We hypothesized that Restless Legs Syndrome (RLS) and the restlessness of opioid withdrawal share common neurobiological mechanisms based on the efficacy of μ-opioid receptor (MOR) agonists in RLS and the common RLS-like phenotype of patients with opioid withdrawal. We also hypothesized this involves an increased sensitivity of the striatal striosomal neurons that co-express MORs and dopamine D1 receptors (D1Rs) and release GABA in the internal segment of the globus pallidus (GPi). This hypothesis was tested in mice with diet-induced brain iron deficiency (BID), a rodent model of RLS. Fiber-photometry experiments were performed in mice with BID using a viral GABA biosensor injected in the entopeduncular nucleus (EPN), the GPi equivalent in rodents. EPN GABA release was measured after the systemic administration of the D1R agonist SKF81297 and the MOR agonist methadone. Locomotor activation and striatal mRNA expression of D1Rs, MORs and adenosine A1 receptors (A1Rs) were also analyzed. A minimal locomotor-activating dose of SKF81297 induced a significant EPN GABA release in mice with BID but not controls, while a maximal locomotor-activating dose of the MOR agonist methadone significantly reduced EPN GABA release in mice with BID after saline or SKF81297 administration. BID was associated with a significant reduction in the striatal expression of MORs and A1Rs. The results indicate that BID induces an increased dopaminergic sensitivity of the opioid-responsive striatal-EPN pathway, which might represent a pivotal pathogenetic mechanism of the restlessness of RLS and opioid withdrawal.

Several dopaminergic compounds, including the clinically used pramipexole, are labelled as preferential dopamine D3 receptor (D3R) agonists based on their moderately higher affinity for the D3R versus other D2-like receptor subtypes. In rodents, these compounds typically produce locomotor depression with low doses and locomotor activation with higher doses, which has been assumed to be mediated by presynaptic D3Rs and postsynaptic striatal D2Rs, respectively. However, studies with selective pharmacological and genetic blockade of each dopamine receptor subtype suggest opposite roles. We address this apparent conundrum by performing a comprehensive in vitro, in vivo and ex vivo pharmacological comparison of several preferential D3R agonists. Their differential properties reveal that their locomotor activating effects in mice are dependent on the striatal postsynaptic D3Rs forming heteromers with D1Rs, via their ability to potentiate β-arrestin recruitment by the D1R in the D1R-D3R heteromer. The results also indicate that the locomotor depressant effects are largely dependent on their ability to activate presynaptic D2Rs. More broadly, it is demonstrated that locomotor activity in mice depends on β-arrestin recruitment by the D1R in the striatal D1R-D3R heteromer. These results can have implications for the treatment of L-dopa-induced dyskinesia and Restless Legs Syndrome.

(R,S)-methadone ((R,S)-MTD) is a µ-opioid receptor (MOR) agonist comprised of (R)-MTD and (S)-MTD enantiomers. (S)-MTD is being developed as an antidepressant and is considered an N-methyl-D-aspartate receptor (NMDAR) antagonist. We compared the pharmacology of (R)-MTD and (S)-MTD and found they bind to MORs, but not NMDARs, and induce full analgesia. Unlike (R)-MTD, (S)-MTD was a weak reinforcer that failed to affect extracellular dopamine or induce locomotor stimulation. Furthermore, (S)-MTD antagonized motor and dopamine releasing effects of (R)-MTD. (S)-MTD acted as a partial agonist at MOR, with complete loss of efficacy at the MOR-galanin Gal 1 receptor (Gal 1 R) heteromer, a key mediator of the dopaminergic effects of opioids. In sum, we report novel and unique pharmacodynamic properties of (S)-MTD that are relevant to its potential mechanism of action and therapeutic use. One-sentence summary: (S)-MTD, like (R)-MTD, binds to and activates MORs in vitro, but (S)-MTD antagonizes the MOR-Gal 1 R heteromer, decreasing its abuse liability.

