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1.  SNARE Complex Dysfunction: A Unifying Hypothesis for Schizophrenia 
Biological psychiatry  2015;78(6):356-358.
PMCID: PMC4703341  PMID: 26296424
2.  APOEε4 and Depression: Following a Winding Road 
Biological psychiatry  2015;78(10):670-671.
PMCID: PMC4699301  PMID: 26497282
3.  Schizophrenia as a Disorder of Molecular Pathways 
Biological psychiatry  2014;77(1):22-28.
Over the last decade transcriptome studies of postmortem tissue from subjects with schizophrenia revealed that synaptic, mitochondrial, immune system, GABA-ergic and oligodendrocytic changes are all integral parts of the disease process. The combined genetic and transcriptomics studies argue that the molecular underpinnings of the disease are even more varied than the symptomatic diversity of schizophrenia. Ultimately, to decipher the pathophysiology of human disorders in general, we will need to understand the function of hundreds of genes and regulatory elements in our genome, and the consequences of their overexpression and reduced expression in a developmental context. Furthermore, integration of knowledge from various data sources remains a monumental challenge that has to be systematically addressed in the upcoming decades. In the end, our success in interpreting the molecular changes in schizophrenia will depend on our ability to understand the biology using innovative ideas and cannot depend on the hope of developing novel, more powerful technologies.
PMCID: PMC4092052  PMID: 24507510
schizophrenia; gene expression; genetics; gene network; convergence; postmortem
4.  From psychiatric disorders to animal models: a bidirectional and dimensional approach 
Biological psychiatry  2014;77(1):15-21.
Psychiatric genetics research is bidirectional in nature, with human and animal studies becoming more closely integrated as techniques for genetic manipulations allow for more subtle exploration of disease phenotypes. This synergy, however, highlights the importance of considering the way in which we approach the genotype-phenotype relationship. In particular, the nosological divide of psychiatric illness, while clinically relevant, is not directly translatable in animal models. For instance, mice will never fully re-capitulate the broad criteria for many psychiatric disorders; nor will they have guilty ruminations, suicidal thoughts, or rapid speech. Instead, animal models have been and continue to provide a means to explore dimensions of psychiatric disorders in order to identify neural circuits and mechanisms underlying disease-relevant phenotypes. Thus, the genetic investigation of psychiatric illness will yield the greatest insights if efforts continue to identify and utilize biologically valid phenotypes across species. In this review we discuss the progress to date and the future efforts that will enhance translation between human and animal studies, including the identification of intermediate phenotypes that can be studied across species, as well as the importance of refined modeling of human disease-associated genetic variation in mice and other animal models.
PMCID: PMC4135025  PMID: 24650688
intermediate phenotype; BDNF; serotonin; dentate gyrus; pattern separation; amygdala; anxiety
5.  Alzheimer's disease risk genes and mechanisms of disease pathogenesis 
Biological psychiatry  2014;77(1):43-51.
Here, we review the genetic risk factors for late onset Alzheimer's disease (AD) and their role in AD pathogenesis. Recent advances in our understanding of the human genome, namely technological advances in methods to analyze millions of polymorphisms in thousands of subjects, have revealed new genes associated with AD risk: ABCA7, BIN1, CASS4, CD33, CD2AP, CELF1, CLU, CR1, DSG2, EPHA1, FERMT2, HLA-DRB5-DBR1, INPP5D, MS4A, MEF2C, NME8, PICALM, PTK2B, SLC24H4 RIN3, SORL1, ZCWPW1. Emerging technologies to analyze the entire genome in large datasets have also revealed coding variants that increase AD risk: PLD3 and TREM2. We review the relationship between these AD risk genes and the cellular and neuropathological features of AD. Together, understanding the mechanisms underlying the association of these genes with risk for disease will provide the most meaningful targets for therapeutic development to date.
PMCID: PMC4234692  PMID: 24951455
Alzheimer's disease; amyloid precursor protein; genome wide association studies; endocytosis; immune response; cholesterol metabolism
6.  Genetic Epidemiology and Insights into Interactive Genetic and Environmental Effects in Autism Spectrum Disorders 
Biological psychiatry  2014;77(1):66-74.
