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Rationale The majority of preclinical studies assessing treatments for alcohol use disorder use singly housed animals. Because social factors affect ethanol intake, studies investigating such treatments in group-housed animals are needed. Objectives We investigated the effects of repeated oxytocin treatment on ethanol intake in socially housed male and female C57BL/6J mice. Methods We used the novel “Herdsman” system implementing radiotracking technology to measure individual ethanol intake in group-housed animals. Mice were housed in same-sex groups of 4 per cage and exposed to 3 and 6% ethanol solutions. After baseline drinking was established, half of the animals in each cage received repeated intraperitoneal injections of 3 mg/kg oxytocin. Results During baseline, females consumed more ethanol than males partly due to greater number of ethanol drinks taken by females. We also observed a gradual development of two peaks of ethanol consumption during the dark phase of the circadian cycle. The effects of oxytocin treatment were short-acting and varied across treatment days. Oxytocin significantly decreased ethanol intake on three out the four treatment days. On the fourth treatment day, oxytocin decreased ethanol intake and water intake. Conclusion The greater intake of ethanol in female mice is associated with the number of drinks taken. Oxytocin treatments not only cause an acute decrease in ethanol consumption, but can also change in efficacy over time. While the oxytocin system remains a promising therapeutic target for alcoholism, studies investigating longer periods of repeated oxytocin treatment and those using additional oxytocin receptor agonists are warranted. Supplementary Information The online version contains supplementary material available at 10.1007/s00213-020-05741-3.

Background: Alzheimer’s disease (AD) is a neurodegenerative disease characterized by the accumulation of amyloid-β (Aβ) peptide in the brain. Objective: To gain a better insight into alterations in major biochemical pathways underlying AD. Methods: We compared metabolomic profiles of hippocampal tissue of 20-month-old female Tg2576 mice expressing the familial AD-associated hAPP695SW transgene with their 20-month-old wild type female littermates. Results: The hAPP695SW transgene causes overproduction and accumulation of Aβ in the brain. Out of 180 annotated metabolites, 54 metabolites differed (30 higher and 24 lower in Tg2576 versus wild-type hippocampal tissue) and were linked to the amino acid, nucleic acid, glycerophospholipid, ceramide, and fatty acid metabolism. Our results point to 1) heightened metabolic activity as indicated by higher levels of urea, enhanced fatty acid β-oxidation, and lower fatty acid levels; 2) enhanced redox regulation; and 3) an imbalance of neuro-excitatory and neuro-inhibitory metabolites in hippocampal tissue of aged hAPP695SW transgenic mice. Conclusion: Taken together, our results suggest that dysregulation of multiple metabolic pathways associated with a concomitant shift to an excitatory-inhibitory imbalance are contributing mechanisms of AD-related pathology in the Tg2576 mouse.

An individual's social environment affects alcohol intake. However, the complex interactions between social context and alcohol intake remain understudied in preclinical models. In the present study, we sought to characterize the effects of social housing on voluntary ethanol intake in male C567BL/6J mice using a continuous access two-bottle choice model. This was accomplished using HM2 cages, which allow for the continuous monitoring of individuals' fluid intake through radiofrequency tracking while they remain undisturbed in a group setting. These cages are moderately environmentally enriched compared to standard shoebox cages. By analyzing the levels of voluntary ethanol intake between socially- and individually-housed mice in HM2 cages, we were able to parse apart the effects of environmental enrichment vs. social enrichment. We found that while intake levels were overall lower than those observed when animals are singly housed in standard shoebox cages, socially-housed males consumed significantly more ethanol compared to individually-housed mice, suggesting that while environmental enrichment attenuates ethanol intake, social enrichment may, in fact, potentiate it. This effect was not specific for alcohol, however, in that ethanol preference did not differ as a product of social context. We also found that the total number of non-consummatory channel entries were consistently higher in individually-housed mice. Additionally, a single corticotropin releasing factor receptor 1 antagonist treatment significantly decreased both water and ethanol intake in socially- and individually-housed mice up to 3 h post-treatment, though the effect on water intake was longer lasting. This treatment also significantly decreased the number of non-consummatory channel entries in individually-housed mice, but not in socially-housed mice, suggesting that increased channel visits may be a stress-related behavior. Lastly, we examined blood ethanol concentrations and FosB immunoreactivity to characterize the physiological responses to ethanol intake in socially- and individually-housed mice. The number of FosB-positive cells in the centrally-projecting Edinger-Westphal nucleus and nucleus accumbens shell positively correlated with average baseline ethanol intake in individually-housed mice, but not in socially-housed mice. Overall, we found that social, but not environmental, enrichment can increase ethanol intake in male C57BL/6J mice. Future studies need to test this phenomenon in female mice and assess the generalizability of this finding.

