Genetic networks controlling retinal injury.

PURPOSE: The present study defines genomic loci underlying coordinate changes in gene expression following retinal injury. METHODS: A group of acute phase genes expressed in diverse nervous system tissues was defined by combining microarray results from injury studies from rat retina, brain, and spinal cord. Genomic loci regulating the brain expression of acute phase genes were identified using a panel of BXD recombinant inbred (RI) mouse strains. Candidate upstream regulators within a locus were defined using single nucleotide polymorphism databases and promoter motif databases. RESULTS: The acute phase response of rat retina, brain, and spinal cord was dominated by transcription factors. Three genomic loci control transcript expression of acute phase genes in brains of BXD RI mouse strains. One locus was identified on chromosome 12 and was highly correlated with the expression of classic acute phase genes. Within the locus we identified the inhibitor of DNA binding 2 (Id2) as a candidate upstream regulator. Id2 was upregulated as an acute phase transcript in injury models of rat retina, brain, and spinal cord. CONCLUSIONS: We defined a group of transcriptional changes associated with the retinal acute injury response. Using genetic linkage analysis of natural transcript variation, we identified regulatory loci and candidate regulators that control transcript levels of acute phase genes.

The volume effect in irradiated mouse colorectum

Damage of the colorectum is the dose-limiting normal tissue complication following radiotherapy of prostate and cervical cancers. One approach for decreasing complications is to physically reduce the treatment volume. Mathematical models have been previously developed to describe the change in associated toxicity with a change in irradiated volume, i.e. the "volume effect", for serial-type normal tissues including the colorectum. The first goal of this thesis was to test the hypothesis that there would not be a threshold length in the development of obstruction after irradiation of mouse colorectum, as predicted by the Probability model of the volume effect. The second goal was to examine if there were differences in the threshold and in the incidence of colorectal obstruction after irradiation of two mouse strains, C57B1/6 (C57) and C3Hf/Kam (C3H), previously found to be fibrosis-prone and-resistant, respectively, after lung irradiation due, in part, to genetic differences. The hypothesis examined was that differences in incidence between strains were due to the differential expression of the fibrogenic cytokines $\rm TGF\beta$ and $\rm TNF\alpha.$ Various lengths of C57 and C3H mouse colorectum were irradiated and the incidence of colorectal obstruction was followed up to 15 months. A threshold length was observed for both mouse strains, in contradiction of model predictions. The mechanism of the threshold was epithelial regeneration after irradiation. C57 mice had significantly higher incidence of colorectal obstruction compared to C3H mice, especially at smaller irradiated lengths. Colorectal tissue was obtained at various times after irradiation and prepared for histology, immunohistochemistry and RNase protection assay for measurement of $\rm TGF\beta 1,$ 2, 3 and $\rm TNF\alpha$ mRNA. Distinct strain differences in the histological time of appearance and spatial locations of fibrosis were observed. However, there were no consistent strain difference in mRNA levels or immunolocalization for any of the cytokines examined. The data indicate the need for volume effect models that account for biologically important processes, such as the effect of epithelial regeneration after irradiation. As well, changes in fibrogenic cytokines at the mRNA level do not contribute to the strain difference in radiation-induced colorectal obstruction. ^

Mouse models and quantitative trait loci of susceptibility to bleomycin- and radiation-induced pulmonary fibrosis