Understanding the pathogenesis of Neurodevelopmental Disorders (NDDs) has proven to be challenging. Using Autism Spectrum Disorder (ASD) as a paradigmatic NDD, this paper reviews the existing literature on the etiologic substrates of ASD and explores how genetic epidemiology approaches including gene-environment interactions (GxE) can play roles in identifying factors associated with ASD etiology. New genetic and bioinformatics strategies have yielded important clues to ASD genetic substrates. Next steps for understanding ASD pathogenesis require significant effort to focus on how genes and environment interact with one another in typical development and its perturbations. Along with larger sample sizes, future study designs should include sample ascertainment that is epidemiologic and population-based to capture the entire ASD spectrum with both categorical and dimensional phenotypic characterization, environmental measurement with accuracy, validity and biomarkers, statistical methods to address population stratification, multiple comparisons and GxE of rare variants, animal models to test hypotheses and, new methods to broaden the capacity to search for GxE, including genome-wide and environment-wide association studies, precise estimation of heritability using dense genetic markers and consideration of GxE both as the disease cause and a disease course modifier. While examination of GxE appears to be a daunting task, tremendous recent progress in gene discovery opens new horizons for advancing our understanding the role of GxE in the pathogenesis of, and ultimately identifying the causes, treatments and even prevention for ASD and other NDDs.
PMCID: PMC4260177  PMID: 25483344
neurodevelopmental disorders; Autism Spectrum Disorders; genetic epidemiology; genes; environment; interactions
7.  Discovering Schizophrenia Endophenotypes in Randomly Ascertained Pedigrees 
Biological psychiatry  2014;77(1):75-83.
Although case-control approaches are beginning to disentangle schizophrenia’s complex polygenic burden, other methods will likely be necessary to fully identify and characterize risk genes. Endophenotypes, traits genetically correlated with an illness, can help characterize the impact of risk genes by providing genetically relevant traits that are more tractable than the behavioral symptoms that classify mental illness. Here we present an analytic approach for discovering and empirically validating endophenotypes in extended pedigrees with very few affected individuals. Our approach indexes each family member’s risk as a function of shared genetic kinship with an affected individual, often referred to as the coefficient of relatedness. To demonstrate the utility of this approach, we search for neurocognitive and neuroanatomic endophenotypes for schizophrenia in large unselected multigenerational pedigrees.
A fixed effect test within the variance component framework was performed on neurocognitive and cortical surface area traits in 1,606 Mexican-American individuals from large, randomly ascertained extended pedigrees who participate in the “Genetics of Brain Structure and Function” study. As affecteds are excluded from analyses, results are not influenced by disease state or medication usage.
Despite having sampled just 6 individuals with schizophrenia, our sample provided 233 individuals at various levels of genetic risk for the disorder. We identified three neurocognitive measures (digit-symbol substitution, facial memory, and emotion recognition) and six medial temporal and prefrontal cortical surfaces associated with liability for schizophrenia.
With our novel analytic approach one can discover and rank endophenotypes for schizophrenia, or any heritable disease, in randomly ascertained pedigrees.
PMCID: PMC4261014  PMID: 25168609
endophenotype; schizophrenia; family study; coefficient of relatedness; cognition; cortical surface area
8.  Genetics of complex traits in psychiatry 
Biological psychiatry  2014;77(1):36-42.
Virtually all psychiatric traits are genetically complex. This article discusses the genetics of complex traits in psychiatry. The complexity is accounted for by numerous factors, including multiple risk alleles, epistasis, and epigenetic effects, such as methylation. Risk alleles can individually be common or rare, and can include, for example, single nucleotide polymorphisms (SNPs) and copy number variants (CNV) that are transmitted or are new mutations, and other kinds of variation. Many different kinds of variation can be important for trait risk, either together in various proportions, or as different factors in different subjects. Until recently, our approaches to complex traits were limited, and consequently only a small number of variants, usually of individually minor effect, were identified. Currently, we have a much richer armamentarium that includes the routine application of genomewide association studies (GWAS) and next-generation high throughput sequencing (NextGen); and the combination of this information with other biologically relevant information, such as expression data. We have also seen the emergence of large meta-analysis and mega-analysis consortia. These developments are extremely important for psychiatric genetics, have moved the field forward substantially, and promise formidable gains in the years to come as they are applied more widely.
PMCID: PMC4282183  PMID: 25444161
genetics; complex traits; GWAS; polymorphisms; GxE; sequencing studies
9.  Is Serum Brain-Derived Neurotrophic Factor a Biomarker for Cognitive Enhancement in Schizophrenia? 
Biological psychiatry  2009;66(6):549-553.
