Molecular examination of the endogenous opioid system in rhesus macaque monkeys with self-injurious behavior.

Self-injurious behavior (SIB) can lead to serious injury and occurs in approximately 1%-4% of the adult population, with higher incidences in adolescent and institutionalized populations, as well as in children with developmental disorders such as Autism. SIB also spontaneously occurs in a low percentage of captive monkeys. Rhesus macaque (Macaca mulatta) monkeys are evolutionarily and physiologically similar to humans, share 93% genetic sequence similarity to humans, and have long been used as testing subjects for vaccine and clinical trials. Previous studies hypothesized that altered endogenous opioid expression occurs in the brains of individuals and animals that self-injure. We examined the regional mRNA expression of opioid signaling genes in sixteen rhesus macaques that exhibited SIB and eight sex- and age- matched controls. The brain regions examined are linked to reward reinforcement and stress adaptation including the hypothalamus, orbital frontal cortex, nucleus accumbens, hippocampus, caudate, and the amygdala. We found decreased µ-opioid receptor (OPRM1) in the amygdala of monkeys with SIB, and reduced prodynorphin (PDYN) in the hypothalamus. Our data suggest dysfunction in the regulation of opioid peptide precursors and calls for further investigation of the endogenous opioid system in SIB.

Sexual differentiation of estrogen receptor alpha subpopulations in the ventromedial nucleus of the hypothalamus.

Estrogen receptor (ER) α-expressing neurons in the ventrolateral area of the ventromedial hypothalamus (VMHvl) are implicated in the control of many behaviors and physiological processes, some of which are sex-specific. Recently, three sex-differentiated ERα subpopulations have been discovered in the VMHvl marked by co-expression with tachikinin1 (Tac1), reprimo (Rprm), or prodynorphin (Pdyn), that may subserve specific functions. These markers show sex differences in adulthood: females have many more Tac1/Esr1 and Rprm/Esr1 co-expressing cells, while males have more Pdyn/Esr1 cells. In this study, we sought to understand the development of these sex differences and pinpoint the sex-differentiating signal. We examined developmental changes in the number of Esr1 cells co-expressing Tac1, Rprm or Pdyn using single-molecule in situ hybridization. We found that both sexes have similarly high numbers of Tac1/Esr1 and Rprm/Esr1 cells at birth, but newborn males have many more Pdyn/Esr1 cells than females. However, the number of cells with Tac1/Esr1 and Rprm/Esr1 co-expression markedly decreases by weaning in males, but not females, leading to sex differences in neurochemical expression. Female mice administered testosterone at birth have expression patterns akin to male mice. Thus, a substantial neurochemical reorganization of the VMHvl occurs in males between birth and weaning that likely underlies the previously reported sex differences in behavioral and physiological responses to estrogens in adulthood.

Regulation of Cortico-Thalamic JNK1/2 and ERK1/2 MAPKs and Apoptosis-Related Signaling Pathways in PDYN Gene-Deficient Mice Following Acute and Chronic Mild Stress.

The crosstalk between the opioidergic system and mitogen-activated protein kinases (MAPKs) has a critical role in mediating stress-induced behaviors related to the pathophysiology of anxiety. The present study evaluated the basal status and stress-induced alterations of cortico-thalamic MAPKs and other cell fate-related signaling pathways potentially underlying the anxiogenic endophenotype of PDYN gene-deficient mice. Compared to littermates, PDYN knockout (KO) mice had lower cortical and or thalamic amounts of the phospho-activated MAPKs c-Jun N-terminal kinase (JNK1/2) and extracellular signal-regulated kinase (ERK1/2). Similarly, PDYN-KO animals displayed reduced cortico-thalamic densities of total and phosphorylated (at Ser191) species of the cell fate regulator Fas-associated protein with death domain (FADD) without alterations in the Fas receptor. Exposure to acute restraint and chronic mild stress stimuli induced the robust stimulation of JNK1/2 and ERK1/2 MAPKs, FADD, and Akt-mTOR pathways, without apparent increases in apoptotic rates. Interestingly, PDYN deficiency prevented stress-induced JNK1/2 and FADD but not ERK1/2 or Akt-mTOR hyperactivations. These findings suggest that cortico-thalamic MAPK- and FADD-dependent neuroplasticity might be altered in PDYN-KO mice. In addition, the results also indicate that the PDYN gene (and hence dynorphin release) may be required to stimulate JNK1/2 and FADD (but not ERK1/2 or Akt/mTOR) pathways under environmental stress conditions.

