Neural Receptors Research Articles

A Role of Endogenous Progesterone in Stroke Cerebroprotection Revealed by the Neural-Specific Deletion of Its Intracellular Receptors.

Treatment with progesterone protects the male and female brain against damage after middle cerebral artery occlusion (MCAO). However, in both sexes, the brain contains significant amounts of endogenous progesterone. It is not known whether endogenously produced progesterone enhances the resistance of the brain to ischemic insult. Here, we used steroid profiling by gas chromatography-tandem mass spectrometry (GC-MS/MS) for exploring adaptive and sex-specific changes in brain levels of progesterone and its metabolites after MCAO. We show that, in the male mouse brain, progesterone is mainly metabolized via 5α-reduction leading to 5α-dihydroprogesterone (5α-DHP), also a progesterone receptor (PR) agonist ligand in neural cells, then to 3α,5α-tetrahydroprogesterone (3α,5α-THP). In the female mouse brain, levels of 5α-DHP and 3α,5α-THP are lower and levels of 20α-DHP are higher than in males. After MCAO, levels of progesterone and 5α-DHP are upregulated rapidly to pregnancy-like levels in the male but not in the female brain. To assess whether endogenous progesterone and 5α-DHP contribute to the resistance of neural cells to ischemic damage, we inactivated PR selectively in the CNS. Deletion of PR in the brain reduced its resistance to MCAO, resulting in increased infarct volumes and neurological deficits in both sexes. Importantly, endogenous PR ligands continue to protect the brain of aging mice. These results uncover the unexpected importance of endogenous progesterone and its metabolites in cerebroprotection. They also reveal that the female reproductive hormone progesterone is an endogenous cerebroprotective neurosteroid in both sexes.SIGNIFICANCE STATEMENT The brain responds to injury with protective signaling and has a remarkable capacity to protect itself. We show here that, in response to ischemic stroke, levels of progesterone and its neuroactive metabolite 5α-dihydroprogesterone are upregulated rapidly in the male mouse brain but not in the female brain. An important role of endogenous progesterone in cerebroprotection was demonstrated by the conditional inactivation of its receptor in neural cells. These results show the importance of endogenous progesterone, its metabolites, and neural progesterone receptors in acute cerebroprotection after stroke. This new concept could be exploited therapeutically by taking into account the progesterone status of patients and by supplementing and reinforcing endogenous progesterone signaling for attaining its full cerebroprotective potential.

Tuning Surface and Topographical Features to Investigate Competitive Guidance of Spiral Ganglion Neurons.

Cochlear Implants (CIs) suffer from limited tonal resolution due, in large part, to spatial separation between stimulating electrode arrays and primary neural receptors. In this work, a combination of physical and chemical micropatterns, formed on acrylate polymers, are used to direct the growth of primary spiral ganglion neurons (SGNs), the inner ear neurons. Utilizing the inherent temporal and spatial control of photopolymerization, physical microgrooves are fabricated using a photomask in a single step process. Biochemical patterns are generated by adsorbing laminin, a cell adhesion protein, to acrylate polymer surfaces followed by irradiation through a photomask with UV light to deactivate protein in exposed areas and generate parallel biochemical patterns. Laminin deactivation was shown increase as a function of UV light exposure while remaining adsorbed to the polymer surface. SGN neurites show alignment to both biochemical and physical patterns when evaluated individually. Competing biochemical and physical patterns were also examined. The relative guiding strength of physical cues was varied by independently changing both the amplitude and the band spacing of the microgrooves, with higher amplitudes and shorter band spacing providing cues that more effective guide neurite growth. SGN neurites aligned to laminin patterns with lower physical pattern amplitude and thus weaker physical cues. Alignment of SGNs shifted toward the physical pattern with higher amplitude and lower periodicity patterns which represent stronger cues. These results demonstrate the ability of photopolymerized microfeatures to modulate alignment of inner ear neurites even in the presence of conflicting physical and biochemical cues laying the groundwork for next generation cochlear implants and neural prosthetic devices.

