Ketamine metabolite (2R,6R)-hydroxynorketamine reverses behavioral despair produced by adolescent trauma.

Early life trauma dramatically increases the risk of developing major depressive disorder (MDD), and is associated with a markedly decreased adult treatment response to antidepressants. Novel treatment approaches are required to treat childhood trauma-associated MDD. Recent studies suggest that the (R,S)-ketamine (ketamine) metabolite, (2R,6R)-hydroxynorketamine (HNK), exerts fast- and long-lasting antidepressant-like effects without ketamine's NMDAR-inhibition-associated adverse side-effect profile. We investigated the therapeutic potential of (2R,6R)-HNK against behavioral despair produced by a novel live-predator stress exposure during adolescence. Male and female C57BL/6J mice were exposed to a live snake or control conditions at post-natal (PND) days 31, 45 and 61. In order to assess the enduring consequences of trauma-exposure, at a minimum of 14 days following the last exposure, mice received inescapable shocks followed by a session with available escape options twenty-four hours later. Mice that manifested enduring escape deficits (helplessness) were treated with vehicle or (2R,6R)-HNK (20 mg/kg, i.p.), 24 h prior to retesting for reversal of escape deficits. We found that a significantly greater number of mice developed the helpless phenotype when they were exposed to the live predator and that the helpless phenotype was reversed in mice treated with (2R,6R)-HNK. There were no sex differences in the response to predator-stress exposure or (2R,6R)-HNK treatment. The live-predator model developed in this study provides an opportunity to further refine our understanding of the neurobiological substrates impacted by adolescent trauma and improve treatment strategies. The demonstrated efficacy of (2R,6R)-HNK in this model suggests a novel therapeutic intervention for a treatment-resistant population.

NMDAR inhibition-independent antidepressant actions of ketamine metabolites.

Major depressive disorder affects around 16 per cent of the world population at some point in their lives. Despite the availability of numerous monoaminergic-based antidepressants, most patients require several weeks, if not months, to respond to these treatments, and many patients never attain sustained remission of their symptoms. The non-competitive, glutamatergic NMDAR (N-methyl-d-aspartate receptor) antagonist (R,S)-ketamine exerts rapid and sustained antidepressant effects after a single dose in patients with depression, but its use is associated with undesirable side effects. Here we show that the metabolism of (R,S)-ketamine to (2S,6S;2R,6R)-hydroxynorketamine (HNK) is essential for its antidepressant effects, and that the (2R,6R)-HNK enantiomer exerts behavioural, electroencephalographic, electrophysiological and cellular antidepressant-related actions in mice. These antidepressant actions are independent of NMDAR inhibition but involve early and sustained activation of AMPARs (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors). We also establish that (2R,6R)-HNK lacks ketamine-related side effects. Our data implicate a novel mechanism underlying the antidepressant properties of (R,S)-ketamine and have relevance for the development of next-generation, rapid-acting antidepressants.

MicroRNAs Are Involved in the Development of Morphine-Induced Analgesic Tolerance and Regulate Functionally Relevant Changes in Serpini1.

Long-term opioid treatment results in reduced therapeutic efficacy and in turn leads to an increase in the dose required to produce equivalent pain relief and alleviate break-through or insurmountable pain. Altered gene expression is a likely means for inducing long-term neuroadaptations responsible for tolerance. Studies conducted by our laboratory (Tapocik et al., 2009) revealed a network of gene expression changes occurring in canonical pathways involved in neuroplasticity, and uncovered miRNA processing as a potential mechanism. In particular, the mRNA coding the protein responsible for processing miRNAs, Dicer1, was positively correlated with the development of analgesic tolerance. The purpose of the present study was to test the hypothesis that miRNAs play a significant role in the development of analgesic tolerance as measured by thermal nociception. Dicer1 knockdown, miRNA profiling, bioinformatics, and confirmation of high value targets were used to test the proposition. Regionally targeted Dicer1 knockdown (via shRNA) had the anticipated consequence of eliminating the development of tolerance in C57BL/6J (B6) mice, thus supporting the involvement of miRNAs in the development of tolerance. MiRNA expression profiling identified a core set of chronic morphine-regulated miRNAs (miR's 27a, 9, 483, 505, 146b, 202). Bioinformatics approaches were implemented to identify and prioritize their predicted target mRNAs. We focused our attention on miR27a and its predicted target serpin peptidase inhibitor clade I (Serpini1) mRNA, a transcript known to be intricately involved in dendritic spine density regulation in a manner consistent with chronic morphine's consequences and previously found to be correlated with the development of analgesic tolerance. In vitro reporter assay confirmed the targeting of the Serpini1 3'-untranslated region by miR27a. Interestingly miR27a was found to positively regulate Serpini1 mRNA and protein levels in multiple neuronal cell lines. Lastly, Serpini1 knockout mice developed analgesic tolerance at a slower rate than wild-type mice thus confirming a role for the protein in analgesic tolerance. Overall, these results provide evidence to support a specific role for miR27a and Serpini1 in the behavioral response to chronic opioid administration (COA) and suggest that miRNA expression and mRNA targeting may underlie the neuroadaptations that mediate tolerance to the analgesic effects of morphine.

