D2 Mice Research Articles

Distinct roles of presynaptic dopamine receptors in the differential modulation of the intrinsic synapses of medium-spiny neurons in the nucleus accumbens

BackgroundIn both schizophrenia and addiction, pathological changes in dopamine release appear to induce alterations in the circuitry of the nucleus accumbens that affect coordinated thought and motivation. Dopamine acts principally on medium-spiny GABA neurons, which comprise 95% of accumbens neurons and give rise to the majority of inhibitory synapses in the nucleus. To examine dopamine action at single medium-spiny neuron synapses, we imaged Ca2+ levels in their presynaptic varicosities in the acute brain slice using two-photon microscopy.ResultsPresynaptic Ca2+ rises were differentially modulated by dopamine. The D1/D5 selective agonist SKF81297 was exclusively facilitatory. The D2/D3 selective agonist quinpirole was predominantly inhibitory, but in some instances it was facilitatory. Studies using D2 and D3 receptor knockout mice revealed that quinpirole inhibition was either D2 or D3 receptor-mediated, while facilitation was mainly D3 receptor-mediated. Subsets of varicosities responded to both D1 and D2 agonists, showing that there was significant co-expression of these receptor families in single medium-spiny neurons. Neighboring presynaptic varicosities showed strikingly heterogeneous responses to DA agonists, suggesting that DA receptors may be differentially trafficked to individual varicosities on the same medium-spiny neuron axon.ConclusionDopamine receptors are present on the presynaptic varicosities of medium-spiny neurons, where they potently control GABAergic synaptic transmission. While there is significant coexpression of D1 and D2 family dopamine receptors in individual neurons, at the subcellular level, these receptors appear to be heterogeneously distributed, potentially explaining the considerable controversy regarding dopamine action in the striatum, and in particular the degree of dopamine receptor segregation on these neurons. Assuming that post-receptor signaling is restricted to the microdomains of medium-spiny neuron varicosities, the heterogeneous distribution of dopamine receptors on individual varicosities is likely to encode patterns in striatal information processing.

Gene expression profiling of Hfe -/-liver and duodenum in mouse strains with differing susceptibilities to iron loading: identification of transcriptional regulatory targets of Hfe and potential hemochromatosis modifiers

BackgroundHfe disruption in mouse leads to experimental hemochromatosis by a mechanism that remains elusive. Affymetrix GeneChip® Mouse Genome 430 2.0 microarrays and bioinformatics tools were used to characterize patterns of gene expression in the liver and the duodenum of wild-type and Hfe-deficient B6 and D2 mice (two inbred mouse strains with divergent iron loading severity in response to Hfe disruption), to clarify the mechanisms of Hfe action, and to identify potential modifier genes.ResultsWe identified 1,343 transcripts that were upregulated or downregulated in liver and 370 in duodenum of Hfe-/- mice, as compared to wild-type mice of the same genetic background. In liver, Hfe disruption upregulated genes involved in antioxidant defense, reflecting mechanisms of hepatoprotection activated by iron overload. Hfe disruption also downregulated the expression of genes involved in fatty acid β-oxidation and cholesterol catabolism, and of genes participating in mitochondrial iron traffic, suggesting a link between Hfe and the mitochondrion in regulation of iron homeostasis. These latter alterations may contribute to the inappropriate iron deficiency signal sensed by the duodenal enterocytes of these mice, and the subsequent upregulation of the genes encoding the ferrireductase Dcytb and several iron transporters or facilitators of iron transport in the duodenum. In addition, for several genes differentially expressed between B6 and D2 mice, expression was regulated by loci overlapping with previously mapped Hfe-modifier loci.ConclusionThe expression patterns identified in this study contribute novel insights into the mechanisms of Hfe action and potential candidate genes for iron loading severity.

Identification of a Novel Candidate Regulator of HSC Aging.