Bupropión: una alternativa farmacológica en cesación tabáquica

There are many reasons for the use of antidepressants in smoking cessation: 1) lack of nicotine can produce depressive symptoms or precipitate a severe depressive episode; 2) nicotine may have antidepressant effects that maintain addiction; 3) some antidepressants have an action on neural pathways or receptors involved in nicotine addiction; and 4) some patients do not want to use other therapies or they have failed with them. Bupropion is first-line therapy for smoking cessation, with long-term effectiveness, serious side effects are rare. The evidence suggests bupropion is similar in effectiveness to nicotine replacement therapy (NRT), and less effective than varenicline, but more studies are needed to confirm this. Bupropion is safe in psychiatric patients compensated over a period of three months or more.

Abstract 1821: Sympathetic nerves regulate a metabolic switch promoting angiogenesis through adrenergic signaling in prostate cancer

Abstract Nerves pattern the vasculature during development and regeneration. Recent studies have shown that the sympathetic nervous system (SNS) is coopted to promote carcinogenesis, and that depletion of SNS β-adrenergic receptors (βARs) in the prostate stromal compartment inhibits tumor growth. As the vasculature is in direct contact with the SNS, and regulates tumor glucose and oxygen delivery, we aimed to identify the stromal populations that mediate SNS signaling and the mechanisms by which loss of βAR signaling inhibits prostate cancer progression. In vivo xenograft tumor growth was measured in Adrb2-/-; Adrb3-/- mice using an orthotopic PC3-luciferase model. Prostate cancer progression in the transgenic autochthonous HI-myc model was assessed after conditional Adrb2 deletion in stromal populations by intercrossing Adrb2fl/fl line to stromal Cre lines. Metabolism was measured ex vivo by flow quantification of the lipophilic cationic dye TMRE whose accumulation reflects mitochondrial membrane potential, and by NADH autofluorescence under UV excitation. In vitro oxidative phosphorylation and glycolytic flux were measured in primary prostate endothelial cells (VeraVec) using the Seahorse bioanalyzer and targeted gas chromatography-mass spectrometry (GC/MS). Deletion of the SNS βAR neural receptors in the microenvironment synergized to arrest exponential tumor growth. Imaging the tumor vasculature revealed a defect in vessel branching, a phenotype also observed after chemical sympathectomy, suggesting altered angiogenesis in the absence of βAR stimulation. Histology revealed an increase in vascular innervation and density during the transition from low to high grade pre-neoplastic PIN. Conditional Adrb2 deletion (Adrb2cKO) in endothelial cells (ECs), but not myeloid cells or pericytes, not only inhibited angiogenesis, but also reduced disease progression throughout all histopathological cancer stages. Transcriptome analysis of flow isolated Adrb2cKO ECs suggested an increase in oxidative metabolism and upregulation of mitochondrial Coa6. Ex vivo analysis of ECs revealed a metabolic switch during the low to high grade PIN transition, and this switch was inhibited by Adrb2cKO. Further in vitro characterization by Adrb2 knockdown revealed a metabolic shift in ECs to oxidative phosphorylation, which was mediated by Coa6 upregulation. As Coa6 cooperates with Cox10 in respiratory complex IV assembly, conditional co-deletion of Adrb2 and Cox10 in the HI-myc mouse rescued aerobic glycolysis, angiogenesis, and cancer progression. Our data thus demonstrates that the SNS mediates a pro-angiogenic switch during the early stages of prostate carcinogenesis. Moreover, we identified ECs as SNS stromal targets, and that abrogation of βAR signaling inhibits the angio-metabolic switch and delays prostate cancer progression, suggesting a novel therapeutic target for prostate cancer treatment. Citation Format: Ali Zahalka, Anna Arnal-Estape, Maria Maryanovich, Fumio Nakahara, Cristian Cruz, Paul S. Frenette. Sympathetic nerves regulate a metabolic switch promoting angiogenesis through adrenergic signaling in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1821. doi:10.1158/1538-7445.AM2017-1821