Brain-derived neurotrophic factor (BDNF) plays a critical role in neurodevelopment and plasticity; decreased BDNF functioning may contribute to the pathogenesis of schizophrenia. However, BDNF levels are not static; in animal experiments, brain BDNF increases during spatial learning, and in clinical depression, successful antidepressant treatment raises serum BDNF. We asked: would neuroplasticity-based cognitive training in schizophrenia result in increased serum BDNF?
Fifty-six schizophrenia outpatients and 16 matched healthy comparison subjects were assessed on baseline cognitive performance and serum BDNF. Schizophrenia subjects were randomly assigned to either 50 hours (10 weeks) of computerized auditory training or a computer game control condition, followed by reassessment of cognition and serum BDNF.
At baseline, schizophrenia participants had significantly lower-than-normal serum BDNF. Schizophrenia subjects who engaged in computerized cognitive training designed to improve auditory processing showed significant cognitive gains and a significant increase in serum BDNF compared with subjects who played computer games. This increase was evident after 2 weeks of training, and after 10 weeks in the active condition, subjects “normalized” their mean serum BDNF levels, whereas the control group showed no change. In the active condition, change in BDNF was significantly associated with improved quality of life.
Serum BDNF levels are significantly increased in clinically stable, chronically ill schizophrenia subjects after neuroplasticity-based cognitive training, but not after computer games. Serum BDNF levels may serve as a peripheral biomarker for the effects of intensive cognitive training and may provide a useful tool for the evaluation of cognitive enhancement methods in schizophrenia.
PMCID: PMC4691262  PMID: 19368899
BDNF; biomarker; cognitive enhancement cognitive remediation; cognitive training; neuroplasticity; schizophrenia
10.  How does stress-induced activation of the kappa opioid system increase addiction risk? 
Biological psychiatry  2014;76(10):760-762.
PMCID: PMC4684190  PMID: 25442057
11.  A Randomized Controlled Trial of Intranasal Ketamine in Major Depressive Disorder 
Biological psychiatry  2014;76(12):970-976.
The N-methyl-d-aspartate glutamate receptor antagonist ketamine, delivered via an intravenous route, has shown rapid antidepressant effects in patients with treatment-resistant depression. The current study was designed to test the safety, tolerability and efficacy of intranasal ketamine in patients with depression who had failed at least one prior antidepressant trial.
Twenty patients with major depression were randomized and 18 completed two treatment days with intranasal ketamine hydrochloride (50 mg) or saline solution in a randomized, double-blind, crossover study. The primary efficacy outcome measure was change in depression severity 24 hours following ketamine or placebo, measured using the Montgomery-Asberg Depression Rating Scale. Secondary outcomes included persistence of benefit, changes in self-reports of depression, changes in anxiety, and proportion of responders. Potential psychotomimetic, dissociative, hemodynamic, and general adverse effects associated with ketamine were also measured.
Patients showed significant improvement in depressive symptoms at 24 hours following ketamine compared to placebo [t=4.39, p<0.001; estimated mean MADRS score difference of 7.6 ± 3.7 (95% CI: 3.9 – 11.3)]. Eight of 18 patients (44%) met response criteria 24 hours following ketamine administration, compared to 1 of 18 (6%) following placebo (p=0.033). Intranasal ketamine was well tolerated with minimal psychotomimetic or dissociative effects and was not associated with clinically significant changes in hemodynamic parameters.
This study provides the first controlled evidence for the rapid antidepressant effects of intranasal ketamine. Treatment was associated with minimal adverse effects. If replicated, these findings may lead to novel approaches to the pharmacologic treatment of patients with major depression.
Trial Registration identifier NCT01304147
PMCID: PMC4185009  PMID: 24821196
depression; treatment resistant; ketamine; antidepressant; intranasal; glutamate
12.  Methylphenidate Exerts Dose-Dependent Effects on Glutamate Receptors and Behaviors 
Biological psychiatry  2014;76(12):953-962.
Methylphenidate (MPH), a psychostimulant drug for the treatment of attention-deficit hyperactivity disorder (ADHD), produces the effects of increasing alertness and improving attention, while its misuse has been associated with an increased risk of aggression and psychosis. In this study, we sought to determine the molecular mechanism underlying the complex actions of MPH.
