Selective refinement of glutamate and GABA synapses on dorsal raphe 5-HT neurons during postnatal life.

Serotonin (5-hydroxytryptamine, 5-HT) neurons are implicated in the etiology and therapeutics of anxiety and depression. Critical periods of vulnerability during brain development enable maladaptive mechanisms to produce detrimental consequences on adult mood and emotional responses. 5-HT plays a crucial role in these mechanisms; however, little is known about how synaptic inputs and modulatory systems that shape the activity of early 5-HT networks mature during postnatal development. We investigated in mice the postnatal trajectory of glutamate and GABA synaptic inputs to dorsal raphe nucleus (DRN) 5-HT neurons, the main source of forebrain 5-HT. High-resolution quantitative analyses with array tomography and ex vivo electrophysiology indicate that cortical glutamate and subcortical GABA synapses undergo a profound refinement process after the third postnatal week, whereas subcortical glutamate inputs do not. This refinement of DRN inputs is not accompanied by changes in 5-HT1A receptor-mediated inhibition over 5-HT neurons. Our study reveals a precise developmental pattern of synaptic refinement of DRN excitatory and inhibitory afferents, when 5-HT-related inhibitory mechanisms are in place. These findings contribute to the understanding of neurodevelopmental vulnerability to psychiatric disorders. This article has an associated 'The people behind the papers' interview.

Tau reduction with artificial microRNAs modulates neuronal physiology and improves tauopathy phenotypes in mice.

Abnormal tau accumulation is the hallmark of several neurodegenerative diseases, named tauopathies. Strategies aimed at reducing tau in the brain are promising therapeutic interventions, yet more precise therapies would require targeting specific nuclei and neuronal subpopulations affected by disease while avoiding global reduction of physiological tau. Here, we developed artificial microRNAs directed against the human MAPT mRNA to dwindle tau protein by engaging the endogenous RNA interference pathway. In human differentiated neurons in culture, microRNA-mediated tau reduction diminished neuronal firing without affecting neuronal morphology or impairing axonal transport. In the htau mouse model of tauopathy, we locally expressed artificial microRNAs in the prefrontal cortex (PFC), an area particularly vulnerable to initiating tau pathology in this model. Tau knockdown prevented the accumulation of insoluble and hyperphosphorylated tau, modulated firing activity of putative pyramidal neurons, and improved glucose uptake in the PFC. Moreover, such tau reduction prevented cognitive decline in aged htau mice. Our results suggest target engagement of designed tau-microRNAs to effectively reduce tau pathology, providing a proof of concept for a potential therapeutic approach based on local tau knockdown to rescue tauopathy-related phenotypes.

Tau mis-splicing correlates with motor impairments and striatal dysfunction in a model of tauopathy.

Tauopathies are neurodegenerative diseases caused by the abnormal metabolism of the microtubule associated protein tau (MAPT), which is highly expressed in neurons and critically involved in microtubule dynamics. In the adult human brain, the alternative splicing of exon 10 in MAPT pre-mRNA produces equal amounts of protein isoforms with either three (3R) or four (4R) microtubule binding domains. Imbalance in the 3R:4R tau ratio is associated with primary tauopathies that develop atypical parkinsonism, such as progressive supranuclear palsy and corticobasal degeneration. Yet, the development of effective therapies for those pathologies is an unmet goal. Here we report motor coordination impairments in the htau mouse model of tauopathy which harbour abnormal 3R:4R tau isoforms content, and in contrast to TauKO mice, are unresponsive to l-DOPA. Preclinical-PET imaging, array tomography and electrophysiological analyses indicated the dorsal striatum as the candidate structure mediating such phenotypes. Indeed, local modulation of tau isoforms by RNA trans-splicing in the striata of adult htau mice, prevented motor coordination deficits and restored basal neuronal firing. Together, these results suggest that abnormal striatal tau isoform content might lead to parkinsonian-like phenotypes and demonstrate a proof of concept that modulation of tau mis-splicing is a plausible disease-modifying therapy for some primary tauopathies.

Presynaptic gating of excitation in the dorsal raphe nucleus by GABA.

