Comparative compositional analysis of the gut microbiome in animal models of addiction and stress

Intro: The microbiome-gut-brain axis (MGBA) has been shown to be instrumental to brain and behaviour, including psychological disorders affecting stress, depression, and anxiety. Many of the aforementioned disorders are comorbid with substance abuse disorders, and on a larger scale with addiction. In this body of work, we seek to characterize addiction-related phenotypes in the microbiome and correlate with measures of addiction-related behaviours, along with changes in the virome following chronic social stress. Aims: We first examined if vapor administration of ethanol is capable of altering the microbiome and to assess if substances of abuse, such as ethanol, can alter the microbiome outside of oral/gastrointestinal administration. To characterize addictive phenotypes behavioural measures of impulsivity, reward learning, and dopaminergic response to novelty were used. Methods: All studies were carried out in rodents. Following behavioural testing, gut microbiota samples were analysed with 16S rRNA gene amplicon sequencing. In the chronic social stress study, the virome was also sequenced via shot-gun metagenomic sequencing of faecal filtrate. Correlations were performed between microbiome measures and biological measures of behaviour, cytokines, corticosterone, and dopamine mRNA expression. Results: Chapter 2) Investigation of microbiome and vapor alcohol administration in mice showed significant alterations in microbiome; Chapter 3) A pre-clinical model of alcohol-addiction showed no significant differences in the microbiome by grouped phenotype. However, low abundance genus-level bacteria correlated to striatal dopamine mRNA expression. Chapter 4) A large heterogenous study of male and female rats revealed that female microbiome composition is more associated to addictive measures. However, within both male and female operational taxonomic units (OTUs) were significantly correlated to impulsivity measures. Chapter 5) Chronic social stress in male mice significantly affected bacterial and viral 1-14 composition. Viral richness was increased alongside decreases in bacterial richness. Discussion: Results from this body of work show that the microbiome may be impacted by addiction and psychosocial stress. Our findings suggest that there are subtle yet significant differences in microbiome composition between rats with different behavioural phenotypes. These findings indicate that associations to addictive-related behaviour occur in low-abundance (<5%) bacteria and are sex-specific. Furthermore, microbiome (bacteriome and virome) is impacted top-down, brain to gut via the hypothalamic-pituitary-adrenal axis, during chronic psychosocial stress. These initial investigations into stress and gut bacteriophage (aka phage) indicate that increases in phage richness are associated to decreases in bacterial richness and alpha diversity during stress. These novel findings seed the foundation for new research investigating MGBA in stress, substance abuse, and addiction. Further research is necessary to elucidate the mechanisms involved in this cross-communication of the MGBA. New insights into these challenging disorders of addiction and stress may provide unique therapies targeting the gut microbiome

Effects of Ethanol Exposure and Withdrawal on Neuronal Morphology in the Agranular Insular and Prelimbic Cortices: Relationship with Withdrawal-Related Structural Plasticity in the Nucleus Accumbens

peer-reviewedThe present study investigated the effects of chronic intermittent ethanol exposure and withdrawal on dendritic morphology and spine density in the agranular insular and prelimbic cortices. Adult male Sprague–Dawley rats were passively exposed to vaporized ethanol (~37 mg/L; 12 h/day) or air (control) for ten consecutive days. Dendritic length, branching, and spine density were quantified in layer II/III pyramidal neurons 24 hours or seven days following the final ethanol exposure. Compared to unexposed control animals there were structural alterations on neurons in the prelimbic cortex, and to a lesser extent the agranular insular cortex. The most prominent ethanol-related differences were the transient increases in dendritic length and branching in prelimbic neurons at 24 h post-cessation, and increased mushroom-shaped spines at seven days post-cessation. The results obtained in the prelimbic cortex are the opposite of those previously reported in the nucleus accumbens core (Peterson, et al. 2015), suggesting that these regions undergo distinct functional adaptations following ethanol exposure and withdrawal

Statin use and adverse effects among adults \u3e 75 years of age: Insights from the Patient and Provider Assessment of Lipid Management (PALM) registry

