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Each cerebral hemisphere is functionally connected to the contralateral side of the body through the decussating neural tracts. The crossed neural pathways set a basis for contralateral effects of brain injury such hemiparesis and hemiplegia as it has been already noted by Hippocrates. Recent studies demonstrated that, in addition to neural mechanisms, the contralateral effects of brain lesions are mediated through the humoral pathway by neurohormones that produce either the left or right side-specific effects. The side-specific humoral signaling defines whether the left or right limbs are affected after a unilateral brain injury. The hormonal signals are released by the pituitary gland and may operate through their receptors that are lateralized in the spinal cord and involved in the side-specific control of symmetric neurocircuits innervating the left and right limbs. Identification of features and a proportion of neurological deficits transmitted by neurohormonal signals vs. those mediated by neural pathways is essential for better understanding of mechanisms of brain trauma and stroke and development of new therapies. In a biological context, the left–right side-specific neuroendocrine signaling may be fundamental for the control of the left- and right-sided processes in bilaterally symmetric animals.

In spite of its apparent symmetry, the spinal cord is asymmetric in its reflexes and gene expression patterns including leftward expression bias of the opioid and glutamate genes. To examine whether this is a general phenomenon for neurotransmitter and neurohormonal genes, we here characterized expression and co-expression (transcriptionally coordinated) patterns of genes of the renin-angiotensin system (RAS) that is involved in neuroprotection and pathological neuroplasticity in the left and right lumbar spinal cord. We also tested whether the RAS expression patterns were affected by unilateral brain injury (UBI) that rewired lumbar spinal neurocircuits. The left and right halves of the lumbar spinal cord were analysed in intact rats, and rats with left- or right-sided unilateral cortical injury, and left- or right-sided sham surgery. The findings were (i) lateralized expression of the RAS genes Ace, Agtr2 and Ren with higher levels on the left side; (ii) the asymmetry in coordination of the RAS gene expression that was stronger on the right side; (iii) the decay in coordination of co-expression of the RAS and neuroplasticity-related genes induced by the right-side but not left-side sham surgery and UBI; and (iv) the UBI-induced shift to negative regulatory interactions between RAS and neuroplasticity-related genes on the contralesional spinal side. Thus, the RAS genes may be a part of lateralized gene co-expression networks and have a role in a side-specific regulation of spinal neurocircuits.

Neuropeptides serve as neurohormones and local paracrine regulators that control neural networks regulating behavior, endocrine system and sensorimotor functions. Their expression is characterized by exceptionally restricted profiles. Circuit-specific and adaptive expression of neuropeptide genes may be defined by transcriptional and epigenetic mechanisms controlled by cell type and subtype sequence-specific transcription factors, insulators and silencers. The opioid peptide dynorphins play a critical role in neurological and psychiatric disorders, pain processing and stress, while their mutations cause profound neurodegeneration in the human brain. In this review, we focus on the prodynorphin gene as a model for the in-depth epigenetic and transcriptional analysis of expression of the neuropeptide genes. Prodynorphin studies may provide a framework for analysis of mechanisms relevant for regulation of neuropeptide genes in normal and pathological human brain.

Traumatic brain injury and stroke result in hemiplegia, hemiparesis, and asymmetry in posture. The effects are mostly contralateral; however, ipsilesional deficits may also develop. We here examined whether ablation brain injury and controlled cortical impact (CCI), a rat model of clinical focal traumatic brain injury, both centered over the left or right sensorimotor cortex, induced hindlimb postural asymmetry (HL-PA) with contralesional or ipsilesional limb flexion. The contralesional hindlimb was flexed after left or right side ablation injury. In contrast, both the left and right CCI unexpectedly produced HL-PA with flexion on left side. The flexion persisted after complete spinal cord transection suggesting that CCI triggered neuroplastic processes in lumbar neural circuits enabling asymmetric muscle contraction. Left limb flexion was exhibited under pentobarbital anesthesia. However, under ketamine anesthesia, the body of the left and right CCI rats bent laterally in the coronal plane to the ipsilesional side suggesting that the left and right injury engaged mirror-symmetrical motor pathways. Thus, the effects of the left and right CCI on HL-PA were not mirror-symmetrical in contrast to those of the ablation brain injury, and to the left and right CCI produced body bending. Ipsilateral effects of the left CCI on HL-PA may be mediated by a lateralized motor pathway that is not affected by the left ablation injury. Alternatively, the left-side-specific neurohormonal mechanism that signals from injured brain to spinal cord may be activated by both the left and right CCI but not by ablation injury. Supplementary Information The online version contains supplementary material available at 10.1007/s00221-021-06118-4.

Effects of environmental factors may be transmitted to the following generation, and cause neuropsychiatric disorders including depression, anxiety, and posttraumatic stress disorder in the offspring. Enhanced synaptic plasticity induced by environmental enrichment may be also transmitted. We here test the hypothesis that the effects of brain injury in pregnant animals may produce neurological deficits in the offspring. Unilateral brain injury (UBI) by ablation of the hindlimb sensorimotor cortex in pregnant rats resulted in the development of hindlimb postural asymmetry (HL-PA), and impairment of balance and coordination in beam walking test in the offspring. The offspring of rats with the left UBI exhibited HL-PA before and after spinal cord transection with the contralesional (i.e., right) hindlimb flexion. The right UBI caused the offspring to develop HL-PA that however was cryptic and not-lateralized; it was evident only after spinalization, and was characterized by similar occurrence of the ipsi- and contralesional hindlimb flexion. The HL-PA persisted after spinalization suggesting that the asymmetry was encoded in lumbar spinal neurocircuits that control hindlimb muscles. Balance and coordination were affected by the right UBI but not the left UBI. Thus, the effects of a unilateral brain lesion in pregnant animals may be intergenerationally transmitted, and this process may depend on the side of brain injury. The results suggest the existence of left-right side-specific mechanisms that mediate transmission of the lateralized effects of brain trauma from mother to fetus.

Alcohol misuse is a major public health problem originating from genetic and environmental risk factors. Alterations in the brain epigenome may orchestrate changes in gene expression that lead to alcohol misuse and dependence. Through epigenome-wide association analysis of DNA methylation from human brain tissues, we identified a differentially methylated region, DMR-DLGAP2, associated with alcohol dependence. Methylation within DMR-DLGAP2 was found to be genotype-dependent, allele-specific and associated with reward processing in brain. Methylation at the DMR-DLGAP2 regulated expression of DLGAP2 in vitro, and Dlgap2-deficient mice showed reduced alcohol consumption compared with wild-type controls. These results suggest that DLGAP2 may be an interface for genetic and epigenetic factors controlling alcohol use and dependence.

Spinal cord as a connection between brain and peripheral nervous system is an essential material for studying neural transmission, especially in pain-related research. This study was the first to investigate pain-related neuropeptide distribution in rat spinal cord using a matrix-assisted laser desorption ionization-time of flight imaging mass spectrometry (MALDI TOF MS) approach. The imaging workflow was evaluated and showed that MALDI TOF MS provides efficient resolution and robustness for neuropeptide imaging in rat spinal cord tissue. The imaging result showed that in naive rat spinal cord the molecular distribution of haeme, phosphatidylcholine, substance P and thymosin beta 4 were well in line with histological features. Three groups of pain-related neuropeptides, which are cleaved from prodynorphin, proenkephalin and protachykinin-1 proteins were detected. All these neuropeptides were found predominantly localized in the dorsal spinal cord and each group had unique distribution pattern. This study set the stage for future MALDI TOF MS application to elucidate signalling mechanism of pain-related diseases in small animal models.