Background: The medicinal herb Centella asiatica has been long been used for its neuroprotective and cognitive enhancing effects. We have previously shown that two weeks of treatment with a water extract of Centella asiatica (CAW) improves cognition and activates the endogenous antioxidant response pathway without altering amyloid-β (Aβ) plaque burden. Objective: Here, we assess the effect of long-term treatment of CAW in the 5xFAD mouse model of Aβ accumulation. Methods: Four-month-old 5xFAD mice were treated with CAW in their drinking water (2 g/L) for three months at which point they underwent cognitive testing as well as analysis of Aβ plaque levels and antioxidant and synaptic gene expression. In order to confirm the involvement of the antioxidant regulatory transcription factor NRF2 on the effects of CAW on synaptic plasticity, neurons isolated from 5xFAD mice were also treated with CAW and the targeted inhibitor ML385. Results: Three months of treatment with CAW improved spatial and contextual memory as well as executive function in 5xFAD mice. This improvement was accompanied by increased antioxidant gene expression and a decrease in Aβ plaque burden relative to untreated 5xFAD animals. In isolated neurons, treatment with ML385 blocked the effects of CAW on dendritic arborization and synaptic gene expression. Conclusion: These results suggest that prolonged CAW exposure could be beneficial in Alzheimer’s disease and that these effects likely involve NRF2 activation. Moreover, these findings suggest that targeting NRF2 itself may be a relevant therapeutic strategy for improving synaptic plasticity and cognitive function in Alzheimer’s disease.

Social hierarchies are ubiquitous features of virtually all animal groups. The varying social ranks of members within these groups have profound effects on both physical and emotional health, with lower-ranked individuals typically being the most adversely affected by their respective ranks. Thus, reliable measures of social dominance in preclinical rodent models are necessary to better understand the effects of an individual’s social rank on other behaviors and physiological processes. In this review, we outline the primary methodologies used to assess social dominance in various rodent species: those that are based on analyses of agonistic behaviors, and those that are based on resource competition. In synthesizing this review, we conclude that assays based on resource competition may be better suited to characterize social dominance in a wider variety of rodent species and strains, and in both males and females. Lastly, albeit expectedly, we demonstrate that similarly to many other areas of preclinical research, studies incorporating female subjects are lacking in comparison to those using males. These findings emphasize the need for an increased number of studies assessing social dominance in females to form a more comprehensive understanding of this behavioral phenomenon.

Centella asiatica (CA) is an edible plant and a popular botanical dietary supplement. It is reputed, in Ayurveda, to mitigate age-related cognitive decline. There is a considerable body of preclinical literature supporting CA’s ability to improve learning and memory. This study evaluated the contribution of CA’s triterpenes (TT), widely considered its active compounds, and caffeoylquinic acids (CQA) to the cognitive effects of CA water extract (CAW) in 5XFAD mice, a model of Alzheimer’s disease. 5XFAD mice were fed a control diet alone, or one containing 1% CAW or compound groups (TT, CQA, or TT + CQA) equivalent to their content in 1% CAW. Wild-type (WT) littermates received the control diet. Conditioned fear response (CFR) was evaluated after 4.5 weeks. Female 5XFAD controls showed no deficit in CFR compared to WT females, nor any effects from treatment. In males, CFR of 5XFAD controls was attenuated compared to WT littermates (p = 0.005). 5XFAD males receiving CQA or TT + CQA had significantly improved CFR (p < 0.05) compared to 5XFAD male controls. CFR did not differ between 5XFAD males receiving treatment diets and WT males. These data confirm a role for CQA in CAW’s cognitive effects.

