Tryptophan hydroxylase 1 knockout and tryptophan hydroxylase 2 polymorphism: effects on hypoxic pulmonary hypertension in mice

Serotonin [5-hydroxytryptamine (5-HT)] biosynthesis depends on two rate-limiting tryptophan hydroxylases (Tph): Tph1, which is expressed in peripheral organs, and Tph2, which is expressed in neurons. Because 5-HT is involved in pulmonary hypertension (PH), we investigated whether genetic variations in Tph1 and/or Tph2 affected PH development in mice. To examine the functional impact of peripheral Tph1 deficiency on hypoxic PH, we used Tph1(-/-) mice characterized by very low 5-HT synthesis rates and contents in the gut and lung and increased 5-HT synthesis in the forebrain. With chronic hypoxia, 5-HT synthesis in the forebrain increased further. Hypoxic PH, right ventricular hypertrophy, and distal pulmonary artery muscularization were less severe (P < 0.001) than in wild-type controls. The Tph inhibitor p-chlorophenylalanine (100 mgxkg(-1)xday(-1)) further improved these parameters. We then investigated whether mouse strains harboring the C1473G polymorphism of the Tph2 gene showed different PH phenotypes during hypoxia. Forebrain Tph activity was greater and hypoxic PH was more severe in C57Bl/6 and 129X1/SvJ mice homozygous for the 1473C allele than in DBA/2 and BALB/cJ mice homozygous for the 1473G allele. p-Chlorophenylalanine reduced PH in all groups and abolished the difference in PH severity across mouse strains. Hypoxia increased 5-hydroxytryptophan accumulation but decreased 5-HT contents in the forebrain and lung, suggesting accelerated 5-HT turnover during hypoxia. These results provide evidence that dysregulation of 5-HT synthesis is closely linked to the hypoxic PH phenotype in mice and that Tph1 and Tph2 may contribute to PH development.     

Association between Tph2 gene polymorphism, brain tryptophan hydroxylase activity and aggressiveness in mouse strains

The brain neurotransmitter serotonin is involved in the regulation of aggressive behavior. The main factor determining the brain serotonin level is the activity of the rate-limiting enzyme in the biosynthesis of the neurotransmitter--tryptophan hydroxylase isoform (TPH) 2 encoded by the Tph2 gene. Recently the C1473G single-nucleotide polymorphism in the Tph2 gene was reported. Here we study the C1473G polymorphism in 10 inbred mouse strains (C57BL/6J, AKR/J, DD/He, C3H/HeJ, YT/Y, BALB/cJLac, CC57BR/Mv and A/He) and demonstrate the association of the polymorphism with brain TPH activity and intermale aggressiveness. TPH activity in the midbrain of mice homozygous for the 1473C allele was higher than that in mice carrying 1473G alleles. A close association of the 1473C allele with increased number of attacks towards another male was found. The results support a link between the C1473G polymorphism in Tph2 gene, tryptophan hydroxylase activity and intensity of intermale aggression.     

Tyrosine hydroxylase in various brain regions of three strains of mice differing in spontaneous activity, learning ability, and emotionality.

Tyrosine hydroxylase has been measured in brains of three inbred strains of mice ; DBA/2J ; C57 BL/6J and BALB/cJ. Compared to C57 BL/6J, DBA/2J showed a higher enzyme activity in hypothalamus, a lower activity in pons-medulla, and no significant changes in cortex or striatum. BALB/cJ showed a higher level of activity in all regions studied (striatum, pons-medulla and hypothalamus). No effect of isolation or of social dominance position were noted on the enzyme activities in C57 BL/6J or BALB/cJ mice.     