Background: Neuropeptide precursors are traditionally viewed as proteins giving rise to small neuropeptide molecules. Prodynorphin (PDYN) is the precursor protein to dynorphins, endogenous ligands for the kappa-opioid receptor. Alternative mRNA splicing of neuropeptide genes may regulate cell- and tissue-specific neuropeptide expression and produce novel protein isoforms. We here searched for novel PDYN mRNA and their protein product in the human brain.Methods: Novel PDYN transcripts were identified using nested PCR amplification of oligo(dT) selected full-length capped mRNA. Gene expression was analyzed by qRT-PCR, PDYN protein by western blotting and confocal imaging, dynorphin peptides by radioimmunoassay. Neuronal nuclei were isolated using fluorescence activated nuclei sorting (FANS) from postmortem human striatal tissue. lmmunofluorescence staining and con focal microscopy was performed for human caudate nucleus.Results: Two novel human PDYN mRNA splicing variants were identified. Expression of one of them was confined to the striatum where its levels constituted up to 30% of total PDYN mRNA. This transcript may be translated into ASP-PDYN protein lacking 13 N-terminal amino acids, a fragment of signal peptide (SP). Delta SP-PDYN was not processed to mature dynorphins and surprisingly, was targeted to the cell nuclei in a model cellular system. The endogenous PDYN protein was identified in the cell nuclei in human striatum by western blotting of isolated neuronal nuclei, and by confocal imaging.Conclusions and general significance: High levels of alternatively spliced Delta SP-PDYN mRNA and nuclear localization of PDYN protein suggests a nuclear function for this isoform of the opioid peptide precursor in human striatum. (C) 2016 Elsevier B.V. All rights reserved.

Brain region-specific expression of proteolytic enzymes can control the biological activity of endogenous neuropeptides and has recently been targeted for the development of novel drugs, for neuropathic pain, cancer, and Parkinson's disease. Rapid and sensitive analytical methods to profile modulators of enzymatic activity are important for finding effective inhibitors with high therapeutic value. Combination of in situ enzyme histochemistry with MALDI imaging mass spectrometry allowed developing a highly sensitive method for analysis of brain-area specific neuropeptide conversion of synthetic and endogenous neuropeptides, and for selection of peptidase inhibitors that differentially target conversion enzymes at specific anatomical sites. Conversion and degradation products of Dynorphin B as model neuropeptide and effects of peptidase inhibitors applied to native brain tissue sections were analyzed at different brain locations. Synthetic dynorphin B (2 pmol) was found to be converted to the N-terminal fragments on brain sections whereas fewer C-terminal fragments were detected. N-ethylmaleimide (NEM), a non-selective inhibitor of cysteine peptidases, almost completely blocked the conversion of dynorphin B to dynorphin B(1-6; Leu-Enk-Arg), (1-9), (2-13), and (7-13). Proteinase inhibitor cocktail, and also incubation with acetic acid displayed similar results. Bioconversion of synthetic dynorphin B was region-specific producing dynorphin B(1-7) in the cortex and dynorphin B (2-13) in the striatum. Enzyme inhibitors showed region-and enzyme-specific inhibition of dynorphin bioconversion. Both phosphoramidon (inhibitor of the known dynorphin converting enzyme neprilysin) and opiorphin (inhibitor of neprilysin and aminopeptidase N) blocked cortical bioconversion to dynorphin B(1-7), wheras only opiorphin blocked striatal bioconversion to dynorphin B(2-13). This method may impact the development of novel therapies with aim to strengthen the effects of endogenous neuropeptides under pathological conditions such as chronic pain. Combining histochemistry and MALDI imaging MS is a powerful and sensitive tool for the study of inhibition of enzyme activity directly in native tissue sections. (C) 2016 The Authors. Published by Elsevier Inc.

Nociceptive discharges caused by the unilateral tissue damage are processed in the spinal cord by both ipsi- and contralateral neuronal circuits. The mechanisms of the neurotransmitter control of this bilateral excitation spread is poorly understood. Spinally administered opiates are known to suppress nociceptive transmission and nociceptive withdrawal reflexes. Here we investigated whether three major types of opioid receptors are involved in the bilateral control of the spinal nociceptive sensorimotor processing. Effects of the µ-, δ- and κ-opioid receptor agonists on the ipsi- and contralateral nociceptive reflexes were studied by recording slow ventral root potentials in an isolated spinal cord preparation of the new-born rat. Absolute levels of expression of the opioid genes were analyzed by the droplet digital PCR. Ipsi- and contralateral slow ventral root potentials were most strongly suppressed by the µ-opioid receptor agonist DAMGO, by 63% and 85%, followed by the κ-opioid receptor agonist U-50488H, by 44% and 73%, and δ-opioid receptor agonist leucine-enkephalin, by 27% and 49%, respectively. All these agonists suppressed stronger contra- than ipsilateral responses. Naloxone prevented effects of the agonists indicating that they act through opioid receptors, which, as we show, are expressed in the neonatal spinal cord at the levels similar to those in adults. Thus, opioid receptor agonists suppress the segmental nociceptive reflexes. Stronger contralateral effects suggest that the endogenous opioid system regulates sensorimotor processing in the spinal commissural pathways. These effects of opioids may be relevant for treatment of symmetric clinical pain symptoms caused by unilateral tissue injury.

The endogenous opioid system (EOS) controls the processing of nociceptive stimuli and is a pharmacological target for opioids. Alterations in expression of the EOS genes under neuropathic pain condition may account for low efficacy of opioid drugs. We here examined whether EOS expression patterns are altered in the lumbar spinal cord of the rats with spinal nerve ligation (SNL) as a neuropathic pain model. Effects of the left- and right-side SNL on expression of EOS genes in the ipsi- and contralateral spinal domains were analysed. The SNL-induced changes were complex and different between the genes; between the dorsal and ventral spinal domains; and between the left and right sides of the spinal cord. Prodynorphin (Pdyn) expression was upregulated in the ipsilateral dorsal domains by each the left and right-side SNL, while changes in expression of μ-opioid receptor (Oprm1) and proenkephalin (Penk) genes were dependent on the SNL side. Changes in expression of the Pdyn and κ-opioid receptor (Oprk1) genes were coordinated between the ipsi- and contralateral sides. Withdrawal response thresholds, indicators of mechanical allodynia correlated negatively with Pdyn expression in the right ventral domain after right side SNL. These findings suggest multiple roles of the EOS gene products in spinal sensitization and changes in motor reflexes, which may differ between the left and right sides.

Here, we describe a molecular switch associated with opioid receptors-linked signalling cascades that provides a dual opioid control over P2X3 purinoceptor in sensory neurones. Leu-enkephalin inhibited P2X3-mediated currents with IC50 ~10 nM in ~25 % of small nociceptive rat dorsal root ganglion (DRG) neurones. In contrast, in neurones pretreated with pertussis toxin leu-enkephalin produced stable and significant increase of P2X3 currents. All effects of opioid were abolished by selective μ-opioid receptor antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), nonselective inhibitor naloxone, and by PLC inhibitor U73122. Thus, we discovered a dual link between purinoceptors and μ-opioid receptors: the latter exert both inhibitory (pertussis toxin-sensitive) and stimulatory (pertussis toxin-insensitive) actions on P2X3 receptors through phospholipase C (PLC)-dependent pathways. This dual opioid control of P2X3 receptors may provide a molecular explanation for dichotomy of opioid therapy. Pharmacological control of this newly identified facilitation/inhibition switch may open new perspectives for the adequate medical use of opioids, the most powerful pain-killing agents known today.

BACKGROUND: Nociceptin is a key regulator linking environmental stress and alcohol drinking. In a genome-wide methylation analysis, we recently identified an association of a methylated region in the OPRL1 gene with alcohol-use disorders. METHODS: Here, we investigate the biological basis of this observation by analysing psychosocial stressors, methylation of the OPRL1 gene, brain response during reward anticipation and alcohol drinking in 660 fourteen-year-old adolescents of the IMAGEN study. We validate our findings in marchigian sardinian (msP) alcohol-preferring rats that are genetically selected for increased alcohol drinking and stress sensitivity. RESULTS: We found that low methylation levels in intron 1 of OPRL1 are associated with higher psychosocial stress and higher frequency of binge drinking, an effect mediated by OPRL1 methylation. In individuals with low methylation of OPRL1, frequency of binge drinking is associated with stronger BOLD response in the ventral striatum during reward anticipation. In msP rats, we found that stress results in increased alcohol intake and decreased methylation of OPRL1 in the nucleus accumbens. CONCLUSIONS: Our findings describe an epigenetic mechanism that helps to explain how psychosocial stress influences risky alcohol consumption and reward processing, thus contributing to the elucidation of biological mechanisms underlying risk for substance abuse.

