Sebastian P. Fernandez

Chronic stress triggers social aversion via glucocorticoid receptor in dopaminoceptive neurons.

Defeat, Distress, and Glucocorticoids Understanding how individuals control emotions and cope with stressful events is a major clinical concern and of importance for the treatment of psychiatric illnesses (see the Perspective by McEwen). Barik et al. (p. 332) discovered that aggressive defeat stress in mice caused glucocortioid release and increased activity in the dopamine system. Deleting the glucocorticoid receptors in dopaminoceptive neurons completely prevented the social avoidance that usually follows aggressive defeat. How the combination of genetic factors and environmental stressors during adolescence determines adult behavior and how their disturbance results in neuropsychiatric disorders is poorly understood. Niwa et al. (p. 335) found that isolation stress during adolescence, which does not cause any long-lasting changes in wild-type mice, induced significant neurochemical and behavioral alterations in mutant mice expressing a dominant-negative variant of the disrupted in schizophrenia 1 gene under the control of the prion protein promoter. These deficits could be reversed by a glucocorticoid receptor antagonist. Aggressive defeat stress in mice causes glucocorticoid release and increased activity in the dopamine system. [Also see Perspective by McEwen] Repeated traumatic events induce long-lasting behavioral changes that are key to organism adaptation and that affect cognitive, emotional, and social behaviors. Rodents subjected to repeated instances of aggression develop enduring social aversion and increased anxiety. Such repeated aggressions trigger a stress response, resulting in glucocorticoid release and activation of the ascending dopamine (DA) system. We bred mice with selective inactivation of the gene encoding the glucocorticoid receptor (GR) along the DA pathway, and exposed them to repeated aggressions. GR in dopaminoceptive but not DA-releasing neurons specifically promoted social aversion as well as dopaminergic neurochemical and electrophysiological neuroadaptations. Anxiety and fear memories remained unaffected. Acute inhibition of the activity of DA-releasing neurons fully restored social interaction in socially defeated wild-type mice. Our data suggest a GR-dependent neuronal dichotomy for the regulation of emotional and social behaviors, and clearly implicate GR as a link between stress resiliency and dopaminergic tone.

A Genetically Defined Morphologically and Functionally Unique Subset of 5-HT Neurons in the Mouse Raphe Nuclei

Heterogeneity of central serotonin (5-HT) raphe neurons is suggested by numerous lines of evidence, but its genetic basis remains elusive. The transcription factor Pet1 is required for the acquisition of serotonergic identity in a majority of neurons in the raphe nuclei. Nevertheless, a subset of 5-HT neurons differentiates in Pet1 knock-out mice. We show here that these residual 5-HT neurons outline a unique subpopulation of raphe neurons with highly selective anatomical targets and characteristic synaptic differentiations. In Pet1 knock-out mice, 5-HT innervation strikingly outlines the brain areas involved in stress responses with dense innervation to the basolateral amygdala, the paraventricular nucleus of the hypothalamus, and the intralaminar thalamic nuclei. In these regions, 5-HT terminals establish asymmetric synaptic junctions. This target selectivity could not be related to altered growth of the remaining 5-HT neurons, as indicated by axon tracing and cell culture analyses. The residual 5-HT axon terminals are functional with maintained release properties in vitro and in vivo. The functional consequence of this uneven distribution of 5-HT innervation on behavior was characterized. Pet1 knock-out mice showed decreased anxiety behavior in novelty exploration and increased fear responses to conditioned aversive cues. Overall, our findings lead us to propose the existence of Pet1-dependent and Pet1-resistant 5-HT neurons targeting different brain centers that might delineate the anatomical basis for a dual serotonergic control on stress responses.

Multiscale single-cell analysis reveals unique phenotypes of raphe 5-HT neurons projecting to the forebrain

