Low-activity Strains Research Articles

Diazepam effects on anxiety-related defensive behavior of male and female high and low open‐field activity inbred mouse strains

Open-field activity is a commonly used measure of anxiety-related behavior in rodents. The inbred High and Low Activity strains of mice, selected for extreme differences in open-field activity, have been used as a genetic model of anxiety-related behaviors. These selected strains have been thoroughly studied through extensive behavioral testing, quantitative trait locus (QTL) mapping, whole-genome sequencing, and RNA sequencing, to uncover phenotypic and genotypic differences related to anxiety-related behavior. However, the effects of anxiolytic drugs on anxiety-related behavior in these strains have not been studied previously. This study allowed us to expand on previous findings to further characterize the anxiety-related behavior of these unique strains, using an anxiolytic drug. The goal of this study was to determine whether the treatment of adult male and female High Activity (low anxiety) and Low Activity (high anxiety) mice with diazepam, an agonist at the benzodiazepine allosteric site on the GABAA receptor and a drug commonly prescribed to treat anxiety disorders in humans, led to decreases in anxiety-like defensive behavioral responses as assessed in the open-field test (OFT) and elevated plus-maze (EPM). We tested the effects of three doses of diazepam (0, 0.5, 1.0, 3.0 mg/kg, i.p.), given 30 min before behavioral testing to one High Activity strain (H2) and two Low Activity strains (L1 and L2). There was an anxiolytic effect of diazepam observed in the High Activity strain, with more entries into the open arms of the elevated plus-maze, an effect similar to that seen in common mouse strains. However, the only anxiolytic effect of diazepam seen in the Low Activity strains was a reduction in stretch attend posture (SAP). Low Activity strains also displayed freezing behavior in both the OFT and EPM. The combination of the observed freezing behavior, that was not reduced by diazepam, and the reduction in SAP seen with diazepam, suggests a more complex phenotype that includes a component of innate fear in addition to anxiety-related risk assessment behaviors. Since fear and anxiety are distinguishable traits, and both contribute to human anxiety disorders, these results provide novel insight about interpretation of previous genetic and phenotypic differences observed between the High and Low Activity strains.

Hippocampal RNA sequencing in mice selectively bred for high and low activity.

High and Low Activity strains of mice were bidirectionally selected for differences in open-field activity (DeFries et al., 1978, Behavior Genetics, 8: 3-13) and subsequently inbred to use as a genetic model for studying anxiety-like behaviors (Booher et al., 2021, Genes, Brain and Behavior, 20: e12730). Hippocampal RNA-sequencing of the High and Low Activity mice identified 3901 differentially expressed protein-coding genes, with both sex-dependent and sex-independent effects. Functional enrichment analysis (PANTHER) highlighted 15 gene ontology terms, which allowed us to create a narrow list of 264 top candidate genes. Of the top candidate genes, 46 encoded four Complexes (I, II, IV and V) and two electron carriers (cytochrome c and ubiquinone) of the mitochondrial oxidative phosphorylation process. The most striking results were in the female high anxiety, Low Activity mice, where 39/46 genes relating to oxidative phosphorylation were upregulated. In addition, comparison of our top candidate genes with two previously curated High and Low Activity gene lists highlight 24 overlapping genes, where Ndufa13, which encodes the supernumerary subunit A13 of complex I, was the only gene to be included in all three lists. Mitochondrial dysfunction has recently been implicated as both a cause and effect of anxiety-related disorders and thus should be further explored as a possible novel pharmaceutical treatment for anxiety disorders.

Anxiety-related defensive behavioral responses in mice selectively bred for High and Low Activity.

