Proenkephalin deletion in hematopoietic cells induces intestinal barrier failure resulting in clinical feature similarities with irritable bowel syndrome in mice.

Opioid-dependent immune-mediated analgesic effects have been broadly reported upon inflammation. In preclinical mouse models of intestinal inflammatory diseases, the local release of enkephalins (endogenous opioids) by colitogenic T lymphocytes alleviate inflammation-induced pain by down-modulating gut-innervating nociceptor activation in periphery. In this study, we wondered whether this immune cell-derived enkephalin-mediated regulation of the nociceptor activity also operates under steady state conditions. Here, we show that chimeric mice engrafted with enkephalin-deficient bone marrow cells exhibit not only visceral hypersensitivity but also an increase in both epithelial paracellular and transcellular permeability, an alteration of the microbial topography resulting in increased bacteria-epithelium interactions and a higher frequency of IgA-producing plasma cells in Peyer's patches. All these alterations of the intestinal homeostasis are associated with an anxiety-like behavior despite the absence of an overt inflammation as observed in patients with irritable bowel syndrome. Thus, our results show that immune cell-derived enkephalins play a pivotal role in maintaining gut homeostasis and normal behavior in mice. Because a defect in the mucosal opioid system remarkably mimics some major clinical symptoms of the irritable bowel syndrome, its identification might help to stratify subgroups of patients.

Lateral hypothalamic proenkephalin neurons drive threat-induced overeating associated with a negative emotional state.

Psychological stressors, like the nearby presence of a predator, can be strong enough to induce physiological/hormonal alterations, leading to appetite changes. However, little is known about how threats can alter feeding-related hypothalamic circuit functions. Here, we found that proenkephalin (Penk)-expressing lateral hypothalamic (LHPenk) neurons of mice exposed to predator scent stimulus (PSS) show sensitized responses to high-fat diet (HFD) eating, whereas silencing of the same neurons normalizes PSS-induced HFD overconsumption associated with a negative emotional state. Downregulation of endogenous enkephalin peptides in the LH is crucial for inhibiting the neuronal and behavioral changes developed after PSS exposure. Furthermore, elevated corticosterone after PSS contributes to enhance the reactivity of glucocorticoid receptor (GR)-containing LHPenk neurons to HFD, whereas pharmacological inhibition of GR in the LH suppresses PSS-induced maladaptive behavioral responses. We have thus identified the LHPenk neurons as a critical component in the threat-induced neuronal adaptation that leads to emotional overconsumption.

Endogenous opioid system modulates conditioned cocaine reward in a sex-dependent manner.

Cocaine predictive cues and contexts exert powerful control over behaviour and can incite cocaine seeking and taking. This type of conditioned behaviour is encoded within striatal circuits, and these circuits and behaviours are, in part, regulated by opioid peptides and receptors expressed in striatal medium spiny neurons. We previously showed that augmenting levels of the opioid peptide enkephalin in the striatum facilitates acquisition of cocaine conditioned place preference (CPP), while opioid receptor antagonists attenuate expression of cocaine CPP. However, whether striatal enkephalin is necessary for acquisition of cocaine CPP and maintenance during extinction remains unknown. To address this, we generated mice with a targeted deletion of enkephalin from dopamine D2-receptor expressing medium spiny neurons and tested them in a cocaine CPP paradigm. Low striatal enkephalin levels did not attenuate acquisition of CPP. However, expression of preference, assessed after acute administration of the opioid receptor antagonist naloxone, was blocked in females, regardless of genotype. When saline was paired with the cocaine context during extinction sessions, females, regardless of genotype, extinguished preference faster than males, and this was prevented by naloxone when paired with the cocaine context. We conclude that while striatal enkephalin is not necessary for acquisition, expression, or extinction of cocaine CPP, expression and extinction of cocaine preference in females is mediated by an opioid peptide other than striatal enkephalin. The unique sensitivity of females to opioid antagonists suggests sex should be a consideration when using these compounds in the treatment of cocaine use disorder.

