Dopamine transporter blockade during adolescence increases adult dopamine function, impulsivity, and aggression.

Sensitive developmental periods shape neural circuits and enable adaptation. However, they also engender vulnerability to factors that can perturb developmental trajectories. An understanding of sensitive period phenomena and mechanisms separate from sensory system development is still lacking, yet critical to understanding disease etiology and risk. The dopamine system is pivotal in controlling and shaping adolescent behaviors, and it undergoes heightened plasticity during that time, such that interference with dopamine signaling can have long-lasting behavioral consequences. Here we sought to gain mechanistic insight into this dopamine-sensitive period and its impact on behavior. In mice, dopamine transporter (DAT) blockade from postnatal (P) day 22 to 41 increases aggression and sensitivity to amphetamine (AMPH) behavioral stimulation in adulthood. Here, we refined this sensitive window to P32-41 and identified increased firing of dopaminergic neurons in vitro and in vivo as a neural correlate to altered adult behavior. Aggression can result from enhanced impulsivity and cognitive dysfunction, and dopamine regulates working memory and motivated behavior. Hence, we assessed these behavioral domains and found that P32-41 DAT blockade increases impulsivity but has no effect on cognition, working memory, or motivation in adulthood. Lastly, using optogenetics to drive dopamine neurons, we find that increased VTA but not SNc dopaminergic activity mimics the increase in impulsive behavior in the Go/NoGo task observed after adolescent DAT blockade. Together our data provide insight into the developmental origins of aggression and impulsivity that may ultimately improve diagnosis, prevention, and treatment strategies for related neuropsychiatric disorders.

Perinatal interference with the serotonergic system affects VTA function in the adult via glutamate co-transmission.

Serotonin and dopamine are associated with multiple psychiatric disorders. How they interact during development to affect subsequent behavior remains unknown. Knockout of the serotonin transporter or postnatal blockade with selective serotonin reuptake inhibitors (SSRIs) leads to novelty-induced exploration deficits in adulthood, potentially involving the dopamine system. Here, we show in the mouse that raphe nucleus serotonin neurons activate ventral tegmental area dopamine neurons via glutamate co-transmission and that this co-transmission is reduced in animals exposed postnatally to SSRIs. Blocking serotonin neuron glutamate co-transmission mimics this SSRI-induced hypolocomotion, while optogenetic activation of dopamine neurons reverses this hypolocomotor phenotype. Our data demonstrate that serotonin neurons modulate dopamine neuron activity via glutamate co-transmission and that this pathway is developmentally malleable, with high serotonin levels during early life reducing co-transmission, revealing the basis for the reduced novelty-induced exploration in adulthood due to postnatal SSRI exposure.

5-HT2C receptor blockade reverses SSRI-associated basal ganglia dysfunction and potentiates therapeutic efficacy.

Serotonin (5-HT) selective reuptake inhibitors (SSRIs) are widely used in the treatment of depression and anxiety disorders, but responsiveness is uncertain and side effects often lead to discontinuation. Side effect profiles suggest that SSRIs reduce dopaminergic (DAergic) activity, but specific mechanistic insight is missing. Here we show in mice that SSRIs impair motor function by acting on 5-HT2C receptors in the substantia nigra pars reticulata (SNr), which in turn inhibits nigra pars compacta (SNc) DAergic neurons. SSRI-induced motor deficits can be reversed by systemic or SNr-localized 5-HT2C receptor antagonism. SSRIs induce SNr hyperactivity and SNc hypoactivity that can also be reversed by systemic 5-HT2C receptor antagonism. Optogenetic inhibition of SNc DAergic neurons mimics the motor deficits due to chronic SSRI treatment, whereas local SNr 5-HT2C receptor antagonism or optogenetic activation of SNc DAergic neurons reverse SSRI-induced motor deficits. Lastly, we find that 5-HT2C receptor antagonism potentiates the antidepressant and anxiolytic effects of SSRIs. Together our findings demonstrate opposing roles for 5-HT2C receptors in the effects of SSRIs on motor function and affective behavior, highlighting the potential benefits of 5-HT2C receptor antagonists for both reduction of motor side effects of SSRIs and augmentation of therapeutic antidepressant and anxiolytic effects.