High and Low Activity strains of mice (displaying low and high anxiety-like behavior, respectively) with 7.8-20 fold differences in open-field activity were selected and subsequently inbred to use as a genetic model for studying anxiety-like behavior in mice (DeFries et al., 1978, Behavior Genetics, 8:3-13). These strains exhibited differences in other anxiety-related behaviors as assessed using the light-dark box, elevated plus-maze, mirror chamber, and elevated square-maze tests (Henderson et al., 2004, Behavior Genetics, 34: 267-293). The purpose of these experiments was three-fold. First, we repeated a 6-day behavioral battery using updated equipment and software to confirm the extreme differences in anxiety-like behaviors. Second, we tested novel object exploration, a measure of anxiety-like behavior that does not rely heavily on locomotion. Third, we conducted a home cage wheel running experiment to determine whether these strains differ in locomotor activity in a familiar, home cage environment. Our behavioral test battery confirmed extreme differences in multiple measures of anxiety-like behaviors. Furthermore, the novel object test demonstrated that the High Activity mice exhibited decreased anxiety-like behaviors (increased nose pokes) compared to Low Activity mice. Finally, male Low Activity mice ran nearly twice as far each day on running wheels compared to High Activity mice, while female High and Low Activity mice did not differ in wheel running. These results support the idea that the behavioral differences between High and Low Activity mice are likely to be due to anxiety-related factors and not simply generalized differences in locomotor activity.

Whole-Genome Sequencing of Inbred Mouse Strains Selected for High and Low Open-Field Activity.

We conducted whole-genome sequencing of four inbred mouse strains initially selected for high (H1, H2) or low (L1, L2) open-field activity (OFA), and then examined strain distribution patterns for all DNA variants that differed between their BALB/cJ and C57BL/6J parental strains. Next, we assessed genome-wide sharing (3,678,826 variants) both between and within the High and Low Activity strains. Results suggested that about 10% of these DNA variants may be associated with OFA, and clearly demonstrated its polygenic nature. Finally, we conducted bioinformatic analyses of functional genomics data from mouse, rat, and human to refine previously identified quantitative trait loci (QTL) for anxiety-related measures. This combination of sequence analysis and genomic-data integration facilitated refinement of previously intractable QTL findings, and identified possible genes for functional follow-up studies.

Functional Differences in Pertussis Toxins from Bordetella pertussis Clinical Isolates as Determined by in vitro and in vivo Assays

Whooping cough caused by Bordetella pertussis is a serious disease especially for infants and young children. Detoxified pertussis toxin is a key component of vaccines used in campaigns worldwide for the prevention of the disease. A biochemical assay system, which measures pertussis toxin enzymatic and carbohydrate binding activities, has been developed to measure the residual toxin activities in vaccine matrix. We report here that B. pertussis clinical isolates show differences in pertussis toxin (PTx) activities in the in vitro biochemical assays and these in vitro activities were also positively related to the in vivo toxic activities. In addition, interesting information on the genetic and possible post-translational changes of PTx produced by the strains included in this study is tentatively discussed. Of the six strains studied in details, three low- and three high-activity strains, all the DNA sequences of the ptx gene clusters were found to be identical, except that in the high-activity strains, there was a silent mutation with a single nucleotide change at base 681 in ptxC which coded for Cys199 of PTx subunit S3. Mass spectrometric analysis of tryptic peptides from PTx produced by these strains detected two peptides in subunits S1 and S3, which were present in the low-activity strains but not in the high-activity strains. This suggests that PTx might differ in structure between these strains and this is possibly due to post-translational modification.

Genetic diversity and expression of carbon monoxide dehydrogenase from Aeropyrum pernix

The aerobic hyperthermophilic archaeon Aeropyrum pernix expresses molybdopterin carbon monoxide dehydrogenase (Mo-CODH). A. pernix strains isolated from Tachibana Bay (TB1–8) were found to exhibit different levels of total Mo-CODH activity (low and high, respectively), and the Mo-CODHs isolated from these strains also exhibit high or low activity. Mo-CODH gene transcription was detected by real-time reverse transcription-PCR, but no relation was found between the expression level of mRNA and the activity level of Mo-CODH. The nucleotide sequences of A. pernix genes encoding the small, large, and medium subunits of Mo-CODH, respectively, and those of the putative promoter region were identified from all TB strains. Amino acid substitutions were found in the sequences of high- and low-activity strains, but no mutation was detected in the putative promoter regions. Homology modeling revealed that all amino acid substitutions were localized on the surface of the Mo-CODH proteins. Based on these findings, we conclude that in A. pernix, the activity level of Mo-CODH may be regulated by translation or post-translational modification rather than by genomic diversity or transcription.