Biased signaling at neural melanocortin receptors in regulation of energy homeostasis

The global prevalence of obesity highlights the importance of understanding on regulation of energy homeostasis. The central melanocortin system is an important intersection connecting the neural pathways controlling satiety and energy expenditure to regulate energy homeostasis by sensing and integrating the signals of external stimuli. In this system, neural melanocortin receptors (MCRs), melanocortin-3 and -4 receptors (MC3R and MC4R), play crucial roles in the regulation of energy homeostasis. Recently, multiple intracellular signaling pathways and biased signaling at neural MCRs have been discovered, providing new insights into neural MCR signaling. This review attempts to summarize biased signaling including biased receptor mutants (both naturally occurring and lab-generated) and biased ligands at neural MCRs, and to provide a better understanding of obesity pathogenesis and new therapeutic implications for obesity treatment.

Tyramide Signal Amplification Permits Immunohistochemical Analyses of Androgen Receptors in the Rat Prefrontal Cortex.

Research on neural androgen receptors (ARs) has traditionally focused on brain regions that regulate reproductive and aggressive behaviors, such as the hypothalamus and amygdala. Although many cells in the prefrontal cortex (PFC) also express ARs, the number of ARs per cell appears to be much lower, and thus, AR immunostaining is often hard to detect and quantify in the PFC. Here, we demonstrate that biotin tyramide signal amplification (TSA) dramatically increases AR immunoreactivity in the rat brain, including critical regions of the PFC such as the medial PFC (mPFC) and orbitofrontal cortex (OFC). We show that TSA is useful for AR detection with both chromogenic and immunofluorescent immunohistochemistry. Double-labeling studies reveal that AR+ cells in the PFC and hippocampus are NeuN+ but not GFAP+ and thus primarily neuronal. Finally, in gonadally intact rats, more AR+ cells are present in the mPFC and OFC of males than of females. Future studies can use TSA to further examine AR immunoreactivity across ages, sexes, strains, and different procedures (e.g., fixation methods). In light of emerging evidence for the androgen regulation of executive function and working memory, these results may help understand the distribution and roles of ARs in the PFC.

Neuronal signaling repertoire in the mammalian sperm functionality.

The common embryonic origin has been a recurrent explanation to understand the presence of "neural receptors" in sperm. However, this designation has conditioned a bias marked by the classical neurotransmission model, dismissing the possibility that neurotransmitters can play specific roles in the sperm function by themselves. For instance, the launching of acrosome reaction, a fundamental sperm function, includes several steps that recall the process of presynaptic secretion. Unlike of postsynaptic neuron, whose activation is mediated by molecular interaction between neurotransmitter and postsynaptic receptors, the oocyte activation is not mediated by receptors, but by cytosolic translocation of sperm phospholipase (PLCζ). Thus, the sperm has a cellular design to access and activate the oocyte and restore the ploidy of the species by an "allogenic pronuclear fusion." At subcellular level, the events controlling sperm function, particularly the capacitation process, are activated by chemical signals that trigger ion fluxes, sterol oxidation, synthesis of cyclic adenosine monophosphate, protein kinase A activation, tyrosine phosphorylations and calcium signaling, which correspond to second messengers similar to those associated with exocytosis and growth cone guidance in neurons. Classically, the sperm function associated with neural signals has been analyzed as a unidimensional approach (single ligand-receptor effect). However, the in vivo sperm are exposed to multidimensional signaling context, for example, the GABAergic, monoaminergic, purinergic, cholinergic, and melatoninergic, to name a few. The aim of this review is to present an overview of sperm functionality associated with "neuronal signaling" and possible cellular and molecular mechanisms involved in their regulation.

