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

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

CSF1R-dependent macrophages control postnatal somatic growth and organ maturation

Homozygous mutation of the Csf1r locus (Csf1rko) in mice, rats and humans leads to multiple postnatal developmental abnormalities. To enable analysis of the mechanisms underlying the phenotypic impacts of Csf1r mutation, we bred a rat Csf1rko allele to the inbred dark agouti (DA) genetic background and to a Csf1r-mApple reporter transgene. The Csf1rko led to almost complete loss of embryonic macrophages and ablation of most adult tissue macrophage populations. We extended previous analysis of the Csf1rko phenotype to early postnatal development to reveal impacts on musculoskeletal development and proliferation and morphogenesis in multiple organs. Expression profiling of 3-week old wild-type (WT) and Csf1rko livers identified 2760 differentially expressed genes associated with the loss of macrophages, severe hypoplasia, delayed hepatocyte maturation, disrupted lipid metabolism and the IGF1/IGF binding protein system. Older Csf1rko rats developed severe hepatic steatosis. Consistent with the developmental delay in the liver Csf1rko rats had greatly-reduced circulating IGF1. Transfer of WT bone marrow (BM) cells at weaning without conditioning repopulated resident macrophages in all organs, including microglia in the brain, and reversed the mutant phenotypes enabling long term survival and fertility. WT BM transfer restored osteoclasts, eliminated osteopetrosis, restored bone marrow cellularity and architecture and reversed granulocytosis and B cell deficiency. Csf1rko rats had an elevated circulating CSF1 concentration which was rapidly reduced to WT levels following BM transfer. However, CD43hi non-classical monocytes, absent in the Csf1rko, were not rescued and bone marrow progenitors remained unresponsive to CSF1. The results demonstrate that the Csf1rko phenotype is autonomous to BM-derived cells and indicate that BM contains a progenitor of tissue macrophages distinct from hematopoietic stem cells. The model provides a unique system in which to define the pathways of development of resident tissue macrophages and their local and systemic roles in growth and organ maturation

Serotonin and noradrenaline signalling in binge-like long-term alcohol intake and associated maladaptive effects in adult mice

Alcohol dependence is a debilitating disorder with current therapies displaying limited efficacy and/or compliance. This thesis investigated the contribution of two key neurotransmitters; noradrenaline and serotonin in alcohol dependence using a mouse model of binge-like alcohol consumption. Using this model, the study demonstrated that long-term alcohol intake causes neuroadaptive changes in the signalling of these molecules leading to alterations in normal emotional states and alcohol dependence. Finally, the thesis highlighted the efficacy of the FDA approved drug for hypertension pindolol and the anxiolytic drug tandospirone to reduce alcohol consumption, thereby offering a novel approach to improve treatments for alcohol addiction

Mapping the connectivity of serotonin transporter immunoreactive axons to excitatory and inhibitory neurochemical synapses in the mouse limbic brain

Serotonin neurons arise from the brainstem raphe nuclei and send their projections throughout the brain to release 5-HT which acts as a modulator of several neuronal populations. Previous electron microscopy studies in rats have morphologically determined the distribution of 5-HT release sites (boutons) in certain brain regions and have shown that 5-HT containing boutons form synaptic contacts that are either symmetric or asymmetric. In addition, 5-HT boutons can form synaptic triads with the pre- and postsynaptic specializations of either symmetrical or asymmetrical synapses. However, due to the labor intensive processing of serial sections required by electron microscopy, little is known about the neurochemical properties or the quantitative distribution of 5-HT triads within whole brain or discrete subregions. Therefore, we used a semi-automated approach that combines immunohistochemistry and high-resolution confocal microscopy to label serotonin transporter (SERT) immunoreactive axons and reconstruct in 3D their distribution within limbic brain regions. We also used antibodies against key pre- (synaptophysin) and postsynaptic components of excitatory (PSD95) or inhibitory (gephyrin) synapses to (1) identify putative 5-HTergic boutons within SERT immunoreactive axons and, (2) quantify their close apposition to neurochemical excitatory or inhibitory synapses. We provide a 5-HTergic axon density map and have determined the ratio of synaptic triads consisting of a 5-HT bouton in close proximity to either neurochemical excitatory or inhibitory synapses within different limbic brain areas. The ability to model and map changes in 5-HTergic axonal density and the formation of triadic connectivity within whole brain regions using this rapid and quantitative approach offers new possibilities for studying neuroplastic changes in the 5-HTergic pathway

Serotonergic neuroplasticity in alcohol addiction

Alcohol addiction is a debilitating disorder producing maladaptive changes in the brain, leading drinkers to become more sensitive to stress and anxiety. These changes are key factors contributing to alcohol craving and maintaining a persistent vulnerability to relapse. Serotonin (5-Hydroxytryptamine, 5-HT) is a monoamine neurotransmitter widely expressed in the central nervous system where it plays an important role in the regulation of mood. The serotonin system has been extensively implicated in the regulation of stress and anxiety, as well as the reinforcing properties of all of the major classes of drugs of abuse, including alcohol. Dysregulation within the 5-HT system has been postulated to underlie the negative mood states associated with alcohol use disorders. This review will describe the serotonergic (5-HTergic) neuroplastic changes observed in animal models throughout the alcohol addiction cycle, from prenatal to adulthood exposure. The first section will focus on alcohol-induced 5-HTergic neuroadaptations in offspring prenatally exposed to alcohol and the consequences on the regulation of stress/anxiety. The second section will compare alterations in 5-HT signalling induced by acute or chronic alcohol exposure during adulthood and following alcohol withdrawal, highlighting the impact on the regulation of stress/anxiety signalling pathways. The third section will outline 5-HTergic neuroadaptations observed in various genetically-selected ethanol preferring rat lines. Finally, we will discuss the pharmacological manipulation of the 5-HTergic system on ethanol- and anxiety/stress-related behaviours demonstrated by clinical trials, with an emphasis on current and potential treatments