The myriad proteins may be involved in the mechanisms underlying the development and maintenance of neuropathic pain, an extremely disabling condition that originates from pathology of the nervous system. To address the mechanisms, we here analyzed proteins and cellular networks in the dorsal spinal cord mediating pain processing in a well-established rat model of neuropathic pain induced by spinal nerve ligation (SNL). Labeling-based proteomic methods together with high-resolution mass spectrometry for proteome analysis were applied. 38 proteins including synapsin 1 and microtubule-associated protein 2 were identified as differently expressed in the SNL group. Pathway analysis suggests that maladaptive changes in the levels of these proteins may contribute to abnormal synaptic transmission and neuronal intracellular signaling underlying the onset and development of neuropathic pain.

Aims In rheumatoid arthritis (RA), pain and inflammation are initial symptoms followed by various degrees of bone and cartilage destruction. Previously, we have shown that reversible proteasome inhibitor MG132 attenuates pain and joint inflammation in a rat model of adjuvant-arthritis. Our present study aims to study the effects of MG132 on molecular changes in the dorsal root ganglia (DRG) and in the spinal cord (SC) using the same animal model. Methods Arthritis was induced by heat-killed Mycobacterium butyricum in rats. The expression of substance P (SP) was analyzed by quantitative reverse transcription polymerase chain reaction and immunohistochemistry in DRG and in the SC. The nuclear factor-κB (NF-κB) DNA-binding activity in the SC was analyzed by electromobility shift assay. Results Arthritic rats treated daily with MG132 demonstrated a marked reduction of SP gene expression in the DRG and number of SP-positive cells was reduced. In the spinal cord of arthritic rats elevated SP messenger RNA levels were normalized and NF-κB-DNA-binding activity was down-regulated in arthritic rats treated with MG132. Conclusion Our results indicate that proteasome inhibitor MG132 attenuates pain in adjuvant arthritis by targeting the sensory neuropeptide substance P in the peripheral and central nervous systems. These effects may be mediated through the inhibition of NF-κB activation.

Excessive alcohol consumption is a major public health problem worldwide. Although drinking habits are known to be inherited, few genes have been identified that are robustly linked to alcohol drinking. We conducted a genome-wide association metaanalysis and replication study among >105,000 individuals of European ancestry and identified beta-Klotho (KLB) as a locus associated with alcohol consumption (rs11940694; P = 9.2 x 10(-12)). beta-Klotho is an obligate coreceptor for the hormone FGF21, which is secreted from the liver and implicated in macronutrient preference in humans. We show that brain-specific beta-Klotho KO mice have an increased alcohol preference and that FGF21 inhibits alcohol drinking by acting on the brain. These data suggest that a liver-brain endocrine axis may play an important role in the regulation of alcohol drinking behavior and provide a unique pharmacologic target for reducing alcohol consumption.

Regulation of the formation and rewiring of neural circuits by neuropeptides may require coordinated production of these signaling molecules and their receptors that may be established at the transcriptional level. Here, we address this hypothesis by comparing absolute expression levels of opioid peptides with their receptors, the largest neuropeptide family, and by characterizing coexpression (transcriptionally coordinated) patterns of these genes. We demonstrated that expression patterns of opioid genes highly correlate within and across functionally and anatomically different areas. Opioid peptide genes, compared with their receptor genes, are transcribed at much greater absolute levels, which suggests formation of a neuropeptide cloud that covers the receptor-expressed circuits. Surprisingly, we found that both expression levels and the proportion of opioid receptors are strongly lateralized in the spinal cord, interregional coexpression patterns are side specific, and intraregional coexpression profiles are affected differently by left- and right-side unilateral body injury. We propose that opioid genes are regulated as interconnected components of the same molecular system distributed between distinct anatomic regions. The striking feature of this system is its asymmetric coexpression patterns, which suggest side-specific regulation of selective neural circuits by opioid neurohormones.-Kononenko, O., Galatenko, V., Andersson, M., Bazov, I., Watanabe, H., Zhou, X. W., Iatsyshyna, A., Mityakina, I., Yakovleva, T., Sarkisyan, D., Ponomarev, I., Krishtal, O., Marklund, N., Tonevitsky, A., Adkins, D. L., Bakalkin, G. Intra- and interregional coregulation of opioid genes: broken symmetry in spinal circuits.

This review represents the focus of a symposium that was presented at the “Alcoholism and Stress: A Framework for Future Treatment Strategies” conference in Volterra, Italy on May 3–6, 2011 and organized / chaired by Dr. Brendan M. Walker. The primary goal of the symposium was to evaluate and disseminate contemporary findings regarding the emerging role of kappa-opioid receptors (KORs) and their endogenous ligands dynorphins (DYNs) in the regulation of escalated alcohol consumption, negative affect and cognitive dysfunction associated with alcohol dependence, as well as DYN / KOR mediation of the effects of chronic stress on alcohol reward and seeking behaviors. Dr. Glenn Valdez described a role for KORs in the anxiogenic effects of alcohol withdrawal. Dr. Jay McLaughlin focused on the role of KORs in repeated stress-induced potentiation of alcohol reward and increased alcohol consumption. Dr. Brendan Walker presented data characterizing the effects of KOR antagonism within the extended amygdala on withdrawal-induced escalation of alcohol self-administration in dependent animals. Dr. Georgy Bakalkin concluded with data indicative of altered DYNs and KORs in the prefrontal cortex of alcohol dependent humans that could underlie diminished cognitive performance. Collectively, the data presented within this symposium identified the multifaceted contribution of KORs to the characteristics of acute and chronic alcohol-induced behavioral dysregulation and provided a foundation for the development of pharmacotherapeutic strategies to treat certain aspects of alcohol use disorders.

Osteoarthritis is a degenerative joint disease with pain and loss of joint function as major pathological features. Recent studies show that proteasome inhibitors reduce pain in various pathological conditions. We evaluated the effects of MG132, a reversible proteasome inhibitor on pain and joint destruction in a rat model of osteoarthritis. Osteoarthritis was induced by intraarticular injection of monosodium iodoacetate into the rat knee. Knee joint stiffness was scored and nociception was evaluated by mechanical pressure applied to the respective hind paw. Knee joint destruction was assessed by radiological and histological analyses. Expression of matrix metalloproteinase-3 (MMP-3) was analyzed by quantitative reverse transcription polymerase chain reaction in the knee articular cartilage. Expression of substance P (SP) and calcitonin gene-related peptide (CGRP) was studied in the dorsal root ganglia (L4-L6) by quantitative reverse transcription polymerase chain reaction and in the knee joints by immunohistochemistry. Our results indicate that daily treatment of osteoarthritic rats with MG132 significantly increases their mobility while the swelling, pain thresholds, and pathological features of the affected joints were reduced. Furthermore, the upregulated expression of MMP-3, SP, and CGRP in the arthritic rats was normalized by MG132 administration. We conclude that the proteasome inhibitor MG132 reduces pain and joint destruction, probably by involving the peripheral nervous system, and that changes in SP and CGRP expression correlate with alterations in behavioural responses. Our findings suggest that nontoxic proteasome inhibitors may represent a novel pharmacotherapy for osteoarthritis.