Mice with compromised 5-HTT function lack phosphotyrosine-mediated inhibitory control over prefrontal 5-HT responses.

The activity of the prefrontal cortex is essential for normal emotional processing and is strongly modulated by serotonin (5-HT). Yet, little is known about the regulatory mechanisms that control the activity of the prefrontal 5-HT receptors. Here, we found and characterized a deregulation of prefrontal 5-HT receptor electrophysiological signaling in mouse models of disrupted serotonin transporter (5-HTT) function, a risk factor for emotional and cognitive disturbances. We identified a novel tyrosine kinase-dependent mechanism that regulates 5-HT-mediated inhibition of prefrontal pyramidal neurons. We report that mice with compromised 5-HTT, resulting from either genetic deletion or brief treatment with selective serotonin reuptake inhibitors during development, have amplified 5-HT1A receptor-mediated currents in adulthood. These greater inhibitory effects of 5-HT are accompanied by enhanced downstream coupling to Kir3 channels. Notably, in normal wild-type mice, we found that these larger 5-HT1A responses can be mimicked through inhibition of Src family tyrosine kinases. By comparison, in our 5-HTT mouse models, the larger 5-HT1A responses were rapidly reduced through inhibition of tyrosine phosphatases. Our findings implicate tyrosine phosphorylation in regulating the electrophysiological effects of prefrontal 5-HT1A receptors with implications for neuropsychiatric diseases associated with emotional dysfunction, such as anxiety and depressive disorders.

Postnatal day 2 to 11 constitutes a 5-HT-sensitive period impacting adult mPFC function.

Early-life serotonin [5-hydroxytryptamine (5-HT)] signaling modulates brain development, which impacts adult behavior, but 5-HT-sensitive periods, neural substrates, and behavioral consequences remain poorly understood. Here we identify the period ranging from postnatal day 2 (P2) to P11 as 5-HT sensitive, with 5-HT transporter (5-HTT) blockade increasing anxiety- and depression-like behavior, and impairing fear extinction learning and memory in adult mice. Concomitantly, P2-P11 5-HTT blockade causes dendritic hypotrophy and reduced excitability of infralimbic (IL) cortex pyramidal neurons that normally promote fear extinction. By contrast, the neighboring prelimbic (PL) pyramidal neurons, which normally inhibit fear extinction, become more excitable. Excitotoxic IL but not PL lesions in adult control mice reproduce the anxiety-related phenotypes. These findings suggest that increased 5-HT signaling during P2-P11 alters adult mPFC function to increase anxiety and impair fear extinction, and imply a differential role for IL and PL neurons in regulating affective behaviors. Together, our results support a developmental mechanism for the etiology and pathophysiology of affective disorders and fear-related behaviors.

Medial Prefrontal Cortex Serotonin Input Regulates Cognitive Flexibility in Mice.

The medial prefrontal cortex (mPFC) regulates cognitive flexibility and emotional behavior. Neurons that release serotonin project to the mPFC, and serotonergic drugs influence emotion and cognition. Yet, the specific roles of endogenous serotonin release in the mPFC on neurophysiology and behavior are unknown. We show that axonal serotonin release in the mPFC directly inhibits the major mPFC output neurons. In serotonergic neurons projecting from the dorsal raphe to the mPFC, we find endogenous activity signatures pre-reward retrieval and at reward retrieval during a cognitive flexibility task. In vivo optogenetic activation of this pathway during pre-reward retrieval selectively improved extradimensional rule shift performance while inhibition impaired it, demonstrating sufficiency and necessity for mPFC serotonin release in cognitive flexibility. Locomotor activity and anxiety-like behavior were not affected by either optogenetic manipulation. Collectively, our data reveal a powerful and specific modulatory role of endogenous serotonin release from dorsal raphe-to-mPFC projecting neurons in cognitive flexibility.

Chronic 5-HT transporter blockade reduces DA signaling to elicit basal ganglia dysfunction.