Naturally Occurring Lipid A Mutants in Neisseria meningitidis from Patients with Invasive Meningococcal Disease Are Associated with Reduced Coagulopathy

Neisseria meningitidis is a major cause of bacterial meningitis and sepsis worldwide. Lipopolysaccharide (LPS), a major component of the Gram-negative bacterial outer membrane, is sensed by mammalian cells through Toll-like receptor 4 (TLR4), resulting in activation of proinflammatory cytokine pathways. TLR4 recognizes the lipid A moiety of the LPS molecule, and the chemical composition of the lipid A determines how well it is recognized by TLR4. N. meningitidis has been reported to produce lipid A with six acyl chains, the optimal number for TLR4 recognition. Indeed, meningococcal sepsis is generally seen as the prototypical endotoxin-mediated disease. In the present study, we screened meningococcal disease isolates from 464 patients for their ability to induce cytokine production in vitro. We found that around 9% of them were dramatically less potent than wild-type strains. Analysis of the lipid A of several of the low-activity strains by mass spectrometry revealed they were penta-acylated, suggesting a mutation in the lpxL1 or lpxL2 genes required for addition of secondary acyl chains. Sequencing of these genes showed that all the low activity strains had mutations that inactivated the lpxL1 gene. In order to see whether lpxL1 mutants might give a different clinical picture, we investigated the clinical correlate of these mutations in a prospective nationwide observational cohort study of adults with meningococcal meningitis. Patients infected with an lpxL1 mutant presented significantly less frequently with rash and had higher thrombocyte counts, consistent with reduced cytokine induction and less activation of tissue-factor mediated coagulopathy. In conclusion, here we report for the first time that a surprisingly large fraction of meningococcal clinical isolates have LPS with underacylated lipid A due to mutations in the lpxL1 gene. The resulting low-activity LPS may have an important role in virulence by aiding the bacteria to evade the innate immune system. Our results provide the first example of a specific mutation in N. meningitidis that can be correlated with the clinical course of meningococcal disease.

Effects of apomorphine on open-field behavior in high- and low-activity mouse strains

The effects of i.p. injections of the non-selective dopamine (DA) agonist apomorphine on open-field behavior were examined in high- (H) and low-activity (L) strains of mice selected for open-field activity. ICR mice used as a foundation population for selection were also included. In all strains, apomorphine (0.01, 0.1, and 1mg/kg) dose-dependently reduced ambulatory activity, defecation, locomotion, and leaning. Significant decreases were found at 0.1-1mg/kg compared to the vehicle control. The occurrence of grooming reached a peak in the second minute at 1mg/kg, whereas it gradually increased during testing at other doses and in the vehicle control. The strains showed different dose responses for stretching and rearing: significant reductions were detected only in L at 1mg/kg for stretching and only in ICR at 0.1-1mg/kg for rearing. These strain differences suggest that the DA systems were genetically modified during selection in both strains.

高・低活動系マウスのオープンフィールド行動の時間構造：ＩＣＲおよび近交系５系統との比較

The present study examined open-field behavior in the high- and low-activity mouse strains, ICR, and five inbred strains of mice (BALB/c, C3H/He, CBA/Ca, C57BL/6, and DBA/2). The high- and low-activity strains were characterized as the highest and the lowest in ambulatory activity, respectively. However, they did not differ for defecation. A principal component analysis applied to observational data based on 11 behavioral items revealed four components, which accounted for 64% of the total variance. The strain differences found in component 1 and 2 could be described from four elements (initial values, changes in 1-3min and 4-10min of the test period, and final values). The high-activity strain contrasted with the low-activity strain in component 1, whereas they resembled each other in component 2. The similar pattern of strain differences was also found between BALB and C57BL. Component 2 might be related to a trait independent of “activity” or “emotionality”. Open-field behavior of the high-activity strain was much more similar to that of ICR (a base population of the selection) than the low-activity strain, which indicated that genetic changes had occurred mainly in the low-activity strain.