Enkephalin-δ Opioid Receptor Signaling Mediates Glucoprivic Suppression of LH Pulse and Gluconeogenesis in Female Rats.

Energy availability is an important regulator of reproductive function at various reproductive phases in mammals. Glucoprivation induced by 2-deoxy-D-glucose (2DG), an inhibitor of glucose utilization, as an experimental model of malnutrition suppresses the pulsatile release of GnRH/LH and induces gluconeogenesis. The present study was performed with the aim of examining whether enkephalin-δ-opioid receptor (DOR) signaling mediates the suppression of pulsatile GnRH/LH release and gluconeogenesis during malnutrition. The administration of naltrindole hydrochloride (NTI), a selective DOR antagonist, into the third ventricle blocked the suppression of LH pulses and part of gluconeogenesis induced by IV 2DG administration in ovariectomized rats treated with a negative feedback level of estradiol-17â€…ß (OVX + low E2). The IV 2DG administration significantly increased the number of Penk (enkephalin gene)-positive cells coexpressing fos (neuronal activation marker gene) in the paraventricular nucleus (PVN), but not in the arcuate nucleus (ARC) in OVX + low E2 rats. Furthermore, double in situ hybridization for Penk/Pdyn (dynorphin gene) in the PVN revealed that approximately 35% of the PVN Penk-expressing cells coexpressed Pdyn. Double in situ hybridization for Penk/Crh (corticotropin-releasing hormone gene) in the PVN and Penk/Kiss1 (kisspeptin gene) in the ARC revealed that few Penk-expressing cells coexpressed Crh and Kiss1. Taken together, these results suggest that central enkephalin-DOR signaling mediates the suppression of pulsatile LH release during malnutrition. Moreover, the current study suggests that central enkephalin-DOR signaling is also involved in gluconeogenesis during malnutrition in female rats.

Organisation of enkephalin inputs and outputs of the central nucleus of the amygdala in mice.

The central nucleus of the amygdala (CeA) is a key hub integrating sensory inputs and modulating behavioural outputs. The CeA is a complex structure with discrete subdivisions, high peptidergic heterogeneity and broad CNS afferent and efferent projections. While several neuropeptide systems within the CeA have been examined in detail, less is known about CeA preproenkephalin (ppENK) cells. Here, we used a recently developed transgenic Penk-Cre mouse line to advance our understanding of the efferent and afferent connectivity of ppENK in the CeA. First, to determine the fidelity of Cre expression in Penk-Cre transgenic mice, we conducted RNAscope in the CeA of Penk-Cre mice. Our analysis revealed that 96.6 % of CeA Cre+ neurons co-expressed pENK mRNA, and 99.7 % of CeA pENK+ neurons co-expressed Cre mRNA, indicating faithful recapitulation of Cre expression in CeA ppENK-expressing cells, supporting the fidelity of the Penk-Cre reporter mouse. Anterograde tracing of CeAPenk cells showed strong efferent projections to the extended amygdala, midbrain and hindbrain PBN and NTS. Retrograde tracing of Penk afferents to the CeA were more restricted, with primary innervation originating within the amygdala complex and bed nucleus of the stria terminalis, and minor innervation from the parabrachial nucleus and nucleus of the solitary tract. Together, our data provide a comprehensive map of ENKergic efferent and afferent connectivity of the CeA in Penk-Cre mice. Further, we highlight both the utility and limitations of the Penk-Cre mice to study the function of CeA, PBN and NTS ppENK cells.

Cellular Profiles of Prodynorphin and Preproenkephalin mRNA-Expressing Neurons in the Anterior Olfactory Tubercle of Mice.