Mining mouse behavior for patterns predicting psychiatric drug classification.

RATIONALE: In psychiatric drug discovery, a critical step is predicting the psychopharmacological effect and therapeutic potential of novel (or repurposed) compounds early in the development process. This process is hampered by the need to utilize multiple disorder-specific and labor-intensive behavioral assays. OBJECTIVES: This study aims to investigate the feasibility of a single high-throughput behavioral assay to classify psychiatric drugs into multiple psychopharmacological classes. METHODS: Using Pattern Array, a procedure for data mining exploratory behavior in mice, we mined ~100,000 complex movement patterns for those that best predict psychopharmacological class and dose. The best patterns were integrated into a classification model that assigns psychopharmacological compounds to one of six clinically relevant classes--antipsychotic, antidepressant, opioids, psychotomimetic, psychomotor stimulant, and α-adrenergic. RESULTS: Surprisingly, only a small number of well-chosen behaviors were required for successful class prediction. One of them, a behavior termed "universal drug detector", was dose-dependently decreased by drugs from all classes, thus providing a sensitive index of psychopharmacological activity. In independent validation in a blind fashion, simulating the process of in vivo pre-clinical drug screening, the classification model correctly classified nine out of 11 "unknown" compounds. Interestingly, even "misclassifications" match known alternate therapeutic indications, illustrating drug "repurposing" potential. CONCLUSIONS: Unlike standard animal models, the discovered classification model can be systematically updated to improve its predictive power and add therapeutic classes and subclasses with each additional diversification of the database. Our study demonstrates the power of data mining approaches for behavior analysis, using multiple measures in parallel for drug screening and behavioral phenotyping.

Neuroplasticity, axonal guidance and micro-RNA genes are associated with morphine self-administration behavior.

Neuroadaptations in the ventral striatum (VS) and ventral midbrain (VMB) following chronic opioid administration are thought to contribute to the pathogenesis and persistence of opiate addiction. In order to identify candidate genes involved in these neuroadaptations, we utilized a behavior-genetics strategy designed to associate contingent intravenous drug self-administration with specific patterns of gene expression in inbred mice differentially predisposed to the rewarding effects of morphine. In a Yoked-control paradigm, C57BL/6J mice showed clear morphine-reinforced behavior, whereas DBA/2J mice did not. Moreover, the Yoked-control paradigm revealed the powerful consequences of self-administration versus passive administration at the level of gene expression. Morphine self-administration in the C57BL/6J mice uniquely up- or down-regulated 237 genes in the VS and 131 genes in the VMB. Interestingly, only a handful of the C57BL/6J self-administration genes (<3%) exhibited a similar expression pattern in the DBA/2J mice. Hence, specific sets of genes could be confidently assigned to regional effects of morphine in a contingent- and genotype-dependent manner. Bioinformatics analysis revealed that neuroplasticity, axonal guidance and micro-RNAs (miRNAs) were among the key themes associated with drug self-administration. Noteworthy were the primary miRNA genes H19 and micro-RNA containing gene (Mirg), processed, respectively, to mature miRNAs miR-675 and miR-154, because they are prime candidates to mediate network-like changes in responses to chronic drug administration. These miRNAs have postulated roles in dopaminergic neuron differentiation and mu-opioid receptor regulation. The strategic approach designed to focus on reinforcement-associated genes provides new insight into the role of neuroplasticity pathways and miRNAs in drug addiction.

Altered spatial learning, cortical plasticity and hippocampal anatomy in a neurodevelopmental model of schizophrenia-related endophenotypes.