Pindolol rescues anxiety-like behavior and neurogenic maladaptations of long-term binge alcohol intake in mice

Long-term binge alcohol consumption alters the signaling of numerous neurotransmitters in the brain including noradrenaline (NE) and serotonin (5-HT). Alterations in the signaling of these neuronal pathways result in dysfunctional emotional states like anxiety and depression which are typically seen during alcohol withdrawal. Interestingly, studies have demonstrated that the development of alcohol-induced negative affective states is linked to disrupted neurogenesis in the dentate gyrus (DG) region of the hippocampus in alcohol-dependent animals. We have previously shown that modulation of NE and 5-HT activity by pharmacological targeting of β-adrenoreceptors (β-ARs) and 5-HT1A/1B receptors with pindolol reduces consumption in long-term alcohol-consuming mice. Since these receptors are also involved in emotional homeostasis and hippocampal neurogenesis, we investigated the effects of pindolol administration on emotional and neurogenic deficits in mice consuming long-term alcohol (18 weeks). We report that acute administration of pindolol (32 mg/kg) reduces anxiety-like behavior in mice at 24 h withdrawal in the marble-burying test (MBT) and the elevated plus-maze (EPM). We also show that chronic (2 weeks) pindolol treatment (32 mg/kg/day) attenuates alcohol-induced impairments in the density of immature neurons (DCX+) but not newborn cells (BrdU+) in the hippocampal DG. Pindolol treatment also restores the normal proportion of newborn proliferating cells (BrdU+/Ki67+/DCX−), newborn proliferating immature neurons (BrdU+/Ki67+/DCX+) and newborn non-proliferating immature neurons (BrdU+/Ki67−/DCX+) following long-term alcohol intake. These results suggest that pindolol, through its unique pharmacology may rescue some but not all deficits of long-term alcohol abuse on the brain, adding further value to its properties as a strong pharmaceutical option for alcohol use disorders (AUDs)

5-HT1A receptor-dependent modulation of emotional and neurogenic deficits elicited by prolonged consumption of alcohol

Repeated episodes of binge-like alcohol consumption produce anxiety, depression and various deleterious effects including alterations in neurogenesis. While the involvement of the serotonin receptor 1 A (5-HT1A) in the regulation of anxiety-like behavior and neurogenesis is well documented, its contribution to alcohol withdrawal-induced anxiety and alcohol-induced deficits in neurogenesis is less documented. Using the Drinking-In-the-Dark (DID) paradigm to model chronic long-term (12 weeks) binge-like voluntary alcohol consumption in mice, we show that the selective partial activation of 5-HT1A receptors by tandospirone (3 mg/kg) prevents alcohol withdrawal-induced anxiety in a battery of behavioral tests (marble burying, elevated-plus-maze, open-field), which is accompanied by a robust decrease in binge-like ethanol intake (1 and 3 mg/kg). Furthermore, using triple immunolabelling of proliferation and neuronal differentiation markers, we show that long-term DID elicits profound deficits in neurogenesis and neuronal fate specification in the dorsal hippocampus that are entirely reversed by a 2-week chronic treatment with the 5-HT1A partial agonist tandospirone (3 mg/kg/day). Together, our results confirm previous observations that 5-HT1A receptors play a pivotal role in alcohol drinking behavior and the associated emotional and neurogenic impairments, and suggest that 5-HT1A partial agonists represent a promising treatment strategy for alcohol abuse

Modulation of serotonin and noradrenaline in the BLA by pindolol reduces long-term ethanol intake

Repeated cycles of binge-like alcohol consumption and abstinence change the activity of several neurotransmitter systems. Some of these changes are consolidated following prolonged alcohol use and are thought to play an important role in the development of dependence. We have previously shown that systemic administration of the dual beta-adrenergic antagonist and 5-HT partial agonist pindolol selectively reduces long-term but not short-term binge-like consumption of ethanol and alters excitatory postsynaptic currents in basolateral amygdala (BLA) principal neurons. The aim of this study was to investigate the effects of pindolol microinfusions in the BLA on long-term ethanol intake using the drinking-in-the-dark paradigm in mice. We also microinfused RU24969 (5-HT receptor partial agonist) and CGP12177 (β adrenergic antagonist) following long-term ethanol intake and determined the densities of 5-HT receptors and β adrenergic in the BLA following short-term (4 weeks) and long-term ethanol (12 weeks) consumption. We show that intra-BLA infusion of pindolol (1000 pmol/0.5 μl), RU24969 (0.3 and 3 pmol/0.5 μl) and CGP12177 (500 pmol/0.5 μl) produce robust decreases in long-term ethanol consumption. Additionally, we identified reduced β adrenergic receptor expression and no change in 5-HT receptor density in the BLA of long-term ethanol-consuming mice. Collectively, our data highlight the effects of pindolol on voluntary, binge-like ethanol consumption behavior following long-term intake