Adolescent (4-week-old) rats were given one injection of MPH at different doses. The impact of MPH on glutamatergic signaling in pyramidal neurons of prefrontal cortex (PFC) was measured. MPH-induced behavioral changes were also examined in parallel.
We found that administration of low-dose (0.5 mg/kg) MPH selectively potentiated NMDAR-mediated excitatory synaptic currents (EPSCs) via adrenergic receptor activation, while the high-dose (10 mg/kg) MPH suppressed both NMDAR- and AMPAR-EPSCs. The dual effects of MPH on EPSCs were associated with bi-directional changes in the surface level of glutamate receptor subunits. Behavioral tests also indicated that low-dose MPH facilitated the PFC-mediated temporal order recognition memory (TORM) and attention, while animals injected with high-dose MPH exhibited significantly elevated locomotive activity. Inhibiting the function of SNAP-25, a key SNARE proteins involved in NMDAR exocytosis, blocked the increase of NMDAR-EPSC by low-dose MPH. In animals exposed to repeated stress, administration of low-dose MPH effectively restored NMDAR function and TORM via a mechanism dependent on SNAP-25.
Our results have provided a potential mechanism underlying the cognitive enhancing effects of low-dose MPH, as well as the psychosis-inducing effects of high-dose MPH.
PMCID: PMC4194277  PMID: 24832867
Methylphenidate; prefrontal cortex; NMDA receptors; AMPA receptors; SNAP-25; stress
13.  Restoring mood balance in depression: ketamine reverses deficit in dopamine-dependent synaptic plasticity 
Biological psychiatry  2014;76(12):927-936.
One of the most novel and exciting findings in major depressive disorder research over the last decade is the discovery of the fast-acting and long-lasting antidepressant effects of ketamine. Indeed, the therapeutic effects of classic antidepressant such as SSRIs require a month or longer to be expressed, with about a third of MDD patients resistant to treatment. Clinical studies have shown that low dose of ketamine exhibits fast-acting relatively sustained antidepressant action even in treatment-resistant patients. However, the mechanisms of ketamine action at a systems level remain unclear.
Wistar-Kyoto rats were exposed to inescapable, uncontrollable footshocks. To evaluate learned helplessness behavior, we used an active avoidance task in a shuttle box equipped with an electrical grid floor. After helplessness assessment, we performed in vivo electrophysiological recordings first from ventral tegmental area dopaminergic (DA) neurons, and second from accumbens neurons responsive to fimbria stimulation. Ketamine was injected and tested on helpless behavior and electrophysiological recordings.
We show that ketamine is able to restore the integrity of a network by acting on the DA system and restoring synaptic dysfunction observed in stress-induced depression. We show that part of the antidepressant effect of ketamine is via the DA system. Indeed injection of ketamine restores a decreased dopamine neuron population activity as well as restores synaptic plasticity (long-term potentiation) in the hippocampus-accumbens pathway, via in part, activation of D1 receptors.
This work provides for a unique systems perspective on the mechanisms of ketamine on a disrupted limbic system.
PMCID: PMC4223010  PMID: 24931705
ketamine; dopamine; learned helplessness; nucleus accumbens; ventral tegmental area; synaptic plasticity
14.  Defining Critical White Matter Pathways Mediating Successful Subcallosal Cingulate Deep Brain Stimulation for Treatment-Resistant Depression 
Biological psychiatry  2014;76(12):963-969.
Deep brain stimulation (DBS) of subcallosal cingulate white matter (SCC) is an evolving investigational treatment for major depression. Mechanisms of action are hypothesized to involve modulation of activity within a structurally defined network of brain regions involved in mood regulation. Diffusion tensor imaging (DTI) was used to model white matter connections within this network to identify those critical for successful antidepressant response to SCC DBS.
Pre-operative high-resolution MRI data, including DTI, were acquired in 16 patients with treatment-resistant depression who then received SCC DBS. Computerized tomography was used post-operatively to locate DBS contacts. The activation volume around the active contacts used for chronic stimulation was modeled for each patient retrospectively. Probabilistic tractography was used to delineate the white matter tracts that traveled through each activation volume. Patient-specific tract maps were calculated using whole-brain analysis. Clinical evaluations of therapeutic outcome from SCC DBS were defined at 6 months and 2 years.