Background: Environmental copper has been implicated in the pathogenesis of Alzheimer’s disease based on evidence that: 1) brain copper levels increase with age, 2) copper promotes misfolding and toxicity of amyloid-β in vitro, 3) copper-modulating interventions reduce amyloid pathology in animal models. However, the effect of copper upon non-amyloid Alzheimer’s pathology is relatively under-explored. Objective: To determine if modulation of brain copper level affects brain tau pathology and/or associated cognitive impairment. Methods: We tested the hypothesis that brain copper modulates tau pathology by manipulating brain levels of copper in the PS19 transgenic mouse model of tau pathology. We treated PS19 and wild-type mice with oral zinc acetate, an established therapy for long term control of excess brain copper, and examined treatment effects upon brain copper, brain tau, NFT-like pathology, and spatial memory. We treated a second cohort of mice with exogenous dietary copper in order to evaluate whether excess environmental copper promotes brain tau pathology. Results: Copper-lowering with oral zinc attenuated spatial memory impairment in female but not male PS19 mice, without a significant effect upon tau pathology. Copper loading increased brain copper, but did not have an effect on brain tau pathology or spatial memory function. Conclusion: These findings suggest that a strategy to lower brain copper may be viable for symptomatic benefit in the setting of tau neuropathology, but unlikely to have robust effects on the underlying pathology. These findings are consistent with dietary or other exogenous copper being unlikely to promote tau pathology.

Centella asiatica has been used for centuries to enhance memory. We have previously shown that a water extract of Centella asiatica (CAW) protects against the deleterious effects of amyloid-β (Aβ) in neuroblastoma cells and attenuates Aβ-induced cognitive deficits in mice. Yet, the neuroprotective mechanism of CAW has yet to be thoroughly explored in neurons from these animals. This study investigates the effects of CAW on neuronal metabolism and oxidative stress in isolated Aβ-expressing neurons. Hippocampal neurons from amyloid precursor protein overexpressing Tg2576 mice and wild-type (WT) littermates were treated with CAW. In both genotypes, CAW increased the expression of antioxidant response genes which attenuated the Aβ-induced elevations in reactive oxygen species (ROS) and lipid peroxidation in Tg2576 neurons. CAW also improved mitochondrial function in both genotypes and increased the expression of electron transport chain enzymes and mitochondrial labeling, suggesting an increase in mitochondrial content. These data show that CAW protects against mitochondrial dysfunction and oxidative stress in Aβ-exposed hippocampal neurons which could contribute to the beneficial effects of the extract observed in vivo. Since CAW also improved mitochondrial function in the absence of Aβ, these results suggest a broader utility for other conditions where neuronal mitochondrial dysfunction occurs.

The water extract of Centella asiatica (CAW) improves cognitive and mitochondrial function and activates the nuclear factor erythroid 2-related factor 2 (NRF2) regulated antioxidant response pathway in aged mice. Here we investigate whether NRF2 activation is required for the cognitive and mitochondrial effects of prolonged CAW exposure during aging. Five-month-old NRF2 knockout (NRF2KO) and wild-type mice were treated with CAW for 1, 7, or 13 months. Each cohort underwent cognitive testing and hippocampal mitochondrial analyses. Age-related cognitive decline was accelerated in NRF2KO mice and while CAW treatment improved cognitive performance in wild-type mice, it had no effect on NRF2KO animals. Hippocampal mitochondrial function also declined further with age in NRF2KO mice and greater hippocampal mitochondrial dysfunction was associated with poorer cognitive performance in both genotypes. Long-term CAW treatment did not affect mitochondrial endpoints in animals of either genotype. These data indicate that loss of NRF2 results in accelerated age-related cognitive decline and worsened mitochondrial deficits. NRF2 also appears to be required for the cognitive enhancing effects of CAW during aging.

