Alcohol-preferring Rats Research Articles

Excessive alcohol intake produces persistent mechanical allodynia and dysregulates the endocannabinoid system in the lumbar dorsal root ganglia of genetically-selected Marchigian Sardinian alcohol-preferring rats

Epidemiological data indicate a strong association between alcohol use disorder (AUD) and neuropathic pain. Genetically-selected Marchigian Sardinian alcohol-preferring (msP) rats exhibit a high preference for alcohol compared with their background strain (Wistar rats), but their sensitivity to mechanical allodynia after chronic alcohol exposure is unknown. The present study compared the development of mechanical allodynia between “low, non-pathological drinker” Wistar rats and “high drinker” msP rats using the two-bottle choice (2BC) free-access procedure. Several studies reported the involvement of endocannabinoids (eCBs) in modulating mechanical allodynia, but there are no data on their role in alcohol-related allodynia. Thus, the present study assessed eCBs and their related lipid species in lumbar dorsal root ganglia (DRG) and correlated them with mechanical allodynia in our model. We found that male and female msP rats developed persistent mechanical allodynia during protracted abstinence from alcohol, presenting no sign of recovery, as opposed to Wistar rats. This effect directly correlated with their total alcohol intake. Notably, we found a correlation between lower lumbar DRG 2-arachidonoylglycerol (2-AG) levels and the development of higher mechanical allodynia during abstinence in msP rats of both sexes but not in Wistar rats. Moreover, alcohol-exposed and abstinent msP and Wistar females but not males exhibited significant alterations of thromboxane B2 and prostaglandin E2/prostaglandin D2 compared with naive rats. These findings demonstrate that DRG 2-AG metabolism is altered in msP rats during prolonged abstinence and represents a potentially interesting pharmacological target for the treatment of mechanical allodynia during alcohol abstinence.

Neurosteroid [3α,5α]3-hydroxypregnan-20-one inhibition of chemokine monocyte chemoattractant protein-1 in alcohol-preferring rat brain neurons, microglia, and astroglia.

Neuroimmune dysfunction in alcohol use disorder (AUD) is associated with activation of myeloid differentiation primary response 88 (MyD88)-dependent Toll-like receptors (TLR) resulting in overexpression of the chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2). MCP-1 overexpression in the brain is linked to anxiety, higher alcohol intake, neuronal death, and activation of microglia observed in AUD. The neurosteroid [3α,5α][3-hydroxypregnan-20-one (3α,5α-THP) has been reported as an inhibitor of MyD88-dependent TLR activation and MCP-1 overexpression in mouse and human macrophages and the brain of alcohol-preferring (P) rats. We investigated how 3α,5α-THP regulates MCP-1 expression at the cellular level in P rat nucleus accumbens (NAc) and central amygdala (CeA). We focused on neurons, microglia, and astrocytes, examining the individual voxel density of MCP-1, neuronal marker NeuN, microglial marker IBA1, astrocytic marker GFAP, and their shared voxel density, defined as intersection. Ethanol-naïve male and female P rats were perfused 1 h after IP injections of 15 mg/kg of 3α,5α-THP, or vehicle. The NAc and CeA were imaged using confocal microscopy following double-immunofluorescence staining for MCP-1 with NeuN, IBA1, and GFAP, respectively. MCP-1 intersected with NeuN predominantly and IBA1/GFAP negligibly. 3α,5α-THP reduced MCP-1 expression in NeuN-labeled cells by 38.27 ± 28.09% in male and 56.11 ± 21.46% in female NAc, also 37.99 ± 19.53% in male and 54.96 ± 30.58% in female CeA. In females, 3α,5α-THP reduced the MCP-1 within IBA1 and GFAP-labeled voxels in the NAc and CeA. Conversely, in males, 3α,5α-THP did not significantly alter the MCP-1 within IBA1 in NAc or with GFAP in the CeA. Furthermore, 3α,5α-THP decreased levels of IBA1 in both regions and sexes with no impact on GFAP or NeuN levels. Secondary analysis performed on data normalized to % control values indicated that no significant sex differences were present. These data suggest that 3α,5α-THP inhibits neuronal MCP-1 expression and decreases the proliferation of microglia in P rats. These results increase our understanding of potential mechanisms for 3α,5α-THP modulation of ethanol consumption.