A main rationale for the role of G protein-coupled receptor (GPCR) heteromers as targets for drug development is the putative ability of selective ligands for specific GPCRs to change their pharmacological properties upon GPCR heteromerization. The present study provides a proof of concept for this rationale by demonstrating that heteromerization of dopamine D1 and D3 receptors (D1R and D3R) influences the pharmacological properties of three structurally similar selective dopamine D3R ligands, the phenylpiperazine derivatives PG01042, PG01037 and VK4–116. By using D1R-D3R heteromer-disrupting peptides, it could be demonstrated that the three D3R ligands display different D1R-D3R heteromer-dependent pharmacological properties: PG01042, acting as G protein-biased agonist, counteracted D1R-mediated signaling in the D1R-D3R heteromer; PG01037, acting as a D3R antagonist cross-antagonized D1R-mediated signaling in the D1R-D3R heteromer; and VK4–116 specifically acted as a ß-arrestin-biased agonist in the D1R-D3R heteromer. Molecular dynamics simulations predicted potential molecular mechanisms mediating these qualitatively different pharmacological properties of the selective D3R ligands that are dependent on D1R-D3R heteromerization. The results of in vitro experiments were paralleled by qualitatively different pharmacological properties of the D3R ligands in vivo. The results supported the involvement of D1R-D3R heteromers in the locomotor activation by D1R agonists in reserpinized mice and L-DOPA-induced dyskinesia in rats, highlighting the D1R-D3R heteromer as a main pharmacological target for L-DOPA-induced dyskinesia in Parkinson’s disease. More generally, the present study implies that when suspecting its pathogenetic role, a GPCR heteromer, and not its individual GPCR units, should be considered as main target for drug development.

Ghrelin receptor, also known as growth hormone secretagogue receptor (GHS-R1a), is coexpressed with its truncated isoform GHS-R1b, which does not bind ghrelin or signal, but oligomerizes with GHS-R1a, exerting a complex modulatory role that depends on its relative expression. D1dopamine receptor (D1R) and D5R constitute the two D1-like receptor subtypes. Previous studies showed that GHS-R1b also facilitates oligomerization of GHS-R1a with D1R, conferring GHS-R1a distinctive pharmacological properties. Those include a switch in the preferred coupling of GHS-R1a from Gq to Gs and the ability of D1R/D5R agonists and antagonists to counteract GHS-R1a signaling. Activation of ghrelin receptors localized in the ventral tegmental area (VTA) seems to play a significant role in the contribution of ghrelin to motivated behavior. In view of the evidence indicating that dopaminergic cells of the VTA express ghrelin receptors and D5R, but not D1R, we investigated the possible existence of functional GHS-R1a:GHS-R1b:D5R oligomeric complexes in the VTA. GHS-R1a:GHS-R1b:D5R oligomers were first demonstrated in mammalian transfected cells, and their pharmacological properties were found to be different from those of GHS-R1a:GHS-R1b:D1R oligomers, including weak Gs coupling and the ability of D1R/D5R antagonists, but not agonists, to counteract the effects of ghrelin. However, analyzing the effect of ghrelin in the rodent VTA on MAPK activation withex vivoexperiments, on somatodendritic dopamine release within vivomicrodialysis and on the activation of dopaminergic cells with patch-clamp electrophysiology, provided evidence for a predominant role of GHS-R1a:GHS-R1b:D1R oligomers in the rodent VTA as main mediators of the dopaminergic effects of ghrelin.SIGNIFICANCE STATEMENTThe activation of ghrelin receptors localized in the ventral tegmental area (VTA) plays a significant role in the contribution of ghrelin to motivated behavior. We present evidence that indicates these receptors form part of oligomeric complexes that include the functional ghrelin receptor GHS-R1a, its truncated nonsignaling isoform GHS-R1b, and the dopamine D1receptor (D1R). The binding of ghrelin to these complexes promotes activation of the dopaminergic neurons of the VTA by activation of adenylyl cyclase–protein kinase A signaling, which can be counteracted by both GHS-R1a and D1R antagonists. Our study provides evidence for a predominant role of GHS-R1a:GHS-R1b:D1R oligomers in rodent VTA as main mediators of the dopaminergic effects of ghrelin.

Akathisia is an urgent need to move that is associated with treatment with dopamine receptor blocking agents (DRBAs) and with restless legs syndrome (RLS). The pathogenetic mechanism of akathisia has not been resolved. This article proposes that it involves an increased presynaptic dopaminergic transmission in the ventral striatum and concomitant strong activation of postsynaptic dopamine D1 receptors, which form complexes (heteromers) with dopamine D3 and adenosine A1 receptors. It also proposes that in DRBA-induced akathisia, increased dopamine release depends on inactivation of autoreceptors, whereas in RLS it depends on a brain iron deficiency-induced down-regulation of striatal presynaptic A1 receptors.

We examined the properties of the coefficients of the approximation of the Γ-function with complex values of the free parameter together with the convergence properties of the approximation when using these coefficients. We report that for fixed real parts of the free parameter that using complex coefficients both increases the computational cost of the Lanczos approximation while drecreasing the accuracy. We conclude that in practical applications of numerical evaluation of the Γ-function only coefficients generated with real values of the free parameter should be used.