Streptobacillus moniliformis epizootic in barrier-maintained C57BL/6J mice and susceptibility to infection of different strains of mice

We report a Streptobacillus moniliformis epizootic in barrier-maintained SPF mice. Although various inbred and F1 hybrid strains of mice have been kept in this animal facility, only C57BL/6J Han [corrected] mice showed clinical signs of disease. During the course of the epizootic, 825 breeding animals (approximately 36% of the breeders) died or had to be killed because of severe clinical signs. Although sequential treatment with ampicillin and chlortetracycline gave good therapeutic results, the animal facility was vacated in order to exclude any risk of cross-contamination of the other rodent colonies in our institute. The source and route of transmission of S. moniliformis could not be elucidated. To investigate strain dependent differences experimental infection of different strains of mice with our S. moniliformis isolate was performed. After oral infection only C57BL/6J showed the typical signs of a cervical lymphadenitis and gave an immunological response. BALB/cJ, C3H/He, DBA/2J, CB6F1 and B6D2F1 mice were not affected except in two cases of DBA/2J and B6D2F1 mice where seroconversion was observed. After intravenous infection of C57BL/6J, DBA/2J [corrected] and BALB/cJ all animals showed positive titers in the indirect immunofluorescence test (IIF). One hundred percent of the C57BL/6J, forty percent of the DBA/2J, and none of the BALB/cJ mice developed severe symptoms. The results demonstrate that the susceptibility to streptobacillosis is predominantly influenced by genetic factors.     

Female-predominant expression of testosterone 16 alpha-hydroxylase ("I"-P-450(16)alpha) and its repression in strain 129/J

Using specific testosterone 16 alpha-hydroxylase activity as the basis for selection of fractions during purification, the cytochrome P-450 ("I"-P-450(16)alpha) has been isolated from livers of phenobarbital-treated 129/J female mice [K. Devore, N. Harada, and M. Negishi (1985) Biochemistry, 24, 5632-5637]. An antibody elicited in rabbits to "I"-P-450(16)alpha was used to determine the amount of hepatic microsomal 16 alpha-hydroxylase activity due to "I"-P-450(16)alpha in untreated females and males of the two mouse strains, 129/J and BALB/cJ. The activities inhibited were 0.03 and 0.3 nmol/min/mg protein in the 129/J and BALB/cJ females, respectively. No significant level of "I"-P-450(16)alpha-dependent activity was detected in the microsomes from males of either mouse strain. Immunoblotting of microsomal proteins with the antibody to "I"-P-450(16)alpha revealed approximately a 10-fold greater amount of a 54-kDa protein in the microsomes from BALB/cJ than from 129/J females (0.03 and 0.26 pmol/micrograms protein, respectively). A cDNA clone (R17) for phenobarbital-inducible rat cytochrome P-450 selected "I"-P-450(16)alpha mRNA of mice, indicating a high degree of homology between the mRNAs of mouse "I"-P-450(16)alpha and phenobarbital-inducible rat cytochrome P-450s. Northern and dot hybridization of total mouse liver poly(A)+ RNA with the R17 cDNA probe indicated that the specific content of the hybridizable mRNA was more than 10 times higher in BALB/cJ females than in males, and that the mRNA level in female 129/J mice was very similar to that of 129/J and BALB/cJ males. The repression of "I"-P-450(16)alpha in 129/J females was inherited as an autosomal recessive trait in 129/J and BALB/cJ pairs as indicated by the levels of mRNA in female F1 offspring and the "I"-P-450(16)alpha-dependent hydroxylase activity. Female and male mice of eight more inbred strains (AKR/J, DBA/2J, C57BL/6J, C3H/HeJ, NZB/J, A/J, CBA/CaJ, and P/J) were tested for levels of mRNA. The results showed that the levels of mRNA were always 5- to 10-fold greater in the females than in the corresponding males, although there was some variation in the mRNA content in the males from the different strains. 129/J females appear to be a genetic variant where the female-predominant expression of the mRNA is repressed.     

