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Alcohol-use-disorders are chronic relapsing illnesses, often co-morbid with anxiety. We have previously shown using the “drinking-in-the-dark” model in mice that the stimulation of the serotonin receptor 1A (5-HT1A) reduces ethanol binge-drinking behaviour and withdrawal-induced anxiety. The 5-HT1A receptor is located either on Raphe neurons as autoreceptors, or on target neurons as heteroreceptors. By combining a pharmacological approach with biased agonists targeting the 5-HT1A auto- or heteroreceptor and a chemogenetic approach (DREADDs), here we identified that ethanol-binge drinking behaviour is dependent on 5-HT1A autoreceptors and 5-HT neuronal function, with a transition from DRN-dependent regulation of short-term (6 weeks) ethanol intake, to MRN-dependent regulation after longer ethanol exposure (12 weeks). We further identified a serotonergic microcircuit (5-HTMRN→DG) originating from the MRN and projecting to the dentate gyrus (DG) of the hippocampus, that is specifically affected by, and modulates long-term ethanol consumption. The present study indicates that targeting Raphe nuclei 5-HT1A autoreceptors with agonists might represent an innovative pharmacotherapeutic strategy to combat alcohol abuse.

While the dire cardiometabolic consequences of the hypercaloric modern ‘Western’ diet are well known, there is not much information on the health impact of a high sucrose diet not inducing weight gain. Here, we tested the hypothesis that rats reared with intermittent binge access to sucrose in addition to normal chow would develop an inflammatory response in brain. To test this hypothesis, we undertook serial PET/MRI scans with the TSPO ligand [18F]DPA714 in a group of (n=9) rats at baseline and again after voluntarily consuming 5% sucrose solution three days a week for three months. Compared to a control group fed with normal chow (n=9), the sucrose rats indeed showed widespread increases in the availability of cerebral binding sites for the microglial marker, despite normal weight gain compared to the control diet group. Subsequent immunofluorescence staining of the brains confirmed the PET findings, showing a widespread 20% increase in the abundance of IBA-1-positive microglia with characteristic ‘semi-activated’ morphology in the binge sucrose rats, which had 23% lower density of microglial endpoints and 25% lower mean process length compared to microglia in the control rats with ordinary feeding. GFAP immunofluorescence showed no difference in astroglial coverage in the sucrose rats, except for a slight reduction in hypothalamus. The binge sucrose diet-induced neuroinflammation was associated with a significant elevation of white blood cell counts. Taking these results together, we find that long-term intake of sucrose in a binge paradigm, similar in sucrose content to the contemporary Western diet, triggered a low-grade systemic and central inflammation in non-obese rats. The molecular mechanism of this phenomenon remains to be established.

Alcohol use disorder is a pervasive and detrimental condition that involves changes in neuroplasticity and neurogenesis. Alcohol activates the neuroimmune system and alters the inflammatory status of the brain. Tumour necrosis factor (TNF) is a well characterised neuroimmune signal but its involvement in alcohol use disorder is unknown. In this review, we discuss the variable findings of TNF’s effect on neuroplasticity and neurogenesis. Acute ethanol exposure reduces TNF release while chronic alcohol intake generally increases TNF levels. Evidence suggests TNF potentiates excitatory transmission, promotes anxiety during alcohol withdrawal and is involved in drug use in rodents. An association between craving for alcohol and TNF is apparent during withdrawal in humans. While anti-inflammatory therapies show efficacy in reversing neurogenic deficit after alcohol exposure, there is no evidence for TNF’s essential involvement in alcohol’s effect on neurogenesis. Overall, defining TNF’s role in alcohol use disorder is complicated by poor understanding of its variable effects on synaptic transmission and neurogenesis. While TNF may be of relevance during withdrawal, the neuroimmune system likely acts through a larger group of inflammatory cytokines to alter neuroplasticity and neurogenesis. Understanding the individual relevance of TNF in alcohol use disorder awaits a more comprehensive understanding of TNF’s effects within the brain.

