Unique pharmacodynamic properties and low abuse liability of the µ-opioid receptor ligand (S)-methadone.

(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 Gal1 receptor (Gal1R) 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-Gal1R heteromer, decreasing its abuse liability.

Humanized dopamine D4.7 receptor male mice display risk-taking behavior and deficits of social recognition and working memory in light/dark-dependent manner.

The dopamine D4 receptor 7-repeat allele (D4.7 R) has been linked with psychiatric disorders such as attention-deficit-hyperactivity disorder, autism, and schizophrenia. However, the highly diverse study populations and often contradictory findings make it difficult to draw reliable conclusions. The D4.7 R has the potential to explain individual differences in behavior. However, there is still a great deal of ambiguity surrounding whether it is causally connected to the etiology of psychiatric disorders. Therefore, humanized D4.7 R mice, with the long third intracellular domain of the human D4.7 R, may provide a valuable tool to examine the relationship between the D4.7 R variant and specific behavioral phenotypes. We report that D4.7 R male mice carrying the humanized D4.7 R variant exhibit distinct behavioral features that are dependent on the light-dark cycle. The behavioral phenotype was characterized by a working memory deficit, delayed decision execution in the light phase, decreased stress and anxiety, and increased risk behavior in the dark phase. Further, D4.7 R mice displayed impaired social recognition memory in both the light and dark phases. These findings provide insight into the potential causal relationship between the human D4.7 R variant and specific behaviors and encourage further consideration of dopamine D4 receptor (DRD4) ligands as novel treatments for psychiatric disorders in which D4.7 R has been implicated.

Complexes of Ghrelin GHS-R1a, GHS-R1b, and Dopamine D1 Receptors Localized in the Ventral Tegmental Area as Main Mediators of the Dopaminergic Effects of Ghrelin.

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. D1 dopamine 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 with ex vivo experiments, on somatodendritic dopamine release with in vivo microdialysis 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 STATEMENT The 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 D1 receptor (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.

Pharmacological targeting of G protein-coupled receptor heteromers.

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.

Preferential Gs protein coupling of the galanin Gal1 receptor in the µ-opioid-Gal1 receptor heterotetramer.

Recent studies have proposed that heteromers of µ-opioid receptors (MORs) and galanin Gal1 receptors (Gal1Rs) localized in the mesencephalon mediate the dopaminergic effects of opioids. The present study reports converging evidence, using a peptide-interfering approach combined with biophysical and biochemical techniques, including total internal reflection fluorescence microscopy, for a predominant homodimeric structure of MOR and Gal1R when expressed individually, and for their preference to form functional heterotetramers when co-expressed. Results show that a heteromerization-dependent change in the Gal1R homodimeric interface leads to a switch in G-protein coupling from inhibitory Gi to stimulatory Gs proteins. The MOR-Gal1R heterotetramer, which is thus bound to Gs via the Gal1R homodimer and Gi via the MOR homodimer, provides the framework for a canonical Gs-Gi antagonist interaction at the adenylyl cyclase level. These novel results shed light on the intense debate about the oligomeric quaternary structure of G protein-coupled receptors, their predilection for heteromer formation, and the resulting functional significance.

Brain Iron Deficiency Changes the Stoichiometry of Adenosine Receptor Subtypes in Cortico-Striatal Terminals: Implications for Restless Legs Syndrome.