Alcohol consumption is a moderately heritable trait, but the genetic basis in humans is largely unknown, despite its clinical and societal importance. We report a genome-wide association study meta-analysis of ∼2.5 million directly genotyped or imputed SNPs with alcohol consumption (gram per day per kilogram body weight) among 12 population-based samples of European ancestry, comprising 26,316 individuals, with replication genotyping in an additional 21,185 individuals. SNP rs6943555 in autism susceptibility candidate 2 gene ( AUTS2 ) was associated with alcohol consumption at genome-wide significance ( P = 4 × 10 −8 to P = 4 × 10 −9 ). We found a genotype-specific expression of AUTS2 in 96 human prefrontal cortex samples ( P = 0.026) and significant ( P < 0.017) differences in expression of AUTS2 in whole-brain extracts of mice selected for differences in voluntary alcohol consumption. Down-regulation of an AUTS2 homolog caused reduced alcohol sensitivity in Drosophila ( P < 0.001). Our finding of a regulator of alcohol consumption adds knowledge to our understanding of genetic mechanisms influencing alcohol drinking behavior.

The essential properties of the primary structure of regulatory peptides, i.e. amino acid residues and their combinations, which are characteristic of the whole population of regulatory peptides, have been revealed using statistical methodology. These properties are as follows: increased content of certain residues (Gly, Pro, Phe, Arg, Tyr, Met and Trp) as well as an increased rate of occurrence of certain pairs of residue as compared with proteins, a random sequence of residues and "nonregulatory" peptides. By representing regulatory peptides as a sequence of hydrophobic (2 types) and hydrophilic (3 types) segments, the pattern for alternation of these segments in regulatory peptides has been determined. The segments were classified into 5 types according to the peculiarities of mutual localization of hydrophobic and hydrophilic residues within the primary structure of regulatory peptides. As compared with proteins, "nonregulatory" peptides and a random sequence of segments, regulatory peptides were characterized by an increased frequency of 4 particular pairs of segments among 12 theoretically possible pairs. These 4 pairs are fragments of the periodic segment sequence with periods of 4 segments. The revealed pattern indicates that there exists a general principle of the regulatory peptide primary structure organization and possibly a common type of the regulatory peptides flexible peptide chain folding at the ligand-receptor complex formation.

Activation of dopamine (DA) D1 receptors (D1Rs) in the nucleus accumbens (Acb) markedly affects the levels of prodynorphin, the precursor of aversion-associated dynorphin peptides. The location of prodynorphin, specifically as related to the dopaminergic inputs and D1Rs in the Acb, is fundamental for establishing the physiologically relevant sites. To determine these sites, we examined the electron microscopic dual-immunolabeling of prodynorphin and D1R or tyrosine hydroxylase (TH), a marker of catecholamine terminals in the rat Acb shell. This subregion is targeted by mesolimbic dopaminergic inputs affecting reward-aversion responses and locomotor activity. Prodynorphin was prominently localized to large (100-200 nm) granular aggregates in somatodendritic and axonal profiles, some of which expressed dynorphin A/B. In somata and dendrites, prodynorphin was often found in punctate clusters in the cytoplasm. Of the total prodynorphin-labeled dendrites, approximately 63% expressed D1Rs, which were largely located on the plasma membranes. In comparison with dendrites, many more axon terminals contained prodynorphin, although only 15% of these terminals contained D1R-labeling. Prodynorphin terminals formed symmetric synapses with D1R-labeled or unlabeled dendrites, and also apposed TH-containing axon terminals. Our results provide ultrastructural evidence that in the Acb shell, the prodynorphin is available for cleavage to physiologically active peptides in both dendrites and terminals of neurons that express D1Rs. They also indicate that dynorphin peptides have distributions that would enable their participation in modulation of DA release or D1R-mediated postsynaptic responses in Acb shell neurons.

Effects of big dynorphin (Big Dyn), a prodynorphin-derived peptide consisting of dynorphin A (Dyn A) and dynorphin B (Dyn B) on memory function, anxiety, and locomotor activity were studied in mice and compared to those of Dyn A and Dyn B. All peptides administered i.c.v. increased step-through latency in the passive avoidance test with the maximum effective doses of 2.5, 0.005, and 0.7 nmol/animal, respectively. Effects of Big Dyn were inhibited by MK 801 (0.1 mg/kg), an NMDA ion-channel blocker whereas those of dynorphins A and B were blocked by the kappa-opioid antagonist nor-binaltorphimine (6 mg/kg). Big Dyn (2.5 nmol) enhanced locomotor activity in the open field test and induced anxiolytic-like behavior both effects blocked by MK 801. No changes in locomotor activity and no signs of anxiolytic-like behavior were produced by dynorphins A and B. Big Dyn (2.5 nmol) increased time spent in the open branches of the elevated plus maze apparatus with no changes in general locomotion. Whereas dynorphins A and B (i.c.v., 0.05 and 7 nmol/animal, respectively) produced analgesia in the hot-plate test Big Dyn did not. Thus, Big Dyn differs from its fragments dynorphins A and B in its unique pattern of memory enhancing, locomotor- and anxiolytic-like effects that are sensitive to the NMDA receptor blockade. The findings suggest that Big Dyn has its own function in the brain different from those of the prodynorphin-derived peptides acting through kappa-opioid receptors.

Dynorphins, endogenous kappa-opioid agonists widely expressed in the central nervous system, have been reported to increase following diverse pathophysiological processes, including excitotoxicity, chronic inflammation, and traumatic injury. These peptides have been implicated in cognitive impairment, especially that associated with aging. To determine whether absence of dynorphin confers any beneficial effect on spatial learning and memory, knockout mice lacking the coding exons of the gene encoding its precursor prodynorphin (Pdyn) were tested in a water maze task. Learning and memory assessment using a 3-day water maze protocol demonstrated that aged Pdyn knockout mice (13-17 months) perform comparatively better than similarly aged wild-type (WT) mice, based on acquisition and retention probe trial indices. There was no genotype effect on performance in the cued version of the swim task nor on average swim speed, suggesting the observed genotype effects are likely attributable to differences in cognitive rather than motor function. Young (3-6 months) mice performed significantly better than aged mice, but in young mice, no genotype difference was observed. To investigate the relationship between aging and brain dynorphin expression in mice, we examined dynorphin peptide levels at varying ages in hippocampus and frontal cortex of WT 129SvEv mice. Quantitative radioimmunoassay demonstrated that dynorphin A levels in frontal cortex, but not hippocampus, of 12- and 24-month mice were significantly elevated compared to 3-month mice. Although the underlying mechanisms have yet to be elucidated, the results suggest that chronic increases in endogenous dynorphin expression with age, especially in frontal cortex, may adversely affect learning and memory.

To understand processes in a living cell, sophisticated and creative approaches are required that can be used for gathering quantitative information about large number of components interacting across temporal and spatial scales without major disruption of the integral network of processes. A physical method of analysis that can meet these requirements is fluorescence correlation spectroscopy (FCS), which is an ultrasensitive and non-invasive detection method capable of single-molecule and real-time resolution. Since its introduction about 3 decades ago, this until recently emerging technology has reached maturity. As commercially built equipment is now available, FCS is extensively applied for extracting biological information from living cells unattainable by other methods, and new biological concepts are formulated based on findings by FCS. In this review, we focus on examples in the field of molecular cellular biology. The versatility of the technique in this field is illustrated in studies of single-molecule dynamics and conformational flexibility of proteins, and the relevance of conformational flexibility for biological functions regarding the multispecificity of antibodies, modulation of activity of C5a receptors in clathrin-mediated endocytosis and multiplicity of functional responses mediated by the p53 tumor suppressor protein; quantitative characterization of physicochemical properties of the cellular interior; protein trafficking; and ligand-receptor interactions. FCS can also be used to study cell-to-cell communication, here exemplified by clustering of apoptotic cells via bystander killing by hydrogen peroxide.