The olfactory tubercle (OT) is a striatal region that receives olfactory inputs. mRNAs of prodynorphin (Pdyn) and preproenkephalin (Penk), precursors of dynorphins and enkephalins, respectively, are strongly expressed in the striatum. Both produce opioid peptides with various physiological effects such as pain relief and euphoria. Recent studies have revealed that OT has anatomical and cytoarchitectonic domains that play different roles in odor-induced motivated behavior. Neuronal subtypes of the OT can be distinguished by their expression of the dopamine receptors D1 (Drd1) and D2 (Drd2). Here, we addressed whether and which type of opioid peptide precursors the D1- and D2-expressing neurons in the OT express. We used multiple fluorescence in situ hybridization for mRNAs of the opioid precursors and dopamine receptors to characterize mouse OT neurons. Pdyn was mainly expressed by Drd1-expressing cells in the dense cell layer (DCL) of the OT, whereas Penk was expressed primarily by Drd2-expressing cells in the DCL. We also confirmed the presence of a larger population of Pdyn-Penk-Drd1 co-expressing cells in the DCL of the anteromedial OT compared with the anterolateral OT. These observations will help understand whether and how dynorphins and enkephalins in the OT are involved in diverse odor-induced motivated behaviors.

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.

The Expression Pattern of Genes Related to Melanogenesis and Endogenous Opioids in Psoriasis.

The melanocortin system is a major regulator of stress responses in the skin and is responsible for the induction of melanin synthesis through activation of melanogenesis enzymes. The expression of both melanocortin system genes and melanogenesis enzyme genes is altered in psoriasis, and the focus here was on twelve genes related to the signal transduction between them. Additionally, five endogenous opioid system genes that are involved in cutaneous inflammation were examined. Quantitative real-time-PCR was utilized to measure mRNA expression in punch biopsies from lesional and non-lesional skin of psoriasis patients and from the skin of healthy control subjects. Most of the genes related to melanogenesis were down-regulated in patients (CREB1, MITF, LEF1, USF1, MAPK14, ICAM1, PIK3CB, RPS6KB1, KIT, and ATRN). Conversely, an up-regulation occurred in the case of opioids (PENK, PDYN, and PNOC). The suppression of genes related to melanogenesis is in agreement with the reported reduction in pigmentation signaling in psoriatic skin and potentially results from the pro-inflammatory environment. The increase in endogenous opioids can be associated with their involvement in inflammatory dysregulation in psoriasis.

Refeeding activates neurons in the dorsomedial hypothalamus to inhibit food intake and promote positive valence.

OBJECTIVE: The regulation of food intake is a major research area in the study of obesity, which plays a key role in the development of metabolic syndrome. Gene targeting studies have clarified the roles of hypothalamic neurons in feeding behavior, but the deletion of a gene has a long-term effect on neurophysiology. Our understanding of short-term changes such as appetite under physiological conditions is therefore still limited. METHODS: Targeted recombination in active populations (TRAP) is a newly developed method for labeling active neurons by using tamoxifen-inducible Cre recombination controlled by the promoter of activity-regulated cytoskeleton-associated protein (Arc/Arg3.1), a member of immediate early genes. Transgenic mice for TRAP were fasted overnight, re-fed with normal diet, and injected with 4-hydroxytamoxifen 1 h after the refeeding to label the active neurons. The role of labeled neurons was examined by expressing excitatory or inhibitory designer receptors exclusively activated by designer drugs (DREADDs). The labeled neurons were extracted and RNA sequencing was performed to identify genes that are specifically expressed in these neurons. RESULTS: Fasting-refeeding activated and labeled neurons in the compact part of the dorsomedial hypothalamus (DMH) that project to the paraventricular hypothalamic nucleus. Chemogenetic activation of the labeled DMH neurons decreased food intake and developed place preference, an indicator of positive valence. Chemogenetic activation or inhibition of these neurons had no influence on the whole-body glucose metabolism. The labeled DMH neurons expressed prodynorphin (pdyn), gastrin-releasing peptide (GRP), cholecystokinin (CCK), and thyrotropin-releasing hormone receptor (Trhr) genes. CONCLUSIONS: We identified a novel cell type of DMH neurons that can inhibit food intake and promote feeding-induced positive valence. Our study provides insight into the role of DMH and its molecular mechanism in the regulation of appetite and emotion.