Neurosteroid [3α,5α]-3-Hydroxy-pregnan-20-one Enhances the CX3CL1-CX3CR1 Pathway in the Brain of Alcohol-Preferring Rats with Sex-Specificity.

This study investigates the impact of allopregnanolone ([3α,5α]3-hydroxypregnan-20-one or 3α,5α-tetrahydroprogesterone (3α,5α-THP); 10 mg/kg, IP) on fractalkine/CX3-C motif chemokine ligand 1 (CX3CL1) levels, associated signaling components, and markers for microglial and astrocytic cells in the nucleus accumbens (NAc) of male and female alcohol-preferring (P) rats. Previous research suggested that 3α,5α-THP enhances anti-inflammatory interleukin-10 (IL-10) cytokine production in the brains of male P rats, with no similar effect observed in females. This study reveals that 3α,5α-THP elevates CX3CL1 levels by 16% in the NAc of female P rats, with no significant changes observed in males. The increase in CX3CL1 levels induced by 3α,5α-THP was observed in females across multiple brain regions, including the NAc, amygdala, hypothalamus, and midbrain, while no significant effect was noted in males. Additionally, female P rats treated with 3α,5α-THP exhibited notable increases in CX3CL1 receptor (CX3CR1; 48%) and transforming growth factor-beta 1 (TGF-β1; 24%) levels, along with heightened activation (phosphorylation) of signal transducer and activator of transcription 1 (STAT1; 85%) in the NAc. Conversely, no similar alterations were observed in male P rats. Furthermore, 3α,5α-THP decreased glial fibrillary acidic protein (GFAP) levels by 19% in both female and male P rat NAc, without affecting microglial markers ionized calcium-binding adaptor molecule 1 (IBA1) and transmembrane protein 119 (TMEM119). These findings indicate that 3α,5α-THP enhances the CX3CL1/CX3CR1 pathway in the female P rat brain but not in males, primarily influencing astrocyte reactivity, with no observed effect on microglial activation.

Effects of MC-100093 on Ethanol Drinking and the Expression of Astrocytic Glutamate Transporters in the Mesocorticolimbic Brain Regions of Male and Female Alcohol-Preferring Rats

Chronic ethanol consumption increased extracellular glutamate concentrations in several reward brain regions. Glutamate homeostasis is regulated in majority by astrocytic glutamate transporter 1 (GLT-1) as well as the interactive role of cystine/glutamate antiporter (xCT). In this study, we aimed to determine the attenuating effects of a novel beta-lactam MC-100093, lacking the antibacterial properties, on ethanol consumption and GLT-1 and xCT expression in the subregions of nucleus accumbens (NAc core and NAc shell) and medial prefrontal cortex (Infralimbic, mPFC-IL and Prelimbic, mPFC-PL) in male and female alcohol-preferring (P) rats. Female and male rats were exposed to free access to ethanol (15% v/v) and (30% v/v) and water for five weeks, and on Week 6, rats were administered 100 mg/kg (i.p) of MC-100093 or saline for five days. MC-100093 reduced ethanol consumption in both male and female P rats from Day 1–5. Additionally, MC-100093 upregulated GLT-1 and xCT expression in the mPFC and NAc subregions as compared to ethanol-saline groups in female and male rats. Chronic ethanol intake reduced GLT-1 and xCT expression in the IL and PL in female and male rats, except there was no reduction in GLT-1 expression in the mPFC-PL in female rats. Although, MC-100093 upregulated GLT-1 and xCT expression in the subregions of NAc, we did not observe any reduction in GLT-1 and xCT expression with chronic ethanol intake in female rats. These findings strongly suggest that MC-100093 treatment effectively reduced ethanol intake and upregulated GLT-1 and xCT expression in the mPFC and NAc subregions in male and female P rats.

Neurosteroid [3α,5α]-3-hydroxy-pregnan-20-one enhances IL-10 production via endosomal TRIF-dependent TLR4 signaling pathway.