Radiotherapy involving the thoracic cavity and chemotherapy with the drug bleomycin are both dose limited by the development of pulmonary fibrosis. From evidence that there is variation in the population in susceptibility to pulmonary fibrosis, and animal data, it was hypothesized that individual variation in susceptibility to bleomycin-induced, or radiation-induced, pulmonary fibrosis is, in part, genetically controlled. In this thesis a three generation mouse genetic model of C57BL/6J (fibrosis prone) and C3Hf/Kam (fibrosis resistant) mouse strains and F1 and F2 (F1 intercross) progeny derived from the parental strains was developed to investigate the genetic basis of susceptibility to fibrosis. In the bleomycin studies the mice received 100 mg/kg (125 for females) of bleomycin, via mini osmotic pump. The animals were sacrificed at eight weeks following treatment or when their breathing rate indicated respiratory distress. In the radiation studies the mice were given a single dose of 14 or 16 Gy (Co$\sp{60})$ to the whole thorax and were sacrificed when moribund. The phenotype was defined as the percent of fibrosis area in the left lung as quantified with image analysis of histological sections. Quantitative trait loci (QTL) mapping was used to identify the chromosomal location of genes which contribute to susceptibility to bleomycin-induced pulmonary fibrosis in C57BL/6J mice compared to C3Hf/Kam mice and to determine if the QTL's which influence susceptibility to bleomycin-induced lung fibrosis in these progenitor strains could be implicated in susceptibility to radiation-induced lung fibrosis. For bleomycin, a genome wide scan revealed QTL's on chromosome 17, at the MHC, (LOD = 11.7 for males and 7.2 for females) accounting for approximately 21% of the phenotypic variance, and on chromosome 11 (LOD = 4.9), in male mice only, adding 8% of phenotypic variance. The bleomycin QTL on chromosome 17 was also implicated for susceptibility to radiation-induced fibrosis (LOD = 5.0) and contributes 7% of the phenotypic variance in the radiation study. In conclusion, susceptibility to both bleomycin-induced and radiation-induced pulmonary fibrosis are heritable traits, and are influenced by a genetic factor which maps to a genomic region containing the MHC. ^

Skeletal abnormalities in mice lacking extracellular matrix proteins, thrombospondin-1, thrombospondin-3, thrombospondin-5, and type IX collagen.

Thrombospondin-5 (TSP5) is a large extracellular matrix glycoprotein found in musculoskeletal tissues. TSP5 mutations cause two skeletal dysplasias, pseudoachondroplasia and multiple epiphyseal dysplasia; both show a characteristic growth plate phenotype with retention of TSP5, type IX collagen (Col9), and matrillin-3 in the rough endoplasmic reticulum. Whereas most studies focus on defining the disease process, few functional studies have been performed. TSP5 knockout mice have no obvious skeletal abnormalities, suggesting that TSP5 is not essential in the growth plate and/or that other TSPs may compensate. In contrast, Col9 knockout mice have diminished matrillin-3 levels in the extracellular matrix and early-onset osteoarthritis. To define the roles of TSP1, TSP3, TSP5, and Col9 in the growth plate, all knockout and combinatorial strains were analyzed using histomorphometric techniques. While significant alterations in growth plate organization were found in certain single knockout mouse strains, skeletal growth was only mildly disturbed. In contrast, dramatic changes in growth plate organization in TSP3/5/Col9 knockout mice resulted in a 20% reduction in limb length, corresponding to similar short stature in humans. These studies show that type IX collagen may regulate growth plate width; TSP3, TSP5, and Col9 appear to contribute to growth plate organization; and TSP1 may help define the timing of growth plate closure when other extracellular proteins are absent.

Selective elevation of c-{\it myc} RNA transcripts during clonal evolution of N-methyl-N-nitrosourea induced thymic lymphomas in AKR/J mice

Untreated AKR mice develop spontaneous thymic lymphomas by 6-12 months of age. Lymphoma development is accelerated when young mice are injected with the carcinogen N-methyl-N-nitrosourea (MNU). Selected molecular and cellular events were compared during the latent period preceding "spontaneous" (retrovirally-induced) and MNU-induced thymic lymphoma development in AKR mice. These studies were undertaken to test the hypothesis that thymic lymphomas induced in the same inbred mouse strain by endogenous retroviruses and by a chemical carcinogen develop by different mechanisms.^ Immunofluorescence analysis of differentiation antigens showed that most MNU-induced lymphomas express an immature CD4-8+ profile. In contrast, spontaneous lymphomas represent each of the major lymphocyte subsets. These data suggest involvement of different target populations in MNU-induced and spontaneous lymphomas. Analyses at intervals after MNU treatment revealed selective expansion of the CD4-8+ J11d+ thymocyte subset at 8-10 weeks post-MNU in 68% of the animals examined, suggesting that these cells are targets for MNU-induced lymphomagenesis. Untreated age-matched animals showed no selective expansion of thymocyte subsets.^ Previous data have shown that both spontaneous and MNU-induced lymphomas are monoclonal or oligoclonal. Distinct rearrangement patterns of the J$\sb2$ region of the T-cell receptor $\beta$-chain showed emergence of clonal thymocyte populations beginning at 6-7 weeks after MNU treatment. However, lymphocytes from untreated animals showed no evidence of clonal expansion at the time intervals investigated.^ Activation of c-myc frequently occurs during development of B- and T- cell lymphomas. Both spontaneous and MNU-induced lymphomas showed increased c-myc transcript levels. Increased c-myc transcription was first detected at 6 weeks post-MNU, and persisted throughout the latent period. However, untreated animals showed no increases in c-myc transcripts at the time intervals examined. Another nuclear oncogene, c-fos, did not display a similar change in RNA transcription during the latent period.^ These results supports the hypothesis that MNU-induced and spontaneous tumors develop by multi-step pathways which are distinct with respect to the target cell population affected. Clonal emergence and c-myc deregulation are important steps in the development of both MNU-induced and spontaneous tumors, but the onset of these events is later in spontaneous tumor development. ^