It is well documented that both quantitative and qualitative changes in the murine hematopoietic stem cell (HSC) population occur with age. In mice, the effect of aging on stem cells is highly strain-specific, thus suggesting genetic regulation plays a role in HSC aging. We have previously mapped a quantitative trait locus (QTL) to murine Chr 2 that is associated with the variation in frequency of HSCs between aged B6 and D2 mice. In C57BL/6 (B6) mice the HSC population steadily increases with age, whereas in DBA/2 mice, this population declines. A QTL regulating the natural variation in lifespan between the two strains was mapped to the same location on mouse Chr 2, thus leading to the hypothesis that stem cell function affects longevity. B6 alleles, associated with expansion of the stem cell pool, are also associated with a ~50% increase in lifespan. Using a congenic mouse model, in which D2 alleles in the QTL interval were introgressed onto a B6 background, genome wide gene expression analyses were performed using sorted lineage negative hematopoietic cells, which are enriched for primitive stem and progenitor cells. Three variables were examined using Affymetrix M430 arrays:the effect of strain--congenic versus background;the effect of age--2 months versus 22 months; andthe effects of 2 Gy of radiation because previous studies indicated that congenic animals were highly sensitive to the effects of mild radiation compared to B6 background animals.Extensive analysis of the expression arrays pointed to a single strong candidate, the gene encoding ribosome binding protein 1 (Rrbp1). Real-time PCR was used to validate the differential expression of Rrbp1 in lineage negative, Sca-1+, c-kit+ (LSK) cells, a population highly enriched for stem and progenitor cells. Further analysis revealed the presence eight non-synonymous, coding single nucleotide polymorphisms (SNPs), and at least one of them because of its location and nature may significantly alter protein structure and function. The Rrbp1 gene consists of 23 exons in mouse and is highly conserved among mammalian species including mouse, human, and canine. The Rrbp1 protein is present on the surface of the rough endoplasmic reticulum where it tethers ribosomes to the membrane, stabilizes mRNA transcripts, and mediates translocation of nascent proteins destined for the cell secretory pathway. It is well established that the interaction of HSCs with microenvironmental niches in the bone marrow is crucial for their maintenance and self-renewal, and that this interaction is mediated in part by the molecular repertoires displayed on the cell surfaces of both HSCs and niche stromal cells. Therefore, we hypothesize that age and strain specific variation in Rrbp1, through its role in the secretory pathway, affects the molecular repertoire at the cell surface of the HSC, thus altering the way stem cells interact with their niches. This altered microenvironmental interaction could have profound effects on fundamental properties relevant to stem cell aging such as pluripotency, self-renewal, and senescence.

Genetic context determines susceptibility to intraocular pressure elevation in a mouse pigmentary glaucoma.

BackgroundDBA/2J (D2) mice develop an age-related form of glaucoma. Their eyes progressively develop iris pigment dispersion and iris atrophy followed by increased intraocular pressure (IOP) and glaucomatous optic nerve damage. Mutant alleles of the Gpnmb and Tyrp1 genes are necessary for the iris disease, but it is unknown whether alleles of other D2 gene(s) are necessary for the distinct later stages of disease. We initiated a study of congenic strains to further define the genetic requirements and disease mechanisms of the D2 glaucoma.ResultsTo further understand D2 glaucoma, we created congenic strains of mice on the C57BL/6J (B6) genetic background. B6 double-congenic mice carrying D2-derived Gpnmb and Tyrp1 mutations develop a D2-like iris disease. B6 single-congenics with only the Gpnmb and Tyrp1 mutations develop milder forms of iris disease. Genetic epistasis experiments introducing a B6 tyrosinase mutation into the congenic strains demonstrated that both the single and double-congenic iris diseases are rescued by interruption of melanin synthesis. Importantly, our experiments analyzing mice at ages up to 27 months indicate that the B6 double-congenic mice are much less prone to IOP elevation and glaucoma than are D2 mice.ConclusionAs demonstrated here, the Gpnmb and Tyrp1 iris phenotypes are both individually dependent on tyrosinase function. These results support involvement of abnormal melanosomal events in the diseases caused by each gene. In the context of the inbred D2 mouse strain, the glaucoma phenotype is clearly influenced by more genes than just Gpnmb and Tyrp1. Despite the outward similarity of pigment-dispersing iris disease between D2 and the B6 double-congenic mice, the congenic mice are much less susceptible to developing high IOP and glaucoma. These new congenic strains provide a valuable new resource for further studying the genetic and mechanistic complexity of this form of glaucoma.

A strain-specific alteration of proteomic expression in mouse liver fructose 1,6-bisphosphatase isoforms by alcohol.