Brain iron deficiency (BID) constitutes a primary pathophysiological mechanism in restless legs syndrome (RLS). BID in rodents has been widely used as an animal model of RLS, since it recapitulates key neurochemical changes reported in RLS patients and shows an RLS-like behavioral phenotype. Previous studies with the BID-rodent model of RLS demonstrated increased sensitivity of cortical pyramidal cells to release glutamate from their striatal nerve terminals driving striatal circuits, a correlative finding of the cortical motor hyperexcitability of RLS patients. It was also found that BID in rodents leads to changes in the adenosinergic system, a downregulation of the inhibitory adenosine A1 receptors (A1Rs) and upregulation of the excitatory adenosine A2A receptors (A2ARs). It was then hypothesized, but not proven, that the BID-induced increased sensitivity of cortico-striatal glutamatergic terminals could be induced by a change in A1R/A2AR stoichiometry in favor of A2ARs. Here, we used a newly developed FACS-based synaptometric analysis to compare the relative abundance on A1Rs and A2ARs in cortico-striatal and thalamo-striatal glutamatergic terminals (labeled with vesicular glutamate transporters VGLUT1 and VGLUT2, respectively) of control and BID rats. It could be demonstrated that BID (determined by measuring transferrin receptor density in the brain) is associated with a selective decrease in the A1R/A2AR ratio in VGLUT1 positive-striatal terminals.

Prefrontal Cortex-Driven Dopamine Signals in the Striatum Show Unique Spatial and Pharmacological Properties.

Dopamine (DA) signals in the striatum are critical for a variety of vital processes, including motivation, motor learning, and reinforcement learning. Striatal DA signals can be evoked by direct activation of inputs from midbrain DA neurons (DANs) as well as cortical and thalamic inputs to the striatum. In this study, we show that in vivo optogenetic stimulation of prelimbic (PrL) and infralimbic (IL) cortical afferents to the striatum triggers an increase in extracellular DA concentration, which coincides with elevation of striatal acetylcholine (ACh) levels. This increase is blocked by a nicotinic ACh receptor (nAChR) antagonist. Using single or dual optogenetic stimulation in brain slices from male and female mice, we compared the properties of these PrL/IL-evoked DA signals with those evoked by stimulation from midbrain DAN axonal projections. PrL/IL-evoked DA signals are undistinguishable from DAN evoked DA signals in their amplitudes and electrochemical properties. However, PrL/IL-evoked DA signals are spatially restricted and preferentially recorded in the dorsomedial striatum. PrL/IL-evoked DA signals also differ in their pharmacological properties, requiring activation of glutamate and nicotinic ACh receptors. Thus, both in vivo and in vitro results indicate that cortical evoked DA signals rely on recruitment of cholinergic interneurons, which renders DA signals less able to summate during trains of stimulation and more sensitive to both cholinergic drugs and temperature. In conclusion, cortical and midbrain inputs to the striatum evoke DA signals with unique spatial and pharmacological properties that likely shape their functional roles and behavioral relevance.SIGNIFICANCE STATEMENT Dopamine signals in the striatum play a critical role in basal ganglia function, such as reinforcement and motor learning. Different afferents to the striatum can trigger dopamine signals, but their release properties are not well understood. Further, these input-specific dopamine signals have only been studied in separate animals. Here we show that optogenetic stimulation of cortical glutamatergic afferents to the striatum triggers dopamine signals both in vivo and in vitro These afferents engage cholinergic interneurons, which drive dopamine release from dopamine neuron axons by activation of nicotinic acetylcholine receptors. We also show that cortically evoked dopamine signals have other unique properties, including spatial restriction and sensitivity to temperature changes than dopamine signals evoked by stimulation of midbrain dopamine neuron axons.

A Randomized, Placebo-Controlled Crossover Study with Dipyridamole for Restless Legs Syndrome.