Several studies found in vitro evidence for heteromerization of dopamine D1 receptors (D1R) and D3 receptors (D3R), and it has been postulated that functional D1R-D3R heteromers that are normally present in the ventral striatum mediate synergistic locomotor-activating effects of D1R and D3R agonists in rodents. Based also on results obtained in vitro, with mammalian transfected cells, it has been hypothesized that those behavioral effects depend on a D1R-D3R heteromer-mediated G protein-independent signaling. Here, we demonstrate the presence on D1R-D3R heteromers in the mouse ventral striatum by using a synthetic peptide that selectively destabilizes D1R-D3R heteromers. Parallel locomotor activity and ex vivo experiments in reserpinized mice and in vitro experiments in D1R-D3R mammalian transfected cells were performed to dissect the signaling mechanisms of D1R-D3R heteromers. Co-administration of D1R and D3R agonists in reserpinized mice produced synergistic locomotor activation and a selective synergistic AKT phosphorylation in the most ventromedial region of the striatum in the shell of the nucleus accumbens. Application of the destabilizing peptide in transfected cells and in the shell of the nucleus accumbens allowed demonstrating that both in vitro and in vivo co-activation of D3R induces a switch from G protein-dependent to G protein-independent D1R-mediated signaling determined by D1R-D3R heteromerization. The results therefore demonstrate that a biased G protein-independent signaling of D1R-D3R heteromers localized in the shell of the nucleus accumbens mediate the locomotor synergistic effects of D1R and D3R agonists in reserpinized mice.

Our working hypothesis is that a hypoadenosinergic state is a main pathogenetic factor that determines the sensory-motor symptoms and hyperarousal of restless legs syndrome (RLS). We have recently demonstrated that brain iron deficiency (BID) in rodents, a well-accepted animal model of RLS, is associated with a generalized downregulation of adenosine A1 receptors (A1R) in the brain and with hypersensitivity of corticostriatal glutamatergic terminals. Here, we first review the experimental evidence for a pivotal role of adenosine and A1R in the control of striatal glutamatergic transmission and the rationale for targeting putative downregulated striatal A1R in RLS patients, which is supported by recent clinical results obtained with dipyridamole, an inhibitor of the nucleoside transporters ENT1 and ENT2. Second, we perform optogenetic-microdialysis experiments in rats to demonstrate that A1R determine the sensitivity of corticostriatal glutamatergic terminals and the ability of dipyridamole to counteract optogeneticallyinduced corticostriatal glutamate release in both animals with BID and controls. Thus, a frequency of optogenetic stimulation that was ineffective at inducing corticostriatal glutamate release in control rats became effective with the local perfusion of a selective A1R antagonist. Furthermore, in animals with and without BID, the striatal application of dipyridamole blocked the optogenetic-induced glutamate release and decreased basal levels of glutamate, which was counteracted by the A1R antagonist. The results support the clinical application of ENT1 inhibitors in RLS.

Identifying non-addictive opioid medications is a high priority in medical sciences, but μ-opioid receptors mediate both the analgesic and addictive effects of opioids. We found a significant pharmacodynamic difference between morphine and methadone that is determined entirely by heteromerization of μ-opioid receptors with galanin Gal1 receptors, rendering a profound decrease in the potency of methadone. This was explained by methadone's weaker proficiency to activate the dopaminergic system as compared to morphine and predicted a dissociation of therapeutic versus euphoric effects of methadone, which was corroborated by a significantly lower incidence of self-report of "high" in methadone-maintained patients. These results suggest that μ-opioid-Gal1 receptor heteromers mediate the dopaminergic effects of opioids that may lead to a lower addictive liability of opioids with selective low potency for the μ-opioid-Gal1 receptor heteromer, exemplified by methadone.

Huntington's disease (HD) is a hereditary neurodegenerative disorder caused by a mutation in the IT15 gene that encodes for the huntingtin protein. Mutated hungtingtin, although widely expressed in the brain, predominantly affects striato-pallidal neurons, particularly enriched with adenosine A2A receptors (A2AR), suggesting a possible involvement of adenosine and A2AR is the pathogenesis of HD. In fact, polymorphic variation in the ADORA2A gene influences the age at onset in HD, and A2AR dynamics is altered by mutated huntingtin. Basal levels of adenosine and adenosine receptors are involved in many processes critical for neuronal function and homeostasis, including modulation of synaptic activity and excitotoxicity, the control of neurotrophin levels and functions, and the regulation of protein degradation mechanisms. In the present review, we critically analyze the current literature involving the effect of altered adenosine tone and adenosine receptors in HD and discuss why therapeutics that modulate the adenosine system may represent a novel approach for the treatment of HD.