Intrathecal (i.t.) administration into mice of N-ethylmaleimide (NEM), a cysteine protease inhibitor, produced a characteristic behavioral response, the biting and/or licking of the hindpaw and the tail along with slight hindlimb scratching directed toward the flank. The behavior induced by NEM was inhibited by the intraperitoneal injection of morphine. We have recently reported that dynorphin A and, more potently big dynorphin, consisting of dynorphins A and B, produce the same type of nociceptive response whereas dynorphin B does not [Tan-No K, Esashi A, Nakagawasai O, Niijima F, Tadano T, Sakurada C, Sakurada T, Bakalkin G, Terenius L, Kisara K. Intrathecally administered big dynorphin, a prodynorphin-derived peptide, produces nociceptive behavior through an N-methyl-d-aspartate receptor mechanism. Brain Res 2002;952:7-14]. The NEM-induced nociceptive behavior was inhibited by pretreatment with dynorphin A- or dynorphin B-antiserum and each antiserum also reduced the nociceptive effects of i.t.-injected synthetic big dynorphin. The characteristic NEM-evoked response was not observed in prodynorphin knockout mice. Naloxone, an opioid receptor antagonist, had no effects on the NEM-induced behavior. Ifenprodil, arcaine and agmatine, antagonists at the polyamine recognition site on the N-methyl-D-aspartate (NMDA) receptor ion-channel complex, and MK-801, an NMDA ion-channel blocker inhibited the NEM-induced effects. Ro25-6981, an antagonist of the NMDA receptor subtype containing NR2B subunit was not active. NEM completely inhibited degradation of dynorphin A by soluble and particulate fractions of mouse spinal cord. Collectively, the results demonstrate that endogenous prodynorphin-derived peptides are pronociceptive in uninjured animals, and required for the NEM-induced behavior. The NEM effects may be mediated through inhibition of the degradation of endogenous dynorphins, presumably big dynorphin that in turn activates the NMDA receptor ion-channel complex by acting on the polyamine recognition site.

A novel soluble non-opioid dynorphin A-binding factor (DABF) was identified and characterized in neuronal cell lines, rat spinal cord, and brain. DABF binds dynorphin A(1–17), dynorphin A(2–17), and the 32 amino acid prodynorphin fragment big dynorphin consisting of dynorphin A and B, but not other opioid and non-opioid peptides, opiates, and benzomorphans. The IC50 for dynorphin A(1–17), dynorphin A(2–17), and big dynorphin is in the 5–10 nM range. Using dynorphin A and big dynorphin fragments a binding epitope was mapped to dynorphin A(6–13). DABF has a molecular mass of about 70 kDa. SH-groups are apparently involved in the binding of dynorphin A since p-hydroxy-mercuribenzoic acid inhibited this process. Upon interaction with DABF dynorphin A was converted into Leu-enkephalin, which remained bound to the protein. These data suggest that DABF functions as an oligopeptidase that forms stable and specific complexes with dynorphin A. The presence of DABF in brain structures and other tissues with low level of prodynorphin expression suggests that DABF as an oligopeptidase may degrade other peptides. Dynorphin A at the sites of its release in the CNS may attenuate this degradation as a competitor when it specifically binds to the enzyme.

The tumor suppressor protein p53 regulates gene transcription through binding to specific DNA-target sites. We here demonstrate that a subset of these sites is targeted by another DNA-binding factor. Binding specificity, reactivity with specific antibodies, and experiments with purified protein identified the factor as the multifunctional transcription regulator YY1. The YY1 core binding sequence ACAT is present in the center of p53-half-binding sites in the p21 and GADD45 genes regulating growth arrest and DNA repair, respectively, but is absent in those of the Bax gene critical for apoptosis. In transfection experiments YY1 inhibits p53-activated transcription from the p53-binding site that contains the ACAT sequence. YY1 and p53 are colocalized around the nucleoli and in discrete nuclear domains in PC12 cells undergoing apoptosis. YY1 might attenuate p53-dependent transcription from a subset of p53-target genes and this may be relevant for directing cells either to growth arrest or apoptosis upon p53 activation.

Neuropeptides induce signal transduction across the plasma membrane by acting through cell-surface receptors. The dynorphins, endogenous ligands for opioid receptors, are an exception; they also produce non-receptor-mediated effects causing pain and neurodegeneration. To understand non-receptor mechanism(s), we examined interactions of dynorphins with plasma membrane. Using fluorescence correlation spectroscopy and patch-clamp electrophysiology, we demonstrate that dynorphins accumulate in the membrane and induce a continuum of transient increases in ionic conductance. This phenomenon is consistent with stochastic formation of giant (~2.7 nm estimated diameter) unstructured non-ion-selective membrane pores. The potency of dynorphins to porate the plasma membrane correlates with their pathogenic effects in cellular and animal models. Membrane poration by dynorphins may represent a mechanism of pathological signal transduction. Persistent neuronal excitation by this mechanism may lead to profound neuropathological alterations, including neurodegeneration and cell death.

Item does not contain fulltext Spinocerebellar ataxia type 23 is caused by mutations in PDYN, which encodes the opioid neuropeptide precursor protein, prodynorphin. Prodynorphin is processed into the opioid peptides, alpha-neoendorphin, and dynorphins A and B, that normally exhibit opioid-receptor mediated actions in pain signalling and addiction. Dynorphin A is likely a mutational hotspot for spinocerebellar ataxia type 23 mutations, and in vitro data suggested that dynorphin A mutations lead to persistently elevated mutant peptide levels that are cytotoxic and may thus play a crucial role in the pathogenesis of spinocerebellar ataxia type 23. To further test this and study spinocerebellar ataxia type 23 in more detail, we generated a mouse carrying the spinocerebellar ataxia type 23 mutation R212W in PDYN. Analysis of peptide levels using a radioimmunoassay shows that these PDYN(R212W) mice display markedly elevated levels of mutant dynorphin A, which are associated with climber fibre retraction and Purkinje cell loss, visualized with immunohistochemical stainings. The PDYN(R212W) mice reproduced many of the clinical features of spinocerebellar ataxia type 23, with gait deficits starting at 3 months of age revealed by footprint pattern analysis, and progressive loss of motor coordination and balance at the age of 12 months demonstrated by declining performances on the accelerating Rotarod. The pathologically elevated mutant dynorphin A levels in the cerebellum coincided with transcriptionally dysregulated ionotropic and metabotropic glutamate receptors and glutamate transporters, and altered neuronal excitability. In conclusion, the PDYN(R212W) mouse is the first animal model of spinocerebellar ataxia type 23 and our work indicates that the elevated mutant dynorphin A peptide levels are likely responsible for the initiation and progression of the disease, affecting glutamatergic signalling, neuronal excitability, and motor performance. Our novel mouse model defines a critical role for opioid neuropeptides in spinocerebellar ataxia, and suggests that restoring the elevated mutant neuropeptide levels can be explored as a therapeutic intervention.

Previous work has shown that oligodendrocytes (OLs) express both μ- and κ-opioid receptors. In developing OLs, μ receptor activation increases OL proliferation, while the κ-antagonist nor-binaltorphimine (NorBNI) affects OL differentiation. Because exogenous opioids were not present in our defined culture medium, we hypothesized that NorBNI blocked endogenous opioids produced by the OLs themselves. To test this, intact and partially processed proenkephalin and prodynorphin-derived peptides were assessed in OLs using immunocytochemistry or Western blot analysis, or both. Immature OLs possessed large amounts of intact and partially processed proenkephalin precursors, as well as posttranslational products of prodynorphin including dynorphin A (1–17). With maturation, however, intact or partially processed proenkephalin was expressed by only about 50% of OLs, while dynorphin A (1–17) was undetectable. To assess the function of OL-derived opioids, the effect of κ-agonists/antagonists on OL differentiation and death was explored. κ-Agonists alone had no effect. In contrast, NorBNI significantly increased OL death. Additive OL losses were evident when NorBNI was paired with toxic levels of glutamate, suggesting that κ-receptor blockade alone is sufficient to induce OL death. Thus, the results indicate that OLs express proenkephalin and prodynorphin peptides in a developmentally regulated manner, and further suggest that opioids produced by OLs modulate OL maturation and survival through local (i.e., autocrine and/or paracrine) mechanisms. GLIA 35:156–165, 2001. © 2001 Wiley-Liss, Inc.