Regulation of polyunsaturated fat induced postprandial hypercholesterolemia by a novel genePHC-2

Inbred mouse strains vary in susceptibility or resistance to dietary induced atherosclerosis. To investigate the effect of polyunsaturated fat feeding on postprandial serum cholesterol levels, in C57BL/67 (B6) and BALB/cJ inbred mice, we fed by stomach gavage previously fasted mice, a mixture containing 30% sunflower oil, 5% cholesterol, 2% sodium cholate and 0.5% choline chloride. The most significant difference in serum cholesterol levels between B6 and BALB/cJ mouse strains was observed at 2 h postfeeding. Susceptible B6 strain mice had a 41% postprandial increment in serum cholesterol. The resistant BALB/cJ strain had an insignificant 16% rise in serum cholesterol, at 2 h. We next examined eight other inbred mouse strains, to identify the gene(s) that regulate the observed 2 h postprandial hypercholesterolemia response, in the susceptible B6 mouse strain. Only the C57BR/cdJ and C57L/J strains developed postprandial hypercholesterolemia, at 2 h. The C57BR/cdJ strain had a 20% increase and the C57L/J strain a 62% increase in postprandial serum cholesterol levels. From this result, we found that the postprandial hypercholesterolemic response to an acute polyunsaturated fat-cholesterol feed, cosegregated with the a allele at the Gpd-1 and Ahd-1 loci, on mouse chromosome 4. In this study, non-responsiveness cosegregated with the b allele at the Gpd-1 and Ahd-1 loci. Thus polyunsaturated fat-cholesterol induced postprandial hypercholesterolemia appeared to be genetically determined by a gene located between the Gpd-1 and Ahd-1 loci, in mice. The putative gene regulating polyunsaturated fat-cholesterol induced post-absorptive hypercholesterolemia was designated Phc-2. Further studies with (BALB/cJ×C57BL/6J) Recombinant Inbred strains mapped the Phc-2 gene to a 2 cM region, within the Gpd-1 and Ahd-1 chromosomal segment, between the Pgd and Gpd-1 loci, on mouse chromosome 4.Abbreviations B6 C57BL/6J - HDL High Density Lipoprotein - LDL Low Density Lipoprotein - Phe Postprandial Hypercholesterolemia - PUFA-C Polyunsaturated Fat with Cholesterol - VLDL Very Low Density Lipoprotein     

Genetic regulation of estrogen-dependent repression of female-specific testosterone 16 alpha-hydroxylase (I-P-450(16 alpha) in male mouse liver: murine Ripr locus

The genetic basis for repression of I-P-450(16 alpha) in livers of male mice was examined in 129/J and BALB/cJ mice. Castration of adult male BALB/cJ but not 129/J mice resulted in derepression of I-P-450(16 alpha) at its mRNA and activity levels. It was further found that the patterns of derepression in (129/J x BALB/cJ) F1 and F2 offspring indicated that the derepression of I-P-450(16 alpha) is inherited as an autosomal additive trait. The distribution of derepression among castrated recombinant inbred strains (9 X A) indicated a close link of a locus repressing I-P-450(16 alpha) in male mice to the Rip locus on chromosome 7. Rip was previously defined as a locus that regulates specific expression of I-P-450(16 alpha) in livers of female mice [Noshiro, M., Lakso, M., Kawajiri, K., & Negishi, M. (1988) Biochemistry (preceding paper in this issue)]. Other tested inbred mice (A/HeJ, C57BL/6J, C3H/HeJ, and DBA/2J) showed the derepression of I-P-450(16 alpha) by castration, such as BALB/cJ. We propose Ripr (repression of an action of Rip locus in male mice) as the name of the locus by which repression of I-P-450(16 alpha) is regulated in male mice. Treatment of castrated male BALB/cJ mice by testosterone propionate, estradiol valerate, or diethylstilbestrol repressed I-P-450(16 alpha) to the levels seen in normal BALB/cJ male mice. Dihydrotestosterone, however, had little effect in repressing I-P-450(16 alpha) in castrated mice. The results suggested that estrogen rather than androgen is a repressor of I-P-450(16 alpha) in livers of male mice.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tetracycline inducible gene manipulation in serotonergic neurons

