Enhanced cognitive flexibility and phasic striatal dopamine dynamics in a mouse model of low striatal tonic dopamine.

The catecholamine neuromodulators dopamine and norepinephrine are implicated in motor function, motivation, and cognition. Although roles for striatal dopamine in these aspects of behavior are well established, the specific roles for cortical catecholamines in regulating striatal dopamine dynamics and behavior are less clear. We recently showed that elevating cortical dopamine but not norepinephrine suppresses hyperactivity in dopamine transporter knockout (DAT-KO) mice, which have elevated striatal dopamine levels. In contrast, norepinephrine transporter knockout (NET-KO) mice have a phenotype distinct from DAT-KO mice, as they show elevated extracellular cortical catecholamines but reduced baseline striatal dopamine levels. Here we evaluated the consequences of altered catecholamine levels in NET-KO mice on cognitive flexibility and striatal dopamine dynamics. In a probabilistic reversal learning task, NET-KO mice showed enhanced reversal learning, which was consistent with larger phasic dopamine transients (dLight) in the dorsomedial striatum (DMS) during reward delivery and reward omission, compared to WT controls. Selective depletion of dorsal medial prefrontal cortex (mPFC) norepinephrine in WT mice did not alter performance on the reversal learning task but reduced nestlet shredding. Surprisingly, NET-KO mice did not show altered breakpoints in a progressive ratio task, suggesting intact food motivation. Collectively, these studies show novel roles of cortical catecholamines in the regulation of tonic and phasic striatal dopamine dynamics and cognitive flexibility, updating our current views on dopamine regulation and informing future therapeutic strategies to counter multiple psychiatric disorders.

Locus coeruleus injury modulates ventral midbrain neuroinflammation during DSS-induced colitis.

Parkinson's disease (PD) is characterized by a decades-long prodrome, consisting of a collection of non-motor symptoms that emerges prior to the motor manifestation of the disease. Of these non-motor symptoms, gastrointestinal dysfunction and deficits attributed to central norepinephrine (NE) loss, including mood changes and sleep disturbances, are frequent in the PD population and emerge early in the disease. Evidence is mounting that injury and inflammation in the gut and locus coeruleus (LC), respectively, underlie these symptoms, and the injury of these systems is central to the progression of PD. In this study, we generate a novel two-hit mouse model that captures both features, using dextran sulfate sodium (DSS) to induce gut inflammation and N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) to lesion the LC. We first confirmed the specificity of DSP-4 for central NE using neurochemical methods and fluorescence light-sheet microscopy of cleared tissue, and established that DSS-induced outcomes in the periphery, including weight loss, gross indices of gut injury and systemic inflammation, the loss of tight junction proteins in the colonic epithelium, and markers of colonic inflammation, were unaffected with DSP-4 pre-administration. We then measured alterations in neuroimmune gene expression in the ventral midbrain in response to DSS treatment alone as well as the extent to which prior LC injury modified this response. In this two-hit model we observed that DSS-induced colitis activates the expression of key cytokines and chemokines in the ventral midbrain only in the presence of LC injury and the typical DSS-associated neuroimmune is blunted by pre-LC lesioning with DSP-4. In all, this study supports the growing appreciation for the LC as neuroprotective against inflammation-induced brain injury and draws attention to the potential for NEergic interventions to exert disease-modifying effects under conditions where peripheral inflammation may compromise ventral midbrain dopaminergic neurons and increase the risk for development of PD.

Computational insights into ligand-induced G protein and ß-arrestin signaling of the dopamine D1 receptor.

