DBA/2J Research Articles

Sex and Strain Variation in Initial Sensitivity and Rapid Tolerance to Δ9-Tetrahydrocannabinol.

Background and Objectives: For cannabis and other drugs of abuse, initial response and/or tolerance to drug effects can predict later dependence and problematic use. Our objective is to identify sex and genetic (strain) differences in initial response and rapid tolerance to Δ9–tetrahydrocannabinol (THC), the main psychoactive ingredient in cannabis, between highly genetically divergent inbred mouse strains—C57BL/6J (B6) and DBA/2J (D2).Experimental Approach: Sex and strain responses relative to baseline were quantified following daily exposure (i.p.) to 10 mg/kg THC or vehicle (VEH) over the course of 5 days. Dependent measures included hypothermia (decreased body temperature) and ataxia (decreased spontaneous activity in the open field), and antinociception (increase in tail withdrawal latency to a thermal stimulus). Initial sensitivity to THC was defined as the difference in response between baseline and day 1. Rapid tolerance to THC was defined as the difference in response between days 1 and 2.Results: B6 exhibited greater THC-induced motor activity suppression and initial sensitivity to ataxia relative to the D2 strain. Females demonstrated greater levels of THC-induced hypothermia and initial sensitivity relative to males. Higher levels of THC-induced antinociception and initial sensitivity were observed for D2 relative to B6. Rapid tolerance to THC was observed for hypothermia and antinociception. Much less tolerance was observed for THC-induced ataxia. D2 exhibited rapid tolerance to THC-induced hypothermia and antinociception at time points associated with peak THC initial response. Likewise, at the peak initial THC response time point, females demonstrated greater levels of rapid tolerance to hypothermic effects relative to males.Conclusions: Both sex and genetic factors drive variation in initial response and rapid tolerance to the ataxic, antinociceptive, and hypothermic effects of THC. As these traits directly result from THC activation of the cannabinoid receptor 1, gene variants between B6 and D2 in cannabinoid signaling pathways are likely to mediate strain differences in response to THC.

Nicotinamide-Rich Diet in DBA/2J Mice Preserves Retinal Ganglion Cell Metabolic Function as Assessed by PERG Adaptation to Flicker.

Flickering light increases metabolic demand in the inner retina. Flicker may exacerbate defective mitochondrial function in glaucoma, which will be reflected in the pattern electroretinogram (PERG), a sensitive test of retinal ganglion cell (RGC) function. We tested whether flicker altered the PERG of DBA/2J (D2) glaucomatous mice and whether vitamin B3-rich diet contributed to the flicker effect. D2 mice fed with either standard chow (control, n = 10) or chow/water enriched with nicotinamide (NAM, 2000 mg/kg per day) (treated, n = 10) were monitored from 3 to 12 months. The PERG was recorded with superimposed flicker (F-PERG) at either 101 Hz (baseline) or 11 Hz (test), and baseline-test amplitude difference (adaptation) evaluated. At endpoint, flat-mounted retinas were immunostained (RBPMS and mito-tracker). F-PERG adaptation was 41% in 3-month-old D2 and decreased with age more in control D2 than in NAM-fed D2 (GEE, p < 0.01). At the endpoint, F-PERG adaptation was 0% in control D2 and 17.5% in NAM-fed D2, together with higher RGC density (2.4×), larger RGC soma size (2×), and greater intensity of mitochondrial staining (3.75×). F-PERG adaptation may provide a non-invasive tool to assess RGC autoregulation in response to increased metabolic demand and test the effect of dietary/pharmacological treatments on optic nerve disorders.

An Age-Related Hearing Protection Locus on Chromosome 16 of BXD Strain Mice.

Inbred mouse models are widely used to study age-related hearing loss (AHL). Many genes associated with AHL have been mapped in a variety of strains. However, little is known about gene variants that have the converse function—protective genes that confer strong resistance to hearing loss. Previously, we reported that C57BL/6J (B6) and DBA/2J (D2) strains share a common hearing loss allele in Cdh23. The cadherin 23 (Cdh23) gene is a key contributor to early-onset hearing loss in humans. In this study, we tested hearing across a large family of 54 BXD strains generated from B6 to D2 crosses. Five of 54 strains maintain the normal threshold (20 dB SPL) even at 2 years old—an age at which both parental strains are essentially deaf. Further analyses revealed an age-related hearing protection (ahp) locus on chromosome 16 (Chr 16) at 57~76 Mb with a maximum LOD of 5.7. A small number of BXD strains at 2 years with good hearing correspond roughly to the percentage of humans who have good hearing at 90 years old. Further studies to define candidate genes in the ahp locus and related molecular mechanisms involved in age-related resilience or resistance to AHL are warranted.

