Biomedical subjects

Andy J Fischer

Publications and source records attributed to Andy J Fischer.

16 recordsLinked to original sources

PubMed2007

Heterogeneity of horizontal cells in the chicken retina.

Despite numerous reports that different markers are expressed by horizontal cells in the avian retina, it remains unknown whether different types of horizontal cells can be defined by differences in their immunocytochemical profiles. The purpose of this study was to rectify this deficiency. We identified horizontal cells by indirect immunofluorescence with antibodies to calretinin, trkA, GABA, Prox1, AP2alpha, Pax6, islet1, and Lim1 + 2. We found two major groups of horizontal cells, those that express trkA and those that express calretinin. The trkA-immunoreactive (-IR) horizontal cells had small, round somata and robust, bulbous dendritic endings, whereas calretinin-IR horizontal cells had large, polygonal cell bodies and fine, diffuse dendritic endings, both contacting the calbindin-IR pedicles of double cones. Weak gamma-aminobutyric acid (GABA) immunoreactivity was observed only in a few of the trkA-IR horizontal cells, whereas the overlap of calretinin and GABA immunoreactivities was 100%. The majority of trkA-IR horizontal cells expressed islet1, and the majority of calretinin-IR horizontal cells expressed Lim1 + 2, AP2alpha, and Pax6. Islet1 immunoreactivity was observed in a small fraction of calretinin-IR/non-trkA-IR cells. In agreement with previous reports, we detected Prox1 immunoreactivity in all types of horizontal cells. These immunolabeling profiles suggest that there are four immunochemically distinct subtypes of horizontal cells in the postnatal chick retina, which may match the four types that have been observed in Golgi-impregnated pigeon and turtle retinas.

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PubMed2006

Evidence for the presence of the type 2 corticotropin releasing factor receptor in the rodent cerebellum.

Corticotropin releasing factor (CRF), localized in afferent inputs to the cerebellum, binds to two receptors defined as the Type 1 (CRF-R1) and the Type 2 (CRF-R2alpha). CRF-R1 has been localized to the cerebellum, as has a truncated isoform of CRF-R2alpha. Evidence for the presence of the full length isoform of CRF-R2alpha in the cerebellum is conflicting. We used RT-PCR, immunohistochemical, and physiologic techniques to resolve this conflict. RT-PCR data show low levels of CRF-R2alpha in the vermis and hemisphere of the cerebellum. These observations were confirmed by the Gene Expression Nervous System Atlas (GENSAT) database. A CRF-R2alpha antibody was used to determine the cellular distribution of the receptor in the cerebellum. The vast majority of the receptors are localized to Bergmann glial cells located throughout the cerebellum, as well as astrocytes in the granule cell layer. Neuronal labeling is present in sub-populations of Purkinje cells, Golgi cells, basket cells, and cerebellar nuclear neurons. Physiologic data show that urocortin II, which binds selectively to CRF-R2alpha, increases the firing rate of both Purkinje cells and nuclear neurons; this response can be blocked by the CRF-R2alpha-specific antagonist, antisauvagine-30. The present results confirm that CRF-R2alpha is present in the cerebellum and functions in circuits that modulate the firing rate of Purkinje cells and cerebellar nuclear neurons. A comparative analysis showed that the patterns of distribution of CRF-R1, CRF-R2alpha and CRF-R2alpha-tr are distinct. These data indicate that the CRF family of peptides modulates cerebellar output by binding to multiple CRF receptors.

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PubMed2006

Characterization of glucagon-expressing neurons in the chicken retina.

We recently identified large glucagon-expressing neurons that densely ramify neurites in the peripheral edge of the retina and regulate the proliferation of progenitors in the circumferential marginal zone (CMZ) of the postnatal chicken eye (Fischer et al. [2005] J Neurosci 25:10157-10166). However, nothing is known about the transmitters and proteins that are expressed by the glucagon-expressing neurons in the avian retina. We used antibodies to cell-distinguishing markers to better characterize the different types of glucagon-expressing neurons. We found that the large glucagon-expressing neurons were immunoreactive for substance P, neurofilament, Pax6, AP2alpha, HuD, calretinin, trkB, and trkC. Colocalization of glucagon and substance P in the large glucagon-expressing neurons indicates that these cells are the "bullwhip cells" that have been briefly described by Ehrlich et al. ([1987] J Comp Neurol 266:220-233). Similar to the bullwhip cells, the conventional glucagon-expressing amacrine cells were immunoreactive for calretinin, HuD, Pax6, and AP2alpha. Unlike bullwhip cells, the conventional glucagon-expressing amacrine cells were immunoreactive for GABA. While glucagon-immunoreactive amacrine cells were negative for substance P in central regions of the retina, a subset of this type of amacrine cell was immunoreactive for substance P in far peripheral regions of the retina. An additional type of glucagon/substance P-expressing neuron, resembling the bullwhip cells, was found in far peripheral and dorsal regions of the retina. Based on morphology, distribution within the retina, and histological markers, we conclude that there may be four different types of glucagon-expressing neurons in the avian retina.

