Contribution of Noradrenaline, Serotonin, and the Basolateral Amygdala to Alcohol Addiction: Implications for Novel Pharmacotherapies for AUDs

Alcohol use disorders (AUDs) constitute one of the 10 leading causes of preventable deaths worldwide. To date, there are only a few Food and Drug Administration (FDA)‐approved medications for AUDs, all of which are only moderately effective. The development of improved and effective strategies for the management of AUDs is greatly needed. This review focuses on understanding the neurobiological basis of alcohol addiction with a special emphasis on the role of serotonin (5‐hydroxytryptamine, 5‐HT) and noradrenaline (NE) in AUDs and sheds light on their complex interplay in the basolateral amygdala (BLA)––a brain region widely implicated in addiction. There is a significant evidence to support the role of the amygdala in stress‐induced negative emotional states resulting from withdrawal from alcohol; in fact, it has been hypothesized that this leads to craving and relapse. Dysregulation of 5‐HT and NE signaling in the BLA have been proposed to alter affective behavior, memory consolidation, and most importantly increase the propensity for addiction to alcohol and other common drugs of abuse. Improving deficits in 5‐HT and NE receptor signaling may provide ideal targets for the treatment of AUDs

Acute Ethanol Administration Upregulates Synaptic α4-Subunit of Neuronal Nicotinic Acetylcholine Receptors within the Nucleus Accumbens and Amygdala

Alcohol and nicotine are two of the most frequently abused drugs, with their comorbidity well described. Previous data show that chronic exposure to nicotine upregulates high-affinity nicotinic acetylcholine receptors (nAChRs) in several brain areas. Effects of ethanol on specific brain nAChR subtypes within the mesolimbic dopaminergic (DA) pathway may be a key element in the comorbidity of ethanol and nicotine. However, it is unknown how alcohol affects the abundance of these receptor proteins. In the present study, we measured the effect of acute binge ethanol on nAChR α4 subunit levels in the prefrontal cortex (PFC), nucleus accumbens (NAc), ventral tegmental area (VTA), and amygdala (Amg) by western blot analysis using a knock-in mouse line, generated with a normally functioning α4 nAChR subunit tagged with yellow fluorescent protein (YFP). We observed a robust increase in α4-YFP subunit levels in the NAc and the Amg following acute ethanol, with no changes in the PFC and VTA. To further investigate whether this upregulation was mediated by increased local mRNA transcription, we quantified mRNA levels of the Chrna4 gene using qRT-PCR. We found no effect of ethanol on α4 mRNA expression, suggesting that the upregulation of α4 protein rather occurs post-translationally. The quantitative counting of YFP immunoreactive puncta further revealed that α4-YFP protein is upregulated in presynaptic boutons of the dopaminergic axons projecting to the shell and the core regions of the NAc as well as to the basolateral amygdala (BLA), but not to the central or lateral Amg. Together, our results demonstrate that a single exposure to binge ethanol upregulates level of synaptic α4∗ nAChRs in dopaminergic inputs to the NAc and BLA. This upregulation could be linked to the functional dysregulation of dopaminergic signalling observed during the development of alcohol dependence

Axonal Non-segregation of the Vesicular Glutamate Transporter VGLUT3 Within Serotonergic Projections in the Mouse Forebrain

A subpopulation of raphe 5-HT neurons expresses the vesicular glutamate transporter VGLUT3 with the co-release of glutamate and serotonin proposed to play a pivotal role in encoding reward- and anxiety-related behaviors. Serotonin axons are identifiable by immunolabeling of either serotonin (5-HT) or the plasma membrane 5-HT transporter (SERT), with SERT labeling demonstrated to be only partially overlapping with 5-HT staining. Studies investigating the colocalization or segregation of VGLUT3 within SERT or 5-HT immunolabeled boutons have led to inconsistent results. Therefore, we combined immunohistochemistry, high resolution confocal imaging, and 3D-reconstruction techniques to map and quantify the distribution of VGLUT3 immunoreactive boutons within 5-HT vs. SERT-positive axons in various regions of the mouse forebrain, including the prefrontal cortex, nucleus accumbens core and shell, bed nucleus of the stria terminalis, dorsal striatum, lateral septum, basolateral and central amygdala, and hippocampus. Our results demonstrate that about 90% of 5-HT boutons are colocalized with SERT in almost all the brain regions studied, which therefore reveals that VGLUT3 and SERT do not segregate. However, in the posterior part of the NAC shell, we confirmed the presence of a subtype of 5-HT immunoreactive axons that lack the SERT. Interestingly, about 90% of the 5-HT/VGLUT3 boutons were labeled for the SERT in this region, suggesting that VGLUT3 is preferentially located in SERT immunoreactive 5-HT boutons. This work demonstrates that VGLUT3 and SERT cannot be used as specific markers to classify the different subtypes of 5-HT axons

Contextual Fear Conditioning Alter Microglia Number and Morphology in the Rat Dorsal Hippocampus