The dopamine D1 receptor (D1R), is a class A G protein coupled-receptor (GPCR) which has been a promising drug target for psychiatric and neurological disorders such as Parkinson's disease (PD). Previous studies have suggested that therapeutic effects can be realized by targeting the ß-arrestin signaling pathway of dopamine receptors, while overactivation of the G protein-dependent pathways leads to side effects, such as dyskinesias. Therefore, it is highly desirable to develop a D1R ligand that selectively regulates the ß-arrestin pathway. Currently, most D1R agonists are signaling-balanced and stimulate both G protein and ß-arrestin pathways, with a few reports of G protein biased ligands. However, identification and characterization of ß-arrestin biased D1R agonists has been a challenge thus far. In this study, we implemented Gaussian accelerated molecular dynamics (GaMD) simulations to provide valuable computational insights into the possible underlying molecular mechanism of the different signaling properties of two catechol and two non-catechol D1R agonists that are either G protein biased or signaling-balanced. Dynamic network analysis further identified critical residues in the allosteric signaling network of D1R for each ligand at different conformational or binding states. Some of these residues are crucial for G protein or arrestin signals of GPCRs based on previous studies. Finally, we provided a molecular design strategy which can be utilized by medicinal chemists to develop potential ß-arrestin biased D1R ligands. The proposed hypotheses are experimentally testable and can guide the development of safer and more effective medications for a variety of CNS disorders.

Structure-Functional Selectivity Relationship Studies on A-86929 Analogs and Small Aryl Fragments toward the Discovery of Biased Dopamine D1 Receptor Agonists.

Dopamine regulates normal functions such as movement, reinforcement learning, and cognition, and its dysfunction has been implicated in multiple psychiatric and neurological disorders. Dopamine acts through D1- (D1R and D5R) and D2-class (D2R, D3R, and D4R) receptors and activates both G protein- and ß-arrestin-dependent signaling pathways. Current dopamine receptor-based therapies are used to ameliorate motor deficits in Parkinson's disease or as antipsychotic medications for schizophrenia. These drugs show efficacy for ameliorating only some symptoms caused by dopamine dysfunction and are plagued by debilitating side effects. Studies in primates and rodents have shown that shifting the balance of dopamine receptor signaling toward the arrestin pathway can be beneficial for inducing normal movement, while reducing motor side effects such as dyskinesias, and can be efficacious at enhancing cognitive function compared to balanced agonists. Several structure-activity relationship (SAR) studies have embarked on discovering ß-arrestin-biased dopamine agonists, focused on D2 partial agonists, noncatechol D1 agonists, and mixed D1/D2R dopamine receptor agonists. Here, we describe an SAR study to identify novel D1R ß-arrestin-biased ligands using A-86929, a high-affinity D1R catechol agonist, as a core scaffold to identify chemical motifs responsible for ß-arrestin-biased activity at both D1 and D2Rs. Most of the A-86929 analogs screened were G protein-biased, but none of them were exclusively arrestin-biased. Additionally, various small-fragment molecular probes displayed weak bias toward the ß-arrestin pathway. Continued in-depth SFSR (structure-functional selectivity relationship) studies informed by structure determination, molecular modeling, and mutagenesis studies will facilitate the discovery of potent and efficacious arrestin-biased dopamine receptor ligands.

A role for cortical dopamine in the paradoxical calming effects of psychostimulants.

Psychostimulants have a paradoxical calming effect in the treatment of attention deficit hyperactivity disorder (ADHD), but their mechanism of action is unclear. Studies using dopamine (DA) transporter (DAT) knockout (KO) mice have suggested that the paradoxical calming effect of psychostimulants might occur through actions on serotonin (5-HT) neurotransmission. However, newer non-stimulant drugs, such as atomoxetine and guanfacine, suggest that targeting the norepinephrine (NE) system in the prefrontal cortex (PFC) might explain this paradoxical calming effect. Thus, we sought to clarify the mechanism of this paradoxical action of psychostimulants. Our ex vivo efflux experiments reveal that the NE transporter (NET) blocker desipramine elevates both norepinephrine (NE) and dopamine (DA), but not 5-HT levels, in PFC tissue slices from wild-type (WT) and DAT-KO, but not NET-KO mice. However, the 5-HT transporter (SERT) inhibitor fluoxetine elevates only 5-HT in all three genotypes. Systemic administration of desipramine or fluoxetine inhibits hyperactivity in DAT-KO mice, whereas local PFC infusion of desipramine alone produced this same effect. In contrast, pharmacological NE depletion and DA elevation using nepicastat also inhibits hyperactivity in DAT-KO mice. Together, these data suggest elevation of PFC DA and not NE or 5-HT, as a convergent mechanism for the paradoxical effects of psychostimulants observed in ADHD therapy.