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.

miR-506-loaded gelatin nanospheres target PENK and inactivate the ERK/Fos signaling pathway to suppress triple-negative breast cancer aggressiveness.

Triple-negative breast cancer (TNBC) is the most malignant subtype of breast cancer. Some microRNAs (miRNAs) were abnormally expressed in TNBC, and they are closely related to the occurrence and progression of TNBC. Here, we found that miR-506 was significantly downregulated in TNBC and relatively lower miR-506 expression predicted a poorer prognosis. Moreover, we found that miR-506 could inhibit MDA-MB-231 cell viability, colony formation, migration, and invasion, and suppress the ERK/Fos oncogenic signaling pathway through upregulating its direct target protein proenkephalin (PENK). Therefore, miR-506 was proposed as a nucleic acid drug for TNBC therapy. However, miRNA is unstable in vivo, which limiting its application as a therapeutic drug via conventional oral or injected therapies. Here, a gelatin nanosphere (GN) delivery system was applied for the first time to load exogenous miRNA. Exogenous miR-506 mimic was loaded on GNs and injected into the in situ TNBC animal model, and the miR-506 could achieve sustained and controlled release. The results confirmed that overexpression of miR-506 and PENK in vivo through loading on GNs inhibited in situ triple-negative breast tumor growth and metastasis significantly in the xenograft model. Moreover, we indicated that the ERK/Fos signaling pathway was intensively inactivated after overexpression of miR-506 and PENK both in vitro and in vivo, which was further validated by the ERK1/2-specific inhibitor SCH772984. In conclusion, this study demonstrates that miR-506-loaded GNs have great potential in anti-TNBC aggressiveness therapy.

Parabrachial opioidergic projections to preoptic hypothalamus mediate behavioral and physiological thermal defenses.

Maintaining stable body temperature through environmental thermal stressors requires detection of temperature changes, relay of information, and coordination of physiological and behavioral responses. Studies have implicated areas in the preoptic area of the hypothalamus (POA) and the parabrachial nucleus (PBN) as nodes in the thermosensory neural circuitry and indicate that the opioid system within the POA is vital in regulating body temperature. In the present study we identify neurons projecting to the POA from PBN expressing the opioid peptides dynorphin and enkephalin. Using mouse models, we determine that warm-activated PBN neuronal populations overlap with both prodynorphin (Pdyn) and proenkephalin (Penk) expressing PBN populations. Here we report that in the PBN Prodynorphin (Pdyn) and Proenkephalin (Penk) mRNA expressing neurons are partially overlapping subsets of a glutamatergic population expressing Solute carrier family 17 (Slc17a6) (VGLUT2). Using optogenetic approaches we selectively activate projections in the POA from PBN Pdyn, Penk, and VGLUT2 expressing neurons. Our findings demonstrate that Pdyn, Penk, and VGLUT2 expressing PBN neurons are critical for physiological and behavioral heat defense.

Precursor fractions of neurotensin and enkephalin might point to molecular mechanisms of cancer risk modulation during a lifestyle-intervention in germline BRCA1/2 gene mutation carriers.

