enkephalins; Parkinson Disease, Secondary; Protein Precursors

Studies showed that increased let-7b-5p microRNA during repeated electroacupuncture (EA) treatment was associated the formation of EA tolerance, which manifested as gradually decreased nociceptive threshold. Proenkephalin (PENK) is the precursor of enkephalin which is a pivot neuropeptide responsible for the decreased nociceptive threshold in EA. The aim of this study was to evaluate the relationship between let-7b-5p and PENK in EA tolerance.. The target gene of let-7b-5p microRNA was determined through the dual-luciferase reporter assay in cortical neurons. Seventy-two Sprague Dawley rats received a combination of EA and intracerebroventricular injection of microRNA (let-7b-5p agomir, antagomir or their controls). The nociceptive thresholds were assessed with radiant heat tail-flick method. PENK and let-7b-5p were measured with Western Blot and qPCR, respectively, after administration of let-7b-5p agomir, antagomir, and their controls at day 1, 4 and 7.. Let-7b-5p targeted the 3' untranslated region of Penk1. The nociceptive thresholds in Let-7b-5p agomir + EA group were decreased (p < 0.05) compared with those in Let-7b-5p antagomir + EA group at day 1 to 7. Compared with Let-7b-5p agomir + EA group, the expression level of PENK in Let-7b-5p antagomir + EA group was increased at days 1, 4, and 7 (p < 0.05) CONCLUSION: Let-7b-5p may be a new potential target for decreasing the EA tolerance effect and facilitating the application of EA in treating chronic nociception of patients.

Topics: Animals; Antagomirs; Disease Models, Animal; Down-Regulation; Electroacupuncture; Enkephalins; Female; Freund's Adjuvant; Humans; Injections, Intraventricular; MicroRNAs; Nociception; Nociceptive Pain; Pain Threshold; Protein Precursors; Rats

Persistent use of prescription opioids beyond the period of surgical recovery is a large part of a public health problem linked to the current opioid crisis in the United States. However, few studies have been conducted to examine whether morphine reward is influenced by acute pain and injury.. In a mouse model of incisional injury and minor trauma, animals underwent conditioning, extinction, and drug-primed reinstatement with morphine to examine the rewarding properties of morphine in the presence of acute incisional injury and drug-induced relapse, respectively. In addition, we sought to determine whether these behaviors were influenced by kappa opioid receptor signaling and measured expression of prodynorphin messenger RNA in the nucleus accumbens and medial prefrontal cortex after conditioning and before reinstatement with morphine and incisional injury.. In the presence of incisional injury, we observed enhancement of morphine reward with morphine-conditioned place preference but attenuated morphine-primed reinstatement to reward. This adaptation was not present in animals conditioned 12 days after incisional injury when nociceptive sensitization had resolved; however, they showed enhancement of morphine-primed reinstatement. Prodynorphin expression was greatly enhanced in the nucleus accumbens and medial prefrontal cortex of mice with incisional injury and morphine conditioning and remained elevated up to drug-primed reinstatement. These changes were not observed in mice conditioned 12 days after incisional injury. Further, kappa opioid receptor blockade with norbinaltorphimine before reinstatement reversed the attenuation induced by injury.. These findings suggest enhancement of morphine reward as a result of incisional injury but paradoxically a protective adaptation with incisional injury from drug-induced relapse resulting from kappa opioid receptor activation in the reward circuitry. Remote injury conferred no such protection and appeared to enhance reinstatement.

Topics: Acute Pain; Animals; Behavior, Animal; Conditioning, Psychological; Disease Models, Animal; Enkephalins; Extinction, Psychological; Male; Mice, Inbred C57BL; Morphine; Narcotic Antagonists; Pain Threshold; Protein Precursors; Receptors, Opioid, kappa; Reward; Signal Transduction; Wounds, Penetrating

T lymphocytes play a pivotal role in endogenous regulation of inflammatory visceral pain. The analgesic activity of T lymphocytes is dependent on their production of opioids, a property acquired on antigen activation. Accordingly, we investigated whether an active recruitment of T lymphocytes within inflamed colon mucosa via a local vaccinal strategy may counteract inflammation-induced visceral pain in mice. Mice were immunized against ovalbumin (OVA). One month after immunization, colitis was induced by adding 3% (wt/vol) dextran sulfate sodium into drinking water containing either cognate antigen OVA or control antigen bovine serum albumin for 5 days. Noncolitis OVA-primed mice were used as controls. Visceral sensitivity was then determined by colorectal distension. Oral administration of OVA but not bovine serum albumin significantly reduced dextran sulfate sodium-induced abdominal pain without increasing colitis severity in OVA-primed mice. Analgesia was dependent on local release of enkephalins by effector anti-OVA T lymphocytes infiltrating the inflamed mucosa. The experiments were reproduced with the bacillus Calmette-Guerin vaccine as antigen. Similarly, inflammatory visceral pain was dramatically alleviated in mice vaccinated against bacillus Calmette-Guerin and then locally administered with live Mycobacterium bovis. Together, these results show that the induction of a secondary adaptive immune response against vaccine antigens in inflamed mucosa may constitute a safe noninvasive strategy to relieve from visceral inflammatory pain.

Topics: Animals; CD11 Antigens; CD4-Positive T-Lymphocytes; Colitis; Disease Models, Animal; Enkephalins; Freund's Adjuvant; Immunization; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mucous Membrane; Ovalbumin; Protein Precursors; Statistics, Nonparametric; Visceral Pain

Expression of the opioid peptide genes proopiomelanocortin (Pomc), proenkephalin (Penk), and prodynorphin (Pdyn), in immune cells plays a key role in endogenous pain control. In a rat model of painful unilateral paw inflammation, we isolated cells from popliteal lymph nodes and evaluated the role of CpG island C5-methylation on the transcriptional activation of those genes. Using methylated DNA immunoprecipitation, we sorted gDNA into methylated (me) and non-me fractions and then determined the CpG island methylation status of each fraction via quantitative Real Time-PCR (qRT-PCR). In silico analysis by MethPrimer software identified one CpG island in Pdyn and three each in Pomc and Penk. No substantial changes in C5-methylation of any gene were observed. In conclusion, the CpG island methylation status does not seem to be a key regulator of opioid gene activation in immune cells during peripheral tissue inflammation.

Topics: Animals; CpG Islands; Disease Models, Animal; DNA Methylation; Enkephalins; Gene Expression Regulation; Inflammation; Male; Pain; Pro-Opiomelanocortin; Protein Precursors; Rats; Rats, Wistar; Real-Time Polymerase Chain Reaction

3,4-Dihydroxy-l-phenylalanine (l-Dopa) remains the most effective drug for treating the motor symptoms of Parkinson's disease (PD). However, its long-term use is limited due to motor complications such as wearing-off and dyskinesia. A clinical study in PD patients with motor complications has demonstrated that selegiline, a monoamine oxidase type B inhibitor, is effective in reducing off time without worsening dyskinesia, although another study has shown worsening dyskinesia. Here, using unilateral 6-hydroxydopamine-lesioned rats showing degeneration of nigrostriatal dopaminergic neurons and l-Dopa-induced motor complications, we determined the efficacy of selegiline in controlling l-Dopa-induced motor fluctuations and exacerbated dyskinesia. Repeated administration of l-Dopa/benserazide (25/6.25 mg/kg, intraperitoneally, twice daily for 22 days) progressively shortened rotational response duration (on time) and augmented peak rotation in lesioned rats. Single subcutaneous injection of selegiline (10 mg/kg) extended l-Dopa-induced shortened on time without augmenting peak rotation. Furthermore, l-Dopa/benserazide (25/6.25 mg/kg, intraperitoneally, once daily for 7 days) progressively increased abnormal involuntary movements (l-Dopa-induced dyskinesia, LID) and peak rotation. Single subcutaneous injection of selegiline (10 mg/kg) did not exacerbate LID or alter mRNA expression of prodynorphin (PDy) and activity-regulated cytoskeleton-associated protein (Arc), both mRNAs associated with LID in the lesioned striatum. Despite undetectable plasma concentrations of selegiline and its metabolites at 24 h post-administration, these on time and LID effects did not decrease, suggesting involvement of irreversible mechanisms. Altogether, these results indicate that selegiline is effective in increasing on time without worsening dyskinesia.

Topics: Animals; Antiparkinson Agents; Benserazide; Cytoskeletal Proteins; Disease Models, Animal; Drug Therapy, Combination; Dyskinesia, Drug-Induced; Enkephalins; Levodopa; Male; Movement; Nerve Tissue Proteins; Oxidopamine; Parkinsonian Disorders; Protein Precursors; Rats, Sprague-Dawley; RNA, Messenger; Selegiline; Time Factors

Animal models have suggested that prenatal ethanol exposure (PEE) alters the κ opioid receptor system. The present study investigated the brain expression of dynorphin and nociceptin/orphanin FQ related genes and assessed anxiety-like behavior in the light-dark box (LDB), shelter-seeking and risk-taking behaviors in the concentric square field (CSF) test, and ethanol-induced locomotion in the open field (OF), in infant or adolescent Wistar rats that were exposed to PEE (0.0 or 2.0 g/kg, intragastrically, gestational days 17-20). We measured brain mRNA levels of prodynorphin (PDYN), κ opioid receptors (KOR), the nociceptin/orphanin FQ opioid peptide precursor prepronociceptin (ppN/OFQ) and nociceptine/orphanin FQ receptors (NOR). Prenatal ethanol exposure upregulated PDYN and KOR mRNA levels in the ventral tegmental area (VTA) in infant and adolescent rats and KOR mRNA levels in the prefrontal cortex in infant rats. The changes in gene expression in the VTA were accompanied by a reduction of DNA methylation at the PDYN gene promoter, and by a reduction of DNA methylation at the KOR gene promoter. The PEE-induced upregulation of PDYN/KOR in the VTA was accompanied by lower NOR gene expression in the VTA, and lower PDYN gene expression in the nucleus accumbens. PEE rats exhibited hypolocomotion in the OF, greater avoidance of the white and brightly lit areas in the LDB and CSF, and greater preference for the sheltered area in the CSF test. These results suggest that PEE upregulates the dynorphin system, resulting in an anxiety-prone phenotype and triggering compensatory responses in the nociceptin/orphanin FQ system. These findings may help elucidate the mechanisms that underlie the effects of PEE and suggest that the dynorphin and nociceptin/orphanin FQ systems may be possible targets for the prevention and treatment of PEE-induced alterations.

Topics: Animals; Animals, Outbred Strains; Anxiety; Brain; Central Nervous System Depressants; Disease Models, Animal; DNA Methylation; Enkephalins; Ethanol; Female; Fetal Alcohol Spectrum Disorders; Gene Expression Regulation, Developmental; Male; Motor Activity; Nociceptin Receptor; Promoter Regions, Genetic; Protein Precursors; Random Allocation; Rats, Wistar; Receptors, Opioid; Receptors, Opioid, kappa; Risk-Taking; RNA, Messenger

The serotonergic system is a well-established modulator of l-dopa-induced dyskinesia. To date, targeting serotonin transporters or serotonin receptor subtype 1A (5-HT. The goal of the present study was to characterize Vilazodone's effects on l-dopa-induced behaviors, neurochemistry and gene expression in unilateral 6-hydroxydopamine-lesioned hemi-parkinsonian rats.. In experiments 1 and 2, l-dopa-naïve and l-dopa-primed animals were coadministered Vilazodone and l-dopa daily for 3 weeks to model subchronic use, and behavioral, neurochemical, and messenger RNA (mRNA) expression changes were measured. In experiment 3, dyskinetic behavior was assessed following 5-HT. Vilazodone significantly suppressed developing and established l-dopa-induced dyskinesia without compromising the promotor effects of l-dopa therapy. In the dopamine-depleted striatum, Vilazodone-l-dopa cotreatment increased dopamine content, suggesting a normalization of dopamine kinetics in dyskinetic brain, and reduced l-dopa-induced c-Fos and preprodynorphin mRNA overexpression, indicative of attenuated dopamine D. Our findings show Vilazodone has a serotonin-dependent effect on rodent l-dopa-induced dyskinesia and implicate the potential for repositioning Vilazodone against l-dopa-induced dyskinesia development and expression in Parkinson's disease patients. © 2018 International Parkinson and Movement Disorder Society.

Topics: Animals; Antiparkinson Agents; Disease Models, Animal; Dynorphins; Dyskinesia, Drug-Induced; Enkephalins; Gene Expression Regulation; Levodopa; Male; Oxidopamine; Parkinsonian Disorders; Piperazines; Protein Precursors; Proto-Oncogene Proteins c-fos; Pyridines; Rats; Rats, Sprague-Dawley; Receptors, Serotonin; RNA, Messenger; Selective Serotonin Reuptake Inhibitors; Serotonin; Serotonin Antagonists; Time Factors; Vilazodone Hydrochloride

Joint pain is a major clinical problem mainly associated to osteoarthritis, and characterized by articular cartilage degradation resulting in a complex chronic pain state that includes nociceptive, emotional and cognitive manifestations. Memory impairment, depressive- and anxiety-like symptoms have been reported to be associated with chronic pain, leading to a decrease of life quality. In this study, we evaluated the involvement of the endogenous dynorphin/kappa opioid receptor (KOR) system on the nociceptive, emotional, cognitive, neurochemical and epigenetic manifestations of joint pain. The murine model of monosodium iodoacetate (MIA) was used to induce joint pain in knockout mice for KOR (KOR-KO), prodynorphin (PDYN-KO) and their wild-type (WT) littermates. KOR-KO and PDYN-KO mice developed mechanical allodynia after intra-articular injection of MIA. This allodynia was significantly increased in both KOR-KO and PDYN-KO when compared to WT mice. Accordingly, both mutants showed increased microglial activation on the lumbar section of the spinal cord after MIA. The emotional responses were evaluated by measuring anxiety-like behaviour in the elevated plus maze and anhedonia as depressive-like behaviour, and cognitive alterations in the object recognition paradigm. Emotional and cognitive impairments after joint pain were differently modified in KOR-KO and PDYN-KO mice. Alterations of corticotropin-releasing factor (CRF) on the amygdala and hippocampus and down regulation of histone 3 acetylation on the amygdala suggest a possible mechanism to explain these emotional and cognitive manifestations. Our results reveal a specific involvement of the dynorphin/KOR system on joint pain manifestations that are usually associated to osteoarthritis.

Topics: Acetylation; Amygdala; Animals; Arthralgia; Chronic Pain; Cognition; Corticotropin-Releasing Hormone; Disease Models, Animal; Emotions; Enkephalins; Epigenesis, Genetic; Hippocampus; Histones; Hyperalgesia; Mice, Inbred C57BL; Mice, Knockout; Microglia; Osteoarthritis; Protein Precursors; Receptors, Opioid, kappa; Spinal Cord

The parabrachial nucleus is important for thermoregulation because it relays skin temperature information from the spinal cord to the hypothalamus. Prior work in rats localized thermosensory relay neurons to its lateral subdivision (LPB), but the genetic and neurochemical identity of these neurons remains unknown. To determine the identity of LPB thermosensory neurons, we exposed mice to a warm (36°C) or cool (4°C) ambient temperature. Each condition activated neurons in distinct LPB subregions that receive input from the spinal cord. Most c-Fos+ neurons in these LPB subregions expressed the transcription factor marker FoxP2. Consistent with prior evidence that LPB thermosensory relay neurons are glutamatergic, all FoxP2+ neurons in these subregions colocalized with green fluorescent protein (GFP) in reporter mice for Vglut2, but not for Vgat. Prodynorphin (Pdyn)-expressing neurons were identified using a GFP reporter mouse and formed a caudal subset of LPB FoxP2+ neurons, primarily in the dorsal lateral subnucleus (PBdL). Warm exposure activated many FoxP2+ neurons within PBdL. Half of the c-Fos+ neurons in PBdL were Pdyn+, and most of these project into the preoptic area. Cool exposure activated a separate FoxP2+ cluster of neurons in the far-rostral LPB, which we named the rostral-to-external lateral subnucleus (PBreL). These findings improve our understanding of LPB organization and reveal that Pdyn-IRES-Cre mice provide genetic access to warm-activated, FoxP2+ glutamatergic neurons in PBdL, many of which project to the hypothalamus.

Topics: Animals; Disease Models, Animal; Enkephalins; Fever; Forkhead Transcription Factors; Gene Expression Regulation; Genes, Reporter; Genotype; Glutamic Acid; Green Fluorescent Proteins; Hypothermia; Integrases; Internal Ribosome Entry Sites; Male; Mice, Transgenic; Neuroanatomical Tract-Tracing Techniques; Neurons; Parabrachial Nucleus; Phenotype; Protein Precursors; Proto-Oncogene Proteins c-fos; Repressor Proteins; Skin Temperature; Thermosensing; Vesicular Glutamate Transport Protein 2; Vesicular Inhibitory Amino Acid Transport Proteins

To determine whether herpes simplex virus-based vectors can efficiently transduce mouse trigeminal ganglion (TG) neurons and attenuate preexisting nerve injury-induced whisker pad mechanical hypersensitivity in a trigeminal inflammatory compression (TIC) neuropathic pain model.. Tissue transduction efficiencies of replication-conditional and replication-defective vectors to mouse whisker pads after topical administration and subcutaneous injection were assessed using quantitative real-time PCR (qPCR). Tissue tropism and transgene expression were assessed using qPCR and reverse-transcriptase qPCR following topical application of the vectors. Whisker pad mechanical sensitivities of TIC-injured mice were determined using graduated von Frey fibers before and after application of human preproenkephalin expressing replication-conditional vector (KHPE). Data were analyzed using one-way analysis of variance (ANOVA) and post hoc tests.. Transduction of target TGs was 8- to 50-fold greater after topical application than subcutaneous injection and ≥ 100-fold greater for replication-conditional than replication-defective vectors. Mean KHPE loads remained constant in TGs (4.5-9.8 × 10(4) copies/TG) over 3 weeks but were below quantifiable levels (10 copies/tissue) within 2 weeks of application in other nontarget cephalic tissues examined. Transgene expression in TGs was maximal during 2 weeks after topical application (100-200 cDNA copies/mL) and was below quantifiable levels (1 cDNA copy/mL) in all nontarget tissues. Topical KHPE administration reduced TIC-related mechanical hypersensitivity on whisker pads 4-fold (P < .05) for at least 1 week.. Topically administered KHPE produced a significant antinociceptive effect in the TIC mouse model of chronic facial neuropathic pain. This is the first report in which a gene therapeutic approach reduced trigeminal pain-related behaviors in an established pain state in mice.