Measuring Nonapoptotic Caspase Activity with a Transgenic Reporter in Mice.

The protease caspase-3 is a key mediator of apoptotic programmed cell death. But weak or transient caspase activity can contribute to neuronal differentiation, axonal pathfinding, and synaptic long-term depression. Despite the importance of sublethal, or nonapoptotic, caspase activity in neurodevelopment and neural plasticity, there has been no simple method for mapping and quantifying nonapoptotic caspase activity (NACA) in rodent brains. We therefore generated a transgenic mouse expressing a highly sensitive and specific fluorescent reporter of caspase activity, with peak signal localized to the nucleus. As a proof of concept, we first obtained evidence that NACA influences neurophysiology in an amygdalar circuit. Then focusing on the amygdala, we were able to quantify a sex-specific persistent elevation in caspase activity in females after restraint stress. This simple in vivo caspase activity reporter will facilitate systems-level studies of apoptotic and nonapoptotic phenomena in behavioral and pathologic models.

Enhanced tyrosine hydroxylase activity induces oxidative stress, causes accumulation of autotoxic catecholamine metabolites, and augments amphetamine effects in vivo.

In Parkinson's disease, dopamine-containing nigrostriatal neurons undergo profound degeneration. Tyrosine hydroxylase (TH) is the rate-limiting enzyme in dopamine biosynthesis. TH increases in vitro formation of reactive oxygen species, and previous animal studies have reported links between cytosolic dopamine build-up and oxidative stress. To examine effects of increased TH activity in catecholaminergic neurons in vivo, we generated TH-over-expressing mice (TH-HI) using a BAC-transgenic approach that results in over-expression of TH with endogenous patterns of expression. The transgenic mice were characterized by western blot, qPCR, and immunohistochemistry. Tissue contents of dopamine, its metabolites, and markers of oxidative stress were evaluated. TH-HI mice had a 3-fold increase in total and phosphorylated TH levels and an increased rate of dopamine synthesis. Coincident with elevated dopamine turnover, TH-HI mice showed increased striatal production of H2 O2 and reduced glutathione levels. In addition, TH-HI mice had elevated striatal levels of the neurotoxic dopamine metabolites 3,4-dihydroxyphenylacetaldehyde and 5-S-cysteinyl-dopamine and were more susceptible than wild-type mice to the effects of amphetamine and methamphetamine. These results demonstrate that increased TH alone is sufficient to produce oxidative stress in vivo, build up autotoxic dopamine metabolites, and augment toxicity.

Targeting ß-Arrestins in the Treatment of Psychiatric and Neurological Disorders.

Therapies for psychiatric and neurological disorders have been in the development and refinement process for the past 5 decades. Yet, most of these therapies lack optimal therapeutic efficacy and have multiple debilitating side effects. Recent advances in understanding the pathophysiological processes of psychiatric and neurological disorders have revealed an important role for ß-arrestins, which are important regulators of G-protein-coupled receptor (GPCR) function, including desensitization and intracellular signaling. These findings have pushed ß-arrestins to the forefront as potential therapeutic targets. Here, we highlight current knowledge on ß-arrestin functions in certain psychiatric and neurological disorders (schizophrenia, Parkinson's disease, and substance abuse disorders), and how this has been leveraged to develop new therapeutic strategies. Furthermore, we discuss the obstacles impacting the field of ß-arrestin-based therapeutic development and future approaches that might help advance strategies to develop optimal ß-arrestin-based therapies.

Retrograde Labeling Illuminates Distinct Topographical Organization of D1 and D2 Receptor-Positive Pyramidal Neurons in the Prefrontal Cortex of Mice.