Ontogeny and regulation of interleukin-7 expressing thymic epithelial cells

T cell development is a multistage process of differentiation that depends on proper thymocyte-thymic epithelial cell (TEC) interactions. Epithelial cells in the thymus are organized in a three-dimensional network that provides support and signals for thymocyte maturation. Concurrently, proper TEC differentiation in the adult thymus relies on thymocyte-derived signals. TECs produce interleukin-7 (IL-7), a non-redundant cytokine that promotes the survival, differentiation, and proliferation of thymocytes. We have identified IL-7 expressing TECs throughout ontogeny and in the adult thymus by in situ hybridization analysis. IL-7 expression is initiated in the thymic fated domain of the thymic primordium by embryonic day 11.5, in a Foxn1 independent pathway. Marked changes occur in the localization and regulation of IL-7 expressing TECs during development. Whereas IL-7 expressing TECs are present throughout the early thymic rudiment, the majority of IL-7 producing TECs are concentrated in the adult thymic medulla. By analyzing mouse strains that sustain blocks at different stages of thymocyte development, we show that IL-7 expression is initiated independently of hematopoietic-derived signals during thymic organogenesis. However, thymocyte-derived signals play an essential role in regulating IL-7 expression in the adult TEC compartment. Furthermore, distinct thymocyte subsets regulate the expression of IL-7 and keratin 5 in adult cortical epithelium. Intraperitoneal injection of Recombination Activating Gene deficient mice (RAG-2−/−) with anti-CD3ϵ monoclonal antibody (mAb) induces CD4− 8− double negative thymocytes to undergo β-selection and differentiate into CD4+8+ cells. Analysis of the thymic stromal compartment reveals that progression through β-selection renders thymocytes competent to alter the pattern of IL-7 expression in the cortical TEC compartment. RAG-2−/− mice do not generate mature T cells and therefore the RAG-2−/− thymus is devoid of organized medullary regions. Histological examination of RAG-2−/− thymus following anti-CD3ϵ stimulation reveals the emergence of mature thymic medullary regions, as assessed by H & E staining and expression of thymic stromal medullary markers. Stromal medullary reorganization occurs in the absence of T cell receptor αβ expression, suggesting that activation of RAG-2−/− thymocytes by CD3ϵ ligation generates thymocyte-derived signals that induce thymic epithelial reorganization, generating a mature medullary compartment. This model provides a tool to assess the mechanisms underlying thymic medullary development. ^

Regulation of T cell function by complement C5A in mice during mycobacterial infection

Tuberculosis is the leading cause of death in the world due to a single infectious agent, making it critical to investigate all aspects of the immune response mounted against the causative agent, Mycobacterium tuberculosis , in order to better treat and prevent disease. Previous observations show a disparity in the ability to control mycobacterial growth between mouse strains sufficient in C5, such as C57BL/6 and B10.D2/nSnJ, and those naturally deficient in C5, such as A/J and B10.D2/nSnJ, with C5 deficient mice being more susceptible. It has been shown that during M. tuberculosis infection, C5 deficient macrophages have a defect in production of interleukin (IL)-12, a cytokine involved in the cyclical activation between infected macrophages and effector T cells. T cells stimulated by IL-12 produce interferon (IFN)-γ, the signature cytokine of T helper type 1 (Th1) cells. It is known that a cell-mediated Th1 response is crucial for control of M. tuberculosis in the lungs of humans and mice. This study demonstrates that murine T cells express detectable levels of CD88, a receptor for C5a (C5aR), following antigen presentation by macrophages infected with mycobacteria. T cells from C5 deficient mice infected with M. tuberculosis were found to secrete less IFN-γ and had a reduced Th1 phenotype associated with fewer cells expressing the transcription factor, T-box expressed in T cells (T-bet). The altered Th1 phenotype in M. tuberculosis infected C5 deficient mice coincided with a rise in IL-4 and IL-10 secretion from Th2 cells and inducible regulatory T cells, respectively. It was found that the ineffective T cell response to mycobacteria in C5 deficient mice was due indirectly to a lack of C5a via poor priming by infected macrophages and possibly by a direct interaction between T cells and C5a peptide. Therefore, these studies show a link between the cells of the innate and adaptive arms of the immune system, macrophages and T cells respectively, that was mediated by C5a using a mouse model of M. tuberculosis infection. ^