To study alcohol-related metabolism across inbred mouse strains, liver tissues from C57BL/6J (B6, an alcohol-preferring mouse) and DBA/2J (D2, an alcohol-avoiding strain) mice were analyzed for proteomic expression patterns over time after a single-dose of alcohol (1.5 g/kg ingestion). Despite no significant difference in the elimination rate of blood ethanol, two-dimensional electrophoresis gel images of liver proteins showed that proteins in B6 mice exhibited faster response and more quantitative (spot numbers) and qualitative (spot densities) changes than in D2 mice. Among the differentially expressed metabolic enzymes, four variants (alpha, beta, gamma and delta) of fructose 1,6-bisphosphatase (FBPase), a key regulatory gluconeogenic enzyme, showed remarkable changes in expression with time across the strains. The degree of spot alteration in alpha- and gamma-variants of FBPase in B6 mice was much higher than in D2 mice, while the beta- and delta-forms were not changed as much. Mass spectrometry (MS) analysis showed that the 1714.9 +/- 1 mass peak from the alpha- and gamma-variants of FBPase was much stronger than that of the beta- and delta-variants in both strains regardless of spot density. This MS peak contains 2-ANHAPFETDISTLTR-16, located at the N-terminal of FBPase, where the N-terminal alanine was found to be trimethylated. Thus, we propose this N-terminal fragment as a potential site for enzyme modification in response to ethanol, allowing for differences in two-dimensional gel spot intensity of variants of FBPase in the two mouse strains.

A simple pattern classification method for alcohol-responsive proteins that are differentially expressed in mouse brain.

Proteomic analysis of brain tissues obtained from two inbred mice, C57BL/6J (B6, an alcohol-preferring strain) and DBA/2J (D2, an alcohol-avoiding strain), that were orally administered 1.5 g/kg ethanol, was performed to investigate alcohol-responsive proteins. To analyze relationships of alcohol-responsive protein spots between B6 and D2 mice, we have developed a simple spot classification method (SCM) for the fully matched spot data sets produced by the Melanie 4 analysis software using the paired two-dimensional (2-D) gels of two strains over time. By applying SCM, 55 protein spots that were differentially expressed in brain tissue were classified into 16 patterns as mirror images (2x8 patterns), and additionally in an ordered fashion such as 'fast turn over' and 'slow turn over' forms, depending on the frequency of repetition and rate of changed expression profile in 2-D gels over time. Searching for any interaction proteins through databases of interacting proteins using the classified data set has led to the construction of a linkage map, which reveals the interrelationship of the alcohol-responsive proteins between different species. Thus, it is suggested that the different responses for alcohol between B6 and D2 may come from differences of the response rates and interactions of different variants of the alcohol-responsive protein family.

Two kinds of ENU-induced scant hair mice and mapping of the mutant genes

To establish mouse models for human diseases through N-ethyl-N-nitrosourea (ENU) mutagenesis, and to provide groundwork to clone genes and study their functions after mapping the mutant genes. 18 male D2 mice (G0) at age of 8-10 weeks old were injected intraperitoneally with ENU (100 mg/kg) once a week for three consecutive weeks. The treated male mice were mated with females of the same strain, and their offspring (G1) were used to screen for dominant and recessive mutation. After breeding the mutant F2 (D2B6 F1 intercrossing) mice, 39 microsatellites that are equally distributed on the mouse genome and are different between B6 and D2 strains were used to scan the genome. According to the log odds score (LODS) we determined whether these microsatellites were linked to the mutant genes and calculated the location of mutant genes based on their recombination ratio. We screened 532 G1 mice, of which 14 exhibited mutation phenotypes. None was dominantly hereditable. Two cases of recessive inheritable scant hair mice were obtained through testing 30 G1 mice with normal phenotype and potential recessive mutant genes. All showed scant coat hair, grew slowly, and hyperkeratoses of epidermis and bollicular horn plug in histological sections. Their visceral organs were not markedly different from normal, and they were named scant hair 1 Baojin (symbol is snthr(-1Bao)) and scant hair 2 Baojin (symbol is snthr(-2Bao)). Through microsatellite screening we found that the LODS between snthr(-1Bao) and D9Mit243 was 7.73, and the linkage was determined. After analyzing the recombination ratio between snthr(-1Bao) and microsatellite D9Mit18 which was near snthr(-1Bao) based on a total number of 126 F2 mice with the scant hair phenotype, we determined that snthr(-1Bao) was located at chromosome 9 and was 71cM from centromere. Using the same technique, snthr(-2Bao) was mapped to the same position as snthr(-1Bao). In our research, two cases of scant hair mice provide good models for the study of dermatology, and the location of mutant genes provides a solid foundation for cloning new mice scant hair genes.