Previous studies demonstrated the inhibitory effect of allopregnanolone (3α,5α-THP) on the activation of inflammatory toll-like receptor 4 (TLR4) signals in RAW264.7 macrophages and the brains of selectively bred alcohol-preferring (P) rats. In the current study, we investigated the impact of 3α,5α-THP on the levels of IL-10 and activation of the TRIF-dependent endosomal TLR4 pathway. The amygdala and nucleus accumbens (NAc) of P rats, which exhibit innately activated TLR4 pathways as well as RAW264.7 cells, were used. Enzyme-linked immunosorbent assays (ELISA) and immunoblotting assays were used to ascertain the effects of 3α,5α-THP on the TRIF-dependent endosomal TLR4 pathway and endosomes were isolated to examine translocation of TLR4 and TRIF. Additionally, we investigated the effects of 3α,5α-THP and 3α,5α-THDOC (0.1, 0.3, and 1.0 µM) on the levels of IL-10 in RAW264.7 macrophages. Finally, we examined whether inhibiting TRIF (using TRIF siRNA) in RAW264.7 cells altered the levels of IL-10. 3α,5α-THP administration facilitated activation of the endosomal TRIF-dependent TLR4 pathway in males, but not female P rats. 3α,5α-THP increased IL-10 levels (+13.2 ± 6.5%) and BDNF levels (+21.1 ± 11.5%) in the male amygdala. These effects were associated with increases in pTRAM (+86.4 ± 28.4%), SP1 (+122.2 ± 74.9%), and PI(3)K-p110δ (+61.6 ± 21.6%), and a reduction of TIRAP (-13.7 ± 6.0%), indicating the activation of the endosomal TRIF-dependent TLR4 signaling pathway. Comparable effects were observed in NAc of these animals. Furthermore, 3α,5α-THP enhanced the accumulation of TLR4 (+43.9 ± 11.3%) and TRIF (+64.8 ± 32.8%) in endosomes, with no significant effect on TLR3 accumulation. Additionally, 3α,5α-THP facilitated the transition from early endosomes to late endosomes (increasing Rab7 levels: +35.8 ± 18.4%). In RAW264.7 cells, imiquimod (30 µg/mL) reduced IL-10 while 3α,5α-THP and 3α,5α-THDOC (0.1, 0.3, and 1.0 µM) restored IL-10 levels. To determine the role of the TRIF-dependent TLR4 signaling pathway in IL-10 production, the downregulation of TRIF (-62.9 ± 28.2%) in RAW264.7 cells led to a reduction in IL-10 levels (-42.3 ± 8.4%). TRIF (-62.9 ± 28.2%) in RAW264.7 cells led to a reduction in IL-10 levels (-42.3 ± 8.4%) and 3α,5α-THP (1.0 µM) no longer restored the reduced IL-10 levels. The results demonstrate 3α,5α-THP enhancement of the endosomal TLR4-TRIF anti-inflammatory signals and elevations of IL-10 in male P rat brain that were not detected in female P rat brain. These effects hold significant implications for controlling inflammatory responses in both the brain and peripheral immune cells.

Early social isolation differentially affects the glucocorticoid receptor system and alcohol-seeking behavior in male and female Marchigian Sardinian alcohol-preferring rats

Adverse early life experiences during postnatal development can evoke long-lasting neurobiological changes in stress systems, thereby affecting subsequent behaviors including propensity to develop alcohol use disorder. Here, we exposed genetically selected male and female Marchigian Sardinian alcohol-preferring (msP) and Wistar rats to mild, repeated social deprivation from postnatal day 14 (PND14) to PND21 and investigated the effect of the early social isolation (ESI) on the glucocorticoid receptor (GR) system and on the propensity to drink and seek alcohol in adulthood. We found that ESI resulted in higher levels of GR gene and protein expression in the prefrontal cortex (PFC) in male but not female msP rats. In female Wistars, ESI resulted in significant downregulation of Nr3c1 mRNA levels and lower GR protein levels. In male and female msP rats, plasma corticosterone levels on PND35 were similar and unaffected by ESI. Wistar females exhibited higher levels of corticosterone compared with males, independently from ESI. In alcohol self-administration experiments we found that the pharmacological stressor yohimbine (0.0, 0.312, 0.625, and 1.25 mg/kg) increased alcohol self-administration in both rat lines, regardless of ESI. After extinction, 0.625 mg/kg yohimbine significantly reinstated alcohol seeking in female rats only. ESI enhanced reinstatement in female msP rats. Overall, the present results indicate that repeated social deprivation during the third week of postnatal life affects GR expression in a strain- and sex-dependent manner: such effect may contribute, at least partially, to the heightened sensitivity of female msP rats to the effects of yohimbine-induced alcohol seeking.