Serotonergic neurons of the raphe nuclei exhibit anatomical, neurochemical and elecrophysiological heterogeneity that likely underpins their specific role in multiple behaviors. However, the precise organization of serotonin (5-HT) neurons to orchestrate 5-HT release patterns throughout the brain is not well understood. We compared the electrophysiological and neurochemical properties of dorsal and median raphe 5-HT neurons projecting to the medial prefrontal cortex (mPFC), amygdala (BLA) and dorsal hippocampus (dHP), combining retrograde tract tracing with brain slice electrophysiology and single-cell RT-PCR in Pet1-EGFP mice. Our results show that 5-HT neurons projecting to the dHP and the mPFC and the BLA form largely non-overlapping populations and that BLA-projecting neurons have characteristic excitability and membrane properties. In addition, using an unbiased clustering method that correlates anatomical, molecular and electrophysiological phenotypes, we find that 5-HT neurons with projections to the mPFC and the dHP segregate from those projecting to the BLA. Single-cell gene profiling showed a restricted expression of the peptide galanin in the population of 5-HT neurons projecting to the mPFC. Finally, cluster analysis allowed identifying an atypical subtype of 5-HT neuron with low excitability, long firing delays and preferential expression of the vesicular glutamate transporter type 3. Overall, these findings allow to define correlated anatomical and physiological identities of serotonin raphe neurons that help understanding how discrete raphe cells subpopulations account for the heterogeneous activities of the midbrain serotonergic system.

A Subpopulation of Serotonergic Neurons That Do Not Express the 5‑HT1A Autoreceptor

5-HT neurons are topographically organized in the hindbrain, and have been implicated in the etiology and treatment of psychiatric diseases such as depression and anxiety. Early studies suggested that the raphe 5-HT neurons were a homogeneous population showing similar electrical properties, and feedback inhibition mediated by 5-HT1A autoreceptors. We utilized histochemistry techniques in ePet1-eGFP and 5-HT1A-iCre/R26R mice to show that a subpopulation of 5-HT neurons do not express the somatodendritic 5-HT1A autoreceptor mRNA. In addition, we performed patch-clamp recordings followed by single-cell PCR in ePet1-eGFP mice. From 134 recorded 5-HT neurons located in the dorsal, lateral, and median raphe, we found lack of 5-HT1A mRNA expression in 22 cells, evenly distributed across raphe subfields. We compared the cellular characteristics of these neuronal types and found no difference in passive membrane properties and general excitability. However, when injected with large depolarizing current, 5-HT1A-negative neurons fired more action potentials, suggesting a lack of autoinhibitory action of local 5-HT release. Our results support the hypothesis that the 5-HT system is composed of subpopulations of serotonergic neurons with different capacity for adaptation.

Constitutive and Acquired Serotonin Deficiency Alters Memory and Hippocampal Synaptic Plasticity

Serotonin (5-HT) deficiency occurs in a number of brain disorders that affect cognitive function. However, a direct causal relationship between 5-HT hypo-transmission and memory and underlying mechanisms has not been established. We used mice with a constitutive depletion of 5-HT brain levels (Pet1KO mice) to analyze the contribution of 5-HT to different forms of learning and memory. Pet1KO mice exhibited a striking deficit in novel object recognition memory, a hippocampal-dependent task. No alterations were found in tasks for social recognition, procedural learning, or fear memory. Viral delivery of designer receptors exclusively activated by designer drugs was used to selectively silence the activity of 5-HT neurons in the raphe. Inhibition of 5-HT neurons in the median raphe, but not the dorsal raphe, was sufficient to impair object recognition in adult mice. In vivo electrophysiology in behaving mice showed that long-term potentiation in the hippocampus of 5-HT-deficient mice was altered, and administration of the 5-HT1A agonist 8-OHDPAT rescued the memory deficits. Our data suggest that hyposerotonergia selectively affects declarative hippocampal-dependent memory. Serotonergic projections from the median raphe are necessary to regulate object memory and hippocampal synaptic plasticity processes, through an inhibitory control mediated by 5-HT1A receptors.

Nicotinic receptors mediate stress-nicotine detrimental interplay via dopamine cells’ activity

Epidemiological studies report strong association between mood disorders and tobacco addiction. This high comorbidity requires adequate treatment but the underlying mechanisms are unknown. We demonstrate that nicotine exposure, independent of drug withdrawal effects, increases stress sensitivity, a major risk factor in mood disorders. Nicotine and stress concur to induce long-lasting cellular adaptations within the dopamine (DA) system. This interplay is underpinned by marked remodeling of nicotinic systems, causing increased ventral tegmental area (VTA) DA neurons’ activity and stress-related behaviors, such as social aversion. Blocking β2 or α7 nicotinic acetylcholine receptors (nAChRs) prevents, respectively, the development and the expression of social stress-induced neuroadaptations; conversely, facilitating α7 nAChRs activation specifically in the VTA promotes stress-induced cellular and behavioral maladaptations. Our work unravels a complex nicotine-stress bidirectional interplay and identifies α7 nAChRs as a promising therapeutic target for stress-related psychiatric disorders.