Genetic regulation of thymic involution

In this review, we have summarized our work using combined complex statistical genetics, bioinformatics, and functional genomics to determine the genetic basis of the age-related thymic involution in C57BL/6J X DBA/2J recombinant inbred mice and the parental B6 and D2 mice. We have shown that these mice provided a valuable genetic model that can permit resampling of thymuses from different aged but genetically identical animals and determination of the relative significance of age-associated changes in the thymus. Our results suggest that the quantitative trait loci (QTL) regulating the Con A-induced thymocyte proliferative response were mapped to mouse chromosome Chr 11 ( D11Mit51 at 18 cM), a region that harbors the IL-12b gene. The importance of IL-12b in maintaining thymic integrity and function during the aging process was confirmed by a more rapid involution of the thymus in IL-12b knockout ( IL-12b −/−) mice compared to wild-type (WT) mice. Functionally, IL-12 provided a strong synergistic effect to augment the IL-7 or IL-2 induced thymocyte proliferative response, especially in both aged WT and IL-12b −/− mice, but not in normal young mice. In contract to the proliferative response, the age-related decline in the total number of thymocytes was determined at different age, and mapped to loci on Chr 9, 62 cM and Chr 10, 32 cM. Using matrix-assisted laser desorption/ionisation–time of flight–mass spectrometry (MALDI–TOF–MS), increased expression of peroxiredoxin was found to be correlated with thymic involution. Our results suggest the possibility to identify the complex molecular network that can be associated with the regulation of thymic involution in aged mice using a high-dimensional functional genomics approach.

640. E1 Bp-Specific CD8+ T Lymphocytes Are Highly Induced in the Liver and Spleen Eight Days after Adenoviral Infection

Top of pageAbstract The use of Adenovirus (Ad) as a gene delivery vector is limited by transient gene expression due to the host cytotoxic T lymphocyte (CTL) response and innate responses. We and others have shown that the cytotoxic response to Ad is only partially inhibited by administration of Ad to mutant mice deficient in producing a cytotoxic immune response, or by treatment with TNF inhibitors. However, analysis of the specific CTL response to Ad has not been possible due to the lack of reagents that recognize the rearranged T cell receptor expressed on Ad-specific cytotoxic T cells. We therefore developed a tetramer that recognizes the immunodominant epitope of E1B (192 VNIRNCCYI), referred to as E1Bp, in the context of class Ib MHC. The tetramer consists of H2-Db, β2-microglobulin and the peptide epitope, and is referred to as Db-E1Bp. First, to test the specificity of this peptide-MHC complex, C57BL/6 (H-2b; B6) mice were injected with 8×108i.u. of wild type (wt) Ad5. As a control, wt Ad5 injected DBA/2 (H-2d; D2) were analyzed. Lymphocytes (105 )isolated 8 days later from spleen or liver were restimulated with E1Bp in vitro and analyzed for IFN-γ production by an ELISPOT assay. There was a significant increase in spots (6 and 63 spots, respectively) compared to cells stimulated with human influenza virus peptide M1(58 GILGFVFTL) (0 spot, p 98%) of the Db-E1Bp+CD8+ T cells are CD44+. No Db-E1Bp+CD8+ T cells were found in immunized D2 or naive B6 mice. To determine peptide-specific lysis of target cells in vivo, we labeled target cells from syngeneic mice with either high (2μM; CFSEHi) or low (0.2μM; CFSELo) concentration of CFSE and then pulsed CFSEHi cells with E1Bp and CFSELo cells remained unpulsed. Target cells were adoptively transferred into B6 or D2 mice that were challenged 8 days earlier with wt Ad5, and the percentage of specific killing was determined 6 hours later. As a peptide specificity control, CFSEHi cells pulsed with M1 peptide and unpulsed CFSELo cells were transferred to identically immunized B6 mice. E1Bp-specific lysis was significantly increased in B6 mice compared to control mice or immunized D2 mice (p<0.001). There was a significant correlation between E1Bp-specific lysis and the frequency of Db-E1Bp+ CD8+ T cells in B6 mice (p<0.01). We next used these methods to measure the decreased Ad-specific CTL response in aged mice. Compared with 2-mo-old mice, there was a 30% decrease in killing efficiency in 18-mo-old mice and a 58% decrease in Db-E1Bp+CD8+ T cells. Our results indicate that this tetramer is novel, sensitive and specific tool to detect and follow the E1Bp-specific CTL response against Ad infection.