Sitagliptin ameliorates renal tubular injury in diabetic kidney disease via STAT3-dependent mitochondrial homeostasis through SDF-1α/CXCR4 pathway.

Mitochondrial abnormalities play critical roles in diabetic tubular injury progression. Dipeptidyl peptidase-4 (DPP4) inhibitors are widely used antihyperglycemicagents that exert renal protective and positive effects against mitochondrial dysfunction in diabetic kidney disease (DKD). However, their underlying mechanism remains unclear. In this study, DPP4 upregulation, mitochondrial fragmentation, and altered mitochondrial dynamics-associated protein expression were observed in the tubules ofDBA2/J (D2) diabetic mice with unilateral nephrectomy and in albumin-stimulated tubular cells. The inhibition of DPP4 by sitagliptin(Sita) ameliorated these mitochondrial perturbations bothin vivoandin vitro, whereas DPP4 overexpression aggravated mitochondrial fusion-fission disorder and tubular cell injury in albumin-treated HK-2 cells. Downstream of DPP4,the SDF-1α/CXCR4 pathway was significantly suppressed in diabetic tubules. After Sitatreatment, this signaling pathway was restored, and themitochondrial dynamics was improved. Furthermore, a direct interaction between STAT3 and OPA1 was found inthe mitochondria oftubular cells, and this effect was weakened by overloading albumin andby CXCR4 siRNA treatment, suggesting a possible link between DPP4-mediated SDF-1α/CXCR4/STAT3 signaling and mitochondrial dysfunction in diabetic tubular cells. The results suggest that a novel mechanism links the DPP4 enzyme to impaired mitochondrial dynamics homeostasis during tubular injury in DKD and highlight that the SDF-1α/CXCR4/STAT3 signaling pathway could become a potential target for managing DKD.

Genetic Background influences Endothelial Function along the Mouse Vascular Tree

The endothelium is crucial for its role in maintaining vascular integrity and protecting against cardiovascular disease. Endothelial dysfunction is associated with a host of disease states including cardiovascular disease, diabetes, chronic kidney disease, and atherosclerosis. However, it is unclear whether endothelial function or dysfunction varies throughout the vasculature. In addition, accumulating evidence suggests that endothelial function, and more generally vasomotor function, is genetically regulated. The purpose of this study was to determine the influence of genetic background on vasomotor function in blood vessels of differing size. Vasomotor performance was measured in aorta (thoracic (TA), and abdominal (AA)) and conduit arteries (carotid artery (CA) and femoral artery (FA)) from four inbred strains (SJL/J (SJL), DBA/2J (DBA), NZW/LacJ (NZW) and C57BL/6J (B6)) of mice. Arteries were dissected, cut into 2 mm segments, and mounted in a wire myograph system. Increasing concentrations of phenylephrine (PE, 1×10−9 – 1×10−5M) were used to measure contractile responses, while increasing concentrations of the endothelium‐dependent vasodilator acetylcholine (ACh, 1×10−9 – 1×10−5M) and endothelium‐independent vasodilator sodium nitroprusside (SNP, 1×10−9 – 1×10−5M) were used to assess relaxation responses. Two way ANOVA revealed a significant main effect of strain in response to PE for AA (P = .0032) and CA (P = .0098). Post hoc analysis for max contractile response showed impaired vasoconstriction in NZW AA compared to B6 (P = .0004). In response to ACh, a significant main effect of strain was found for all vessels (TA: P = &lt;.0001, AA: P = .0156, CA: P = .0069, FA: P = &lt;.0001). Post hoc analysis showed that TA max vasorelaxation was impaired for all strains (NZW: P = &lt; .0001, DBA: P = .0140, SJL: P = &lt; .0001) and that NZW FA were impaired when compared to B6 ( P = .0015). A significant main effect of strain in response to SNP was found in TA (P = .0011), AA (P = .0086), and CA (P = &lt; .0001). Relaxation responses to SNP were not significantly different between strains. These findings indicate a strong influence of genetic background on endothelial function and could be important in understanding the implications that genetics have on vascular response to vasoactive agents.Support or Funding InformationThis work was supported by a Texas A&amp;M Triads for Transformation grant (MPM), a Texas A&amp;M Merit Fellowship (DH), a Texas A&amp;M College of Education Strategic Research Award (SYS), and J.L. Huffines Institute of Sports Medicine and Human Performance (SYS).

Systems Genetics of Optic Nerve Axon Necrosis During Glaucoma.