Topics: Administration, Topical; Animals; Chronic Pain; Disease Models, Animal; Enkephalins; Facial Pain; Genetic Therapy; Genetic Vectors; Humans; Injections, Subcutaneous; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Protein Precursors; Simplexvirus; Touch; Transduction, Genetic; Transfection; Transgenes; Trigeminal Ganglion; Trigeminal Neuralgia

The opioidergic neuropeptides dynorphin (DYN) and enkephalin (ENK) and the D1 and D2 dopaminergic receptors (D1R, D2R) are involved in the striatal control of motor and behavioral function. In Parkinson's disease, motor disturbances such as "on-off" motor fluctuations and involuntary movements (dyskinesia) are severe complications that often arise after chronic l-dihydroxyphenylalanine (l-DOPA) treatment. Changes in the striatal expression of preproENK (PPENK), proDYN (PDYN), D1R, and D2R mRNA have been observed in parkinsonian animals treated with l-DOPA. Enhanced opioidergic transmission has been found in association with l-DOPA-induced dyskinesia, but the connection of PPENK, PDYN, D1R, and D2R mRNA expression with locomotor activity remains unclear. In this study, we measured PPENK, PDYN, D1R and D2R mRNA levels by in situ hybridization in the striatum of 6-OHDA hemi-parkinsonian rats treated with l-DOPA (PD+l-DOPA group), along with two control groups (PD+saline and naive+l-DOPA). We found different levels of expression of PPENK, PDYN, D1R and D2R mRNA across the experimental groups and correlated the changes in mRNA expression with dyskinesia and locomotor variables assessed by open field test during several phases of l-DOPA treatment. Both PDYN and PPENK mRNA levels were correlated with the severity of dyskinesia, while PPENK mRNA levels were also correlated with the frequency of contralateral rotational movements and with locomotor variables. Moreover, a strong correlation was found between D1R mRNA expression and D2R mRNA expression in the PD+l-DOPA group. These findings suggest that, in parkinsonian animals treated with l-DOPA, high levels of PPENK are a prerequisite for a locomotor sensitization to l-DOPA treatment, while PDYN overexpression is responsible only for the development of dyskinesia.

Topics: Analysis of Variance; Animals; Antiparkinson Agents; Disease Models, Animal; Dynorphins; Dyskinesia, Drug-Induced; Enkephalins; Female; Gene Expression Regulation; Levodopa; Oxidopamine; Parkinson Disease; Protein Precursors; Rats; Rats, Sprague-Dawley; Receptors, Dopamine; RNA, Messenger; Statistics as Topic; Sympatholytics; Tyrosine 3-Monooxygenase

l-3,4-Dihydroxyphenylalanine (l-DOPA) is the therapeutic gold standard in Parkinson's disease. However, most patients develop debilitating abnormal involuntary movements termed l-DOPA-induced dyskinesia (LID) as therapy-complicating side effects. The underlying mechanisms of LID pathogenesis are still not fully understood. Recent evidence suggests an involvement of striatal tyrosine hydroxylase (TH) protein-expressing neurons, as they are capable of endogenously producing l-DOPA and possibly dopamine. The aim of this study was to elucidate changes of TH transcription in the striatum and nucleus accumbens that occur under experimental conditions of LID. Mice with a unilateral 6-hydroxydopamine-induced lesion of the medial forebrain bundle were treated daily with l-DOPA for 15days to provoke dyskinesia. In situ hybridization analysis revealed a significant numerical decrease of TH mRNA-positive neurons in the striatum and nucleus accumbens of mice not exhibiting LID, whereas dyskinetic animals failed to show this reduction of TH transcription. Interestingly, similar changes were observed in intact non-deafferentiated striata, demonstrating an l-DOPA-responsive transcriptional TH regulation independently from nigrostriatal lesion severity. Consolidation with our previous study on TH protein level (Keber et al., 2015) impressively highlights that LID is associated with both a deficient downregulation of TH transcription and an excessive translation of TH protein in intrastriatal neurons. As TH protein levels in comparison to mRNA levels showed a stronger correlation with development and severity of LID, antidyskinetic treatment strategies should focus on translational and posttranslational modulations of TH as a promising target.

Topics: Animals; Antiparkinson Agents; Corpus Striatum; Disease Models, Animal; Down-Regulation; Dyskinesia, Drug-Induced; Enkephalins; Levodopa; Male; Medial Forebrain Bundle; Mice, Inbred C57BL; Neuronal Plasticity; Neurons; Oxidopamine; Protein Precursors; RNA, Messenger; Tyrosine 3-Monooxygenase

The association between morphine withdrawal and depressive-like symptoms is well documented, however, the role of dynorphin/κ opioid receptor system and the underlying neural substrates have not been fully understood. In the present study, we found that four weeks morphine abstinence after a chronic escalating morphine regimen significantly induced depressive-like behaviors in mice. Prodynorphin mRNA and protein levels were increased in the nucleus accumbens (NAc) after four weeks of morphine withdrawal. Local injection of κ opioid receptor antagonist nor-Binaltorphimine (norBNI) in the NAc significantly blocked the expression of depressive-like behaviors without influencing general locomotor activity. Thus, the present study extends previous findings by showing that prolonged morphine withdrawal-induced depressive-like behaviors are regulated by dynorphin/κ opioid receptor system, and shed light on the κ opioid receptor antagonists as potential therapeutic agents for the treatment of depressive-like behaviors induced by opiate withdrawal.

Topics: Animals; Antidepressive Agents; Depressive Disorder; Disease Models, Animal; Dose-Response Relationship, Drug; Enkephalins; Male; Mice, Inbred C57BL; Morphine; Motor Activity; Naltrexone; Narcotic Antagonists; Narcotics; Nucleus Accumbens; Protein Precursors; Receptors, Opioid, kappa; RNA, Messenger; Substance Withdrawal Syndrome

Maternal obesity during pregnancy increases the risk for offspring obesity, in part through effects on the developing brain. Previous research has shown that perinatal consumption of highly palatable foods by the mother can influence the development of offspring taste preferences and alter gene expression within the central nervous system (CNS) reward system. Opioids stimulate consumption of both fats and carbohydrates, and overconsumption of these energy dense foods increases the risk for obesity. What has remained unclear is whether this risk can be transmitted to the offspring before gestation or if it is wholly the gestational exposure that affects offspring brain development. Utilizing an embryo transfer experimental design, 2-cell embryos were obtained from obese or control dams, and transferred to obese or control gestational carriers. Expression of the mu-opioid receptor (MOR), preproenkephalin (PENK), and the dopamine transporter was evaluated in the hypothalamus and reward circuitry (ventral tegmental area, prefrontal cortex, and nucleus accumbens) in adult and late embryonic brains. Obesity before pregnancy altered expression levels of both MOR and PENK, with males relatively more affected than females. These data are the first to demonstrate that obesity at conception, in addition to during gestation, can program the brain reward system.

Topics: Animals; Brain; Disease Models, Animal; DNA Methylation; Dopamine Plasma Membrane Transport Proteins; Embryo Disposition; Enkephalins; Female; Gene Expression Regulation, Developmental; Genes, sry; Green Fluorescent Proteins; Male; Maternal-Fetal Relations; Mice; Mice, Inbred C57BL; Mice, Transgenic; Obesity; Pregnancy; Prenatal Exposure Delayed Effects; Protein Precursors; Receptors, Opioid, mu

The endogenous opioid enkephalin is known to inhibit spinal nociceptive transmission. Here we investigated activation of spinal enkephalinergic neurons by determining the proportions of c-Fos expressing (activated) spinal neurons that were enkephalinergic after different acute and chronic peripheral nociceptive stimuli. The number of c-Fos-activated neurons in the dorsal horn was increased after hind paw injection of capsaicin, formalin or complete Freund's adjuvant (CFA, 1.5 hrs - 4 days). The numbers of these neurons that were enkephalinergic increased after paraformaldehyde, and at 20 hrs, but not 1.5 hrs or 4 days post-CFA as compared to saline. In the spared nerve injury (SNI) model of neuropathic pain, c-Fos expression was increased acutely (2 hrs) and chronically (2 weeks), and a greater number of these were enkephalinergic in the nerve-injured animals acutely compared to controls (sham-SNI). Combining all acute (=2 hrs) versus chronic (≥20 hrs) treatment groups, there was a significant decrease in the percentage of activated neurons that were enkephalinergic in superficial layers, but a significant increase in the deeper layers of the dorsal horn in the chronic treatment group. It is concluded that the overall percentage of c-Fos activated neurons that contained enkephalin was not significantly different between acute and chronic pain phases. However, the shift in localization of these neurons within the spinal dorsal horn indicates a noxious stimulus directed activation pattern.

Topics: Analysis of Variance; Animals; Capsaicin; Disease Models, Animal; Enkephalins; Formaldehyde; Freund's Adjuvant; Hyperalgesia; Male; Neurons; Pain; Pain Threshold; Polymers; Protein Precursors; Proto-Oncogene Proteins c-fos; Rats; Rats, Wistar; RNA, Messenger; Spinal Cord; Time Factors

μ-opioid receptors (MORs) are necessary for the analgesic and addictive effects of opioids such as morphine, but the MOR-expressing neuronal populations that mediate the distinct opiate effects remain elusive. Here we devised a new conditional bacterial artificial chromosome rescue strategy to show, in mice, that targeted MOR expression in a subpopulation of striatal direct-pathway neurons enriched in the striosome and nucleus accumbens, in an otherwise MOR-null background, restores opiate reward and opiate-induced striatal dopamine release and partially restores motivation to self administer an opiate. However, these mice lack opiate analgesia or withdrawal. We used Cre-mediated deletion of the rescued MOR transgene to establish that expression of the MOR transgene in the striatum, rather than in extrastriatal sites, is needed for the restoration of opiate reward. Our study demonstrates that a subpopulation of striatal direct-pathway neurons is sufficient to support opiate reward-driven behaviors and provides a new intersectional genetic approach to dissecting neurocircuit-specific gene function in vivo.

Topics: Analysis of Variance; Animals; Conditioning, Operant; Corpus Striatum; Disease Models, Animal; Dopamine; Enkephalins; Exploratory Behavior; Flow Cytometry; Green Fluorescent Proteins; Mice; Mice, Transgenic; Microdialysis; Morphine; Naloxone; Narcotic Antagonists; Narcotics; Neural Pathways; Neurons; Pain; Pain Measurement; Protein Precursors; Receptors, Opioid, mu; Reward; Substance Withdrawal Syndrome

To observe the influence of different moxibustion durations on hypothalamic pro-opiomelanocortin (POMC) and prodynorphin (PDYN) mRNA expressions and plasma beta-endorphin (beta-EP) content in rheumatoid arthritis (RA) rats, to understand the mechanism of moxibustion analgesia and its dose-effect relationship.. Twelve male Wistar rats were randomly selected from 48 male Wistar rats as a normal control group. The RA model was created by raising rats in a windy (blowing with electric fan), cold (6 degrees C +/- 2 degrees C), and wet (80%-90% humidity) environment for 20 days, 12 h each day. This was followed by injection of Freund's complete adjuvant (0.15 mL) into the ankle. Then, rats were randomly divided into a model group, moxibustion group I, and moxibustion group II, with 12 rats in each group. In moxibustion groups I and II, moxibustion was given at Shenshu (BL 23) and Zusanli (ST 36) for 20 and 40 min, respectively, once daily for 15 days. Hypothalamic POMC and PDYN mRNA expression levels and plasma beta-EP content were determined.. Compared with the normal group, the pressure pain threshold decreased, while the hypothalamic POMC and PDYN mRNA expression levels and plasma beta-EP content increased in the moxibustion groups (P < 0.01). Compared with the model group, the pressure pain threshold, hypothalamic POMC and PDYN mRNA expression levels and plasma beta-EP content in the moxibustion groups increased significantly (P < 0.01). Compared the moxibustion group I, the pain threshold, hypothalamic POMC and PDYN mRNA expression levels and plasma beta-EP content in moxibustion group II significantly increased (P < 0.01).. Moxibustion has an analgesic effect and increases hypothalamic POMC and PDYN mRNA expression levels and plasma beta-EP content in RA rats.The analgesic effect in moxibustion group II is betterthan that in moxibustion group I.

Topics: Acupuncture Points; Analgesia; Animals; Arthralgia; Arthritis, Rheumatoid; beta-Endorphin; Disease Models, Animal; Enkephalins; Humans; Hypothalamus; Male; Moxibustion; Pain Threshold; Pro-Opiomelanocortin; Protein Precursors; Rats; Rats, Wistar

Expression of dynorphin, an endogenous opioid peptide, increases with age and has been associated with memory impairments in rats. In human, prodynorphin (Pdyn) gene polymorphisms might be linked to cognitive function in the elderly. Moreover, elevated dynorphin levels have been reported in postmortem samples from Alzheimer's disease patients. However, the cellular and molecular processes affected by higher dynorphin levels during aging remain unknown. Using Pdyn(-/-) mice, we observed significant changes in the function and expression of Group 1 metabotropic glutamate receptor (mGluR). Compared with age-matched wild-type (WT) littermates, we found increased expression of mGluR1α and mGluR5 in the hippocampus and cortex of old, but not young, Pdyn(-/-) mice. Increased Group 1 mGluR expression in aged Pdyn(-/-) mice was associated with enhanced mGluR-mediated long-term depression, a form of synaptic plasticity. Notably, whereas aged WT mice developed spatial and recognition memory deficits, aged Pdyn(-/-) mice performed similarly as young mice. Pharmacological treatments with 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide, a positive modulator of mGlu5 receptors, or norbinaltorphimine, an antagonist for dynorphin-targeted κ-opioid receptor, rescued memory in old WT mice. Conversely, mGlu5 receptor antagonist 2-methyl-6-(phenylethynyl)pyridine hydrochloride impaired spatial memory of old Pdyn(-/-) mice. Intact cognition in aged Pdyn(-/-) mice paralleled with increased expression of Group 1 mGluR-related genes Homer 1a and Arc. Finally, aged Pdyn(-/-) mice displayed less anxiety-related behaviors than age-matched WT mice. Together, our results suggest that elevated Pdyn expression during normal aging reduces mGluR expression and signaling, which in turn impairs cognitive functions and increases anxiety.

Topics: Aging; Animals; Anxiety; Benzamides; Benzphetamine; Central Nervous System Stimulants; Cerebral Cortex; Cognition Disorders; Disease Models, Animal; Enkephalins; Excitatory Amino Acid Antagonists; Exploratory Behavior; Gene Expression Regulation; Hippocampus; In Vitro Techniques; Long-Term Synaptic Depression; Memory Disorders; Mice; Mice, Inbred C57BL; Mice, Knockout; Protein Precursors; Pyrazoles; Pyridines; Receptors, Metabotropic Glutamate; Recognition, Psychology

Dystonia has traditionally been considered as a basal ganglia disorder, but there is growing evidence that impaired function of the cerebellum may also play a crucial part in the pathogenesis of this disorder. We now demonstrate that chronic application of kainic acid into the cerebellar vermis of rats results in a prolonged and generalized dystonic motor phenotype and provide detailed characterization of this new animal model for dystonia. c-fos expression, as a marker of neuronal activation, was increased not only in the cerebellum itself, but also in the ventro-anterior thalamus, further supporting the assumption of a disturbed neuronal network underlying the pathogenesis of this disorder. Preproenkephalin expression in the striatum was reduced, but prodynorphin expression remained unaltered, suggesting secondary changes in the indirect, but not in the direct basal ganglia pathway in our model system. Hsp70 expression was specifically increased in the Purkinje cell layer and the red nucleus. This new rat model of dystonia may be useful not only for further studies investigating the role of the cerebellum in the pathogenesis of dystonia, but also to assess compounds for their beneficial effect on dystonia in a rodent model of prolonged, generalized dystonia.

Topics: Animals; Brain; Cerebellum; Disease Models, Animal; Dystonia; Enkephalins; HSP72 Heat-Shock Proteins; Kainic Acid; Neurons; Protein Precursors; Proto-Oncogene Proteins c-fos; Rats

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.

Topics: Amygdala; Animals; Disease Models, Animal; Electroshock; Enkephalins; Genetic Predisposition to Disease; Genotype; Hippocampus; I-kappa B Proteins; Mice; Mice, Inbred Strains; NF-KappaB Inhibitor alpha; Phenotype; Protein Precursors; Species Specificity; Stress Disorders, Post-Traumatic; Transcription Factors

Adenosine A(₂A) receptor antagonism provides a promising approach to developing nondopaminergic therapy for Parkinson's disease (PD). Clinical trials of A(₂A) antagonists have targeted PD patients with L-3,4-dihydroxyphenylalanine (L-DOPA)-induced dyskinesia (LID) in an effort to improve parkinsonian symptoms. The role of adenosine in the development of LID is little known, especially regarding its actions via A₁ receptors. We aimed to examine the effects of genetic deletion and pharmacological blockade of A₁ and/or A(₂A) receptors on the development of LID, on the induction of molecular markers of LID including striatal preprodynorphin and preproenkephalin (PPE), and on the integrity of dopaminergic nigrostriatal neurons in hemiparkinsonian mice. Following a unilateral 6-hydroxydopamine lesion A₁, A(₂A) and double A₁-A(₂A) knockout (KO) and wild-type littermate mice, and mice pretreated with caffeine (an antagonist of both A₁ and A(₂A) receptors) or saline were treated daily for 18-21 days with a low dose of L-DOPA. Total abnormal involuntary movements (AIMs, a measure of LID) were significantly attenuated (p<0.05) in A₁ and A(₂A) KOs, but not in A₁-A(₂A) KOs and caffeine-pretreated mice. An elevation of PPE mRNA ipsilateral to the lesion in WT mice was reduced in all KO mice. In addition, neuronal integrity assessed by striatal dopamine content was similar in all KOs and caffeine-pretreated mice following 6-hydroxydopamine lesioning. Our findings raise the possibility that A₁ or A(₂A) receptors blockade might also confer a disease-modifying benefit of reduced risk of disabling LID, whereas the effect of their combined inactivation is less clear.

Topics: Adrenergic Agents; Animals; Antiparkinson Agents; Caffeine; Corpus Striatum; Disease Models, Animal; Dynorphins; Dyskinesia, Drug-Induced; Enkephalins; Gene Expression Regulation; Levodopa; Mice; Mice, Inbred C57BL; Mice, Knockout; Oxidopamine; Parkinson Disease; Protein Precursors; Purinergic P1 Receptor Antagonists; Receptor, Adenosine A1; Receptor, Adenosine A2A; RNA, Messenger; Statistics, Nonparametric; Time Factors

Botulinum neurotoxin serotype A (BoNT/A) acts by cleaving synaptosome-associated-protein-25 (SNAP-25) in nerve terminals to inhibit neuronal release and shows long-lasting antinociceptive action in neuropathic pain. However, its precise mechanism of action remains unclear. Our study aimed to characterize BoNT/A-induced neuroimmunological changes after chronic constriction injury (CCI) of the sciatic nerve. In the ipsilateral lumbar spinal cords of CCI-exposed rats, the mRNA of microglial marker (complement component 1q, C1q), astroglial marker (glial fibrillary acidic protein, GFAP), and prodynorphin were upregulated, as measured by reverse transcription-polymerase chain reaction (RT-PCR). No changes appeared in mRNA for proenkephalin, pronociceptin, or neuronal and inducible nitric oxide synthase (NOS1 and NOS2, respectively). In the dorsal root ganglia (DRG), an ipsilateral upregulation of prodynorphin, pronociceptin, C1q, GFAP, NOS1 and NOS2 mRNA and a downregulation of proenkephalin mRNA were observed. A single intraplantar BoNT/A (75 pg/paw) injection induced long-lasting antinociception in this model. BoNT/A diminished the injury-induced ipsilateral spinal upregulation of C1q mRNA. In the ipsilateral DRG a significant decrease of C1q-positive cell activation and of the upregulation of prodynorphin, pronociceptin and NOS1 mRNA was also observed following BoNT/A admistration. BoNT/A also diminished the injury-induced upregulation of SNAP-25 expression in both structures. We provide evidence that BoNT/A impedes injury-activated neuronal function in structures distant from the injection site, which is demonstrated by its influence on NOS1, prodynorphin and pronociceptin mRNA levels in the DRG. Moreover, the silence of microglia/macrophages after BoNT/A administration could be secondary to the inhibition of neuronal activity, but this decrease in neuroimmune interactions could be the key to the long-lasting BoNT/A effect on neuropathic pain.