Background: Current statin use and symptoms among older adults in routine community practice have not been well characterized since the release of the 2013 American College of Cardiology/American Heart Association guideline. Methods and results: We compared statin use and dosing between adults \u3e75 and ≤75 years old who were eligible for primary or secondary prevention statin use without considering guideline-recommended age criteria. The patients were treated at 138 US practices in the Patient and Provider Assessment of Lipid Management (PALM) registry in 2015. Patient surveys also evaluated reported symptoms while taking statins. Multivariable logistic regression models examined the association between older age and statin use and dosing. Among 6717 people enrolled, 1704 (25%) were \u3e75 years old. For primary prevention, use of any statin or high-dose statin did not vary by age group: any statin, 62.6% in those \u3e75 years old versus 63.1% in those ≤75 years old (P=0.83); high-dose statin, 10.2% versus 12.3% in the same groups (P=0.14). For secondary prevention, older patients were slightly less likely to receive any statin (80.1% versus 84.2% [P=0.003]; adjusted odds ratio, 0.81; 95% confidence interval, 0.66-1.01 [P=0.06]), but were much less likely to receive a high-intensity statin (23.5% versus 36.2% [PP=0.0001]). Among current statin users, older patients were slightly less likely to report any symptoms (41.3% versus 46.6%; P=0.003) or myalgias (27.3% versus 33.3%; Conclusions: Overall use of statins was similar for primary prevention in those aged \u3e75 years versus younger patients, yet older patients were less likely to receive high-intensity statins for secondary prevention. Statins appear to be similarly tolerated in older and younger adult

Patient-reported reasons for declining or discontinuing statin therapy: Insights from the PALM registry

Background: Many adults eligible for statin therapy for cardiovascular disease prevention are untreated. Our objective was to investigate patient‐reported reasons for statin underutilization, including noninitiation, refusal, and discontinuation.Methods and Results: This study included the 5693 adults recommended for statin therapy in the PALM (Patient and Provider Assessment of Lipid Management) registry. Patient surveys evaluated statin experience, reasons for declining or discontinuing statins, and beliefs about statins and cardiovascular disease risk. Overall, 1511 of 5693 adults (26.5%) were not on treatment. Of those not on a statin, 894 (59.2%) reported never being offered a statin, 153 (10.1%) declined a statin, and 464 (30.7%) had discontinued therapy. Women (relative risk: 1.22), black adults (relative risk: 1.48), and those without insurance (relative risk: 1.38) were most likely to report never being offered a statin. Fear of side effects and perceived side effects were the most common reasons cited for declining or discontinuing a statin. Compared with statin users, those who declined or discontinued statins were less likely to believe statins are safe (70.4% of current users vs. 36.9% of those who declined and 37.4% of those who discontinued) or effective (86.3%, 67.4%, and 69.1%, respectively). Willingness to take a statin was high; 67.7% of those never offered and 59.7% of patients who discontinued a statin would consider initiating or retrying a statin.Conclusions: More than half of patients eligible for statin therapy but not on treatment reported never being offered one by their doctor. Concern about side effects was the leading reason for statin refusal or discontinuation. Many patients were willing to reconsider statin therapy if offered

Measurement of low‐density lipoprotein cholesterol levels in primary and secondary prevention patients: Insights from the PALM registry

Background The 2013 American College of Cardiology/American Heart Association Guideline on the Treatment of Blood Cholesterol to Reduce Atherosclerotic Cardiovascular Risk in Adults recommended testing low-density lipoprotein cholesterol ( LDL -C) to identify untreated patients with LDL -C ≥190 mg/dL, assess lipid-lowering therapy adherence, and consider nonstatin therapy. We sought to determine whether clinician lipid testing practices were consistent with these guidelines. Methods and Results The PALM (Patient and Provider Assessment of Lipid Management) registry enrolled primary and secondary prevention patients from 140 US cardiology, endocrinology, and primary care offices in 2015 and captured demographic data, lipid treatment history, and the highest LDL -C level in the past 2 years. Core laboratory lipid levels were drawn at enrollment. Among 7627 patients, 2787 (36.5%) had no LDL -C levels measured in the 2 years before enrollment. Patients without chart-documented LDL -C levels were more often women, nonwhite, uninsured, and non-college graduates (all P\u3c0.01). Patients without prior lipid testing were less likely to receive statin treatment (72.6% versus 76.0%; P=0.0034), a high-intensity statin (21.5% versus 24.3%; P=0.016), nonstatin lipid-lowering therapy (24.8% versus 27.3%; P=0.037), and had higher core laboratory LDL -C levels at enrollment (median 97 versus 92 mg/dL; P\u3c0.0001) than patients with prior LDL -C testing. Of 166 individuals with core laboratory LDL -C levels ≥190 mg/dL, 36.1% had no LDL -C measurement in the prior 2 years, and 57.2% were not on a statin at the time of enrollment. Conclusions In routine clinical practice, LDL -C testing is associated with higher-intensity lipid-lowering treatment and lower achieved LDL -C level