The serotonergic (5-HT) neuronal system has important and diverse physiological functions throughout development and adulthood. Its dysregulation during development or later in adulthood has been implicated in many neuropsychiatric disorders. Transgenic animal models designed to study the contribution of serotonergic susceptibility genes to a pathological phenotype should ideally allow to study candidate gene overexpression or gene knockout selectively in serotonergic neurons at any desired time during life. For this purpose, conditional expression systems such as the tet-system are preferable. Here, we generated a transactivator (tTA) mouse line (TPH2-tTA) that allows temporal and spatial control of tetracycline (Ptet) controlled transgene expression as well as gene deletion in 5-HT neurons. The tTA cDNA was inserted into a 196 kb PAC containing a genomic mouse Tph2 fragment (177 kb) by homologous recombination in E. coli. For functional analysis of Ptet-controlled transgene expression, TPH2-tTA mice were crossed to a Ptet-regulated lacZ reporter line (Ptet-nLacZ). In adult double-transgenic TPH2-tTA/Ptet-nLacZ mice, TPH2-tTA founder line L62-20 showed strong serotonergic β-galactosidase expression which could be completely suppressed with doxycycline (Dox). Furthermore, Ptet-regulated gene expression could be reversibly activated or inactivated when Dox was either withdrawn or added to the system. For functional analysis of Ptet-controlled, Cre-mediated gene deletion, TPH2-tTA mice (L62-20) were crossed to double transgenic Ptet-Cre/R26R reporter mice to generate TPH2-tTA/Ptet-Cre/R26R mice. Without Dox, 5-HT specific recombination started at E12.5. With permanent Dox administration, Ptet-controlled Cre-mediated recombination was absent. Dox withdrawal either postnatally or during adulthood induced efficient recombination in serotonergic neurons of all raphe nuclei, respectively. In the enteric nervous system, recombination could not be detected. We generated a transgenic mouse tTA line (TPH2-tTA) which allows both inducible and reversible transgene expression and inducible Cre-mediated gene deletion selectively in 5-HT neurons throughout life. This will allow precise delineation of serotonergic gene functions during development and adulthood.     

Genetic control of immune responses to the 18-kDa protein of Mycobacterium leprae. Different TH1 subsets may be involved in proliferative and delayed-type hypersensitivity responses.

In vitro and in vivo responses to the 18-kDa protein of Mycobacterium leprae have been analysed in different strains of mice. Lymphocytes from BALB/cJ (H-2d), BALB.B (H-2b), B10.BR (H-2k), and B10.M (H-2f) mice primed with 18-kDa protein yielded high T cell proliferative responses, while those from C57BL/10J (H-2b) mice yielded lower responses. Both H-2 and non-H-2 genes contributed to the magnitude of responsiveness. F1 mice from high and low responder strains showed high responsiveness to the 18-kDa protein. Supernatants from lymph node cell cultures prepared from 18-kDa protein-immunised BALB/cJ, B10.BR, and C57BL/10J mice contained IL-2 but no IL-4, indicating that activated T cells from both high and low responder mice were of a TH1 phenotype. Cell cultures from low responder C57BL/10J mice produced less IL-2 than those from high responders. The low responsiveness to the 18-kDa protein in proliferative assays might be due to a low frequency of antigen-specific T cells in the C57BL/10J mouse strain. BALB/cJ, C57BL/10J, and F1 (BALB/cJ x B10.BR) mouse strains were tested for in vivo DTH reactions to the 18-kDa protein. All strains, including C57BL/10J, were high DTH responders. Although DTH effector cells and 18-kDa protein-specific proliferative T cells belong to the TH1 subset, our data comparing high and low responder status indicate that distinct TH1 subpopulations are stimulated in response to the 18-kDa protein of M. leprae.     