Serotonin (5-HT)-selective reuptake inhibitors (SSRIs) are widely administered for the treatment of depression, anxiety, and other neuropsychiatric disorders, but response rates are low, and side effects often lead to discontinuation. Side effect profiles suggest that SSRIs inhibit dopaminergic activity, but mechanistic insight remains scarce. Here we show that in mice, chronic 5-HT transporter (5-HTT) blockade during adulthood but not during development impairs basal ganglia-dependent behaviors in a dose-dependent and reversible fashion. Furthermore, chronic 5-HTT blockade reduces striatal dopamine (DA) content and metabolism. A causal relationship between reduced DA signaling and impaired basal ganglia-dependent behavior is indicated by the reversal of behavioral deficits through L-DOPA administration. Our data suggest that augmentation of DA signaling would reduce side effects and increase efficacies of SSRI-based therapy.

Tianeptine, but not fluoxetine, decreases avoidant behavior in a mouse model of early developmental exposure to fluoxetine.

Depression and anxiety, two of the most common mental health disorders, share common symptoms and treatments. Most pharmacological agents available to treat these disorders target monoamine systems. Currently, finding the most effective treatment for an individual is a process of trial and error. To better understand how disease etiology may predict treatment response, we studied mice exposed developmentally to the selective serotonin reuptake inhibitor (SSRI) fluoxetine (FLX). These mice show the murine equivalent of anxiety- and depression-like symptoms in adulthood and here we report that these mice are also behaviorally resistant to the antidepressant-like effects of adult SSRI administration. We investigated whether tianeptine (TIA), which exerts its therapeutic effects through agonism of the mu-opioid receptor instead of targeting monoaminergic systems, would be more effective in this model. We found that C57BL/6J pups exposed to FLX from postnatal day 2 to 11 (PNFLX, the mouse equivalent in terms of brain development to the human third trimester) showed increased avoidant behaviors as adults that failed to improve, or were even exacerbated, by chronic SSRI treatment. By contrast, avoidant behaviors in these same mice were drastically improved following chronic treatment with TIA. Overall, this demonstrates that TIA may be a promising alternative treatment for patients that fail to respond to typical antidepressants, especially in patients whose serotonergic system has been altered by in utero exposure to SSRIs.

Dopamine promotes aggression in mice via ventral tegmental area to lateral septum projections.

Septal-hypothalamic neuronal activity centrally mediates aggressive behavior and dopamine system hyperactivity is associated with elevated aggression. However, the causal role of dopamine in aggression and its target circuit mechanisms are largely unknown. To address this knowledge gap, we studied the modulatory role of the population- and projection-specific dopamine function in a murine model of aggressive behavior. We find that terminal activity of ventral tegmental area (VTA) dopaminergic neurons selectively projecting to the lateral septum (LS) is sufficient for promoting aggression and necessary for establishing baseline aggression. Within the LS, dopamine acts on D2-receptors to inhibit GABAergic neurons, and septal D2-signaling is necessary for VTA dopaminergic activity to promote aggression. Collectively, our data reveal a powerful modulatory influence of dopaminergic synaptic input on LS function and aggression, effectively linking the clinically pertinent hyper-dopaminergic model of aggression with the classic septal-hypothalamic aggression axis.

Impact of antenatal selective serotonin reuptake inhibitor exposure on pregnancy outcomes in mice.

OBJECTIVE: This study investigates fluoxetine (FLX) exposure as an etiology for altered gestational length and adverse pregnancy outcomes. STUDY DESIGN: Two experiments were performed exposing mice to drinking water (H(2)O) or H(2)O+FLX. Primary outcomes included gestational length, litter size, and live birth rate. In experiment 1, time-mated dams were monitored for spontaneous birth, and gestational length was calculated. In experiment 2, dams were dissected on day 14 to verify litter size and qualities of embryo implantation. RESULTS: There was no difference in gestational length between H(2)O dams (480.7 ± 13.2 hours) and H(2)O+FLX dams (483.5 ± 10.1 hours), P = .70. Mean litter size was decreased in H(2)O+FLX dams (4.1 ± 1.3/litter) compared to H(2)O dams (5.5 ± 1.9/litter), P = .04. H(2)O+FLX dams were less likely to have live births (25.4%) compared to H(2)O dams (49.3%), P = .01. CONCLUSION: Antenatal FLX exposure did not statistically alter gestational length, but did affect litter size and spontaneous loss in mice. This warrants further investigation.

