Contribution of the Opioid System to the Antidepressant Effects of Fluoxetine.

BACKGROUND: Selective serotonin reuptake inhibitors such as fluoxetine have a limited treatment efficacy. The mechanism by which some patients respond to fluoxetine while others do not remains poorly understood, limiting treatment effectiveness. We have found the opioid system to be involved in the responsiveness to fluoxetine treatment in a mouse model for anxiety- and depressive-like behavior. METHODS: We analyzed gene expression changes in the dentate gyrus of mice chronically treated with corticosterone and fluoxetine. After identifying a subset of genes of interest, we studied their expression patterns in relation to treatment responsiveness. We further characterized their expression through in situ hybridization and the analysis of a single-cell RNA sequencing dataset. Finally, we behaviorally tested mu and delta opioid receptor knockout mice in the novelty suppressed feeding test and the forced swim test after chronic corticosterone and fluoxetine treatment. RESULTS: Chronic fluoxetine treatment upregulates proenkephalin expression in the dentate gyrus, and this upregulation is associated with treatment responsiveness. The expression of several of the most significantly upregulated genes, including proenkephalin, is localized to an anatomically and transcriptionally specialized subgroup of mature granule cells in the dentate gyrus. We have also found that the delta opioid receptor contributes to some, but not all, of the behavioral effects of fluoxetine. CONCLUSIONS: These data indicate that the opioid system is involved in the antidepressant effects of fluoxetine, and this effect may be mediated through the upregulation of proenkephalin in a subpopulation of mature granule cells.

A Role for Serotonin in Modulating Opposing Drive and Brake Circuits of Impulsivity.

Impulsivity generally refers to a deficit in inhibition, with a focus on understanding the neural circuits which constitute the "brake" on actions and gratification. It is likely that increased impulsivity can arise not only from reduced inhibition, but also from a heightened or exaggerated excitatory "drive." For example, an action which has more vigor, or is fueled by either increased incentive salience or a stronger action-outcome association, may be harder to inhibit. From this perspective, this review focuses on impulse control as a competition over behavioral output between an initially learned response-reward outcome association, and a subsequently acquired opposing inhibitory association. Our goal is to present a synthesis of research from humans and animal models that supports this dual-systems approach to understanding the behavioral and neural substrates that contribute to impulsivity, with a focus on the neuromodulatory role of serotonin. We review evidence for the role of serotonin signaling in mediating the balance of the "drive" and "brake" circuits. Additionally, we consider parallels of these competing instrumental systems in impulsivity within classical conditioning processes (e.g., extinction) in order to point us to potential behavioral and neural mechanisms that may modulate the competing instrumental associations. Finally, we consider how the balance of these competing associations might contribute to, or be extracted from, our experimental assessments of impulsivity. A careful understanding of the underlying behavioral and circuit level contributions to impulsivity is important for understanding the pathogenesis of increased impulsivity present in a number of psychiatric disorders. Pathological levels of impulsivity in such disorders are likely subserved by deficits in the balance of motivational and inhibitory processes.

Mu opioid receptors on hippocampal GABAergic interneurons are critical for the antidepressant effects of tianeptine.

Tianeptine is an atypical antidepressant used in Europe to treat patients who respond poorly to selective serotonin reuptake inhibitors (SSRIs). The recent discovery that tianeptine is a mu opioid receptor (MOR) agonist has provided a potential avenue for expanding our understanding of antidepressant treatment beyond the monoamine hypothesis. Thus, our studies aim to understand the neural circuits underlying tianeptine's antidepressant effects. We show that tianeptine induces rapid antidepressant-like effects in mice after as little as one week of treatment. Critically, we also demonstrate that tianeptine's mechanism of action is distinct from fluoxetine in two important aspects: (1) tianeptine requires MORs for its chronic antidepressant-like effect, while fluoxetine does not, and (2) unlike fluoxetine, tianeptine does not promote hippocampal neurogenesis. Using cell-type specific MOR knockouts we further show that MOR expression on GABAergic cells-specifically somatostatin-positive neurons-is necessary for the acute and chronic antidepressant-like responses to tianeptine. Using central infusion of tianeptine, we also implicate the ventral hippocampus as a potential site of antidepressant action. Moreover, we show a dissociation between the antidepressant-like phenotype and other opioid-like phenotypes resulting from acute tianeptine administration such as analgesia, conditioned place preference, and hyperlocomotion. Taken together, these results suggest a novel entry point for understanding what circuit dysregulations may occur in depression, as well as possible targets for the development of new classes of antidepressant drugs.