The rise in obesity prevalence has been linked to overconsumption of high-sugar containing food and beverages. Recent evidence suggests that chronic sucrose consumption leads to changes in serotonergic neuroplasticity within the neural circuits involved in feeding control. Although there is a relationship between serotonin signalling in the brain and diet-induced obesity, the specific serotonin (5-HT) receptors or pathways involved remain unknown. The 5-HT1A receptor subtype plays a role in regulating mood, anxiety, and appetite, and has been associated with reversing addiction to substances of abuse. However, the respective role of 5-HT1A auto- vs heteroreceptors in sucrose consumption has not been examined. Mice were given controlled access to either 5%, 10% or 25% w/v sucrose, or water as a control, for 12 weeks using the well-established “drinking in the dark” protocol (n = 6–8 mice per group). Ligands selectively targeting 5-HT1A auto- and/or heteroreceptors (NLX-112, unbiased 5-HT1A receptor agonist; NLX-101, preferential heteroreceptor agonist; F13714, preferential autoreceptor agonist) were administered i.p. acutely after 6 and 12 weeks of sucrose consumption. The specific involvement of 5-HT1A receptors in these effects was verified by blockade with the selective 5-HT1A receptors antagonist WAY-100,635. The specific subpopulation of 5-HT1A receptors involved in sucrose consumption was dependent on the concentration of sucrose solution and the duration of exposure to sucrose (6 weeks vs 12 weeks). Long-term sucrose consumption leads to accentuated 5-HT1A autoreceptor function. Thus, targeting 5-HT1A autoreceptors might represent an effective therapeutic strategy to combat the rise in obesity resulting from the overconsumption of high-sugar diet.

Purpose This study aimed to assess the activity of controlled release nicotine from dry powder inhaler formulation via locomotor activity of C57BL/6 mice. Methods To achieve this we built a nose-only inhalation device for pulmonary administration of nicotine to mice and determined the optimal operational parameters. We used the locomotor activity test to compare the effects of the inhaled nicotine hydrogen tartrate-loaded chitosan nanoparticles (NHT-CS) with NHT in C57BL/6 mice. The minimum inhaled dose of NHT-CS required to alter locomotor activity was compared with inhaled and subcutaneously (s.c) injected NHT. Finally, histological examination of lung tissues was performed to ensure inhalation of NHT-CS did not cause lung damage. Results We found a flow rate of 0.9 L/min and an exposure time of 5 min achieved optimal delivery of nicotine. A minimum of 0.88 mg inhaled of NHT-CS or 0.59 mg inhaled of NHT was required to alter locomotor activity similarly to injection of 0.5 mg/kg nicotine, suggesting the reformulation process did not alter the activity of NHT-CS. No differences between untreated and NHT-CS treated lung tissue upon histological examination were observed. Conclusions The results indicated the inhaled NHT-CS is a viable preclinical option for developing novel inhalation formulations as a potential anti-smoking therapeutic.

The brain forms robust associations between odors and emotionally salient memories, making odors especially effective at triggering fearful or traumatic memories. Using Pavlovian olfactory fear conditioning (OFC), a variant of the traditional tone-shock paradigm, this study explored the changes involved in its processing. We assessed the expression of neuronal plasticity markers phosphorylated cyclic adenosine monophosphate response element binding protein (pCREB) and phosphorylated mitogen-activated protein kinase (pMAPK) 24 h and 14 days following OFC, in newborn neurons (EdU+) and in brain regions associated with olfactory memory processing; the olfactory bulb, piriform cortex, amygdale, and hippocampus. Here, we show that all proliferating neurons in the dentate gyrus of the hippocampus and glomerular layer of the olfactory bulb were colocalized with pCREB at 24 h and 14 days post-conditioning, and the number of proliferating neurons at both time points were statistically similar. This suggests the occurrence of long-term potentiation within the neurons of this pathway. Finally, OFC significantly increased the density of pCREB- and pMAPK-positive immunoreactive neurons in the medial and cortical subnuclei of the amygdala and the posterior piriform cortex, suggesting their key involvement in its processing. Together, our investigation identifies changes in neuroplasticity within critical neural circuits responsible for olfactory fear memory.