Delving into the reducing effects of the GABAB positive allosteric modulator, KK-92A, on alcohol-related behaviors in rats.

Recent studies have demonstrated the ability of the positive allosteric modulator (PAM) of the GABAB receptor (GABAB PAM), KK-92A, to suppress operant alcohol self-administration and reinstatement of alcohol seeking in selectively bred Sardinian alcohol-preferring (sP) rats. The present study was designed to scrutinize the suppressing effects of KK-92A on alcohol-related behaviors; to this end, four separate experiments were conducted to address just as many new research questions, some of which bear translational value. Experiment 1 found that 7-day treatment with KK-92A (0, 5, 10, and 20 mg/kg, intraperitoneally [i.p.]) effectively reduced alcohol intake in male sP rats exposed to the home-cage 2-bottle "alcohol (10% v/v) vs. water" choice regimen with 1 hour/day limited access, extending to excessive alcohol drinking the ability of KK-92A to suppress operant alcohol self-administration. Experiment 2 demonstrated that the ability of KK-92A to reduce lever-responding for alcohol was maintained also after acute, intragastric treatment (0, 20, and 40 mg/kg) in female sP rats trained to lever-respond for 15% (v/v) alcohol under the fixed ratio 5 schedule of reinforcement. In Experiment 3, acutely administered KK-92A (0, 5, 10, and 20 mg/kg, i.p.) dampened alcohol-seeking behavior in female sP rats exposed to a single session under the extinction responding schedule. Experiment 4 used a taste reactivity test to demonstrate that acute treatment with KK-92A (0 and 20 mg/kg, i.p.) did not alter either hedonic or aversive reactions to a 15% (v/v) alcohol solution in male sP rats, ruling out that KK-92A-induced reduction of alcohol drinking and self-administration could be due to alterations in alcohol palatability. Together, these results enhance the behavioral pharmacological profile of KK-92A and further strengthen the notion that GABAB PAMs may represent a novel class of ligands with therapeutic potential for treating alcohol use disorder.

Sexually dimorphic effects of monoacylglycerol lipase inhibitor MJN110 on stress-related behaviour and drinking in Marchigian Sardinian alcohol-preferring rats.

The endocannabinoid (eCB) system plays an important homeostatic role in the regulation of stress circuits and has emerged as a therapeutic target to treat stress disorders and alcohol use disorder (AUD). Extensive research has elucidated a role for the eCB anandamide (AEA), but less is known about 2-arachidonoylglycerol (2-AG) mediated signalling. We pharmacologically enhanced eCB signalling by inhibiting the 2-AG metabolizing enzyme, monoacylglycerol lipase (MAGL), in male and female Marchigian Sardinian alcohol-preferring (msP) rats, a model of innate alcohol preference and stress hypersensitivity, and in control Wistar rats. We tested the acute effect of the selective MAGL inhibitor MJN110 in alleviating symptoms of alcohol drinking, anxiety, irritability and fear. A single systemic administration of MJN110 increased 2-AG levels in the central amygdala, prelimbic and infralimbic cortex but did not acutely alter alcohol drinking. MAGL inhibition reduced aggressive behaviours in female msPs, and increased defensive behaviours in male msPs, during the irritability test. Moreover, in the novelty-induced hypophagia test, MJN110 selectively enhanced palatable food consumption in females, mitigating stress-induced food suppression. Lastly, msP rats showed increased conditioned fear behaviour compared with Wistar rats, and MJN110 reduced context-associated conditioned fear responses, but not cue-probed fear expression, in male msPs. Acute inhibition of MAGL attenuated some stress-related responses in msP rats but not voluntary alcohol drinking. Our results provide new insights into the sex dimorphism documented in stress-induced responses. Sex-specific eCB-based approaches should be considered in the clinical development of therapeutics.