Serotonin 1B receptor effects on response inhibition are independent of inhibitory learning.

Impulsivity is defined in terms of deficits in instrumental response inhibition, when the inability to withhold an action produces a negative outcome. However, there are many behavioral and cognitive constructs which theoretically could contribute to disordered impulsivity, including Pavlovian responding, which few studies have considered in this context. In the present set of studies, we examine Pavlovian inhibitory learning and excitatory responding in a mouse model for dysregulated impulsivity, specifically, mice lacking the serotonin 1B receptor (5-HT1BR). Consistent with previous results, we show that these mice display increased impulsivity as measured by premature responding in the operant 5-choice serial reaction time test. In a Pavlovian conditioned inhibition paradigm, they also show a decreased ability to withhold responding, but importantly have an intact ability to learn inhibitory associations. In a Pavlovian appetitive conditioning experiment, 5-HT1BR knockout mice show normal responding under a positive contingency schedule, however, they display increased responding to cues presented on an independent schedule from reinforcement in a zero contingency schedule. Interestingly this difference does not occur when the cues are explicitly unpaired in a negative contingency schedule, nor during a 25% reinforcement schedule. Overall, while our results show that the deficits in operant response inhibition in mice lacking 5-HT1BR are likely not due to Pavlovian inhibitory or excitatory learning, it is relevant to consider associative learning in the context of dysregulated impulsive behavior.

Synaptic mechanism underlying serotonin modulation of transition to cocaine addiction.

Compulsive drug use despite adverse consequences defines addiction. While mesolimbic dopamine signaling is sufficient to drive compulsion, psychostimulants such as cocaine also boost extracellular serotonin (5-HT) by inhibiting reuptake. We used SERT Met172 knockin (SertKI) mice carrying a transporter that no longer binds cocaine to abolish 5-HT transients during drug self-administration. SertKI mice showed an enhanced transition to compulsion. Conversely, pharmacologically elevating 5-HT reversed the inherently high rate of compulsion transition with optogenetic dopamine self-stimulation. The bidirectional effect on behavior is explained by presynaptic depression of orbitofrontal cortex­to­dorsal striatum synapses induced by 5-HT via 5-HT1B receptors. Consequently, in projection-specific 5-HT1B receptor knockout mice, the fraction of individuals compulsively self-administering cocaine was elevated.

A role for reward valuation in the serotonergic modulation of impulsivity.

RATIONALE: Impulsive behavior is a deleterious component of a number of mental health disorders but has few targeted pharmacotherapies. One contributing factor to the difficulty in understanding the neural substrates of disordered impulsivity is the diverse presentations of impulsive behavior. Defining the behavioral and cognitive processes which contribute to different subtypes of impulsivity is important for understanding the neural underpinnings of dysregulated impulsive behavior. METHODS: Using a mouse model for disordered impulsivity, our goal was to identify behavioral and cognitive processes that are associated with increased impulsivity. Specifically, we were interested in the facets of impulsivity modulated by serotonin signaling. We used mice lacking the serotonin 1B receptor (5-HT1BR) and measured different types of impulsivity as well as goal-directed responding, extinction, habitual-like behavior, cue reactivity, and reward reactivity. RESULTS: Mice lacking expression of 5-HT1BR had increased levels of impulsive action, goal-directed responding, and motivation, with no differences seen in rate of extinction, development of habitual behavior, delay discounting, or effort-based discounting. Interestingly, mice lacking 5-HT1BR expression also showed an overall increase in the choice of higher value rewards, increased hedonic responses to sweet rewards, and responded more for cues that predict reward. We developed a novel paradigm to demonstrate that increasing anticipated reward value could directly increase impulsive action. Furthermore, we found that 5-HT1BR KO-induced impulsivity could be ameliorated by decreasing the reward value relative to controls, suggesting that the increased 5-HT1BR-associated impulsive action may be a result of increased reward valuation. CONCLUSIONS: Taken together, these data show that the effects of serotonin on impulsive action are mediated through the modulation of hedonic value, which may alter the reward representations that motivate action. Overall, this data supports a role for reward value as an important substrate in impulsive action which may drive clinically relevant increases in impulsivity.