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PubMed2006

Retinal stem cells.

During the embryonic development of the eye, a group of founder cells in the optic vesicle gives rise to multipotent progenitor cells that generate all the neurons and the Müller glia of the mature retina. In most vertebrates, a small group of retinal stem cells persists at the margin of the retina, near the junction with the ciliary epithelium. In fish and amphibians, the retinal stem cells continue to produce progenitors throughout life, adding new retina to the periphery of the existing retina as the eye grows. In birds the new retinal addition is more limited, and it is absent in those mammals that have been analyzed. Nevertheless, cells from the retinal periphery and ciliary body of mammals can be isolated and grown in vitro for extended periods. Methods for the study of both embryonic progenitors and adult retinal stem cells in vitro and in vivo have led to a better understanding of retinal development, allowed for the screening of factors important in retinal growth and differentiation, and enabled the development of methods to direct stem and progenitor cells to specific fates. These methods may ultimately lead to the development of strategies for retinal repair.

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PubMed2005

Ultrasound-mediated gene transfer into neuronal cells.

A new field of gene transfer is emerging as a simple, effective means to drive the expression foreign genes in cells: ultrasound-mediated gene transfer or sonoporation. We report here that sonoporation is an effective means of gene transfer for cultured neurons, a cell type that has been difficult to transfect. Neuronal cell types that are effectively sonoporated include chick retinal neurons, chick dorsal forebrain, chick optic tectum, PC12 cells, rat cerebellar neurons and mouse hippocampal neurons. Depending on the type of cell and conditions of sonoporation the transfection efficacy was as high as 20%. Sonoporation of plasmid DNA was effective for cells adherent to a substrate and for free-floating cells that were freshly dissociated. In the free-floating preparations, between 60 and 95% of the cells that were transfected were neuronal, as much as 90% higher than that observed for other methods of gene transfer including adenovirus and lipid-based transfection methods. We conclude that sonoporation is a simple, effective and inexpensive means by which to preferentially transfect DNA into neuronal cells.

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PubMed2005

Glucagon-expressing neurons within the retina regulate the proliferation of neural progenitors in the circumferential marginal zone of the avian eye.

Glucagon-expressing retinal amacrine cells have been implicated in regulating postnatal ocular growth. Furthermore, experimentally accelerated rates of ocular growth increase the number of neurons added to the peripheral edge of the retina. Accordingly, we assayed whether glucagon-expressing neurons within the retina regulate the proliferation of progenitors in the circumferential marginal zone (CMZ) of the postnatal chicken eye. We found that glucagon-containing neurites are heavily clustered within the CMZ at the peripheral edge of the retina. Many of these neurites originate from a cell type that is distinct from other types of retinal neurons, which we termed large glucagon-expressing neurons (LGENs). The LGENs are immunoreactive for glucagon and glucagon-like peptide 1 (GLP1), have a unipolar morphology, produce an axon that projects into the CMZ, and are found only in ventral regions of the retina. In dorsal regions of the retina, a smaller version of the LGENs densely ramifies neurites in the CMZ. Intraocular injections of glucagon or GLP1 suppressed the proliferation of progenitors in the CMZ, whereas a glucagon-receptor antagonist promoted proliferation. In addition, we found that glucagon, GLP1, and glucagon antagonist influenced the number of progenitors in the CMZ. We conclude that the LGENs may convey visual information to the CMZ to control the addition of new cells to the edge of the retina. We propose that glucagon/GLP1 released from LGENs acts in opposition to insulin (or insulin-like growth factor) to regulate precisely the proliferation of retinal progenitors in the CMZ.

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PubMed2005

Transitin, a nestin-related intermediate filament, is expressed by neural progenitors and can be induced in Müller glia in the chicken retina.