Optimal N-ethyl-N-nitrosourea (ENU) doses for inbred mouse strains

ENU is a powerful germline mutagen in the mouse, providing the opportunity to analyze the functions of large numbers of genes in the mammalian genome. In many mutagenesis experiments, it would be beneficial to exploit the advantages of inbred mouse strains. To perform an effective ENU mutagenesis screen using inbred mice, a dosage regimen is required to determine the optimal dose of ENU for that inbred strain, a time-consuming preliminary process. We have carried out dosage regimens for mutagenizing doses of ENU in ten inbred strains of mouse: 129X1/SvJ, 129S6/SvEv, A/J, BALB/cJ, BTBR/N, C3He/J, C3HeB/FeJ, C57BL/6J, C57BR/cdJ, and CBA/CaJ, and determined an optimal dose for each strain, defined by length of sterile period and number of males to survive treatment. Three strains: A/J, BALB/cJ and C57BL/6J, are able to tolerate high doses, up to 300 mg/kg body weight, and are highly recommended for mutagenesis studies.     

Effects of acute tryptophan depletion on brain serotonin function and concentrations of dopamine and norepinephrine in C57BL/6J and BALB/cJ mice

Acute tryptophan depletion (ATD) is a method of lowering brain serotonin (5-HT). Administration of large neutral amino acids (LNAA) limits the transport of endogenous tryptophan (TRP) across the blood brain barrier by competition with other LNAAs and subsequently decreases serotonergic neurotransmission. A recent discussion on the specificity and efficacy of the ATD paradigm for inhibition of central nervous 5-HT has arisen. Moreover, side effects such as vomiting and nausea after intake of amino acids (AA) still limit its use. ATD Moja-De is a revised mixture of AAs which is less nauseating than conventional protocols. It has been used in preliminary clinical studies but its effects on central 5-HT mechanisms and other neurotransmitter systems have not been validated in an animal model. We tested ATD Moja-De (TRP-) in two strains of mice: C57BL/6J, and BALB/cJ, which are reported to have impaired 5-HT synthesis and a more anxious phenotype relative to other strains of mice. ATD Moja-De lowered brain TRP, significantly decreased 5-HT synthesis as indexed by 5-HTP levels after decarboxlyase inhibition, and lowered 5-HT and 5-HIAA in both strains of mice, however more so in C57BL/6J than in BALB/cJ. Dopamine and its metabolites as well as norepinephrine were not affected. A balanced (TRP+) control mixture did not raise 5-HT or 5-HIAA. The present findings suggest that ATD Moja-De effectively and specifically suppresses central serotonergic function. These results also demonstrate a strain-specific effect of ATD Moja-De on anxiety-like behavior.     

Plasma levels of proteins of the alternative complement pathway in inbred mice that differ in resistance to Trypanosoma congolense infections.

Inbred BALB/c, A/J, and C57B1/6J mice were infected with Trypanosoma congolense (Trans Mara strain), clone TC13, and monitored for parasitemia, survival times, and plasma levels of complement components C3, C5, factor B, and factor H. Parasitemia was highest in BALB/c, intermediate in A/J, and lowest in C57Bl/6J mice. The mean survival times were 11.5 +/- 0.9, 23.8 +/- 2.3, and 119 +/- 26 days for BALB/c, A/J, and C57Bl/6J mice, respectively. Preinfection levels of factor H were significantly correlated with survival times (r = 0.7722, P less than 0.001). Marked differences were observed between the plasma levels of C3, factor B, and factor H in the 3 mouse strains following infection. Complement C5 levels showed the fewest changes. In the initial postinfection period, BALB/c mice had highest increases in the levels of the 4 complement proteins but also had the greatest declines toward the end of the infection. Factor H levels showed a biphasic increase in BALB/c and C57Bl/6J, but not in A/J mice, with peaks at days 3 and 9. Complement C3 levels declined in all mice toward the terminal stage of the disease. In the late stages of infection, factor B levels markedly decreased in BALB/c but significantly increased in C57Bl/6J mice. Factor B levels measured at the terminal stages in BALB/c, A/J, and C57Bl/6J were correlated positively with their respective survival times (r = 0.714, P less than 0.01). The results suggest that genetic differences in the alternative complement pathway might affect the resistance to T. congolense infections.     