Evolution of the functionally conserved DCC gene in birds

Understanding the loss of conserved genes is critical for determining how phenotypic diversity is generated. Here we focus on the evolution of DCC, a gene that encodes a highly conserved neural guidance receptor. Disruption of DCC in animal models and humans results in major neurodevelopmental defects including commissural axon defects. Here we examine DCC evolution in birds, which is of particular interest as a major model system in neurodevelopmental research. We found the DCC containing locus was disrupted several times during evolution, resulting in both gene losses and faster evolution rate of salvaged genes. These data suggest that DCC had been lost independently twice during bird evolution, including in chicken and zebra finch, whereas it was preserved in many other closely related bird species, including ducks. Strikingly, we observed that commissural axon trajectory appeared similar regardless of whether DCC could be detected or not. We conclude that the DCC locus is susceptible to genomic instability leading to independent disruptions in different branches of birds and a significant influence on evolution rate. Overall, the phenomenon of loss or molecular evolution of a highly conserved gene without apparent phenotype change is of conceptual importance for understanding molecular evolution of key biological processes.

Effects of Short Term Hypoxia-Preconditioning on Glial Phenotype Induction of Human Mesenchymal Stem Cells

Development of successful clinical treatments for peripheral nerve injury is limited due to the complications behind neural physiology. Human mesenchymal stem cells (hMSCs) have the ability to directly promote tissue repair and protect cells at the injury site. Studies have shown that hMSCs can be transplanted to improve nerve regeneration. Hypoxic culture condition has been proven to maintain the stemness of hMSCs for later differentiation. In this study, we investigated the effects of low oxygen (O2) (2% and 5% O2) pre-treatment and initial seeding density (500, 1000, and 2000 cells/cm2) on glial protein expression during glial differentiation of hMSCs. Results showed that the secretion of glial proteins was tunable by modifying the seeding density. Moreover, glial induction of hMSCs, characterized by the glial fibrillary acidic protein (GFAP) and S100β expressing phenotype, were enhanced by short-term hypoxia pretreatment. The significantly increased gene expression, including GFAP (10 folds in 2% O2, 25 folds in 5% O2), 2’,3’-Cyclic Nucleotide 3’ Phosphodiesterase (CNP) (600 folds in 2% O2, 800 folds in 5% O2), and neural growth factor receptor (NGFR) (4 folds in 5% O2), indicated that low oxygen, especially 5% O2 pretreated hMSCs had an improved potential for peripheral nerve regeneration.

Association of SNPs in dopamine and serotonin pathway genes and their interacting genes with temperament traits in Charolais cows.

Cattle temperament is a complex trait, and molecular studies aimed at defining this trait are scarce. We used an interaction networks approach to identify new genes (interacting genes) and to estimate their effects and those of 19 dopamine- and serotonin-related genes on the temperament traits of Charolais cattle. The genes proopiomelanocortin (POMC), neuropeptide Y (NPY), solute carrier family 18, member 2 (SLC18A2) and FBJ murine osteosarcoma viral oncogene homologue (FOSFBJ) were identified as new candidates. Their potential to be associated with temperament was estimated according to their reported biological activities, which included interactions with neural activity, receptor function, targeting or synthesis of neurotransmitters and association with behaviour. Pen score (PS) and exit velocity (EV) measures were determined from 412 Charolais cows to calculate their temperament score (TS). Based on the TS, calm (n = 55; TS, 1.09 ± 0.33) and temperamental (n = 58; TS, 2.27 ± 0.639) cows were selected and genotyped using a 248 single-nucleotide variation (SNV) panel. Of the 248 variations in the panel, only 151 were confirmed to be polymorphic (single-nucleotide polymorphisms; SNPs) in the tested population. Single-marker association analyses between genotypes and temperament measures (EV, PS and/or TS) indicated significant associations of six SNPs from four candidate genes. The markers rs109576799 and rs43696138, located in the DRD3 and HTR2A genes, respectively, were significantly associated with both EV and TS traits. Four markers, rs110365063 and rs137756569 from the POMC gene and rs110365063 and rs135155082 located in SLC18A2 and DRD2, respectively, were associated with PS. The variant rs110365063 located in bovine SLC18A2 causes a change in the amino acid sequence from Ala to Thr. Further studies are needed to confirm the association of genetic profile with cattle temperament; however, our study represents important progress in understanding the regulation of cattle temperament by different genes with divergent functions.