Positive regulation of raphe serotonin neurons by serotonin 2B receptors

Serotonin is a neurotransmitter involved in many psychiatric diseases. In humans, a lack of 5-HT2B receptors is associated with serotonin-dependent phenotypes, including impulsivity and suicidality. A lack of 5-HT2B receptors in mice eliminates the effects of molecules that directly target serotonergic neurons including amphetamine derivative serotonin releasers, and selective serotonin reuptake inhibitor antidepressants. In this work, we tested the hypothesis that 5-HT2B receptors directly and positively regulate raphe serotonin neuron activity. By ex vivo electrophysiological recordings, we report that stimulation by the 5-HT2B receptor agonist, BW723C86, increased the firing frequency of serotonin Pet1-positive neurons. Viral overexpression of 5-HT2B receptors in these neurons increased their excitability. Furthermore, in vivo 5-HT2B-receptor stimulation by BW723C86 counteracted 5-HT1A autoreceptor-dependent reduction in firing rate and hypothermic response in wild-type mice. By a conditional genetic ablation that eliminates 5-HT2B receptor expression specifically and exclusively from Pet1-positive serotonin neurons (Htr2b5-HTKO mice), we demonstrated that behavioral and sensitizing effects of MDMA (3,4-methylenedioxy-methamphetamine), as well as acute behavioral and chronic neurogenic effects of the antidepressant fluoxetine, require 5-HT2B receptor expression in serotonergic neurons. In Htr2b5-HTKO mice, dorsal raphe serotonin neurons displayed a lower firing frequency compared to control Htr2blox/lox mice as assessed by in vivo extracellular recordings and a stronger hypothermic effect of 5-HT1A-autoreceptor stimulation was observed. The increase in head-twitch response to DOI (2,5-dimethoxy-4-iodoamphetamine) further confirmed the lower serotonergic tone resulting from the absence of 5-HT2B receptors in serotonin neurons. Together, these observations indicate that the 5-HT2B receptor acts as a direct positive modulator of serotonin Pet1-positive neurons in an opposite way as the known 5-HT1A-negative autoreceptor.

Serotonin 2B Receptors in Mesoaccumbens Dopamine Pathway Regulate Cocaine Responses

Addiction is a maladaptive pattern of behavior following repeated use of reinforcing drugs in predisposed individuals, leading to lifelong changes. Common among these changes are alterations of neurons releasing dopamine in the ventral and dorsal territories of the striatum. The serotonin 5-HT2B receptor has been involved in various behaviors, including impulsivity, response to antidepressants, and response to psychostimulants, pointing toward putative interactions with the dopamine system. Despite these findings, it remains unknown whether 5-HT2B receptors directly modulate dopaminergic activity and the possible mechanisms involved. To answer these questions, we investigated the contribution of 5-HT2B receptors to cocaine-dependent behavioral responses. Male mice permanently lacking 5-HT2B receptors, even restricted to dopamine neurons, developed heightened cocaine-induced locomotor responses. Retrograde tracing combined with single-cell mRNA amplification indicated that 5-HT2B receptors are expressed by mesolimbic dopamine neurons. In vivo and ex vivo electrophysiological recordings showed that 5-HT2B-receptor inactivation in dopamine neurons affects their neuronal activity and increases AMPA-mediated over NMDA-mediated excitatory synaptic currents. These changes are associated with lower ventral striatum dopamine activity and blunted cocaine self-administration. These data identify the 5-HT2B receptor as a pharmacological intermediate and provide mechanistic insight into attenuated dopamine tone following exposure to drugs of abuse. SIGNIFICANCE STATEMENT Here we report that mice lacking 5-HT2B receptors totally or exclusively in dopamine neurons exhibit heightened cocaine-induced locomotor responses. Despite the sensitized state of these mice, we found that associated changes include lower ventral striatum dopamine activity and lower cocaine operant self-administration. We described the selective expression of 5-HT2B receptors in a subpopulation of dopamine neurons sending axons to the ventral striatum. Increased bursting in vivo properties of these dopamine neurons and a concomitant increase in AMPA synaptic transmission to ex vivo dopamine neurons were found in mice lacking 5-HT2B receptors. These data support the idea that the chronic 5-HT2B-receptor inhibition makes mice behave like animals already exposed to cocaine with higher cocaine-induced locomotion associated with changes in dopamine neuron reactivity.