Alcohol Use Disorder: Neurobiology and Therapeutics.

Alcohol use disorder (AUD) encompasses the dysregulation of multiple brain circuits involved in executive function leading to excessive consumption of alcohol, despite negative health and social consequences and feelings of withdrawal when access to alcohol is prevented. Ethanol exerts its toxicity through changes to multiple neurotransmitter systems, including serotonin, dopamine, gamma-aminobutyric acid, glutamate, acetylcholine, and opioid systems. These neurotransmitter imbalances result in dysregulation of brain circuits responsible for reward, motivation, decision making, affect, and the stress response. Despite serious health and psychosocial consequences, this disorder still remains one of the leading causes of death globally. Treatment options include both psychological and pharmacological interventions, which are aimed at reducing alcohol consumption and/or promoting abstinence while also addressing dysfunctional behaviours and impaired functioning. However, stigma and social barriers to accessing care continue to impact many individuals. AUD treatment should focus not only on restoring the physiological and neurological impairment directly caused by alcohol toxicity but also on addressing psychosocial factors associated with AUD that often prevent access to treatment. This review summarizes the impact of alcohol toxicity on brain neurocircuitry in the context of AUD and discusses pharmacological and non-pharmacological therapies currently available to treat this addiction disorder.

Effects of prenatal ethanol exposure on choline-induced long-term depression in the hippocampus.

Choline is an essential nutrient under evaluation as a cognitive enhancing treatment for fetal alcohol spectrum disorders (FASD) in clinical trials. As a result, there is increased pressure to identify therapeutic mechanism(s) of action. Choline is not only a precursor for several essential cell membrane components and signaling molecules but also has the potential to directly affect synaptic mechanisms that are believed important for cognitive processes. In the current work, we study how the direct application of choline can affect synaptic transmission in the dentate gyrus (DG) of hippocampal slices obtained from adolescent (postnatal days 21-28) Sprague-Dawley rats (Rattus norvegicus). The acute administration of choline chloride (2 mM) reliably induced a long-term depression (LTD) of field excitatory postsynaptic potentials (fEPSPs) in the DG in vitro. The depression required the involvement of M1 receptors, and the magnitude of the effect was similar in slices obtained from male and female animals. To further study the impact of choline in an animal model of FASD, we examined offspring from dams fed an ethanol-containing diet (35.5% ethanol-derived calories) throughout gestation. In slices from the adolescent animals that experienced prenatal ethanol exposure (PNEE), we found that the choline induced an LTD that uniquely involved the activation of N-methyl-d-aspartate (NMDA) and M1 receptors. This study provides a novel insight into how choline can modulate hippocampal transmission at the level of the synapse and that it can have unique effects following PNEE.NEW & NOTEWORTHY Choline supplementation is a nutraceutical therapy with significant potential for a variety of developmental disorders; however, the mechanisms involved in its therapeutic effects remain poorly understood. Our research shows that choline directly impacts synaptic communication in the brain, inducing a long-term depression of synaptic efficacy in brain slices. The depression is equivalent in male and female animals, involves M1 receptors in control animals, but uniquely involves NMDA receptors in a model of FASD.

Endocannabinoid receptors contribute significantly to multiple forms of long-term depression in the rat dentate gyrus.

Cannabinoid receptors are widely expressed throughout the hippocampal formation, but are particularly dense in the dentate gyrus (DG) subregion. We, and others, have shown in mice that cannabinoid type 1 receptors (CB1Rs) are involved in a long-term depression (LTD) that can be induced by prolonged 10 Hz stimulation of the medial perforant path (MPP)-granule cell synaptic input to the DG. Here, we extend this work to examine the involvement of CB1Rs in other common forms of LTD in the hippocampus of juvenile male and female Sprague-Dawley rats (Rattus norvegicus). We found, as in mice, that prolonged 10 Hz stimulation (6000 pulses) could reliably induce a form of LTD that was dependent upon CB1R activation. In addition, we also discovered a role for both CB1R and mGluR proteins in LTD induced with 1 Hz low-frequency stimulation (1 Hz-LTD; 900 pulses) and in LTD induced by bath application of the group I mGluR agonist (RS)-3,5-Dihydroxyphenylglycine (DHPG; DHPG-LTD). This study elucidates an essential role for endocannabinoid receptors in a number of forms of LTD in the rat DG, and identifies a novel role for CB1Rs as potential therapeutic targets for conditions that involve impaired LTD in the DG.