DIY-NAMIC Behavior: A High-Throughput Method to Measure Complex Phenotypes in the Homecage.

Complex behavioral assessment is necessary to comprehensively assess in vivo manipulations in rodent models for neuropsychiatric disorders. Operant behavioral paradigms provide rich datasets and allow for the careful analysis of behavioral phenotypes. However, one major limitation in these studies is the expense and work-load that are required using traditional methods. The equipment for commercial operant boxes can be prohibitively expensive, and the daily experimenter effort and mouse costs required for these studies is extensive. Rodents are generally trained on task-specific paradigms for months, tested every day for 5-7 d/week. Additionally, appetitive paradigms usually require food restriction and are also commonly run in the non-active light phase of the rodent circadian rhythm. These limitations make operant behavioral testing especially difficult during adolescence, a time period of interest with regards to the development of adult-like phenotypes and a high-risk period for the development of neuropsychiatric disorders, including those which involve impulsive behavior. In order to address these issues, we developed an automated, inexpensive, open-source method which allows the implementation of most standard operant paradigms in the homecage of rodents in shorter time frames without food restriction, and with much less experimenter effort. All construction and code for the do-it-yourself Nautiyal Automated Modular Instrumental Conditioning (DIY-NAMIC) system are open source. We demonstrate their utility here by measuring impulsive behavior in a pharmacology experiment, as well as in adolescent mice.

OCD candidate gene SLC1A1/EAAT3 impacts basal ganglia-mediated activity and stereotypic behavior.

Obsessive-compulsive disorder (OCD) is a chronic, disabling condition with inadequate treatment options that leave most patients with substantial residual symptoms. Structural, neurochemical, and behavioral findings point to a significant role for basal ganglia circuits and for the glutamate system in OCD. Genetic linkage and association studies in OCD point to SLC1A1, which encodes the neuronal glutamate/aspartate/cysteine transporter excitatory amino acid transporter 3 (EAAT3)/excitatory amino acid transporter 1 (EAAC1). However, no previous studies have investigated EAAT3 in basal ganglia circuits or in relation to OCD-related behavior. Here, we report a model of Slc1a1 loss based on an excisable STOP cassette that yields successful ablation of EAAT3 expression and function. Using amphetamine as a probe, we found that EAAT3 loss prevents expected increases in (i) locomotor activity, (ii) stereotypy, and (iii) immediate early gene induction in the dorsal striatum following amphetamine administration. Further, Slc1a1-STOP mice showed diminished grooming in an SKF-38393 challenge experiment, a pharmacologic model of OCD-like grooming behavior. This reduced grooming is accompanied by reduced dopamine D1 receptor binding in the dorsal striatum of Slc1a1-STOP mice. Slc1a1-STOP mice also exhibit reduced extracellular dopamine concentrations in the dorsal striatum both at baseline and following amphetamine challenge. Viral-mediated restoration of Slc1a1/EAAT3 expression in the midbrain but not in the striatum results in partial rescue of amphetamine-induced locomotion and stereotypy in Slc1a1-STOP mice, consistent with an impact of EAAT3 loss on presynaptic dopaminergic function. Collectively, these findings indicate that the most consistently associated OCD candidate gene impacts basal ganglia-dependent repetitive behaviors.

Gambling disorder: an integrative review of animal and human studies.

Gambling disorder (GD), previously called pathological gambling and classified as an impulse control disorder in DSM-III and DSM-IV, has recently been reclassified as an addictive disorder in the DSM-5. It is widely recognized as an important public health problem associated with substantial personal and social costs, high rates of psychiatric comorbidity, poor physical health, and elevated suicide rates. A number of risk factors have been identified, including some genetic polymorphisms. Animal models have been developed in order to study the underlying neural basis of GD. Here, we discuss recent advances in our understanding of the risk factors, disease course, and pathophysiology. A focus on a phenotype-based dissection of the disorder is included in which known neural correlates from animal and human studies are reviewed. Finally, current treatment approaches are discussed, as well as future directions for GD research.