Whole brain activation volume tractography (AVT) demonstrated that all DBS responders at six months (n=6) and 2 years (n=12) shared bilateral pathways from their activation volumes to (1) medial frontal cortex via forceps minor and uncinate fasciculus, (2) rostral and dorsal cingulate cortex via the cingulum bundle, and (3) subcortical nuclei. Non-responders did not consistently show these connections. Specific anatomical coordinates of the active contacts did not discriminate responders from non-responders.
Patient-specific AVT modeling may identify critical tracts that mediate SCC DBS antidepressant response. This suggests a novel method for patient-specific target and stimulation parameter selection.
PMCID: PMC4487804  PMID: 24832866
deep brain stimulation; major depressive disorder; bipolar disorder; treatment-resistant depression; subcallosal cingulate; subgenual cingulate; diffusion tensor imaging; tractography; antidepressant response
15.  Traumatic Stress Is Heartbreaking 
Biological psychiatry  2013;74(11):790-792.
PMCID: PMC4667744  PMID: 24188697
16.  Neuropeptide Y Receptor Gene Expression in the Primate Amygdala Predicts Anxious Temperament and Brain Metabolism 
Biological psychiatry  2013;76(11):850-857.
Anxious temperament (AT) is identifiable early in life and predicts the later development of anxiety disorders and depression. Neuropeptide Y (NPY) is a putative endogenous anxiolytic neurotransmitter that adaptively regulates responses to stress and may confer resilience to stress-related psychopathology. Using a well-validated non-human primate model of AT, we examined expression of the NPY system in the central nucleus (Ce) of the amygdala, a critical neural substrate for extreme anxiety.
In twenty-four young rhesus monkeys, we measured Ce mRNA levels of all members of the NPY system that are detectable in the Ce using quantitative real time polymerase chain reaction (qRT-PCR). We then examined the relationship between these mRNA levels and both AT expression and brain metabolism.
Lower mRNA levels of NPY receptor 1 (NPY1R) and NPY receptor 5 (NPY5R), but not NPY or NPY receptor 2 (NPY2R) in the Ce predicted elevated AT; mRNA levels for NPY1R and NPY5R in the motor cortex were not related to AT. In situ hybridization analysis provided for the first time a detailed description of NPY1R and NPY5R mRNA distribution in the rhesus amygdala and associated regions. Lastly, mRNA levels for these two receptors in the Ce predicted metabolic activity in several regions that have the capacity to regulate the Ce.
Decreased NPY signaling in the Ce may contribute to the altered metabolic activity that is a component of the neural substrate underlying AT. This suggests that enhancement of NPY signaling may reduce the risk to develop psychopathology.
PMCID: PMC4022724  PMID: 24342924
Anxiety; behavioral inhibition; depression; rhesus macaque; stress; prefrontal cortex
17.  The Human Ortholog of Acid-Sensing Ion Channel Gene ASIC1a Is Associated With Panic Disorder And Amygdala Structure And Function 
Biological psychiatry  2014;76(11):902-910.
Individuals with panic disorder (PD) exhibit a hypersensitivity to inhaled carbon dioxide (CO2), possibly reflecting a lowered threshold for sensing signals of suffocation. Animal studies have shown that CO2-mediated fear behavior depends on chemosensing of acidosis in the amygdala via the acid sensing ion channel ASIC1a. We examined whether the human ortholog of the ASIC1a gene, ACCN2, is associated with the presence of PD and with amygdala structure and function.
We conducted a case-control analysis (N=414 PD cases, 846 healthy controls) of ACCN2single nucleotide polymorphisms (SNPs) and PD. We then tested whether variants showing significant association with PD are also associated with amygdala volume (n=1,048) and/or task-evoked reactivity to emotional stimuli (n=103) in healthy individuals.
Two SNPs at the ACCN2 locus showed evidence of association with PD: rs685012 (OR=1.32, gene-wise corrected p=0.011) and rs10875995 (OR=1.26, gene-wise corrected p=0.046). The association appeared to be stronger when early-onset (age ≤ 20) PD cases and when cases with prominent respiratory symptoms were compared to controls. The PD risk allele at rs10875995 was associated with increased amygdala volume (p=0.035), as well as task-evoked amygdala reactivity to fearful and angry faces (p=0.0048).
Genetic variation at ACCN2 appears to be associated with PD and with amygdala phenotypes that have been linked to anxiety proneness. These results support the possibility that modulation of acid-sensing ion channels may have therapeutic potential for PD.