The symptomatology of Restless Legs Syndrome (RLS) includes periodic leg movements during sleep (PLMS), dysesthesias, and hyperarousal. Alterations in the dopaminergic system, a presynaptic hyperdopaminergic state, seem to be involved in PLMS, while alterations in glutamatergic neurotransmission, a presynaptic hyperglutamatergic state, seem to be involved in hyperarousal and also PLMS. Brain iron deficiency (BID) is well-recognized as a main initial pathophysiological mechanism of RLS. BID in rodents have provided a pathogenetic model of RLS that recapitulates the biochemical alterations of the dopaminergic system of RLS, although without PLMS-like motor abnormalities. On the other hand, BID in rodents reproduces the circadian sleep architecture of RLS, indicating the model could provide clues for the hyperglutamatergic state in RLS. We recently showed that BID in rodents is associated with changes in adenosinergic transmission, with downregulation of adenosine A1 receptors (A1R) as the most sensitive biochemical finding. It was hypothesized that A1R downregulation leads to hypersensitive striatal glutamatergic terminals and facilitation of striatal dopamine release. Hypersensitivity of striatal glutamatergic terminals was demonstrated by an optogenetic-microdialysis approach in the rodent with BID, indicating that it could represent a main pathogenetic factor that leads to PLMS in RLS. In fact, the dopaminergic agonists pramipexole and ropinirole and the α2δ ligand gabapentin, used in the initial symptomatic treatment of RLS, completely counteracted optogenetically-induced glutamate release from both normal and BID-induced hypersensitive corticostriatal glutamatergic terminals. It is a main tenet of this essay that, in RLS, a single alteration in the adenosinergic system, downregulation of A1R, disrupts the adenosine-dopamine-glutamate balance uniquely controlled by adenosine and dopamine receptor heteromers in the striatum and also the A1R-mediated inhibitory control of glutamatergic neurotransmission in the cortex and other non-striatal brain areas, which altogether determine both PLMS and hyperarousal. Since A1R agonists would be associated with severe cardiovascular effects, it was hypothesized that inhibitors of nucleoside equilibrative transporters, such as dipyridamole, by increasing the tonic A1R activation mediated by endogenous adenosine, could represent a new alternative therapeutic strategy for RLS. In fact, preliminary clinical data indicate that dipyridamole can significantly improve the symptomatology of RLS.

The two highly homologous subtypes of stimulatory G proteins Gαs (Gs) and Gαolf (Golf) display contrasting expression patterns in the brain. Golf is predominant in the striatum, while Gs is predominant in the cortex. Yet, little is known about their functional distinctions. The dopamine D1 receptor (D1R) couples to Gs/olf and is highly expressed in cortical and striatal areas, making it an important therapeutic target for neuropsychiatric disorders. Using novel drug screening methods that allow analysis of specific G-protein subtype coupling, we found that, relative to dopamine, dihydrexidine and N-propyl-apomorphine behave as full D1R agonists when coupled to Gs, but as partial D1R agonists when coupled to Golf. The Gs/Golf-dependent biased agonism by dihydrexidine was consistently observed at the levels of cellular signaling, neuronal function, and behavior. Our findings of Gs/Golf-dependent functional selectivity in D1R ligands open a new avenue for the treatment of cortex-specific or striatum-specific neuropsychiatric dysfunction.

This study uses Monte Carlo experiments to produce new evidence on the performance of a wide range of panel data estimators. It focuses on estimators that are readily available in statistical software packages such as Stata and Eviews, and for which the number of cross-sectional units (N) and time periods (T) are small to moderate in size. The goal is to develop practical guidelines that will enable researchers to select the best estimator for a given type of data. It extends a previous study on the subject (Reed and Ye, Which panel data estimator should I use? 2011), and modifies their recommendations. The new recommendations provide a (virtually) complete decision tree: When it comes to choosing an estimator for efficiency, it uses the size of the panel dataset (N and T) to guide the researcher to the best estimator. When it comes to choosing an estimator for hypothesis testing, it identifies one estimator as superior across all the data scenarios included in the study. An unusual finding is that researchers should use different estimators for estimating coefficients and testing hypotheses. The authors present evidence that bootstrapping allows one to use the same estimator for both.