BACKGROUND: New pharmacological targets are needed for restless legs syndrome. Preclinical data suggest that a hypoadenosinergic state plays an important pathogenetic role. OBJECTIVE: The objective of this study was to determine whether inhibitors of equilibrative nucleoside transporters, for example, dipyridamole, could provide effective symptomatic treatment. METHODS: A 2-week double-blind, placebo-controlled crossover study assessed the efficacy of dipyridamole (possible up-titration to 300 mg) in untreated patients with idiopathic restless legs syndrome. Multiple suggested immobilization tests and polysomnography were performed after each treatment phase. Severity was assessed weekly using the International Restless Legs Rating Scale, Clinical Global Impression, and the Medical Outcomes Study Sleep scale. The primary end point was therapeutic response. RESULTS: Twenty-eight of 29 patients recruited were included. International Restless Legs Rating Scale scores improved from a mean ± standard deviation of 24.1 ± 3.1 at baseline to 11.1 ± 2.3 at the end of week 2, versus 23.7 ± 3.4 to 18.7 ± 3.2 under placebo (P < 0.001). Clinical Global Impression, Medical Outcomes Study Sleep, and Multiple Suggested Immobilization Test scores all improved (P < 0.001). The mean effective dose of dipyridamole was 217.8 ± 33.1 mg/d. Sleep variables improved. The mean periodic leg movement index at the end of treatment with dipyridamole was 8.2 ± 3.5 versus. 28.1 ± 6.7 under placebo. Side effects (dipyridamole vs placebo) included abdominal distension (18% vs. 7%), dizziness (10.7% vs 7.1%), diarrhea, and asthenia (each 7.1% vs 3.6%). CONCLUSIONS: Dipyridamole has significant therapeutic effects on both sensory and motor symptoms of restless legs syndrome and on sleep. Our findings confirm the efficacy of dipyridamole in restless legs syndrome predicted from preclinical studies and support a key role of adenosine in restless legs syndrome. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Consensus Guidelines on Rodent Models of Restless Legs Syndrome.

Restless legs syndrome (RLS) is a chronic sensorimotor disorder diagnosed by clinical symptoms. It is challenging to translate the diagnostic self-reported features of RLS to animals. To help researchers design their experiments, a task force was convened to develop consensus guidelines for experimental readouts in RLS animal models. The RLS clinical diagnostic criteria were used as a starting point. After soliciting additional important clinical features of RLS, a consensus set of methods and outcome measures intent on capturing these features-in the absence of a face-to-face interview-was generated and subsequently prioritized by the task force. These were, in turn, translated into corresponding methods and outcome measures for research on laboratory rats and mice and used to generate the final recommendations. The task force recommended activity monitoring and polysomnography as principal tools in assessing RLS-like behavior in rodents. Data derived from these methods were determined to be the preferred surrogate measures for the urge to move, the principal defining feature of RLS. The same tools may be used to objectively demonstrate sleep-state features highly associated with RLS, such as sleep disturbance and number and periodicity of limb movements. Pharmacological challenges and dietary or other manipulations that affect iron availability are desirable to aggravate or improve RLS-like behavior and lend greater confidence that the animal model being proffered replicates key clinical features of RLS. These guidelines provide the first consensus experimental framework for researchers to use when developing new rodent models of RLS. © 2020 International Parkinson and Movement Disorder Society.

Heteromerization between α2A adrenoceptors and different polymorphic variants of the dopamine D4 receptor determines pharmacological and functional differences. Implications for impulsive-control disorders.

Polymorphic alleles of the human dopamine D4 receptor gene (DRD4) have been consistently associated with individual differences in personality traits and neuropsychiatric disorders, particularly between the gene encoding dopamine D4.7 receptor variant and attention deficit hyperactivity disorder (ADHD). The α2A adrenoceptor gene has also been associated with ADHD. In fact, drugs targeting the α2A adrenoceptor (α2AR), such as guanfacine, are commonly used in ADHD treatment. In view of the involvement of dopamine D4 receptor (D4R) and α2AR in ADHD and impulsivity, their concurrent localization in cortical pyramidal neurons and the demonstrated ability of D4R to form functional heteromers with other G protein-coupled receptors, in this study we evaluate whether the α2AR forms functional heteromers with D4R and weather these heteromers show different properties depending on the D4R variant involved. Using cortical brain slices from hD4.7R knock-in and wild-type mice, here, we demonstrate that α2AR and D4R heteromerize and constitute a significant functional population of cortical α2AR and D4R. Moreover, in cortical slices from wild-type mice and in cells transfected with α2AR and D4.4R, we detect a negative crosstalk within the heteromer. This negative crosstalk is lost in cortex from hD4.7R knock-in mice and in cells expressing the D4.7R polymorphic variant. We also show a lack of efficacy of D4R ligands to promote G protein activation and signaling only within the α2AR-D4.7R heteromer. Taken together, our results suggest that α2AR-D4R heteromers play a pivotal role in catecholaminergic signaling in the brain cortex and are likely targets for ADHD pharmacotherapy.