Genetic analysis of tumor progression susceptibility in the mouse skin model

In the field of chemical carcinogenesis the use of animal models has proved to be a useful tool in dissecting the multistage process of tumor formation. In this regard the outbred SENCAR mouse has been the strain of choice in the analysis of skin carcinogenesis given its high sensitivity to the chemically induced acquisition of premalignant lesions, papillomas, and the later progression of these lesions into squamous cell carcinomas (SCC).^ The derivation of an inbred strain from the SENCAR stock called SSIN, that in spite of a high sensitivity to the development of papillomas lack the ability to transform these premalignant lesions into SCC, suggested that tumor promotion and progression were under the genetic control of different sets of genes.^ In the present study the nature of susceptibility to tumor progression was investigated. Analysis of F1 hybrids between the outbred SENCAR and SSIN mice suggested that there is at least one dominant gene responsible for susceptibility to tumor progression.^ Later development of another inbred strain from the outbred SENCAR stock, that had sensitivity to both tumor promotion and progression, allowed the formulation of a more accurate genetic model. Using this newly derived line, SENCAR B/Pt. and SSIN it was determined that there is one dominant tumor progression susceptibility gene. Linkage analysis showed that this gene maps to mouse chromosome 14 and it was possible to narrow the region to a 16 cM interval.^ In order to better characterize the nature of the progression susceptibility differences between these two strains, their proliferative pattern was investigated. It was found that SENCAR B/Pt, have an enlarged proliferative compartment with overexpression of cyclin D1, p16 and p21. Further studies showed an aberrant overexpression of TGF-$\beta$ in the susceptible strain, an increase in apoptosis, p53 protein accumulation and early loss of connexin 26. These results taken together suggest that papillomas in the SENCAR B/Pt. mice have higher proliferation and may have an increase in genomic instability, these two factors would contribute to a higher sensitivity to tumor progression. ^

Genetically controlled variation of "acid" beta-galactosidase detected in Rattus norvegicus by isoelectric focusing.

Two genetically variant forms of rat "acid" beta-galactosidase were found to differ in isoelectric point and pH dependence, but not in thermostability or sensitivity to inhibition by p-mercuribenzoate (PMB). The results of two backcrosses and an intercross indicated that the isoelectric focusing phenotypes are controlled by two codominant alleles at a single autosomal locus, for which we propose the name Glb-1. No significant linkage between Glb-1 and albino (LG I), brown (LG II), or hooded (LG VI) was observed. Strain-specific differences in total levels of kidney beta-galactosidase were detected, but it is not yet known whether the variation is controlled by genes linked to Glb-1. Experiments in which organ homogenates were incubated with neuraminidase indicated that the genetically variant forms do not result from differences in sialylation, though sialylation does appear to be largely responsible for the presence of multiple bands within each phenotype and for differences in the banding patterns of beta-galactosidases derived from different organs. The beta-galactosidase present in the bands used for Glb-1 typing resembles human GM1 gangliosidase (GLB1) with respect to pH optimum, substrate specificity, and susceptibility to inhibition by PMB. It also appears that Glb-1 is homologous with the Bgl-e locus of the mouse. In rats as in mice the genetically variant bands of beta-galactosidase are active at acid pH and have relatively high isoelectric points. In both species these bands are readily detectable in kidney homogenates, and can be revealed in homogenates of liver or spleen following treatment with neuraminidase. The presence of the same beta-galactosidase bands in homogenates of rat kidney and small intestine as well as in neuraminidase-treated homogenates of liver and spleen suggests that the Glb-1 variants differ by one or more point mutations in the structural gene for "acid" beta-galactosidase.

Detailed analysis of sequence changes occurring during vlsE antigenic variation in the mouse model of Borrelia burgdorferi infection.