The rise in obesity prevalence has been linked to overconsumption of high-sugar containing food and beverages. Recent evidence suggests that chronic sucrose consumption leads to changes in serotonergic neuroplasticity within the neural circuits involved in feeding control. Although there is a relationship between serotonin signalling in the brain and diet-induced obesity, the specific serotonin (5-HT) receptors or pathways involved remain unknown. The 5-HT

Sugar has become embedded in modern food and beverages. This has led to overconsumption of sugar in children, adolescents, and adults, with more than 60 countries consuming more than four times (>100 g/person/day) the WHO recommendations (25 g/person/day). Recent evidence suggests that obesity and impulsivity from poor dietary habits leads to further overconsumption of processed food and beverages. The long-term effects on cognitive processes and hyperactivity from sugar overconsumption, beginning at adolescence are not known. Using a well-validated mouse model of sugar consumption, we found that long-term sugar consumption, at a level that significantly augments weight gain, elicits an abnormal hyperlocomotor response to novelty and alters both episodic and spatial memory. Our results are similar to those reported in attention deficit and hyperactivity disorders. The deficits in hippocampal-dependent learning and memory were accompanied by altered hippocampal neurogenesis, with an overall decrease in the proliferation and differentiation of newborn neurons within the dentate gyrus. This suggests that long-term overconsumption of sugar, as that which occurs in the Western Diet might contribute to an increased risk of developing persistent hyperactivity and neurocognitive deficits in adulthood.

Serotonin neurons originate from the brainstem raphe nuclei and innervate the entire brain to regulate mood, emotion, sleep, appetite and aggression. Previous electron microscopy (EM) studies have revealed that 5-HT boutons directly contact several neuronal populations via asymmetrical (excitatory) or symmetrical (inhibitory) synapses. Additionally, 5-HT boutons sometimes form “triads” with the pre and postsynaptic components of asymmetrical or symmetrical synapses to modulate their activity. However, the exact proportion and distribution of 5-HT excitatory/inhibitory synapses and triads within the entire brain remains poorly described. Recently, we have published a novel semi-quantitative approach which combines fluorescent confocal microscopy and 3D reconstruction of 5-HT fibers apposed to excitatory and inhibitory neurochemical synapses (triads). Here, we review the similarities and differences in the distribution of 5-HT asymmetrical/symmetrical synapses observed in EM and the distribution of 5-HT excitatory/inhibitory triads quantified in our recent study. We further put into perspective the possible physiological role played by 5-HT triads in the regulation of glutamate and GABA signaling in these various brain regions.

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).

Odors have proven to be the most resilient trigger for memories of high emotional saliency. Fear associated olfactory memories pose a detrimental threat of potentially transforming into severe mental illness such as fear and anxiety-related disorders. Many studies have deliberated on auditory, visual and general contextual fear memory (CFC) processes; however, fewer studies have investigated mechanisms of olfactory fear memory. Evidence strongly suggests that the neuroanatomical representation of olfactory fear memory differs from that of auditory and visual fear memory. The aim of this review article is to revisit the literature regarding the understanding of the neurobiological process of fear conditioning and to illustrate the circuitry of olfactory fear memory.

International audience; 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 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.

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. Electronic supplementary material The online version of this article (doi:10.1007/s00429-016-1278-x) contains supplementary material, which is available to authorized users.

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.

BACKGROUND: Pharmacotherapies for alcohol use disorder have been shown to reduce hazardous drinking and improve overall health. The effect sizes for the effectiveness of these medications, however, are small, underscoring the need to expand the range of therapeutics and develop personalized treatment approaches. Recent studies have suggested that varenicline, an α4β2-nicotinic partial agonist widely used for smoking cessation, can help alcoholics reduce drinking, but the neurocognitive underpinnings of its effectiveness remain largely unexplored. METHODS: In this double-blind study, 32 heavy drinkers were randomized to receive varenicline (2 mg/d) or placebo. After 2 weeks of dosing, participants underwent functional MRI scans, during which they viewed images of faces with either neutral or fearful expressions at baseline and following an intravenous alcohol infusion to a target breath alcohol concentration of 80 mg%. Blood oxygen level-dependent (BOLD) response was analyzed with Analysis of Functional Neuroimaging software. Linear mixed-effects models were used to examine the effects of facial expression (fearful vs. neutral) and medication (placebo vs. varenicline) on BOLD response. The effect of medication on measures of subjective response to alcohol was also examined. RESULTS: Results indicated a significant facial expression-by-medication interaction in the left amygdala. The groups showed equivalent activation to neutral faces, but, whereas the placebo group showed increased activation to fearful faces, the varenicline group showed no change in activation. Amygdala activation to fearful faces correlated with number of drinks in the previous 90 days and Obsessive Compulsive Drinking Scale scores. There was no effect of varenicline on subjective response to alcohol. CONCLUSIONS: Our results indicate that varenicline may disrupt amygdala response to fearful faces in heavy drinkers. Further, amygdala activation correlated with alcohol consumption, suggesting that the effects of varenicline may be related to aspects of drinking behavior. These results suggest that amygdala response to fearful faces may be developed as a biomarker of the effectiveness of medications being developed for the treatment of alcohol use disorder.