Adult rats exposed to the DNA-methylating agent methylazoxymethanol on embryonic day 17 show a pattern of neurobiological deficits that model some of the neuropathological and behavioral changes observed in schizophrenia. Although it is generally assumed that these changes reflect targeted disruption of embryonic neurogenesis, it is unknown whether these effects generalise to other antimitotic agents administered at different stages of development. In the present study, neurochemical, behavioral and electrophysiological techniques were used to determine whether exposure to the antimitotic agent Ara-C later in development recapitulates some of the changes observed in methylazoxymethanol (MAM)-treated animals and in patients with schizophrenia. Male rats exposed to Ara-C (30 mg/kg/day) at embryonic days 19.5 and 20.5 show reduced cell numbers and heterotopias in hippocampal CA1 and CA2/3 regions, respectively, as well as cell loss in the superficial layers of the pre- and infralimbic cortex. Birth date labeling with bromodeoxyuridine reveals that the cytoarchitectural changes in CA2/3 are a consequence rather that a direct result of disrupted cortical neurogenesis. Ara-C-treated rats possess elevated levels of cortical dopamine and DOPAC (3,4-didyhydroxypheylacetic acid) but no change in norepinephrine or serotonin. Ara-C-treated rats are impaired in their ability to learn the Morris water maze task and showed diminished synaptic plasticity in the hippocampocortical pathway. These data indicate that disruption of neurogenesis at embryonic days 19.5 and 20.5 constitutes a useful model for the comparative study of deficits observed in other gestational models and their relationship to cognitive changes observed in schizophrenia.

Identification of candidate genes and gene networks specifically associated with analgesic tolerance to morphine.

Chronic morphine administration may alter the expression of hundreds to thousands of genes. However, only a subset of these genes is likely involved in analgesic tolerance. In this report, we used a behavior genetics strategy to identify candidate genes specifically linked to the development of morphine tolerance. Two inbred genotypes [C57BL/6J (B6), DBA2/J (D2)] and two reciprocal congenic genotypes (B6D2, D2B6) with the proximal region of chromosome 10 (Chr10) introgressed into opposing backgrounds served as the behavior genetic filter. Tolerance after therapeutically relevant doses of morphine developed most rapidly in the B6 followed by the B6D2 genotype and did not develop in the D2 mice and only slightly in the D2B6 animals indicating a strong influence of the proximal region of Chr10 in the development of tolerance. Gene expression profiling and pattern matching identified 64, 53, 86, and 123 predisposition genes and 81, 96, 106, and 82 tolerance genes in the periaqueductal gray (PAG), prefrontal cortex, temporal lobe, and ventral striatum, respectively. A potential gene network was identified in the PAG in which 19 of the 34 genes were strongly associated with tolerance. Eleven of the network genes were found to reside in quantitative trait loci previously associated with morphine-related behaviors, whereas seven were predictive of tolerance (morphine-naive condition). Overall, the genes modified by chronic morphine administration show a strong presence in canonical pathways representative of neuroadaptation. A potentially significant role for the micro-RNA and epigenetic mechanisms in response to chronic administration of pharmacologically relevant doses of morphine was highlighted by candidate genes Dicer and H19.

Combined application of behavior genetics and microarray analysis to identify regional expression themes and gene-behavior associations.

In this report we link candidate genes to complex behavioral phenotypes by using a behavior genetics approach. Gene expression signatures were generated for the prefrontal cortex, ventral striatum, temporal lobe, periaqueductal gray, and cerebellum in eight inbred strains from priority group A of the Mouse Phenome Project. Bioinformatic analysis of regionally enriched genes that were conserved across all strains revealed both functional and structural specialization of particular brain regions. For example, genes encoding proteins with demonstrated anti-apoptotic function were over-represented in the cerebellum, whereas genes coding for proteins associated with learning and memory were enriched in the ventral striatum, as defined by the Expression Analysis Systematic Explorer (EASE) application. Association of regional gene expression with behavioral phenotypes was exploited to identify candidate behavioral genes. Phenotypes that were investigated included anxiety, drug-naive and ethanol-induced distance traveled across a grid floor, and seizure susceptibility. Several genes within the glutamatergic signaling pathway (i.e., NMDA/glutamate receptor subunit 2C, calmodulin, solute carrier family 1 member 2, and glutamine synthetase) were identified in a phenotype-dependent and region-specific manner. In addition to supporting evidence in the literature, many of the genes that were identified could be mapped in silico to surrogate behavior-related quantitative trait loci. The approaches and data set described herein serve as a valuable resource to investigate the genetic underpinning of complex behaviors.

The interaction of methylphenidate and benztropine with the dopamine transporter is different than other substrates and ligands.