Topics: Animals; Botulinum Toxins, Type A; Disease Models, Animal; Down-Regulation; Enkephalins; Ganglia, Spinal; Male; Neuralgia; Neuroimmunomodulation; Neurotoxins; Posterior Horn Cells; Protein Precursors; Rats; Rats, Wistar; Sciatic Neuropathy; Up-Regulation

Opioid peptides are involved in various pathophysiological processes, including algesia, epilepsy, and drug dependence. A strong association between L-DOPA-induced dyskinesia (LID) and elevated prodynorphin mRNA levels has been established in both patients and in animal models of Parkinson's disease, but to date the endogenous prodynorphin peptide products have not been determined. Here, matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) was used for characterization, localization, and relative quantification of striatal neuropeptides in a rat model of LID in Parkinson's disease. MALDI IMS has the unique advantage of high sensitivity and high molecular specificity, allowing comprehensive detection of multiple molecular species in a single tissue section. Indeed, several dynorphins and enkephalins could be detected in the present study, including dynorphin A(1-8), dynorphin B, α-neoendorphin, MetEnkRF, MetEnkRGL, PEnk (198-209, 219-229). IMS analysis revealed elevated levels of dynorphin B, α-neoendorphin, substance P, and PEnk (220-229) in the dorsolateral striatum of high-dyskinetic animals compared with low-dyskinetic and lesion-only control rats. Furthermore, the peak-intensities of the prodynorphin derived peptides, dynorphin B and α-neoendorphin, were strongly and positively correlated with LID severity. Interestingly, these LID associated dynorphin peptides are not those with high affinity to κ opioid receptors, but are known to bind and activate also μ- and Δ-opioid receptors. In addition, the peak intensities of a novel endogenous metabolite of α-neoendorphin lacking the N-terminal tyrosine correlated positively with dyskinesia severity. MALDI IMS of striatal sections from Pdyn knockout mice verified the identity of fully processed dynorphin peptides and the presence of endogenous des-tyrosine α-neoendorphin. Des-tyrosine dynorphins display reduced opioid receptor binding and this points to possible novel nonopioid receptor mediated changes in the striatum of dyskinetic rats. Because des-tyrosine dynorphins can only be detected by mass spectrometry, as no antibodies are available, these findings highlight the importance of MALDI IMS analysis for the study of molecular dynamics in neurological diseases.

Topics: Animals; Antiparkinson Agents; Disease Models, Animal; Dynorphins; Dyskinesia, Drug-Induced; Enkephalins; Female; Humans; Levodopa; Mice; Neostriatum; Parkinson Disease; Protein Precursors; Rats; Rats, Sprague-Dawley; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Dopamine denervation in Parkinson's disease and repeated Levodopa (L-DOPA) administration that induces dyskinesias are associated with an enhancement of basal ganglia neuropeptide transmission. Various adjunct non-dopaminergic treatments to Levodopa were shown to reduce and/or prevent dyskinesias. The aim of this study was to seek if non-dopaminergic drug treatments to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) lesioned monkeys combined with L-DOPA to prevent dyskinesia were associated with changes of striatal neuropeptides. Chronic treatment with Ro 61-8048 a kynurenine hydroxylase inhibitor, docosahexaenoic acid (DHA) a polyunsaturated fatty acid (omega-3), naltrexone an opioidergic antagonist and CI-1041 an N-methyl-D-aspartate (NMDA) glutamate receptor antagonist with L-DOPA prevented dyskinesias to various extents except naltrexone whereas all MPTP monkeys treated with L-DOPA alone developed dyskinesias. Striatal preproenkephalin (PPE), preprodynorphin (PPD) and preprotachykinin A (PPT-A) mRNA levels were measured by in situ hybridization. An increase of PPE and PPD mRNA levels was observed in anterior caudate nucleus of L-DOPA treated MPTP monkeys compared to controls and to Saline-treated MPTP monkeys whereas PPT-A mRNA levels were unchanged. Striatal PPE and PPD mRNA levels remained elevated in L-DOPA plus naltrexone-treated MPTP monkeys, while co-treatment with DHA, CI-1041 or Ro 61-8048 prevented their increase to various extents. Maximal dyskinesias scores of MPTP monkeys correlated significantly with striatal PPE and PPD mRNA levels but not with PPT-A mRNA levels. These results show that drugs displaying a wide range of pharmacological activities can modulate L-DOPA induced dyskinesias and this activity is correlated with striatal PPD and PPE mRNA levels suggesting a convergent mechanism.

Topics: Animals; Antiparkinson Agents; Benzoxazoles; Cocaine; Corpus Striatum; Disease Models, Animal; Docosahexaenoic Acids; Dopamine; Dopamine Uptake Inhibitors; Dynorphins; Dyskinesia, Drug-Induced; Enkephalins; Female; Iodine Isotopes; Levodopa; Macaca fascicularis; Naltrexone; Neuropeptides; Ovariectomy; Parkinsonian Disorders; Piperidines; Protein Precursors; RNA, Messenger; Sulfonamides; Tachykinins; Thiazoles; Time Factors

A role of neuropeptides in neuropathic pain development has been implicated; however, the neuroimmune interactions that are involved in the underlying mechanisms may be more important than previously thought. To examine a potential role of relations between glia cells and neuropeptides in neuropathic pain, we performed competitive reverse-transcription polymerase chain reaction (RT-PCR) from the dorsal lumbar spinal cord and the dorsal root ganglion (DRG) after chronic constriction injury (CCI) in the rat sciatic nerve. The RT-PCR results indicated that complement component 1, q subcomponent (C1q) mRNA expression was higher than glial fibrillary acidic protein (GFAP) in the spinal cord 3 and 7 days post-CCI, suggesting that spinal microglia and perivascular macrophages are more activated than astrocytes. In parallel, we observed a strong upregulation of prodynorphin mRNA in the spinal cord after CCI, with no changes in the expression of proenkephalin or pronociceptin. Conversely, the expression of GFAP mRNA in the DRG was higher than C1q, which suggests that the satellite cells are activated shortly after injury, followed by the macrophages and polymorphonuclear leukocytes infiltrating the DRG. In the DRG, we also observed a very strong upregulation of prodynorphin (1387%) as well as pronociceptin (122%) and a downregulation of proenkephalin (47%) mRNAs. Interestingly, preemptive and repeated i.p. injection of minocycline reversed the activation of microglia/macrophages in the spinal cord and the trafficking of peripheral immune cells into the DRG, and markedly diminished the upregulation of prodynorphin and pronociceptin in the DRG. We thus provide novel findings that inhibition of C1q-positive cells by minocycline can diminish injury-induced neuropeptide changes in the DRG. This suggests that immune cells-derived pronociceptive factors may influence opioid peptide expression. Therefore, the injury-induced activation of microglia and leukocytes and the subsequent activation of neuropeptides involved in nociception processes are potential targets for the attenuation of neuropathic pain.

Topics: Animals; Central Nervous System Agents; Disease Models, Animal; Enkephalins; Ganglia, Spinal; Lumbar Vertebrae; Male; Minocycline; Neuroimmunomodulation; Pain; Protein Precursors; Rats; Rats, Wistar; Receptors, Opioid; RNA, Messenger; Sciatic Neuropathy; Spinal Cord

Although our understanding of the molecular mechanisms controlling osteoblast differentiation and function is steadily increasing, there are still many open questions, especially regarding the regulation of bone matrix mineralization. For instance, while there is hallmark evidence for the importance of the endopeptidase Phex, whose inactivation in Hyp mice or human patients causes X-linked hypophosphatemic rickets, it is still largely unknown how Phex controls bone mineralization since a physiological substrate for its endopeptidase activity has not been identified yet. Using a genome-wide expression analysis comparing primary calvarial osteoblasts, we have identified preproenkephalin (Penk) as a gene that is selectively expressed in mineralized cultures. Since a role of enkephalin in the regulation of bone remodeling has been suggested previously and since Leu-enkephalin is known to be cleaved by Phex, we analyzed whether Penk expression in osteoblasts is physiologically relevant. Through skeletal analysis of a Penk-deficient mouse model, we found that Penk expression is dispensable for bone development and remodeling since we could not detect any defect following nondecalcified bone histology and histomorphometry compared to wild-type littermates. When Penk was deleted in Phex-deficient Hyp mice, however, we observed a significant reduction of the osteoid enrichment at 24 weeks of age, whereas their disturbance of mineral homeostasis was not affected by the additional absence of the Penk gene. Taken together, our data provide the first in vivo analysis concerning the role of Penk in osteoblasts.

Topics: Animals; Bone Demineralization, Pathologic; Calcification, Physiologic; Cell Differentiation; Cells, Cultured; Disease Models, Animal; Enkephalins; Epistasis, Genetic; Familial Hypophosphatemic Rickets; Female; Gene Deletion; Gene Expression; Genetic Diseases, X-Linked; Male; Mice; Mice, Transgenic; Osteoblasts; PHEX Phosphate Regulating Neutral Endopeptidase; Protein Precursors

The present study used the preproenkephalin knockout (ppENK) mice to test whether the endogenous enkephalins deficit could facilitate the anxiety- and depressive-like symptoms of posttraumatic stress disorder (PTSD). On Day 1, sixteen wildtype (WT) and sixteen ppENK male mice were given a 3 mA or no footshock treatment for 10 seconds in the footshock apparatus, respectively. On Days 2, 7, and 13, all mice were given situational reminders for 1 min per trial, and the freezing response was assessed. On Day 14, all mice were tested in the open field test, elevated plus maze, light/dark avoidance test, and forced swim test. Two hours after the last test, brain tissues were stained to examine c-fos expression in specific brain areas. The present results showed that the conditioned freezing response was significant for different genotypes (ppENK vs WT). The conditioned freezing effect of the ppENK mice was stronger than those of the WT mice. On Day 14, the ppENK mice showed more anxiety- and depressive-like responses than WT mice. The magnitude of Fos immunolabeling was also significantly greater in the primary motor cortex, bed nucleus of the stria terminalis-lateral division, bed nucleus of the stria terminalis-supracapsular division, paraventricular hypothalamic nucleus-lateral magnocellular part, central nucleus of the amygdala, and basolateral nucleus of the amygdala in ppENK mice compared with WT mice. In summary, animals with an endogenous deficit in enkephalins might be more sensitive to PTSD-like aversive stimuli and elicit stronger anxiety and depressive PTSD symptoms, suggesting an oversensitivity hypothesis of enkephalin deficit-induced PTSD.

Topics: Animals; Anxiety; Avoidance Learning; Behavior, Animal; Brain; Depression; Disease Models, Animal; Electroshock; Enkephalins; Male; Maze Learning; Mice; Mice, Inbred C57BL; Mice, Knockout; Models, Biological; Protein Precursors; Proto-Oncogene Proteins c-fos; Stress Disorders, Post-Traumatic; Tissue Distribution

Neuroplasticity induced by neonatal inflammation is the consequence of a combination of activity-dependent changes in neurons. We investigated neuronal sensitivity to a noxious stimulus in a rat model of neonatal hind-paw peripheral inflammation and assessed changes in pain behaviour at the physiological and molecular levels after peripheral reinflammation in adulthood.. A decrease in paw withdrawal latency (PWL) after a heat stimulus was documented in rats that received inflammatory injections in their left hind paws on postnatal day one (P1) and a reinflammation stimulus at postnatal 6-8 weeks of age, compared with normal rats. An increase in the expression of the prodynorphin (proDYN) gene was noted after reinflammation in the spinal cord ipsilateral to the afferents of the neonatally treated hind paw. The involvement of the activation of extracellular signal-regulated kinases (ERK) in peripheral inflammatory pain hypersensitivity was evidenced evident by the increase in phospho-ERK (pERK) activity after reinflammation.. Our results indicate that peripheral inflammation in neonates can permanently alter the pain processing pathway during the subsequent sensory stimulation of the region. Elucidation of the mechanism underlying the developing pain circuitry will provide new insights into the understanding of the early pain behaviours and the subsequent adaptation to pain.

Topics: Animals; Animals, Newborn; Disease Models, Animal; Enkephalins; Gene Expression Regulation; Hindlimb; Hot Temperature; Hyperalgesia; Inflammation; Physical Stimulation; Protein Precursors; Rats; Rats, Sprague-Dawley; Spinal Cord; Up-Regulation

L-DOPA-induced dyskinesia (LID) is among the motor complications that arise in Parkinson patients after a prolonged treatment with levodopa (L-DOPA). Since previous transcriptome and proteomic studies performed in the rat model of LID suggested important changes in striatal energy-related components, we hypothesize that oral creatine supplementation could prevent or attenuate the occurrence of LID. In this study, 6-hydroxydopamine-lesioned rats received a 2% creatine-supplemented diet for 1 month prior to L-DOPA therapy. During the 21 days of L-DOPA treatment, significant reductions in abnormal involuntary movements (AIMs) have been observed in the creatine-supplemented group, without any worsening of parkinsonism. In situ hybridization histochemistry and immunohistochemistry analysis of the striatum also showed a reduction in the levels of prodynorphin mRNA and FosB/DeltaFosB-immunopositive cells in creatine-supplemented diet group, an effect that was dependant on the development of AIMs. Further investigation of the bioenergetics' status of the denervated striatum revealed significant changes in the levels of creatine both after L-DOPA alone and with the supplemented diet. In conclusion, we demonstrated that combining L-DOPA therapy with a diet enriched in creatine could attenuate LID, which may represent a new way to control the motor complications associated with L-DOPA therapy.

Topics: Administration, Oral; Analysis of Variance; Animals; Creatine; Dietary Supplements; Disease Models, Animal; Dyskinesia, Drug-Induced; Energy Metabolism; Enkephalins; Female; In Vitro Techniques; Levodopa; Neostriatum; Neuroprotective Agents; Oxidopamine; Parkinsonian Disorders; Phosphocreatine; Protein Precursors; Proto-Oncogene Proteins c-fos; Rats; Rats, Sprague-Dawley; RNA, Messenger; Statistics, Nonparametric

Chronic L-3,4-dihydroxyphenylalanine (L-DOPA) treatment of Parkinson's disease (PD) leads to debilitating involuntary movements, termed L-DOPA-induced dyskinesia. Striatofugal medium spiny neurons (MSN) lose their dendritic spines and cortico-striatal glutamatergic synapses in PD and in experimental models of DA depletion. This loss of connectivity is triggered by a dysregulation of intraspine Cav1.3 L-type Ca2+ channels. Here we address the possible implication of DA denervation-induced spine pruning in the development of L-DOPA-induced dyskinesia.. The L-type Ca2+ antagonist, isradipine was subcutaneously delivered to rats at the doses of .05, .1, or .2 mg/kg/day, for 4 weeks, starting the day after a unilateral nigrostriatal 6-hydroxydopamine (6-OHDA) lesion. Fourteen days later, L-DOPA treatment was initiated.. Isradipine-treated animals displayed a dose-dependent reduction in L-DOPA-induced rotational behavior and abnormal involuntary movements. Dendritic spine counting at electron microscopy level showed that isradipine (.2 mg/kg/day) prevented the 6-OHDA-induced spine loss and normalized preproenkephalin-A messenger RNA expression. Involuntary movements were not reduced when isradipine treatment was started concomitantly with L-DOPA.. These results indicate that isradipine, at a therapeutically relevant dose, might represent a treatment option for preventing L-DOPA-induced dyskinesia in PD.

Topics: Animals; Calcium Channel Blockers; Calcium Channels, L-Type; Cerebrum; Dendritic Spines; Disease Models, Animal; Dose-Response Relationship, Drug; Dyskinesia, Drug-Induced; Enkephalins; Isradipine; Levodopa; Male; Motor Activity; Nimodipine; Oxidopamine; Protein Precursors; Rats; Rats, Wistar; RNA, Messenger; Sympatholytics

A higher prevalence and incidence of Parkinson's disease is observed in men, and beneficial motor effects of estrogens are observed in parkinsonian women. In rodents, an effect of estradiol on dopamine systems is documented, whereas much less is known in monkeys. Enkephalin was shown to exert a compensatory modulatory effect on the denervated dopamine nigrostriatal pathway in monkeys and in humans. Moreover in rodents, striatal preproenkephalin mRNA is increased by estrogen treatment. Hence, we investigated the responsiveness of striatal dopamine to estradiol in long-term ovariectomized monkeys bearing a unilateral lesion with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to mimic parkinsonian postmenopausal women. Seven ovariectomized female monkeys received a unilateral MPTP lesion; 4 years after ovariectomy, three received 1-month treatment with 17beta-estradiol and the others received vehicle. The lesioned striata showed extensive denervation in all monkeys as measured with dopamine and metabolite concentrations assayed by high-performance liquid chromatography and by autoradiography of the dopamine transporter. The lesioned and intact striata of estradiol-treated monkeys had increased 3-methoxytyramine, and lesioned putamen increased dopamine concentrations compared with vehicle-treated monkeys. Estradiol treatment increased the dopamine transporter in subregions of the intact caudate and putamen compared with the intact striata of vehicle-treated monkeys, but not in the lesioned striata. Preproenkephalin mRNA levels measured by in situ hybridization were increased in the lesioned striata of vehicle treated monkeys and were not further enhanced in estradiol-treated monkeys. These results show that long after ovariectomy, modeling postmenopausal hormonal conditions, brain dopamine metabolism, and transporter are still responsive to estradiol.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Animals; Autoradiography; Biogenic Amines; Chromatography, High Pressure Liquid; Corpus Striatum; Disease Models, Animal; Dopamine; Dopamine Plasma Membrane Transport Proteins; Enkephalins; Estradiol; Estrogens; Female; In Situ Hybridization; Iodine Radioisotopes; Macaca fascicularis; Ovariectomy; Parkinsonian Disorders; Protein Precursors; RNA, Messenger

Development of L-DOPA-induced dyskinesia (LID) remains a major problem in the long-term treatment of Parkinson's disease (PD). Sensitization to L-DOPA correlates with ectopic expression of D3 dopamine receptors in the striatum, implicating D3 receptors in development of LID. We demonstrate that the selective D3 antagonist S33084 abolishes development of LID over 30 days in MPTP-lesioned marmosets without effecting the anti-parkinsonian actions of L-DOPA. Furthermore, following a 14 day washout, when challenged with L-DOPA in the absence of S33084, these animals continued to exhibit reduced LID. In the 6-OHDA-lesioned rat, S33084 similarly attenuated development of behavioural sensitization to L-DOPA. Additionally, L-DOPA-induced elevations in striatal pre-proenkephalin-A (PPE-A) (but not PPE-B, phospho[Thr(34)]DARPP-32, D1, and D2 receptor mRNA or D3 receptor levels) were reduced in S33084 treated animals. Our data suggest a role for D3 receptors in the development of LID and suggest that initiating L-DOPA treatment with a D3 antagonist may reduce the development of LID in PD.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Animals; Antiparkinson Agents; Benzopyrans; Callithrix; Corpus Striatum; Disease Models, Animal; Dopamine and cAMP-Regulated Phosphoprotein 32; Dopamine Antagonists; Dyskinesia, Drug-Induced; Enkephalins; Female; Levodopa; Male; Oxidopamine; Parkinsonian Disorders; Protein Precursors; Pyrroles; Rats; Rats, Sprague-Dawley; Receptors, Dopamine D1; Receptors, Dopamine D2; Receptors, Dopamine D3; RNA, Messenger

The principal causative pathology of Parkinson disease is the progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta projecting to the striatum in the brain. The information regarding the expression of neuropeptides in parkinsonism is very limited. Here we have elucidated striatal neuropeptide mechanisms in experimental parkinsonism using the unilateral 6-hydroxydopamine model to degenerate dopamine neurons. A thoroughly controlled sample preparation technique together with a peptidomics approach and targeted neuropeptide sequence collections enabled sensitive detection, identification, and relative quantitation of a great number of endogenous neuropeptides. Previously not recognized alterations in neuropeptide levels were identified in the unilateral lesioned mice with or without subchronic 3,4-dihydroxy-L-phenylalanine administration, the conventional treatment of Parkinson disease. Several of these peptides originated from the same precursor such as secretogranin-1, somatostatin, prodynorphin, and cholecystokinin. Disease-related biotransformation of precursors into individual peptides was observed in the experimental model of Parkinson disease. Several previously unreported potentially biologically active peptides were also identified from the striatal samples. This study provides further evidence that neuropeptides take part in mediating the central nervous system failure associated with Parkinson disease.