Alcohol dependence is a debilitating disorder with current therapies displaying limited efficacy and/or compliance. Consequently, there is a critical need for improved pharmacotherapeutic strategies to manage alcohol use disorders (AUDs). Previous studies have shown that the development of alcohol dependence involves repeated cycles of binge-like ethanol intake and abstinence. Therefore, we used a model of binge ethanol consumption (drinking-in-the-dark) in mice to test the effects of compounds known to modify the activity of neurotransmitters implicated in alcohol addiction. From this, we have identified the FDA-approved antihypertensive drug pindolol, as a potential candidate for the management of AUDs. We show that the efficacy of pindolol to reduce ethanol consumption is enhanced following long-term (12 weeks) binge ethanol intake, compared with short-term (4 weeks) intake. Furthermore, pindolol had no effect on locomotor activity or consumption of the natural reward sucrose. Because pindolol acts as a dual beta-adrenergic antagonist and 5-HT1A/1B partial agonist, we examined its effect on spontaneous synaptic activity in the basolateral amygdala (BLA), a brain region densely innervated by serotonin and norepinephrine-containing fibres. Pindolol increased spontaneous excitatory post-synaptic current frequency of BLA principal neurons from long-term ethanol-consuming mice but not naive mice. Additionally, this effect was blocked by the 5-HT1A/1B receptor antagonist methiothepin, suggesting that altered serotonergic activity in the BLA may contribute to the efficacy of pindolol to reduce ethanol intake following long-term exposure. Although further mechanistic investigations are required, this study demonstrates the potential of pindolol as a new treatment option for AUDs that can be fast-tracked into human clinical studies.

In 2016 the World Health Organization reported 39% of the world's adult population (over 18 y) was overweight, with western countries such as Australia and the United States of America at 64.5% and 67.9% respectively. Overconsumption of high fat/sugar containing food and beverages contribute to the development of obesity. Neural plasticity that occurs as a result of long term sugar consumption has been shown to reduce impulse control and therefore lower the ability to resist the high fat/sugar foods contributing to the obesity epidemic. There is significant overlap between the neural pathways involved in emotions that guide behavioural responses to survival situations with those regulating overconsumption of highly palatable food. This suggests that having a clearer understanding of the role of stress and emotions in the development of obesity will lead to the development of novel therapeutic strategies. Sucrose consumption activates the mesocorticolimbic system in a manner synonymous with substances of abuse. There is overwhelming evidence to support the hypothesis that sucrose consumption results in pathophysiological consequences such as morphological neuronal changes, altered emotional processing and modified behaviour in rodent and human models. In this comprehensive review, we examined >300 studies investigating the interaction between sugar consumption, stress and emotions. Preclinical and clinical trials investigating highly palatable foods and stress, anxiety, depression and fear are reviewed. Importantly, the synergy between sugar consumption and neurobiology is addressed. This review summarizes the neurochemical changes and neural adaptations o including changes in the dopaminergic system o that influence emotion and behaviour following sugar consumption.

Debilitating and persistent fear memories can rapidly form in humans following exposure to traumatic events. Fear memories can also be generated and studied in animals via Pavlovian fear conditioning. The current study was designed to evaluate basolateral amygdala complex (BLC) involvement following the formation of different fear memories (two contextual fear memories and one adjusted auditory fear memory). Fear memories were created in the same context with five 1.0 mA (0.50 s) foot-shocks and, where necessary, five auditory tones (5 kHz, 75 dB, 20 s). The adjusted auditory fear conditioning protocol was employed to remove background contextual fear and produce isolated auditory fear memories. Immunofluorescent labeling was utilized to identify neurons expressing immediate early genes (IEGs). We found the two contextual fear conditioning (CFC) procedures to produce similar levels of fear-related freezing to context. Contextual fear memories produced increases in BLC IEG expression with distinct and separate patterns of expression. These data suggest contextual fear memories created in slightly altered contexts, can produce unique patterns of amygdala activation. The adjusted auditory fear conditioning procedure produced memories to a tone, but not to a context. This group, where no contextual fear was present, had a significant reduction in BLC IEG expression. These data suggest background contextual fear memories, created in standard auditory fear conditioning protocols, contribute significantly to increases in amygdala activation.