Reduction of nicotine and ethanol intake in alcohol-preferring (UChB) female rats by the α4β2 nicotinic acetylcholine receptor partial agonists 5-bromocytisine and cytisine

RationaleNeuronal nicotinic acetylcholine receptors (nAChRs) are implicated in the reinforcing effects of nicotine and ethanol. Previous studies have shown that cytisine and its 5-bromo derivative are partial agonists at the α4β2 nAChRs and that the parent molecule cytisine is effective in reducing both nicotine- and ethanol-self-administration in rats. However, whether 5-bromocytisine affects nicotine or ethanol self-administration was unknown. ObjectivesThe present study compared the effects of 5-bromocytisine and cytisine on nicotine self-administration and further assessed the effect of daily drug injection on voluntary ethanol consumption in alcohol-preferring female rats. Animals were administered a 1.5mg/kg i.p. dose of 5-bromocytisine or cytisine every day for 15–16 days. ResultsThe initial efficacy of 5-bromocytisine and cytisine in reducing nicotine intake was similar (−80%) while for voluntary ethanol intake 5-bromocytisine was a superior inhibitor over cytisine (−78% and −40% respectively). The efficacy of cytisine began to diminish after 10 days of daily administration, which was attributed to tolerance development to its inhibitory effects both on nicotine and ethanol self-administration. Tolerance did not develop for 5-bromocytisine. Conclusion5-Bromocytisine, a weaker α4β2 nAChR partial agonist than cytisine, also produces a sustained inhibition of both nicotine and ethanol self-administration, and unlike cytisine, it does not develop tolerance.

Exposure to an enriched environment exerts anxiolytic effects in Sardinian alcohol-preferring rats

Exposure to an enriched environment (EE) has been reported to generate multiple beneficial effects in rodents, including – among the many – amelioration of anxiety-related behaviors. The present study investigated whether living in an EE produced anxiolytic effects also in selectively bred Sardinian alcohol-preferring (sP) rats. The relevance of this research question relied on two factors: sP rats displayed an inherent, high anxiety-like state under different experimental conditions; exposure to EE reduced operant, oral alcohol self-administration in sP rats. Starting from weaning, male sP rats were kept under 3 different housing conditions: impoverished environment (IE; single housing with no environmental enrichment); standard environment (SE; 3 rats/cage with no environmental enrichment); EE (6 rats/cage with various elements of environmental enrichment). At the age of approximately 80 days, rats were exposed to an elevated plus maze test for assessment of anxiety-related behaviors. Compared to IE and SE rats, EE rats displayed higher basal levels of exploratory activity (i.e., increased number of entries into closed arms). Compared to IE and SE rats, EE rats also displayed a less “anxious” profile, as suggested by the increase in percent number of entries into open arms (OAs), percent time spent in OAs, number of head dips, and number of end-arm explorations in OAs. These data extend the protective (anxiolytic) effects of EE to a proposed animal model of comorbid alcohol use disorder and anxiety disorders.

The administration of Alda-1, an activator of ALDH2, inhibits relapse-like ethanol intake in female alcohol-preferring UChB rats

AimsAlcohol relapse is a main limitation for the treatment of alcohol use disorders. Previous studies have shown that Alda-1, a pharmacological activator of ALDH2, inhibits both acquisition and chronic ethanol intake in rats; however, its effects on relapse-like ethanol intake are unknown. The aim of this study was to assess the effect of Alda-1 on post-deprivation and reaccess relapse-like ethanol intake in alcohol-preferring UChB rats. We also aimed to assess the possible mechanisms associated with the effects of Alda-1 by measuring the levels of glutamate transporter (GLT-1), oxidative stress and neuroinflammation markers in different regions of the mesocorticolimbic system. Main methodsIn Experiment I, UChB female rats were exposed for 100 days to voluntary ethanol intake followed by 2-weeks of ethanol withdrawal and 1 week of ethanol reaccess. Alda-1 (25 mg/kg, intragastric, i.g) or vehicle was administered daily for 14 days during the withdrawal/re-access period. Experiment II was similar to Experiment I, but after the withdrawal period, ethanol re-access was not allowed, and Alda-1 was administered during the last week of withdrawal. At the end of both experiments, the levels of GLT-1, oxidative stress (GSH, MDA), and neuroinflammation markers (GFAP, Iba-1) were assessed in nucleus accumbens and/or hippocampus. Key findingsThe results showed that Alda-1 administration markedly blocked (90 %, p < 0.001) relapse-like ethanol intake in UChB rats. Alda-1 increased Iba-1 reactivity (microglial marker) in the NAc of ethanol-deprived rats. Alda-1 administration did not influence the levels of GLT-1, oxidative stress markers (MDA, GSH) or GFAP reactivity in the mesocorticolimbic system. SignificanceThese preclinical findings support the use of activators of ALDH2, such as Alda-1, as a potential pharmacological strategy in the treatment of alcohol relapse.