Topics: Amino Acid Sequence; Animals; Cholecystokinin; Chromogranin B; Disease Models, Animal; Enkephalins; Mice; Molecular Sequence Data; Neostriatum; Oxidopamine; Parkinson Disease; Peptides; Postmortem Changes; Protein Precursors; Reproducibility of Results; Somatostatin; Tissue Extracts; Tyrosine 3-Monooxygenase

Critically ill neonates experience multiple stressors during hospitalization. Opioids are commonly prescribed to ameliorate their pain and stress. However, the enduring effects of stress and opioids are not understood. The kappa opioid system is important in the mediation of stress in adults, but little is known about its function in neonates.. To characterize kappa opioid receptor (KOR) distribution in the neonatal mouse brain and test whether neonatal exposure to morphine, stress, or both, change KOR signaling.. Five groups of wild-type C57BL/6 or prodynorphin (Pdyn) knockout mice were tested: (1) untreated control (dam-reared, no handling), (2) saline-injected control, (3) morphine-injected control, (4) stressed with saline injections and (5) stressed with morphine injections. Mice were treated from postnatal day 5 to postnatal day 9, after which their brains were immunolabeled with a phospho-specific KOR antibody (KOR-P), glial fibrillary acidic protein or glutamic acid decarboxylase.. There were no effects of saline or morphine injection on KOR-P immunoreactivity. Neonatal stress increased KOR-P labeling in wild-type brains (p < 0.05), but not in Pdyn(-/-) animals. Mice exposed to stress and morphine showed region-specific increases in KOR-P immunoreactivity from 38 to 500% (p < 0.05 to p < 0.001), with marked gliosis. In stressed morphine-treated Pdyn(-/-) animals, KOR-P immunoreactivity was absent, but gliosis increased compared to wild-type animals.. Neonatal stress increases KOR activation via the dynorphin system. Neonatal stress plus morphine treatment further increased this response and also resulted in hippocampal gliosis. Enhanced gliosis noted in Pdyn(-/-) animals suggests that the endogenous dynorphin may play a role in downregulating this inflammatory response.

Topics: Analgesics, Opioid; Animals; Animals, Newborn; Brain; Disease Models, Animal; Enkephalins; Fluorescent Antibody Technique, Direct; Gene Silencing; Glial Fibrillary Acidic Protein; Gliosis; Glutamate Decarboxylase; Mice; Mice, Inbred C57BL; Mice, Knockout; Morphine; Nerve Tissue Proteins; Oxidative Stress; Protein Precursors; Receptors, Opioid, kappa; Signal Transduction

Dynorphins, endogenous neuropeptides found in striatonigral neurons, have been observed to exhibit dopamine-inhibitory actions and under some circumstances possess intrinsic neurotoxic activity. To test the hypothesis that dynorphin suppression mitigates effects of aging on the striatal dopaminergic system, HPLC quantitation of dopamine and related amines was performed on striatal homogenates of wild-type (WT) mice and mice lacking the prodynorphin (Pdyn) gene at varying ages. Pdyn knockout (KO) mice at 10 and 20 months show significant elevations in striatal dopamine compared to 3-month mice. Differences in tyrosine hydroxylase (TH) immunoreactivity could not account for these findings, but phosphorylation of TH at Ser40, but not Ser31, was enhanced in aged Pdyn KO mice. Systemic administration of MPTP produced significant dopamine depletion in an age-dependent manner, but Pdyn deletion conferred no protection against MPTP-induced dopamine loss, arguing against a mechanism by which Pdyn deletion enhances dopaminergic neuron survival. The above findings demonstrate an age-dependent inhibitory effect of dynorphins on striatal dopamine synthesis via modulation of TH activity.

Topics: Aging; Amino Acid Sequence; Animals; Corpus Striatum; Disease Models, Animal; Dopamine; Enkephalins; Female; Immunohistochemistry; Male; Mice; Mice, Knockout; Neural Pathways; Parkinsonian Disorders; Phosphorylation; Protein Precursors; Serine; Substantia Nigra; Tyrosine 3-Monooxygenase; Up-Regulation

Interstitial cystitis/painful bladder syndrome (IC/PBS) is a major challenge to treat. We studied the effect of targeted and localized expression of enkephalin in afferent nerves that innervate the bladder by gene transfer using replication-defective herpes simplex virus (HSV) vectors in a rat model of bladder hyperactivity and pain. Replication-deficient HSV vectors encoding preproenkephalin, which is a precursor for Met- and Leu-enkephalin, or control vector encoding the lacZ reporter gene, were injected into the bladder wall of female rats. After viral vector injection, quantitative polymerase chain reaction showed high preproenkephalin transgene levels in bladder and dorsal root ganglia innervating the bladder in enkephalin vector-treated animals. Functionally, enkephalin vector-treated animals showed reductions in bladder hyperactivity and nociceptive behavior induced by intravesical application of capsaicin; however, vector-mediated expression of enkephalin did not alter normal voiding. This antinociceptive effect of enkephalin gene therapy was antagonized by naloxone hydrochloride administration. Together, our results with HSV vectors encoding preproenkephalin demonstrated physiological improvement in visceral pain induced by bladder irritation. Thus, gene therapy may represent a potentially useful treatment modality for bladder hypersensitive disorders such as IC/PBS.

Topics: Animals; Cystitis, Interstitial; Disease Models, Animal; Enkephalins; Female; Ganglia, Spinal; Genetic Therapy; Genetic Vectors; Humans; Neurons, Afferent; Protein Precursors; Rats; Rats, Sprague-Dawley; Simplexvirus; Treatment Outcome; Urinary Bladder

Prior activation of the kappa opioid system by repeated stress or agonist administration has been previously shown to potentiate the rewarding properties of subsequently administered cocaine. In the present study, intermittent and uncontrollable footshock, a single session of forced swim, or acute administration of the kappa agonist U50,488 (5 mg/kg) were found to reinstate place preference in mice previously conditioned with cocaine (15 mg/kg) and subsequently extinguished by repeated training sessions without drug.. Stress-induced reinstatement did not occur for mice pretreated with the kappa opioid receptor antagonist norbinaltorphimine (10 mg/kg) and did not occur in mice lacking either kappa opioid receptors (KOR -/-) or prodynorphin (Dyn -/-). In contrast, the initial cocaine conditioning and extinction rates were not significantly affected by disruption of the kappa opioid system. Cocaine-injection also reinstated conditioned place preference in extinguished mice; however, cocaine-primed reinstatement was not blocked by kappa opioid system disruption.. The results suggest that stress-induced drug craving in mice may require activation of the dynorphin/kappa opioid system.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Behavior, Animal; Cocaine-Related Disorders; Conditioning, Operant; Disease Models, Animal; Enkephalins; Extinction, Psychological; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Protein Precursors; Receptors, Opioid, kappa; Stress, Psychological; Swimming

OFF-L-dopa dyskinesias have been a surprising side-effect of intrastriatal foetal ventral mesencephalic transplantation in patients with Parkinson's disease. It has been proposed that excessive and unregulated dopaminergic stimulation of host post-synaptic striatal neurons by the grafts could be responsible for these dyskinesias. To address this issue we transplanted foetal dopaminergic neurons from mice lacking the dopamine transporter (DATKO) or from wild-type mice, into a rat model of Parkinson's disease and L-dopa-induced dyskinesias. Both wild-type and DATKO grafts reinnervated the host striatum to a similar extent, but DATKO grafts produced a greater and more diffuse increase in extra-cellular striatal dopamine levels. Interestingly, grafts containing wild-type dopaminergic neurons improved parkinsonian signs to a similar extent as DATKO grafts, but provided a more complete reduction of L-dopa induced dyskinesias. Neither DATKO nor wild-type grafts induced OFF-L-dopa dyskinesias. Behavioural and receptor autoradiography analyses demonstrated that DATKO grafts induced a greater normalization of striatal dopaminergic receptor supersensitivity than wild-type grafts. Both graft types induced a similar downregulation and normalization of PEnk and fosb/Deltafosb in striatal neurons. In summary, DATKO grafts causing high and diffuse extra-cellular dompamine levels do not per se alter graft-induced recovery or produce OFF-L-dopa dyskinesias. Wild-type dopaminergic neurons appear to be the most effective neuronal type to restore function and reduce L-dopa-induced dyskinesias.

Topics: Animals; Antiparkinson Agents; Brain Tissue Transplantation; Corpus Striatum; Disease Models, Animal; Dopamine; Dopamine Plasma Membrane Transport Proteins; Dyskinesia, Drug-Induced; Enkephalins; Female; Fetal Tissue Transplantation; Gene Expression Regulation; Graft Survival; Levodopa; Mesencephalon; Microdialysis; Neurons; Parkinson Disease; Protein Precursors; Rats; Rats, Sprague-Dawley; Receptors, Dopamine D1; Receptors, Dopamine D2; Receptors, N-Methyl-D-Aspartate; RNA, Messenger

The enkephalin signaling pathway regulates various neural functions and can be altered by neurodegenerative disorders. In Alzheimer's disease (AD), elevated enkephalin levels may reflect compensatory processes or contribute to cognitive impairments. To differentiate between these possibilities, we studied transgenic mice that express human amyloid precursor protein (hAPP) and amyloid-beta (Abeta) peptides in neurons and exhibit key aspects of AD. Met-enkephalin levels in neuronal projections from the entorhinal cortex and dentate gyrus (brain regions important for memory that are affected in early stages of AD) were increased in hAPP mice, as were preproenkephalin mRNA levels. Genetic manipulations that exacerbate or prevent excitotoxicity also exacerbated or prevented the enkephalin alterations. In human AD brains, enkephalin levels in the dentate gyrus were also increased. In hAPP mice, enkephalin elevations correlated with the extent of Abeta-dependent neuronal and behavioral alterations, and memory deficits were reduced by irreversible blockade of mu-opioid receptors with the antagonist beta-funaltrexamine. We conclude that enkephalin elevations may contribute to cognitive impairments in hAPP mice and possibly in humans with AD. The therapeutic potential of reducing enkephalin production or signaling merits further exploration.

Topics: Aged; Aged, 80 and over; Alzheimer Disease; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Animals; Behavior, Animal; Dentate Gyrus; Disease Models, Animal; Enkephalin, Methionine; Enkephalins; Entorhinal Cortex; Female; Humans; Male; Mice; Mice, Transgenic; Neural Pathways; Neurons; Protein Precursors; RNA, Messenger; Up-Regulation

The Fischer 344 (F344) rat strain differs from the Lewis strain in the response to neuropathic pain. Recently, we found that F344 rats totally recover from mechanical allodynia induced by chronic constriction injury (CCI) of the sciatic nerve 28 days after surgery whereas Lewis rats are initiating their recovery at this time point. Thus, the use of this neuropathic pain model in these different rat strains constitutes a good strategy to identify possible target genes involved in the development of neuropathic pain. Since differences between Lewis and F344 rats in their response to pain stimuli in acute pain models have been related to differences in the endogenous opioid and noradrenergic systems, we aimed to determine the levels of expression of key genes of both systems in the spinal cord and dorsal root ganglia (DRG) of both strains 28 days after CCI surgery. Real time RT-PCR revealed minimal changes in gene expression in the spinal cord after CCI despite the strain considered, but marked changes in DRG were observed. A significant upregulation of prodynorphin gene expression occurred only in injured DRG of F344 rats, the most resistant strain to neuropathic pain. In addition, we found a significant downregulation of tyrosine hydroxylase and proenkephalin gene expression levels in both strains whereas delta-opioid receptor was found to be significantly downregulated only in injured DRG of Lewis rats although the same trend was observed in F344 rats. The data strongly suggest that dynorphins could be involved in strain differences concerning CCI resistance.

Topics: Animals; Chronic Disease; Denervation; Disease Models, Animal; Down-Regulation; Dynorphins; Enkephalins; Ganglia, Spinal; Gene Expression Regulation; Hyperalgesia; Ligation; Male; Neurons, Afferent; Norepinephrine; Peripheral Nerve Injuries; Peripheral Nerves; Peripheral Nervous System Diseases; Protein Precursors; Rats; Rats, Inbred F344; Rats, Inbred Lew; Receptors, Opioid, delta; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Species Specificity; Spinal Cord; Tyrosine 3-Monooxygenase

Clinical, preclinical, and pharmacological studies have suggested that decreased enkephalin tone is associated with depression-like symptoms and increase in enkephalin signaling could have a therapeutic value in the treatment of depression. In this study we demonstrate that, surprisingly, animals lacking enkephalin (preproenkephalin, Penk1(-/-)) showed no depression-related phenotype in the Porsolt forced swimming or tail suspension tests. Moreover, Penk1(-/-) mice had a lower frequency of depression-related behavior in stress-induced hypoactivity and ultrasonic vocalization models of depression, similar to animals treated with antidepressant drugs, although this effect was specific to the genetic background. In addition, there was no significant difference in the efficacy of antidepressant reference compounds in wild-type and knockout animals. Nialamide and amitriptyline were even slightly more effective in animals with genetic deletion of Penk1, whereas the minimal effective dose of imipramine and fluoxetine was the same in the two genotypes. The dual peptidase inhibitor RB-101 was also effective in Penk1(-/-) as well as in Penk1(-/-)/Pdyn(-/-) animals, although its efficacy was somewhat reduced compared with wild-type animals. This result was also surprising because the antidepressant effects of RB-101 were thought to be due to the elevation of enkephalin levels.

Topics: Animals; Antidepressive Agents; Behavior, Animal; Depression; Disease Models, Animal; Disulfides; Dose-Response Relationship, Drug; Enkephalins; Enzyme Inhibitors; Hindlimb Suspension; Immobility Response, Tonic; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Mice, Knockout; Motor Activity; Nialamide; Phenotype; Phenylalanine; Protein Precursors; Swimming; Vocalization, Animal

L-DOPA-induced dyskinesia (LID) is one of the main limitations of long term L-DOPA use in Parkinson's disease (PD) patients. We show that chronic L-DOPA treatment induces novel dyskinetic behaviors in aphakia mouse with selective nigrostriatal deficit mimicking PD. The stereotypical abnormal involuntary movements were induced by dopamine receptor agonists and attenuated by antidyskinetic agents. The development of LID was accompanied by preprodynorphin and preproenkephalin expression changes in the denervated dorsal striatum. Increased FosB-expression was also noted in the dorsal striatum. In addition, FosB expression was noted in the pedunculopontine nucleus and the zona incerta, structures previously not examined in the setting of LID. The aphakia mouse is a novel genetic model with behavioral and biochemical characteristics consistent with those of PD dyskinesia and provides a more consistent, convenient, and physiologic model than toxic lesion models to study the mechanism of LID and to test therapeutic approaches for LID.

Topics: Afferent Pathways; Animals; Antiparkinson Agents; Aphakia; Corpus Striatum; Disease Models, Animal; Dynorphins; Dyskinesia, Drug-Induced; Enkephalins; Levodopa; Mice; Mice, Inbred C57BL; Mice, Neurologic Mutants; Parkinsonian Disorders; Protein Precursors; Proto-Oncogene Proteins c-fos; Substantia Nigra

Psalmotoxin 1, a peptide extracted from the South American tarantula Psalmopoeus cambridgei, has very potent analgesic properties against thermal, mechanical, chemical, inflammatory and neuropathic pain in rodents. It exerts its action by blocking acid-sensing ion channel 1a, and this blockade results in an activation of the endogenous enkephalin pathway. The analgesic properties of the peptide are suppressed by antagonists of the mu and delta-opioid receptors and are lost in Penk1-/- mice.

Topics: Acid Sensing Ion Channels; Analgesics; Animals; Area Under Curve; Behavior, Animal; Disease Models, Animal; Dose-Response Relationship, Drug; Enkephalins; Membrane Proteins; Mice; Mice, Knockout; Morphine; Naloxone; Naltrexone; Narcotic Antagonists; Nerve Tissue Proteins; Neurons; Pain; Pain Measurement; Peptides; Protein Precursors; Reaction Time; Sodium Channels; Spider Venoms; Spinal Cord; Time Factors

The dopamine precursor 3,4-dihydroxyphenyl-L-alanine (L-DOPA) is currently the most efficacious noninvasive therapy for Parkinson's disease. A major complication of this therapy, however, is the appearance of the abnormal involuntary movements known as dyskinesias. We have developed a model of L-DOPA-induced dyskinesias in mice that reproduces the main clinical features of dyskinesia in humans.. Dyskinetic symptoms were triggered by repetitive administration of a constant dose of L-DOPA (25 mg/kg, twice a day, for 25 days) in unilaterally 6-hydroxydopamine (6-OHDA) lesioned mice. Mice were examined for behavior, expression of FosB, neuropeptides, and externally regulated kinase (ERK) phosphorylation.. Dyskinetic symptoms appear toward the end of the first week of treatment and are associated with L-DOPA-induced changes in DeltaFosB and prodynorphin expression. L-DOPA also induces activation of ERK1/2 in the dopamine-depleted striatum. Interestingly, elevated FosB/DeltaFosB expression occurs exclusively within completely lesioned regions of the striatum, displaying an inverse correlation with remaining dopaminergic terminals. Following acute L-DOPA treatment, FosB expression occurs in direct striatal output neurons, whereas chronic L-DOPA also induces FosB expression in nitric oxide synthase-positive striatal interneurons.. This model provides a system in which genetic manipulation of individual genes can be used to elucidate the molecular mechanisms responsible for the development and expression of dyskinesia.