The Leu-enkephalin-encoding sequence DNA-binding factor (LEF) with high affinity for the Leu-enkephalin-encoding sequences in the prodynorphin and proenkephalin genes has earlier been identified. This factor is composed of three subunits of about 60, 70 (the major DNA-binding subunit), and 95 kDa, respectively. Estimated molecular mass, sequence specificity of DNA-binding, and supershift/inhibition with specific antibodies in a band shift assay showed that the DNA-binding subunit of LEF is identical to the multifunctional transcription factor YY1. However, an antibody against the C-terminus of YY1 distinguished the YY1 complexes with a Leu-enkephalin-encoding sequence and canonical YY1 binding site oligonucleotides, suggesting different protein conformations in complexes with these two DNA fragments.

In this paper we describe a simple, fast, and inexpensive approach for quantitative analysis of proteins originated from small central nervous system (CNS) samples, i.e., rat spinal cord. The presented sample preparation protocol and quantification results from isotope dimethyl labeling were statistically evaluated and approved as a reliable and robust method for animal model studies of neurological disorders. Combined with the biopathway analysis tool IPA, the method was applied for comparative analysis of proteins in the dorsal and ventral segments of the rat spinal cord. The results are in agreement with the previously published protein patterns in these tissues. A majority (73%) of proteins identified as "related with CNS development and functions" were found to be overexpressed in the dorsal section compared to the ventral segment. The pathway related to neuropathic pain was overrepresented in the dorsal tissue samples. The developed novel approach may be applied for analyses of the spinal cord mediated neurological dysfunctions and pathological pain.

A DNA-binding factor with high affinity and specificity for the [Leu5]enkephalin-encoding sequences in the prodynorphin and proenkephalin genes has been characterized. The factor has the highest affinity for the [Leu5]-enkephalin-encoding sequence in the dynorphin B-encoding region of the prodynorphin gene, has relatively high affinity for other [Leu5]enkephalin-encoding sequences in the prodynorphin and proenkephalin genes, but has no apparent affinity for similar DNA sequences coding for [Met5]-enkephalin in the prodynorphin or proopiomelanocortin genes. The factor has been named [Leu5]enkephalin-encoding sequence DNA-binding factor (LEF). LEF has a nuclear localization and is composed of three subunits of about 60, 70, and 95 kDa, respectively. The highest levels were observed in rat testis, cerebellum, and spleen and were generally higher in late embryonal compared to newborn or adult animals. LEF activity was also recorded in human clonal tumor cell lines. LEF inhibited the transcription of reporter genes in artificial gene constructs where a [Leu5]enkephalin-encoding DNA fragment had been inserted between the transcription initiation site and the coding region of the reporter genes. These observations suggest that the [Leu5]enkephalin-encoding sequences in the prodynorphin and proenkephalin genes also have regulatory functions realized through interaction with a specific DNA-binding factor.

Traumatic brain injury (TBI) induces a cascade of primary and secondary events resulting in impairment of neuronal networks that eventually determines clinical outcome. The dynorphins, endogenous opioid peptides, have been implicated in secondary injury and neurodegeneration in rodent and human brain. To gain insight into the role of dynorphins in the brain's response to trauma, we analyzed short-term (1-day) and long-term (7-day) changes in dynorphin A (Dyn A) levels in the frontal cortex, hippocampus, and striatum, induced by unilateral left-side or right-side cortical TBI in mice. The effects of TBI were significantly different from those of sham surgery (Sham), while the sham surgery also produced noticeable effects. Both sham and TBI induced short-term changes and long-term changes in all three regions. Two types of responses were generally observed. In the hippocampus, Dyn A levels were predominantly altered ipsilateral to the injury. In the striatum and frontal cortex, injury to the right (R) hemisphere affected Dyn A levels to a greater extent than that seen in the left (L) hemisphere. The R-TBI but not L-TBI produced Dyn A changes in the striatum and frontal cortex at 7 days after injury. Effects of the R-side injury were similar in the two hemispheres. In naive animals, Dyn A was symmetrically distributed between the two hemispheres. Thus, trauma may reveal a lateralization in the mechanism mediating the response of Dyn A-expressing neuronal networks in the brain. These networks may differentially mediate effects of left and right brain injury on lateralized brain functions.

We previously identified four missense mutations in the prodynorphin gene that cause human neurodegenerative disorder spinocerebellar ataxia type 23 (SCA23). Three mutations substitute Leu(5), Arg(6), and Arg(9) to Ser (L5S), Trp (R6W) and Cys (R9C) in dynorphin A(1-17) (Dyn A), a peptide with both opioid activities and non-opioid neurodegenerative actions. It has been reported that Dyn A administered intrathecally (i.t.) in femtomolar doses into mice produces nociceptive behaviors consisting of hindlimb scratching along with biting and licking of the hindpaw and tail (SBL responses) through a non-opioid mechanism. We here evaluated the potential of the three mutant peptides to produce similar behaviors. Compared to the wild type (WT)-peptide, the relative potency of Dyn A R6W, L5S and R9C peptides for SBL responses was 50-, 33- and 2-fold higher, and Dyn A R6W and L5S induced the SBL responses at a 10-30-fold lower doses. Dyn A R6W was the most potent peptide. The SBL responses induced by Dyn A R6W were dose dependently inhibited by morphine (i.p.; 0.1-1 mg/kg) or MK-801, an NMDA ion channel blocker (i.t. co-administration; 5-7.5 nmol). CP-99,994, a tachykinin NK1 receptor antagonist (i.t. co-administration; 2 nmol) and naloxone (i.p.; 5 mg/kg) failed to block effects of Dyn A R6W. Thus, similarly to Dyn A WT, the SBL responses induced by Dyn A R6W may involve the NMDA receptor but are not mediated through the opioid and tachykinin NK1 receptors. Enhanced non-opioid excitatory activities of Dyn A mutants may underlie in part development of SCA23.

Objective: Circadian and stress-response systems mediate environmental changes that affect alcohol drinking. Psychosocial stress is an environmental risk factor for alcohol abuse. Circadian rhythm gene period 1 (Per1) is targeted by stress hormones and is transcriptionally activated in corticotropin releasing factor-expressing cells. The authors hypothesized that Per1 is involved in integrating stress response and circadian rhythmicity and explored its relevance to alcohol drinking.Method: In mice, the effects of stress on ethanol intake in mPer1-mutant and wild-type mice were assessed. In humans, single nucleotide polymorphisms (SNPs) in hPer1 were tested for association with alcohol drinking behavior in 273 adolescents and an adult case-control sample of 1,006 alcohol-dependent patients and 1,178 comparison subjects. In vitro experiments were conducted to measure genotype-specific expression and transcription factor binding to hPer1.Results: The mPer1-mutant mice showed enhanced alcohol consumption in response to social defeat stress relative to their wild-type littermates. An association with the frequency of heavy drinking in adolescents with the hPer1 promoter SNP rs3027172 and with psychosocial adversity was found. There was significant interaction between the rs3027172 genotype and psychosocial adversity on this drinking measure. In a confirmatory analysis, association of hPer1 rs3027172 with alcohol dependence was shown. Cortisol- induced transcriptional activation of hPer1 was reduced in human B- lymphoblastoid cells carrying the risk genotype of rs3027172. Binding affinity of the transcription factor Snail1 to the risk allele of the hPer1 SNP rs3027172 was also reduced.Conclusions: The findings indicate that the hPer1 gene regulates alcohol drinking behavior during stressful conditions and provide evidence for underlying neurobiological mechanisms.

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

Neuropeptide-plasma membrane interactions in the absence of a corresponding specific receptor may result in neuropeptide translocation into the cell. Translocation across the plasma membrane may represent a previously unknown mechanism by which neuropeptides can signal information to the cell interior. We introduce here two complementary optical methods with single-molecule sensitivity, fluorescence imaging with avalanche photodiode detectors (APD imaging) and fluorescence correlation spectroscopy (FCS), and demonstrate how they may be applied for the analysis of neuropeptide ability to penetrate into live cells in real time. APD imaging enables us to visualize fluorescently labeled neuropeptide molecules at very low, physiologically relevant concentrations, whereas FCS enables us to characterize quantitatively their concentration and diffusion properties in different cellular compartments. Application of these methodologies for the analysis of the endogenous opioid peptide dynorphin A (Dyn A), a ligand for the kappa-opioid receptor (KOP), demonstrated that this neuropeptide may translocate across the plasma membrane of living cells and enter the cellular interior without binding to its cognate receptor.