Moderation of buprenorphine therapy for cocaine dependence efficacy by variation of the Prodynorphin gene.

PURPOSE: The aim of this secondary analysis was to identify prodynorphin (PDYN) genetic markers moderating the therapeutic response to treatment of cocaine dependence with buprenorphine/naloxone (Suboxone®; BUP). METHODS: Cocaine-dependent participants (N = 302) were randomly assigned to a platform of injectable, extended-release naltrexone (XR-NTX) and one of three daily medication arms: 4 mg BUP (BUP4), 16 mg BUP (BUP16), or placebo (PLB) for 8 weeks (Parent Trial Registration: Protocol ID: NIDA-CTN-0048, Clinical Trials.gov ID: NCT01402492). DNA was obtained from 277 participants. Treatment response was determined from weeks 3 to 7 over each 1-week period by the number of cocaine-positive urines per total possible urines. RESULTS: In the cross-ancestry group, the PLB group had more cocaine-positive urines than the BUP16 group (P = 0.0021). The interactions of genetic variant × treatment were observed in the rs1022563 A-allele carrier group where the BUP16 group (N = 35) had fewer cocaine-positive urines (P = 0.0006) than did the PLB group (N = 26) and in the rs1997794 A-allele carrier group where the BUP16 group (N = 49) had fewer cocaine-positive urines (P = 0.0003) than did the PLB group (N = 58). No difference was observed in the rs1022563 GG or rs1997794 GG genotype groups between the BUP16 and PLB groups. In the African American-ancestry subgroup, only the rs1022563 A-allele carrier group was associated with treatment response. CONCLUSION: These results suggest that PDYN variants may identify patients who are best suited to treatment with XR-NTX plus buprenorphine for cocaine use disorder pharmacotherapy.

Considerations on Using Antibodies for Studying the Dynorphins/Kappa Opioid Receptor System.

Antibodies are important tools for protein and peptide research, including for the kappa opioid receptor (KOR) and dynorphins (Dyns). Well-characterized antibodies are essential for rigorous and reproducible research. However, lack of validation of antibody specificity has been thought to contribute significantly to the reproducibility crisis in biomedical research. Since 2003, many scientific journals have required documentation of validation of antibody specificity and use of knockout mouse tissues as a negative control is strongly recommended. Lack of specificity of antibodies against many G protein-coupled receptors (GPCRs) after extensive testing has been well-documented, but antibodies generated against partial sequences of the KOR have not been similarly investigated. For the dynorphins, differential processing has been described in distinct brain areas, resulting in controversial findings in immunohistochemistry (IHC) when different antibodies were used. In this chapter, we summarized accepted approaches for validation of antibody specificity. We discussed two KOR antibodies most commonly used in IHC and described generation and characterization of KOR antibodies and phospho-KOR specific antibodies in western blotting or immunoblotting (IB). In addition, applying antibodies targeting prodynorphin or mature dynorphin A illustrates the diversity of results obtained regarding the distribution of dynorphins in distinct brain areas.

Mechanisms for the Sour Taste.

Sour, the taste of acids, provides important sensory information to prevent the ingestion of unripe, spoiled, or fermented foods. In mammals, acids elicit disgust and pain by simultaneously activating taste and somatosensory neurons innervating the oral cavity. Early researchers detected electrical activity in taste nerves upon presenting acids to the tongue, establishing this as the bona fide sour taste. Recent studies have made significant contributions to our understanding of the mechanisms underlying acid sensing in the taste receptor cells at the periphery and the neural circuitry that convey this information to the brain. In this chapter, we discuss the characterization of sour taste receptor cells, the twists and turns eventually leading to the identification of Otopetrin1 (OTOP1) as the sour taste receptor, the pathway of sour taste signaling from the tongue to the brainstem, and other roles sour taste receptor cells play in the taste bud.