644. Decreased Generation and Cytotoxic Function of Adenovirus-Specific CD8 T Cells in Both CD28 and FasL Deficient Mice

The development of conditionally replicative adenoviruses (CRAds) has presented a new platform for cancer gene therapy. The relative contribution of CRAd-related toxicity, and the cytotoxic immune response, especially the antigen specific cytotoxic T lymphocyte (CTL) response, in tumor cell killing is unclear. The anti-CRAd CTL may destroy tumor cells by specific killing; on the other hand, the spread of viral progeny may be limited by the clearance of infected cells. A better understanding of the effector mechanism of the CTL response would have implications in design of adenovirus-derived vectors, in particular, the potent CRAds. Previous studies indicated that FasL and TNF-α but not perforin, play a central role in immune-mediated adenoviral vector clearance, whereas CD28 is thought to be a critical molecule for the generation of effector cells. However, it has been difficult to clearly distinguish effector cell generation from its function due to the inability to specifically recognize Ad-specific CTLs. To investigate the effector mechanisms directly, we employed two novel approaches: tetramer staining and an antigen specific in vivo CTL assay. The Db-E1Bp MHC-I tetramer, that recognizes the immunodominant epitope of Ad E1B (192 VNIRNCCYI) is the first Ad-mouse tetramer. B6 mice, CD28KO mice and FasL deficient gld mice which are all on the H-2b background, as well as D2 mice (H-2d), were immunized with 4×108 i.u. of wild type Ad5. Eight days later, target cells from syngeneic mice were labeled with high concentration of CFSE (2μM; CFSEHi) and pulsed with E1Bp, or labeled with low concentration of CFSE (0.2μM; CFSELo), but unpulsed. Immunized mice were then adoptively transferred with identical numbers of CFSEHi and CFSELo cells. After 6 hours, lymphocytes were isolated from the spleen and liver and stained with fluorescent anti-CD8 and anti-CD44 antibodies and the Db-E1Bp tetramer. Flow cytometry results showed that in B6 mice, approximately 33% of the CD8+ T cells in liver and 11% in spleen are Db-E1Bp positive. The E1Bp-specific killing in the spleen of B6 mice was 93% whereas no Db-E1Bp+ CD8+ T cells or E1Bp-specific killing were observed in D2 mice. E1Bp-specific lysis was significantly correlated with the frequency of Db-E1Bp+CD8+ T cells in B6 mice (p<0.01). As expected, CD28 deficient mice exhibited an 80% reduction in generation of Db-E1Bp+CD8+ efffector T cells and E1Bp specific killing efficiency was reduced by 65% compared to B6 mice. Surprisingly, the E1Bp-specific killing efficiency in gld mice was only minimally reduced by 25% compared to B6 mice. There was also a 25% decrease in generation of Db-E1Bp+CD8+ T cells in gld mice. However, the specific cytotoxicity of individual CTL in both CD28KO and gld mice was compromised in that the percentage lysis per cell was lower than in B6. Our results indicate 1) CD28 plays an important role in generation of the specific CTL response to Ad; 2) FasL-dependent pathway plays a minor role in CTL response and also affects generation of CTLs; and 3) tetramer analysis of Ad-specific CTL and specific in vivo CTL assay can be used to distinguish generation and effector functions of CTLs.

Alternative mammary oncogenic pathways are induced by D-type cyclins; MMTV-cyclin D3 transgenic mice develop squamous cell carcinoma.

The three human D-type cyclins, cyclin D1, D2 and D3 share the ability to bind to and activate cdk4 and 6. MMTV-cyclin D1 transgenic mice develop mainly adenocarcinoma, while MMTV-cyclin D2 mice show a lack of alveologenesis during pregnancy and only develop carcinoma at low frequency. The effect of cyclin D3 overexpression in mammary glands remains hitherto unknown. We generated MMTV-cyclin D3 transgenic mice and report here that they develop exclusively squamous cell carcinoma. We show that although cyclin D3 transgene expression was detected early in puberty, postnatal development and mammary gland proliferation were normal in virgin animals. In contrast, multiparous mice develop multiple foci of abnormal growth that correspond to various stages of squamous metaplasia. Therefore, our results support a role for cyclin D3 in squamous differentiation. In addition, we found that p16 expression during involution is abolished, while p27 expression increased in MMTV-cyclin D3 mice, two modifications that have been reported in the other MMTV-D-type cyclin transgenic models. Our observations indicate that despite biochemical redundancy in vitro and in vivo, D-type cyclins promote distinct oncogenic pathways.