Partial characterisation of dimethylsulfoniopropionate (DMSP) lyase isozymes in 6 strains of Emiliania huxleyi

We characterised and compared dimethylsulfoniopropionate (DMSP) lyase isozymes in crude extracts of 6 axenic Emiliania huxleyi cultures (CCMP 370, 373, 374, 379, 1516, and strain L). This enzyme cleaves DMSP to form dimethyl sulfide (DMS), acrylate and a proton, but the function of this reaction in algae is still poorly understood. Most of the cultures produced high concentrations of intracellular DMSP, which was constant over the growth cycle and ranged from 157 to 242 mM, except for 1516 which had 50 mM DMSP cell-1. Extracts of all strains produced DMS from exogenous DMSP in vitro. DMSP lyases appeared constitutive, but enzyme activity and behaviour varied greatly among strains, and did not correlate with intracellular DMSP concentration. Strains 373 and 379 showed high DMSP lyase activities (12.5 and 6.1 fmol DMS cell-1 min-1, respectively), whereas DMS production was more than 100-fold lower in 370, 374, 1516 and L. This difference was intrinsic and the general pattern of high- and low-activity strains remained true over more than a 1 yr cultivation period. The cleavage reaction was optimal at pH 6 in the strains with high lyase activity and pH 5 was optimal for 374, 1516 and L. Strain 370 showed increasing activity with increasing pH. Experiments with additions of 0.125 to 2 M NaCl indicated halotolerant DMSP lyases in 373, 379 and 374. However, the halophilic DMSP lyases in 370 and L required 1 M NaCl addition for optimal DMSP cleavage, and 1516 showed optimal activity at 2 M NaCl. These results suggest that there are several structurally different DMSP lyase isozymes within E. huxleyi. However, it cannot be ruled out that varying concentrations of DMSP lyase per cell may have contributed to the differences in enzyme activity per cell. Comparison with other algal taxa indicates several families of DMSP lyases, hinting at possibly different cellular locations and functions, and varying DMS production under natural conditions.

Genotype- and gender-dependent hepatic alcohol dehydrogenase (ADH) activity in developing mice

Ethanol is metabolized in human and rodent liver primarily by the enzyme alcohol dehydrogenase (ADH). Hepatic ADH activity in adult males and females of seven inbred strains of mice was determined to examine genotype- and sex-dependent variability among the strains and the level of sexual dimorphism in each of the strains. ADH activity varied considerably among the strains, which could be categorized into high-activity strains C57BL/6, C57Brcd, and Swiss, and relatively low-activity strains C3H, CBA, and DBA. Adult Swiss, AKR, C57BL/6, and DBA females exhibited significantly higher levels of hepatic ADH than their male counterparts, whereas no gender differences were seen in C3H, CBA, and C57Brcd. Young mice of high and/or low ADH activity strains viz. C57BL/6 and C3H did not exhibit gender differences in ADH activity at weanling but the enzyme levels increased by sixth week in females and remained higher thereafter. The progeny of a high-activity strain with sexual dimorphism (C57BL/6) and a low-activity strain lacking gender difference (C3H) exhibited intermediate levels of ADH and age-dependent sexual dimorphism.

Structure and Function of a Long Alternating Purine-Pyrimidine Sequence in the Mouse Alcohol Dehydrogenase Adh-1 Gene

The mouse alcohol dehydrogenase gene Adh-1 contains an unusually long alternating purine-pyrimidine sequence within its first intron. This alternating sequence differs in length between strains that differ in the extent of Adh-1 expression, and it has been suggested that it plays a role in gene expression. We demonstrate that this alternating sequence can form Z-DNA in vitro. The alternating sequence can act as a positive regulatory element in transient transfection assays in hepatoma cell lines, but not in CV-1 (monkey kidney) cells, suggesting that it can act as a tissue-specific regulatory element. Nuclear run-on experiments showed that the differential expression of Adh-1 from high- and low-activity strains is, however, controlled at the post-transcription level.