Genetic analysis of strains C57BL/6J and BALB/cJ for Ath-1, a gene determining atherosclerosis susceptibility in mice

We previously reported that mice have at least one major gene determining atherosclerosis susceptibility, Ath-1. Susceptible alleles of Ath-1 are found in strain C57BL/6J and are associated with relatively low levels of high-density lipoprotein cholesterol (HDL-C) when these mice are fed an atherogenic diet. Resistant alleles of Ath-1 are found in strains C3H/HeJ and BALB/cJ and are associated with relatively high levels of HDL-C. Data reported earlier from the set of seven recombinant inbred (RI) strains, derived from C57BL/6By and BALB/cBy, showed that these parental strains differed at Ath-1. However, due to the limited number of RI strains, it was not possible to determine with certainty whether Ath-1 was the only major gene determining atherosclerosis susceptibility in these two strains or to determine its map position accurately. In this report, examination of F1, F2, and backcross progeny from a cross between C57BL/6J and BALB/cJ demonstrates that Ath-1 is the major gene determining atherosclerotic lesion formation and HDL-C levels in female mice. The data from male animals suggest that environmental factors or modifying genes also influence male HDL-C levels and thus partly obscure the Ath-1 phenotype. HDL-C levels in F1 progeny resemble the BALB/c parent. The data from the cross provide confirmatory evidence that Ath-1 is linked to Alp-2 on chromosome 1 with a map distance of 4.8 +/- 2.3 (SE). Combining these data with a previous cross between strain C57BL/6 and strain C3H/HeJ gives a map distance between Ath-1 and Alp-2 of 4.9 +/- 1.8 based on 7 crossovers found among 144 tested chromosomes.     

Rip locus: regulation of female-specific isozyme (I-P-450(16 alpha) of testosterone 16 alpha-hydroxylase in mouse liver, chromosome localization, and cloning of P-450 cDNA

The constitutive expression of phenobarbital-inducible mouse cytochrome P-450 (I-P-450(16 alpha) at the mRNA level and its associated testosterone 16 alpha-hydroxylase activity in liver microsomes was a female characteristic in many inbred mice, including BALB/cJ, A/HeJ, and C57BL/6J. This sex-dependent constitutive expression of the mRNA and enzyme activity was severely reduced in females of mouse strain 129/J. The distribution patterns of the mRNA and activity levels in individual offspring of F1, F2, and F1 backcrosses to progenitors, generated from crosses between 129/J and BALB/cJ mice, indicated that the female-specific expression of I-P-450(16 alpha) is an autosomal dominant trait under the regulation of a sex-limited single locus. It was found that the genotypes of this locus exhibited concordance with that of the coumarin hydroxylase locus (Coh locus) in eight out of nine 9 X A recombinant inbred strains, suggesting the localization of this sex-limited locus on chromosome 7. We propose Rip (regulation of sex-dependent, constitutive expression of phenobarbital-inducible P-450) as the name of this sex-limited locus. With the use of the rat P-450e cDNA probe, a cDNA library from liver poly(A+) RNA of BALB/cJ was screened, and three distinct cDNAs (pf3, pf26, and pf46) were selected on the basis of their restriction patterns. Nucleotide sequences of the cDNAs revealed that pf3 and pf46 are clones overlapped, with the exception that the 27-bp DNA is inserted in the coding region of pf46. The nucleotide sequence (named pf3/46) obtained from the overlapping sequences of pf3 and pf46 contained 1473 or 1500 bp of open-reading frame, and the deduced amino acid sequence shared 93% similarity with those of rat P-450b. The 27-bp insertion resulted in nine extra amino acids just in front of the cysteine residue, the fifth ligand for heme binding. The mRNA with 27-bp insertion was ubiquitously present in other inbred mice such as A/HeJ and C57BL/6J, but not in 129/J. S-1 nuclease analysis estimated a ratio of p46 and pf3 to be 1:50. Nucleotide and deduced amino acid sequences of the 1473-bp open-reading frame in pf26 possessed 83% similarity to those of pf3/46. Hybridizations of oligonucleotide probes (pf26-cu and pf3/46-cu) specific to either pf26 or pf3/46 with liver poly(A+) RNA from males and females of BALB/cJ, 129/F, and F1 offspring demonstrated that the expression of pf26, but not pf3/46, mRNA was associated with the autosomal dominant inheritance of I-P-450(16 alpha).(ABSTRACT TRUNCATED AT 400 WORDS)     