Kappa Opioid Receptors in the Pathology and Treatment of Major Depressive Disorder.

The kappa opioid receptor (KOR) is thought to regulate neural systems associated with anhedonia and aversion and mediate negative affective states that are associated with a number of psychiatric disorders, but especially major depressive disorder (MDD). Largely because KOR antagonists mitigate the effects of stress in preclinical studies, KOR antagonists have been recommended as novel drugs for treating MDD. The purpose of this review is to examine the role of KORs and its endogenous ligand dynorphins (DYNs) in the pathology and treatment of MDD derived from different types of clinical studies. Evidence pertaining to the role of KOR and MDD will be reviewed from (1) post mortem mRNA expression patterns in MDD, (2) the utility of KOR neuroimaging agents and serum biomarkers in MDD, and (3) evidence from the recent Fast Fail clinical trial that established KOR antagonism as a potential therapeutic strategy for the alleviation of anhedonia, a core feature of MDD. These findings are compared with a focused evaluation of stress-induced alterations in OPRK and PDYN mRNA expression. Finally, the current status of the effects of KOR antagonists on behavioral phenotypes of stress in preclinical studies related to MDD is summarized.

Prodynorphin and Proenkephalin in Cerebrospinal Fluid of Sporadic Creutzfeldt-Jakob Disease.

Proenkephalin (PENK) and prodynorphin (PDYN) are endogenous opioid peptides mainly produced in the striatum and, to a lesser extent, in the cerebral cortex. Dysregulated metabolism and altered cerebrospinal fluid (CSF) levels of PENK and PDYN have been described in several neurodegenerative diseases. However, no study to date investigated these peptides in the CSF of sporadic Creutzfeldt-Jakob disease (sCJD). Using liquid chromatography-multiple reaction monitoring mass spectrometry, we evaluated the CSF PDYN- and PENK-derived peptide levels in 25 controls and 63 patients with sCJD belonging to the most prevalent molecular subtypes (MM(V)1, VV2 and MV2K). One of the PENK-derived peptides was significantly decreased in each sCJD subtype compared to the controls without a difference among subtypes. Conversely, PDYN-derived peptides were selectively decreased in the CSF of sCJD MV2K, a subtype with a more widespread overall pathology compared to the sCJD MM(V)1 and the VV2 subtypes, which we confirmed by semiquantitative analysis of cortical and striatal neuronal loss and astrocytosis. In sCJD CSF PENK and PDYN were associated with CSF biomarkers of neurodegeneration but not with clinical variables and showed a poor diagnostic performance. CSF PDYN and PENK-derived peptides had no significant diagnostic and prognostic values in sCJD; however, the distinct marker levels between molecular subtypes might help to better understand the basis of phenotypic heterogeneity determined by divergent neuronal targeting.

Prodynorphin (PDYN) gene polymorphisms in Turkish patients with methamphetamine use disorder, changes in PDYN serum levels in withdrawal and the relationship between PDYN, temperament and depression.

Aim of the study is to compare prodynorphin (PDYN) rs1997794, rs1022563, rs6045819, rs2235749 polymorphisms in individuals with methamphetamine use disorder (MD) to that of healthy controls (HC), and to investigate the differences in serum PDYN levels in methamphetamine withdrawal. It is also aimed to explore the temperament characteristics and depression and their relationship with PDYN polymorphisms and PDYN serum levels in MD group. PDYN gene and serum levels were studied in 134 patients with MD and 97 HC. Patients with MD were administered Beck Depression Inventory (BDI) and Temperament Evaluation of Memphis, Pisa, Paris and San Diego Autoquestionnaire (TEMPS-A). For rs1022563 polymorphism, TT and CT genotype frequency and T allele frequency were significantly higher in the MD group than the frequencies in HC. It was found that rs2235749 polymorphism AA genotype was associated with increased risk of MD. PDYN rs1997794 CT genotypes had significantly higher scores of TEMPS-A irritable than CC genotypes and PDYN rs1022563 CC genotypes had significantly higher scores of TEMPS-A irritable than TT genotypes. PDYN levels among persons with MD were significantly higher than among the HC group when the withdrawal level increased and withdrawal symptoms improved. During the period in which the withdrawal level increased, there was a negative correlation between PDYN level and BDI and a positive relationship between PDYN level and TEMPS-A hyperthymic. It may be beneficial to screen temperament characteristics associated with increased risk of addiction in patients with MD and develop interventions based on temperament characteristics and the effects of PDYN.