Contextual fear conditioning is a Pavlovian conditioning paradigm capable of rapidly creating fear memories to contexts, such as rooms or chambers. Contextual fear conditioning protocols have long been utilized to evaluate how fear memories are consolidated, maintained, expressed, recalled, and extinguished within the brain. These studies have identified the lateral portion of the amygdala and the dorsal portion of the hippocampus as essential for contextual fear memory consolidation. The current study was designed to evaluate how two different contextual fear memories alter amygdala and hippocampus microglia, brain derived neurotrophic factor (BDNF), and phosphorylated cyclic-AMP response element binding (pCREB). We find rats provided with standard contextual fear conditioning to have more microglia and more cells expressing BDNF in the dentate gyrus as compared to a context only control group. Additionally, standard contextual fear conditioning altered microglia morphology to become amoeboid in shape – a common response to central nervous system insult, such as traumatic brain injury, infection, ischemia, and more. The unpaired fear conditioning procedure (whereby non-reinforced and non-overlapping auditory tones were provided at random intervals during conditioning), despite producing equivalent levels of fear as the standard procedure, did not alter microglia, BDNF or pCREB number in any dorsal hippocampus or lateral amygdala brain regions. Despite this, the unpaired fear conditioning protocol produced some alterations in microglia morphology, but less compared to rats provided with standard contextual fear conditioning. Results from this study demonstrate that contextual fear conditioning is capable of producing large alterations to dentate gyrus plasticity and microglia, whereas unpaired fear conditioning only produces minor changes to microglia morphology. These data show, for the first time, that Pavlovian fear conditioning protocols can induce similar responses as trauma, infection or other insults within the central nervous system

Rôle des récepteurs 5-HT2B

Le rôle du récepteur 5-HT2B dans le système nerveux central est encore méconnu. En invalidant ce récepteur totalement, spécifiquement dans les neurones sérotoninergiques ou spécifiquement dans les neurones dopaminergiques, nous avons mis en évidence un rôle essentiel des récepteurs 5-HT2B présynaptiques et postsynaptiques. Les récepteurs 5-HT2B présynaptiques apparaissent être nécessaires aux effets biochimiques et comportementaux des molécules dérivées d amphétamine et des antidépresseurs qui agissent sur le transporteur de la sérotonine (SERT). L étude de la régulation du SERT par les récepteurs 5-HT2B dans l action de ces molécules a identifié deux sites potentiels de phosphorylation impliqués dans les mécanismes de recapture ou d efflux de sérotonine. Ce travail a également mis en évidence que les récepteurs 5-HT2B postsynaptiques, localisés sur les neurones dopaminergiques de l aire tegmentale ventrale, modulent l activité de ces neurones dans les mécanismes à l origine de l impulsivité et de l addiction. Ainsi, deux polymorphismes perte de fonction ont été identifiés chez l homme et associés à l impulsivité d une part et à l addiction d autre part. Enfin, l étude structurelle des récepteurs 5-HT2B a révélé un rôle allostérique important de l extrémité N-terminale dans la fonction de ce récepteur. Un double polymorphisme dans l extrémité N-terminale, associé à une vulnérabilité à l addiction, s est ainsi révélé être un gain de fonction du récepteur 5-HT2B. L identification d un rôle prépondérant du récepteur 5-HT2B dans les interactions sérotonine/dopamine ouvre de nouvelles perspectives dans l étude de la dépression, l impulsivité et l addictionThe role of the 5-HT2B receptor in the central nervous system is still unclear. By invalidating this receptor totally, specifically in serotonergic neurons or specifically in dopaminergic neurons, we have demonstrated an essential role of both presynaptic and postsynaptic 5-HT2B receptors. The presynaptic 5-HT2B receptors appear to be necessary to the biochemical and behavioral effects of amphetamine-derived molecules and antidepressants that act on the serotonin transporter (SERT). The study of the regulation of SERT by 5-HT2B receptors in the action of these molecules identified two potential phosphorylation sites involved in the mechanisms of serotonin uptake or efflux. This work also demonstrated that postsynaptic 5-HT2B receptors, located on dopaminergic neurons of the VTA, modulate the activity of these neurons in the mechanisms underlying impulsivity and addiction. Thus, two loss-of-function polymorphisms have been identified in humans and associated with impulsivity in the one hand and with addiction in the other. Finally, the structural study of 5-HT2B receptors revealed an important allosteric role of the N-terminus in the function of this receptor. A double polymorphism in the N-terminus, associated with a vulnerability to addiction, has been caracterised as a gain-of-function of the 5-HT2B receptor. The identification of a role of 5-HT2B receptor in serotonin / dopamine interactions opens new perspectives in the study of depression, impulsivity and addictionPARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF

5-HTT

The first evidence of an active transport of serotonin (5-hydroxytryptamine, 5-HT) came from pioneer studies in the late 1950s showing an uptake of 5-HT into the blood platelets from rabbit or guinea pig. A few years later, a similar mechanism was observed in the rat brain (Schanberg 1963). The mechanism of this transport, involving the sodium and potassium ionic gradients, was rapidly elucidated, and the existence of a dedicated transporter protein postulated. In addition, the antidepressant imipramine was shown to bind to this putative transporter to reduce 5-HT uptake into the blood platelet, and, therefore, the serotonin transporter was named the “serotonin transporter-imipramine receptor.” Using the binding properties of imipramine to the serotonin transporter, Jane Talvenheimo and Gary Rudnick have purified the transporter protein from porcine blood platelet preparations (Talvenheimo and Rudnick 1980). Following that, studies have shown that most of the antidepressant drugs bind to the serotonin transporter and inhibit 5-HT uptake, further supporting the 5-HT hypothesis of depression. Since the early 1990s, the gene encoding the serotonin transporter (5-HTT) was identified in rat, human, drosophila, mouse, guinea pig, and cow..

Acute alcohol exposure alters the distribution Of alpha4 nicotinic receptors in the mouse brain

A growing body of evidence suggests that nicotinic acetylcholine receptors are important mediators of the effects of alcohol and represent significant pharmacotherapeutic targets for the treatment of alcohol use disorders (AUDs). In particular, the nicotinic alpha4 subunit has been shown to control the brain reward pathway and modulate its response to alcohol. Here we present evidence that a single exposure to alcohol is sufficient to elicit a profound redistribution of alpha4 subunits within the mouse brain reward circuit, including prefrontal-subcortical circuits. Using intraperitoneal bolus injection of a sedative dose of ethanol (3.6 g/kg) in alpha4-YFP transgenic mice, we have quantified the changes in the expression of nicotinic alpha4 subunits by using a combination of western blots and immunohistological techniques combined with 3D reconstruction. Our results show that 24 hr after a single ethanol injection, the expression of alpha4-containing nicotinic receptors is significantly upregulated in the nucleus accumbens (217%) and the amygdala (175%) but not in the prefrontal cortex. This data suggests that acute alcohol exposure alters alpha4 nicotinic receptor signalling principally in the mesolimbic pathway which in turn, might modulate the activity of several neuronal pathways in this brain region

SOMATO-DENDRITIC REGULATION OF RAPHE SEROTONIN NEURONS; A KEY TO ANTIDEPRESSANT ACTION. Serotonin volume transmission

International audienceSeveral lines of evidence implicate serotonin (5-hydroxytryptamine, 5-HT) in regulating personality traits and mood control. Serotonergic neurons are classically thought to be tonic regular-firing, 'clock-like' neurons. Neurotransmission by serotonin is tightly regulated by the serotonin transporter (SERT) and by autoreceptors (serotonin receptors expressed by serotonin neurons) through negative feedback inhibition at the cell bodies and dendrites (5-HT 1A receptors) of the dorsal raphe nuclei or at the axon terminals (5-HT 1B receptors). In dorsal raphe neurons, the release of serotonin from vesicles in the soma, dendrites, and/or axonal varicosities is independent of classical synapses and can be induced by neuron depolarization, by the stimulation of L-type calcium channels, by activation of glutamatergic receptors, and/or by activation of 5-HT 2 receptors. The resulting serotonin release displays a slow kinetic and a large diffusion. This process called volume transmission may ultimately affect the rate of discharge of serotonergic neurons, and their tonic activity. The therapeutic effects induced by serotonin-selective reuptake inhibitor (SSRI) antidepressants are initially triggered by blocking SERT but rely on consequences of chronic exposure, i.e. a selective desensitization of somatodendritic 5-HT 1A autoreceptors. Agonist stimulation of 5-HT 2B receptors mimicked behavioral and neurogenic SSRI actions, and increased extracellular serotonin in dorsal raphe. By contrast, a lack of effects of SSRIs was observed in the absence of 5-HT 2B receptors (knockout-KO), even restricted to serotonergic neurons (Htr2b 5-HTKO mice). The absence of 5-HT 2B receptors in serotonergic neurons is associated with a higher 5-HT 1A-autoreceptor reactivity and thus a lower firing activity of these neurons. In agreement, mice with overexpression of 5-HT 1A autoreceptor show decreased neuronal activity and increased depression-like behavior that is resistant to SSRI treatment. We propose thus that the serotonergic tone results from the opposite control exerted by somatodendritic (Gi-coupled) 5-HT 1A and (Gq-coupled) 5-HT 2B receptors on dorsal raphe neurons. Therefore, 5-HT 2B receptors may contribute to SSRI therapeutic effects by their positive regulation of adult raphe serotonergic neurons. Deciphering the molecular mechanism controlling extrasynaptic release of serotonin, and how autoreceptors interact in regulating the tonic activity of serotonergic neurons, is critical to fully understand the therapeutic effect of SSRIs