PMCID: PMC4103972  PMID: 24529281
Panic disorder; ASIC1a; ACCN2; amygdala; genetic; association
19.  An unconditioned stimulus retrieval-extinction procedure to prevent the return of fear memory 
Biological psychiatry  2014;76(11):895-901.
Conditioned fear memories can be updated by extinction during reconsolidation, and this effect is specific to the reactivated conditioned stimulus (CS). However, a traumatic event can be associated with several cues, and each cue can potentially trigger recollection of the event. In the present study, we introduced a technique to target all diverse cues associated with an aversive event that causes fear.
In the human experiments, the subjects underwent modified fear conditioning, in which they were exposed to an unconditioned stimulus (US) or unreinforced CS to reactivate the memory and then underwent extinction, spontaneous recovery, and reinstatement. In the animal experiments, rats underwent contextual fear conditioning under a similar protocol as the used in the human experiments. We also explored the molecular alterations after US reactivation in rats.
We found that presentation of a lower-intensity US following extinction disrupted the associations between the different CSs and reactivated US in both humans and rats. This disruptive effect persisted for at least 6 months in humans and was selective to the reactivated US. This procedure was also effective for remote memories in both humans and rats. Compared with the CS, the US induced stronger endocytosis of AMPA glutamate receptors 1 and 2 and stronger activation of protein kinase A, p70S6 kinase, and cyclic adenosine monophosphate response element binding protein in the dorsal hippocampus in rats.
These findings demonstrate that a modified US retrieval-extinction strategy may have a potential impact on therapeutic approaches to prevent the return of fear.
PMCID: PMC4480632  PMID: 24813334
fear memory; unconditioned stimulus; retrieval; extinction; reconsolidation; hippocampus
20.  Reductions in Brain 5-HT1B Receptor Availability in Primarily Cocaine-Dependent Humans 
Biological psychiatry  2013;76(10):816-822.
Preclinical evidence implicates the 5-HT1B receptor in cocaine’s effects. This study explores 5-HT1B in humans by examining receptor availability in vivo with primary cocaine-dependent (CD) subjects using positron emission tomography (PET).
Fourteen medically healthy CD subjects (mean age=41±6 yrs) were compared to 14 age-matched healthy control subjects (41±8 yrs) with no past or current history of cocaine or other illicit substance abuse. Participants received an MRI and then a PET scan with the highly selective 5HT1B tracer, [11C]P943, for purposes of quantifying regional binding potential (BPND). Voxel-based morphometry (VBM) and gray matter masking (GMM) were also employed to control for potential partial volume effects.
[11C]P943 PET imaging data in nine candidate regions (amygdala, anterior cingulate cortex, caudate, frontal cortex, hypothalamus, pallidum, putamen, thalamus and ventral striatum) showed significant or nearly significant reductions of BPND in CD subjects in three regions, including the anterior cingulate (−16%; P<0.01), hypothalamus (−16%, P=0.03) and frontal cortex (−7%, P=0.08). VBM showed significant gray matter reductions in the frontal cortex of CD subjects. After GMM, statistically significant reductions in [11C]P943 BPND were either retained (anterior cingulate, −14%, p=0.01; hypothalamus, −20%, P<0.01) or achieved (frontal cortex, −14%, p<0.01). Whole brain voxel-wise parameter estimation confirmed these results. Secondary analyses were also significant in some regions for years of cocaine and daily tobacco use.
The reductions found in this study suggest that 5-HT1B receptors may contribute to the etiology and/or expression of cocaine dependence and potentially represent a target for medication development.
PMCID: PMC4037398  PMID: 24433854
cocaine; 5-HT1B; serotonin; PET; VBM; human
21.  Loss of Morphine Reward and Dependence in Mice Lacking G Protein-Coupled Receptor Kinase 5 
Biological psychiatry  2014;76(10):767-774.
The clinical benefits of opioid drugs are counteracted by the development of tolerance and addiction. Here, we provide in vivo evidence for the involvement of G protein-coupled receptor kinases (GRKs) in opioid dependence in addition to their roles in agonist-selective μ-opioid receptor phosphorylation.
In vivo μ-opioid receptor (MOR) phosphorylation was examined by immunoprecipitation and NanoLC-MS/MS analysis. Using the hot-plate and conditioned place preference (CPP) test we investigated opioid-related antinociception and reward effects in mice lacking GRK3 or GRK5.