Topics: Animals; Antiparkinson Agents; Behavior, Animal; Blotting, Western; Corpus Striatum; Disease Models, Animal; Dopamine; Drug Administration Schedule; Dyskinesia, Drug-Induced; Dyskinesias; Enkephalins; Extracellular Signal-Regulated MAP Kinases; Gene Expression; Immunohistochemistry; In Situ Hybridization; Levodopa; Locomotion; Male; Mice; Mice, Inbred C57BL; Motor Activity; Oxidopamine; Parkinsonian Disorders; Phosphorylation; Protein Precursors; Proto-Oncogene Proteins c-fos; RNA, Messenger; Statistics, Nonparametric; Time Factors

The aim of this study was to determine whether inhibition of the extracellular-regulated kinase signaling pathway decreases acute amphetamine-induced behavioral activity and neuropeptide gene expression in the rat striatum. Western blotting revealed that extracellular-regulated kinase1/2 phosphorylation was highly induced in the rat striatum 15 min after an acute amphetamine (2.5 mg/kg, i.p.) injection without altering the total amount of extracellular-regulated kinase protein. In a separate experiment, the systemic injection of SL327, a selective inhibitor of extracellular regulated kinase kinase that crosses the blood-brain barrier, 1 h prior to amphetamine administration decreased amphetamine-induced vertical and horizontal activity. Quantitative in situ hybridization histochemistry showed that SL327 abolished the high levels of preproenkephalin and preprodynorphin mRNA induced by amphetamine in the striatum with no alteration of their basal levels. In another set of experiments, the hyperlocomotor activity induced by amphetamine was reduced by pretreatment with intra-striatal infusion of U0126. U0126 also blocked the amphetamine-induced increases in phospho-extracellular-regulated kinase and preproenkephalin and preprodynorphin gene expression in the striatum. These data indicate that activation of the extracellular-regulated kinase cascade contributes to the behavioral effects and changes in striatal neuropeptide gene expression induced by acute amphetamine.

Topics: Aminoacetonitrile; Amphetamine; Amphetamine-Related Disorders; Animals; Butadienes; Corpus Striatum; Disease Models, Animal; Dynorphins; Enkephalins; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation; Male; MAP Kinase Signaling System; Motor Activity; Neuropeptides; Nitriles; Phosphorylation; Protein Precursors; Rats; Rats, Sprague-Dawley; RNA, Messenger; Up-Regulation

In the present study, we first observed up-regulation in preproenkephalin (PPE)-like immunoreactivity (-LIR), a precursor of Met- and Leu-enkephalin, in the rat ipsilateral medial vestibular nucleus (ipsi-MVN) after unilateral labyrinthectomy (UL). By means of double-staining immunohistochemistry with PPE and Fos, a putative regulator of PPE gene expression, we revealed that some of these PPE-LIR neurons were also Fos immunopositive. The time course of decay of these double-stained neurons was quite parallel to that of UL-induced behavioral deficits. This suggests that these double-labeled neurons could have something to do with development of vestibular compensation. We next examined correlation between Fos and PPE expression in the ipsi-MVN by means of a 15-min pre-UL application of antisense oligonucleotide probes against c-fos mRNA into the ipsi-MVN. Gel shift assay and Western blotting revealed that elimination of Fos expression significantly reduced both AP-1 DNA binding activity and PPE expression in the ipsi-MVN after UL. C-fos antisense study also revealed that depression of Fos-PPE signaling in the ipsi-MVN caused significantly more severe behavioral deficits during vestibular compensation. Furthermore, studies with PPE antisense and naloxone, an opioid receptor antagonist, demonstrated that specific depression of enkephalinergic effects in the ipsi-MVN significantly delayed vestibular compensation. All these findings suggest that, immediately after UL, Fos induced in some of the ipsi-MVN neurons could regulate consequent PPE expression via the AP-1 activation and facilitate the restoration of balance between bilateral MVN activities via the opioid receptor activation, resulting in progress of vestibular compensation.

Topics: Adaptation, Physiological; Animals; Denervation; Disease Models, Animal; Enkephalins; Functional Laterality; Gene Expression Regulation; Immunohistochemistry; Male; Oligonucleotides, Antisense; Postural Balance; Protein Binding; Protein Precursors; Proto-Oncogene Proteins c-fos; Rats; Rats, Wistar; Receptors, Opioid; Recovery of Function; RNA, Messenger; Transcription Factor AP-1; Up-Regulation; Vestibular Diseases; Vestibular Nuclei

Transcriptional dysregulation is a central pathogenic mechanism in Huntington's disease (HD); HD and transgenic mouse models of HD demonstrate down-regulation of specific genes at the level of mRNA expression. Furthermore, neuronal intranuclear inclusions (NIIs) have been identified in the brains of R6/2 mice and HD patients. One possibility is that NIIs contribute to transcriptional dysregulation by sequestering transcription factors. We therefore assessed the relationship between NIIs and transcriptional dysregulation in the R6/2 mouse, using double-label in situ hybridization combined with immunohistochemistry, and laser capture microdissection combined with quantitative real-time PCR. There was no difference in transcript levels of specific genes between NII-positive and NII-negative neurons. These results demonstrate that NIIs do not cause decreases in D2, PPE and PSS mRNA levels in R6/2 striatum and therefore are not involved in the down-regulation of these specific genes in this HD model. In addition, these observations argue against the notion that NIIs protect against transcriptional dysregulation in HD.

Topics: Animals; Disease Models, Animal; Down-Regulation; Enkephalins; Gene Expression Regulation; Humans; Huntingtin Protein; Huntington Disease; Immunohistochemistry; In Situ Hybridization; Intranuclear Inclusion Bodies; Mice; Mice, Transgenic; Nerve Tissue Proteins; Neurons; Nuclear Proteins; Protein Precursors; Receptors, Dopamine D2; Somatostatin

Levels of preproenkephalin mRNA expression in trigeminal subnucleus complex by noxious tooth movement stimuli were examined using in situ hybridization. At 24 h, preproenkephalin mRNA expression was significantly upregulated in the ipsilateral trigeminal subnucleus caudalis (P<0.05), and in the subnucleus oralis (P<0.05). These findings suggested that enkephalinergic inhibitory systems could be activated during tooth movement, and that subnucleus oralis may be involved in modulation of the nociception, as well as the subnucleus caudalis.

Topics: Afferent Pathways; Animals; Disease Models, Animal; Enkephalins; Functional Laterality; Male; Neural Inhibition; Nociceptors; Orthodontic Appliances; Pain; Protein Precursors; Rats; Rats, Sprague-Dawley; RNA, Messenger; Tooth; Trigeminal Caudal Nucleus; Up-Regulation

The Flinders sensitive line (FSL) rat is a proposed genetic hypercholinergic animal model of human depression. Considering the strong comorbidity between depression and cocaine dependence we investigated the well-documented behavioral and molecular effects of cocaine in the FSL and their control Flinders resistant line (FRL) rats. First, we found no difference between the two lines to establish cocaine self-administration; both lines reached stable responding within 10 days of training at a fixed ratio-1 schedule of reinforcement (1.5 mg/kg/injection). However, the FSL rats exhibited reduced cocaine intake at a dose of 0.09 mg/kg/injection in a within-session dose-response curve (0.02, 0.09, 0.38, 1.5 mg/kg/injection). Second, we examined the effects of repeated cocaine administration on locomotor activity, dopamine overflow and striatal prodynorphin mRNA expression. We found the FSL rats to be low responders to novelty and to exhibit less locomotor activation after repeated cocaine administration (30 mg/kg, i.p., daily injections for 10 days) than their controls. Microdialysis sampling from the nucleus accumbens shell revealed no significant difference in the dopamine overflow between the rat lines, neither during baseline nor after cocaine stimulation. Postmortem analyses of striatal prodynorphin mRNA expression (using in situ hybridization histochemistry) revealed a differentiated response to the cocaine exposure. In contrast to control FRL rats, the FSL rats showed no typical cocaine-evoked elevation of prodynorphin mRNA levels in rostral subregions of the striatum whereas both strains expressed increased prodynorphin mRNA levels in the caudal striatum after cocaine administration. In conclusion, the FSL animal model of depression demonstrates marked blunting of the locomotor and dynorphin neuroadaptative responses to cocaine in accordance with its enhanced cholinergic sensitivity.

Topics: Acetylcholine; Animals; Chromatography, High Pressure Liquid; Cocaine; Cocaine-Related Disorders; Corpus Striatum; Depression; Disease Models, Animal; Dopamine; Dopamine Uptake Inhibitors; Enkephalins; In Situ Hybridization; Male; Microdialysis; Motor Activity; Nucleus Accumbens; Protein Precursors; Rats; RNA, Messenger

Abrupt withdrawal from chronic alcohol exposure can produce convulsions that are likely due to ethanol (EtOH) neuroadaptations. While significant efforts have focused on elucidating dependence mechanisms, the alterations contributing to EtOH withdrawal severity are less well characterized. The present studies examined the kappa-opioid receptor (KOP-R) system in Withdrawal Seizure-Prone (WSP) and Withdrawal Seizure-Resistant (WSR) mice, selected lines that display severe and mild convulsions upon removal from chronic EtOH exposure. Previous data demonstrated significant increases in whole brain prodynorphin (Pdyn) mRNA in WSP mice only during EtOH withdrawal. No significant effects of EtOH exposure or withdrawal were observed in WSR mice. The present study characterized Pdyn mRNA and the KOP-R in WSP and WSR mice during EtOH withdrawal using in situ hybridization (ISH) and KOP-R autoradiography. Analyses were performed in brain regions that express Pdyn mRNA and/or KOP-R and that might participate in seizure circuitry: the piriform cortex, olfactory tubercle, nucleus accumbens, caudate-putamen, claustrum, dorsal endopiriform nucleus, and cingulate cortex. ISH analyses confirmed previous findings; EtOH withdrawal increased Pdyn mRNA in multiple brain regions of WSP mice, but not WSR. Basal KOP-R binding was higher in WSR mice than in WSP mice, suggesting an anti-convulsant role for receptor activation. Finally, increased KOP-R density was present during EtOH withdrawal in WSP mice. These data suggest that differences in the KOP-R system among the lines might contribute to their selected difference in EtOH withdrawal severity.

Topics: Alcohol Withdrawal Seizures; Analysis of Variance; Animals; Basal Ganglia; Brain; Caudate Nucleus; Central Nervous System Depressants; Disease Models, Animal; Enkephalins; Ethanol; Genetic Predisposition to Disease; Gyrus Cinguli; In Situ Hybridization; Male; Mice; Mice, Inbred Strains; Nucleus Accumbens; Olfactory Pathways; Protein Precursors; Putamen; Receptors, Opioid, kappa; RNA, Messenger; Species Specificity; Tissue Distribution

An increase in preprodynorphin (ppdyn) mRNA was detected in the caudate putamen of chronically cocaine-treated and 3-h withdrawn rats. An increase in mu-opioid receptor (MOP) mRNA levels was observed in the frontal cortex of 3-h withdrawn rats. Naloxone had no effect on the increase of MOP or ppdyn mRNA levels. The results indicate that the opioid system is altered during early withdrawal from chronic cocaine administration.

Topics: Animals; Brain Chemistry; Cocaine; Cocaine-Related Disorders; Disease Models, Animal; Dopamine Uptake Inhibitors; Down-Regulation; Drug Administration Schedule; Enkephalins; Frontal Lobe; Male; Narcotic Antagonists; Protein Precursors; Rats; Rats, Inbred F344; Receptors, Opioid, mu; RNA, Messenger; Substance Withdrawal Syndrome; Time Factors

To observe the analgesic effect of intrathecal transplant of immortalized rat astrocyte genetically modified by human preproenkephalin gene (IAST/hPPE) on chronic neuropathic pain.. 40 adult male Sprague-Dawley rats were randomly divided into four groups, 10 rats for each group. Naive group, SNI group, SNI + IAST group and SNI + IAST/hPPE group. The immortalized rat astrocyte (IAST) or IAST/hPPE co-incubated with bromodeoxyuridine (BrdU) in vitro were transplanted in the lumbar 4 to 6 subarachnoid space near the spinal cord 1 week after right side spared nerve injury (SNI). All animals were tested for bilateral 50% hindpaw withdrawal threshold (PWT) to a graded series of Von Frey hairs stimulation once a week from one week before SNI to six weeks after transplant, the difference value for right 50% PWT minus left 50% PWT was calculated and the effect of intraperitoneal naloxone on the analgesic efficacies was also observed. The content of L-EK in the spinal cord of L4 - 6 and was determined using immunohistochemistry and radioimmunoassay, and the expression of BrdU in grafts was determined using immunohistochemistry.. Allodynia-like behaviour after 1 week following SNI was observed. As compared with Naive group, the difference value for the right 50% PWT minus left 50% PWT in the other three groups was higher significantly (P < 0.01). The tactile allodynia induced by SNI was significantly alleviated during the 1 to 6 week period after transplantation of IAST/hPPE cells, but transplants of IAST cells had no effect on the allodynia-like behaviour. The difference value for the right 50% PWT minus left 50% PWT in SNI + IAST/hPPE group was lower significantly than that in the SNI and SNI +I AST group (P < 0.01), but there was no significant difference between SNI and SNI + IAST group (P > 0.05). The efficacies were reversed by intraperitoneal naloxone in SNI + IAST/hPPE group. The content of L-EK in the lumbar spinal cord in IAST/hPPE group (108.1 pg/mg +/- 12.5 pg/mg) was significantly higher than that in other three groups (P < 0.01), but there was no significant difference between SNI and SNI + IAST group (25.4 pg/mg +/- 1.9 pg/mg vs 28.0 pg/mg +/- 2.1 pg/mg, P > 0.05). Furthermore, The grafts in the surface of dorsal horn were still stained positively for BrdU, they survived greater than 6 weeks on the pia mater around the spinal cord.. Intrathecal transplant of IAST/hPPE cells could alleviate the allodynia-like behaviour after chronic neuropathic pain, which is associated with enkephalin secreted continuously from the grafts and conducted via opiate receptors.

Topics: Animals; Astrocytes; Cells, Cultured; Chronic Pain; Disease Models, Animal; Enkephalins; Genetic Therapy; Humans; Male; Neuralgia; Protein Precursors; Rats; Rats, Sprague-Dawley

The dopamine precursor, L-3,4-dihydroxyphenylalanine (L-DOPA), remains the most common treatment for Parkinson's disease. However, following long-term treatment, disabling side effects, particularly L-DOPA-induced dyskinesias, are encountered. Conversely, D2/D3 dopamine receptor agonists, such as ropinirole, exert an anti-parkinsonian effect while eliciting less dyskinesia when administered de novo in Parkinson's disease patients. Parkinson's disease and L-DOPA-induced dyskinesia are both associated with changes in mRNA and peptide levels of the opioid peptide precursors preproenkephalin-A (PPE-A) and preproenkephalin-B (PPE-B). Furthermore, a potential role of abnormal opioid peptide transmission in dyskinesia is suggested due to the ability of opioid receptor antagonists to reduce the L-DOPA-induced dyskinesia in animal models of Parkinson's disease. In this study, the behavioural response, striatal topography and levels of expression of the opioid peptide precursors PPE-A and PPE-B were assessed, following repeated vehicle, ropinirole, or L-DOPA administration in the 6-OHDA-lesioned rat model of Parkinson's disease. While repeated administration of L-DOPA significantly elevated PPE-B mRNA levels (313% cf. vehicle, 6-OHDA-lesioned rostral striatum; 189% cf. vehicle, 6-OHDA-lesioned caudal striatum) in the unilaterally 6-OHDA-lesioned rat model of Parkinson's disease, ropinirole did not. These data and previous studies suggest the involvement of enhanced opioid transmission in L-DOPA-induced dyskinesia and that part of the reason why D2/D3 dopamine receptor agonists have a reduced propensity to elicit dyskinesia may reside in their reduced ability to elevate opioid transmission.

Topics: Animals; Antiparkinson Agents; Behavior, Animal; Corpus Striatum; Disease Models, Animal; Dopamine Agonists; Dyskinesia, Drug-Induced; Enkephalins; Gene Expression; In Situ Hybridization; Indoles; Levodopa; Male; Motor Activity; Opioid Peptides; Oxidopamine; Parkinsonian Disorders; Protein Precursors; Rats; Rats, Sprague-Dawley; RNA, Messenger; Synaptic Transmission

Release of endogenous dynorphin opioids within the spinal cord after partial sciatic nerve ligation (pSNL) is known to contribute to the neuropathic pain processes. Using a phosphoselective antibody [kappa opioid receptor (KOR-P)] able to detect the serine 369 phosphorylated form of the KOR, we determined possible sites of dynorphin action within the spinal cord after pSNL. KOR-P immunoreactivity (IR) was markedly increased in the L4-L5 spinal dorsal horn of wild-type C57BL/6 mice (7-21 d) after lesion, but not in mice pretreated with the KOR antagonist nor-binaltorphimine (norBNI). In addition, knock-out mice lacking prodynorphin, KOR, or G-protein receptor kinase 3 (GRK3) did not show significant increases in KOR-P IR after pSNL. KOR-P IR was colocalized in both GABAergic neurons and GFAP-positive astrocytes in both ipsilateral and contralateral spinal dorsal horn. Consistent with sustained opioid release, KOR knock-out mice developed significantly increased tactile allodynia and thermal hyperalgesia in both the early (first week) and late (third week) interval after lesion. Similarly, mice pretreated with norBNI showed enhanced hyperalgesia and allodynia during the 3 weeks after pSNL. Because sustained activation of opioid receptors might induce tolerance, we measured the antinociceptive effect of the kappa agonist U50,488 using radiant heat applied to the ipsilateral hindpaw, and we found that agonist potency was significantly decreased 7 d after pSNL. In contrast, neither prodynorphin nor GRK3 knock-out mice showed U50,488 tolerance after pSNL. These findings suggest that pSNL induced a sustained release of endogenous prodynorphin-derived opioid peptides that activated an anti-nociceptive KOR system in mouse spinal cord. Thus, endogenous dynorphin had both pronociceptive and antinociceptive actions after nerve injury and induced GRK3-mediated opioid tolerance.