Intermittent ethanol exposure during adolescence impairs cannabinoid type 1 receptor-dependent long-term depression and recognition memory in adult mice.

Binge drinking is a significant problem in adolescent populations, and because of the reciprocal interactions between ethanol (EtOH) consumption and the endocannabinoid (eCB) system, we sought to determine if adolescent EtOH intake altered the localization and function of the cannabinoid 1 (CB1) receptors in the adult brain. Adolescent mice were exposed to a 4-day-per week drinking in the dark (DID) procedure for a total of 4 weeks and then tested after a 2-week withdrawal period. Field excitatory postsynaptic potentials (fEPSPs), evoked by medial perforant path (MPP) stimulation in the dentate gyrus molecular layer (DGML), were significantly smaller. Furthermore, unlike control animals, CB1 receptor activation did not depress fEPSPs in the EtOH-exposed animals. We also examined a form of excitatory long-term depression that is dependent on CB1 receptors (eCB-eLTD) and found that it was completely lacking in the animals that consumed EtOH during adolescence. Histological analyses indicated that adolescent EtOH intake significantly reduced the CB1 receptor distribution and proportion of immunopositive excitatory synaptic terminals in the medial DGML. Furthermore, there was decreased binding of [35S]guanosine-5*-O-(3-thiotriphosphate) ([35S] GTPγS) and the guanine nucleotide-binding (G) protein Gαi2 subunit in the EtOH-exposed animals. Associated with this, there was a significant increase in monoacylglycerol lipase (MAGL) mRNA and protein in the hippocampus of EtOH-exposed animals. Conversely, deficits in eCB-eLTD and recognition memory could be rescued by inhibiting MAGL with JZL184. These findings indicate that repeated exposure to EtOH during adolescence leads to long-term deficits in CB1 receptor expression, eCB-eLTD, and reduced recognition memory, but that these functional deficits can be restored by treatments that increase endogenous 2-arachidonoylglycerol.

Impaired Bidirectional Synaptic Plasticity in Juvenile Offspring Following Prenatal Ethanol Exposure.

BACKGROUND: The hippocampus is particularly vulnerable to the teratogenic effects of prenatal ethanol exposure (PNEE), and hippocampal structural and functional deficits are thought to contribute to the learning and memory deficits that are a hallmark feature of fetal alcohol spectrum disorders. METHODS: Sprague Dawley dams were exposed to a liquid diet that contained EtOH (35.5% EtOH-derived calories) throughout gestation, and then, PNEE juvenile (P21-28) male and female offspring were used for in vitro electrophysiological recordings. We examined long-term potentiation (LTP), long-term depression (LTD), and depotentiation in the medial perforant path input to the dentate gyrus (DG) to determine the impact of PNEE on the dynamic range of bidirectional synaptic plasticity in both sexes. RESULTS: PNEE reduced the responsiveness of the DGs of male but not in female offspring, and this effect was no longer apparent when GABAergic signaling was inhibited. There was also a sex-specific LTD impairment in males, but increasing the duration of the conditioning stimulus could overcome this deficit. The magnitude of LTP was also reduced, but in both sexes following PNEE. This appears to be an increase in the threshold for induction, not in capacity, as the level of LTP induced in PNEE animals was increased to control levels when additional conditioning stimuli were administered. CONCLUSIONS: These data are the first to describe, in a single study, the impact of PNEE on the dynamic range of bidirectional synaptic plasticity in the juvenile DG in both males and in females. The data suggest that PNEE increases the threshold for LTP in the DG in both sexes, but produces a sex-specific increase in the threshold for LTD in males These alterations reduce the dynamic range for synaptic plasticity in both sexes.

Endocannabinoid long-term depression revealed at medial perforant path excitatory synapses in the dentate gyrus.