Lyme disease Borrelia can infect humans and animals for months to years, despite the presence of an active host immune response. The vls antigenic variation system, which expresses the surface-exposed lipoprotein VlsE, plays a major role in B. burgdorferi immune evasion. Gene conversion between vls silent cassettes and the vlsE expression site occurs at high frequency during mammalian infection, resulting in sequence variation in the VlsE product. In this study, we examined vlsE sequence variation in B. burgdorferi B31 during mouse infection by analyzing 1,399 clones isolated from bladder, heart, joint, ear, and skin tissues of mice infected for 4 to 365 days. The median number of codon changes increased progressively in C3H/HeN mice from 4 to 28 days post infection, and no clones retained the parental vlsE sequence at 28 days. In contrast, the decrease in the number of clones with the parental vlsE sequence and the increase in the number of sequence changes occurred more gradually in severe combined immunodeficiency (SCID) mice. Clones containing a stop codon were isolated, indicating that continuous expression of full-length VlsE is not required for survival in vivo; also, these clones continued to undergo vlsE recombination. Analysis of clones with apparent single recombination events indicated that recombinations into vlsE are nonselective with regard to the silent cassette utilized, as well as the length and location of the recombination event. Sequence changes as small as one base pair were common. Fifteen percent of recovered vlsE variants contained "template-independent" sequence changes, which clustered in the variable regions of vlsE. We hypothesize that the increased frequency and complexity of vlsE sequence changes observed in clones recovered from immunocompetent mice (as compared with SCID mice) is due to rapid clearance of relatively invariant clones by variable region-specific anti-VlsE antibody responses.

Acute and chronic methylphenidate dose-response assessment on three adolescent male rat strains.

Methylphenidate (MPD), commonly known as Ritalin, is the most frequently prescribed drug to treat children and adults with attention deficit hyperactivity disorder (ADHD). Adolescence is a period of development involving numerous neuroplasticities throughout the central nervous system (CNS). Exposure to a psychostimulant such as MPD during this crucial period of neurodevelopment may cause transient or permanent changes in the CNS. Genetic variability may also influence these differences. Thus, the objective of the present study was to determine whether acute and chronic administration of MPD (0.6, 2.5, or 10.0mg/kg, i.p.) elicit effects among adolescent WKY, SHR, and SD rats and to compare whether there were strain differences. An automated, computerized, open-field activity monitoring system was used to study the dose-response characteristics of acute and repeated MPD administration throughout the 11-day experimental protocol. Results showed that all three adolescent rat groups exhibited dose-response characteristics following acute and chronic MPD administration, as well as strain differences. These strain differences depended on the MPD dose and locomotor index. Chronic treatment of MPD in these animals did not elicit behavioral sensitization, a phenomenon described in adult rats that is characterized by the progressive augmentation of the locomotor response to repeated administration of the drug. These results suggest that the animal's age at time of drug treatment and strain/genetic variability play a crucial role in the acute and chronic effect of MPD and in the development of behavioral sensitization.

Cardiomyocyte PDGFR-beta signaling is an essential component of the mouse cardiac response to load-induced stress.

PDGFR is an important target for novel anticancer therapeutics because it is overexpressed in a wide variety of malignancies. Recently, however, several anticancer drugs that inhibit PDGFR signaling have been associated with clinical heart failure. Understanding this effect of PDGFR inhibitors has been difficult because the role of PDGFR signaling in the heart remains largely unexplored. As described herein, we have found that PDGFR-beta expression and activation increase dramatically in the hearts of mice exposed to load-induced cardiac stress. In mice in which Pdgfrb was knocked out in the heart in development or in adulthood, exposure to load-induced stress resulted in cardiac dysfunction and heart failure. Mechanistically, we showed that cardiomyocyte PDGFR-beta signaling plays a vital role in stress-induced cardiac angiogenesis. Specifically, we demonstrated that cardiomyocyte PDGFR-beta was an essential upstream regulator of the stress-induced paracrine angiogenic capacity (the angiogenic potential) of cardiomyocytes. These results demonstrate that cardiomyocyte PDGFR-beta is a regulator of the compensatory cardiac response to pressure overload-induced stress. Furthermore, our findings may provide insights into the mechanism of cardiotoxicity due to anticancer PDGFR inhibitors.

Genetic and hormonal factors modulate spreading depression and transient hemiparesis in mouse models of familial hemiplegic migraine type 1.