Does N,N-Dimethyltryptamine (DMT) Adequately Explain Near-Death Experiences?

Abstract: Some NDE researchers have suggested that because some users of psychedelic drugs have experiences purportedly similar to near-death experiences (NDEs), neural receptors and neurotransmitters affected by a particular drug may underlie out-of-body experiences and NDEs. One of the most recent psychedelic candidates that allegedly causes NDE-like experiences is N,N-dimethyltryptamine (DMT), a natural substance that the body produces in small amounts. If DMT experiences are phenomenologically similar to NDEs, then it is possible that the human body in extremis may produce larger amounts of DMT that reach psychedelic experience-causing levels in the blood. In this paper, I explore the issue of whether DMT might play a causal role in the production of NDEs. The first section summarizes basic information of about NDEs, focusing on their phenomenological aspects. The second scetion classifies theories of NDEs to place the DMT theory in some context of the history of the debate over the cause of NDEs. The following section discusses DMT's chemical composition, physical effects, and psychological effects. The final section explores whether NDE and DMT experiences have a sufficient degree of phenomenological similarity to justify a causal role for DMT in the production of NDEs and concludes that such similarity is lacking.

Spinal cord injury induces astroglial conversion towards neuronal lineage.

BackgroundNeurons have intrinsic capability to regenerate after lesion, though not spontaneously. Spinal cord injury (SCI) causes permanent neurological impairments partly due to formation of a glial scar that is composed of astrocytes and microglia. Astrocytes play both beneficial and detrimental roles on axonal re-growth, however, their precise role after SCI is currently under debate.MethodsWe analyzed molecular changes in astrocytes at multiple stages after two SCI severities using cell-specific transcriptomic analyses.ResultsWe demonstrate that astrocyte response after injury depends on both time after injury and lesion severity. We then establish that injury induces an autologous astroglial transdifferentiation where over 10 % of astrocytes express classical neuronal progenitor markers including βIII-tubulin and doublecortin with typical immature neuronal morphology. Lineage tracing confirmed that the origin of these astrocytes is resident mature, rather than newly formed astrocytes. Astrocyte-derived neuronal progenitors subsequently express GABAergic, but not glutamatergic-specific markers. Furthermore, we have identified the neural stem cell marker fibroblast growth factor receptor 4 (Fgfr4) as a potential autologous modulator of astrocytic transdifferentiation following SCI. Finally, we establish that astroglial transdifferentiation into neuronal progenitors starts as early as 72 h and continues to a lower degrees up to 6 weeks post-lesion.ConclusionWe thus demonstrate for the first time autologous injury-induced astroglial conversion towards neuronal lineage that may represent a therapeutic strategy to replace neuronal loss and improve functional outcomes after central nervous system injury.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-016-0133-0) contains supplementary material, which is available to authorized users.

A point mutation in the extracellular domain of L1 blocks its capacity to confer metastasis in colon cancer cells via CD10.

The neural L1 transmembrane cell adhesion receptor of the immunoglobulin-like family is a target gene of Wnt-β-catenin signaling in human colorectal cancer (CRC) cells and is expressed at the invasive edge of the tumor tissue. L1 overexpression in cultured CRC cells confers enhanced proliferation, motility and liver metastasis. We have analyzed the mechanisms of L1-mediated signaling in CRC cells by using various point mutations in the L1 ectodomain that are known to cause severe genetically inherited mental retardation disorders in patients. We found that all such L1 ectodomain mutations abolish the ability of L1 to confer metastatic properties in CRC cells. Using gene array analysis, we identified L1-mutation-specific gene expression signatures for the L1/H210Q and L1/D598N mutations. We identified CD10, a metalloprotease (neprilysin, neutral endopeptidase) and a gene that is specifically induced in CRC cells by L1 in an L1/H210Q mutation-specific manner. CD10 expression was required for the L1-mediated induction of cell proliferation, motility and metastasis, as suppression of CD10 levels in L1-expressing CRC cells abolished the L1 effects on CRC progression. The signaling from L1 to CD10 was mediated through the L1-ezrin-NF-κB pathway. In human CRC tissue L1 and CD10 were localized in partially overlapping regions in the more invasive areas of the tumor tissue. The results suggest that CD10 is a necessary component conferring the L1 effects in CRC cells. The identification of gene expression patterns of L1-domain-specific point mutations may provide novel markers and targets for interfering with L1-mediated CRC progression.