The endocannabinoid system modulates synaptic plasticity in the hippocampus, but a link between long-term synaptic plasticity and the type 1 cannabinoid (CB1) receptor at medial perforant path (MPP) synapses remains elusive. Here, immuno-electron microscopy in adult mice showed that ∼26% of the excitatory synaptic terminals in the middle 1/3 of the dentate molecular layer (DML) contained CB1 receptors, and field excitatory postsynaptic potentials evoked by MPP stimulation were inhibited by CB1 receptor activation. In addition, MPP stimulation at 10 Hz for 10 min triggered CB1 receptor-dependent excitatory long-term depression (eCB-eLTD) at MPP synapses of wild-type mice but not on CB1-knockout mice. This eCB-eLTD was group I mGluR-dependent, required intracellular calcium influx and 2-arachydonoyl-glycerol (2-AG) synthesis but did not depend on N-methyl-d-aspartate (NMDA) receptors. Overall, these results point to a functional role for CB1 receptors with eCB-eLTD at DML MPP synapses and further involve these receptors in memory processing within the adult brain.

Fetal alcohol spectrum disorder (FASD) affects the hippocampal levels of histone variant H2A.Z-2.

Fetal alcohol spectrum disorder (FASD) is caused by prenatal exposure to ethanol and has been linked to neurodevelopmental impairments. Alcohol has the potential to alter some of the epigenetic components that play a critical role during development. Previous studies have provided evidence that prenatal exposure to ethanol results in abnormal epigenetic patterns (i.e., hypomethylation) of the genome. The aim of this study was to determine how prenatal exposure to ethanol in rats affects the hippocampal levels of expression of two important brain epigenetic transcriptional regulators involved in synaptic plasticity and memory consolidation: methyl CpG-binding protein 2 (MeCP2) and histone variant H2A.Z. Unexpectedly, under the conditions used in this work we were not able to detect any changes in MeCP2. Interestingly, however, we observed a significant decrease in H2A.Z, accompanied by its chromatin redistribution in both female and male FASD rat pups. Moreover, the data from reverse-transcription qPCR later confirmed that this decrease in H2A.Z is mainly due to down-regulation of its H2A.Z-2 isoform gene expression. Altogether, these data provide strong evidence that prenatal exposure to ethanol alters histone variant H2A.Z during neurogenesis of rat hippocampus.

Cognitive and neurobehavioral benefits of an enriched environment on young adult mice after chronic ethanol consumption during adolescence.

Binge drinking (BD) is a common pattern of ethanol (EtOH) consumption by adolescents. The brain effects of the acute EtOH exposure are well-studied; however, the long-lasting cognitive and neurobehavioral consequences of BD during adolescence are only beginning to be elucidated. Environmental enrichment (EE) has long been known for its benefits on the brain and may serve as a potential supportive therapy following EtOH exposure. In this study, we hypothesized that EE may have potential benefits on the cognitive deficits associated with BD EtOH consumption. Four-week-old C57BL/6J male mice were exposed to EtOH following an intermittent 4-day drinking-in-the-dark procedure for 4 weeks. Then they were exposed to EE during EtOH withdrawal for 2 weeks followed by a behavioral battery of tests including novel object recognition, novel location, object-in-place, rotarod, beam walking balance, tail suspension, light-dark box and open field that were run during early adulthood. Young adult mice exposed to EE significantly recovered recognition, spatial and associative memory as well as motor coordination skills and balance that were significantly impaired after adolescent EtOH drinking with respect to controls. No significant permanent anxiety or depressive-like behaviors were observed. Taken together, an EE exerts positive effects on the long-term negative cognitive deficits as a result of EtOH consumption during adolescence.

Localization of the cannabinoid type-1 receptor in subcellular astrocyte compartments of mutant mouse hippocampus.