Hippocampal neurogenesis is not required for behavioral effects of environmental enrichment.

Environmental enrichment increases adult hippocampal neurogenesis and alters hippocampal-dependent behavior in rodents. To investigate a causal link between these two observations, we analyzed the effect of enrichment on spatial learning and anxiety-like behavior while blocking adult hippocampal neurogenesis. We report that environmental enrichment alters behavior in mice regardless of their hippocampal neurogenic capability, providing evidence that the newborn cells do not mediate these effects of enrichment.

Chemogenetic activation of adrenocortical Gq signaling causes hyperaldosteronism and disrupts functional zonation.

The mineralocorticoid aldosterone is produced in the adrenal zona glomerulosa (ZG) under the control of the renin-angiotensin II (AngII) system. Primary aldosteronism (PA) results from renin-independent production of aldosterone and is a common cause of hypertension. PA is caused by dysregulated localization of the enzyme aldosterone synthase (Cyp11b2), which is normally restricted to the ZG. Cyp11b2 transcription and aldosterone production are predominantly regulated by AngII activation of the Gq signaling pathway. Here, we report the generation of transgenic mice with Gq-coupled designer receptors exclusively activated by designer drugs (DREADDs) specifically in the adrenal cortex. We show that adrenal-wide ligand activation of Gq DREADD receptors triggered disorganization of adrenal functional zonation, with induction of Cyp11b2 in glucocorticoid-producing zona fasciculata cells. This result was consistent with increased renin-independent aldosterone production and hypertension. All parameters were reversible following termination of DREADD-mediated Gq signaling. These findings demonstrate that Gq signaling is sufficient for adrenocortical aldosterone production and implicate this pathway in the determination of zone-specific steroid production within the adrenal cortex. This transgenic mouse also provides an inducible and reversible model of hyperaldosteronism to investigate PA therapeutics and the mechanisms leading to the damaging effects of aldosterone on the cardiovascular system.

Inhibition of serotonin but not norepinephrine transport during development produces delayed, persistent perturbations of emotional behaviors in mice.

Serotonin (5-HT) acts as a neurotransmitter, but also modulates brain maturation during early development. The demonstrated influence of genetic variants on brain function, personality traits, and susceptibility to neuropsychiatric disorders suggests a critical importance of developmental mechanisms. However, little is known about how and when developmentally perturbed 5-HT signaling affects circuitry and resulting behavior. The 5-HT transporter (5-HTT) is a key regulator of extracellular 5-HT levels and we used pharmacologic strategies to manipulate 5-HTT function during development and determine behavioral consequences. Transient exposure to the 5-HTT inhibitors fluoxetine, clomipramine, and citalopram from postnatal day 4 (P4) to P21 produced abnormal emotional behaviors in adult mice. Similar treatment with the norepinephrine transporter (NET) inhibitor, desipramine, did not adversely affect adult behavior, suggesting that 5-HT and norepinephrine (NE) do not share the same effects on brain development. Shifting our period of treatment/testing to P90/P185 failed to mimic the effect of earlier exposure, demonstrating that 5-HT effects on adult behavior are developmentally specific. We have hypothesized that early-life perturbations of 5-HT signaling affect corticolimbic circuits that do not reach maturity until the peri-adolescent period. In support of this idea, we found that abnormal behaviors resulting from postnatal fluoxetine exposure have a post-pubescent onset and persist long after reaching adult age. A better understanding of the underlying 5-HT sensitive circuits and how they are perturbed should lead to new insights into how various genetic polymorphisms confer their risk to carriers. Furthermore, these studies should help determine whether in utero exposure to 5-HTT blocking drugs poses a risk for behavioral abnormalities in later life.

Neurodevelopmental origins of depressive disorders.