Functional analysis of cyclin D2 and p27(Kip1) in cyclin D2 transgenic mouse mammary gland during development.

Two mammary gland phenotypes were detected in pregnant MMTV-cyclin D2 transgenic mice; line D2-53 exhibited a lack of alveologenesis and failure to nurse, whereas line D2-58 featured a reduction in alveologenesis, but retained normal nursing behavior. In pregnant mammary glands, cyclin D2 protein levels were twofold (P<0.107) and 3.8-fold (P<0.0076) higher in line D2-58 and D2-53, respectively, compared to wild type. Concomitantly with the increase in cyclin D2 was a fivefold decrease in cyclin D1 hyper-phosphorylated isoform in mammary glands of pregnant cyclin D2-58 mice. Because cyclin D1 is a critical molecule in normal mammary lobuloalveolar development, these data suggest that overexpression of cyclin D2 may block mammary lobuloalveolar development through inhibition of cyclin D1 phosphorylation. During mammary gland development, p27(kip1) protein level oscillated in a similar profile in wild type and cyclin D2 transgenic mice, but was consistently higher in the cyclin D2 mice suggesting that p27(kip1) functions downstream of cyclin D2. The ratio of p27(kip1)-cdk4/p27(kip1)-cdk2 was 6.5-fold (P<0.0003) higher in cyclin D2 mammary glands compared to wild type in pregnant animals. This ratio reversed to 2.2-fold (P<0.005) higher in wild type compared to cyclin D2 mammary glands in involution suggesting that overexpression of cyclin D2 moderately induced apoptosis during pregnancy but accelerated involution. Collectively, the effects of cyclin D2 overexpression on mammary gland development during pregnancy and involution are attributed to two major factors, altered p27(kip1) protein level and inhibition of cyclin D1 phosphorylation.

Immunization with combined HSV-2 glycoproteins B2 : D2 gene DNAs: protection against lethal intravaginal challenges in mice.

The immunity of a combined DNA vaccine of HSV-2 glycoproteins B2 (gB2) and D2 (gD2) genes in comparison to individual vaccines was studied with regard to protecting against the HSV infection. Two recombinant DNA vaccines of the pHS2-gB2 or pHS2-gD2 were constructed and formulated. The neutralizing antibody titers appeared higher in the B2 : D2 gene cocktail-vaccinated mice than that of the individual B2 or D2 gene-vaccinated group alone, and the positive KOS control induced higher titer of the neutralizing antibody than combined or individual gene vaccines. The mock-immunized mice failed to induce enough. The ranks for the CTL activity and the protection rates against the lethal intravaginal challenge were shown as KOS > B2:D2 cocktail > D2 > B2 gene vaccines. The vaginal external diseases in the B2 : D2 or D-vaccinated mice were significantly reduced against the challenging dosages. The virus titers in the vaginal secretions of the vaccinated mice significantly reduced with time, and the B2 : D2 gene vaccine decreased more than each individual vaccine alone. It can be concluded that the cocktailed vaccines are more effective in the humoral and cellular immune responses in the mice, and in the protection of the mice against the intravaginal challenging dosages when compared with individual gene vaccines. All the DNA vaccines failed to block the latent infection in sensory nerves.

Amphetamine Distorts Stimulation-Dependent Dopamine Overflow: Effects on D2 Autoreceptors, Transporters, and Synaptic Vesicle Stores