Topics: Animals; Astrocytes; Disease Models, Animal; Disease Progression; Drug Tolerance; Dynorphins; Enkephalins; G-Protein-Coupled Receptor Kinase 3; Hyperalgesia; Lumbosacral Region; Mice; Mice, Inbred C57BL; Mice, Knockout; Narcotic Antagonists; Narcotics; Neuralgia; Neurons; Protein Precursors; Protein Serine-Threonine Kinases; Receptors, Opioid; Receptors, Opioid, kappa; Sciatic Neuropathy; Spinal Cord

The dtsz mutant hamster represents a model of primary paroxysmal dystonia, in which dystonic episodes occur in response to stress. Previous examinations demonstrated striatal dysfunctions in dtsz hamsters. In the present study, in situ hybridization was used to examine preproenkephalin and prodynorphin expression as potential indices of imbalances between the striatopallidal and striatonigral pathways. Brain analyses were performed in dtsz hamsters under basal conditions, i.e., in the absence of dystonia, as well as mutant hamsters that exhibited severe stress-induced dystonic attacks immediately prior to sacrifice. In the striatum the basal expression of prodynorphin tended to be higher, while that of preproenkephalin tended to be lower in mutant hamsters in comparison to non-dystonic control hamsters. Significant basal changes were restricted to higher levels of prodynorphin in the ventrolateral striatum and lower prodynorphin and preproenkephalin mRNA expression in the hippocampus and/or in subregions of the hypothalamus. After stressful stimulation, the neuropeptides increased in several regions in both animals groups. In comparison to stimulated control hamsters, a significantly lower prodynorphin expression was found in several limbic areas of stimulated mutant hamsters during the manifestation of dystonia, while preproenkephalin mRNA was significantly lower in the anterior and dorsal striatal subregions and in nucleus accumbens. Since changes in the expression of these opioid peptides have been suggested to be related to abnormal dopaminergic activity, the present findings may reflect disturbances in striatal dopaminergic systems, and also in limbic structures in the dtsz mutant, particularly during the expression of dystonia.

Topics: Analysis of Variance; Animals; Basal Ganglia; Brain; Chorea; Cricetinae; Disease Models, Animal; Dystonia; Enkephalins; Gene Expression Regulation; In Situ Hybridization; Mutation; Protein Precursors; Reference Values; RNA, Messenger; Tissue Distribution

Identifying genes for bipolar mood disorders through classic genetics has proven difficult. Here, we present a comprehensive convergent approach that translationally integrates brain gene expression data from a relevant pharmacogenomic mouse model (involving treatments with a stimulant--methamphetamine, and a mood stabilizer--valproate), with human data (linkage loci from human genetic studies, changes in postmortem brains from patients), as a bayesian strategy of crossvalidating findings. Topping the list of candidate genes, we have DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of 32 kDa) located at 17q12, PENK (preproenkephalin) located at 8q12.1, and TAC1 (tachykinin 1, substance P) located at 7q21.3. These data suggest that more primitive molecular mechanisms involved in pleasure and pain may have been recruited by evolution to play a role in higher mental functions such as mood. The analysis also revealed other high-probability candidates genes (neurogenesis, neurotrophic, neurotransmitter, signal transduction, circadian, synaptic, and myelin related), pathways and mechanisms of likely importance in pathophysiology.

Topics: Animals; Antimanic Agents; Bayes Theorem; Bipolar Disorder; Brain; Central Nervous System Stimulants; Disease Models, Animal; Dopamine and cAMP-Regulated Phosphoprotein 32; Enkephalins; Gene Expression Profiling; Genetic Linkage; Genetic Predisposition to Disease; Genetic Testing; Genomics; Humans; Male; Methamphetamine; Mice; Mice, Inbred C57BL; Microarray Analysis; Nerve Tissue Proteins; Pharmacogenetics; Phosphoproteins; Protein Precursors; Substance P; Tachykinins; Valproic Acid

The hph-1 mice have defective tetrahydrobiopterin biosynthesis and share many neurochemical similarities with l-dopa-responsive dystonia (DRD) in humans. In both, there are deficiencies in GTP cyclohydrolase I and low brain levels of dopamine (DA). Striatal tyrosine hydroxylase (TH) levels are decreased while the number of DA neurones in substantia nigra (SN) appears normal. The hph-1 mouse is therefore a useful model in which to investigate the biochemical mechanisms underlying dystonia in DRD. In the present study, the density of striatal DA terminals and DA receptors and the expression of D-1, D-2, and D-3 receptors, preproenkephalin (PPE-A), preprotachykinin (PPT), and nitric oxide synthase (NOS) mRNAs in the striatum and nucleus accumbens and nigral TH mRNA expression were examined. Striatal DA terminal density as judged by specific [3H]mazindol binding was not altered while the levels of TH mRNA were elevated in the SN of hph-1 mice compared to control (C57BL) mice. Total and subregional analysis of the striatum and nucleus accumbens showed that D-2 receptor ([3H]spiperone) binding density was increased while D-1 receptor ([3H]SCH 23390) and D-3 receptor ([3H]7-OH-DPAT) binding density was not altered. In the striatum and nucleus accumbens, expression of PPT mRNA was elevated but PPE-A mRNA, D-1, D-2 receptor, and nNOS mRNA were not changed in hph-1 mice compared to controls. These findings suggest that an imbalance between the direct strionigral and indirect striopallidal output pathways may be relevant to the genesis of DRD. However, the pattern of changes observed is not that expected as a result of striatal dopamine deficiency and suggests that other effects of GTP cyclohydrolase I deficiency may be involved.

Topics: Animals; Autoradiography; Brain; Disease Models, Animal; Dystonia; Enkephalins; GTP Cyclohydrolase; In Situ Hybridization; Mice; Neuropeptides; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Presynaptic Terminals; Protein Precursors; Receptors, Dopamine; RNA, Messenger; Tachykinins; Tyrosine 3-Monooxygenase

While levodopa-induced neurochemical changes have been studied in animal models of Parkinson's disease, very little is known regarding the effects of levodopa administration in normal animals. The present study investigates the effects normal and MPTP-lesioned mice chronically treated with two different doses of levodopa. We assess changes in striatal dopamine (DA) receptor binding, striatal DA receptor mRNA levels and striatal neuropeptide precursor levels (preproenkephalin-A [PPE-A]; preprotachykinin [PPT]; preproenkephalin-B [PPE-B]). The extent of the lesion was measured by striatal DA transporter binding and stereological estimation of the number of tyrosine hydroxylase immunoreactive neurones in the substantia nigra pars compacta (SNc). In non-lesioned animals, chronic levodopa treatment induced an increase in PPE-A mRNA, whereas both D3R binding and PPE-B mRNA levels were dramatically increased in the lesioned animals in a dose dependent manner. The present results show that chronic levodopa administration may induce pathophysiological changes, even in the absence of a lesion of the nigro-striatal pathway, suggesting that the sensitization process involves predominantly the indirect striatofugal pathway in non-lesioned animals, whereas the direct pathway is primarily involved in lesioned animals.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Animals; Antiparkinson Agents; Binding, Competitive; Chronic Disease; Corpus Striatum; Disease Models, Animal; Dopamine Plasma Membrane Transport Proteins; Enkephalins; Levodopa; Male; Membrane Glycoproteins; Membrane Transport Proteins; Mice; Mice, Inbred C57BL; Nerve Tissue Proteins; Neural Pathways; Neurons; Parkinsonian Disorders; Protein Precursors; Receptors, Dopamine D1; Receptors, Dopamine D2; Receptors, Dopamine D3; RNA, Messenger; Substantia Nigra; Tachykinins; Tyrosine 3-Monooxygenase

We have examined the ability of KW-6002, an adenosine A2a antagonist, to modulate the dyskinetic effects of L-DOPA in 6-hydroxydopamine-lesioned rats. In animals rendered dyskinetic by a previous course of L-DOPA treatment, KW-6002 did not elicit any abnormal involuntary movements on its own, but failed to reduce the severity of dyskinesia when coadministered with L-DOPA. A second experiment was undertaken in order to study the effects of KW-6002 in L-DOPA-naive rats. Thirty-five animals were allotted to four groups to receive a 21-day treatment with: (i) KW-6002 (10 mg/kg/day); (ii) L-DOPA (6 mg/kg/day) i.p.; (iii) KW-6002 plus L-DOPA (same doses as above) or (iv) vehicle. Chronic treatment with KW-6002-only produced a significant relief of motor disability in the rotarod test in the absence of any abnormal involuntary movements. Combined treatment with L-DOPA and KW-6002 improved rotarod performance to a significantly higher degree than did each of the two drugs alone. However, this combined treatment induced dyskinesia to about the same degree as did L-DOPA alone. In situ hybridization histochemistry showed that KW-6002 treatment alone caused an approximately 20% reduction in the striatal levels of preproenkephalin mRNA, whereas neither the coadministration of KW-6002 and L-DOPA nor L-DOPA alone significantly altered the expression of this transcript in the dopamine-denervated striatum. Either alone or in combination with L-DOPA, KW-6002 did not have any modulatory effect on prodynorphin mRNA expression or FosB/DeltaFosB-like immunoreactivity in the dopamine-denervated striatum. These results show that monotreatment with an adenosine A2a receptor antagonist can relieve motor disability without inducing behavioural and cellular signs of dyskinesia in rats with 6-hydroxydopamine lesions. Cotreatment with KW-6002 and L-DOPA potentiates the therapeutic effect but not the dyskinesiogenic potential of the latter drug.

Topics: Animals; Behavior, Animal; Corpus Striatum; Disease Models, Animal; Drug Therapy, Combination; Dyskinesia, Drug-Induced; Enkephalins; Female; Levodopa; Motor Activity; Oxidopamine; Parkinsonian Disorders; Protein Precursors; Proto-Oncogene Proteins c-fos; Purinergic P1 Receptor Antagonists; Purines; Rats; Rats, Sprague-Dawley; Receptor, Adenosine A2A; RNA, Messenger; Treatment Outcome

Previous studies have shown that intrastriatal transplants of dopamine (DA)-rich fetal ventral mesencephalic (VM) tissue can correct denervation-induced changes in the cellular expression of neuropeptide and receptor mRNAs in the rat Parkinson model. However, with the standard transplantation approach normalization of all cellular parameters has not been obtained. This may be due either to the incomplete striatal reinnervation achieved by these transplants, or to the ectopic placement of the grafts. In the present study we have used a microtransplantation approach to obtain a more complete reinnervation of the denervated striatum (20 micrograft deposits spread over the entire structure). Neurons were also implanted directly into the substantia nigra. In rats with multiple intrastriatal VM transplants the lesion-induced upregulation of mRNAs encoding for preproenkephalin (PPE), the D(2)-type DA-receptor, and the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD(67)) was normalized throughout the striatum, whereas the lesion-induced downregulation of preprotachykinin mRNA was unaffected. Intranigral grafts of either fetal DA-rich VM tissue or GABA-rich striatal tissue did not induce any changes in striatal neuropeptide and D(2)-receptor mRNA expression despite significant behavioral improvement. Comparison of the behavioral data with levels of neuropeptide expression showed that in rats with intrastriatal VM transplants a complete normalization of striatal PPE and GAD(67) mRNA expression did not translate into a complete recovery of spontaneous motor behaviors. The results show that extensive DA reinnervation of the host striatum by multiple VM microtransplants is insufficient to obtain full recovery of all lesion-induced changes at both the cellular and the behavioral level. A full reconstruction of the nigrostriatal pathway or, alternatively, modulation of basal ganglia function by grafting in non-striatal regions may be required to further improve the functional outcome in the DA-denervated brain.

Topics: Adrenergic Agents; Animals; Autoradiography; Basal Ganglia; Brain Mapping; Disease Models, Animal; Embryo, Mammalian; Enkephalins; Entopeduncular Nucleus; Female; Fetal Tissue Transplantation; Gene Expression Regulation; Glutamate Decarboxylase; Immunohistochemistry; In Situ Hybridization; Neuropeptides; Oxidopamine; Parkinsonian Disorders; Protein Precursors; Rats; Rats, Sprague-Dawley; Receptors, Dopamine D2; Regression Analysis; RNA, Messenger; Septal Nuclei; Substantia Nigra; Tachykinins

Compensatory mechanisms delay the appearance of parkinsonian symptoms. However, both the order of appearance and potential interactions of compensatory mechanisms acting within the nigrostriatal pathway as well as inside and outside the basal ganglia are not clear. We hypothesize that, after the striatal dopaminergic homeostasis breakdown, a modification in the expression of several striatal markers (neuropeptide precursors and dopamine receptors) may occur before a change in the activity of both globus pallidus (GP) and substantia nigra pars reticulata (SNr) in response to a partial nigrostriatal lesion. The present data show, in MPTP-treated mice, that preproenkephalin-A and preprotachykinin mRNA expression and D(3) receptor binding are modified without changes in cytochrome oxidase metabolic activity in both GP and SNr, respectively. These changes in neuropeptide expression would compensate for the dopamine depletion-induced changes in inhibitory GABAergic input from the striatum to GP and SNr. It also indicates that nondopaminergic compensatory mechanisms inherent to the basal ganglia are activated before those residing outside the basal ganglia.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Animals; Basal Ganglia; Corpus Striatum; Disease Models, Animal; Disease Progression; Electron Transport Complex IV; Enkephalins; Gene Expression Regulation; In Situ Hybridization; Male; Mice; Mice, Inbred C57BL; Neuropeptides; Parkinsonian Disorders; Protein Precursors; Receptors, Dopamine D1; Receptors, Dopamine D2; Receptors, Dopamine D3; RNA, Messenger; Tachykinins

Inactivation of the subthalamic nucleus (STN) or the internal segment of the pallidum (GPi)/entopeduncular nucleus (EP) by deep brain stimulation or lesioning alleviates clinical manifestations of Parkinson's disease (PD) as well as reducing the side-effects of levodopa treatment. However, the effects of STN or entopeduncular nucleus (EP) lesion on levodopa-related motor fluctuations and on neurochemical changes induced by levodopa remain largely unknown. The effects of such lesions on levodopa-induced motor alterations were studied in 6-hydroxydopamine (6-OHDA)-lesioned rats and were assessed neurochemically by analyzing the functional activity of the basal ganglia nuclei, using the expression levels of the mRNAs coding for glutamic acid decarboxylase and cytochrome oxidase as molecular markers of neuronal activity. At the striatal level, preproenkephalin (PPE) mRNA levels were analyzed. We found in 6-OHDA-lesioned rats that a unilateral STN or EP lesion ipsilateral to the 6-OHDA lesion had no effect on either the shortening in the duration of the levodopa-induced rotational response or the levodopa-induced biochemical changes in the basal ganglia nuclei. In contrast, overexpression of PPE mRNA due to levodopa treatment was reversed by the STN or EP lesion. Our study thus shows that lesion of the EP or STN may counteract some of the neurochemical changes induced by levodopa treatment within the striatum.

Topics: Animals; Basal Ganglia; Corpus Striatum; Disease Models, Animal; Electron Transport Complex IV; Enkephalins; Entopeduncular Nucleus; Globus Pallidus; Glutamate Decarboxylase; In Situ Hybridization; Isoenzymes; Levodopa; Male; Motor Activity; Neurons; Oxidopamine; Parkinsonian Disorders; Protein Precursors; Rats; Rats, Sprague-Dawley; RNA, Messenger; Substantia Nigra; Subthalamic Nucleus

Nerve injury-induced afferent discharge is thought to elicit spinal sensitization and consequent abnormal pain. Experimental neuropathic pain, however, also depends on central changes, including descending facilitation arising from the rostral ventromedial medulla (RVM) and upregulation of spinal dynorphin. A possible intersection of these influences at the spinal level was explored by measuring evoked, excitatory transmitter release in tissues taken from nerve-injured animals with or without previous manipulation of descending modulatory systems. Spinal nerve ligation (SNL) produced expected tactile and thermal hyperesthesias. Capsaicin-evoked calcitonin gene-related peptide (CGRP) release was markedly enhanced in lumbar spinal tissue from SNL rats when compared with sham-operated controls. Enhanced, evoked CGRP release from SNL rats was blocked by anti-dynorphin A(1-13) antiserum; this treatment did not alter evoked release in tissues from sham-operated rats. Dorsolateral funiculus lesion (DLF) or destruction of RVM neurons expressing mu-opioid receptors with dermorphin-saporin, blocked tactile and thermal hypersensitivity, as well as SNL-induced upregulation of spinal dynorphin. Spinal tissues from these DLF-lesioned or dermorphin-saporin-treated SNL rats did not exhibit enhanced capsaicin-evoked CGRP-IR release. These data demonstrate exaggerated release of excitatory transmitter from primary afferents after injury to peripheral nerves, supporting the likely importance of increased afferent input as a driving force of neuropathic pain. The data also show that modulatory influences of descending facilitation are required for enhanced evoked transmitter release after nerve injury. Thus, convergence of descending modulation, spinal plasticity, and afferent drive in the nerve-injured state reveals a mechanism by which some aspects of nerve injury-induced hyperesthesias may occur.

Topics: Afferent Pathways; Analgesics, Opioid; Animals; Calcitonin Gene-Related Peptide; Capsaicin; Disease Models, Animal; Dynorphins; Enkephalins; Hyperesthesia; Ligation; Lumbosacral Region; Male; Medulla Oblongata; Microinjections; N-Glycosyl Hydrolases; Nerve Compression Syndromes; Neuralgia; Neuronal Plasticity; Neurotransmitter Agents; Oligopeptides; Opioid Peptides; Pain Measurement; Protein Precursors; Rats; Rats, Sprague-Dawley; Receptors, Opioid, mu; Recombinant Fusion Proteins; Ribosome Inactivating Proteins, Type 1; Saporins; Sensory Thresholds; Spinal Cord; Spinal Nerves

Long-term treatment of Parkinson's disease with levodopa is compromised by the development of motor complications, including on-off fluctuations and involuntary movements termed dyskinesia. The neural mechanisms underlying treatment-related dyskinesias may involve underactivity of the output regions of the basal ganglia, i.e., the medial segment of the globus pallidus (GPm) and substantia nigra pars reticulata (SNR). Increased activity of GABAergic neurons of the "direct" striatopallidal pathway has been implicated in the suppression of the GPm and SNR and thus the development of dyskinesia. The direct pathway uses opioids as a co-neurotransmitter. These opioid peptides are products of the high-molecular weight opioid precursor pre-proenkephalin B (PPE-B). In situ hybridisation studies were employed to investigate PPE-B mRNA expression in postmortem striatal tissue from patients with a clinicopathological diagnosis of Parkinson's disease, all of whom displayed levodopa-induced motor complications, including dyskinesia prior to death and in the caudate-putamen (striatum) of the MPTP-lesioned macaque model of Parkinson's disease with treatment-related dyskinesia. Striatal PPE-B mRNA expression was significantly increased by 172% in dyskinetic Parkinson's disease patients compared to age-matched controls. This increase was heterogeneous with increased expression within the striosomes compared to matrix compartments of the striatum. Striatal PPE-B mRNA expression was significantly increased by 185% in the MPTP-lesioned macaque exhibiting dyskinesia, compared to parkinsonian, nondyskinetic MPTP-lesioned macaques, and by 146% compared to non-parkinsonian, nondyskinetic controls. Increased PPE-B mRNA expression, with subsequent elevations in opioid peptide transmission within the direct striatal output pathways, may underlie treatment-related dyskinesia in Parkinson's disease.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Aged; Aged, 80 and over; Animals; Antiparkinson Agents; Corpus Striatum; Disease Models, Animal; Dyskinesia, Drug-Induced; Enkephalins; Female; Humans; Macaca mulatta; Male; Parkinson Disease; Parkinsonian Disorders; Protein Precursors; RNA, Messenger

In order to assess the role of striatal dopamine (DA) afferents in L-DOPA-induced dyskinesia, we have studied a large series of rats sustaining 2, 3, or 4 unilateral injections of 6-hydroxydopamine (6-OHDA) in the lateral striatum. This type of lesion produced a dose-dependent depletion of DA fibers in the caudate-putamen, which was most pronounced in the lateral aspects of this structure. An additional group of rats was injected with 6-OHDA in the medial forebrain bundle to obtain complete DA denervation on one side of the brain. During a course of chronic L-DOPA treatment, rats with intrastriatal 6-OHDA lesions developed abnormal involuntary movements (AIMs), which mapped onto striatal domains exhibiting at least approximately 90% denervation, as judged by DA transporter autoradiography. The denervated areas showed local upregulation of preproenkephalin and prodynorphin mRNA, and FosB-like immunoreactivity, which were highly correlated with the rats' AIM scores. When compared to completely DA-denervated animals, the rats with intrastriatal 6-OHDA lesions showed an overall lower incidence, lower severity and different topographic distribution of AIMs. The involvement of proximal limb and axial muscles in the abnormal movements was proportional to the spreading of the lesion from lateral towards medial aspects of the caudate-putamen. Locomotive AIMs were only seen in rats with complete lesions, but not in any of the animals with intrastriatal 6-OHDA (which showed > 5% DA fiber sparing in the medial striatum). Intrastriatally 6-OHDA-lesioned rats had a larger therapeutic window for L-DOPA than did rats with complete bundle lesions, since they exhibited an overall lower predisposition to dyskinesia but a similar degree of drug-induced motor improvement in a test of forelimb stepping. Our results are the first to demonstrate that selective and partial DA denervation in the sensorimotor part of the striatum can confer cellular and behavioral supersensitivity to L-DOPA, and that the phenomenology of L-DOPA-induced rat AIMs can be accounted for by the topography of DA denervation within the caudate-putamen.