The dorsal raphe nucleus (DR) controls forebrain serotonin neurotransmission to influence emotional states. GABA neurotransmission in the DR has been implicated in regulating sleep/wake states and influencing anxiety and aggression. To gain insight into how GABA regulates DR activity, we analyzed the organization of both GABA and glutamate axons in the rat DR using a high-resolution immunofluorescence technique, array tomography, as well as EM. This analysis revealed that a third or more of GABA-containing axons are organized in synaptic triads with a glutamatergic axon and a common postsynaptic target. Electrophysiological recordings showed that GABA has the capacity to presynaptically gate glutamate release in the DR through a combination of GABA-A and GABA-B receptor-mediated effects. Thus, GABA-glutamate synaptic triads are a common feature of the network architecture of the DR with the potential to regulate excitation of the nucleus.

Transcriptional preservation of serotonergic connectivity may shed light on neurodegeneration.

In a recent study, Kitt, Tabuchi, and colleagues unveiled a novel function of an early-stage transcriptional network to maintain the adult integrity of serotonergic connectivity. Reported axonal and synaptic morphological alterations in serotonin (5-HT) neurons after selective inactivation of Lmx1b/Pet1 transcriptional networks may help to understand aging and neurodegenerative processes.

Stress-driven potentiation of lateral hypothalamic synapses onto ventral tegmental area dopamine neurons causes increased consumption of palatable food.

Stress can cause overconsumption of palatable high caloric food. Despite the important role of stress eating in obesity and (binge) eating disorders, its underlying neural mechanisms remain unclear. Here we demonstrate in mice that stress alters lateral hypothalamic area (LHA) control over the ventral tegmental area (VTA), thereby promoting overconsumption of palatable food. Specifically, we show that glutamatergic LHA neurons projecting to the VTA are activated by social stress, after which their synapses onto dopamine neurons are potentiated via AMPA receptor subunit alterations. We find that stress-driven strengthening of these specific synapses increases LHA control over dopamine output in key target areas like the prefrontal cortex. Finally, we demonstrate that while inducing LHA-VTA glutamatergic potentiation increases palatable fat intake, reducing stress-driven potentiation of this connection prevents such stress eating. Overall, this study provides insights in the neural circuit adaptations caused by stress that drive overconsumption of palatable food.

Novel role of the synaptic scaffold protein Dlgap4 in ventricular surface integrity and neuronal migration during cortical development.

Subcortical heterotopias are malformations associated with epilepsy and intellectual disability, characterized by the presence of ectopic neurons in the white matter. Mouse and human heterotopia mutations were identified in the microtubule-binding protein Echinoderm microtubule-associated protein-like 1, EML1. Further exploring pathological mechanisms, we identified a patient with an EML1-like phenotype and a novel genetic variation in DLGAP4. The protein belongs to a membrane-associated guanylate kinase family known to function in glutamate synapses. We showed that DLGAP4 is strongly expressed in the mouse ventricular zone (VZ) from early corticogenesis, and interacts with key VZ proteins including EML1. In utero electroporation of Dlgap4 knockdown (KD) and overexpression constructs revealed a ventricular surface phenotype including changes in progenitor cell dynamics, morphology, proliferation and neuronal migration defects. The Dlgap4 KD phenotype was rescued by wild-type but not mutant DLGAP4. Dlgap4 is required for the organization of radial glial cell adherens junction components and actin cytoskeleton dynamics at the apical domain, as well as during neuronal migration. Finally, Dlgap4 heterozygous knockout (KO) mice also show developmental defects in the dorsal telencephalon. We hence identify a synapse-related scaffold protein with pleiotropic functions, influencing the integrity of the developing cerebral cortex.

Serotonin-related rodent models of early-life exposure relevant for neurodevelopmental vulnerability to psychiatric disorders.

Mental disorders including depression and anxiety are continuously rising their prevalence across the globe. Early-life experience of individuals emerges as a main risk factor contributing to the developmental vulnerability to psychiatric disorders. That is, perturbing environmental conditions during neurodevelopmental stages can have detrimental effects on adult mood and emotional responses. However, the possible maladaptive neural mechanisms contributing to such psychopathological phenomenon still remain poorly understood. In this review, we explore preclinical rodent models of developmental vulnerability to psychiatric disorders, focusing on the impact of early-life environmental perturbations on behavioral aspects relevant to stress-related and psychiatric disorders. We limit our analysis to well-established models in which alterations in the serotonin (5-HT) system appear to have a crucial role in the pathophysiological mechanisms. We analyze long-term behavioral outcomes produced by early-life exposures to stress and psychotropic drugs such as the selective 5-HT reuptake inhibitor (SSRI) antidepressants or the anticonvulsant valproic acid (VPA). We perform a comparative analysis, identifying differences and commonalities in the behavioral effects produced in these models. Furthermore, this review discusses recent advances on neurodevelopmental substrates engaged in these behavioral effects, emphasizing the possible existence of maladaptive mechanisms that could be shared by the different models.