Insertional activation of cepA leads to high-level beta-lactamase expression in Bacteroides fragilis clinical isolates

Bacteroides fragilis is an important opportunistic pathogen of humans and is resistant to many drugs commonly used to treat anaerobic infections, including beta-lactams. A strain set comprised of B. fragilis isolates producing either low or high levels of the endogenous cephalosporinase activity, CepA, has been described previously (M. B. Rogers, A. C. Parker, and C. J. Smith, Antimicrob. Agents Chemother. 37:2391-2400, 1993). Clones containing cepA genes from each of seven representative strains were isolated, and the DNA sequences were determined. Nucleotide sequence comparisons revealed that there were few differences between the cepA coding sequences of the low- and high-activity strains. The cepA coding sequences were cloned into an expression vector, pFD340, and analyzed in a B. fragilis 638 cepA mutant. The results of beta-lactamase assays and ampicillin MICs showed that there was no significant difference in the enzymatic activity of structural genes from the high- or low-activity strains. Comparison of sequences upstream of the cepA coding region revealed that 50 bp prior to the translation start codon, the sequence for high-activity strains change dramatically. This region of the high-activity strains shared extensive homology with IS21, suggesting that an insertion was responsible for the increased expression of cepA in these isolates. Northern (RNA) blot analysis of total RNA by using cepA-specific DNA probes supported the idea that differential cepA expression in low- and high-activity strains was controlled at the level of transcription. However, the insertion did not alter the cepA transcription start site, which occurred 27 bp upstream of the ATG translation start codon in both expression classes. Possible mechanisms of cepA activation are discussed.

Physiological and genetic analyses of inbred mouse strains with a type I iodothyronine 5' deiodinase deficiency.

Inbred mouse strains differ in their capacity to deiodinate iododioxin and iodothyronines, with strains segregating into high or low activity groups. Metabolism of iododioxin occurs via the type I iodothyronine 5'deiodinase (5'DI), one of two enzymes that metabolize thyroxine (T4) to 3,5,3'-triiodothyronine (T3). Recombinant inbred strains derived from crosses between high and low activity strains exhibit segregation characteristic of a single allele difference. Hepatic and renal 5'DI mRNA in a high (C57BL/6J) and low (C3H/HeJ) strain paralleled enzyme activity and concentration, in agreement with a recent report. 5'DI-deficient mice had twofold higher serum free T4 but normal free T3 and thyrotropin. Brown adipose tissue 5'DII was invariant between the two strains. Southern analyses using a 5'DI probe identified a restriction fragment length variant that segregated with 5'DI activity in 33 of 35 recombinant inbred strains derived from four different pairs of high and low activity parental strains. Recombination frequencies using previously mapped loci allowed assignment of the 5'DI gene to mouse chromosome 4 and identified its approximate chromosomal position. We propose the symbol Dio1 to denote the mouse 5'DI gene. Conserved linkage between this segment of mouse chromosome 4 and human HSA1p predicts this location for human Dio1.

A comparison of hepatic glucokinase gene expression in high- and low-activity strains of mice

1. 1. Compared with the rat, mouse liver glucokinase activities show a different sensitivity to changes in insulin concentrations. When animals from a high glucokinase-high insulin level strain C3H/He are crossed with those from a low glucokinase-low insulin strain C58, individuals from recombinant inbred lines show non-parental phenotypes with high glucokinase activity but low insulin and vice versa. 2. 2. Messenger RNA levels are greater in high-enzyme-activity mice strains than in low-activity animals, suggesting that differences in either transcription of the glucokinase gene or in mRNA stability occur between the two strains. 3. 3. There is no evidence of a different number of glucokinase genes in the high- and low-activity strains. Differences in activity therefore suggest that dissimilarities in the regulation of the expression of these genes may well occur.

Molecular characterization of the murine Coh locus: an amino acid difference at position 117 confers high and low coumarin 7-hydroxylase activity in P450coh.