Topics: Afferent Pathways; Animals; Bacterial Proteins; Behavior, Animal; Biomarkers; Brain Mapping; Caudate Nucleus; Corpus Striatum; Disease Models, Animal; Dopamine; Dose-Response Relationship, Drug; Dyskinesia, Drug-Induced; Enkephalins; Female; Image Processing, Computer-Assisted; Levodopa; Motor Activity; Nerve Tissue Proteins; Oxidopamine; Parkinsonian Disorders; Protein Precursors; Proto-Oncogene Proteins c-fos; Putamen; Rats; Rats, Sprague-Dawley; RNA, Messenger; Severity of Illness Index; Substantia Nigra; Sympathectomy, Chemical; Transcription Factors

L-DOPA-induced dyskinesia (abnormal involuntary movements) is one of the most debilitating complications of chronic L-DOPA pharmacotherapy in Parkinson's disease. It is generally agreed that dyskinesia arises as a consequence of pulsatile dopamine-receptor stimulation in the brain, causing downstream changes in genes and proteins. Advance in our understanding of such changes is critically dependent on the availability of suitable animal models. We have introduced a new method to classify and rate L-DOPA-induced abnormal involuntary movements (AIMs) in 6-hydroxydopamine (6-OHDA) lesioned rats. This method allows us to dissect the molecular correlates of a dyskinetic motor response to L-DOPA in this species. One of the most prominent molecular changes underlying the development of dyskinesia in the rat consists in the striatal induction of prodynorphin gene expression by L-DOPA. This effect is mediated by FosB-related transcription factors of 32-37 kDa, which are co-induced with prodynophin in striatal neurons of the "direct pathway". Both AIM development and the associated upregulation of prodynorphin mRNA by L-DOPA are significantly inhibited by the intrastriatal infusion of fosB antisense. Antisense-mediated knockdown of CREB (cyclic AMP response-element binding proteins) has however no effect. Our results identify fosB as a potential target for adjunctive antiparkinsonian therapies.

Topics: Animals; Antiparkinson Agents; Corpus Striatum; Disease Models, Animal; Dyskinesia, Drug-Induced; Enkephalins; Gene Expression Regulation; Levodopa; Oxidopamine; Parkinsonian Disorders; Protein Precursors; Proto-Oncogene Proteins c-fos; Rats; Sympatholytics; Transcription Factors

Striatal neurons that contain GABA and enkephalin and project to the external segment of the pallidum are thought to be overactive in Parkinson's disease. Furthermore, it has been shown that the appearance of L-dopa-induced dyskinesias is correlated to an increase of preproenkephalin (PPE) mRNA expression and that some antagonists of glutamate receptors can prevent and reverse L-dopa-induced dyskinesias in parkinsonian rats. The aim of this study was therefore to analyse the effect of a systemic treatment with glutamate receptor antagonists, alone or in combination with L-dopa, on the PPE mRNA level in rats with a 6-hydroxydopamine-induced unilateral lesion of the nigrostriatal pathway. In vehicle-treated animals, PPE mRNA levels were markedly increased in the striatum on the lesioned side. Sub-chronic L-dopa treatment, with bi-daily injections for 22 days, induced a further increase in PPE mRNA expression in the denervated striatum. Administration of the AMPA receptor antagonist, LY293558, partially reversed the lesion-induced and L-dopa-induced increases in PPE mRNA expression. However, although the administration of the NMDA receptor antagonist MK801 showed a tendency to decrease this L-dopa induced overexpression, it did not reach significance. This study provides evidence that glutamatergic antagonists, and particularly AMPA antagonists, tend to reverse PPE neurochemical changes at the striatal level induced by L-dopa in hemiparkinsonian rats.

Topics: Animals; Disease Models, Animal; Dizocilpine Maleate; Dopamine; Dopamine Plasma Membrane Transport Proteins; Dyskinesia, Drug-Induced; Enkephalins; Excitatory Amino Acid Antagonists; Gene Expression Regulation; Isoquinolines; Levodopa; Male; Membrane Glycoproteins; Membrane Transport Proteins; Neostriatum; Nerve Tissue Proteins; Oxidopamine; Parkinsonian Disorders; Protein Precursors; Rats; Rats, Sprague-Dawley; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate; RNA, Messenger; Tetrazoles

In early adult-onset Huntington's disease (HD), enkephalinergic striatopallidal projection neurons show preferential loss, reduced preproenkephalin (PPE) expression in surviving striatopallidal neurons, and loss of fibers in their projection target area. We examined PPE and PPT (preprotachykinin) gene expression in striatal projection neurons and in striatal projection fibers immunoreactive for the PPE product enkephalin (ENK) and the PPT product substance P (SP) in a transgenic HD model, the R6/2 mouse, to see if changes occur in these neuron types similar to those seen in early adult-onset HD. The results show that PPE mRNA level, the number of striatal neurons expressing PPE, and the staining intensity of fibers immunoreactive for ENK in the pallidum were all decreased. By contrast, the SP-containing striatal projection systems to the pallidum and substantia nigra were relatively normal in R6/2 mice. The selective reduction in striatal PPE in R6/2 mice is reminiscent of adult-onset HD, but the preservation of the striatonigral projection system is not. Thus, R6/2 mice do not strictly mimic adult-onset HD in their striatal pathology.

Topics: Age Factors; Animals; Autoradiography; Corpus Striatum; Disease Models, Animal; Enkephalins; Gene Expression; Genotype; Globus Pallidus; Huntington Disease; Immunohistochemistry; In Situ Hybridization; Mice; Mice, Transgenic; Neural Pathways; Neurons; Nucleus Accumbens; Protein Precursors; RNA, Messenger; Substance P; Substantia Nigra; Tachykinins

In a previous study, we have shown in unilaterally dopamine-depleted rats that increased behavioral responsiveness to the dopamine D1-receptor agonist SKF-38393, which was induced by pretreatment with L-DOPA, is paralleled by specific alterations in striatal neuropeptide mRNA levels. The behavioral 'priming' effect of L-DOPA is prevented if L-DOPA is preceded by the NMDA-receptor antagonist MK-801. In the present study, the question is addressed whether blockade of the increased behavioral responsiveness with MK-801 also prevents the observed changes in striatal neuropeptide mRNA levels. After a challenge with SKF-38393 (3 mg/kg, s.c.), the striatal levels of preprodynorphin, preprotachykinin, and preproenkephalin mRNA were compared between unilaterally dopamine-depleted rats that were either primed with a single administration of L-DOPA (50 mg/kg, i.p.) or with L-DOPA preceded by MK-801 (0.1 mg/kg, i.p.). Priming with L-DOPA enhanced the increase in dynorphin mRNA levels in the dorsolateral part of the dopamine-depleted striatum that occurred after SKF-38393. On the other hand, it had no significant effect on substance P or enkephalin mRNA levels. MK-801 prior to L-DOPA prevented the increased responsiveness of dynorphin regulation. However, it induced a decreased response to dopamine D1-receptor stimulation in the substance P mRNA levels in dorsal regions of the dopamine-depleted striatum. The levels of enkephalin mRNA after challenge with SKF-38393 were not affected by the MK-801 administration. These results demonstrate that the increased behavioral responsiveness to the D1-receptor agonist SKF-38393 after priming with L-DOPA is primarily related to the upregulation of dynorphin mRNA levels in the dopamine-depleted striatum.

Topics: 2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine; Animals; Disease Models, Animal; Dizocilpine Maleate; Dopamine Agents; Dopamine Agonists; Drug Interactions; Dynorphins; Dyskinesia, Drug-Induced; Enkephalins; Excitatory Amino Acid Antagonists; Immunohistochemistry; Levodopa; Male; Motor Activity; Neostriatum; Neurons; Neuropeptides; Oxidopamine; Parkinsonian Disorders; Protein Precursors; Rats; Rats, Wistar; Receptors, Dopamine D1; Receptors, N-Methyl-D-Aspartate; RNA, Messenger; Tachykinins; Tyrosine 3-Monooxygenase

Activation of ERK (extracellular signal-regulated kinase) MAP (mitogen-activated protein) kinase in dorsal horn neurons of the spinal cord by peripheral noxious stimulation contributes to short-term pain hypersensitivity. We investigated ERK activation by peripheral inflammation and its involvement in regulating gene expression in the spinal cord and in contributing to inflammatory pain hypersensitivity. Injection of complete Freund's adjuvant (CFA) into a hindpaw produced a persistent inflammation and a sustained ERK activation in neurons in the superficial layers (laminae I-IIo) of the dorsal horn. CFA also induced an upregulation of prodynorphin and neurokinin-1 (NK-1) in dorsal horn neurons, which was suppressed by intrathecal delivery of the MEK (MAP kinase kinase) inhibitor U0126. CFA-induced phospho-ERK primarily colocalized with prodynorphin and NK-1 in superficial dorsal horn neurons. Although intrathecal injection of U0126 did not affect basal pain sensitivity, it did attenuate both the establishment and maintenance of persistent inflammatory heat and mechanical hypersensitivity. Activation of the ERK pathway in a subset of nociceptive spinal neurons contributes, therefore, to persistent pain hypersensitivity, possibly via transcriptional regulation of genes, such as prodynorphin and NK-1.

Topics: Animals; Butadienes; Disease Models, Animal; Enkephalins; Enzyme Activation; Enzyme Inhibitors; Freund's Adjuvant; Hindlimb; Hyperalgesia; Inflammation; Injections, Spinal; Male; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Nitriles; Pain; Posterior Horn Cells; Protein Precursors; Rats; Rats, Sprague-Dawley; RNA, Messenger; Spinal Cord; Substance P; Up-Regulation

Excitotoxicity has been involved in the pathogenesis of several neurodegenerative disorders. Using intrastriatal quinolinic acid (QUIN) injection as an animal model of Huntington's disease, we attempt to identify the neurotransmitter phenotype of striatal projection neurons protected by neurturin (NRTN). Control or NRTN-secreting cell lines were grafted in the striatum before QUIN injection and striatal projection neurons were examined by retrograde Fluorogold labeling and in situ hybridization. Intrastriatal grafting of NRTN-secreting cell line selectively prevented the loss of striatopallidal neurons and also the decrease in the mRNA levels for their markers (glutamic acid decarboxylase 67 and preproenkephalin) induced by QUIN, without affecting striatonigral neurons. Thus, our findings show that NRTN is a selective neuroprotective factor for striatopallidal neurons, suggesting that it might be a candidate for the treatment of movement disorders in which this neuronal population is affected.

Topics: Animals; Cell Survival; Cells, Cultured; Disease Models, Animal; Enkephalins; Globus Pallidus; Glutamate Decarboxylase; Huntington Disease; Isoenzymes; Male; Neostriatum; Nerve Growth Factors; Neural Pathways; Neurons; Neuroprotective Agents; Neurotoxins; Neurturin; Protein Precursors; Quinolinic Acid; Rats; Rats, Inbred F344; RNA, Messenger; Tachykinins

Parkinson's disease (PD) is associated with a progressive loss of dopamine neurons in the substantia nigra and degeneration of dopaminergic terminals in the striatum. Although L-DOPA treatment provides the most effective symptomatic relief for PD it does not prevent the progression of the disease, and its long-term use is associated with the onset of dyskinesia. In rodent and primate studies, glial cell line-derived neurotrophic factor (GDNF) may prevent 6-OHDA- or MPTP-induced nigral degeneration and so may be beneficial in the treatment of PD. In this study, we investigate the effects of GDNF on the expression of dyskinesia in L-DOPA-primed MPTP-treated common marmosets, exhibiting dyskinesia. GDNF or saline was administered by two intraventricular injections, 4 weeks apart, to MPTP-treated, L-DOPA-treated common marmosets primed to exhibit dyskinesia. Prior to GDNF or saline administration, all animals displayed marked dyskinesia when treated with L-DOPA. GDNF administration produced a significant improvement in motor disability and, following the second injection of GDNF, a significant improvement in the locomotor activity was observed. Following the administration of L-DOPA there was a greater reversal of disability and a reduction in the intensity of L-DOPA-induced dyskinesia in GDNF-treated animals compared to saline-treated controls. However, there was no significant difference in L-DOPA's ability to increase locomotor activity between GDNF-treated and saline-treated animals. GDNF treatment caused a significant increase in the number of tyrosine hydroxylase-positive neurons in the substantia nigra, but no change in [(3)H]mazindol binding to dopamine terminals was found in the striatum of GDNF-treated animals compared to saline-treated controls. In GDNF-treated animals a small but significant reduction in enkephalin mRNA was observed in the caudate nucleus but not in the putamen or the nucleus accumbens. Substance P mRNA expression was equally reduced in the caudate nucleus and the putamen of the GDNF-treated animals but not in the nucleus accumbens. Intraventricular administration of GDNF improved MPTP-induced disability and reversed dopamine cell loss in the substantia nigra. GDNF also diminished L-DOPA-induced dyskinesia, which may relate to its ability to partly restore nigral dopaminergic transmission or to modify the activity of striatal output pathways.

Topics: Animals; Antiparkinson Agents; Body Weight; Callithrix; Corpus Striatum; Disease Models, Animal; Dopamine Uptake Inhibitors; Dyskinesia, Drug-Induced; Enkephalins; Female; Gene Expression; Glial Cell Line-Derived Neurotrophic Factor; Levodopa; Locomotion; Male; Mazindol; MPTP Poisoning; Nerve Growth Factors; Nerve Tissue Proteins; Neuroprotective Agents; Protein Precursors; Recovery of Function; RNA, Messenger; Substantia Nigra; Tachykinins; Tritium; Tyrosine 3-Monooxygenase

Opioid receptor-binding autoradiography was used as a way to map sites of altered opioid transmission in a rat model of l-DOPA-induced dyskinesia. Rats with unilateral 6-hydroxydopamine lesions of the nigrostriatal pathways sustained a 3-week treatment with l-DOPA (6 mg/kg/day, combined with 12 mg/kg/day benserazide), causing about half of them to develop dyskinetic-like movements on the side of the body contralateral to the lesion. Autoradiographic analysis of mu-, delta-, and kappa-opioid binding sites was carried out in the caudate-putamen (CPu), the globus pallidus (GP), the substantia nigra (SN), the primary motor area, and the premotor-cingulate cortex. The dopamine-denervating lesion alone caused an ipsilateral reduction in opioid radioligand binding in the CPu, GP, and SN, but not in the cerebral cortex. Chronic l-DOPA treatment affected opioid receptor binding in both the basal ganglia and the cerebral cortex, producing changes that were both structure- and receptor-type specific, and closely related to the motor response elicited by the treatment. In the basal ganglia, the most clear-cut differences between dyskinetic and nondyskinetic rats pertained to kappa opioid sites. On the lesioned side, both striatal and nigral levels of kappa binding densities were significantly lower in the dyskinetic group, showing a negative correlation with the rats' dyskinesia scores on one hand and with the striatal expression of opioid precursor mRNAs on the other hand. In the cerebral cortex, levels of mu and delta binding site densities were bilaterally elevated in the dyskinetic group, whereas kappa radioligand binding was specifically increased in the nondyskinetic cases and showed a negative correlation with the rats' dyskinesia scores. These data demonstrate that bilateral changes in cortical opioid transmission are closely associated with l-DOPA-induced dyskinesia in the rat. Moreover, the fact that dyskinetic and nondyskinetic animals often show opposite changes in opioid radioligand binding suggests that the motor response to l-DOPA is determined, at least in part, by compensatory adjustments of brain opioid receptors.

Topics: Animals; Basal Ganglia; Behavior, Animal; Binding Sites; Cerebral Cortex; Diprenorphine; Disease Models, Animal; Dopamine Agents; Dyskinesia, Drug-Induced; Enkephalins; Female; Levodopa; Narcotic Antagonists; Oxidopamine; Protein Precursors; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; RNA, Messenger; Sympatholytics; Tritium

The substituted phenylpiperidine (-)-OSU6162 is a novel modulator of the dopaminergic systems with low affinity for dopamine D(2) receptors and potent normalizing effects on l-DOPA-induced dyskinesias. We studied the effects of coadministration of (-)-OSU6162 with l-DOPA on the regulation of striatal preproenkephalin (PPE) and prodynorphin (PDyn) mRNA expression in the primate brain by in situ hybridization histochemistry. Common marmoset monkeys sustaining unilateral 6-hydroxydopamine lesions of the nigrostriatal pathway received l-DOPA/carbidopa, l-DOPA/carbidopa plus (-)-OSU6162, or vehicle over 14 days. In vehicle-treated animals, PPE mRNA levels were markedly increased in the sensorimotor territory of the lesioned striatum. By contrast, a rather uniform lesion-induced reduction of PDyn mRNA levels was found in the vehicle group. Subchronic l-DOPA treatment induced a further increase in PPE mRNA expression in a number of sensorimotor and associative subregions of the denervated striatum. Coadministration of (-)-OSU6162 with l-DOPA partially reversed the lesion- and l-DOPA-induced elevation of PPE expression and, by affecting PPE mRNA expression differentially on the intact and lesioned striatum, markedly reduced the side-to-side difference in PPE mRNA expression. The effects on PPE mRNA expression were apparent throughout the rostrocaudal extent of the putamen and the dorsal portions of the caudate nucleus. l-DOPA treatment resulted in an enhancement in PDyn mRNA expression in all functional compartments of the striatum. Coadministration of (-)-OSU6162 had no apparent influence on these l-DOPA-induced changes in PDyn mRNA expression. The present results suggest that (-)-OSU6162 acts primarily by modifying striatal output via the indirect pathway.