A tryptophan hydroxlyase 1 reporter that directs Cre recombinase extinguishable placental alkaline phosphatase expression in serotonergic (5-HT) neurons and peripheral tissues

The serotonergic (5-HT) system modulates many behaviors and has been implicated in psychiatric disorders, but the density of 5-HT processes has complicated analyses. We have used regulatory regions from the Tryptophan hydroxylase 1 (Tph1) gene to drive expression of LoxP-flanked placental alkaline phosphatase (PLAP) to generate the Tph1-Lox-PLAP reporter mouse line. In these mice, PLAP is expressed in the hindbrain raphe nuclei and in peripheral tissues known to express Tph1. Tph1 is expressed at low levels in neurons. While, in Tph1-Lox-PLAP mice, most PLAP-expressing neurons are monoaminergic, PLAP was expressed in only 5-10% of neurons expressing the predominant neuronal 5-HT biosynthetic enzyme Tph2, serotonin transporter (SERT) or aromatic amino acid decarboxylase (AADC). To test this reporter further, we examined the brains of mice carrying the anorexia (anx) mutation, in which increased overall density of 5-HT immunoreactivity had been previously observed at P21. PLAP-labeling of processes in anx/anx and anx/+ mice was reduced at P0. By P10, distribution of PLAP-labeled processes in anx/+ and +/+ cortices was indistinguishable, but differed markedly from that seen in the cortical layers of anx/anx mice. Thus, the Tph1-LoxP-PLAP reporter revealed a dosage sensitive role of the anx mutation in the early 5-HT system and later cortical layer-specific differences in 5-HT process distribution in anx/anx mice. Thus, the Tph1-LoxP-PLAP reporter provides a sensitive indicator for analyses of serotonergic cells in the brain and periphery.     

Differential intra-epithelial lymphocyte phenotypes following Cryptosporidium parvum challenge in susceptible and resistant athymic strains of mice

The lack of immunocompetent laboratory animal models has limited our understanding of functional immune responses to Cryptosporidium parvum infection, but such responses have been studied in susceptible laboratory rodents with genetic, acquired, or induced immunodeficiencies. We previously observed that athymic C57BL/6J nude mice inoculated with C. parvum oocysts had lower or absent fecal oocyst excretion when compared to inoculated athymic BALB/cJ nude mice. This discrepancy prompted us to explore potential differences in intestinal immune responses in both strains. Prior to and after C. parvum challenge, BALB/cJ nude and C57BL/6J nude mice did not differ in either spleen cell numbers or in parasite-specific proliferation. However, both strains of mice exhibited a significant increase in intra-epithelial lymphocyte (IEL) numbers prior to and following C. parvum inoculation when compared to uninoculated controls (P<0.05). Prior to challenge, C57BL/6J nude mice had a higher percentage of both CD8+ and CD8+ gammadelta+ IEL than BALB/cJ nude mice. Following challenge, resistant C57BL/6J nude mice had a higher percentage of gammadelta+, CD4+, and CD8+ gammadelta+ IEL than uninoculated C57BL/6J nude mice and than susceptible BALB/cJ nude mice (P<0.05). Conversely, inoculated C57BL/6J nude mice had a significantly lower percentage of alphabeta+ IEL than inoculated BALB/cJ nude mice (P<0.05). We conclude that gammadelta+, CD4+, and/or CD8+ gammadelta+ IEL may influence responses to cryptosporidiosis in athymic murine models, and that the increased percentage of alphabeta+ IEL in susceptible BALB/cJ nude mice could reflect a preferential expression during chronic C. parvum infection and/or might downregulate local protective responses.     