Recombinant Incretin-Secreting Microbe Improves Metabolic Dysfunction in High-Fat Diet Fed Rodents

peer-reviewedThe gut hormone glucagon-like peptide (GLP)-1 and its analogues represent a new generation of anti-diabetic drugs, which have also demonstrated propensity to modulate host lipid metabolism. Despite this, drugs of this nature are currently limited to intramuscular administration routes due to intestinal degradation. The aim of this study was to design a recombinant microbial delivery vector for a GLP-1 analogue and assess the efficacy of the therapeutic in improving host glucose, lipid and cholesterol metabolism in diet induced obese rodents. Diet-induced obese animals received either Lactobacillus paracasei NFBC 338 transformed to express a long-acting analogue of GLP-1 or the isogenic control microbe which solely harbored the pNZ44 plasmid. Short-term GLP-1 microbe intervention in rats reduced serum low-density lipoprotein cholesterol, triglycerides and triglyceride-rich lipoprotein cholesterol substantially. Conversely, extended GLP-1 microbe intervention improved glucose-dependent insulin secretion, glucose metabolism and cholesterol metabolism, compared to the high-fat control group. Interestingly, the microbe significantly attenuated the adiposity associated with the model and altered the serum lipidome, independently of GLP-1 secretion. These data indicate that recombinant incretin-secreting microbes may offer a novel and safe means of managing cholesterol metabolism and diet induced dyslipidaemia, as well as insulin sensitivity in metabolic dysfunction

Targeting the microbiota-gut-brain axis: prebiotics have anxiolytic and antidepressant-like effects and reverse the impact of chronic stress in mice

Background: The realization that the microbiota-gut-brain axis plays a critical role in health and disease, including neuropsychiatric disorders, is rapidly advancing. Nurturing a beneficial gut microbiome with prebiotics, such as fructo-oligosaccharides (FOS) and galacto-oligosaccharides (GOS), is an appealing but underinvestigated microbiota manipulation. Here we tested whether chronic prebiotic treatment modifies behavior across domains relevant to anxiety, depression, cognition, stress response, and social behavior. Methods: C57BL/6J male mice were administered FOS, GOS, or a combination of FOS+GOS for 3 weeks prior to testing. Plasma corticosterone, microbiota composition, and cecal short-chain fatty acids were measured. In addition, FOS+GOS- or water-treated mice were also exposed to chronic psychosocial stress, and behavior, immune, and microbiota parameters were assessed. Results: Chronic prebiotic FOS+GOS treatment exhibited both antidepressant and anxiolytic effects. Moreover, the administration of GOS and the FOS+GOS combination reduced stress-induced corticosterone release. Prebiotics modified specific gene expression in the hippocampus and hypothalamus. Regarding short-chain fatty acid concentrations, prebiotic administration increased cecal acetate and propionate and reduced isobutyrate concentrations, changes that correlated significantly with the positive effects seen on behavior. Moreover, FOS+GOS reduced chronic stress-induced elevations in corticosterone and proinflammatory cytokine levels and depression-like and anxiety-like behavior in addition to normalizing the effects of stress on the microbiota. Conclusions: Taken together, these data strongly suggest a beneficial role of prebiotic treatment for stress-related behaviors. These findings strengthen the evidence base supporting therapeutic targeting of the gut microbiota for brain-gut axis disorders, opening new avenues in the field of nutritional neuropsychopharmacology

Drunk bugs: chronic vapour alcohol exposure induces marked changes in the gut microbiome in mice