Cell-type specific expression and behavioral impact of galanin and GalR1 in the locus coeruleus during opioid withdrawal.

The neuropeptide galanin is reported to attenuate opioid withdrawal symptoms, potentially by reducing neuronal hyperactivity in the noradrenergic locus coeruleus (LC) via galanin receptor 1 (GalR1). We evaluated this mechanism by using RNAscope in situ hybridization to characterize GalR1 mRNA distribution in the dorsal pons and to compare galanin and GalR1 mRNA expression in tyrosine hydroxylase-positive (TH+) LC cells at baseline and following chronic morphine or precipitated withdrawal. We then used genetically altered mouse lines and pharmacology to test whether noradrenergic galanin (NE-Gal) modulates withdrawal symptoms. RNAscope revealed that, while GalR1 signal was evident in the dorsal pons, 80.7% of the signal was attributable to TH- neurons outside the LC. Galanin and TH mRNA were abundant in LC cells at baseline and were further increased by withdrawal, whereas low basal GalR1 mRNA expression was unaltered by chronic morphine or withdrawal. Naloxone-precipitated withdrawal symptoms in mice lacking NE-Gal (GalcKO-Dbh ) were largely similar to WT littermates, indicating that loss of NE-Gal does not exacerbate withdrawal. Complementary experiments using NE-Gal overexpressor mice (NE-Gal OX) and systemic administration of the galanin receptor agonist galnon revealed that increasing galanin signaling also failed to alter behavioral withdrawal, while suppressing noradrenergic transmission with the alpha-2 adrenergic receptor agonist clonidine attenuated multiple symptoms. These results indicate that galanin does not acutely attenuate precipitated opioid withdrawal via an LC-specific mechanism, which has important implications for the general role of galanin in regulation of somatic and affective opioid responses and LC activity.

Control of glutamate release by complexes of adenosine and cannabinoid receptors.

BACKGROUND: It has been hypothesized that heteromers of adenosine A2A receptors (A2AR) and cannabinoid CB1 receptors (CB1R) localized in glutamatergic nerve terminals mediate the integration of adenosine and endocannabinoid signaling involved in the modulation of striatal excitatory neurotransmission. Previous studies have demonstrated the existence of A2AR-CB1R heteromers in artificial cell systems. A dependence of A2AR signaling for the Gi protein-mediated CB1R signaling was described as one of its main biochemical characteristics. However, recent studies have questioned the localization of functionally significant A2AR-CB1R heteromers in striatal glutamatergic terminals. RESULTS: Using a peptide-interfering approach combined with biophysical and biochemical techniques in mammalian transfected cells and computational modeling, we could establish a tetrameric quaternary structure of the A2AR-CB1R heterotetramer. This quaternary structure was different to the also tetrameric structure of heteromers of A2AR with adenosine A1 receptors or dopamine D2 receptors, with different heteromeric or homomeric interfaces. The specific quaternary structure of the A2A-CB1R, which depended on intermolecular interactions involving the long C-terminus of the A2AR, determined a significant A2AR and Gs protein-mediated constitutive activation of adenylyl cyclase. Using heteromer-interfering peptides in experiments with striatal glutamatergic terminals, we could then demonstrate the presence of functionally significant A2AR-CB1R heteromers with the same biochemical characteristics of those studied in mammalian transfected cells. First, either an A2AR agonist or an A2AR antagonist allosterically counteracted Gi-mediated CB1R agonist-induced inhibition of depolarization-induced glutamate release. Second, co-application of both an A2AR agonist and an antagonist cancelled each other effects. Finally, a CB1R agonist inhibited glutamate release dependent on a constitutive activation of A2AR by a canonical Gs-Gi antagonistic interaction at the adenylyl cyclase level. CONCLUSIONS: We demonstrate that the well-established cannabinoid-induced inhibition of striatal glutamate release can mostly be explained by a CB1R-mediated counteraction of the A2AR-mediated constitutive activation of adenylyl cyclase in the A2AR-CB1R heteromer.