The cortex plays an important role in regulating motivation and cognition, and does so by regulating multiple subcortical brain circuits. Glutamatergic pyramidal neurons in the prefrontal cortex (PFC) are topographically organized in different subregions such as the prelimbic, infralimbic (IL), and orbitofrontal and project to topographically-organized subcortical target regions. Dopamine D1 and D2 receptors are expressed on glutamatergic pyramidal neurons in the PFC. However, it is unclear whether D1 and D2 receptor-expressing pyramidal neurons in the PFC are also topographically organized. We used a retrograde adeno-associated virus (AAVRG)-based approach to illuminate the topographical organization of D1 and D2 receptor-expressing neurons, projecting to distinct striatal and midbrain subregions. Our experiments reveal that AAVRG injection in the nucleus accumbens (NAcc) or dorsal striatum (dSTR) of D1Cre mice labeled distinct neuronal subpopulations in medial orbitofrontal or prelimbic PFC, respectively. However, AAVRG injection in NAcc or dSTR of D2Cre mice labeled medial orbitofrontal, but not medial prelimbic PFC, respectively. Additionally, D2R+ but not D1R+ PFC neurons were labeled on injection of AAVRG in substantia nigra pars compacta (SNpc). Thus, our data are the first to highlight a unique dopamine receptor-specific topographical pattern in the PFC, which could have profound implications for corticostriatal signaling in the basal ganglia.

Structure-Functional-Selectivity Relationship Studies of Novel Apomorphine Analogs to Develop D1R/D2R Biased Ligands.

Loss of dopamine neurons is central to the manifestation of Parkinson's disease motor symptoms. The dopamine precursor L-DOPA, the most commonly used therapeutic agent for Parkinson's disease, can restore normal movement yet cause side-effects such as dyskinesias upon prolonged administration. Dopamine D1 and D2 receptors activate G-protein- and arrestin-dependent signaling pathways that regulate various dopamine-dependent functions including locomotion. Studies have shown that shifting the balance of dopamine receptor signaling toward the arrestin pathway can be beneficial for inducing normal movement, while reducing dyskinesias. However, simultaneous activation of both D1 and D2Rs is required for robust locomotor activity. Thus, it is desirable to develop ligands targeting both D1 and D2Rs and their functional selectivity. Here, we report structure-functional-selectivity relationship (SFSR) studies of novel apomorphine analogs to identify structural motifs responsible for biased activity at both D1 and D2Rs.

Deletion of Glycogen Synthase Kinase-3ß in D2 Receptor-Positive Neurons Ameliorates Cognitive Impairment via NMDA Receptor-Dependent Synaptic Plasticity.

BACKGROUND: Cortical dopaminergic systems are critically involved in prefrontal cortex (PFC) functions, especially in working memory and neurodevelopmental disorders such as schizophrenia. GSK-3ß (glycogen synthase kinase-3ß) is highly associated with cAMP (cyclic adenosine monophosphate)-independent dopamine D2 receptor (D2R)-mediated signaling to affect dopamine-dependent behaviors. However, the mechanisms underlying the GSK-3ß modulation of cognitive function via D2Rs remains unclear. METHODS: This study explored how conditional cell-type-specific ablation of GSK-3ß in D2R+ neurons (D2R-GSK-3ß-/-) in the brain affects synaptic function in the medial PFC (mPFC). Both male and female (postnatal days 60-90) mice, including 140 D2R, 24 D1R, and 38 DISC1 mice, were used. RESULTS: This study found that NMDA receptor (NMDAR) function was significantly increased in layer V pyramidal neurons in mPFC of D2R-GSK-3ß-/- mice, along with increased dopamine modulation of NMDAR-mediated current. Consistently, NR2A and NR2B protein levels were elevated in mPFC of D2R-GSK-3ß-/- mice. This change was accompanied by a significant increase in enrichment of activator histone mark H3K27ac at the promoters of both Grin2a and Grin2b genes. In addition, altered short- and long-term synaptic plasticity, along with an increased spine density in layer V pyramidal neurons, were detected in D2R-GSK-3ß-/- mice. Indeed, D2R-GSK-3ß-/- mice also exhibited a resistance of working memory impairment induced by injection of NMDAR antagonist MK-801. Notably, either inhibiting GSK-3ß or disrupting the D2R-DISC1 complex was able to reverse the mutant DISC1-induced decrease of NMDAR-mediated currents in the mPFC. CONCLUSIONS: This study demonstrates that GSK-3ß modulates cognition via D2R-DISC1 interaction and epigenetic regulation of NMDAR expression and function.