In this study, we identify genomic regions that modulate the number of necrotic axons in optic nerves of a family of mice, some of which have severe glaucoma, and define a set of high priority positional candidate genes that modulate retinal ganglion cell (RGC) axonal degeneration. A large cohort of the BXD family were aged to greater than 13 months of age. Optic nerves from 74 strains and the DBA/2J (D2) parent were harvested, sectioned, and stained with p-phenylenediamine. Numbers of necrotic axons per optic nerve cross-section were counted from 1 to 10 replicates per genotype. Strain means and standard errors were uploaded into GeneNetwork 2 for mapping and systems genetics analyses (Trait 18614). The number of necrotic axons per nerve ranged from only a few hundred to more than 4,000. Using conventional interval mapping as well as linear mixed model mapping, we identified a single locus on chromosome 12 between 109 and 112.5 Mb with a likelihood ratio statistic (LRS) of ~18.5 (p genome-wide ~0.1). Axon necrosis is not linked to locations of major known glaucoma genes in this family, including Gpnmb, Tyrp1, Cdh11, Pou6f2, and Cacna2d1. This indicates that although these genes contribute to pigmentary dispersion or elevated IOP, none directly modulates axon necrosis. Of 156 positional candidates, eight genes—CDC42 binding protein kinase beta (Cdc42bpb); eukaryotic translation initiation factor 5 (Eif5); BCL2-associated athanogene 5 (Bag5); apoptogenic 1, mitochondrial (Apopt1); kinesin light chain 1 (Klc1); X-ray repair cross complementing 3 (Xrcc3); protein phosphatase 1, regulatory subunit 13B (Ppp1r13b); and transmembrane protein 179 (Tmem179)—passed stringent criteria and are high priority candidates. Several candidates are linked to mitochondria and/or axons, strengthening their plausible role as modulators of ON necrosis. Additional studies are required to validate and/or eliminate plausible candidates. Surprisingly, IOP and ON necrosis are inversely correlated across the BXD family in mice >13 months of age and these two traits share few genes among their top ocular and retinal correlates. These data suggest that the two traits are independently modulated or that a more complex and multifaceted approach is required to reveal their association.

Meox2 Haploinsufficiency Accelerates Axonal Degeneration in DBA/2J Glaucoma.

PurposeGlaucoma is a complex disease with major risk factors including advancing age and increased intraocular pressure (IOP). Dissecting these earliest events will likely identify new avenues for therapeutics. Previously, we performed transcriptional profiling in DBA/2J (D2) mice, a widely used mouse model relevant to glaucoma. Here, we use these data to identify and test regulators of early gene expression changes in DBA/2J glaucoma.MethodsUpstream regulator analysis (URA) in Ingenuity Pathway Analysis was performed to identify potential master regulators of differentially expressed genes. The function of one putative regulator, mesenchyme homeobox 2 (Meox2), was tested using a combination of genetic, biochemical, and immunofluorescence approaches.ResultsURA identified Meox2 as a potential regulator of early gene expression changes in the optic nerve head (ONH) of DBA/2J mice. Meox2 haploinsufficiency did not affect the characteristic diseases of the iris or IOP elevation seen in DBA/2J mice but did cause a significant increase in the numbers of eyes with axon damage compared to controls. While young mice appeared normal, aged Meox2 haploinsufficient DBA/2J mice showed a 44% reduction in MEOX2 protein levels. This correlated with modulation of age- and disease-specific vascular and myeloid alterations.ConclusionsOur data support a model whereby Meox2 controls IOP-dependent vascular remodeling and neuroinflammation to promote axon survival. Promoting these earliest responses prior to IOP elevation may be a viable neuroprotective strategy to delay or prevent human glaucoma.

Sex and Genetic Factors Involved in Alterations of Behavior and Trace Element Neurobiology Due to Diet-Induced Obesity (P14-029-19)

ObjectivesObesity has been linked to cognitive deficits, behavior issues, and neurodegenerative processes with trace element dysregulation as a potential trigger for their development. Sex and genetics are important factors to consider when evaluating the effects of diet-induced obesity (DIO) on neurophysiology and behavior changes. The objective of this study was to investigate sex and genetic differences on the disturbance of trace element brain biology and behavior due to DIO in male and female C57BL/6J (C57) and DBA/2J (DBA) mice. We hypothesize that alterations due to DIO will be more evident in males than females, and the DBA strain will show more resistance to DIO effects compared to the C57 strain. MethodsMale and female C57 (n = 36) and DBA (n = 36) mice at 21 days old were randomly assigned a control fat diet (CFD) with 10% kcal fat/g or mineral-matched high fat diet (HFD) with 60% kcal fat/g for 16 weeks. Behavior tests for anxiety, cognition, and memory included open field (OF), novel object recognition (NOR), and nestlet shredding (NS). Trace elements iron (Fe), copper (Cu), and manganese (Mn) were measured using graphite furnace atomic absorption spectroscopy in five brain regions. Statistical analysis was conducted using factorial ANOVA and repeated measures ANOVA. ResultsAfter 16 weeks, both strains fed the HFD were confirmed obese by body mass comparison (P < 0.001). A statistically significant interaction between diet and sex was observed for C57 mice in the OF evaluation of center time (P = 0.019), with obese females exhibiting more anxiety. Differences in NS due to DIO were found in male C57 mice only (P = 0.008). NOR performance was similar for all mice. There was a statistically significant diet by sex interaction for Fe in the striatum (P = 0.032) with opposite trends by sex. Diet by strain interactions were observed for Cu in the cortex and hippocampus, and for Mn in the thalamus. ConclusionsMale and female mice exhibit different behavior patterns and trace element distribution in the brain due to DIO, with more pronounced effects in the C57 strain compared to the DBA strain. Based on these findings, sex and genetics are important influences to consider when studying the consequences of obesity. Funding SourcesUNCG Health and Human Sciences Research Grant and Faculty First Award.