Dorsal raphe serotonin neurotransmission is required for the expression of nursing behavior and for pup survival.

Proper maternal care is an essential factor of reproductive success in mammals, involving a repertoire of behaviors oriented toward the feeding and care of the offspring. Among the neurotransmitters involved in the initiation of these behaviors, serotonin (5-HT) seems to play an important role. Here we compared pup-oriented maternal behaviors in mice with constitutive 5-HT depletion, the tryptophan hydroxylase 2-knock-out (Tph2-KO) and the Pet1-KO mice. We report that the only common pup-oriented defect in these 2 hyposerotoninergic models is a defective nursing in parturient mice and altered nursing-like (crouching) behavior in virgin mice, while pup retrieval defects are only present in Tph2-KO. Despite a normal mammary gland development and milk production, the defect in appropriate nursing is responsible for severe growth retardation and early lethality of pups born to hyposerotonergic dams. This nursing defect is due to acute rather constitutive 5-HT depletion, as it is reproduced by adult knockdown of Tph2 in the dorsal raphe nucleus in mothers with a prior normal maternal experience. We conclude that 5-HT innervation from the dorsal raphe is required for both the initiation and maintenance of a normal nursing behavior. Our findings may be related to observations of reduced maternal/infant interactions in human depression.

Stress undermines reward-guided cognitive performance through synaptic depression in the lateral habenula.

Weighing alternatives during reward pursuit is a vital cognitive computation that, when disrupted by stress, yields aspects of neuropsychiatric disorders. To examine the neural mechanisms underlying these phenomena, we employed a behavioral task in which mice were confronted by a reward and its omission (i.e., error). The experience of error outcomes engaged neuronal dynamics within the lateral habenula (LHb), a subcortical structure that supports appetitive behaviors and is susceptible to stress. A high incidence of errors predicted low strength of habenular excitatory synapses. Accordingly, stressful experiences increased error choices while decreasing glutamatergic neurotransmission onto LHb neurons. This synaptic adaptation required a reduction in postsynaptic AMPA receptors (AMPARs), irrespective of the anatomical source of glutamate. Bidirectional control of habenular AMPAR transmission recapitulated and averted stress-driven cognitive deficits. Thus, a subcortical synaptic mechanism vulnerable to stress underlies behavioral efficiency during cognitive performance.

Implication of 5-HT7 receptor in prefrontal circuit assembly and detrimental emotional effects of SSRIs during development.

Altered development of prefrontal cortex (PFC) circuits can have long-term consequences on adult emotional behavior. Changes in serotonin homeostasis during critical periods produced by genetic or pharmacological inactivation of the serotonin transporter (SERT, or Slc6a4), have been involved in such developmental effects. In mice, selective serotonin reuptake inhibitors (SSRIs), administered during postnatal development cause exuberant synaptic connectivity of the PFC to brainstem dorsal raphe nucleus (DRN) circuits, and increase adult risk for developing anxiety and depressive symptoms. SERT is transiently expressed in the glutamate neurons of the mouse PFC, that project to the DRN. Here, we find that 5-HTR7 is transiently co-expressed with SERT by PFC neurons, and it plays a key role in the maturation of PFC-to-DRN synaptic circuits during early postnatal life. 5-HTR7-KO mice show reduced PFC-to-DRN synaptic density (as measured by array-tomography and VGLUT1/synapsin immunocytochemistry). Conversely, 5-HTR7 over-expression in the developing PFC increased PFC-to-DRN synaptic density. Long-term consequences on depressive-like and anxiogenic behaviors were observed in adults. 5-HTR7 over-expression in the developing PFC, results in depressive-like symptoms in adulthood. Importantly, the long-term depressive-like and anxiogenic effects of SSRIs (postnatal administration of fluoxetine from P2 to P14) were not observed in 5-HTR7-KO mice, and were prevented by co-administration of the selective inhibitor of 5-HTR7, SB269970. This study identifies a new role 5-HTR7 in the postnatal maturation of prefrontal descending circuits. Furthermore, it shows that 5-HTR7 in the PFC is crucially required for the detrimental emotional effects caused by SSRI exposure during early postnatal life.