Background: The mechanisms leading from traumatic stress to social, emotional and cognitive impairment and the development of mental illnesses are still undetermined and consequently there remains a critical need to develop therapies for preventing the adverse consequences of traumatic stress. Research indicates nicotinic acetylcholine receptors containing α4 subunits (α4*nAChRs) are both impacted by stress and capable of modulating the stress response. In this study, we investigated whether varenicline, a partial α4β2*nAChR agonist which reduces nicotine, alcohol and sucrose consumption, can reduce stress, a driving factor in substance use disorders. We also examined the effect of stress on nucleus accumbens (NAc) α4*nAChR expression. Methods: Transgenic mice with fluorescent tags attached to α4*nAChRs were administered varenicline and/or yohimbine (a pharmacological stressor) and plasma corticosterone and NAc α4*nAChR expression were measured. A separated group of mice were exposed to maternal separation (MS) during post-natal day (P) 2–14, then restraint stressed (30 min) at six weeks of age. Body weight, anxiety-like behaviours (elevated plus maze), plasma corticosterone and NAc α4*nAChR levels were measured. Results: Varenicline attenuated yohimbine-induced plasma corticosterone increases with no effect on NAc α4*nAChR expression. MS reduced unrestrained plasma corticosterone levels in both sexes. In females, MS increased body weight and NAc α4*nAChR expression, whereas, in males, MS and restraint caused a greater change in anxiety-like behaviours and plasma corticosterone levels. Restraint altered NAc α4*nAChR expression in both male and female MS mice. Conclusions: The effects of stress on NAc α4*nAChR are sex-dependent. While varenicline attenuated acute stress-induced rises in corticosterone levels, future studies are required to determine whether varenicline is effective for relieving the effects of stress.

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.

Quantitative assessments of neuronal subtypes in numerous brain regions show large variations in dendritic arbor size. A critical experimental factor is the method used to visualize neurons. We chose to investigate quantitative differences in basolateral amygdala (BLA) principal neuron morphology using two of the most common visualization methods: Golgi-Cox staining and neurobiotin (NB) filling. We show in 8-week-old Wistar rats that NB-filling reveals significantly larger dendritic arbors and different spine densities, compared to Golgi-Cox-stained BLA neurons. Our results demonstrate important differences and provide methodological insights into quantitative disparities of BLA principal neuron morphology reported in the literature.

The basolateral amygdala (BLA) is a complex brain region associated with processing emotional states, such as fear, anxiety, and stress. Some aspects of these emotional states are driven by the network activity of synaptic connections, derived from both local circuitry and projections to the BLA from other regions. Although the synaptic physiology and general morphological characteristics are known for many individual cell types within the BLA, the combination of morphological, electrophysiological, and distribution of neurochemical GABAergic synapses in a three-dimensional neuronal arbor has not been reported for single neurons from this region. The aim of this study was to assess differences in morphological characteristics of BLA principal cells and interneurons, quantify the distribution of GABAergic neurochemical synapses within the entire neuronal arbor of each cell type, and determine whether GABAergic synaptic density correlates with electrophysiological recordings of inhibitory postsynaptic currents. We show that BLA principal neurons form complex dendritic arborizations, with proximal dendrites having fewer spines but higher densities of neurochemical GABAergic synapses compared with distal dendrites. Furthermore, we found that BLA interneurons exhibited reduced dendritic arbor lengths and spine densities but had significantly higher densities of putative GABAergic synapses compared with principal cells, which was correlated with an increased frequency of spontaneous inhibitory postsynaptic currents. The quantification of GABAergic connectivity, in combination with morphological and electrophysiological measurements of the BLA cell types, is the first step toward a greater understanding of how fear and stress lead to changes in morphology, local connectivity, and/or synaptic reorganization of the BLA.