Depression is a common and devastating neuropsychiatric disorder, and a better understanding of its pathophysiology is needed to improve diagnosis, treatment and prevention. By considering the developmental dimensions of genetic and environmental factors, new insights have been found into the etiology and pathophysiology of depression. Specifically, we begin to understand how certain vulnerability factors affect the maturation of brain circuits involved in emotional function to increase the risk for depressive disorders later in life. These new findings might help us to better categorize and manage this complex disease.

Dopamine neuron glutamate cotransmission evokes a delayed excitation in lateral dorsal striatal cholinergic interneurons.

Dopamine neurons have different synaptic actions in the ventral and dorsal striatum (dStr), but whether this heterogeneity extends to dStr subregions has not been addressed. We have found that optogenetic activation of dStr dopamine neuron terminals in mouse brain slices pauses the firing of cholinergic interneurons in both the medial and lateral subregions, while in the lateral subregion the pause is shorter due to a subsequent excitation. This excitation is mediated mainly by metabotropic glutamate receptor 1 (mGluR1) and partially by dopamine D1-like receptors coupled to transient receptor potential channel 3 and 7. DA neurons do not signal to spiny projection neurons in the medial dStr, while they elicit ionotropic glutamate responses in the lateral dStr. The DA neurons mediating these excitatory signals are in the substantia nigra (SN). Thus, SN dopamine neurons engage different receptors in different postsynaptic neurons in different dStr subregions to convey strikingly different signals. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

Toward Serotonin Fluorescent False Neurotransmitters: Development of Fluorescent Dual Serotonin and Vesicular Monoamine Transporter Substrates for Visualizing Serotonin Neurons.

Ongoing efforts in our laboratories focus on design of optical reporters known as fluorescent false neurotransmitters (FFNs) that enable the visualization of uptake into, packaging within, and release from individual monoaminergic neurons and presynaptic sites in the brain. Here, we introduce the molecular probe FFN246 as an expansion of the FFN platform to the serotonergic system. Combining the acridone fluorophore with the ethylamine recognition element of serotonin, we identified FFN54 and FFN246 as substrates for both the serotonin transporter and the vesicular monoamine transporter 2 (VMAT2). A systematic structure-activity study revealed the basic structural chemotype of aminoalkyl acridones required for serotonin transporter (SERT) activity and enabled lowering the background labeling of these probes while maintaining SERT activity, which proved essential for obtaining sufficient signal in the brain tissue (FFN246). We demonstrate the utility of FFN246 for direct examination of SERT activity and SERT inhibitors in 96-well cell culture assays, as well as specific labeling of serotonergic neurons of the dorsal raphe nucleus in the living tissue of acute mouse brain slices. While we found only minor FFN246 accumulation in serotonergic axons in murine brain tissue, FFN246 effectively traces serotonin uptake and packaging in the soma of serotonergic neurons with improved photophysical properties and loading parameters compared to known serotonin-based fluorescent tracers.

Hippocampal 5-HT Input Regulates Memory Formation and Schaffer Collateral Excitation.

The efficacy and duration of memory storage is regulated by neuromodulatory transmitter actions. While the modulatory transmitter serotonin (5-HT) plays an important role in implicit forms of memory in the invertebrate Aplysia, its function in explicit memory mediated by the mammalian hippocampus is less clear. Specifically, the consequences elicited by the spatio-temporal gradient of endogenous 5-HT release are not known. Here we applied optogenetic techniques in mice to gain insight into this fundamental biological process. We find that activation of serotonergic terminals in the hippocampal CA1 region both potentiates excitatory transmission at CA3-to-CA1 synapses and enhances spatial memory. Conversely, optogenetic silencing of CA1 5-HT terminals inhibits spatial memory. We furthermore find that synaptic potentiation is mediated by 5-HT4 receptors and that systemic modulation of 5-HT4 receptor function can bidirectionally impact memory formation. Collectively, these data reveal powerful modulatory influence of serotonergic synaptic input on hippocampal function and memory formation.

New Insights into How Serotonin Selective Reuptake Inhibitors Shape the Developing Brain.