The purpose of this study was to test whether transitin, the avian homologue of nestin, is expressed by retinal progenitors in the developing and postnatal chicken. Because nestin has been widely used as a cell-distinguishing marker of neural progenitors in the mammalian nervous system, we expected to find transitin expressed specifically by the neural progenitors of the retina. In early stages of development, transitin is expressed by neural progenitors in the retina and by cells in the developing ciliary body. During later stages of development, transitin expression persists in differentiating Müller glia but is down-regulated by these cells as maturation proceeds. In the postnatal chick, transitin expression is restricted to neural progenitors at the peripheral edge of the retina. We found that the expression of transitin in mature Müller glia was induced by intraocular injections of insulin and fibroblast growth factor-2 (FGF2) but not by ciliary neurotrophic factor. In response to insulin and FGF2, the expression of transitin was induced in the nonpigmented epithelium (NPE) of the ciliary body. In the postnatal retina, acute retinal damage transiently induces transitin expression in Müller glia. We propose that the expression of transitin by retinal Müller glia and NPE cells in the postnatal animal represents a state of de-differentiation and a step toward becoming neurogenic progenitor cells. Taken together, our findings indicate that transitin is expressed by neural progenitors in the embryonic and postnatal chicken retina. However, transitin is not exclusively expressed by neural progenitors and is also expressed by non-neurogenic cells.

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PubMed2004

Different aspects of gliosis in retinal Muller glia can be induced by CNTF, insulin, and FGF2 in the absence of damage.

PURPOSE: In response to acute damage, Muller glia in the retina have been shown to dramatically alter their expression of filamentous proteins. Since damaged retinal cells are known to produce growth factors such as insulin-like growth factor (IGF), ciliary neurotrophic factor (CNTF) and fibroblast growth factor (FGF), the altered expression of filaments in Muller glia in response to retinal damage may be induced by some of these factors. The purpose of this study was to assay whether growth factors influence the expression of filamentous proteins in Muller glia in the intact retinas of postnatal chickens. METHODS: We assayed for changes in expression levels of IGF-I, IGF-II, CNTF, FGF1, and FGF2 in N-methyl-D-aspartate(NMDA) damaged retinas by using quantitative PCR. In undamaged retinas, we assayed whether intraocular injections of insulin, CNTF, or FGF2 influenced glial expression of glial fibrillary acidic protein (GFAP), neurofilament, RA4, vimentin and beta3 tubulin by using immunocytochemistry on frozen sections. RESULTS: We demonstrated that levels of mRNA for IGF-II, FGF1, FGF2, and CNTF were increased in the postnatal chicken retina in response to neurotoxic damage. This was coincident with increased glial expression of GFAP and filamentous neuronal proteins. The combination of insulin and FGF2 caused postmitotic Muller glia to transiently increase their expression of vimentin and putative neuron specific filamentous proteins such as neurofilament, beta3 tubulin and RA4. By comparison, insulin or FGF2 alone had minor effects on glial expression of cytoskeletal proteins. Although neurofilament expression was not induced by CNTF, this growth factor stimulated Muller glia to express GFAP. CONCLUSIONS: We conclude that the phenotype of postmitotic Muller glia is plastic and can be regulated by retinal damage, and these damage induced changes in phenotype can be induced by exogenous growth factors in the absence of damage.

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PubMed2004

Neural regeneration in the chick retina.

In warm-blooded vertebrates, possibilities for retinal regeneration have recently become reality with the discovery of neural stem cells in the mature eye. A number of different cellular sources of neural stem cells have been identified. These sources include stem cells at the retinal margin, pigmented cells in the ciliary body and iris, non-pigmented cells in the ciliary body and Müller glia within the retina. This review focuses on recent reports of neural stem cells and regeneration in the postnatal chicken retina. In the chicken eye sources of neurogensis and regeneration include: (1) retinal stem cells at the peripheral edge of the retina; (2) Müller glia in central regions of the retina; (3) non-pigmented epithelial cells in the posterior portion of the ciliary body; and (4) possibly pigmented cells in the pars plana of the ciliary body. This review discusses the similarities between the retinal progenitor cells in the postnatal eye and those found in the embryo. In addition, I discuss combinations of growth factors, (insulin, IGF-I, EGF and FGF2) that are capable of stimulating the proliferation and production of neurons from neural progenitors, non-neural epithelial cells, and postmitotic support cells in the avian eye. In summary, the mechanisms that regulate the proliferation and differentiation of cells with neurogenic potential are beginning to be understood and the postnatal chicken eye has proven to be a useful model system to study retinal regeneration.