Stress is a major driving force in alcohol use disorders (AUDs). It influences how much one consumes, craving intensity and whether an abstinent individual will return to harmful alcohol consumption. We are most vulnerable to the effects of stress during early development, and exposure to multiple traumatic early life events dramatically increases the risk for AUDs. However, not everyone exposed to early life stress will develop an AUD. The mechanisms determining whether an individual’s brain adapts and becomes resilient to the effects of stress or succumbs and is unable to cope with stress remain elusive. Emerging evidence suggests that neuroplastic changes in the nucleus accumbens (NAc) following early life stress underlie the development of AUDs. This review discusses the impact of early life stress on NAc structure and function, how these changes affect cholinergic signaling within the mesolimbic reward pathway and the role nicotinic acetylcholine receptors (nAChRs) play in this process. Understanding the neural pathways and mechanism determining stress resilience or susceptibility will improve our ability to identify individuals susceptible to developing AUDs, formulate cognitive interventions to prevent AUDs in susceptible individuals and to elucidate and enhance potential therapeutic targets, such as the nAChRs, for those struggling to overcome an AUD.

Background Factors leading to the harmful consumption of substances, like alcohol and sucrose, involve a complex interaction of genes and the environment. While we cannot control the genes we inherit, we can modify our environment. Understanding the role that social and environmental experiences play in alcohol and sucrose consumption is critical for developing preventative interventions and treatments for alcohol use disorders and obesity. Methods We used the drinking in the dark two‐bottle choice (2BC) model of ethanol and sucrose consumption to compare male C57BL/6 mice housed in the IntelliCage (an automated device capable of simultaneously measuring behaviors of up to 16 mice living in an enriched social environment) with mice housed in standard isolated and social environments. Results Consistent with previous publications on ethanol‐naïve and ‐experienced mice, social and environmental enrichment reduced ethanol preference. Isolated mice had the highest ethanol preference and IntelliCage mice the least, regardless of prior ethanol experience. In mice with no prior sucrose experience, the addition of social and environmental enrichment increased sucrose preference. However, moving isolated mice to enriched conditions did not affect sucrose preference in sucrose‐experienced mice. Conclusions The impact of social and environmental enrichment on ethanol consumption differs from sucrose consumption suggesting that interventions and treatments developed for alcohol use disorders may not be suitable for sucrose consumption disorders.

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.

Addiction is a devastating disorder that affects 15.3 million people worldwide. While prevalent, few effective treatments exist. Orexin receptors have been proposed as a potential target for anti-craving medications. Orexins, also known as hypocretins, are neuropeptides produced in neurons of the lateral and dorsomedial hypothalamus and perifornical area, which project widely throughout the brain. The absence of orexins in rodents and humans leads to narcolepsy. However, orexins also have an established role in reward seeking. This review will discuss some of the original studies describing the roles of the orexins in reward seeking as well as specific works that were presented at the 2013 International Narcotics Research Conference. Orexin signalling can promote drug-induced plasticity of glutamatergic synapses onto dopamine neurons of the ventral tegmental area (VTA), a brain region implicated in motivated behaviour. Additional evidence suggests that orexin signalling can also promote drug seeking by initiating an endocannabinoid-mediated synaptic depression of GABAergic inputs to the VTA, and thereby disinhibiting dopaminergic neurons. Orexin neurons co-express the inhibitory opioid peptide dynorphin. It has been proposed that orexin in the VTA may not mediate reward per se, but rather occludes the ‘anti-reward’ effects of dynorphin. Finally, orexin signalling in the prefrontal cortex and the central amygdala is implicated in reinstatement of reward seeking. This review will highlight recent work describing the role of orexin signalling in cellular processes underlying addiction-related behaviours and propose novel hypotheses for the mechanisms by which orexin signalling may impart drug seeking. LINKED ARTICLES This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2

Opioids are important endogenous ligands that exist in both invertebrates and vertebrates and signal by activation of opioid receptors to produce analgesia and reward or pleasure. The μ-opioid receptor is the best known of the opioid receptors and mediates the acute analgesic effects of opiates, while the δ-opioid receptor (DOR) has been less well studied and has been linked to effects that follow from chronic use of opiates such as stress, inflammation and anxiety. Recently, DORs have been shown to play an essential role in emotions and increasing evidence points to a role in learning actions and outcomes. The process of learning and memory in addiction has been proposed to involve strengthening of specific brain circuits when a drug is paired with a context or environment. The DOR is highly expressed in the hippocampus, amygdala, striatum and other basal ganglia structures known to participate in learning and memory. In this review, we will focus on the role of the DOR and its potential role in learning and memory underlying the development of addiction.