Development passes through sensitive periods, during which plasticity allows for genetic and environmental factors to exert indelible influence on the maturation of the organism. In the context of central nervous system (CNS) development, such sensitive periods shape the formation of neuro-circuits that mediate, regulate, and control behavior. This general mechanism allows for development to be guided by both the genetic blueprint, as well as the environmental context. While allowing for adaptation, such sensitive periods are also windows of vulnerability during which external and internal factors can confer risk to brain disorders by derailing adaptive developmental programs. Our group has been particularly interested in developmental periods that are sensitive to serotonin (5-HT) signaling, and impact behavior and cognition relevant to psychiatry. Specifically, we review a 5-HT-sensitive period that impacts fronto-limbic system development, resulting in cognitive, anxiety, and depression-related behaviors. We discuss preclinical data to establish biological plausibility and mechanistic insights. We also summarize epidemiological findings that underscore the potential public health implications resulting from the current practice of prescribing 5-HT reuptake inhibiting antidepressants during pregnancy. These medications enter the fetal circulation, likely perturb 5-HT signaling in the brain, and may be affecting circuit maturation in ways that parallel our findings in the developing rodent brain. More research is needed to better disambiguate the dual effects of maternal symptoms on fetal and child development from the effects of 5-HT reuptake inhibitors on clinical outcomes in the offspring. Birth Defects Research 109:924-932, 2017. © 2017 Wiley Periodicals, Inc.

Serotonin signaling modulates the effects of familial risk for depression on cortical thickness.

Depression is a highly familial and a heritable illness that is more prevalent in the biological offspring of the depressed individuals than in the general population. In a 3-generation, 30-year, longitudinal study of individuals at either a high(HR) or a low(LR) familial risk for depression, we previously showed cortical thinning in the right hemisphere was an endophenotype for the familial risk. In this study, we assessed whether the effects of familial risk were modulated by the serotonin-transporter-linked polymorphic region (5-HTTLPR). We measured cortical thickness using MRI of the brain and associated it with 5-HTTLPR polymorphism in 76 HR and 53 LR individuals. We studied the effects of genotype and gene-by-risk interaction on cortical thickness while controlling for the confounding effects of age and gender, and for the familial relatedness by applying a variance component model with random effects for genotype. The results showed significant effects of gene-by-risk interaction on thickness: The "s" allele was associated with thinner cortex in the LR individuals whereas with thicker cortex in the HR individuals. The opposing gene effects across the two risk groups were likely due to either epistatic effects and/or differing modulation of the neural plasticity by the altered 5-HT signaling in utero.

Monoamine-sensitive developmental periods impacting adult emotional and cognitive behaviors.

Development passes through sensitive periods, during which plasticity allows for genetic and environmental factors to exert indelible influence on the maturation of the organism. In the context of central nervous system development, such sensitive periods shape the formation of neurocircuits that mediate, regulate, and control behavior. This general mechanism allows for development to be guided by both the genetic blueprint as well as the environmental context. While allowing for adaptation, such sensitive periods are also vulnerability windows during which external and internal factors can confer risk to disorders by derailing otherwise resilient developmental programs. Here we review developmental periods that are sensitive to monoamine signaling and impact adult behaviors of relevance to psychiatry. Specifically, we review (1) a serotonin-sensitive period that impacts sensory system development, (2) a serotonin-sensitive period that impacts cognition, anxiety- and depression-related behaviors, and (3) a dopamine- and serotonin-sensitive period affecting aggression, impulsivity and behavioral response to psychostimulants. We discuss preclinical data to provide mechanistic insight, as well as epidemiological and clinical data to point out translational relevance. The field of translational developmental neuroscience has progressed exponentially providing solid conceptual advances and unprecedented mechanistic insight. With such knowledge at hand and important methodological innovation ongoing, the field is poised for breakthroughs elucidating the developmental origins of neuropsychiatric disorders, and thus understanding pathophysiology. Such knowledge of sensitive periods that determine the developmental trajectory of complex behaviors is a necessary step towards improving prevention and treatment approaches for neuropsychiatric disorders.