Loss of ß-arrestin2 in D2 cells alters neuronal excitability in the nucleus accumbens and behavioral responses to psychostimulants and opioids.

Psychostimulants and opioids increase dopamine (DA) neurotransmission, activating D1 and D2 G protein-coupled receptors. ß-arrestin2 (ßarr2) desensitizes and internalizes these receptors and initiates G protein-independent signaling. Previous work revealed that mice with a global or cell-specific knockout of ßarr2 have altered responses to certain drugs; however, the effects of ßarr2 on the excitability of medium spiny neurons (MSNs), and its role in mediating the rewarding effects of drugs of abuse are unknown. D1-Cre and D2-Cre transgenic mice were crossed with floxed ßarr2 mice to eliminate ßarr2 specifically in cells containing either D1 (D1ßarr2-KO ) or D2 (D2ßarr2-KO ) receptors. We used slice electrophysiology to characterize the role of ßarr2 in modulating D1 and D2 nucleus accumbens MSN intrinsic excitability in response to DA and tested the locomotor-activating and rewarding effects of cocaine and morphine in these mice. Eliminating ßarr2 attenuated the ability of DA to inhibit D2-MSNs and altered the DA-induced maximum firing rate in D1-MSNs. While D1ßarr2-KO mice had mostly normal drug responses, D2ßarr2-KO mice showed dose-dependent reductions in acute locomotor responses to cocaine and morphine, attenuated locomotor sensitization to cocaine, and blunted cocaine reward measured with conditioned place preference. Both D2ßarr2-KO and D1ßarr2-KO mice displayed an enhanced conditioned place preference for the highest dose of morphine. These results indicate that D1- and D2-derived ßarr2 functionally contribute to DA-induced changes in MSN intrinsic excitability and behavioral responses to psychostimulants and opioids dose-dependently.

Slow-release delivery enhances the pharmacological properties of oral 5-hydroxytryptophan: mouse proof-of-concept.

5-hydroxytryptophan (5-HTP) has shown therapeutic promise in a range of human CNS disorders. But native 5-HTP immediate release (IR) is poorly druggable, as rapid absorption causes rapid onset of adverse events, and rapid elimination causes fluctuating exposure. Recently, we reported that 5-HTP delivered as slow-release (SR) in mice augmented the brain pro-serotonergic effect of selective serotonin reuptake inhibitors (SSRIs), without the usual adverse events associated with 5-HTP IR. However, our previous study entailed translational limitations, in terms of route, dose, and duration. Here we modeled oral 5-HTP SR in mice by administering 5-HTP via the food. We modeled oral SSRI treatment via fluoxetine in the water, in a regimen recapitulating clinical pharmacokinetics and pharmacodynamics. 5-HTP SR produced plasma 5-HTP levels well within the range enhancing brain 5-HT function in humans. 5-HTP SR robustly increased brain 5-HT synthesis and levels. When administered with an SSRI, 5-HTP SR enhanced 5-HT-sensitive behaviors and neurotrophic mRNA expression. 5-HTP SR's pro-serotonergic effects were stronger in mice with endogenous brain 5-HT deficiency. In a comprehensive screen, 5-HTP SR was devoid of overt toxicological effects. The present preclinical data, appreciated in the context of published 5-HTP clinical data, suggest that 5-HTP SR could represent a new therapeutic approach to the plethora of CNS disorders potentially treatable with a pro-serotonergic drug. 5-HTP SR might in particular be therapeutically relevant when brain 5-HT deficiency is pathogenic and as an adjunctive augmentation therapy to SSRI therapy.