Psilocybin prevents reinstatement of alcohol seeking by disrupting the reconsolidation of alcohol-related memories.

For most psychiatric conditions, including alcohol use disorder (AUD), FDA-approved pharmacological treatments are limited and their efficacy is restricted to only certain subgroups of patients. Scientific interest in the potential of psychedelic drugs has dramatically increased because of clinical preliminary evidence of efficacy in treating various psychiatric disorders. One of the most promising compounds belonging to this class of molecules is psilocybin. Here, to elucidate the therapeutic potential and treatment modalities of this drug, we investigated the effect of psilocybin on alcohol drinking and seeking in genetically selected Marchigian Sardinian alcohol-preferring (msP) rats, a well validated animal model of AUD characterized by excessive drinking and seeking. Using male and female msP rats, we tested the effect of psilocybin on home cage voluntary alcohol consumption. We also tested the effect of the drug on the alcohol deprivation effect (ADE) model of relapse and on cue-induced reinstatement of alcohol seeking after a period of abstinence. Finally, we evaluated if psilocybin may disrupt the reconsolidation process of alcohol-related memory. Psilocybin did not reduce alcohol consumption, nor it prevented increased alcohol drinking after a period of forced abstinence and cue-induced reinstatement of alcohol-seeking. Noteworthy, in a memory retrieval-reconsolidation paradigm, psilocybin markedly attenuated resumption of alcohol seeking. Altogether these data suggest that, despite psilocybin does not affect alcohol drinking and relapse, it may be highly effective if used to block the reconsolidation process of alcohol-related memories. This opens to the possibility of using this psychedelic drug in clinical settings in which AUD patients undergo procedures to recall the memory of alcohol and are then treated with psilocybin during the memory reconsolidation phase.

Reducing Effect of the Novel Positive Allosteric Modulator of the GABAB Receptor, KK-92A, on Multiple Alcohol-Related Behaviors in Alcohol-Preferring Rats

Reducing Effect of the Novel Positive Allosteric Modulator of the GABAB Receptor, KK-92A, on Multiple Alcohol-Related Behaviors in Alcohol-Preferring Rats

Genetically Selected Marchigian Sardinian Alcohol Preferring (msP) Rats to Study Excessive Alcohol Drinking Associated with Innate Stress Hypersensitivity: The Role of the Nociceptin/Orphanin FQ NOP Receptor System

Genetically Selected Marchigian Sardinian Alcohol Preferring (msP) Rats to Study Excessive Alcohol Drinking Associated with Innate Stress Hypersensitivity: The Role of the Nociceptin/Orphanin FQ NOP Receptor System

Comorbid Anxiety and Heightened Stress Response are Associated with Excessive Alcohol Intake in Male and Female Genetically Selected Marchigian Sardinian Alcohol Preferring Rats

Comorbid Anxiety and Heightened Stress Response are Associated with Excessive Alcohol Intake in Male and Female Genetically Selected Marchigian Sardinian Alcohol Preferring Rats

Inhibitory and excitatory alcohol-seeking cues distinct roles in behavior, neurochemistry, and mesolimbic pathway in alcohol preferring (P) rats