A substantial body of evidence suggests that the dopamine transporter (DAT) is the principal site for cocaine-induced reward and euphoria. Interactions between the DAT and its substrates and ligands may therefore be of clinical relevance. The pharmacological characteristics of DAT compounds were compared in wild type (WT) and mutant DATs. The DAT mutants chosen for study were those with reduced binding and uptake activities (aspartic acid 79 mutated to alanine, termed D79A), reduced binding but normal uptake (tyrosine 251 mutated to alanine, termed Y251A; tyrosine 273 mutated to alanine, termed, Y273A), and normal binding but reduced uptake (a double mutation: serines 356 and 359 mutated to alanine, termed S356,359A). The WT and mutant DATs were transfected into COS-7 cells, and their pharmacological activities were examined 3 days later. Different patterns of pharmacological activity emerged. GBR 12909, cocaine, and mazindol each showed reduced affinity for the Y251A and the Y273A mutants, but their affinity for the S356,359A mutant was similar to that of the WT DAT. d-Amphetamine, MPP+, and dopamine each showed reduced affinity for the S356,359A mutant. Benztropine and methylphenidate had a different effect. Relative to the WT DAT, they both showed reduced affinity for the S356,359A mutant when displacing radioactive carboxyfluorotropane (CFT) binding, but similar affinity when inhibiting radioactive dopamine uptake. These results indicate that methylphenidate and benztropine may interact with the DAT in a different fashion then other substrates and ligands.

Prenatal exposure to a repeated variable stress paradigm elicits behavioral and neuroendocrinological changes in the adult offspring: potential relevance to schizophrenia.

Exposure to stress during gestation induces marked changes in the behavior of the affected offspring. Examining the consequences of prenatal stress may prove useful in understanding more about the origins of schizophrenia because a number of clinical investigations have suggested that developmental insults are associated with an increased incidence of schizophrenia. The purpose of these studies is to investigate the effects of stress during gestation on the behaviors of the adult male rat offspring with an emphasis on developing a heuristic animal model of schizophrenia. Pregnant female Sprague-Dawley rats were exposed to a novel variable stress paradigm during either the second or third week of gestation. Behavioral and neuroendocrinological consequences of prenatal stress exposure were evaluated in the male offspring on postnatal day 35 or 56. Prenatal stress exposure during the third week of pregnancy caused adult male rats to exhibit prolonged elevation in plasma glucocorticoid levels following acute exposure to restraint stress indicative of diminished glucocorticoid negative feedback. Similarly, exposure to stress during the third week of pregnancy elicited an enhanced locomotor response to the psychomotor stimulant amphetamine on postnatal day 56 but not on postnatal day 35. In addition, prepulse inhibition of the acoustic startle response was diminished across a range of prepulse stimulus intensities in prenatally stressed adult male rats. Similarly, prenatally stressed rats showed evidence of a disruption in auditory sensory gating as measured by the N40 response. Taken together, these findings suggest that prenatal stress exposure significantly changed many facets of adult rat behavior. Interestingly, the behaviors that are altered have been used to validate animal models of schizophrenia and therefore, suggest that this preparation may be useful to learn more about some aspects of the pathophysiology of schizophrenia.

SEE locomotor behavior test discriminates C57BL/6J and DBA/2J mouse inbred strains across laboratories and protocol conditions.

Conventional tests of behavioral phenotyping frequently have difficulties differentiating certain genotypes and replicating these differences across laboratories and protocol conditions. This study explores the hypothesis that automated tests can be designed to quantify ethologically relevant behavior patterns that more readily characterize heritable and replicable phenotypes. It used SEE (Strategy for the Exploration of Exploration) to phenotype the locomotor behavior of the C57BL/6 and DBA/2 mouse inbred strains across 3 laboratories. The 2 genotypes differed in 15 different measures of behavior, none of which had a significant genotype-laboratory interaction. Within the same laboratory, most of these differences were replicated in additional experiments despite the test photoperiod phase being changed and saline being injected. Results suggest that well-designed tests may considerably enhance replicability across laboratories.

Darting behavior: a quantitative movement pattern designed for discrimination and replicability in mouse locomotor behavior.

In the open-field behavior of rodents, Software for Exploring Exploration (SEE) can be used for an explicit design of behavioral endpoints with high genotype discrimination and replicability across laboratories. This ability is demonstrated here in the development of a measure for darting behavior. The behavior of two common mouse inbred strains, C57BL/6J (B6) and DBA/2J (D2), was analyzed across three different laboratories, and under the effect of cocaine or amphetamine. "Darting" was defined as having higher acceleration during progression segments while moving less during stops. D2 mice darted significantly more than B6 mice in each laboratory, despite being significantly less active. These differences were maintained following cocaine administration (up to 20mg/kg) and only slightly altered by amphetamine (up to 5mg/kg) despite a several fold increase in activity. The replicability of darting behavior was confirmed in additional experiments distinct from those used for its design. The strategy leading to the darting measure may be used to develop additional discriminative and replicable endpoints of open-field behavior.