Comparison and Functional Genetic Analysis of Striatal Protein Expression Among Diverse Inbred Mouse Strains.

C57BL/6J (B6) and DBA/2J (D2) inbred mouse strains are highly variable genetically and differ in a large number of behavioral traits related to striatal function, including depression, anxiety, stress response, and response to drugs of abuse. The genetic basis of these phenotypic differences are, however, unknown. Here, we present a comparison of the striatal proteome between B6 and D2 and relate differences at the protein level to strain differences at the mRNA level. We also leverage a recombinant inbred BXD population derived from B6 and D2 strains to investigate the role of genetic variation on the regulation of mRNA and protein levels. Finally, we test the hypothesis that differential protein expression contributes to differential behavioral responses between the B6 and D2 strain. We detected the expression of over 2,500 proteins in membrane-enriched protein fractions from B6 and D2 striatum. Of these, 160 proteins demonstrated significant differential expression between B6 and D2 strains at a 10% false discovery level, including COMT, GABRA2, and cannabinoid receptor 1 (CNR1)—key proteins involved in synaptic transmission and behavioral response. Similar to previous reports, there was little overlap between protein and transcript levels (25%). However, the overlap was greater (51%) for proteins demonstrating genetic regulation of cognate gene expression. We also found that striatal proteins with significantly higher or lower relative expression in B6 and D2 were enriched for dopaminergic and glutamatergic synapses and processes involved in synaptic plasticity [e.g., long-term potentiation (LTP) and long-term depression (LTD)]. Finally, we validated higher expression of CNR1 in B6 striatum and demonstrated greater sensitivity of this strain to the locomotor inhibiting effects of the CNR1 agonist, Δ9-tetrahydrocannabinol (THC). Our study is the first comparison of differences in striatal proteins between the B6 and D2 strains and suggests that alterations in the striatal proteome may underlie strain differences in related behaviors, such as drug response. Furthermore, we propose that genetic variants that impact transcript levels are more likely to also exhibit differential expression at the protein level.

Impact of Genetic Variation on Stress-Related Ethanol Consumption.

The effect of stress on alcohol consumption in humans is highly variable, and the underlying processes are not yet understood. Attempts to model a positive relationship between stress and increased ethanol (EtOH) consumption in animals have been only modestly successful. Our hypothesis is that individual differences in stress effects on EtOH consumption are mediated by genetics. We measured alcohol consumption, using the drinking-in-the-dark (DID) paradigm in females from 2 inbred mouse strains, C57BL/6J (B6) and DBA/2J (D2), and 35 of their inbred progeny (the BXD family). A control group was maintained in normal housing and a stress group was exposed to chronic mild stress (CMS), consisting of unpredictable stressors over 7weeks. These included predator, social, and environmental perturbations. Alcohol intake was measured over 16weeks in both groups during baseline (preceding 5-week period), CMS (intervening 7-week period), and post-CMS (final 4-week period). We detected a strong effect of CMS on alcohol intake. A few strains demonstrated CMS-related increased alcohol consumption; however, most showed decreased intake. We identified 1 nearly significant quantitative trait locus on chromosome 5 that contains the neuronal nitric oxide synthase gene (Nos1). The expression of Nos1 is frequently changed following alcohol exposure, and variants in this gene segregating among the BXD population may modulate alcohol intake in response to stress. The results we present here represent the first study to combine chronic stress and alcohol consumption in a genetic reference population of mice. Differences in susceptibility to the effects of stressful environments vis-à-vis alcohol use disorders would suggest that the differences have at least some basis in genetic constitution. We have also nominated a likely candidate gene underlying the large individual differences in effects of stress on alcohol consumption.