Reinterpreting anomalous competitive binding experiments within G protein-coupled receptor homodimers using a dimer receptor model.

An increasing number of G protein-coupled receptors (GPCRs) have been reported to be expressed in the plasma membrane as dimers. Since most ligand binding data are currently fitted by classical equations developed only for monomeric receptors, the interpretation of data could be misleading in the presence of GPCR dimers. On the other hand, the equations developed from dimer receptor models assuming the existence of two orthosteric binding sites within the dimeric molecule offer the possibility to directly calculate macroscopic equilibrium dissociation constants for the two sites, an index of cooperativity (DC) that reflects the molecular communication within the dimer and, importantly, a constant of radioligand-competitor allosteric interaction (KDAB) in competitive assays. Here, we provide a practical way to fit competitive binding data that allows the interpretation of apparently anomalous results, such as competition curves that could be either bell-shaped, monophasic or biphasic depending on the assay conditions. The consideration of a radioligand-competitor allosteric interaction allows fitting these curve patterns both under simulation conditions and in real radioligand binding experiments, obtaining competitor affinity parameters closer to the actual values. Our approach is the first that, assuming the formation of receptor homodimers, is able to explain several experimental results previously considered erroneous due to their impossibility to be fitted. We also deduce the radioligand concentration responsible for the conversion of biphasic to monophasic or to bell-shaped curves in competitive radioligand binding assays. In conclusion, bell-shaped curves in competitive binding experiments constitute evidence for GPCR homodimerization.

Cross-communication between Gi and Gs in a G-protein-coupled receptor heterotetramer guided by a receptor C-terminal domain.

BACKGROUND: G-protein-coupled receptor (GPCR) heteromeric complexes have distinct properties from homomeric GPCRs, giving rise to new receptor functionalities. Adenosine receptors (A1R or A2AR) can form A1R-A2AR heteromers (A1-A2AHet), and their activation leads to canonical G-protein-dependent (adenylate cyclase mediated) and -independent (ß-arrestin mediated) signaling. Adenosine has different affinities for A1R and A2AR, allowing the heteromeric receptor to detect its concentration by integrating the downstream Gi- and Gs-dependent signals. cAMP accumulation and ß-arrestin recruitment assays have shown that, within the complex, activation of A2AR impedes signaling via A1R. RESULTS: We examined the mechanism by which A1-A2AHet integrates Gi- and Gs-dependent signals. A1R blockade by A2AR in the A1-A2AHet is not observed in the absence of A2AR activation by agonists, in the absence of the C-terminal domain of A2AR, or in the presence of synthetic peptides that disrupt the heteromer interface of A1-A2AHet, indicating that signaling mediated by A1R and A2AR is controlled by both Gi and Gs proteins. CONCLUSIONS: We identified a new mechanism of signal transduction that implies a cross-communication between Gi and Gs proteins guided by the C-terminal tail of the A2AR. This mechanism provides the molecular basis for the operation of the A1-A2AHet as an adenosine concentration-sensing device that modulates the signals originating at both A1R and A2AR.

Functional µ-Opioid-Galanin Receptor Heteromers in the Ventral Tegmental Area.