Centella asiatica (CA) herb is a traditional medicine, long reputed to provide cognitive benefits. We have reported that CA water extract (CAW) treatment improves cognitive function of aged Alzheimer&rsquo, s disease (AD) model Tg2576 and wild-type (WT) mice, and induces an NRF2-regulated antioxidant response in aged WT mice. Here, CAW was administered to AD model 5XFAD female and male mice and WT littermates (age: 7.6 +/ &minus, 0.6 months), and object recall and contextual fear memory were tested after three weeks treatment. CAW&rsquo, s impact on amyloid-&beta, plaque burden, and markers of neuronal oxidative stress and synaptic density, was assessed after five weeks treatment. CAW antioxidant activity was evaluated via nuclear transcription factor (erythroid-derived 2)-like 2 (NRF2) and NRF2-regulated antioxidant response element gene expression. Memory improvement in both genders and genotypes was associated with dose-dependent CAW treatment without affecting plaque burden, and marginally increased synaptic density markers in the hippocampus and prefrontal cortex. CAW treatment increased Nrf2 in hippocampus and other NRF2 targets (heme oxygenase-1, NAD(P)H quinone dehydrogenase 1, glutamate-cysteine ligase catalytic subunit). Reduced plaque-associated SOD1, an indicator of oxidative stress, was observed in the hippocampi and cortices of CAW-treated 5XFAD mice. We postulate that CAW treatment leads to reduced oxidative stress, contributing to improved neuronal health and cognition.

Introduction The phytochemical composition of plant material governs the bioactivity and potential health benefits as well as the outcomes and reproducibility of laboratory studies and clinical trials. Objective The objective of this work was to develop an efficient method for the in‐depth characterisation of plant extracts and quantification of marker compounds that can be potentially used for subsequent product integrity studies. Centella asiatica (L.) Urb., an Ayurvedic herb with potential applications in enhancing mental health and cognitive function, was used as a case study. Methods A quadrupole time‐of‐flight analyser in conjunction with an optimised high‐performance liquid chromatography (HPLC) separation was used for in‐depth untargeted fingerprinting and post‐acquisition precursor ion quantification to determine levels of distinct phytochemicals in various C. asiatica extracts. Results We demonstrate the utility of this workflow for the characterisation of extracts of C. asiatica. This integrated workflow allowed the identification or tentative identification of 117 compounds, chemically interconnected based on Tanimoto chemical similarity, and the accurate quantification of 24 phytochemicals commonly found in C. asiatica extracts. Conclusion We report a phytochemical analysis method combining liquid chromatography, high resolution mass spectral data acquisition, and post‐acquisition interrogation that allows chemical fingerprints of botanicals to be obtained in conjunction with accurate quantification of distinct phytochemicals. The variability in the composition of specialised metabolites across different C. asiatica accessions was substantial, demonstrating that detailed characterisation of plant extracts is a prerequisite for reproducible use in laboratory studies, clinical trials and safe consumption. The methodological approach is generally applicable to other botanical products., Botanical products are popular for promoting resilience, maintaining health and preventing or treating disease. Challenges in defining effects of botanical products reside in achieving batch‐to‐batch reproducibility regarding their composition and product stability, i.e. providing consistent products for clinical trials and consumers. In this work, using Centella asiatica (L.) Urb. as a case study, we demonstrate the efficacy of integrating untargeted high‐resolution mass spectrometry with post acquisition precursor ion extraction for determining chemical profiles and quantifying selected marker compounds.