The gut microbiota includes a community of bacteria that play an integral part in host health and biological processes. Pronounced and repeated findings have linked gut microbiome to stress, anxiety, and depression. Currently, however, there remains only a limited set of studies focusing on microbiota change in substance abuse, including alcohol use disorder. To date, no studies have investigated the impact of vapour alcohol administration on the gut microbiome. For research on gut microbiota and addiction to proceed, an understanding of how route of drug administration affects gut microbiota must first be established. Animal models of alcohol abuse have proven valuable for elucidating the biological processes involved in addiction and alcohol-related diseases. This is the first study to investigate the effect of vapour route of ethanol administration on gut microbiota in mice. Adult male C57BL/6J mice were exposed to 4 weeks of chronic intermittent vapourized ethanol (CIE, N = 10) or air (Control, N = 9). Faecal samples were collected at the end of exposure followed by 16S sequencing and bioinformatic analysis. Robust separation between CIE and Control was seen in the microbiome, as assessed by alpha (p < 0.05) and beta (p < 0.001) diversity, with a notable decrease in alpha diversity in CIE. These results demonstrate that CIE exposure markedly alters the gut microbiota in mice. Significant increases in genus Alistipes (p < 0.001) and significant reductions in genra Clostridium IV and XIVb (p < 0.001), Dorea (p < 0.01), and Coprococcus (p < 0.01) were seen between CIE mice and Control. These findings support the viability of the CIE method for studies investigating the microbiota-gut-brain axis and align with previous research showing similar microbiota alterations in inflammatory states during alcoholic hepatitis and psychological stress

Revisiting Metchnikoff: Age-related alterations in microbiota-gut-brain axis in the mouse

Over the last decade, there has been increased interest in the role of the gut microbiome in health including brain health. This is by no means a new theory; Elie Metchnikoff proposed over a century ago that targeting the gut by consuming lactic acid bacteria such as those in yogurt, could improve or delay the onset of cognitive decline associated with ageing. However, there is limited information characterising the relationship between the behavioural and physiological sequelae of ageing and alterations in the gut microbiome. To this end, we assessed the behavioural, physiological and caecal microbiota profile of aged male mice. Older mice (20–21 months old) exhibited deficits in spatial memory and increases in anxiety-like behaviours compared to younger mice (2–3 months old). They also exhibited increased gut permeability, which was directly correlated with elevations in peripheral pro-inflammatory cytokines. Furthermore, stress exacerbated the gut permeability of aged mice. Examination of the caecal microbiota revealed significant increases in phylum TM7, family Porphyromonadaceae and genus Odoribacter of aged mice. This represents a shift of aged microbiota towards a profile previously associated with inflammatory disease, particularly gastrointestinal and liver disorders. Furthermore, Porphyromonadaceae, which has also been associated with cognitive decline and affective disorders, was directly correlated with anxiety-like behaviour in aged mice. These changes suggest that changes in the gut microbiota and associated increases in gut permeability and peripheral inflammation may be important mediators of the impairments in behavioural, affective and cognitive functions seen in ageing

Sex-dependent associations between addiction-related behaviors and the microbiome in outbred rats.

BackgroundMultiple factors contribute to the etiology of addiction, including genetics, sex, and a number of addiction-related behavioral traits. One behavioral trait where individuals assign incentive salience to food stimuli ("sign-trackers", ST) are more impulsive compared to those that do not ("goal-trackers", GT), as well as more sensitive to drugs and drug stimuli. Furthermore, this GT/ST phenotype predicts differences in other behavioral measures. Recent studies have implicated the gut microbiota as a key regulator of brain and behavior, and have shown that many microbiota-associated changes occur in a sex-dependent manner. However, few studies have examined how the microbiome might influence addiction-related behaviors. To this end, we sought to determine if gut microbiome composition was correlated with addiction-related behaviors determined by the GT/ST phenotype.MethodsOutbred male (N=101) and female (N=101) heterogeneous stock rats underwent a series of behavioral tests measuring impulsivity, attention, reward-learning, incentive salience, and locomotor response. Cecal microbiome composition was estimated using 16S rRNA gene amplicon sequencing. Behavior and microbiome were characterized and correlated with behavioral phenotypes. Robust sex differences were observed in both behavior and microbiome; further analyses were conducted within sex using the pre-established goal/sign-tracking (GT/ST) phenotype and partial least squares differential analysis (PLS-DA) clustered behavioral phenotype.ResultsOverall microbiome composition was not associated to the GT/ST phenotype. However, microbial alpha diversity was significantly decreased in female STs. On the other hand, a measure of impulsivity had many significant correlations to microbiome in both males and females. Several measures of impulsivity were correlated with the genus Barnesiella in females. Female STs had notable correlations between microbiome and attentional deficient. In both males and females, many measures were correlated with the bacterial families Ruminocococcaceae and Lachnospiraceae.ConclusionsThese data demonstrate correlations between several addiction-related behaviors and the microbiome specific to sex