The neuropeptide galanin has been shown to interact with the opioid system. More specifically, galanin counteracts the behavioral effects of the systemic administration of µ-opioid receptor (MOR) agonists. Yet the mechanism responsible for this galanin-opioid interaction has remained elusive. Using biophysical techniques in mammalian transfected cells, we found evidence for selective heteromerization of MOR and the galanin receptor subtype Gal1 (Gal1R). Also in transfected cells, a synthetic peptide selectively disrupted MOR-Gal1R heteromerization as well as specific interactions between MOR and Gal1R ligands: a negative cross talk, by which galanin counteracted MAPK activation induced by the endogenous MOR agonist endomorphin-1, and a cross-antagonism, by which a MOR antagonist counteracted MAPK activation induced by galanin. These specific interactions, which represented biochemical properties of the MOR-Gal1R heteromer, could then be identified in situ in slices of rat ventral tegmental area (VTA) with MAPK activation and two additional cell signaling pathways, AKT and CREB phosphorylation. Furthermore, in vivo microdialysis experiments showed that the disruptive peptide selectively counteracted the ability of galanin to block the dendritic dopamine release in the rat VTA induced by local infusion of endomorphin-1, demonstrating a key role of MOR-Gal1R heteromers localized in the VTA in the direct control of dopamine cell function and their ability to mediate antagonistic interactions between MOR and Gal1R ligands. The results also indicate that MOR-Gal1R heteromers should be viewed as targets for the treatment of opioid use disorders. SIGNIFICANCE STATEMENT: The µ-opioid receptor (MOR) localized in the ventral tegmental area (VTA) plays a key role in the reinforcing and addictive properties of opioids. With parallel in vitro experiments in mammalian transfected cells and in situ and in vivo experiments in rat VTA, we demonstrate that a significant population of these MORs form functional heteromers with the galanin receptor subtype Gal1 (Gal1R), which modulate the activity of the VTA dopaminergic neurons. The MOR-Gal1R heteromer can explain previous results showing antagonistic galanin-opioid interactions and offers a new therapeutic target for the treatment of opioid use disorder.

Development of novel biosensors to study receptor-mediated activation of the G-protein α subunits Gs and Golf.

Gαs (Gs) and Gαolf (Golf) are highly homologous G-protein α subunits that activate adenylate cyclase, thereby serving as crucial mediators of intracellular signaling. Because of their dramatically different brain expression patterns, we studied similarities and differences between their activation processes with the aim of comparing their receptor coupling mechanisms. We engineered novel luciferase- and Venus-fused Gα constructs that can be used in bioluminescence resonance energy transfer assays. In conjunction with molecular simulations, these novel biosensors were used to determine receptor activation-induced changes in conformation. Relative movements in Gs were consistent with the crystal structure of ß2 adrenergic receptor in complex with Gs Conformational changes in Golf activation are shown to be similar to those in Gs Overall the current study reveals general similarities between Gs and Golf activation at the molecular level and provides a novel set of tools to search for Gs- and Golf-specific receptor pharmacology. In view of the wide functional and pharmacological roles of Gs- and Golf-coupled dopamine D1 receptor and adenosine A2A receptor in the brain and other organs, elucidating their differential structure-function relationships with Gs and Golf might provide new approaches for the treatment of a variety of neuropsychiatric disorders. In particular, these novel biosensors can be used to reveal potentially therapeutic dopamine D1 receptor and adenosine A2A receptor ligands with functionally selective properties between Gs and Golf signaling.

Targeting hypersensitive corticostriatal terminals in restless legs syndrome.

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.

Allosteric interactions between agonists and antagonists within the adenosine A2A receptor-dopamine D2 receptor heterotetramer.

Adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heteromers are key modulators of striatal neuronal function. It has been suggested that the psychostimulant effects of caffeine depend on its ability to block an allosteric modulation within the A2AR-D2R heteromer, by which adenosine decreases the affinity and intrinsic efficacy of dopamine at the D2R. We describe novel unsuspected allosteric mechanisms within the heteromer by which not only A2AR agonists, but also A2AR antagonists, decrease the affinity and intrinsic efficacy of D2R agonists and the affinity of D2R antagonists. Strikingly, these allosteric modulations disappear on agonist and antagonist coadministration. This can be explained by a model that considers A2AR-D2R heteromers as heterotetramers, constituted by A2AR and D2R homodimers, as demonstrated by experiments with bioluminescence resonance energy transfer and bimolecular fluorescence and bioluminescence complementation. As predicted by the model, high concentrations of A2AR antagonists behaved as A2AR agonists and decreased D2R function in the brain.