Cardiac phenotyping in the BXD strains as a genetic reference population for cardiovascular diseases

IntroductionDiscovery of genetic modifiers in cardiomyopathy (CM) has proved difficult in human cohorts because the wide range of clinical manifestations due to joint effects of causal and multiple modifier genes and environment. Therefore, combining human GWAS findings with additional animal model and molecular data using systems genetics is powerful approach to understand complex diseases by focusing on how genes work together in groups rather than singly. The main challenge for a systems genetics analysis is the need for comparatively large sample sizes drawn from a genetic reference population (GRP) and the use of advanced statistical and computational methods and models. The study was aimed to establish a murine GRP for CM systems genetics analysis. The BXD recombinant inbred (RI) strains derived from C57BL/6J and DBA/2J (D2) mice is the largest murine GRP. The parental D2 strain expresses CM key features, which make BXD strains particularly suited for systems genetics analysis for CMs.HypothesisBXD strains can be used to study genetic influences and modifiers in CMs.MethodsCM‐associated traits (heart function, rhythm and mass, cardiomyocyte size, fibrosis) and heart transcriptome were assessed in male and female BXD RI mice (40 lines, N&gt;5) at 5–6 months of age. Echocardiography, ECG and blood pressure was performed followed by cardiac histology and microarray analysis (Affymetrix Mouse Gene 2.0 ST).ResultsEchocardiography revealed more than two‐fold variance in LV end‐systolic and end‐diastolic volumes among tested BXD strains. BXD155 has the largest end‐systolic LV volume (42.8±9.0 μl) and BXD74 has the smallest volume (12.1±4.8 μl). Accordingly, %EF also varied greatly among BXDs. Severe hypertension was found in many BXD strains with highest systolic and diastolic blood pressure (200/159 mmHg) in D2. Atrioventricular (AV) block, bradycardia, ST elevation, and inverted T wave was revealed in several BXD lines. The lowest heart rate was found in BXD171 mice (369±20 beat/min). Heart weight, cardiomyocyte size, and cardiac collagen volume fraction significantly varied among BXD strains as well. D2 strain has the largest hearts (185.1 mg) and B6 has the smallest hearts (129.8 mg), while the average heart weight among BXD strains is 144.7mg. Several CM causal and candidate genes such as Mybpc3, Prkag2, Ankrd1, Myl3, and Mypn have mutation in D2 mouse or they are trans‐regulated by the gene with mutation in D2.ConclusionsCM phenotypes from D2 mouse are segregated among the BXD family of strains and CM‐associated traits vary among BXD strains, demonstrating that the BXD family as an excellent platform to identify genetic factors and modifiers that influence CM phenotypes. Identification of these factors will facilitate improved understanding of gene modifications and phenotypes in human CMs.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Sitagliptin Ameliorates Renal Tubular Injury in Diabetic Kidney Disease &lt;i&gt;via&lt;/i&gt; STAT3-Dependent Mitochondrial Homeostasis Through SDF-1α/CXCR4 Pathway

Background: Mitochondrial abnormalities play critical roles in diabetic tubular injury progression. Dipeptidyl peptidase-4 (DPP4) inhibitors are widely used antihyperglycaemic agents that exert renal protective and positive effects against mitochondrial dysfunction in diabetic kidney disease (DKD). Methods: We examined whether DPP4 inhibitor sitagliptin ameliorate mitochondrial altered fusion-fission processes and fragmentation in the tubules of diabetic mice and albumin-overloaded tubular cells. The possible role of sitagliptin-mediated SDF-1α/CXCR4 signalling and mitochondrial STAT3 in protecting against mitochondrial abnormalities was also studied. Findings: DPP4 upregulation, accompanied by mitochondrial fragmentation and altered mitochondrial dynamic protein expressions, were observed in the tubules of DBA2/J (D2) diabetic mice with unilateral nephrectomy and in albumin-stimulated tubular cells. Sitagliptin ameliorated these mitochondrial perturbations both in vivo and in vitro, whereas DPP4 overexpression aggravated mitochondrial fusion-fission disorder and tubular cell injury in albumin-treated HK-2 cells. DPP4 downstream SDF-1α/CXCR4 pathway was significantly suppressed in diabetic tubules, which was restored after sitagliptin treatment. Furthermore, a direct interaction between STAT3 and OPA1 was found in mitochondria in tubular cells, and this effect was weakened by overloaded albumin and CXCR4 siRNA, suggesting a possible link between DPP4-mediated SDF-1α/CXCR4/STAT3 signalling and mitochondrial dysfunction in diabetic tubular cells. Interpretation: The results suggest a novel mechanism linking the DPP4 enzyme to impaired mitochondrial dynamics homeostasis during tubular injury in DKD and highlight that the SDF-1α/CXCR4/STAT3 signalling pathway could become a potential target for managing DKD. Funding Statement: This study was supported by National Nature Science Foundation of China (81670657,81870504,81870468,81400735), National “Twelfth Five-Year” Plan for Science & Technology Support (2013BAI02B01-01), Medical and Engineering Cross funding of Shanghai Jiao Tong University (YG2017MS10), Shanghai Municipal Commission of Science and Technology (Grant No.18DZ2260200), Open Project of Shanghai Key Laboratory of Sleep Disordered Breathing (SHKSDB-KF-19-04), Science and Technology Commission of Shanghai Municipality (14DZ2260200, the project of Shanghai Key Laboratory of Kidney and Blood Purification). Declaration of Interests: No potential conflicts of interest relevant to this article are reported. Ethics Approval Statement: All animal procedures used in this study were approved by the Animal Care and Use Committee of Shanghai Jiao Tong University.