BACKGROUND: Germline BRCA1/2 mutation carriers (gBMC) face increased cancer risks that are modulated via non-genetic lifestyle factors whose underlying molecular mechanisms are unknown. The peptides Neurotensin (NT) and Enkephalin (ENK)-involved in tumorigenesis and obesity-related diseases-are of interest. We wanted to know whether these biomarkers differ between gBMC and women from the general population and what effect a 1-year lifestyle-intervention has in gBMC. METHODS: The stable precursor fragments pro-NT and pro-ENK were measured at study entry (SE), after 3 and 12 months for 68 women from LIBRE-1 (a controlled lifestyle-intervention feasibility trial for gBMC involving structured endurance training and the Mediterranean Diet). The SE values were compared with a cohort of the general population including female subjects with and without previous cancer disease, non-suggestive for hereditary breast and ovarian cancer (OMA-reference). For LIBRE-1, we analysed the association between the intervention-related change in the two biomarkers and certain lifestyle factors. RESULTS: At SE, gBMC had a higher median pro-NT than OMA-reference (in the subgroups with previous cancer 117 vs. 91 pmol/L, p = 0.002). Non-diseased gBMC had lower median pro-ENK levels when compared to the non-diseased reference group. VO2peak and pro-NT 1-year change in LIBRE-1 were inversely correlated (r = - 0.435; CI - 0.653 to - 0.151; p = 0.004). Pro-ENK correlated positively with VO2peak at SE (r = 0.323; CI 0.061-0.544; p = 0.017). Regression analyses showed an inverse association of 1-year changes for pro-NT and Omega-6/Omega-3 (Estimate: - 37.9, p = 0.097/0.080) in multivariate analysis. CONCLUSION: Our results give first indications for lifestyle-related modification particularly of pro-NT in gBMC.

Endogenous Opioid Signaling in the Mouse Retina Modulates Pupillary Light Reflex.

Opioid peptides and their receptors are expressed in the mammalian retina; however, little is known about how they might affect visual processing. The melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs), which mediate important non-image-forming visual processes such as the pupillary light reflex (PLR), express ß-endorphin-preferring, µ-opioid receptors (MORs). The objective of the present study was to elucidate if opioids, endogenous or exogenous, modulate pupillary light reflex (PLR) via MORs expressed by ipRGCs. MOR-selective agonist [D-Ala2, MePhe4, Gly-ol5]-enkephalin (DAMGO) or antagonist D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) was administered via intravitreal injection. PLR was recorded in response to light stimuli of various intensities. DAMGO eliminated PLR evoked by light with intensities below melanopsin activation threshold but not that evoked by bright blue irradiance that activated melanopsin signaling, although in the latter case, DAMGO markedly slowed pupil constriction. CTAP or genetic ablation of MORs in ipRGCs slightly enhanced dim-light-evoked PLR but not that evoked by a bright blue stimulus. Our results suggest that endogenous opioid signaling in the retina contributes to the regulation of PLR. The slowing of bright light-evoked PLR by DAMGO is consistent with the observation that systemically applied opioids accumulate in the vitreous and that patients receiving chronic opioid treatment have slow PLR.

Endogenous enkephalin is necessary for cocaine-induced alteration in glutamate transmission within the nucleus accumbens.

Altered glutamate transmission within the nucleus accumbens (NAc) has been proposed as a central mechanism underlying behavioural sensitisation associated with repeated cocaine exposure. In addition to glutamate, enkephalin, an endogenous opioid peptide derived from proenkephalin, is necessary for the neuroadaptations associated with chronic cocaine. However, the influence of enkephalin on long-term changes in glutamate transmission within the NAc associated with cocaine-induced sensitisation has not been described. This study used knockout proenkephalin mice (KO) to study the influence of endogenous enkephalin on the adaptations in glutamate neurotransmission associated with repeated cocaine treatment. Wild-type (WT) and KO mice were treated with daily cocaine injections for 9 days to induce sensitisation. On days 15 and 21, the animals received a cocaine challenge and locomotor sensitisation was evaluated, and microdialysis was performed to determine accumbens glutamate content on day 21. No expression of behavioural sensitisation to cocaine was evidenced in the KO mice. Consistently, these showed no changes in glutamate transmission in the NAc associated with repeated cocaine. This study reveals the central role of enkephalin in regulating the glutamate mechanisms associated with cocaine sensitisation.

Efferent projections of Vglut2, Foxp2, and Pdyn parabrachial neurons in mice.