Cerebrospinal Fluid Levels of Prodynorphin-Derived Peptides are Decreased in Huntington's Disease.

RESULTS: Huntington's disease (HD) is a devastating neurodegenerative disorder characterized by a selective loss of striatal medium spiny projection neurons (MSNs). Prodynorphin (PDYN) is enriched in a subpopulation of striatal MSNs. Postmortem brains of HD patients and rodent models have been demonstrated to have reduced levels of PDYN transcripts and the neuropeptide dynorphin. RESULTS: Given the unmet need for novel pharmacodynamic HD biomarkers in the context of experimental huntingtin (htt)-lowering therapies, we investigated the levels of PDYN-derived peptides and neurofilament light (NfL) chain in the cerebrospinal fluid (CSF) from HD patients (n = 16), matched controls (n = 55), and patients with other neurodegenerative disorders (n = 70). RESULTS: PDYN-derived peptide levels were found to be substantially decreased in HD patients (P < 0.0001 in comparison to controls), whereas the NfL levels were elevated in all neurodegenerative disorders. CONCLUSIONS: Our study suggests decreased PDYN-derived peptide levels in the CSF as a more specific biomarker for HD in comparison to NfL. © 2020 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Epigenetic and Transcriptional Control of the Opioid Prodynorphine Gene: In-Depth Analysis in the Human Brain.

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.

Association between prodynorphin gene polymorphisms and opioid dependence susceptibility: a meta-analysis.

BACKGROUND: Prodynorphin (PDYN) gene polymorphisms have been linked with opioid dependence (OD) with conflicting outcomes, the aim of this study is to synthesize the existing evidence of the association between PDYN polymorphisms and OD susceptibility. METHODS: Four databases including PubMed, EMBASE, Web of Science, and Wanfang were retrieved for relevant studies before August, 2018. All identified studies were evaluated using predetermined inclusion and exclusion criteria. Summary odds ratio (OR) and 95% confidence interval (95%CI) were calculated to appraise the association. Statistical analysis was performed using RevMan 5.3 software. RESULTS: A total of seven case-control studies with 3129 cases and 3289 controls were recruited in the meta-analysis. For rs910080, rs1997794, rs1022563, and rs2235749 polymorphisms of PDYN gene, there were six, four, five, and four studies eventually included, respectively. The findings indicated that rs910080 polymorphism was significantly correlated with OD among Asian population under allelic model (A vs. G, OR = 1.30, 95% CI 1.04-1.62, P = 0.02, FDR = 0.05) and dominant model (AA+AG vs. GG, OR = 1.25, 95% CI 1.04-1.51, P = 0.02, FDR = 0.05). However, rs1022563, rs1997794 and rs2235749 polymorphisms did not appear to associate with OD susceptibility. CONCLUSIONS: There existed a significant association between rs1022563 polymorphism and OD among Asian population. As the included studies were not adequate to guarantee a robust and convincing conclusion, future studies with larger sample size among more ethnicities are recommended.

Intracrine Endorphinergic Systems in Modulation of Myocardial Differentiation.

A wide variety of peptides not only interact with the cell surface, but govern complex signaling from inside the cell. This has been referred to as an "intracrine" action, and the orchestrating molecules as "intracrines". Here, we review the intracrine action of dynorphin B, a bioactive end-product of the prodynorphin gene, on nuclear opioid receptors and nuclear protein kinase C signaling to stimulate the transcription of a gene program of cardiogenesis. The ability of intracrine dynorphin B to prime the transcription of its own coding gene in isolated nuclei is discussed as a feed-forward loop of gene expression amplification and synchronization. We describe the role of hyaluronan mixed esters of butyric and retinoic acids as synthetic intracrines, controlling prodynorphin gene expression, cardiogenesis, and cardiac repair. We also discuss the increase in prodynorphin gene transcription and intracellular dynorphin B afforded by electromagnetic fields in stem cells, as a mechanism of cardiogenic signaling and enhancement in the yield of stem cell-derived cardiomyocytes. We underline the possibility of using the diffusive features of physical energies to modulate intracrinergic systems without the needs of viral vector-mediated gene transfer technologies, and prompt the exploration of this hypothesis in the near future.