Astroglial type-1 cannabinoid (CB1 ) receptors are involved in synaptic transmission, plasticity and behavior by interfering with the so-called tripartite synapse formed by pre- and post-synaptic neuronal elements and surrounding astrocyte processes. However, little is known concerning the subcellular distribution of astroglial CB1 receptors. In particular, brain CB1 receptors are mostly localized at cells' plasmalemma, but recent evidence indicates their functional presence in mitochondrial membranes. Whether CB1 receptors are present in astroglial mitochondria has remained unknown. To investigate this issue, we included conditional knock-out mice lacking astroglial CB1 receptor expression specifically in glial fibrillary acidic protein (GFAP)-containing astrocytes (GFAP-CB1 -KO mice) and also generated genetic rescue mice to re-express CB1 receptors exclusively in astrocytes (GFAP-CB1 -RS). To better identify astroglial structures by immunoelectron microscopy, global CB1 knock-out (CB1 -KO) mice and wild-type (CB1 -WT) littermates were intra-hippocampally injected with an adeno-associated virus expressing humanized renilla green fluorescent protein (hrGFP) under the control of human GFAP promoter to generate GFAPhrGFP-CB1 -KO and -WT mice, respectively. Furthermore, double immunogold (for CB1 ) and immunoperoxidase (for GFAP or hrGFP) revealed that CB1 receptors are present in astroglial mitochondria from different hippocampal regions of CB1 -WT, GFAP-CB1 -RS and GFAPhrGFP-CB1 -WT mice. Only non-specific gold particles were detected in mouse hippocampi lacking CB1 receptors. Altogether, we demonstrated the existence of a precise molecular architecture of the CB1 receptor in astrocytes that will have to be taken into account in evaluating the functional activity of cannabinergic signaling at the tripartite synapse.

Effects of Voluntary Exercise on Cell Proliferation and Neurogenesis in the Dentate Gyrus of Adult FMR1 Knockout Mice.

Fragile X syndrome (FXS) is the most common cause of inherited intellectual disability that can be traced to a single gene mutation. This disorder is caused by the hypermethylation of the Fmr1 gene, which impairs translation of Fragile X Mental Retardation Protein (FMRP). In Fmr1 knockout (KO) mice, the loss of FMRP has been shown to negatively impact adult hippocampal neurogenesis, and to contribute to learning, memory, and emotional deficits. Conversely, physical exercise has been shown to enhance cognitive performance, emotional state, and increase adult hippocampal neurogenesis. In the current experiments, we used two different voluntary running paradigms to examine how exercise impacts adult neurogenesis in the dorsal and ventral hippocampal dentate gyrus (DG) of Fmr1 KO mice. Immunohistochemical analyses showed that short-term (7 day) voluntary running enhanced cell proliferation in both wild-type (WT) and Fmr1 KO mice. In contrast, long-term (28 day) running only enhanced cell proliferation in the whole DG of WT mice, but not in Fmr1 KO mice. Interestingly, cell survival was enhanced in both WT and Fmr1 KO mice following exercise. Interestingly we found that running promoted cell proliferation and survival in the ventral DG of WTs, but promoted cell survival in the dorsal DG of Fmr1 KOs. Our data indicate that long-term exercise has differential effects on adult neurogenesis in ventral and dorsal hippocampi in Fmr1 KO mice. These results suggest that physical training can enhance hippocampal neurogenesis in the absence of FMRP, may be a potential intervention to enhance learning and memory and emotional regulation in FXS.

Revisiting the flip side: Long-term depression of synaptic efficacy in the hippocampus.

Synaptic plasticity is widely regarded as a putative biological substrate for learning and memory processes. While both decreases and increases in synaptic strength are seen as playing a role in learning and memory, long-term depression (LTD) of synaptic efficacy has received far less attention than its counterpart long-term potentiation (LTP). Never-the-less, LTD at synapses can play an important role in increasing computational flexibility in neural networks. In addition, like learning and memory processes, the magnitude of LTD can be modulated by factors that include stress and sex hormones, neurotrophic support, learning environments, and age. Examining how these factors modulate hippocampal LTD can provide the means to better elucidate the molecular underpinnings of learning and memory processes. This is in turn will enhance our appreciation of how both increases and decreases in synaptic plasticity can play a role in different neurodevelopmental and neurodegenerative conditions.

Effects of pre-natal alcohol exposure on hippocampal synaptic plasticity: Sex, age and methodological considerations.

The consumption of alcohol during gestation is detrimental to the developing central nervous system (CNS). The severity of structural and functional brain alterations associated with alcohol intake depends on many factors including the timing and duration of alcohol consumption. The hippocampal formation, a brain region implicated in learning and memory, is highly susceptible to the effects of developmental alcohol exposure. Some of the observed effects of alcohol on learning and memory may be due to changes at the synaptic level, as this teratogen has been repeatedly shown to interfere with hippocampal synaptic plasticity. At the molecular level alcohol interferes with receptor proteins and can disrupt hormones that are important for neuronal signaling and synaptic plasticity. In this review we examine the consequences of prenatal and early postnatal alcohol exposure on hippocampal synaptic plasticity and highlight the numerous factors that can modulate the effects of alcohol. We also discuss some potential mechanisms responsible for these changes as well as emerging therapeutic avenues that are beginning to be explored.