Convergence of inhibitory neural inputs regulate motor activity in the murine and monkey stomach.

Inhibitory motor neurons regulate several gastric motility patterns including receptive relaxation, gastric peristaltic motor patterns, and pyloric sphincter opening. Nitric oxide (NO) and purines have been identified as likely candidates that mediate inhibitory neural responses. However, the contribution from each neurotransmitter has received little attention in the distal stomach. The aims of this study were to identify the roles played by NO and purines in inhibitory motor responses in the antrums of mice and monkeys. By using wild-type mice and mutants with genetically deleted neural nitric oxide synthase (Nos1-/-) and P2Y1 receptors (P2ry1-/-) we examined the roles of NO and purines in postjunctional inhibitory responses in the distal stomach and compared these responses to those in primate stomach. Activation of inhibitory motor nerves using electrical field stimulation (EFS) produced frequency-dependent inhibitory junction potentials (IJPs) that produced muscle relaxations in both species. Stimulation of inhibitory nerves during slow waves terminated pacemaker events and associated contractions. In Nos1-/- mice IJPs and relaxations persisted whereas in P2ry1-/- mice IJPs were absent but relaxations persisted. In the gastric antrum of the non-human primate model Macaca fascicularis, similar NO and purine neural components contributed to inhibition of gastric motor activity. These data support a role of convergent inhibitory neural responses in the regulation of gastric motor activity across diverse species.

Enhanced detection performance in electrosense through capacitive sensing

Weakly electric fish emit an AC electric field into the water and use thousands of sensors on the skin to detect field perturbations due to surrounding objects. The fish’s active electrosensory system allows them to navigate and hunt, using separate neural pathways and receptors for resistive and capacitive perturbations. We have previously developed a sensing method inspired by the weakly electric fish to detect resistive perturbations and now report on an extension of this system to detect capacitive perturbations as well. In our method, an external object is probed by an AC field over multiple frequencies. We present a quantitative framework that relates the response of a capacitive object at multiple frequencies to the object’s composition and internal structure, and we validate this framework with an electrosense robot that implements our capacitive sensing method. We define a metric for comparing the electrosensory range of different underwater electrosense systems. For detecting non-conductive objects, we show that capacitive sensing performs better than resistive sensing by almost an order of magnitude using this measure, while for conductive objects there is a four-fold increase in performance. Capacitive sensing could therefore provide electric fish with extended sensing range for capacitive objects such as prey, and gives artificial electrolocation systems enhanced range for targets that are capacitive.

Dopamine induces inhibitory effects on the circular muscle contractility of mouse distal colon via D1- and D2-like receptors.