Background and Purpose Varenicline, a neuronal nicotinic acetylcholine receptor (nAChR) modulator, decreases ethanol consumption in rodents and humans. The proposed mechanism of action for varenicline to reduce ethanol consumption has been through modulation of dopamine (DA) release in the nucleus accumbens (NAc) via α4*-containing nAChRs in the ventral tegmental area (VTA). However, presynaptic nAChRs on dopaminergic terminals in the NAc have been shown to directly modulate dopaminergic signalling independently of neuronal activity from the VTA. In this study, we determined whether nAChRs in the NAc play a role in varenicline's effects on ethanol consumption. Experimental Approach Rats were trained to consume ethanol using the intermittent-access two-bottle choice protocol for 10 weeks. Ethanol intake was measured after varenicline or vehicle was microinfused into the NAc (core, shell or core-shell border) or the VTA (anterior or posterior). The effect of varenicline treatment on DA release in the NAc was measured using both in vivo microdialysis and in vitro fast-scan cyclic voltammetry (FSCV). Key Results Microinfusion of varenicline into the NAc core and core-shell border, but not into the NAc shell or VTA, reduced ethanol intake following long-term ethanol consumption. During microdialysis, a significant enhancement in accumbal DA release occurred following systemic administration of varenicline and FSCV showed that varenicline also altered the evoked release of DA in the NAc. Conclusion and Implications Following long-term ethanol consumption, varenicline in the NAc reduces ethanol intake, suggesting that presynaptic nAChRs in the NAc are important for mediating varenicline's effects on ethanol consumption.

Long-term alcohol use causes a multitude of neurochemical changes in cortical regions that facilitate the transition to dependence. Therefore, we used a model of long-term, binge-like ethanol consumption in rats to determine the effects on morphology and synaptic physiology of medial prefrontal cortex (mPFC) layer V pyramidal neurons. Following 10 weeks of ethanol consumption, we recorded synaptic currents from mPFC neurons and used neurobiotin filling to analyze their morphology. We then compared these data to measurements obtained from age-matched, water-drinking control rats. We found that long-term ethanol consumption caused a significant increase in total dendrite arbor length of mPFC layer V pyramidal neurons. Dendritic restructuring was primarily observed in basal dendrite arbors, with mPFC neurons from animals engaged in long-term ethanol drinking having significantly larger and more complex basal arbors compared with controls. These changes were accompanied by significantly increased total spine densities and spontaneous postsynaptic excitatory current frequency, suggesting that long-term binge-like ethanol consumption enhances basal excitatory synaptic transmission in mPFC layer V pyramidal neurons. Our results provide insights into the morphological and functional changes in mPFC layer V pyramidal neuronal physiology following prolonged exposure to ethanol and support changes in mPFC activity during the development of alcohol dependence.

Layer V pyramidal neurons (LVPNs) within the motor cortex integrate sensory cues and co-ordinate voluntary control of motor output. In amyotrophic lateral sclerosis (ALS) LVPNs and spinal motor neurons degenerate. The pathogenesis of neural degeneration is unknown in ALS; 10% of cases have a genetic cause, whereas 90% are sporadic, with most of the latter showing TDP-43 inclusions. Clinical and experimental evidence implicate excitotoxicity as a prime aetiological candidate. Using patch clamp and dye-filling techniques in brain slices, combined with high-resolution confocal microscopy, we report increased excitatory synaptic inputs and dendritic spine densities in early presymptomatic mice carrying a TDP-43Q331K mutation. These findings demonstrate substantive alterations in the motor cortex neural network, long before an overt degenerative phenotype has been reported. We conclude that increased excitatory neurotransmission is a common pathophysiology amongst differing genetic cases of ALS and may be of relevance to the 95% of sporadic ALS cases that exhibit TDP-43 inclusions.