Genotype Differences in Sensitivity to the Anticonvulsant Effect of the Synthetic Neurosteroid Ganaxolone during Chronic Ethanol Withdrawal

Sensitivity to anticonvulsant effects of the γ-aminobutyric acidA receptor-active neurosteroid allopregnanolone (ALLO) during ethanol withdrawal varies across genotypes, with high sensitivity in genotypes with mild withdrawal and low sensitivity in genotypes with high withdrawal. The present studies determined whether the resistance to ALLO during withdrawal in mouse genotypes with high handling-induced convulsions (HICs) during withdrawal could be overcome with use of ganaxolone (GAN), the metabolically stable derivative of ALLO. In separate studies, male and female Withdrawal Seizure-Prone (WSP-1) and DBA/2J (D2) mice were exposed to air (controls) or 72-h ethanol vapor and then were scored for HICs during withdrawal (hourly for the first 12 h, then at hours 24 and 25). After the HIC scoring at hours 5 and 9, mice were injected with 10 mg/kg GAN or vehicle. Area under the HIC curve (AUC) for hours 5–12 was analyzed. In control WSP-1 mice, GAN significantly reduced AUC by 52% (males) and 63% (females), with effects that were absent or substantially reduced during withdrawal. In contrast, GAN significantly reduced AUC in both control and ethanol-withdrawing male and female D2 mice. AUC was decreased by 81% (males) and 70% (females) in controls and by 35% (males) and 21% (females) during withdrawal. The significant anticonvulsant effect of GAN during withdrawal in D2 but not WSP-1 mice suggests that different mechanisms may contribute to ALLO insensitivity during withdrawal in these two genotypes. Importantly, the results in D2 mice suggest that GAN may be a useful treatment for ethanol withdrawal-induced seizures.

Reduced AMPK activation and increased HCAR activation drive anti-inflammatory response and neuroprotection in glaucoma

BackgroundGlaucoma is a chronic degenerative disease for which inflammation is considered to play a pivotal role in the pathogenesis and progression. In this study, we examined the impact of a ketogenic diet on the inflammation evident in glaucoma as a follow-up to a recent set of experiments in which we determined that a ketogenic diet protected retinal ganglion cell structure and function.MethodsBoth sexes of DBA/2J (D2) mice were placed on a ketogenic diet (keto) or standard rodent chow (untreated) for 8 weeks beginning at 9 months of age. DBA/2J-Gpnmb+ (D2G) mice were also used as a non-pathological genetic control for the D2 mice. Retina and optic nerve (ON) tissues were micro-dissected and used for the analysis of microglia activation, expression of pro- and anti-inflammatory molecules, and lactate- or ketone-mediated anti-inflammatory signaling. Data were analyzed by immunohistochemistry, quantitative RT-PCR, ELISA, western blot, and capillary tube-based electrophoresis techniques.ResultsMicroglia activation was observed in D2 retina and ON as documented by intense microglial-specific Iba1 immunolabeling of rounded-up and enlarged microglia. Ketogenic diet treatment reduced Iba1 expression and the activated microglial phenotype. We detected low energy-induced AMP-activated protein kinase (AMPK) phosphorylation in D2 retina and ON that triggered NF-κB p65 signaling through its nuclear translocation. NF-κB induced pro-inflammatory TNF-α, IL-6, and NOS2 expression in D2 retina and ON. However, treatment with the ketogenic diet reduced AMPK phosphorylation, NF-κB p65 nuclear translocation, and expression of pro-inflammatory molecules. The ketogenic diet also induced expression of anti-inflammatory agents Il-4 and Arginase-1 in D2 retina and ON. Increased expression of hydroxycarboxylic acid receptor 1 (HCAR1) after ketogenic diet treatment was observed. HCAR1 stimulation by lactate or ketones from the ketogenic diet reduced inflammasome formation, as shown by reduced mRNA and protein expression of NLRP3 and IL-1β. We also detected increased levels of Arrestin β-2 protein, an adapter protein required for HCAR1 signaling.ConclusionOur data demonstrate that the AMPK activation apparent in the glaucomatous retina and ON triggers NF-κB signaling and consequently induces a pro-inflammatory response. The ketogenic diet resolves energy demand and ameliorates the inflammation by inhibition of AMPK activation and stimulation of HCAR1-ARRB2 signaling that inhibits NLRP3 inflammasome-mediated inflammation. Thus, these findings depict a neuroprotective mechanism of the ketogenic diet in controlling inflammation and suggest potential therapeutic targets for inflammatory neurodegenerative diseases, including glaucoma.