Dopamine (DA) acts as gut motility modulator, via D1- and D2-like receptors, but its effective role is far from being clear. Since alterations of the dopaminergic system could lead to gastrointestinal dysfunctions, a characterization of the enteric dopaminergic system is mandatory. In this study, we investigated the role of DA and D1- and D2-like receptors in the contractility of the circular muscle of mouse distal colon by organ-bath technique. DA caused relaxation in carbachol-precontracted circular muscle strips, sensitive to domperidone, D2-like receptor antagonist, and mimicked by bromocriptine, D2-like receptor agonist. 7-Chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (SCH-23390), D1-like receptor antagonist, neural toxins, L-NAME (nitric oxide (NO) synthase inhibitor), 2'-deoxy-N6-methyl adenosine 3',5'-diphosphate diammonium salt (MRS 2179), purinergic P2Y1 antagonist, or adrenergic antagonists were ineffective. DA also reduced the amplitude of neurally evoked cholinergic contractions. The effect was mimicked by (±)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrobromide (SKF-38393), D1-like receptor agonist and antagonized by SCH-23390, MRS2179, or L-NAME. Western blotting analysis determined the expression of DA receptor proteins in mouse distal colon. Notably, SCH-23390 per se induced an increase in amplitude of spontaneous and neurally evoked cholinergic contractions, unaffected by neural blockers, L-NAME, MRS 2179, muscarinic, adrenergic, or D2-like receptor antagonists. Indeed, SCH-23390-induced effects were antagonized by an adenylyl cyclase blocker. In conclusion, DA inhibits colonic motility in mice via D2- and D1-like receptors, the latter reducing acetylcholine release from enteric neurons, involving nitrergic and purinergic systems. Whether constitutively active D1-like receptors, linked to adenylyl cyclase pathway, are involved in a tonic inhibitory control of colonic contractility is questioned.

Selective activation of α7 nicotinic acetylcholine receptors augments hippocampal oscillations

Neural α7 nicotinic acetylcholine receptors (α7 nAChRs) emerged as a potential pharmacologic target for treating cognitive deficits in schizophrenia and Alzheimer’s disease. Experiments modeling these dysfunctions, as well as clinical evidence, demonstrate the relatively consistent procognitive effects of α7 nAChR agonists. One preclinical observation supporting the procognitive role of α7 nAChRs is their ability to modulate neuronal network oscillations closely associated with learning and memory, especially hippocampal oscillations. Due to the high degree of structural similarity between α7 nACh and 5-HT receptors, the majority of α7 nAChR agonists to date also act as 5-HT3 antagonists. To address this confounding property and determine the relevance of α7 nAChR agonist binding to 5-HT3 receptors in modulating hippocampal activity, we tested two well-described α7 nAChR agonists, PNU-282987 and FRM-17874, in mice lacking α7 nAChRs (α7 knock-out, α7KO) using the brainstem simulation-elicited hippocampal theta oscillation assay. Under urethane anesthesia both agonists at equivalent doses demonstrated efficacy in wild-type (WT) mice, significantly enhancing theta power and theta phase-gamma amplitude coupling as compared to saline treated control mice. These effects are comparable to those seen with drugs clinically used to treat Alzheimer’s disease. Although α7KO mice showed no alterations in elicited hippocampal oscillations, both α7 nAChR agonists failed to enhance theta power or theta phase – gamma amplitude coupling in these mice. Our findings demonstrate that selective activation of α7 nAChRs can modulate hippocampal oscillation, and these receptors are the primary targets of the tested agonists, PNU-282987 and FRM-17874 and likely underlies their observed procognitive activity.

Biased signaling initiated by agouti-related peptide through human melanocortin-3 and -4 receptors

The neural melanocortin receptors (MCRs), melanocortin-3 and -4 receptors (MC3R and MC4R), have been increasingly recognized as important regulators of energy homeostasis. The orexigenic agouti-related peptide (AgRP), initially identified as an endogenous antagonist for both neural MCRs, has been suggested to be a biased agonist of MC4R independent of its antagonizing effects. In the present study, we sought to determine the potential of AgRP to regulate the activation of intracellular kinases, including extracellular signal-regulated kinase 1 and 2 (ERK1/2), AKT and AMP-activated protein kinase (AMPK), through neural MCRs. We showed that AgRP acted as a biased agonist in human MC3R (hMC3R), decreasing cAMP activity of constitutively active mutant (F347A) hMC3R but stimulating ERK1/2 activation in both wide type and F347A hMC3Rs. AgRP-stimulated ERK1/2 phosphorylation through MC3R was abolished by protein kinase A (PKA) inhibitor H-89 but not Rp-cAMPS, whereas AgRP-initiated ERK1/2 activation through MC4R was inhibited by phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002. Both NDP-MSH and AgRP treatment induced significant AKT phosphorylation in GT1-7 cells but not in MC3R- or MC4R-transfected HEK293T cells. The phosphorylated AMPK levels in both GT1-7 cells and HERK293T cells transfected with neural MCRs were significantly decreased upon stimulation with NDP-MSH but not with AgRP. In summary, we provided novel data for AgRP-initiated multiple intracellular signaling pathways, demonstrating biased agonism of AgRP in both neural MCRs, leading to a better understanding of neural MCR pharmacology.