Author Correction: Shifted pallidal co-release of GABA and glutamate in habenula drives cocaine withdrawal and relapse.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Serotonin Subsystems Modulate Diverse and Opposite Behavioral Functions.

Pioneering work showed that serotonin (5-HT) neurons have the unique capacity to engage in different and opposed aspects of motivated behaviors such as reward and punishment responses. These findings provided strong evidence about the functional heterogeneity of 5-HT neurons, and their possible engagement in multiple and behaviorally distinct neural subsystems. A recent study provides further compelling evidence supporting this notion, in which two ascending 5-HT circuits modulate opposed aspects of motivated behaviors.

Mesopontine cholinergic inputs to midbrain dopamine neurons drive stress-induced depressive-like behaviors.

Stressful life events are primary environmental factors that markedly contribute to depression by triggering brain cellular maladaptations. Dysregulation of ventral tegmental area (VTA) dopamine neurons has been causally linked to the appearance of social withdrawal and anhedonia, two classical manifestations of depression. However, the relevant inputs that shape these dopamine signals remain largely unknown. We demonstrate that chronic social defeat (CSD) stress, a preclinical paradigm of depression, causes marked hyperactivity of laterodorsal tegmentum (LDTg) excitatory neurons that project to the VTA. Selective chemogenetic-mediated inhibition of cholinergic LDTg neurons prevent CSD-induced VTA DA neurons dysregulation and depressive-like behaviors. Pro-depressant outcomes are replicated by pairing activation of LDTg cholinergic terminals in the VTA with a moderate stress. Prevention of CSD outcomes are recapitulated by blocking corticotropin-releasing factor receptor 1 within the LDTg. These data uncover a neuro-circuitry of depressive-like disorders and demonstrate that stress, via a neuroendocrine signal, profoundly dysregulates the LDTg.

Refining the Role of 5-HT in Postnatal Development of Brain Circuits.

Changing serotonin (5-hydroxytryptamine, 5-HT) brain levels during critical periods in development has long-lasting effects on brain function, particularly on later anxiety/depression-related behaviors in adulthood. A large part of the known developmental effects of 5-HT occur during critical periods of postnatal life, when activity-dependent mechanisms remodel neural circuits. This was first demonstrated for the maturation of sensory brain maps in the barrel cortex and the visual system. More recently this has been extended to the 5-HT raphe circuits themselves and to limbic circuits. Recent studies overviewed here used new genetic models in mice and rats and combined physiological and structural approaches to provide new insights on the cellular and molecular mechanisms controlled by 5-HT during late stages of neural circuit maturation in the raphe projections, the somatosensory cortex and the visual system. Similar mechanisms appear to be also involved in the maturation of limbic circuits such as prefrontal circuits. The latter are of particular relevance to understand the impact of transient 5-HT dysfunction during postnatal life on psychiatric illnesses and emotional disorders in adult life.

Neural circuit adaptations during drug withdrawal - Spotlight on the lateral habenula.

Withdrawal after drug intake triggers a wealth of affective states including negative feelings reminiscent of depressive symptoms. This negative state can ultimately be crucial for relapse, a hallmark of addiction. Adaptations in a wide number of neuronal circuits underlie aspects of drug withdrawal, however causality between cellular modifications within these systems and precise behavioral phenotypes remains poorly described. Recent advances point to an instrumental role of the lateral habenula in driving depressive-like states during drug withdrawal. In this review we will discuss the general behavioral features of drug withdrawal, the importance of plasticity mechanisms in the mesolimbic systems, and the latest discoveries highlighting the implications of lateral habenula in drug addiction. We will further stress how specific interventions in the lateral habenula efficiently ameliorate depressive symptoms. Altogether, this work aims to provide a general knowledge on the cellular and circuit basis underlying drug withdrawal, ultimately speculating on potential treatment for precise aspects of addiction.

Shifted pallidal co-release of GABA and glutamate in habenula drives cocaine withdrawal and relapse.