Amphetamine (AMPH) is known to raise extracellular dopamine (DA) levels by inducing stimulation-independent DA efflux via reverse transport through the DA transporter and by inhibiting DA re-uptake. In contrast, recent studies indicate that AMPH decreases stimulation-dependent vesicular DA release. One candidate mechanism for this effect is the AMPH-mediated redistribution of DA from vesicles to the cytosol. In addition, the inhibition of stimulation-dependent release may occur because of D2 autoreceptor activation by DA that is released via reverse transport. We used the D2 receptor antagonist sulpiride and mice lacking the D2 receptor to address this issue. To evaluate carefully AMPH effects on release and uptake, we recorded stimulated DA overflow in striatal slices by using continuous amperometry and cyclic voltammetry. Recordings were fit by a random walk simulation of DA diffusion, including uptake with Michaelis-Menten kinetics, that provided estimates of DA concentration and uptake parameters. AMPH (10 microm) promoted the overflow of synaptically released DA by decreasing the apparent affinity for DA uptake (K(m) increase from 0.8 to 32 microm). The amount of DA released per pulse, however, was decreased by 82%. This release inhibition was prevented partly by superfusion with sulpiride (47% inhibition) and was reduced in D2 mutant mice (23% inhibition). When D2 autoreceptor activation was minimal, the combined effects of AMPH on DA release and uptake resulted in an enhanced overflow of exocytically released DA. Such enhancement of stimulation-dependent DA overflow may occur under conditions of low D2 receptor activity or expression, for example as a result of AMPH sensitization.

The Role of Dopamine Receptors in Regulating the Size of Axonal Arbors

Factors that regulate terminal arbor size of substantia nigra pars compacta (SNpc) neurons during development and after injury are not well understood. This study examined the role of dopamine receptors in regulating arbor size. Terminal arbors were examined in mice with targeted deletion of the D1 or D2 dopamine receptor [D1(-/-) and D2(-/-) mice, respectively]. Terminal trees were also examined after treatment with receptor blockers and after partial SNpc lesions. Immunohistochemistry was performed, and the number of SNpc neurons and dopaminergic terminals in the striatum was estimated. The number of dopaminergic SNpc neurons were reduced in D1(-/-) and D2(-/-) mice. Density of dopaminergic terminals was unchanged in D1(-/-) mice and increased in D2 (-/-) mice. Steady-state striatal DA and DOPAC levels revealed that dopamine activity was enhanced in D2(-/-) mice but reduced in D1(-/-) mice. Two months after partial SNpc lesions, striatal terminal density was normal in both wild-type and D1(-/-) mice but reduced in D2(-/-) mice. Administration of DA receptor antagonists resulted in larger terminal arbors in D1(-/-) and wild-type mice, whereas D2(-/-) mice showed no change in terminal density. Functional blockade of the D2R during development or in the adult brain results in increased axonal sprouting. Partial SNpc lesions resulted in compensatory sprouting, only in mice with functional D2R. These results suggest that individual dopaminergic axons in D2(-/-) mice have reached maximal arbor size. We conclude that the D2 receptor may play a role in modulating the extent of the terminal arbor of SNpc neurons.

Cyclin D2 is essential for BCR-mediated proliferation and CD5 B cell development.

Progression into G(1) in B lymphocytes is regulated by cyclins D2 and D3, components of the cell cycle machinery currently believed to have overlapping and potentially redundant roles in cell cycle control. To study the specific role of cyclin D2 in B lymphocyte proliferation, we examined B cells from cyclin D2(-/-) mice and demonstrate a specific requirement for cyclin D2 in BCR- but not CD40- or lipopolysaccharide-induced proliferation. Furthermore, conventional B cell development proceeds normally in the mutant mice; however, the CD5 B cell compartment is dramatically reduced, suggesting that cyclin D2 is important in CD5 B cell development as well as antigen-dependent B cell clonal expansion.

Cyclin D3 Compensates for Loss of Cyclin D2 in Mouse B-lymphocytes Activated via the Antigen Receptor and CD40

Cyclin D2 is the only D-type cyclin expressed in mature mouse B-lymphocytes, and its expression is associated with retinoblastoma protein (pRB) and pRB-related protein phosphorylation and induction of E2F activity, as B-cells enter the cell cycle following stimulation via surface IgM and/or CD40. Cyclin D-dependent kinase activity is required for cell proliferation, yet cyclin D2(-/-) mice have normal levels of mature B-lymphocytes. Here we show that B-lymphocytes from cyclin D2(-/-) mice can proliferate in response to anti-IgM and anti-CD40, but the time taken to enter S-phase is longer than for the corresponding cyclin D2(+/+) cells. This is due to the compensatory induction of cyclin D3, but not cyclin D1, which causes pRb phosphorylation on CDK4-specific sites. This is the first demonstration that loss of a D-type cyclin causes specific expression and functional compensation by another member of the family in vivo and provides a rationale for the presence of mature B-lymphocytes in cyclin D2(-/-) mice.