Genetic dissection of intermale aggressive behavior in BALB/cJ and A/J mice

Aggressive behaviors are disabling, treatment refractory, and sometimes lethal symptoms of several neuropsychiatric disorders. However, currently available treatments for patients are inadequate, and the underlying genetics and neurobiology of aggression is only beginning to be elucidated. Inbred mouse strains are useful for identifying genomic regions, and ultimately the relevant gene variants (alleles) in these regions, that affect mammalian aggressive behaviors, which, in turn, may help to identify neurobiological pathways that mediate aggression. The BALB/cJ inbred mouse strain exhibits relatively high levels of intermale aggressive behaviors and shows multiple brain and behavioral phenotypes relevant to neuropsychiatric syndromes associated with aggression. The A/J strain shows very low levels of aggression. We hypothesized that a cross between BALB/cJ and A/J inbred strains would reveal genomic loci that influence the tendency to initiate intermale aggressive behavior. To identify such loci, we conducted a genomewide scan in an F2 population of 660 male mice bred from BALB/cJ and A/J inbred mouse strains. Three significant loci on chromosomes 5, 10 and 15 that influence aggression were identified. The chromosome 5 and 15 loci are completely novel, and the chromosome 10 locus overlaps an aggression locus mapped in our previous study that used NZB/B1NJ and A/J as progenitor strains. Haplotype analysis of BALB/cJ, NZB/B1NJ and A/J strains showed three positional candidate genes in the chromosome 10 locus. Future studies involving fine genetic mapping of these loci as well as additional candidate gene analysis may lead to an improved biological understanding of mammalian aggressive behaviors.     

Dysregulation of the inflammatory response to the parasite, Toxoplasma gondii, in P2X7 receptor-deficient mice

The P2X(7) receptor (P2X(7)R) is a two transmembrane receptor that is highly expressed on the surface of immune cells. Loss of function polymorphisms in this receptor have been linked to increased susceptibility to intracellular pathogens. P2X(7)R gene knockout (P2X(7)R(-/-); on a C57Bl/6J background), C57Bl/6J and BALB/c mice were infected with the avirulent ME49 strain of the intracellular parasite, Toxoplasma gondii, and susceptibility determined by monitoring weight loss. P2X(7)R(-/-) mice lost significantly more weight than C57Bl/6J mice from day 8p.i. C57Bl/6J, in turn, lost significantly more weight than BALB/c mice. Thus, by day 10p.i., P2X(7)R(-/-) mice had lost 5.7 ± 0.7% of their weight versus 2.4 ± 0.6% for C57Bl/6J mice, whereas BALB/c mice had gained 1.9 ± 0.5%; by day 12p.i., P2X(7)R(-/-) mice had lost 15.1±0.6%, C57Bl/6J had lost 10.1±0.8% and BALB/c had lost 4.8 ± 0.8% of their weight. Neither parasite burden nor liver pathology was greater in the P2X(7)R(-/-) mice than in C57Bl/6J mice but BALB/c mice had significantly smaller numbers of parasites and less pathology in their livers than these strains. Absence of the P2X(7) receptor did not affect IFN-γ, IL-12, IL-1β, monocyte chemoattractant protein-1 (MCP-1) or TNF production. However, both P2X(7)R(-/-) and C57Bl/6J mice produced more IL-1β and TNF than BALB/c mice. There was one important point of differentiation between the P2X(7)R(-/-) and C57Bl/6J mice, namely the significantly enhanced and prolonged production of nitric oxide, accompanied by delayed production of IL-10 in the P2X(7)R-deficient mice.