The Behavioral Effects of the Antidepressant Tianeptine Require the Mu-Opioid Receptor.

Depression is a debilitating chronic illness that affects around 350 million people worldwide. Current treatments, such as selective serotonin reuptake inhibitors, are not ideal because only a fraction of patients achieve remission. Tianeptine is an effective antidepressant with a previously unknown mechanism of action. We recently reported that tianeptine is a full agonist at the mu opioid receptor (MOR). Here we demonstrate that the acute and chronic antidepressant-like behavioral effects of tianeptine in mice require MOR. Interestingly, while tianeptine also produces many opiate-like behavioral effects such as analgesia and reward, it does not lead to tolerance or withdrawal. Furthermore, the primary metabolite of tianeptine (MC5), which has a longer half-life, mimics the behavioral effects of tianeptine in a MOR-dependent fashion. These results point to the possibility that MOR and its downstream signaling cascades may be novel targets for antidepressant drug development.

Serotonin receptors in depression: from A to B.

The role of serotonin in major depressive disorder (MDD) is the focus of accumulating clinical and preclinical research. The results of these studies reflect the complexity of serotonin signaling through many receptors, in a large number of brain regions, and throughout the lifespan. The role of the serotonin transporter in MDD has been highlighted in gene by environment association studies as well as its role as a critical player in the mechanism of the most effective antidepressant treatments - selective serotonin reuptake inhibitors. While the majority of the 15 known receptors for serotonin have been implicated in depression or depressive-like behavior, the serotonin 1A (5-HT 1A) and 1B (5-HT 1B) receptors are among the most studied. Human brain imaging and genetic studies point to the involvement of 5-HT 1A and 5-HT 1B receptors in MDD and the response to antidepressant treatment. In rodents, the availability of tissue-specific and inducible knockout mouse lines has made possible the identification of the involvement of 5-HT 1A and 5-HT 1B receptors throughout development and in a cell-type specific manner. This, and other preclinical pharmacology work, shows that autoreceptor and heteroreceptor populations of these receptors have divergent roles in modulating depression-related behavior as well as responses to antidepressants and also have different functions during early postnatal development compared to during adulthood.

Genetic and Modeling Approaches Reveal Distinct Components of Impulsive Behavior.

Impulsivity is an endophenotype found in many psychiatric disorders including substance use disorders, pathological gambling, and attention deficit hyperactivity disorder. Two behavioral features often considered in impulsive behavior are behavioral inhibition (impulsive action) and delayed gratification (impulsive choice). However, the extent to which these behavioral constructs represent distinct facets of behavior with discrete biological bases is unclear. To test the hypothesis that impulsive action and impulsive choice represent statistically independent behavioral constructs in mice, we collected behavioral measures of impulsivity in a single cohort of mice using well-validated operant behavioral paradigms. Mice with manipulation of serotonin 1B receptor (5-HT1BR) expression were included as a model of disordered impulsivity. A factor analysis was used to characterize correlations between the measures of impulsivity and to identify covariates. Using two approaches, we dissociated impulsive action from impulsive choice. First, the absence of 5-HT1BRs caused increased impulsive action, but not impulsive choice. Second, based on an exploratory factor analysis, a two-factor model described the data well, with measures of impulsive action and choice separating into two independent factors. A multiple-indicator multiple-causes analysis showed that 5-HT1BR expression and sex were significant covariates of impulsivity. Males displayed increased impulsivity in both dimensions, whereas 5-HT1BR expression was a predictor of increased impulsive action only. These data support the conclusion that impulsive action and impulsive choice are distinct behavioral phenotypes with dissociable biological influences that can be modeled in mice. Our work may help inform better classification, diagnosis, and treatment of psychiatric disorders, which present with disordered impulsivity.

A Lack of Serotonin 1B Autoreceptors Results in Decreased Anxiety and Depression-Related Behaviors.