The Benefits of Exercise on Structural and Functional Plasticity in the Rodent Hippocampus of Different Disease Models.

In this review, the benefits of physical exercise on structural and functional plasticity in the hippocampus are discussed. The evidence is clear that voluntary exercise in rats and mice can lead to increases in hippocampal neurogenesis and enhanced synaptic plasticity which ultimately result in improved performance in hippocampal-dependent tasks. Furthermore, in models of neurological disorders, including fetal alcohol spectrum disorders, traumatic brain injury, stroke, and neurodegenerative disorders including Alzheimer's, Parkinson's and Huntington's disease exercise can also elicit beneficial effects on hippocampal function. Ultimately this review highlights the multiple benefits of exercise on hippocampal function in both the healthy and the diseased brain.

A comparison of the different animal models of fetal alcohol spectrum disorders and their use in studying complex behaviors.

Prenatal ethanol exposure (PNEE) has been linked to widespread impairments in brain structure and function. There are a number of animal models that are used to study the structural and functional deficits caused by PNEE, including, but not limited to invertebrates, fish, rodents, and non-human primates. Animal models enable a researcher to control important variables such as the route of ethanol administration, as well as the timing, frequency and amount of ethanol exposure. Each animal model and system of exposure has its place, depending on the research question being undertaken. In this review, we will examine the different routes of ethanol administration and the various animal models of fetal alcohol spectrum disorders (FASD) that are commonly used in research, emphasizing their strengths and limitations. We will also present an up-to-date summary on the effects of prenatal/neonatal ethanol exposure on behavior across the lifespan, focusing on learning and memory, olfaction, social, executive, and motor functions. Special emphasis will be placed where the various animal models best represent deficits observed in the human condition and offer a viable test bed to examine potential therapeutics for human beings with FASD.

A lateralized odor learning model in neonatal rats for dissecting neural circuitry underpinning memory formation.

Rat pups during a critical postnatal period (≤ 10 days) readily form a preference for an odor that is associated with stimuli mimicking maternal care. Such a preference memory can last from hours, to days, even life-long, depending on training parameters. Early odor preference learning provides us with a model in which the critical changes for a natural form of learning occur in the olfactory circuitry. An additional feature that makes it a powerful tool for the analysis of memory processes is that early odor preference learning can be lateralized via single naris occlusion within the critical period. This is due to the lack of mature anterior commissural connections of the olfactory hemispheres at this early age. This work outlines behavioral protocols for lateralized odor learning using nose plugs. Acute, reversible naris occlusion minimizes tissue and neuronal damages associated with long-term occlusion and more aggressive methods such as cauterization. The lateralized odor learning model permits within-animal comparison, therefore greatly reducing variance compared to between-animal designs. This method has been used successfully to probe the circuit changes in the olfactory system produced by training. Future directions include exploring molecular underpinnings of odor memory using this lateralized learning model; and correlating physiological change with memory strength and durations.

Unlearning: NMDA receptor-mediated metaplasticity in the anterior piriform cortex following early odor preference training in rats.

Here we demonstrate metaplastic effect of a change in NMDA receptor (NMDAR) number in the anterior piriform cortex (aPC) in rat induced by a 10 min pairing of peppermint odor + stroking, which significantly modifies later learning and memory. Using isolated synaptoneurosomes, we found NR1 receptor downregulation 3 h after training and upregulation at 24 h. Consistent with the NR1 pattern, the NMDAR-mediated EPSP was smaller at 3 h and larger at 24 h. Subunit composition was unchanged. Whereas LTP was reduced at both times by training, LTD was facilitated only at 3 h. Behaviorally, pups, given a pairing of peppermint + stroking 3 h after an initial peppermint + stroking training, lost the normally acquired peppermint preference 24 h later. To probe the pathway specificity of this unlearning effect, pups were trained first with peppermint and then, at 3 h, given a second training with peppermint or vanillin. Pups given peppermint training at both times lost the learned peppermint preference. Pups given vanillin retraining at 3 h had normal peppermint preference. Downregulating NR1 with siRNA prevented odor preference learning. Finally, the NMDAR antagonist MK-801 blocked the LTD facilitation seen 3 h after training, and giving MK-801 before the second peppermint training trial eliminated the loss of peppermint odor preference. A training-associated reduction in NMDARs facilitates LTD 3 h later; training at the time of LTD facilitation reverses an LTP-dependent odor preference. Experience-dependent, pathway-specific metaplastic effects in a cortical structure have broad implications for the optimal spacing of learning experiences.