Methods to Investigate the Role of ß-Arrestin Signaling in Parkinson's Disease.

ß-Arrestin function has largely been investigated in cell culture-based systems. Here we describe methods to investigate ß-arrestin2 signaling in vivo by developing novel mouse models and viral methods to overexpress ß-arrestin2 in the mouse brain. The methods and concepts described here are in the context of Parkinson's disease (PD) and developing automated in vivo drug screening platforms.

Ghrelin receptor antagonism of hyperlocomotion in cocaine-sensitized mice requires ßarrestin-2.

The "brain-gut" peptide ghrelin, which mediates food-seeking behaviors, is recognized as a very strong endogenous modulator of dopamine (DA) signaling. Ghrelin binds the G protein-coupled receptor GHSR1a, and administration of ghrelin increases the rewarding properties of psychostimulants while ghrelin receptor antagonists decrease them. In addition, the GHSR1a signals through ßarrestin-2 to regulate actin/stress fiber rearrangement, suggesting ßarrestin-2 participation in the regulation of actin-mediated synaptic plasticity for addictive substances like cocaine. The effects of ghrelin receptor ligands on reward strongly suggest that modulation of ghrelin signaling could provide an effective strategy to ameliorate undesirable behaviors arising from addiction. To investigate this possibility, we tested the effects of ghrelin receptor antagonism in a cocaine behavioral sensitization paradigm using DA neuron-specific ßarrestin-2 KO mice. Our results show that these mice sensitize to cocaine as well as wild-type littermates. The ßarrestin-2 KO mice, however, no longer respond to the locomotor attenuating effects of the GHSR1a antagonist YIL781. The data presented here suggest that the separate stages of addictive behavior differ in their requirements for ßarrestin-2 and show that pharmacological inhibition of ßarrestin-2 function through GHSR1a antagonism is not equivalent to the loss of ßarrestin-2 function achieved by genetic ablation. These data support targeting GHSR1a signaling in addiction therapy but indicate that using signaling biased compounds that modulate ßarrestin-2 activity differentially from G protein activity may be required.

Hepatic ß-arrestin 2 is essential for maintaining euglycemia.

An increase in hepatic glucose production (HGP) represents a key feature of type 2 diabetes. This deficiency in metabolic control of glucose production critically depends on enhanced signaling through hepatic glucagon receptors (GCGRs). Here, we have demonstrated that selective inactivation of the GPCR-associated protein ß-arrestin 2 in hepatocytes of adult mice results in greatly increased hepatic GCGR signaling, leading to striking deficits in glucose homeostasis. However, hepatocyte-specific ß-arrestin 2 deficiency did not affect hepatic insulin sensitivity or ß-adrenergic signaling. Adult mice lacking ß-arrestin 1 selectively in hepatocytes did not show any changes in glucose homeostasis. Importantly, hepatocyte-specific overexpression of ß-arrestin 2 greatly reduced hepatic GCGR signaling and protected mice against the metabolic deficits caused by the consumption of a high-fat diet. Our data support the concept that strategies aimed at enhancing hepatic ß-arrestin 2 activity could prove useful for suppressing HGP for therapeutic purposes.

ß-arrestin-2 is an essential regulator of pancreatic ß-cell function under physiological and pathophysiological conditions.

ß-arrestins are critical signalling molecules that regulate many fundamental physiological functions including the maintenance of euglycemia and peripheral insulin sensitivity. Here we show that inactivation of the ß-arrestin-2 gene, barr2, in ß-cells of adult mice greatly impairs insulin release and glucose tolerance in mice fed with a calorie-rich diet. Both glucose and KCl-induced insulin secretion and calcium responses were profoundly reduced in ß-arrestin-2 (barr2) deficient ß-cells. In human ß-cells, barr2 knockdown abolished glucose-induced insulin secretion. We also show that the presence of barr2 is essential for proper CAMKII function in ß-cells. Importantly, overexpression of barr2 in ß-cells greatly ameliorates the metabolic deficits displayed by mice consuming a high-fat diet. Thus, our data identify barr2 as an important regulator of ß-cell function, which may serve as a new target to improve ß-cell function.