Background: The intermittent access 2-bottle choice (IA2BC) and drinking in the dark (DID) models were developed for studying rodent binge-like consumption. Traditionally, IA2BC was used with rats and DID with mice. Recently, IA2BC was adapted to study mouse ethanol consumption. However, it is unknown whether DID is suitable for rats or if one rat model is more advantageous than another for studying binge-like consumption. Methods: Male Wistar rats consumed 20% ethanol or 5% sucrose using IA2BC or DID for 12 weeks. IA2BC drinking sessions occurred on alternate days (Mondays–Fridays) and lasted 24 h, whereas DID sessions ran 4 h/day, 5 days/week (Monday–Friday). Average consumption/session, week and hour was measured. To explore DID model suitability for screening novel compounds for controlling ethanol and sucrose intake, varenicline (2 mg/kg) or vehicle was administered to DID rats. Results: IA2BC rats consume more ethanol/session and similar amounts of ethanol/week than DID rats. While, IA2BC rats consume more sucrose/session and week than DID rats. Although IA2BC rats had more ethanol and sucrose access time, DID rats had greater ethanol and sucrose intake/hour. Varenicline significantly reduced ethanol and sucrose consumption in DID rats, consistent with previously published IA2BC studies. Conclusions: Despite the shorter access time, the rat DID model induced higher initial intake and greater consumption/hour for both ethanol and sucrose. The shorter duration of DID sessions did not prevent detection of varenicline-induced reductions in ethanol or sucrose consumption, suggesting the DID model may be suitable for studying binge-like ethanol and sucrose consumption.

This study reports the development of nanoparticles in the form of inhalable micro-aggregates of biodegradable chitosan (CS) loaded with nicotine hydrogen tartrate (NHT) for potential pulmonary delivery of nicotine from dry powder inhaler (DPI) formulations with prolonged release profile. The NHT-loaded CS particles were prepared using a water-in-oil emulsion crosslinking method. The prepared particles were characterized using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) for morphological studies; Zetasizer and Mastersizer were applied for particle size analysis. The in vitro aerosolization of the formulations was studied using a twin-stage-impinger (TSI) and promising aerosolization characteristics were shown. The nanoparticles were spherical with size ranges between 167 and 411 nm while micro-aggregates (3.73–4.73 μm) were formed among nanoparticles. According to differential scanning calorimetry (DSC), X-ray diffraction (XRD) analysis and attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy, the NHT crystallinity was lost when in the particles, indicating it was uniformly dispersed as a solid solution. On the basis of X-ray photoelectron spectroscopy (XPS) analysis, the amount of NHT loaded on the surface of CS increased proportionally with increasing drug loading in the bulk so there was no surface enhancement. The fine particle doses (FPD) of NHT ranging between 1.7 and 3.2 mg from DPI formulations were concentration dependent and increased with increased drug loading. Based on the in vitro release study, NHT released from CS particles with a burst release in the first 8 h and subsequent prolonged release of nicotine.

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 hypothe‐ sized 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

BACKGROUND: Our laboratory has previously shown that the smoking-cessation agent varenicline, an agonist/partial agonist of alpha4beta2*, alpha3beta4*, alpha3beta2*, alpha6beta2* (* indicates the possibility of additional subunits) and alpha7 subunits of nicotinic acetylcholine receptors (nAChRs), reduces ethanol consumption in rats only after long-term exposure (12 weeks). As compounds having partial agonistic activity on alpha3beta4* nAChRs were shown to decrease ethanol consumption in rodents, we assessed here the involvement of the beta4 subunit in the effect of varenicline in the reduction of short- and long-term binge-like ethanol drinking in mice. METHODS: We used the well-validated drinking-in-the-dark (DID) paradigm to model chronic binge-like ethanol drinking in beta4-/- and beta4+/+ littermate mice and compare the effect of intraperitoneal injection of varenicline (2mg/kg) on ethanol intake following short- (4 weeks) or long-term (12 weeks) exposure. RESULTS: Drinking pattern and amounts of ethanol intake were similar in beta4-/- and beta4+/+ mice. Interestingly, our results showed that varenicline reduces ethanol consumption following short- and long-term ethanol exposure in the DID. Although the effect of varenicline on the reduction of ethanol consumption was slightly more pronounced in beta4-/- mice than their beta4+/+ littermates no significant differences were observed between genotypes. CONCLUSION: In mice, varenicline reduces binge-like ethanol consumption both after short- and long-term exposure in the DID and this effect is independent of beta4 nAChR subunit.