The present study used conventional and quantitative microdialysis to assess glutamatergic and GABAergic neurotransmission in the hippocampal CA3 area of the rat following a moderate-dose ethanol treatment regimen. Male Wistar rats received 3.4 g/kg of ethanol or water for 6 days via gastric gavage. Microdialysis experiments commenced 2 days later. Basal and depolarization-induced glutamate overflow were significantly elevated in ethanol-treated animals. Basal and depolarization-induced gamma-aminobutyric acid (GABA) overflow were unaltered. Quantitative no-net-flux microdialysis was used to determine if changes in dialysate glutamate levels following ethanol administration are due to an increase in release or a decrease in uptake. To confirm the validity of this method for quantifying basal glutamate dynamics, extracellular concentrations of glutamate and the extraction fraction, which reflects changes in analyte clearance, were quantified in response to retro-dialysis of the glutamate uptake blocker trans-pyrrolidine-2,4-dicarboxylic acid (tPDC). tPDC significantly decreased the extraction fraction for glutamate, resulting in augmented extracellular glutamate concentrations. Repeated ethanol administration did not alter the glutamate extraction fraction. However, extracellular glutamate concentrations were significantly elevated, indicating that glutamate release is increased as a consequence of repeated ethanol administration. These data demonstrate that repeated bouts of moderate ethanol consumption alter basal glutamate dynamics in the CA3 region of the dorsal hippocampus. Basal glutamate release is augmented, whereas glutamate uptake is unchanged. Furthermore, they suggest that dysregulation of glutamate transmission in this region may contribute to the previously documented deficits in cognitive function associated with moderate dose ethanol use.

Objective In rheumatoid arthritis (RA), pain and joint destruction are initiated and propagated by the production of proinflammatory mediators. Synthesis of these mediators is regulated by the transcription factor NF-κB, which is controlled by the ubiquitin proteasome system (UPS). The present study explored the effects of the proteasome inhibitor MG132 on inflammation, pain, joint destruction, and expression of sensory neuropeptides as markers of neuronal response in a rat model of arthritis. Methods Arthritis was induced in rats by injection of heat-killed Mycobacterium butyricum. Arthritis severity was scored, and nociception was evaluated by mechanical pressure applied to the hind paw. Joint destruction was assessed by radiologic and histologic analyses. NF-κB DNA-binding activity was analyzed by electromobility shift assay, and changes in the expression of the p50 NF-κB subunit and the proinflammatory neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) were detected by immunohistochemistry. Results Arthritic rats treated with MG132 demonstrated a marked reduction in inflammation, pain, and joint destruction. The elevated DNA-binding activity of the NF-κB/p50 homodimer and p50, as well as the neuronal expression of SP and CGRP, observed in the ankle joints of arthritic rats were normalized after treatment with MG132. Conclusion In arthritic rats, inhibition of proteasome reduced the severity of arthritis and reversed the pain behavior associated with joint inflammation. These effects may be mediated through the inhibition of NF-κB activation and may possibly involve the peripheral nervous system. New generations of nontoxic proteasome inhibitors may represent a novel pharmacotherapy for RA.

Dynorphins, the endogenous opioid peptides derived from prodynorphin may participate not only in the inhibition, but also in facilitation of spinal nociceptive transmission. However, the mechanism of pronociceptive dynorphin actions, and the comparative potential of prodynorphin processing products to induce these actions were not fully elucidated. In our studies, we examined pronociceptive effects of prodynorphin fragments dynorphins A and B and big dynorphin consisting of dynorphins A and B, and focused on the mechanisms underlying these effects. Our principal finding was that big dynorphin was the most potent pronociceptive dynorphin; when administered intrathecally into mice at extremely low doses (1-10fmol), big dynorphin produced nociceptive behavior through the activation of the NMDA receptor ion-channel complex by acting on the polyamine recognition site. We next examined whether the endogenous dynorphins participate in the spinal nociceptive transmission using N-ethylmaleimide (NEM) that blocks dynorphin degradation by inhibiting cysteine proteases. Similar to big dynorphin and dynorphin A, NEM produced nociceptive behavior mediated through inhibition of the degradation of endogenous dynorphins, presumably big dynorphin that in turn activates the NMDA receptor ion-channel complex by acting on the polyamine recognition site. Our findings support the notion that endogenous dynorphins are critical neurochemical mediators of spinal nociceptive transmission in uninjured animals. This chapter will review above-described phenomena and their mechanism.

Astroglia are key cellular sites where opiate drug signals converge with the proinflammatory effects of HIV-1 Tat signals to exacerbate HIV encephalitis. Despite this understanding, the molecular sites of convergence driving opiate-accelerated neuropathogenesis have not been deciphered. We therefore explored potential points of interaction between the signaling pathways initiated by HIV-1 Tat and opioids in striatal astrocytes. Profiling studies screening 152 transcription factors indicated that the nuclear factor-kappa B (NF-kappaB) subunit, c-Rel, was a likely candidate for Tat or Tat plus opiate-induced increases in cytokine and chemokine production by astrocytes. Pretreatment with the NF-kappaB inhibitor parthenolide provided evidence that Tat+/-morphine-induced release of MCP-1, IL-6 and TNF-alpha by astrocytes is NF-kappaB dependent. The nuclear export inhibitor, leptomycin B, blocked the nucleocytoplasmic shuttling of NF-kappaB; causing p65 (RelA) accumulation in the nucleus, and significantly attenuated cytokine production in Tat+/-morphine exposed astrocytes. Similarly, chelating intracellular calcium ([Ca(2+)](i)) blocked Tat+/-morphine-evoked MCP-1 and IL-6 release, while artificially increasing the concentration of extracellular Ca(2+) reversed this effect. Taken together, these results demonstrate that: 1) exposure to Tat+/-morphine is sufficient to activate NF-kappaB and cytokine production, 2) the release of MCP-1 and IL-6 by Tat+/-morphine are highly Ca(2+)-dependent, while TNF-alpha appears to be less affected by the changes in [Ca(2+)](i), and 3) in the presence of Tat, exposure to opiates augments Tat-induced NF-kappaB activation and cytokine release through a Ca(2+)-dependent pathway.

Dynorphins are endogenous opioid peptide products of the prodynorphin gene. An extensive literature suggests that dynorphins have deleterious effects on CNS injury outcome. We thus examined whether a deficiency of dynorphin would protect against tissue damage after spinal cord injury (SCI), and if individual cell types would be specifically affected. Wild-type and prodynorphin(-/-) mice received a moderate contusion injury at 10th thoracic vertebrae (T10). Caspase-3 activity at the injury site was significantly decreased in tissue homogenates from prodynorphin(-/-) mice after 4 h. We examined frozen sections at 4 h post-injury by immunostaining for active caspase-3. At 3-4 mm rostral or caudal to the injury, >90% of all neurons, astrocytes and oligodendrocytes expressed active caspase-3 in both wild-type and knockout mice. At 6-7 mm, there were fewer caspase-3(+) oligodendrocytes and astrocytes than at 3-4 mm. Importantly, caspase-3 activation was significantly lower in prodynorphin(-/-) oligodendrocytes and astrocytes, as compared with wild-type mice. In contrast, while caspase-3 expression in neurons also declined with further distance from the injury, there was no effect of genotype. Radioimmunoassay showed that dynorphin A(1-17) was regionally increased in wild-type injured versus sham-injured tissues, although levels of the prodynorphin processing product Arg(6)-Leu-enkephalin were unchanged. Our results indicate that dynorphin peptides affect the extent of post-injury caspase-3 activation, and that glia are especially sensitive to these effects. By promoting caspase-3 activation, dynorphin peptides likely increase the probability of glial apoptosis after SCI. While normally beneficial, our findings suggest that prodynorphin or its peptide products become maladaptive following SCI and contribute to secondary injury.