We show that the last few components in the principal component analysis of the correlation matrix of a group of stocks may contain useful financial insights by identifying highly correlated pairs or larger groups of stocks. The results of this type of analysis can easily be included in the information an investor uses to manage an investment portfolio.

We present four methods of assessing the diversification potential within a stock market, and two of these are based on principal component analysis. They were applied to the Australian stock exchange for the years 2000 to 2014 and all show a consistent picture. The potential for diversification declined almost monotonically in the three years prior to the 2008 financial crisis, leaving investors poorly diversified at the onset of the Global Financial Crisis. On one of the four measures, the diversification potential declined even further in the 2011 European debt crisis and the American credit downgrade.

OBJECTIVE:The first aim was to demonstrate a previously hypothesized increased sensitivity of corticostriatal glutamatergic terminals in the rodent with brain iron deficiency (BID), a pathogenetic model of restless legs syndrome (RLS). The second aim was to determine whether these putative hypersensitive terminals could constitute a significant target for drugs effective in RLS, including dopamine agonists (pramipexole and ropinirole) and α2 δ ligands (gabapentin). METHODS:A recently introduced in vivo optogenetic-microdialysis approach was used, which allows the measurement of the extracellular concentration of glutamate upon local light-induced stimulation of corticostriatal glutamatergic terminals. The method also allows analysis of the effect of local perfusion of compounds within the same area being sampled for glutamate. RESULTS:BID rats showed hypersensitivity of corticostriatal glutamatergic terminals (lower frequency of optogenetic stimulation to induce glutamate release). Both hypersensitive and control glutamatergic terminals were significant targets for locally perfused pramipexole, ropinirole, and gabapentin, which significantly counteracted optogenetically induced glutamate release. The use of selective antagonists demonstrated the involvement of dopamine D4 and D2 receptor subtypes in the effects of pramipexole. INTERPRETATION:Hypersensitivity of corticostriatal glutamatergic terminals can constitute a main pathogenetic mechanism of RLS symptoms. Selective D4 receptor agonists, by specifically targeting these terminals, should provide a new efficient treatment with fewer secondary effects. Ann Neurol 2017;82:951-960.

The initial goal of this study was to investigate alterations in adenosine A2A receptor (A2AR) density or function in a rat model of Huntington disease (HD) with reported insensitivity to an A2AR antagonist. Unsuspected negative results led to the hypothesis of a low striatal adenosine tone and to the search for the mechanisms involved. Extracellular striatal concentrations of adenosine were measured with in vivo microdialysis in two rodent models of early neuropathological stages of HD disease, the Tg51 rat and the zQ175 knock-in mouse. In view of the crucial role of the equilibrative nucleoside transporter (ENT1) in determining extracellular content of adenosine, the binding properties of the ENT1 inhibitor [3H]-S-(4-Nitrobenzyl)-6-thioinosine were evaluated in zQ175 mice and the differential expression and differential coexpression patterns of the ENT1 gene (SLC29A1) were analyzed in a large human cohort of HD disease and controls. Extracellular striatal levels of adenosine were significantly lower in both animal models as compared with control littermates and striatal ENT1 binding sites were significantly upregulated in zQ175 mice. ENT1 transcript was significantly upregulated in HD disease patients at an early neuropathological severity stage, but not those with a higher severity stage, relative to non-demented controls. ENT1 transcript was differentially coexpressed (gained correlations) with several other genes in HD disease subjects compared to the control group. The present study demonstrates that ENT1 and adenosine constitute biomarkers of the initial stages of neurodegeneration in HD disease and also predicts that ENT1 could constitute a new therapeutic target to delay the progression of the disease.

It is now widely recognized that the statistical property of long memory may be due to reasons other than the data generating process being fractionally integrated. We propose a new procedure aimed at distinguishing between a null hypothesis of unifractal fractionally integrated processes and an alternative hypothesis of other processes which display the long memory property. The procedure is based on a pair of empirical, but consistently defined, statistics namely the number of breaks reported by Atheoretical Regression Trees (ART) and the range of the Empirical Fluctuation Process (EFP) in the CUSUM test. The new procedure establishes through simulation the bivariate distribution of the number of breaks reported by ART with the CUSUM range for simulated fractionally integrated series. This bivariate distribution is then used to empirically construct a test which rejects the null hypothesis for a candidate series if its pair of statistics lies on the periphery of the bivariate distribution determined from simulation under the null. We apply these methods to the realized volatility series of 16 stocks in the Dow Jones Industrial Average and show that the rejection rate of the null is higher than if either statistic was used as a univariate test.