"Of Mice and Measures": A Project to Improve How We Advance Duchenne Muscular Dystrophy Therapies to the Clinic.

A new line of dystrophic mdx mice on the DBA/2J (D2) background has emerged as a candidate to study the efficacy of therapeutic approaches for Duchenne muscular dystrophy (DMD). These mice harbor genetic polymorphisms that appear to increase the severity of the dystropathology, with disease modifiers that also occur in DMD patients, making them attractive for efficacy studies and drug development. This workshop aimed at collecting and consolidating available data on the pathological features and the natural history of these new D2/mdx mice, for comparison with classic mdx mice and controls, and to identify gaps in information and their potential value. The overall aim is to establish guidance on how to best use the D2/mdx mouse model in preclinical studies.

Genetic differences in the behavioral organization of binge eating, conditioned food reward, and compulsive-like eating in C57BL/6J and DBA/2J strains

Binge eating (BE) is a heritable symptom of eating disorders associated with anxiety, depression, malnutrition, and obesity. Genetic analysis of BE could facilitate therapeutic discovery. We used an intermittent, limited access BE paradigm involving sweetened palatable food (PF) to examine genetic differences in BE, conditioned food reward, and compulsive-like eating between C57BL/6J (B6J) and DBA/2J (D2J) inbred mouse strains. D2J mice showed a robust escalation in intake and conditioned place preference for the PF-paired side. D2J mice also showed a unique style of compulsive-like eating in the light/dark conflict test where they rapidly hoarded and consumed PF in the preferred unlit environment. BE and compulsive-like eating exhibited narrow-sense heritability estimates between 56 and 73%. To gain insight into the genetic basis, we phenotyped and genotyped a small cohort of 133 B6J × D2J-F2 mice at the peak location of three quantitative trait loci (QTL) previously identified in F2 mice for sweet taste (chromosome 4: 156 Mb), bitter taste (chromosome 6: 133 Mb) and behavioral sensitivity to drugs of abuse (chromosome 11: 50 Mb). The D2J allele on chromosome 6 was associated with greater PF intake on training days and greater compulsive-like PF intake, but only in males, suggesting that decreased bitter taste may increase BE in males. The D2J allele on chromosome 11 was associated with an increase in final PF intake and slope of escalation across days. Future studies employing larger crosses and genetic reference panels comprising B6J and D2J alleles will identify causal genes and neurobiological mechanisms.

Effects of sex on ethanol conditioned place preference, activity and variability in C57BL/6J and DBA/2J mice

Previous studies of ethanol drinking in rodents have shown greater intake in females than in males, but the reasons behind this difference are unknown. To address one possible interpretation of the drinking difference, these studies tested the hypothesis that female and male mice differ in sensitivity to the rewarding effects of ethanol using the conditioned place preference (CPP) procedure. To increase the generalizability of the results, sex differences were examined in two inbred mouse strains known to differ in their sensitivity to ethanol reward: C57BL/6J (B6) and DBA/2J (D2). Mice were conditioned in an unbiased CPP procedure using either 1 or 2 g/kg ethanol. To detect possible differences in learning rate, they were tested once at the midpoint of conditioning and again after conditioning ended. As expected, CPP was stronger with 2 g/kg than with 1 g/kg, and D2 mice generally showed stronger CPP than B6 mice. However, there were no sex differences in the rate of CPP acquisition or in CPP magnitude, suggesting no sex difference in ethanol reward sensitivity as indexed by CPP. Nevertheless, there were sex differences in locomotor activity. B6 females were generally more active than B6 males during CPP acquisition whereas D2 females were slightly less active than D2 males during both CPP acquisition and preference testing. Unexpectedly, female mice showed more variability than males in the behavioral measures recorded in these studies, encouraging greater attention to variability in the design, analysis and interpretation of future studies of sex differences in mice.

Jnk2 deficiency increases the rate of glaucomatous neurodegeneration in ocular hypertensive DBA/2J mice