Chronic nicotine-induced changes in gene expression of delta and kappa-opioid receptors and their endogenous ligands in the mesocorticolimbic system of the rat.

Delta and kappa opioid receptors (DOR and KOR, respectively) and their endogenous ligands, proenkephalin (PENK) and prodynorphin (PDYN)-derived opioid peptides are proposed as important mediators of nicotine reward. This study investigated the regulatory effect of chronic nicotine treatment on the gene expression of DOR, KOR, PENK and PDYN in the mesocorticolimbic system. Three groups of rats were injected subcutaneously with nicotine at doses of 0.2, 0.4, or 0.6 mg/kg/day for 6 days. Rats were decapitated 1 hr after the last dose on day six, as this timing coincides with increased dopamine release in the mesocorticolimbic system. mRNA levels in the ventral tegmental area (VTA), lateral hypothalamic area (LHA), amygdala (AMG), dorsal striatum (DST), nucleus accumbens, and medial prefrontal cortex were measured by quantitative real-time PCR. Our results showed that nicotine upregulated DOR mRNA in the VTA at all of the doses employed, in the AMG at the 0.4 and 0.6 mg/kg doses, and in the DST at the 0.4 mg/kg dose. Conversely, PDYN mRNA was reduced in the LHA with 0.6 mg/kg nicotine and in the AMG with 0.4 mg/kg nicotine. KOR mRNA was also decreased in the DST with 0.6 mg/kg nicotine. Nicotine did not regulate PENK mRNA in any brain region studied.

Opioid precursor protein isoform is targeted to the cell nuclei in the human brain.

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 κ-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. Immunofluorescence staining and confocal 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 ∆SP-PDYN protein lacking 13 N-terminal amino acids, a fragment of signal peptide (SP). ∆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 ∆SP-PDYN mRNA and nuclear localization of PDYN protein suggests a nuclear function for this isoform of the opioid peptide precursor in human striatum.

Elevated mutant dynorphin A causes Purkinje cell loss and motor dysfunction in spinocerebellar ataxia type 23.

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, α-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.

Endogenous regulation of visceral pain via production of opioids by colitogenic CD4(+) T cells in mice.

BACKGROUND & AIMS: A dysregulated response of CD4(+) T cells against the microbiota contributes to the development of inflammatory bowel disease. Effector CD4(+) T cells, generated in response to microbe-derived antigens, can reduce somatic inflammatory pain through the local release of opioids. We investigated whether colitogenic CD4(+) T cells that accumulate in the inflamed colon also produce opioids and are able to counteract inflammation-induced visceral pain in mice. METHODS: Colitis was induced via transfer of naive CD4(+)CD45RB(high) T cells to immune-deficient mice or by administration of dextran sulfate sodium. Mice without colitis were used as controls. Samples of colon tissue were collected, and production of opioids by immune cells from inflamed intestine was assessed by quantitative polymerase chain reaction and cytofluorometry analyses. The role of intestinal opioid tone in inflammation-induced visceral hypersensitivity was assessed by colorectal distention. RESULTS: In mice with T cell- or dextran sulfate sodium-induced colitis, colitogenic CD4(+) T cells (T-helper 1 and Th17 cells) accumulated in the inflamed intestine and expressed a high level of endogenous opioids. In contrast, macrophages and epithelial cells did not express opioids; opioid synthesis in the myenteric plexus was not altered on induction of inflammation. In mice with colitis, the local release of opioids by colitogenic CD4(+) T cells led to significant reduction of inflammation-associated visceral hypersensitivity. CONCLUSIONS: In mice, colitogenic Th1 and Th17 cells promote intestinal inflammation and colonic tissue damage but have simultaneous opioid-mediated analgesic activity, thereby reducing abdominal pain.