Familial hemiplegic migraine type 1 (FHM1) is an autosomal dominant subtype of migraine with aura that is associated with hemiparesis. As with other types of migraine, it affects women more frequently than men. FHM1 is caused by mutations in the CACNA1A gene, which encodes the alpha1A subunit of Cav2.1 channels; the R192Q mutation in CACNA1A causes a mild form of FHM1, whereas the S218L mutation causes a severe, often lethal phenotype. Spreading depression (SD), a slowly propagating neuronal and glial cell depolarization that leads to depression of neuronal activity, is the most likely cause of migraine aura. Here, we have shown that transgenic mice expressing R192Q or S218L FHM1 mutations have increased SD frequency and propagation speed; enhanced corticostriatal propagation; and, similar to the human FHM1 phenotype, more severe and prolonged post-SD neurological deficits. The susceptibility to SD and neurological deficits is affected by allele dosage and is higher in S218L than R192Q mutants. Further, female S218L and R192Q mutant mice were more susceptible to SD and neurological deficits than males. This sex difference was abrogated by ovariectomy and senescence and was partially restored by estrogen replacement, implicating ovarian hormones in the observed sex differences in humans with FHM1. These findings demonstrate that genetic and hormonal factors modulate susceptibility to SD and neurological deficits in FHM1 mutant mice, providing a potential mechanism for the phenotypic diversity of human migraine and aura.

Synaptic vesicle dynamics in mouse rod bipolar cells.

To better understand synaptic signaling at the mammalian rod bipolar cell terminal and pave the way for applying genetic approaches to the study of visual information processing in the mammalian retina, synaptic vesicle dynamics and intraterminal calcium were monitored in terminals of acutely isolated mouse rod bipolar cells and the number of ribbon-style active zones quantified. We identified a releasable pool, corresponding to a maximum of 7 s. The presence of a smaller, rapidly releasing pool and a small, fast component of refilling was also suggested. Following calcium channel closure, membrane surface area was restored to baseline with a time constant that ranged from 2 to 21 s depending on the magnitude of the preceding Ca2+ transient. In addition, a brief, calcium-dependent delay often preceded the start of onset of membrane recovery. Thus, several aspects of synaptic vesicle dynamics appear to be conserved between rod-dominant bipolar cells of fish and mammalian rod bipolar cells. A major difference is that the number of vesicles available for release is significantly smaller in the mouse rod bipolar cell, both as a function of the total number per neuron and on a per active zone basis.

Cyan fluorescent protein expression in ganglion and amacrine cells in a thy1-CFP transgenic mouse retina.

PURPOSE: To characterize cyan fluorescent protein (CFP) expression in the retina of the thy1-CFP (B6.Cg-Tg(Thy1-CFP)23Jrs/J) transgenic mouse line. METHODS: CFP expression was characterized using morphometric methods and immunohistochemistry with antibodies to neurofilament light (NF-L), neuronal nuclei (NeuN), POU-domain protein (Brn3a) and calretinin, which immunolabel ganglion cells, and syntaxin 1 (HPC-1), glutamate decarboxylase 67 (GAD(67)), GABA plasma membrane transporter-1 (GAT-1), and choline acetyltransferase (ChAT), which immunolabel amacrine cells. RESULTS: CFP was extensively expressed in the inner retina, primarily in the inner plexiform layer (IPL), ganglion cell layer (GCL), nerve fiber layer, and optic nerve. CFP fluorescent cell bodies were in all retinal regions and their processes ramified in all laminae of the IPL. Some small, weakly CFP fluorescent somata were in the inner nuclear layer (INL). CFP-containing somata in the GCL ranged from 6 to 20 microm in diameter, and they had a density of 2636+/-347 cells/mm2 at 1.5 mm from the optic nerve head. Immunohistochemical studies demonstrated colocalization of CFP with the ganglion cell markers NF-L, NeuN, Brn3a, and calretinin. Immunohistochemistry with antibodies to HPC-1, GAD(67), GAT-1, and ChAT indicated that the small, weakly fluorescent CFP cells in the INL and GCL were cholinergic amacrine cells. CONCLUSIONS: The total number and density of CFP-fluorescent cells in the GCL were within the range of previous estimates of the total number of ganglion cells in the C57BL/6J line. Together these findings suggest that most ganglion cells in the thy1-CFP mouse line 23 express CFP. In conclusion, the thy1-CFP mouse line is highly useful for studies requiring the identification of ganglion cells.