The cJun N-terminal kinases (JNKs; JNK1, JNK2, and JNK3) promote degenerative processes after neuronal injury and in disease. JNK2 and JNK3 have been shown to promote retinal ganglion cell (RGC) death after optic nerve injury. In their absence, long-term survival of RGC somas is significantly increased after mechanical optic nerve injury. In glaucoma, because optic nerve damage is thought to be a major cause of RGC death, JNKs are an important potential target for therapeutic intervention. To assess the role of JNK2 and JNK3 in an ocular hypertensive model of glaucoma, null alleles of Jnk2 and Jnk3 were backcrossed into the DBA/2J (D2) mouse. JNK activation occurred in RGCs following increased intraocular pressure in D2 mice. However, deficiency of both Jnk2 and Jnk3 together did not lessen optic nerve damage or RGC death. These results differentiate the molecular pathways controlling cell death in ocular hypertensive glaucoma compared with mechanical optic nerve injury. It is further shown that JUN, a pro-death component of the JNK pathway in RGCs, can be activated in glaucoma in the absence of JNK2 and JNK3. This implicates JNK1 in glaucomatous RGC death. Unexpectedly, at younger ages, Jnk2-deficient mice were more likely to develop features of glaucomatous neurodegeneration than D2 mice expressing Jnk2. This appears to be due to a neuroprotective effect of JNK2 and not due to a change in intraocular pressure. The Jnk2-deficient context also unmasked a lesser role for Jnk3 in glaucoma. Jnk2 and Jnk3 double knockout mice had a modestly increased risk of neurodegeneration compared with mice only deficient in Jnk2. Overall, these findings are consistent with pleiotropic effects of JNK isoforms in glaucoma and suggest caution is warranted when using JNK inhibitors to treat chronic neurodegenerative conditions.

Affective Disturbances During Withdrawal from Chronic Intermittent Ethanol Inhalation in C57BL/6J and DBA/2J Male Mice.

Alcohol use disorders are characterized by a complex behavioral symptomatology, which includes the loss of control over alcohol consumption and the emergence of a negative affective state when alcohol is not consumed. Some of these symptoms can be recapitulated in rodent models, for instance following chronic intermittent ethanol (EtOH; CIE) vapor inhalation. However, the detection of negative affect in mice withdrawn from CIE has proven challenging and variable between strains. This study aimed to detect reliable indices of negative emotionality in CIE-exposed C57BL/6J (C57) and DBA/2J (DBA) mice. Males were used because they are known to escalate their voluntary EtOH consumption upon CIE exposure, which is hypothesized to be driven by negative reinforcement (relief from negative affect). Adult male mice were exposed to 4 to 6weeks of CIE and were evaluated 3 to 10days into withdrawal in the social approach, novelty-suppressed feeding, digging, marble burying, and bottle brush tests. Withdrawal from CIE decreased sociability in DBA mice but not in C57 mice. Conversely, hyponeophagia was exacerbated by CIE in C57 mice but not in DBA mice. Withdrawal from CIE robustly increased digging activity in both strains, even in the absence of marbles. Aggressive responses to bottle brush attacks were elevated in both C57 and DBA mice following CIE exposure, but CIE had an opposite effect on defensive responses in the 2 strains (increase in C57 vs. decrease in DBA). Our results indicate that withdrawal from CIE elicits negative emotionality in both C57 and DBA mice, but different tests need to be used to measure the anxiogenic-like effects of withdrawal in each strain. Increased digging activity and irritability-like behavior represent novel indices of affective dysfunction associated with withdrawal from CIE in both mouse strains. Our findings enrich the characterization of the affective symptomatology of protracted withdrawal from CIE in mice.

Structural and Functional Rescue of Chronic Metabolically Stressed Optic Nerves through Respiration.

Axon degeneration can arise from metabolic stress, potentially a result of mitochondrial dysfunction or lack of appropriate substrate input. In this study, we investigated whether the metabolic vulnerability observed during optic neuropathy in the DBA/2J (D2) model of glaucoma is due to dysfunctional mitochondria or impaired substrate delivery to axons, the latter based on our observation of significantly decreased glucose and monocarboxylate transporters in D2 optic nerve (ON), human ON, and mice subjected to acute glaucoma injury. We placed both sexes of D2 mice destined to develop glaucoma and mice of a control strain, the DBA/2J-Gpnmb+, on a ketogenic diet to encourage mitochondrial function. Eight weeks of the diet generated mitochondria, improved energy availability by reversing monocarboxylate transporter decline, reduced glial hypertrophy, protected retinal ganglion cells and their axons from degeneration, and maintained physiological signaling to the brain. A robust antioxidant response also accompanied the response to the diet. These results suggest that energy compromise and subsequent axon degeneration in the D2 is due to low substrate availability secondary to transporter downregulation.SIGNIFICANCE STATEMENT We show axons in glaucomatous optic nerve are energy depleted and exhibit chronic metabolic stress. Underlying the metabolic stress are low levels of glucose and monocarboxylate transporters that compromise axon metabolism by limiting substrate availability. Axonal metabolic decline was reversed by upregulating monocarboxylate transporters as a result of placing the animals on a ketogenic diet. Optic nerve mitochondria responded capably to the oxidative phosphorylation necessitated by the diet and showed increased number. These findings indicate that the source of metabolic challenge can occur upstream of mitochondrial dysfunction. Importantly, the intervention was successful despite the animals being on the cusp of significant glaucoma progression.