Lateralized odor preference training in rat pups reveals an enhanced network response in anterior piriform cortex to olfactory input that parallels extended memory.

The present study examines synaptic plasticity in the anterior piriform cortex (aPC) using ex vivo slices from rat pups given lateralized odor preference training. In the early odor preference learning model, a brief 10 min training session yields 24 h memory, while four daily sessions yield 48 h memory. Odor preference memory can be lateralized through naris occlusion as the anterior commissure is not yet functional. AMPA receptor-mediated postsynaptic responses in the aPC to lateral olfactory tract input, shown to be enhanced at 24 h, are no longer enhanced 48 h after a single training session. Following four spaced lateralized trials, the AMPA receptor-mediated fEPSP is enhanced in the trained aPC at 48 h. Calcium imaging of aPC pyramidal cells within 48 h revealed decreased firing thresholds in the pyramidal cell network. Thus multiday odor preference training induced increased odor input responsiveness in previously weakly activated aPC cells. These results support the hypothesis that increased synaptic strength in olfactory input networks mediates odor preference memory. The increase in aPC network activation parallels behavioral memory.

A role for the anterior piriform cortex in early odor preference learning: evidence for multiple olfactory learning structures in the rat pup.

cFos activation in the anterior piriform cortex (aPC) occurs in early odor preference learning in rat pups (Roth and Sullivan 2005). Here we provide evidence that the pairing of odor as a conditioned stimulus and ß-adrenergic activation in the aPC as an unconditioned stimulus generates early odor preference learning. ß-Adrenergic blockade in the aPC prevents normal preference learning. Enhancement of aPC cAMP response element-binding protein (CREB) phosphorylation in trained hemispheres is consistent with a role for this cascade in early odor preference learning in the aPC. In vitro experiments suggested theta-burst-mediated long-term potentiation (LTP) at the lateral olfactory tract (LOT) to aPC synapse depends on N-methyl-D-aspartate (NMDA) receptors and can be significantly enhanced by ß-adrenoceptor activation, which causes increased glutamate release from LOT synapses during LTP induction. NMDA receptors in aPC are also shown to be critical for the acquisition, but not expression, of odor preference learning, as would be predicted if they mediate initial ß-adrenoceptor-promoted aPC plasticity. Ex vivo experiments 3 and 24 h after odor preference training reveal an enhanced LOT-aPC field excitatory postsynaptic potential (EPSP). At 3 h both presynaptic and postsynaptic potentiations support EPSP enhancement while at 24 h only postsynaptic potentiation is seen. LOT-LTP in aPC is excluded by odor preference training. Taken together with earlier work on the role of the olfactory bulb in early odor preference learning, these outcomes suggest early odor preference learning is normally supported by and requires multiple plastic changes at least at two levels of olfactory circuitry.

Strain differences in a high response-cost daily time-place learning task.

Previous research has shown that rats, unlike birds, do not readily demonstrate daily time-place learning (TPL). It has been suggested, however, that rats are more successful at these tasks if the response cost (RC) is increased. Widman et al. (2000) found that female Sprague Dawley (SD) rats learned a daily TPL task in which they were required to climb different towers depending on the time of day to find a food reward. Using a similar apparatus, we found that male SD rats learned the task, while male Long Evans rats did not. While all rats quickly learned to restrict the majority of their searching to the two towers that provided food, only the SD rats learned to go to the correct location at the correct time of day. Thus, there appears to be a strain difference in the effectiveness of a high RC task to promote learning. Tests of the timing strategies used revealed individual differences with one rat using a circadian strategy and another using an ordinal strategy. Post criterion decrements in performance did not allow sufficient testing to determine the timing strategies of the remaining rats. Possible interactions between strain, response cost, species typical behaviors and dependent measures are discussed.