New Concepts in Dopamine D2 Receptor Biased Signaling and Implications for Schizophrenia Therapy.

The dopamine D2 receptor (D2R) is a G protein-coupled receptor that is a common target for antipsychotic drugs. Antagonism of D2R signaling in the striatum is thought to be the primary mode of action of antipsychotic drugs in alleviating psychotic symptoms. However, antipsychotic drugs are not clinically effective at reversing cortical-related symptoms, such as cognitive deficits in schizophrenia. While the exact mechanistic underpinnings of these cognitive deficits are largely unknown, deficits in cortical dopamine function likely play a contributing role. It is now recognized that similar to most G protein-coupled receptors, D2Rs signal not only through canonical G protein pathways but also through noncanonical beta-arrestin2-dependent pathways. We review the current mechanistic bases for this dual signaling mode of D2Rs and how these new concepts might be leveraged for therapeutic gain to target both cortical and striatal dysfunction in dopamine neurotransmission and hence have the potential to correct both positive and cognitive symptoms of schizophrenia.

Distinct cortical and striatal actions of a ß-arrestin-biased dopamine D2 receptor ligand reveal unique antipsychotic-like properties.

The current dopamine (DA) hypothesis of schizophrenia postulates striatal hyperdopaminergia and cortical hypodopaminergia. Although partial agonists at DA D2 receptors (D2Rs), like aripiprazole, were developed to simultaneously target both phenomena, they do not effectively improve cortical dysfunction. In this study, we investigate the potential for newly developed ß-arrestin2 (ßarr2)-biased D2R partial agonists to simultaneously target hyper- and hypodopaminergia. Using neuron-specific ßarr2-KO mice, we show that the antipsychotic-like effects of a ßarr2-biased D2R ligand are driven through both striatal antagonism and cortical agonism of D2R-ßarr2 signaling. Furthermore, ßarr2-biased D2R agonism enhances firing of cortical fast-spiking interneurons. This enhanced cortical agonism of the biased ligand can be attributed to a lack of G-protein signaling and elevated expression of ßarr2 and G protein-coupled receptor (GPCR) kinase 2 in the cortex versus the striatum. Therefore, we propose that ßarr2-biased D2R ligands that exert region-selective actions could provide a path to develop more effective antipsychotic therapies.

ML314: A Biased Neurotensin Receptor Ligand for Methamphetamine Abuse.

Pharmacological treatment for methamphetamine addiction will provide important societal benefits. Neurotensin receptor NTR1 and dopamine receptor distributions coincide in brain areas regulating methamphetamine-associated reward, and neurotensin peptides produce behaviors opposing psychostimulants. Therefore, undesirable methamphetamine-associated activities should be treatable with druggable NTR1 agonists, but no such FDA-approved therapeutics exist. We address this limitation with proof-of-concept data for ML314, a small-molecule, brain penetrant, ß-arrestin biased, NTR1 agonist. ML314 attenuates amphetamine-like hyperlocomotion in dopamine transporter knockout mice, and in C57BL/6J mice it attenuates methamphetamine-induced hyperlocomotion, potentiates the psychostimulant inhibitory effects of a ghrelin antagonist, and reduces methamphetamine-associated conditioned place preference. In rats, ML314 blocks methamphetamine self-administration. ML314 acts as an allosteric enhancer of endogenous neurotensin, unmasking stoichiometric numbers of hidden NTR1 binding sites in transfected-cell membranes or mouse striatal membranes, while additionally supporting NTR1 endocytosis in cells in the absence of NT peptide. These results indicate ML314 is a viable, preclinical lead for methamphetamine abuse treatment and support an allosteric model of G protein-coupled receptor signaling.