Cues associated with alcohol use can readily enhance self-reported cravings for alcohol, which increases the likelihood of reusing alcohol. Understanding the neuronal mechanisms involved in alcohol-seeking behavior is important for developing strategies to treat alcohol use disorder. In all experiments, adult female alcohol-preferring (P) rats were exposed to three conditioned odor cues; CS+ associated with EtOH self-administration, CS- associated with the absence of EtOH (extinction training), and a CS0, a neutral stimulus. The data indicated that presentation of an excitatory conditioned cue (CS+) can enhance EtOH- seeking while the CS- can inhibit EtOH-seeking under multiple test conditions. Presentation of the CS+ activates a subpopulation of dopamine neurons within the interfascicular nucleus of the posterior ventral tegmental area (posterior VTA) and basolateral amygdala (BLA). Pharmacological inactivation of the BLA with GABA agonists inhibits the ability of the CS+ to enhance EtOH-seeking but does not alter context-induced EtOH-seeking or the ability of the CS- to inhibit EtOH-seeking. Presentation of the conditioned odor cues in a non-drug-paired environment indicated that presentation of the CS+ increased dopamine levels in the BLA. In contrast, presentation of the CS- decreased both glutamate and dopamine levels in the BLA. Further analysis revealed that presentation of a CS+ EtOH-associated conditioned cue activates GABA interneurons but not glutamate projection neurons. Overall, the data indicate that excitatory and inhibitory conditioned cues can contrarily alter EtOH-seeking behaviors and that different neurocircuitries are mediating these distinct cues in critical brain regions. Pharmacotherapeutics for craving should inhibit the CS+ and enhance the CS- neurocircuits.

MRI texture-based radiomics analysis for the identification of altered functional networks in alcoholic patients and animal models.

Alcohol use disorder (AUD) is a complex condition representing a leading risk factor for death, disease and disability. Its high prevalence and severe health consequences make necessary a better understanding of the brain network alterations to improve diagnosis and treatment. The purpose of this study was to evaluate the potential of resting-state fMRI 3D texture features as a novel source of biomarkers to identify AUD brain network alterations following a radiomics approach. A longitudinal study was conducted in Marchigian Sardinian alcohol-preferring msP rats (N=36) who underwent resting-state functional and structural MRI before and after 30 days of alcohol or water consumption. A cross-sectional human study was also conducted among 33 healthy controls and 35 AUD patients. The preprocessed functional data corresponding to control and alcohol conditions were used to perform a probabilistic independent component analysis, identifying seven independent components as resting-state networks. Forty-three radiomic features extracted from each network were compared using a Wilcoxon signed-rank test with Holm correction to identify the network most affected by alcohol consumption. Features extracted from this network were then used in the machine learning process, evaluating two feature selection methods and six predictive models within a nested cross-validation structure. The classification was evaluated by computing the area under the ROC curve. Images were quantized using different numbers of gray-levels to test their influence on the results. The influence of ageing, data preprocessing, and brain iron accumulation were also analyzed. The methodology was validated using structural scans. The striatal network in alcohol-exposed msP rats presented the most significant number of altered features. The radiomics approach supported this result achieving good classification performance in animals (AUC = 0.915±0.100, with 12 features) and humans (AUC = 0.724±0.117, with 9 features) using a random forest model. Using the structural scans, high accuracy was achieved with a multilayer perceptron in both species (animals: AUC > 0.95 with 2 features, humans: AUC > 0.82 with 18 features). The best results were obtained using a feature selection method based on the p-value. The proposed radiomics approach is able to identify AUD patients and alcohol-exposed rats with good accuracy, employing a subset of 3D features extracted from fMRI. Furthermore, it can help identify relevant networks in drug addiction.

Pharmacological administration of 3α,5α-THP into the nucleus accumbens core increases 3α,5α-THP expression and reduces alcohol self-administration.