Coumarin 7-hydroxylase (P450coh) and steroid 15 alpha-hydroxylase (P450(15 alpha) are encoded by members within the mouse 2A subfamily. Since P450coh activity is regulated by the Coh locus, we characterized P450coh cDNAs in strains having high coumarin 7-hydroxylase activity (CohH homozygote) including 129/J and DBA/2J, and compared them with P450coh cDNAs in low activity strains (CohL homozygote) C57BL/6J, C3H/HeJ and AKR/J. The nucleotide sequences of these two cDNAs differ by a single base, which results in an amino acid difference at position 117 (Val in P450cohH and Ala in P450cohL). The CohH phenotype exhibits approximately 10-fold greater Vmax and four-fold lower Km values than those in the CohL. Male 129 AKF1/J expresses approximately equal amounts of P450cohH and P450cohL mRNAs, associated with two Coh alleles. The levels of P450coh and P450(15 alpha) mRNAs in the F1 offspring suggested that a trans-acting factor(s) appeared to regulate the expressions of the P450 genes. A recent duplication in the ancestral mouse established the line of descent to P450(15 alpha) from the ancestral P450coh gene. During evolution, amino acid substitutions have selectively occurred at positions which alter the enzyme's substrate specificity and increase in the specific activity. Consistent with an important role of natural selection in the evolution of these genes is the relatively high nonsynonomous substitution rates on the P450(15 alpha) and the P450coh branches. As a result of these evolution events, the gene family consists of members which exhibit an extremely high degree of structural similarity, but very divergent hydroxylase activities and modes of regulation.

Distribution of meprin in kidneys from mice with high- and low-meprin activity.

An inherited deficiency of a metalloendopeptidase (meprin) activity occurs in kidneys of many inbred mouse strains. To clarify whether meprin protein is present in low-activity strains and determine the distribution of meprin in kidneys of mice with high- and low-meprin activities, kidney slices were stained through the use of the indirect immunoperoxidase technique and examined by light and electron microscopy. Light microscopy at high dilutions of anti-meprin IgG confirmed the brush border localization of meprin in high-meprin activity strains and revealed no detectable cross-reactive material in low-meprin activity strains. However, light and electron microscopy studies that use lower dilutions of anti-meprin immunoglobulin G (IgG) revealed cross-reactivity in low-activity strains, also at the luminal surface of the proximal tubules. Studies at lower magnifications indicated that meprin is primarily associated with the juxtamedullary region of the kidney in both high- and low-activity strains. Western blots of urinary proteins showed significant amounts of meprin-like proteins, but only in the urine of mice with high-meprin activity. The low activity of meprin in some inbred mouse strains is not associated with the presence of the protein in compartments of kidney cells other than the brush border or with secretion of the protein into the urine.

Purification and properties of arylsulfatase B from high- and low-activity mouse strains

Purification and properties of arylsulfatase B from high- and low-activity mouse strains

Studies on the relationship between dimethyl-nitrosamine-demethylase activity and dimethyl-nitrosamine dependent mutagenesis in, drosophila melanogaster

The relationship between dimethylnitrosamine (DMN) demethylase activity and DMN-induced mutagenesis was investigated in Drosophila melanogaster. The activity of DMN-demethylase was at least 10-fold greater in the Hikone-R strain than in three other Drosophila strains. However, the sex-linked recessive lethal (SLRL) mutations induced by DMN in the four strains differed by less than 2-fold. Several possibilities to explain the lack of correlation between DMN-demethylase activity and DMN-induced mutations were tested and eliminated. They include: (i) the presence of inhibitors of DMN-demethylase in extracts of low-activity strains, (ii) a sex bias in the Hikone-R strain in which the enzyme activity is confined to the females, (iii) the possibility that DMN treatment induces DMN-demethylase activity in the low-activity strains and (iv) the possibility that Hikone-R has a much more efficient DNA repair system than the other strains. The results are discussed in terms of what is known about the role of DMN-demethylase in the metabolic activation of DMN in other systems.

Regulation of mitochondrial malic enzyme synthesis in mouse brain.

In a previous study [Bernstine, E.G. (1979) J. Biol. Chem. 254, 83-87] it was shown that inbred strains of mice fall into two classes based on the specific activity of mitochondrial malic enzyme [L-malate:NADP+ oxidoreductase (oxaloacetate-decarboxylating), EC 1.1.1.40] in brain. In this report we demonstrate differences between high- and low-activity strains in the development of enzyme activity levels in adult mice and show that the rate of enzyme synthesis quantitatively accounts for the inherited level of the brain enzyme. Genetic analysis has established that the locus controlling the amount of enzyme in brain (Mdr-1) is located on chromosome 7. Its linkage to Hbb and c places it in the same region of the chromosome as Mod-2, the structural gene for mitochondrial malic enzyme. By making use of deletions and a duplication that include Mod-2, evidence for cis action of Mdr-1 was obtained.