Neural endocannabinoid CB1 receptor expression, social status, and behavior in male European starlings

Many species modify behavior in response to changes in resource availability or social status; however, the neural mechanisms underlying these modifications are not well understood. Prior work in male starlings demonstrates that status-appropriate changes in behavior involve brain regions that regulate social behavior and vocal production. Endocannabinoids are ubiquitously distributed neuromodulators that are proposed to play a role in adjusting behavior to match social status. As an initial step to provide insight into this hypothesis we observed flocks of male starlings in outdoor aviaries during the breeding season. We used quantitative real-time PCR to measure expression of endocannabinoid CB1 receptors in brain regions involved in social behavior and motivation (lateral septum [LS], ventral tegmental area [VTA], medial preoptic nucleus [POM]) and vocal behavior (Area X and robust nucleus of the arcopallium; RA). Males with nesting sites sang to females and displaced other males more than males without nesting sites. They also had higher levels of CB1 receptor expression in LS and RA. CB1 expression in LS correlated positively with agonistic behaviors. CB1 expression in RA correlated positively with singing behavior. CB1 in VTA also correlated positively with singing when only singing birds were considered. These correlations nicely map onto the well-established role of LS in agonistic behavior and the known role of RA in song production and VTA in motivation and song production. Studies are now needed to precisely characterize the role of CB1 receptors in these regions in the production of status-appropriate social behaviors.

Role of muscarinic-3 receptor antibody in systemic sclerosis: correlation with disease duration and effects of IVIG.

Gastrointestinal dysmotility in systemic sclerosis (SSc) is associated with autoantibodies against muscarinic-3 receptor (M3-R). We investigated the temporal course of the site of action of these autoantibodies at the myenteric neurons (MN) vs. the smooth muscle (SM) M3-R in relation to disease duration, and determined the role of intravenous immunoglobulin (IVIG) in reversing these changes. Immunoglobulins purified from SSc patients (SScIgG) were used to assess their differential binding to MN and SM (from rat colon) employing immunohistochemistry (IHC). Effect of SScIgG on neural and direct muscle contraction was determined by cholinergic nerve stimulation and bethanechol-induced SM contraction. Effects of IVIG and its antigen-binding fragment F(ab')2 on SScIgG binding were studied by enzyme-linked immunosorbent assay (ELISA) of rat colonic longitudinal SM myenteric plexus (LSMMP) lysate and to second extracellular loop peptide of M3-R (M3-RL2). SScIgG from all patients demonstrated significantly higher binding to MN than to SM. With progression of SSc duration, binding at MN and SM increased in a linear fashion with a correlation coefficient of 0.696 and 0.726, respectively (P < 0.05). SScIgG-mediated attenuation of neural and direct SM contraction also increased with disease duration. ELISA analysis revealed that IVIG and F(ab')2 significantly reduced SScIgG binding to LSMMP lysate and M3-RL2. Dysmotility in SSc occurs sequentially, beginning with SScIgG-induced blockage of cholinergic neurotransmission (neuropathy), which progresses to inhibition of acetylcholine action at the SM cell (myopathy). IVIG reverses this cholinergic dysfunction at the neural and myogenic receptors by anti-idiotypic neutralization of SScIgG.