Alcohol affects multiple circuits in the brain, mainly disrupting the delicate balance between inhibitory γ-aminobutyric acid (GABA) transmission and excitatory glutamate signaling in brain areas involved in reward circuits. These include the amygdala, nucleus accumbens (Acb), and ventral tegmental area (VTA). This action impairs circuits that regulate behavioral control of craving and alcohol seeking and intake. Studies in both rodent models and postmortem human brain of patients with alcohol use disorder (AUD) have highlighted the association between the loss of GABAergic inhibition and the development of addiction. The neurosteroid (3α,5α)-3-hydroxypregnan-20-one (3α,5α-THP) is a potent positive modulator of GABAA receptors. Chronic alcohol consumption reduces 3α,5α-THP levels, resulting in decreased GABA inhibition. We previously demonstrated that enhancing neurosteroid biosynthesis by overexpression of the cholesterol side-chain cleavage enzyme P450scc decreased alcohol intake in male alcohol-preferring rats (P-rats). While most of the evidence of alcohol-induced alterations comes from studies in male subjects, some data show that females are more vulnerable to alcohol's effects than males. In this study, we investigated the ability of 3α,5α-THP direct infusions in two brain regions that contribute to alcohol reinforcement, the VTA and Acb core (AcbC), to regulate alcohol self-administration in female P-rats. Administration of 3α,5α-THP into the AcbC increased 3α,5α-THP-positive cell expression in this area and reduced alcohol self-administration. By contrast, 3α,5α-THP infusion into the VTA did not significantly affect alcohol self-administration, though trends for a reduction were found. Our results show that local increases in 3α,5α-THP in the AcbC may alter mesolimbic activity that drives a reduction in alcohol self-administration.

Environmental enrichment augments binge-like alcohol drinking in Sardinian alcohol-preferring rats.

Exposure of Sardinian alcohol-preferring (sP) rats to an enriched environment (EE) reduced different aspects of operant alcohol self-administration. The present study was aimed at expanding investigation of the effect of EE exposure upon a model of binge drinking composed of daily 1-h drinking sessions with unpredictable access to multiple alcohol concentrations; binge-like alcohol intakes were observed when the drinking session occurred at the last hours of the dark phase of the light/dark cycle. Starting from postnatal day (PND) 21, male sP rats were kept under three different housing conditions: impoverished environment (IE; single housing with no environmental enrichment); standard environment (SE; 3 rats/cage and no environmental enrichment); EE (6 rats/cage and multiple elements of environmental enrichment). From PND 69, rats were exposed daily to a 1-hour drinking session under the 4-bottle "alcohol (10%, 20%, and 30%, v/v) vs. water" choice regimen, during the dark phase, and with timing of alcohol exposure changed each day. In all three rat groups (IE, SE, and EE), alcohol intake increased progressively as the drinking session moved from the first to last hours of the dark phase. The slope of the regression line was steeper in EE than IE and SE rats, suggestive of higher intakes of alcohol in EE than IE and SE rats when the drinking session occurred over the last hours of the dark phase. These results are discussed hypothesizing that the stressful attributes of alcohol expectation were potentiated by the increased "emotionality" that rats living in a comfortable environment (i.e., EE) may experience when facing new, challenging events or environments. Blood alcohol levels, assessed at the end of a final drinking session occurring at the 12th hour of the dark phase, did not differ among the three rat groups and averaged approximately 150mg%, confirming that this experimental procedure may generate intoxicating levels of alcohol drinking in sP rats.

The Effects of Transcranial Focused Ultrasound Stimulation of Nucleus Accumbens on Neuronal Gene Expression and Brain Tissue in High Alcohol-Preferring Rats.

We investigated the effect of low-intensity focused ultrasound (LIFU) on gene expression related to alcohol dependence and histological effects on brain tissue. We also aimed at determining the miRNA-mRNA relationship and their pathways in alcohol dependence-induced expression changes after focused ultrasound therapy. We designed a case-control study for 100days of observation to investigate differences in gene expression in the short-term stimulation group (STS) and long-term stimulation group (LTS) compared with the control sham group (SG). The study was performed in our Experimental Research Laboratory. 24 male high alcohol-preferring rats 63 to 79days old, weighing 270 to 300g, were included in the experiment. LTS received 50-day LIFU and STS received 10-day LIFU and 40-day sham stimulation, while the SG received 50-day sham stimulation. In miRNA expression analysis, it was found that LIFU caused gene expression differences in NAc. Significant differences were found between the groups for gene expression. Compared to the SG, the expression of 454 genes in the NAc region was changed in the STS while the expression of 382 genes was changed in the LTS. In the LTS, the expression of 32 genes was changed in total compared to STS. Our data suggest that LIFU targeted on NAc may assist in the treatment of alcohol dependence, especially in the long term possibly through altering gene expression. Our immunohistochemical studies verified that LIFU does not cause any tissue damage. These findings may lead to new studies in investigating the efficacy of LIFU for the treatment of alcohol dependence and also for other psychiatric disorders.