Cytotoxic metabolite of acetaminophen, N-acetyl-p-benzoquinone imine, produces cataract in DBA2 mice.

Acetaminophen, or N-acetyl-p-aminophenol (APAP), is metabolized to N-acetyl-p-benzoquinone imine (NAPQI) by cytochrome P450 enzymes in the liver. The biotransformation of APAP is enhanced in P450-inducible C57BL6 (B6) mice but not in non-inducible DBA2 (D2) mice. Our previous studies showed that high doses of APAP administered to B6 mice pretreated with beta-naphthoflavone (BNF), a P450 inducer, produced ocular tissue damage but not in D2 mice similarly treated. We then proposed that the ocular toxicity of APAP is due to accumulation of its metabolite, NAPQI. In the present work, we tested this hypothesis by injecting NAPQI (50 microg in 2 microl propyleneglycol/eye) intracamerally into B6 and D2 mice. NAPQI produced cataract within a few hours (mean = 4 hr) both in B6 and D2 mice. Lower concentrations of NAPQI did not produce lens opacification. Injection of the solvent propyleneglycol only did not cause cataract. Thus, when NAPQI was injected, P450 inducibility was not essential for cataract formation. In addition to vacuole formation in the lens epithelial cells, alterations were observed in the corneal endothelium and ciliary epithelium. The retinal cell layers remained intact. Extensive mitochondrial damage and changes in chromatin structure in the nucleus were evident in the affected lens epithelial cells. The present result dissociates APAP ocular toxicity from its metabolic potentiation by P450 enzymes and will allow us to investigate the mechanism of cataractogenesis in in vitro lens culture systems.

The Dopamine D2, but not D3or D4, Receptor Subtype is Essential for the Disruption of Prepulse Inhibition Produced by Amphetamine in Mice

Brain dopamine (DA) systems are involved in the modulation of the sensorimotor gating phenomenon known as prepulse inhibition (PPI). The class of D2-like receptors, including the D2, D3, and D4 receptor subtypes, have all been implicated in the control of PPI via studies of DA agonists and antagonists in rats. Nevertheless, the functional relevance of each receptor subtype remains unclear because these ligands are not specific. To determine the relevance of each receptor subtype, we used genetically altered strains of "knock-out" mice lacking the DA D2, D3, or D4 receptors. We tested the effects of each knock-out on both the phenotypic expression of PPI and the disruption of PPI produced by the indirect DA agonist d-amphetamine (AMPH). No phenotypic differences in PPI were observed at baseline. AMPH significantly disrupted PPI in the D2 (+/+) mice but had no effect in the D2 (-/-) mice. After AMPH treatment, both DA D3 and D4 receptor (+/+) and (-/-) mice had significant disruptions in PPI. These findings indicate that the AMPH-induced disruption of PPI is mediated via the DA D2 receptor and not the D3 or D4 receptor subtypes. Uncovering the neural mechanisms involved in PPI will further our understanding of the substrates of sensorimotor gating and could lead to better therapeutics to treat gating disorders, such as schizophrenia.

S10/L1 - Immunotoxicology assessment in the pharmaceutical industry

The effects of the route of benzo[a]pyrene administration on sister chromatid exchange (SCE) and B[a]P diol epoxide (B[a]PDE)-DNA adducts formation in bone marrow cells of Ah responsive (C57BL/6; B6) and Ah non-responsive (DBA/2; D2) mice were determined. Animals were treated intraperitoneally (i.p.), intragastrically (i.g.) or topically with two 100 mg/kg doses of benzo[a]pyrene 24 h apart and killed 96 h after the first treatment. Significant increase in the frequencies of SCE and the level of B[a]PDE-DNA adducts as measured by synchronous fluorescence spectrophotometry were detected in D2 mice as compared to B6 mice. The route of administration had little effect on SCE levels in bone marrow cells in D2 mice. In B6 mice higher levels of SCE were observed following i.p. administration as compared to i.g. or topical administration. In both strains the highest level of B[a]PDE-DNA adducts was formed after i.p. administration of B[a]P. We conclude that the i.p. route of B[a]P administration is the most effective in inducing SCE and B[a]P-DNA adducts formation. SCE induction does not correlate linearly with the amount of B[a]PDE-DNA adducts formed in these cells after administration of the above dose of B[a]P.