Activation of the antioxidant regulatory transcription factor NRF2 (Nuclear factor erythroid-derived 2) regulates cellular bioenergetics and improves neuronal health in aging. Yet how NRF2 participates in maintaining synaptic, mitochondrial and cognitive function has not been fully elucidated. This study investigates how loss of NRF2 affects neuronal metabolism, synaptic density and cognitive performance in aged mice. Dendritic arborization as well as synaptic and mitochondrial gene expression was evaluated in hippocampal neurons isolated from mice lacking NRF2 (NRF2KO) and from wild-type (WT) C57BL6 mice. Mitochondrial function of these neurons was evaluated using the Seahorse XF platform. Additionally learning, memory and executive function were assessed in 20 month old NRF2KO and age-matched WT mice using conditioned fear response (CFR) and odor discrimination reversal learning (ODRL) tests. Hippocampal bioenergetics was profiled using mitochondria isolated from these animals and tissue was harvested for assessment of mitochondrial and synaptic genes. NRF2KO neurons had reduced dendritic complexity and diminished synaptic gene expression. This was accompanied by impaired mitochondrial function and decreased mitochondrial gene expression. Similar mitochondrial deficits were observed in the brains of aged NRF2KO mice. These animals also had significantly impaired cognitive performance and reduced synaptic gene expression as well. These data point to a role for NRF2 in maintaining mitochondrial and cognitive function during aging and suggest that the transcription factor may be a viable target for cognitive enhancing interventions. Because mitochondrial dysfunction and cognitive impairment also occur together in many neurodegenerative conditions there may be broad therapeutic potential of NRF2 activating agents.

BACKGROUND: Diabetic neuropathy (DN), a common complication of diabetes mellitus, results from hyperglycemia, poor microcirculation and attendant nerve damage. Currently available treatments relieve symptoms, but do not modify the neurodegeneration underlying DN. Centella asiatica (CA) triterpenes improved microcirculation in earlier clinical studies, and showed neurotropic effects in preclinical models suggesting a potential disease modifying effect in DN. This 52-week, randomized, double-blind, placebo-controlled trial examined the effects of CAST, a standardized CA extract containing triterpenes, on neuropathy symptoms in Type II diabetic subjects. PATIENTS AND METHODS: The study enrolled patients with a history of Type II diabetes, with evidence of symptomatic symmetrical DN with total symptom score (TSS) ≥4, and stable HbA1c level

Introduction Centella asiatica is a plant used for centuries to enhance memory. We have previously shown that a water extract of Centella asiatica (CAW) attenuates age‐related spatial memory deficits in mice and improves neuronal health. Yet the effect of CAW on other cognitive domains remains unexplored as does its mechanism of improving age‐related cognitive impairment. This study investigates the effects of CAW on a variety of cognitive tasks as well as on synaptic density and mitochondrial and antioxidant pathways. Methods Twenty‐month‐old CB6F1 mice were treated with CAW (2 mg/ml) in their drinking water for 2 weeks prior to behavioral testing. Learning, memory, and executive function were assessed using the novel object recognition task (NORT), object location memory task (OLM), and odor discrimination reversal learning (ODRL) test. Tissue was collected for Golgi analysis of spine density as well as assessment of mitochondrial, antioxidant, and synaptic proteins. Results CAW improved performance in all behavioral tests suggesting effects on hippocampal and cortical dependent memory as well as on prefrontal cortex mediated executive function. There was also an increase in synaptic density in the treated animals, which was accompanied by increased expression of the antioxidant response gene NRF2 as well as the mitochondrial marker porin. Conclusions These data show that CAW can increase synaptic density as well as antioxidant and mitochondrial proteins and improve multiple facets of age‐related cognitive impairment. Because mitochondrial dysfunction and oxidative stress also accompany cognitive impairment in many pathological conditions this suggests a broad therapeutic utility of CAW.