Cocaine withdrawal produces aversive states and vulnerability to relapse, hallmarks of addiction. The lateral habenula (LHb) encodes negative stimuli and contributes to aversive withdrawal symptoms. However, it remains unclear which inputs to LHb promote this and what the consequences are for relapse susceptibility. We report, using rabies-based retrolabeling and optogenetic mapping, that the entopeduncular nucleus (EPN, the mouse equivalent of the globus pallidus interna) projects to an LHb neuronal subset innervating aversion-encoding midbrain GABA neurons. EPN-to-LHb excitatory signaling is limited by GABAergic cotransmission. This inhibitory component decreases during cocaine withdrawal as a result of reduced presynaptic vesicular GABA transporter (VGAT). This shifts the EPN-to-LHb GABA/glutamate balance, disinhibiting EPN-driven LHb activity. Selective virally mediated VGAT overexpression at EPN-to-LHb terminals during withdrawal normalizes GABAergic neurotransmission. This intervention rescues cocaine-evoked aversive states and prevents stress-induced reinstatement, used to model relapse. This identifies diminished inhibitory transmission at EPN-to-LHb GABA/glutamate synapses as a mechanism contributing to the relapsing feature of addictive behavior.

Conditional anterograde tracing reveals distinct targeting of individual serotonin cell groups (B5-B9) to the forebrain and brainstem.

Serotoninergic innervation of the central nervous system is provided by hindbrain raphe nuclei (B1-B9). The extent to which each raphe subdivision has distinct topographic organization of their projections is still unclear. We provide a comprehensive description of the main targets of the rostral serotonin (5-HT) raphe subgroups (B5-B9) in the mouse brain. Adeno-associated viruses that conditionally express GFP under the control of the 5-HT transporter promoter were used to label small groups of 5-HT neurons in the dorsal (B7d), ventral (B7v), lateral (B7l), and caudal (B6) subcomponents of the dorsal raphe (DR) nucleus as well as in the rostral and caudal parts of the median raphe (MR) nucleus (B8 and B5, respectively), and in the supralemniscal (B9) cell group. We illustrate the distinctive and largely non-overlapping projection areas of these cell groups: for instance, DR (B7) projects to basal parts of the forebrain, such as the amygdala, whereas MR (B8) is the main 5-HT source to the hippocampus, septum, and mesopontine tegmental nuclei. Distinct subsets of B7 have preferential brain targets: B7v is the main source of 5-HT for the cortex and amygdala while B7d innervates the hypothalamus. We reveal for the first time the target areas of the B9 cell group, demonstrating projections to the caudate, prefrontal cortex, substantia nigra, locus coeruleus and to the raphe cell groups. The broad topographic organization of the different raphe subnuclei is likely to underlie the different functional roles in which 5-HT has been implicated in the brain. The present mapping study could serve as the basis for genetically driven specific targeting of the different subcomponents of the mouse raphe system.

Enhanced 5-HT1A receptor-dependent feedback control over dorsal raphe serotonin neurons in the SERT knockout mouse.

5-HT1A receptors are widely expressed in the brain and play a critical role in feedback inhibition of serotonin (5-HT) neurons through multiple mechanisms. Yet, it remains poorly understood how these feedback mechanisms, particularly those involving long-range projections, adapt in mood disorders. Here, we examined several aspects of 5-HT1A receptor function in the 5-HT transporter knockout mouse (SERT-KO), a model of vulnerability to stress and mood disorders. We found that in comparison to wild-type (WT) mice, SERT-KO mice had more passive coping in response to acute swim stress and this was accompanied by hypo-activation of medial prefrontal cortex (mPFC) Fos expression. Both of these effects were reversed by systemically blocking 5-HT1A receptors. Ex-vivo electrophysiological experiments showed that 5-HT exerted greater 5-HT1A-mediated inhibitory effects in the mPFC of SERT-KO mice compared to WT. Since 5-HT1A receptors in the mPFC provide a key feedback regulation of the dorsal raphe nucleus (DRN), we used a disinhibition strategy to examined endogenous feedback control of 5-HT neurons. Blocking 5-HT1A receptors disinhibited several fold more 5-HT neurons in the DRN of SERT-KO than in WT mice, revealing the presence of enhanced feedback inhibition of 5-HT neurons in the SERT-KO. Taken together our results indicate that increased stress sensitivity in the SERT-KO is associated with the enhanced capacity of 5-HT1A receptors to inhibit neurons in the mPFC as well as to exert feedback inhibition of DRN 5-HT neurons.