Topics: Animals; Autoradiography; Callithrix; Caudate Nucleus; Corpus Striatum; Disease Models, Animal; Dopamine Agents; Drug Administration Schedule; Drug Therapy, Combination; Enkephalins; Female; In Situ Hybridization; Injections, Subcutaneous; Levodopa; Ligands; Male; Oxidopamine; Parkinson Disease, Secondary; Piperidines; Protein Precursors; Putamen; RNA, Messenger; Tritium

Rheumatoid arthritis is characterized by erosive inflammation of the joints, new bone proliferation, and ankylosis, leading to severely reduced locomotion and intense chronic pain. In a model of this disease, adjuvant-induced polyarthritis in the rat, neurons involved in pain transmission and control undergo plastic changes, especially at the spinal level. These changes affect notably neurons that contain opioids, such as enkephalins deriving from preproenkephalin A (PA) precursor protein. Using recombinant herpes simplex virus containing rat PA cDNA, we enhanced enkephalin synthesis in sensory neurons of polyarthritic rats. This treatment markedly improved locomotion and reduced hyperalgesia. Furthermore, the progression of bone destruction slowed down, which is the most difficult target to reach in the treatment of patients suffering from arthritis. These data demonstrate the therapeutic efficacy of enkephalin overproduction in a model of systemic inflammatory and painful chronic disorder.

Topics: Animals; Arthritis; Arthritis, Experimental; Disease Models, Animal; Disease Progression; Enkephalins; Freund's Adjuvant; Ganglia, Spinal; Genes, Reporter; Genetic Therapy; Genetic Vectors; Herpesvirus 1, Human; Hindlimb; Hyperalgesia; Male; Neurons, Afferent; Pain Measurement; Protein Precursors; Rats; Rats, Sprague-Dawley; Severity of Illness Index; Terminal Repeat Sequences; Treatment Outcome

Current knowledge of the molecular changes induced by dopamine denervation and subsequent treatment with L-DOPA is based on studies performed on relatively acute and young animal models of parkinsonism. It is highly warranted to ask how well these models simulate the state of chronic denervation and sustained L-DOPA pharmacotherapy which are typical of advanced Parkinson's disease. This study investigates the effects of time postdenervation and L-dopa treatment duration on the striatal expression of opioid precursor mRNAs and FosB/DeltaFosB-related proteins. Unilaterally 6-hydroxydopamine-lesioned rats were treated with therapeutical doses of L-DOPA for one year (long-term group) or a few weeks (short-term group). Age-matched lesioned rats received injections of vehicle or bromocriptine, an antiparkinsonian compound which does not produce dyskinesia when administered de novo. The lesion-induced up-regulation of preproenkephalin mRNA expression persisted at more than one year postlesion, and was unaffected by the pharmacological treatments applied. L-DOPA, but not bromocriptine, induced high striatal levels of FosB/DeltaFosB immunoreactivity and prodynorphin mRNA, and these did not differ between short-term and long-term L-DOPA-treated rats. The present data provide the first demonstration that L-DOPA maintains high striatal levels of fosB and prodynorphin gene expression during a prolonged course of treatment, which simulates the clinical practice in Parkinson's disease more closely than the short-treatment paradigms studied thus far.

Topics: Animals; Bromocriptine; Denervation; Disease Models, Animal; Dopamine Agents; Dopamine Agonists; Drug Administration Schedule; Enkephalins; Female; Gene Expression Regulation; Immunohistochemistry; Levodopa; Neostriatum; Oxidopamine; Parkinsonian Disorders; Protein Precursors; Proto-Oncogene Proteins c-fos; Rats; Rats, Sprague-Dawley; RNA, Messenger; Sympatholytics

Common marmosets (Callithrix jacchus) with near-complete unilateral 6-hydroxydopamine denervation of the dopaminergic input received a single injection of saline or L-DOPA (15mg/kg plus 6.25mg/kg benserazide). Using in situ hybridization, the effects of these treatments on c-fos messenger RNA expression in the cerebral cortex, the striatal complex and the external layer of the pallidum were studied. Moreover, receptor autoradiography was used to determine the levels of dopamine D(1) and D(2) receptors in these areas. In the cerebral cortex, animals treated with L-DOPA displayed a high expression of c-fos messenger RNA restricted to the dopamine-denervated hemisphere. No changes in the levels of cortical D(1) and D(2) receptors were found in the dopamine-denervated hemisphere. L-DOPA treatment also induced a strong expression of c-fos messenger RNA in the striatal complex in the dopamine-denervated hemisphere. The levels of striatal D(2), but not D(1), receptors were increased in the dopamine-denervated hemisphere. In the external pallidum, the major terminal region for D(2) dopamine receptor-containing striatal projection neurons, L-DOPA treatment induced c-fos messenger RNA expression in both the intact and the dopamine-denervated hemispheres.Thus, using c-fos messenger RNA as a biochemical marker of postsynaptic neuronal activation, these results provide evidence that near-complete dopamine depletion causes a profound supersensitization to L-DOPA treatment in the cerebral cortex and in the striatal complex, but not in the external layer of the pallidum, of the primate brain. The cortical response may be unique to the primate brain, but c-fos messenger RNA activation within the striatum has also been reported in the rodent. The effects of L-DOPA probably depend both on a direct activation of supersensitized dopamine receptors by dopamine produced in the few remaining, but hyperactive, dopaminergic nerve terminals and in serotonergic nerve terminals, as well as on indirect actions of L-DOPA related to activation of circuitries connecting cerebral cortex and basal ganglia structures. These results provide novel information on the mechanisms underlying L-DOPA's action in the cerebral cortex, striatum and external pallidum in a primate model of Parkinson's disease.

Topics: Animals; Benzazepines; Blotting, Western; Callithrix; Cerebral Cortex; Corpus Striatum; Denervation; Disease Models, Animal; Dopamine; Dopamine Agents; Dopamine Antagonists; Enkephalins; Gene Expression; Genes, Immediate-Early; Levodopa; Nerve Degeneration; Oxidopamine; Parkinson Disease; Protein Precursors; Proto-Oncogene Proteins c-fos; Raclopride; Radioligand Assay; Receptors, Dopamine D1; Receptors, Dopamine D2; RNA, Messenger; Substance P; Substantia Nigra; Sympatholytics; Tritium

Intrastriatal injection of quinolinate has been proven to be a very useful animal model to study the pathogenesis and treatment of Huntington's disease. To determine whether growth factors of the neurotrophin family are able to prevent the degeneration of striatal projection neurons, cell lines expressing brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), or neurotrophin-4/5 (NT-4/5) were grafted in the adult rat striatum before quinolinate injection. Three days after lesioning, ongoing cell death was assessed by in situ detection of DNA fragmentation. In animals grafted with the control cell line, quinolinate injection induced a gradual cell loss that was differentially prevented by intrastriatal grafting of BDNF-, NT-3-, or NT-415-secreting cells. Seven days after lesioning, we characterized striatal projection neurons that were protected by neurotrophins. Quinolinate injection, alone or in combination with the control cell line, induced a selective loss of striatal projection neurons. Grafting of a BDNF-secreting cell line pre-vented the loss of all types of striatal projection neurons analyzed. Glutamic acid decarboxylase 67-, preproenkephalin-, and preprotachykinin A- but not prodynorphin-expressing neurons were protected by grafting of NT-3- or NT-4/5-secreting cells but with less efficiency than the BDNF-secreting cells. Our findings show that neurotrophins are able to promote the survival of striatal projection neurons in vivo and suggest that BDNF might be beneficial for the treatment of striatonigral degenerative disorders, including Huntington's disease.

Topics: Animals; Brain-Derived Neurotrophic Factor; Cell Death; Cell Line; Cell Transplantation; Corpus Striatum; Disease Models, Animal; Enkephalins; Fibroblasts; Glutamate Decarboxylase; Huntington Disease; Isoenzymes; Male; Nerve Growth Factors; Neurons; Neurotrophin 3; Phosphorylation; Protein Precursors; Quinolinic Acid; Rats; Rats, Inbred F344; Receptor, trkB; Tachykinins

Neuroleptic-induced oral dyskinesias in rats, a putative analogue to human tardive dyskinesia, may be due to excitotoxic degeneration within the striatum. Haloperidol treatment for 34 weeks increased the optical density of preproenkephalin messenger RNA in individual striatal neurons and enkephalin peptide in the neuropil, regardless of the level of oral dyskinesia produced. However, using unbiased stereological methods, an increased number of striatal neurons expressing preproenkephalin messenger RNA was observed only in rats that did not develop pronounced oral dyskinesias during haloperidol treatment. Said in another manner, the haloperidol-treated animals that developed pronounced oral dyskinesias, failed to produce an increase in the number of neurons expressing preproenkephalin messenger RNA. These results indicate that the mechanism by which neuroleptics induce oral dyskinesias in rats, and perhaps tardive dyskinesia in humans, involves a functional disturbance or even damage to a subpopulation of enkephalinergic neurons in the striatum.

Topics: Animals; Corpus Striatum; Disease Models, Animal; Dyskinesia, Drug-Induced; Enkephalins; Fasciculation; Female; Gene Expression Regulation; Haloperidol; Humans; Motor Activity; Mouth; Neurons; Protein Precursors; Rats; Rats, Sprague-Dawley; RNA, Messenger; Transcription, Genetic; Videotape Recording

Rats with unilateral dopamine-denervating lesions sustained a 3-week treatment with a daily l-DOPA dose that is in the therapeutic range for Parkinson's disease. In most of the treated animals, chronic l-DOPA administration gradually induced abnormal involuntary movements affecting cranial, trunk, and limb muscles on the side of the body contralateral to the lesion. This effect was paralleled by an induction of FosB-like immunoreactive proteins in striatal subregions somatotopically related to the types of movements that had been elicited by l-DOPA. The induced proteins showed both regional and cellular colocalization with prodynorphin mRNA. Intrastriatal infusion of fosB antisense inhibited the development of dyskinetic movements that were related to the striatal subregion targeted and produced a local specific downregulation of prodynorphin mRNA. These data provide compelling evidence of a causal role for striatal fosB induction in the development of l-DOPA-induced dyskinesia in the rat and of a positive regulation of prodynorphin gene expression by FosB-related transcription factors.

Topics: Animals; Antisense Elements (Genetics); Bacterial Proteins; Biomarkers; Cell Count; Disease Models, Animal; Dyskinesias; Enkephalins; Female; Levodopa; Neostriatum; Nerve Degeneration; Neurons; Parkinsonian Disorders; Protein Precursors; Proto-Oncogene Proteins c-fos; Rats; Rats, Sprague-Dawley; RNA, Messenger; Substantia Nigra; Transcription Factors; Ventral Tegmental Area

Many studies have previously described changes in preproenkephalin-A (PPE-A) and preproenkephalin-B (PPE-B) gene expression in the striatum of the 6-hydroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (both with or without dopamine replacement treatment). To date, these studies have either taken the striatum as a whole or have focused on a single subregion of the striatum. However, the striatum is organized into anatomically discrete parallel circuits serving different functions (motor, associative, and limbic). We have therefore employed in situ hybridization to examine the detailed topography of changes in opioid precursor expression following dopamine depletion and subsequent treatment with apomorphine (5 mg/kg twice daily for 10 days). In the untreated 6-OHDA-lesioned striatum PPE-A expression was elevated only in the dorsal (sensorimotor) caudate-putamen. Following apomorphine treatment PPE-A mRNA levels were further raised in the sensorimotor striatum (

Topics: Animals; Apomorphine; Brain; Caudate Nucleus; Disease Models, Animal; Enkephalins; Gene Expression Regulation; In Situ Hybridization; Male; Motor Activity; Nucleus Accumbens; Organ Specificity; Oxidopamine; Parkinson Disease; Protein Precursors; Putamen; Rats; Rats, Sprague-Dawley; Reference Values; Time Factors

Levels of mRNA for c-fos and prodynorphin were studied by in situ hybridization in adjacent coronal sections taken from kindled rats 30-60 min after the last seizure. Within this time frame, expression of both genes was induced in multiple brain areas. Anatomical colocalization of the induced gene expressions was found in the hippocampus. Induction of c-fos in the dentate gyrus was bilateral and symmetrical in a subgroup of rats, ipsilateral in another subgroup and absent in a third subgroup. However, no relative increase was observed in the ipsilateral compared with the contralateral prodynorphin expression in the dentate gyrus when c-fos expression was induced ipsilaterally only. These observations suggest that, at variance with other experimental situations, Fos is not involved in the mechanisms of kindled seizure-induced activation of prodynorphin transcription in the rat forebrain.

Topics: Animals; Brain; Disease Models, Animal; Electric Stimulation; Enkephalins; Epilepsy, Temporal Lobe; Gene Expression; In Situ Hybridization; Kindling, Neurologic; Male; Nerve Tissue Proteins; Protein Precursors; Proto-Oncogene Proteins c-fos; Rats; Rats, Sprague-Dawley; RNA, Messenger

Several studies have demonstrated that cocaine increases preprodynorphin, c-fos, and zif/268 mRNAs in rat dorsal striatum. Multiple, closely spaced exposures to cocaine appear to elicit the greatest increases in dynorphin. However, the response of preproenkephalin, c-fos and zif/268 mRNAs to such a dosing regimen is unknown. Therefore, we used a 'binge' paradigm to evaluate changes in mRNA for preprodynorphin, preproenkephalin, c-fos and zif/268. Male Wistar rats received three hourly i.p. injections of saline or 10 or 20 mg/kg cocaine for 1, 5, or 10 days. Although cocaine-induced locomotor and stereotypical behaviors were significantly increased as compared to saline on days 1, 5 and 10, these behaviors were significantly less on day 10 than on days 1 and 5. One hour after the last injection on days 1, 5, or 10, the rats were anesthetized and decapitated for quantitative in situ hybridization histochemistry. C-fos mRNA was undetectable in all treatment groups whereas zif/268 mRNA in the dorsal striatum was increased in a dose-dependent manner (20 mg/kg > 10 mg/kg) but the intensity of hybridization signal decreased over time (1 day >> 5 days > 10 days) as compared to that in saline-treated controls. In contrast, 10 mg/kg cocaine binges caused an increase in preprodynorphin, but not preproenkephalin, mRNA in the dorsal, but not ventral, striatum in a time-dependent manner (day 10 >> day 5 > day 1) whereas 20 mg/kg cocaine binges caused an increase in striatal preprodynorphin that was greater on day 1 and day 5 than on day 10. These data indicate that (1) c-fos, zif/268 and preprodynorphin mRNAs are differentially regulated in dorsal striatum, (2) behavioral tolerance results from chronic binges with 10 and 20 mg/kg cocaine and (3) the preprodynorphin genomic response exhibits tolerance to chronic high dose, but not low dose, cocaine binges.

Topics: Analysis of Variance; Animals; Behavior, Animal; Cocaine; Corpus Striatum; Disease Models, Animal; DNA-Binding Proteins; Drug Administration Schedule; Dynorphins; Early Growth Response Protein 1; Enkephalins; Immediate-Early Proteins; Male; Nerve Tissue Proteins; Protein Precursors; Proto-Oncogene Proteins c-fos; Rats; Rats, Wistar; RNA, Messenger; Transcription Factors

Expression of the opioid peptides dynorphin and enkephalin is altered within the first 24 h after acutely induced seizures in certain experimental models of epilepsy. Using in situ hybridization, we examined the expression of prodynorphin and preproenkephalin messenger RNA acutely following induction of kindling with recurrent seizures and in two models of chronic temporal lobe epilepsy: (i) rats fully kindled with rapidly recurring hippocampal seizures; and (ii) rats surviving after self-sustaining limbic status epilepticus induced with focal electrical stimulation of the hippocampus. In naive animals, a ventral-dorsal gradient was identified in the expression of both prodynorphin and preproenkephalin messenger RNA in the dentate gyrus and expression of prodynorphin message was demonstrated for the first time in the ventral portion of cornu Ammonis regio superior. After stimulation producing rapidly recurring hippocampal seizures, acute decreases in prodynorphin messenger RNA were seen in the dentate gyrus and cornu Ammonis regio superior at 24 h after the last seizure. In contrast, increases in preproenkephalin messenger RNA expression were seen acutely in the dentate gyrus, with a decrease seen in the entorhinal cortex. The change in prodynorphin message expression in cornu Ammonis regio superior persisted in kindled animals that were studied after one month seizure-free period. There were no changes in preproenkephalin message in kindled animals studied after the one month seizure-free interval. No statistically significant changes were found for either prodynorphin or preproenkephalin message in the post-self-sustaining limbic status epilepticus group at one month following induced seizures. Acute changes in peptide expression may contribute to increased excitation in the dentate gyrus during induction of kindling, while the chronic change identified in cornu Ammonis regio superior may contribute directly to persistently increased excitability in this region.

Topics: Animals; Autoradiography; Basal Ganglia; Disease Models, Animal; Enkephalins; Epilepsy, Temporal Lobe; Gene Expression; Hippocampus; Male; Opioid Peptides; Protein Precursors; Rats; RNA, Messenger

In the present study, we examined the expression of the gene encoding for the production of the opioid peptide enkephalin in specific brain regions during hemorrhage in the conscious rat. Rats were subjected to fixed-volume (8 ml/300 g) bleeding via jugular vein within 10 min. Total RNA was isolated from rat brain tissue using the hot phenol/chloroform method. Preproenkephalin transcript levels were quantified using Northern blot hybridization of total RNA with alpha-32P-labeled cDNA probe, and autoradiograms were scanned with a densitometer. The ppENK transcript in midbrain was significantly elevated following one and two hours of hemorrhage compared to control group (P < 0.01). The ppENK transcript in spinal cord was also significantly increased after two hours of hemorrhage, when compared with control group (P < 0.01). In the brainstem, ppENK transcript was significantly decreased after both 1 and 2 hr of hemorrhage compared with control values (P < 0.01). These results indicate that hemorrhage acutely changes levels of the transcript of ppENK in specific brain regions. It is possible that the changes in brainstem ppENK transcript may be a mechanism by which certain inhibitory effects of enkephalins on important brain cardiovascular regulatory centers are mediated.

Topics: Animals; Brain Chemistry; Brain Stem; Disease Models, Animal; Enkephalins; Gene Expression Regulation; Hemorrhage; Male; Mesencephalon; Protein Precursors; Rats; Rats, Sprague-Dawley; RNA, Messenger; Spinal Cord