The effects of serotonin (5-HT) on anxiety and depression are mediated by a number of 5-HT receptors, including autoreceptors that act to inhibit 5-HT release. While the majority of anxiety and depression-related research has focused on the 5-HT1A receptor, the 5-HT1B receptor has a lesser known role in modulating emotional behavior. 5-HT1B receptors are inhibitory GPCRs located on the presynaptic terminal of both serotonin and non-serotonin neurons, where they act to inhibit neurotransmitter release. The autoreceptor population located on the axon terminals of 5-HT neurons is a difficult population to study due to their diffuse localization throughout the brain that overlaps with 5-HT1B heteroreceptors (receptors located on non-serotonergic neurons). In order to study the contribution of 5-HT1B autoreceptors to anxiety and depression-related behaviors, we developed a genetic mouse model that allows for selective ablation of 5-HT1B autoreceptors. Mice lacking 5-HT1B autoreceptors displayed the expected increases in extracellular serotonin levels in the ventral hippocampus following administration of a selective serotonin reuptake inhibitor. In behavioral studies, they displayed decreased anxiety-like behavior in the open field and antidepressant-like effects in the forced swim and sucrose preference tests. These results suggest that strategies aimed at blocking 5-HT1B autoreceptors may be useful for the treatment of anxiety and depression.

Distinct Circuits Underlie the Effects of 5-HT1B Receptors on Aggression and Impulsivity.

Impulsive and aggressive behaviors are both modulated by serotonergic signaling, specifically through the serotonin 1B receptor (5-HT1BR). 5-HT1BR knockout mice show increased aggression and impulsivity, and 5-HT1BR polymorphisms are associated with aggression and drug addiction in humans. To dissect the mechanisms by which the 5-HT1BR affects these phenotypes, we developed a mouse model to spatially and temporally regulate 5-HT1BR expression. Our results demonstrate that forebrain 5-HT1B heteroreceptors expressed during an early postnatal period contribute to the development of the neural systems underlying adult aggression. However, distinct heteroreceptors acting during adulthood are involved in mediating impulsivity. Correlating with the impulsivity, dopamine in the nucleus accumbens is elevated in the absence of 5-HT1BRs and normalized following adult rescue of the receptor. Overall, these data show that while adolescent expression of 5-HT1BRs influences aggressive behavior, a distinct set of 5-HT1B receptors modulates impulsive behavior during adulthood.

Combining small-volume metabolomic and transcriptomic approaches for assessing brain chemistry.

The integration of disparate data types provides a more complete picture of complex biological systems. Here we combine small-volume metabolomic and transcriptomic platforms to determine subtle chemical changes and to link metabolites and genes to biochemical pathways. Capillary electrophoresis-mass spectrometry (CE-MS) and whole-genome gene expression arrays, aided by integrative pathway analysis, were utilized to survey metabolomic/transcriptomic hippocampal neurochemistry. We measured changes in individual hippocampi from the mast cell mutant mouse strain, C57BL/6 Kit(W-sh/W-sh). These mice have a naturally occurring mutation in the white spotting locus that causes reduced c-Kit receptor expression and an inability of mast cells to differentiate from their hematopoietic progenitors. Compared with their littermates, the mast cell-deficient mice have profound deficits in spatial learning, memory, and neurogenesis. A total of 18 distinct metabolites were identified in the hippocampus that discriminated between the C57BL/6 Kit(W-sh/W-sh) and control mice. The combined analysis of metabolite and gene expression changes revealed a number of altered pathways. Importantly, results from both platforms indicated that multiple pathways are impacted, including amino acid metabolism, increasing the confidence in each approach. Because the CE-MS and expression profiling are both amenable to small-volume analysis, this integrated analysis is applicable to a range of volume-limited biological systems.

Genetic approaches for understanding the role of serotonin receptors in mood and behavior.