The parabrachial nucleus (PB) is a complex structure located at the junction of the midbrain and hindbrain. Its neurons have diverse genetic profiles and influence a variety of homeostatic functions. While its cytoarchitecture and overall efferent projections are known, we lack comprehensive information on the projection patterns of specific neuronal subtypes in the PB. In this study, we compared the projection patterns of glutamatergic neurons here with a subpopulation expressing the transcription factor Foxp2 and a further subpopulation expressing the neuropeptide Pdyn. To do this, we injected an AAV into the PB region to deliver a Cre-dependent anterograde tracer (synaptophysin-mCherry) in three different strains of Cre-driver mice. We then analyzed 147 neuroanatomical regions for labeled boutons in every brain (n = 11). Overall, glutamatergic neurons in the PB region project to a wide variety of sites in the cerebral cortex, basal forebrain, bed nucleus of the stria terminalis, amygdala, diencephalon, and brainstem. Foxp2 and Pdyn subpopulations project heavily to the hypothalamus, but not to the cortex, basal forebrain, or amygdala. Among the few differences between Foxp2 and Pdyn cases was a notable lack of Pdyn projections to the ventromedial hypothalamic nucleus. Our results indicate that genetic identity determines connectivity (and therefore, function), providing a framework for mapping all PB output projections based on the genetic identity of its neurons. Using genetic markers to systematically classify PB neurons and their efferent projections will enhance the translation of research findings from experimental animals to humans.

Cerebellar developmental deficits underlie neurodegenerative disorder spinocerebellar ataxia type 23.

Spinocerebellar ataxia type 23 (SCA23) is a late-onset neurodegenerative disorder characterized by slowly progressive gait and limb ataxia, for which there is no therapy available. It is caused by pathogenic variants in PDYN, which encodes prodynorphin (PDYN). PDYN is processed into the opioid peptides α-neoendorphin and dynorphins (Dyn) A and B; inhibitory neurotransmitters that function in pain signaling, stress-induced responses and addiction. Variants causing SCA23 mostly affect Dyn A, leading to loss of secondary structure and increased peptide stability. PDYNR212W mice express human PDYN containing the SCA23 variant p.R212W. These mice show progressive motor deficits from 3 months of age, climbing fiber (CF) deficits from 3 months of age, and Purkinje cell (PC) loss from 12 months of age. A mouse model for SCA1 showed similar CF deficits, and a recent study found additional developmental abnormalities, namely increased GABAergic interneuron connectivity and non-cell autonomous disruption of PC function. As SCA23 mice show a similar pathology to SCA1 mice in adulthood, we hypothesized that SCA23 may also follow SCA1 pathology during development. Examining PDYNR212W cerebella during development, we uncovered developmental deficits from 2 weeks of age, namely a reduced number of GABAergic synapses on PC soma, possibly leading to the observed delay in early phase CF elimination between 2 and 3 weeks of age. Furthermore, CFs did not reach terminal height, leaving proximal PC dendrites open to be occupied by parallel fibers (PFs). The observed increase in vGlut1 protein-a marker for PF-PC synapses-indicates that PFs indeed take over CF territory and have increased connectivity with PCs. Additionally, we detected altered expression of several critical Ca2+ channel subunits, potentially contributing to altered Ca2+ transients in PDYNR212W cerebella. These findings indicate that developmental abnormalities contribute to the SCA23 pathology and uncover a developmental role for PDYN in the cerebellum.

Identification of new enterosynes using prebiotics: roles of bioactive lipids and mu-opioid receptor signalling in humans and mice.

OBJECTIVE: The enteric nervous system (ENS) plays a key role in controlling the gut-brain axis under normal and pathological conditions, such as type 2 diabetes. The discovery of intestinal actors, such as enterosynes, able to modulate the ENS-induced duodenal contraction is considered an innovative approach. Among all the intestinal factors, the understanding of the role of gut microbes in controlling glycaemia is still developed. We studied whether the modulation of gut microbiota by prebiotics could permit the identification of novel enterosynes. DESIGN: We measured the effects of prebiotics on the production of bioactive lipids in the intestine and tested the identified lipid on ENS-induced contraction and glucose metabolism. Then, we studied the signalling pathways involved and compared the results obtained in mice to human. RESULTS: We found that modulating the gut microbiota with prebiotics modifies the actions of enteric neurons, thereby controlling duodenal contraction and subsequently attenuating hyperglycaemia in diabetic mice. We discovered that the signalling pathway involved in these effects depends on the synthesis of a bioactive lipid 12-hydroxyeicosatetraenoic acid (12-HETE) and the presence of mu-opioid receptors (MOR) on enteric neurons. Using pharmacological approaches, we demonstrated the key role of the MOR receptors and proliferator-activated receptor γ for the effects of 12-HETE. These findings are supported by human data showing a decreased expression of the proenkephalin and MOR messanger RNAs in the duodenum of patients with diabetic. CONCLUSIONS: Using a prebiotic approach, we identified enkephalin and 12-HETE as new enterosynes with potential real beneficial and safety impact in diabetic human.