Upregulation of the kappa opioidergic system in left ventricular rat myocardium in response to volume overload: Adaptive changes of the cardiac kappa opioid system in heart failure.

Opioids have long been known for their analgesic effects and are therefore widely used in anesthesia and intensive care medicine. However, in the last decade research has focused on the opioidergic influence on cardiovascular function. This project thus aimed to detect the precise cellular localization of kappa opioid receptors (KOR) in left ventricular cardiomyocytes and to investigate putative changes in KOR and its endogenous ligand precursor peptide prodynorphin (PDYN) in response to heart failure. After IRB approval, heart failure was induced using a modified infrarenal aortocaval fistula (ACF) in male Wistar rats. All rats of the control and ACF group were characterized by their morphometrics and hemodynamics. In addition, the existence and localization as well as adaptive changes of KOR and PDYN were investigated using radioligand binding, double immunofluorescence confocal analysis, RT-PCR and Western blot. Similar to the brain and spinal cord, [(3)H]U-69593 KOR selective binding sites were detected the left ventricle (LV). KOR colocalized with Cav1.2 of the outer plasma membrane and invaginated T-tubules and intracellular with the ryanodine receptor of the sarcoplasmatic reticulum. Interestingly, KOR could also be detected in mitochondria of rat LV cardiomyocytes. As a consequence of heart failure, KOR and PDYN were up-regulated on the mRNA and protein level in the LV. These findings suggest that the cardiac kappa opioidergic system might modulate rat cardiomyocyte function during heart failure.

Estrogen promotes gonadotropin-releasing hormone expression by regulating tachykinin 3 and prodynorphin systems in chicken.

The "KNDy neurons" located in the hypothalamic arcuate nucleus (ARC) of mammals are known to co-express kisspeptin, neurokinin B (NKB), and dynorphin (DYN), and have been identified as key mediators of the feedback regulation of steroid hormones on gonadotropin-releasing hormone (GnRH). However, in birds, the genes encoding kisspeptin and its receptor GPR54 are genomic lost, leaving unclear mechanisms for feedback regulation of GnRH by steroid hormones. Here, the genes tachykinin 3 (TAC3) and prodynorphin (PDYN) encoding chicken NKB and DYN neuropeptides were successfully cloned. Temporal expression profiling indicated that TAC3, PDYN and their receptor genes (TACR3, OPRK1) were mainly expressed in the hypothalamus, with significantly higher expression at 30W than at 15W. Furthermore, overexpression or interference of TAC3 and PDYN can regulate the GnRH mRNA expression. In addition, in vivo and in vitro assays showed that estrogen (E2) could promote the mRNA expression of TAC3, PDYN, and GnRH, as well as the secretion of GnRH/LH. Mechanistically, E2 could dimerize the nuclear estrogen receptor 1 (ESR1) to regulate the expression of TAC3 and PDYN, which promoted the mRNA and protein expression of GnRH gene as well as the secretion of GnRH. In conclusion, these results revealed that E2 could regulate the GnRH expression through TAC3 and PDYN systems, providing novel insights for reproductive regulation in chickens.

Endogenous opioids in the olfactory tubercle and their roles in olfaction and quality of life.

Olfactory dysfunctions decrease daily quality of life (QOL) in part by reducing the pleasure of eating. Olfaction plays an essential role in flavor sensation and palatability. The decreased QOL due to olfactory dysfunction is speculated to result from abnormal neural activities in the olfactory and limbic areas of the brain, as well as peripheral odorant receptor dysfunctions. However, the specific underlying neurobiological mechanisms remain unclear. As the olfactory tubercle (OT) is one of the brain's regions with high expression of endogenous opioids, we hypothesize that the mechanism underlying the decrease in QOL due to olfactory dysfunction involves the reduction of neural activity in the OT and subsequent endogenous opioid release in specialized subregions. In this review, we provide an overview and recent updates on the OT, the endogenous opioid system, and the pleasure systems in the brain and then discuss our hypothesis. To facilitate the effective treatment of olfactory dysfunctions and decreased QOL, elucidation of the neurobiological mechanisms underlying the pleasure of eating through flavor sensation is crucial.