Chronic pain is maintained in part by long-lasting neuroplastic changes in synapses and several proteins critical for synaptic plasticity are degraded by the ubiquitin–proteasome system (UPS). Here, we show that proteasome inhibitors administered intrathecally or subcutaneously prevented the development and reversed nerve injury-induced pain behavior. They also blocked pathological pain induced by sustained administration of morphine or spinal injection of dynorphin A, an endogenous mediator of chronic pain. Proteasome inhibitors blocked mechanical allodynia and thermal hyperalgesia in all three pain models although they did not modify responses to mechanical stimuli, but partially inhibited responses to thermal stimuli in control rats. In the spinal cord, these compounds abolished the enhanced capsaicin-evoked calcitonin gene-related peptide (CGRP) release and dynorphin A upregulation, both elicited by nerve injury. Model experiments demonstrated that the inhibitors may act directly on dynorphin-producing cells, blocking dynorphin secretion. Thus, the effects of proteasome inhibitors on chronic pain were apparently mediated through several cellular mechanisms indispensable for chronic pain, including those of dynorphin A release and postsynaptic actions, and of CGRP secretion. Levels of several UPS proteins were reduced in animals with neuropathic pain, suggesting that UPS downregulation, like effects of proteasome inhibitors, counteracts the development of chronic pain. The inhibitors did not produce marked or disabling motor disturbances at doses that were used to modify chronic pain. These results suggest that the UPS is a critical intracellular regulator of pathological pain, and that UPS-mediated protein degradation is required for maintenance of chronic pain and nociceptive, but not non-nociceptive responses in normal animals.

The classical view postulates that neuropeptide precursors in neurons are processed into mature neuropeptides in the somatic trans-Golgi network (TGN) and in secretory vesicles during axonal transport. Here we show that prodynorphin (PDYN), precursor to dynorphin opioid peptides, is predominantly located in axon terminals and dendrites in hippocampal and striatal neurons. The molar content of unprocessed PDYN was much greater than that of dynorphin peptides in axon terminals of PDYN-containing neurons projecting to the CA3 region of the hippocampus and in the striatal projections to the ventral tegmental area. Electron microscopy showed coexistence of PDYN and dynorphins in the same axon terminals with occasional codistribution in individual dense core vesicles. Thus, the precursor protein is apparently stored at presynaptic sites. In comparison with the hippocampus and striatum, PDYN and dynorphins were more equally distributed between neuronal somata and processes in the amygdala and cerebral cortex, suggesting regional differences in the regulation of trafficking and processing of the precursor protein. Potassium-induced depolarization activated PDYN processing and secretion of opioid peptides in neuronal cultures and in a model cell line. Regulation of PDYN storage and processing at synapses by neuronal activity or extracellular stimuli may provide a local mechanism for regulation of synaptic transmission.

Effects of the opioid receptor like-1 (ORL-1) receptor agonist Ro 64-6198 (0.1, 0.3, and 1.0 mg/kg intraperitoneally (i.p.)) on operant ethanol self-administration and activation of self-administration by ethanol deprivation were studied in male Wistar rats. Acute administration of Ro 64-6198 caused a dose-dependent reduction of ethanol self-administration. In comparison, the opioid antagonist naltrexone (0.1, 0.3, and 1.0 mg/kg i.p.) inhibited ethanol self-administration at all doses tested. Ethanol deprivation for 10 days significantly increased ethanol self-administration during the first 2 days after deprivation. Daily pretreatment with Ro 64-6198 (0.3 mg/kg) or naltrexone (0.3 mg/kg) during the last 3 days of ethanol deprivation abolished the deprivation-induced increase in ethanol intake. Thus, stimulation of the ORL-1 receptors by Ro 64-6198 reduced the acute reinforcing effects of ethanol and prevented relapse-like behavior in the ethanol-deprivation model in a similar manner as a blockade of opioid receptors by naltrexone. Ro 64-6198 at 0.1 and 0.3 mg/kg doses did not alter self-administration of 0.2% saccharin solution, indicating an apparent selectivity of this compound in modification of ethanol reward. These findings add further support to the idea that Ro 64-6198 and potentially other synthetic ORL-1 receptor agonists are as effective as naltrexone in blocking the actions of ethanol important for its addictive potential in animal experiments, and therefore may have therapeutic value in the treatment of alcoholism.

Prodynorphin - derived peptides elicit various pathological effects including neurological dysfunction and cell death. These actions are reduced by N-methyl-D-aspartate receptor (NMDAR) but not opioid receptor antagonists suggesting NMDAR - mediation. Here we show that a conserved epitope (KVNSEEEEEDA) of the NR1 subunit of the NMDAR binds dynorphin peptides (DYNp) non-covalently. Synthetic peptides containing this epitope form stable complexes with DYNp and prevent the potentiation of NMDAR - gated currents produced by DYNp. They attenuate DYNp - evoked cell death in spinal cord and prevent, as well as reverse, DYNp - induced paralysis and allodynia. The data reveal a novel mechanism whereby prodynorphin-derived peptides facilitate NMDAR function and produce neurotoxicity. Furthermore, they suggest that synthetic peptides that bind DYNp, thus preventing their interaction with NMDAR, may be novel therapeutic agents for the treatment of spinal cord injury.

International audience; The diversity of peptide ligands for a particular receptor may provide a greater dynamic range of functional responses, while maintaining selectivity in receptor activation. Dynorphin A (Dyn A), and dynorphin B (Dyn B) are endogenous opioid peptides that activate the kappa-opioid receptor (KOR). Here, we characterized interactions of big dynorphin (Big Dyn), a 32-amino acid prodynorphin-derived peptide consisting of Dyn A and Dyn B, with human KOR, mu- (hMOR) and delta- (hDOR) opioid receptors and opioid receptor-like receptor 1 (hORL1) expressed in cells transfected with respective cDNA. Big Dyn and Dyn A demonstrated roughly similar affinity for binding to hKOR that was higher than that of Dyn B. Dyn A was more selective for hKOR over hMOR, hDOR and hORL1 than Big Dyn, while Dyn B demonstrated low selectivity. In contrast, Big Dyn activated G proteins through KOR with much greater potency, efficacy and selectivity than other dynorphins. There was no correlation between the rank order of the potency for the KOR-mediated activation of G proteins and the binding affinity of dynorphins for KOR. The rank of the selectivity for the activation of G proteins through hKOR and of the binding to this receptor also differed. Immunoreactive Big Dyn was detected using the combination of radioimmunoassay (RIA) and HPLC in the human nucleus accumbens, caudate nucleus, hippocampus and cerebrospinal fluid (CSF) with the ratio of Big Dyn and Dyn B being approximately 1:3. The presence in the brain implies that Big Dyn, along with other dynorphins, is processed from prodynorphin and secreted from neurons. Collectively, the high potency and efficacy and the relative abundance suggest that Big Dyn may play a role in the KOR-mediated activation of G proteins.

Changes in genomic DNA methylation are recognized as important events in normal and pathological cellular processes, contributing both to normal development and differentiation as well as cancer and other diseases. Here, we report a novel method to estimate genome-wide DNA methylation, referred to as LUminometric Methylation Assay (LUMA). The method is based on combined DNA cleavage by methylation-sensitive restriction enzymes and polymerase extension assay by Pyrosequencing. The method is quantitative, highly reproducible and easy to scale up. Since no primary modification of genomic DNA, such as bisulfite treatment, is needed, the total assay time is only 6 h. In addition, the assay requires only 200-500 ng of genomic DNA and incorporates an internal control to eliminate the problem of varying amounts of starting DNA. The accuracy and linearity of LUMA were verified by in vitro methylated lambda DNA. In addition, DNA methylation levels were assessed by LUMA in DNA methyltransferase knock-out cell lines and after treatment with the DNA methyltransferase inhibitor (5-AzaCytidine). The LUMA assay may provide a useful method to analyze genome-wide DNA methylation for a variety of physiological and pathological conditions including etiologic, diagnostic and prognostic aspects of cancer.