Pre-locus coeruleus neurons in rat and mouse.

Previously, we identified a population of neurons in the hindbrain tegmentum, bordering the locus coeruleus (LC). We named this population the pre-locus coeruleus (pre-LC) because in rats its neurons lie immediately rostral to the LC. In mice, however, pre-LC and LC neurons intermingle, making them difficult to distinguish. Here, we use molecular markers and anterograde tracing to clarify the location and distribution of pre-LC neurons in mice, relative to rats. First, we colocalized the transcription factor FoxP2 with the activity marker Fos to identify pre-LC neurons in sodium-deprived rats and show their distribution relative to surrounding catecholaminergic and cholinergic neurons. Next, we used sodium depletion and chemogenetic activation of the aldosterone-sensitive HSD2 neurons in the nucleus of the solitary tract (NTS) to identify the homologous population of pre-LC neurons in mice, along with a related population in the central lateral parabrachial nucleus. Using Cre-reporter mice for Pdyn, we confirmed that most of these sodium-depletion-activated neurons are dynorphinergic. Finally, after confirming that these neurons receive excitatory input from the NTS and paraventricular hypothalamic nucleus, plus convergent input from the inhibitory AgRP neurons in the arcuate hypothalamic nucleus, we identify a major, direct input projection from the medial prefrontal cortex. This new information on the location, distribution, and input to pre-LC neurons provides a neuroanatomical foundation for cell-type-specific investigation of their properties and functions in mice. Pre-LC neurons likely integrate homeostatic information from the brainstem and hypothalamus with limbic, contextual information from the cerebral cortex to influence ingestive behavior.

Cocaine Self-administration Regulates Transcription of Opioid Peptide Precursors and Opioid Receptors in Rat Caudate Putamen and Prefrontal Cortex.

The brain opioid system plays an important role in cocaine reward. Altered signaling in the opioid system by chronic cocaine exposure contributes to cocaine-seeking and taking behavior. The current study investigated concurrent changes in the gene expression of multiple components in rat brain opioid system following cocaine self-administration. Animals were limited to 40 infusions (1.5 mg/kg/infusion) within 6 h per day for five consecutive days. We then examined the mRNA levels of opioid receptors including mu (Oprm), delta (Oprd), and kappa (Oprk), and their endogenous opioid peptide precursors including proopiomelanocortin (Pomc), proenkephalin (Penk), prodynorphin (Pdyn) in the dorsal striatum (CPu) and the prefrontal cortex (PFC) 18 h after the last cocaine infusion. We found that cocaine self-administration significantly increased the mRNA levels of Oprm and Oprd in both the CPu and PFC, but had no effect on Oprk mRNA levels in either brain region. Moreover, cocaine had a greater influence on the mRNA levels of opioid peptide precursors in rat CPu than in the PFC. In the CPu, cocaine self-administration significantly increased the mRNA levels of Penk and Pdyn and abolished the mRNA levels of Pomc. In the PFC, cocaine self-administration only increased Pdyn mRNA levels without changing the mRNA levels of Pomc and Penk. These data suggest that cocaine self-administration influences the expression of multiple genes in the brain opioid system, and the concurrent changes in these targets may underlie cocaine-induced reward and habitual drug-seeking behavior.