We show that etonitazene and fentanyl stimulate the in vivo phosphorylation of multiple carboxyl-terminal phosphate acceptor sites including threonine370 (T370), serine375 (S375), and threonine379 (T379), which is predominantly mediated by GRK3. By contrast, morphine promotes a selective phosphorylation of S375 that is predominantly mediated by GRK5. Unlike GRK3 knock-out mice, GRK5 knock-out mice exhibit reduced antinociceptive responses after morphine administration and develop morphine tolerance similar to wild-type mice but fewer signs of physical dependence. Also, morphine is not effective in inducing CPP in GRK5 knock-out mice, whereas cocaine CPP is retained. The rewarding properties of morphine, however, are evident in knock-in mice expressing a phosphorylation-deficient S375A mutation of the μ-opioid receptor.
These findings show, for the first time, that μ-opioid receptor phosphorylation in vivo is regulated by agonist-selective recruitment of distinct GRK isoforms that influence different opioid-related behaviors. Therefore, modulation of GRK5 function could serve as a new approach for preventing addiction to opioids while maintaining the analgesic properties of opioid drugs at a still effective level.
PMCID: PMC4119866  PMID: 24629717
μ-opioid receptor; G protein-coupled receptor kinase; phosphorylation; conditioned place preference; dependence; bar code
22.  A Shift in the Role of Glutamatergic Signaling in the Nucleus Accumbens Core with the Development of an Addicted Phenotype 
Biological psychiatry  2014;76(10):810-815.
While dopamine signaling in the nucleus accumbens (NAc) plays a well-established role in motivating cocaine use in early “non-addicted” stages, recent evidence suggests that other signaling pathways may be critical once addiction has developed. Given the importance of glutamatergic signaling in the NAc for drug-seeking and relapse, here we examined its role in motivating cocaine self-administration under conditions known to produce either a “non-addicted” or an “addicted” phenotype.
Following acquisition, male and female Sprague Dawley rats were given either short access (3 fixed-ratio 1 sessions, 20 infusions/day) or extended 24-hr access (10 days; 4 trials/hr; up to 96 infusions/day) to cocaine. Following a 14-day abstinence period, motivation for cocaine was assessed under a progressive-ratio schedule, and once stable, the effects of intra-NAc infusions of the glutamate AMPA/KA receptor antagonist CNQX (0.0, 0.01, 0.03, 0.1 μg/side) were determined. As an additional measure for the development of an addicted phenotype, separate groups of rats were screened under an extinction/cue-induced reinstatement procedure following abstinence from short versus extended access self-administration.
Motivation for cocaine and levels of extinction and reinstatement responding were markedly higher following extended versus short access self-administration confirming the development of an addicted phenotype in the extended access group. CNQX dose-dependently reduced motivation for cocaine in the extended access group, but was without effect in the short access group.
These results suggest that the role of glutamatergic signaling in the NAc, though not essential for motivating cocaine use in “non-addicted” stages, becomes critical once addiction has developed.
PMCID: PMC4133320  PMID: 24629536
cocaine; CNQX; extended access; nucleus accumbens; glutamate; motivation
23.  Cell type specific expression of p11 controls cocaine reward 
Biological psychiatry  2014;76(10):794-801.
The high rate of comorbidity between depression and cocaine addiction suggests shared molecular mechanisms and anatomical pathways. Limbic structures, such as the Nucleus Accumbens (NAc), play a crucial role in both disorders, yet how different cell types within these structures contribute to the pathogenesis remains elusive. Downregulation of p11 (S100A10) specifically in the NAc elicits depressive like behaviors in mice but its role in drug addiction is unknown.
We combine mouse genetics and viral strategies to determine how the titration of p11 levels within the entire NAc affects the rewarding actions of cocaine on behavior (6 to 8 mice per group) and molecular correlates (3 experiments, 5 to 8 mice per group). Finally, the manipulation of p11 expression in distinct NAc dopaminoceptive neuronal subsets distinguished cell type specific effects of p11 on cocaine reward (5 to 8 mice per group).
We demonstrate that p11 knockout mice have enhanced cocaine conditioned place preference (CPP), which is reproduced by the focal downregulation of p11 in the NAc of wild-type mice. In wild-type mice, cocaine reduced p11 expression in the NAc, while p11 overexpression exclusively in the NAc reduced cocaine CPP. Finally, we identify dopamine receptor-1 (D1) expressing medium spiny neurons (MSNs) as key mediators of p11’s effects on cocaine reward.