Local Control of Extracellular Dopamine Levels in the Medial Nucleus Accumbens by a Glutamatergic Projection from the Infralimbic Cortex.

It is generally assumed that infralimbic cortex (ILC) and prelimbic cortex, two adjacent areas of the medial prefrontal cortex (mPFC) in rodents, provide selective excitatory glutamatergic inputs to the nucleus accumbens (NAc) shell and core, respectively. It is also generally believed that mPFC influences the extracellular levels of dopamine in the NAc primarily by an excitatory collateral to the ventral tegmental area (VTA). In the present study, we first established the existence of a selective functional connection between ILC and the posteromedial portions of the VTA (pmVTA) and the mNAc shell (pmNAc shell), by measuring striatal neuronal activation (immunohistochemical analysis of ERK1/2 phosphorylation) and glutamate release (in vivo microdialysis) upon ILC electrical stimulation. A novel optogenetic-microdialysis approach allowed the measurement of extracellular concentrations of glutamate and dopamine in the pmNAc shell upon local light-induced stimulation of glutamatergic terminals from ILC. Cortical electrical and local optogenetic stimulation produced significant increases in the extracellular concentrations of glutamate and dopamine in the pmNAc shell. Local blockade of glutamate release by perfusion of an adenosine A2A receptor antagonist in the pmNAc shell blocked the dopamine release induced by local optogenetic stimulation but only partially antagonized dopamine release induced by cortical electrical stimulation. The results demonstrate that ILC excitatory afferents directly modulate the extracellular concentration of dopamine in the pmNAc shell, but also support the involvement of an indirect mechanism of dopamine control, through a concomitant ILC-mediated activation of the pmVTA. Significance statement: We established the existence of a functional connection between the infralimbic cortex (ILC) and the posteromedial portions of the ventral tegmental area (pmVTA) and the medial nucleus acumbens shell (pmNAc shell). A novel optogenetic-microdialysis approach allowed us to demonstrate that local glutamate release from glutamatergic terminals from the ILC exert a significant modulation of extracellular concentration of dopamine in the pmNAc shell. This mechanism provides the frame for a selective cortical-mediated tonic dopaminergic modulation of specific striatal compartments.

A Significant Role of the Truncated Ghrelin Receptor GHS-R1b in Ghrelin-induced Signaling in Neurons.

The truncated non-signaling ghrelin receptor growth hormone secretagogue R1b (GHS-R1b) has been suggested to simply exert a dominant negative role in the trafficking and signaling of the full and functional ghrelin receptor GHS-R1a. Here we reveal a more complex modulatory role of GHS-R1b. Differential co-expression of GHS-R1a and GHS-R1b, both in HEK-293T cells and in striatal and hippocampal neurons in culture, demonstrates that GHS-R1b acts as a dual modulator of GHS-R1a function: low relative GHS-R1b expression potentiates and high relative GHS-R1b expression inhibits GHS-R1a function by facilitating GHS-R1a trafficking to the plasma membrane and by exerting a negative allosteric effect on GHS-R1a signaling, respectively. We found a preferential Gi/o coupling of the GHS-R1a-GHS-R1b complex in HEK-293T cells and, unexpectedly, a preferential Gs/olf coupling in both striatal and hippocampal neurons in culture. A dopamine D1 receptor (D1R) antagonist blocked ghrelin-induced cAMP accumulation in striatal but not hippocampal neurons, indicating the involvement of D1R in the striatal GHS-R1a-Gs/olf coupling. Experiments in HEK-293T cells demonstrated that D1R co-expression promotes a switch in GHS-R1a-G protein coupling from Gi/o to Gs/olf, but only upon co-expression of GHS-R1b. Furthermore, resonance energy transfer experiments showed that D1R interacts with GHS-R1a, but only in the presence of GHS-R1b. Therefore, GHS-R1b not only determines the efficacy of ghrelin-induced GHS-R1a-mediated signaling but also determines the ability of GHS-R1a to form oligomeric complexes with other receptors, promoting profound qualitative changes in ghrelin-induced signaling.