Serotonin (5-hydroxytryptamine; 5-HT) is an ancient signaling molecule that has a conserved role in modulating mood and behavior. Integral to its pleiotropic actions is the existence of multiple receptors, expressed in distinct but often overlapping patterns within the brain and the periphery. The existence of ∼14 mammalian receptor subtypes, many of which possess similar pharmacological profiles, has made assigning functional roles for these receptors challenging. This challenge has been further compounded by the revelation that a single receptor can have several different functions depending upon where and when it is expressed and activated, that is, in brain versus periphery, or at different developmental time points. This review highlights the contribution of genetic techniques to dissect the specific function of distinct serotonin receptor populations across the life course, with an emphasis on the contribution of different serotonin 1A receptor populations to mood and behavior. Similar approaches hold the promise to elucidate the functional roles of other receptors, as well as the interaction of serotonin with other neuroendocrine modulators of mood and behavior.

Serotonin of mast cell origin contributes to hippocampal function.

In the central nervous system, serotonin, an important neurotransmitter and trophic factor, is synthesized by both mast cells and neurons. Mast cells, like other immune cells, are born in the bone marrow and migrate to many tissues. We show that they are resident in the mouse brain throughout development and adulthood. Measurements based on capillary electrophoresis with native fluorescence detection indicate that a significant contribution of serotonin to the hippocampal milieu is associated with mast cell activation. Compared with their littermates, mast cell-deficient C57BL/6 Kit(W-sh/W-sh) mice have profound deficits in hippocampus-dependent spatial learning and memory and in hippocampal neurogenesis. These deficits are associated with a reduction in cell proliferation and in immature neurons in the dentate gyrus, but not in the subventricular zone - a neurogenic niche lacking mast cells. Chronic treatment with fluoxetine, a selective serotonin reuptake inhibitor, reverses the deficit in hippocampal neurogenesis in mast cell-deficient mice. In summary, the present study demonstrates that mast cells are a source of serotonin, that mast cell-deficient C57BL/6 Kit(W-sh/W-sh) mice have disrupted hippocampus-dependent behavior and neurogenesis, and that elevating serotonin in these mice, by treatment with fluoxetine, reverses these deficits. We conclude that mast cells contribute to behavioral and physiological functions of the hippocampus and note that they play a physiological role in neuroimmune interactions, even in the absence of inflammatory responses.

Blood-borne donor mast cell precursors migrate to mast cell-rich brain regions in the adult mouse.

Mast cells are hematopoietic immune cells located throughout the body, including within the brain. Reconstitution of mast cell deficient Kit(W-sh/W-sh) mice has proven valuable in determining peripheral mast cell function. Here we study the brain mast cell population using a novel method of blood transfusion for reconstitution. We show that blood transfusion results in mast cells of donor origin in the WT mouse, including in the brain where they are restricted to regions bearing host mast cells. In contrast, in Kit(W-sh/W-sh) mice, transfusion results in mast cells in the pinna of the ear, but not the brain.

Mast cells are necessary for the hypothermic response to LPS-induced sepsis.

As central nervous system residents, mast cells contain many cytokines and are localized primarily near large blood vessels in the diencephalon and within the leptomeninges, making them candidates for immune to neural "cross talk." Using mast cell-deficient Kit(W-sh/W-sh) mice, we assessed the role of these cells in the thermoregulatory component of the immune response to lipopolysaccharide (LPS). Kit(W-sh/W-sh) and wild-type (WT) mice differed in several respects in response to injection of a high dose of LPS (1 mg/kg ip). Core temperature (T(c)) of WT mice decreased by approximately 3 degrees C, whereas Kit(W-sh/W-sh) mice did not become hypothermic but instead exhibited pronounced low-frequency T(c) oscillations around their baseline temperature. In addition, Kit(W-sh/W-sh) mice had lower levels of whole brain TNF-alpha but no differences in IL-1beta, IL-6, IFN-gamma, or histamine compared with WT mice following injection of the high dose of LPS, consistent with the role of TNF-alpha in sepsis. Kit(W-sh/W-sh) mice had increased resistance to LPS, and some survived a dose of LPS that was lethal in littermate controls. In contrast, Kit(W-sh/W-sh) and WT mice were similar in other aspects, namely, in the hyperthermia following injection of TNF-alpha (1.5 microg icv), reduced nighttime T(c) and locomotor activity (to 1 mg/kg LPS), response to a low dose of LPS (10 microg/kg ip), and response to subcutaneous turpentine injection. These results indicate that mast cells play a role in the regulation of thermoregulatory responses and survival following sepsis induction and suggest a brain site of action.