Our data provide evidence that disruption of p11 homeostasis in the NAc particularly in D1 expressing MSNs may underlie pathophysiological mechanisms of cocaine rewarding action. Treatments to counter maladaptation of p11 levels may provide novel therapeutic opportunities for cocaine addiction.
PMCID: PMC4145045  PMID: 24725970
Cocaine reward; p11 (s100a10); Nucleus Accumbens; conditional knock down; striato nigral pathway; striatopallidal pathway
24.  Post-Stress Block of Kappa Opioid Receptors Rescues Long-Term Potentiation of Inhibitory Synapses and Prevents Reinstatement of Cocaine Seeking 
Biological psychiatry  2014;76(10):785-793.
Dopaminergic neurons in the ventral tegmental area (VTA) of the brain are an important site of convergence of drugs and stress. We previously identified a form of long-term potentiation of GABAergic synapses on these neurons (LTPGABA). Our studies have shown that exposure to acute stress blocks this LTP, and that reversal of the block of LTPGABA is correlated with prevention of stress-induced reinstatement of cocaine-seeking.
Sprague-Dawley rats were subjected to cold-water swim stress. Midbrain slices were prepared following stress, and whole-cell patch clamp recordings of IPSCs were performed from VTA dopamine neurons. Antagonists of glucocorticoid and kappa opioid receptors were administered at varying time points after stress. Additionally, the ability of a post-stress kappa antagonist to block FSS-induced reinstatement of cocaine self-administration was tested.
We report that an acute stressor blocks LTPGABA for five days after stress through a transient activation of glucocorticoid receptors and more lasting contribution of kappa opioid receptors. Pharmacological block of kappa opioid receptors beginning as late as 4 days after stress has occurred can reverse the block of LTPGABA. Furthermore, post-stress administration of a kappa opioid antagonist prevents reinstatement of cocaine-seeking.
Our results show that a brief stressor can cause days-long changes in the reward circuitry and reveal roles for glucocorticoid and kappa opioid receptors as mediators of the lasting effects of stress on synaptic plasticity. These results indicate that kappa opioid receptor antagonists reverse the neuroadaptations underlying stress-induced drug-seeking behavior and may be useful in the treatment of cocaine addiction.
PMCID: PMC4240751  PMID: 24957331
Stress; kappa opioid receptor; glucocorticoid receptor; synaptic plasticity; ventral tegmental area; reinstatement
Biological psychiatry  2014;76(10):802-809.
Subjective effects related to cocaine abuse are primarily mediated by blockade of the dopamine (DA) transporter (DAT). The present study assessed the hypothesis that different conformational equilibria of the DAT regulate differences in extracellular DA induced by structurally diverse DA uptake inhibitors (DUI) and their cocaine-like subjective effects.
The relationship between cocaine-like subjective effects and stimulation of mesolimbic-DA levels by standard-DUIs (cocaine, methylphenidate, WIN35,428), and atypical-DUIs (benztropine analogs: AHN1-055, AHN2-005, JHW-007) was investigated using cocaine-discrimination and DA-microdialysis procedures in rats.
All drugs stimulated DA-levels showing different time-courses and maximal effects. Standard-DUIs, which preferentially bind to the outward-facing DAT-conformation, fully substituted for cocaine, consistently producing those subjective effects at DA levels of 100-125% over basal values, regardless of dose or pretreatment time. The atypical-DUIs, with DAT binding minimally affected by DAT conformation, produced inconsistent cocaine-like subjective effects. Full effects were obtained, if at all, only at a few doses and pretreatment times, and at DA-levels 600-700% greater than basal values. Importantly, the linear, time-independent, relationship between cocaine-like subjective effects and stimulation of DA-levels, obtained with standard DUIs was not obtained with the atypical-DUIs.
These results suggest a time-related desensitization process underlying the reduced cocaine subjective effects of atypical-DUIs that may be differentially induced by the binding modalities identified using molecular approaches. Since the DAT is the target of several drugs for treating neuropsychiatric disorders, such as ADHD, these results help to identify safe and effective medications with minimal cocaine-like subjective effects that contribute to abuse liability.
PMCID: PMC4353924  PMID: 24853388
cocaine discrimination; dopamine microdialysis; nucleus accumbens shell; benztropine analogues; ADHD; drug abuse and addiction

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