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PubMed2004

NeuroD induces the expression of visinin and calretinin by proliferating cells derived from toxin-damaged chicken retina.

Müller glia have been shown to be a potential source of neural regeneration in the avian retina. In response to acute damage Müller glia de-differentiate, proliferate, express transcription factors found in embryonic retinal progenitors, and some of the progeny differentiate into neurons and glia (Fischer and Reh [2001a] Nat. Neurosci. 4:247-252). However, most of the cells produced by proliferating Müller cells appear to remain undifferentiated. The purpose of this study was to test whether the neurogenic gene NeuroD can promote the differentiation of proliferating cells derived from the postnatal chick retina. We used recombinant avian retroviruses to transfect green fluorescent protein (GFP) or NeuroD. The majority of cells transfected with GFP remained undifferentiated, with a few cells differentiating into calretinin-immunoreactive neurons. Many cells transfected with the NeuroD-virus expressed calretinin, neurofilament, or visinin, while most cells remained undifferentiated. The number of calretinin-expressing cells that were generated was increased approximately 20-fold with forced expression of NeuroD. In addition, we found that cells transfected with NeuroD never expressed glutamine synthetase, a marker of mature Müller glia, suggesting that NeuroD suppresses glial differentiation. We conclude that NeuroD stimulates cells from the toxin-damaged chicken retina to acquire some neuronal phenotypes. We propose that most of these cells were derived from Müller glia.

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PubMed2004

BMP4 and CNTF are neuroprotective and suppress damage-induced proliferation of Müller glia in the retina.

In response to acute damage, Müller glia in the chicken retina have been shown to be a source of proliferating progenitor-like cells. The secreted factors and signaling pathways that regulate this process remain unknown. The purpose of this study was to test whether secreted factors, which are known to promote glial differentiation during development, regulate the ability of Müller glia to proliferate and become retinal progenitors in response to acute damage in mature retina. We made intraocular injections of BMP4, BMP7, EGF, NGF, BDNF, or CNTF before or after a single, toxic dose of N-methyl-d-aspartate (NMDA) and assayed for proliferating progenitor-like cells within the retina. We found that injections of BMP4, BMP7, or CNTF, but not EGF, NGF, or BDNF, before NMDA treatment reduced the number of Müller glia that proliferated and gave rise to progenitor-like cells. CNTF and BMP4, but not NGF or BDNF, greatly reduced the number of cells destroyed by toxin treatment indicating that these factors protect retinal neurons from a severe excitotoxic insult. Injections of CNTF 5 days before NMDA treatment prevented neurotoxin-induced cell death and Müller glial proliferation, while injections of BMP4 had no protective effect. In addition, CNTF injected after NMDA treatment suppressed glial proliferation, while BMP4 did not. We conclude that BMP4 and CNTF, when applied before a toxic insult, act as neuroprotective agents and likely suppress the proliferative response of Müller glia to retinal damage by attenuating the retinal damage; protecting bipolar and amacrine neurons from NMDA-induced cell death. When applied after a toxic insult, CNTF suppressed glial proliferation independent of levels of retinal damage.

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PubMed2003

Growth factors induce neurogenesis in the ciliary body.

The ciliary body of the eye is a nonneural tissue that is derived from the anterior rim of the optic cup, an extension of the neural tube. This tissue normally does not contain neurons and functions to produce components of the aqueous humor. We found that intraocular injections of insulin, EGF, or FGF2 stimulate NPE cells to proliferate and differentiate into neurons. These growth factors had region-specific effects along the radial axis of the ciliary body, with insulin and EGF stimulating proliferation of NPE cells close to the retina, while FGF2 stimulated the proliferation of NPE cells further toward the lens. Similar region-specific effects were observed for accumulations of neurons in the NPE in response to injections of different growth factors. The neurons derived from NPE cells express neurofilament, beta3 tubulin, RA4, calretinin, Islet1, or Hu, and a few produced long axonal projections, several millimeters in length that extend across the ciliary body. Our results suggest that the ciliary body has the capacity to generate retinal neurons, but normally neurogenesis is actively inhibited.

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PubMed2003

Potential of Müller glia to become neurogenic retinal progenitor cells.

The possibility of neural regeneration has gained credence with the identification of neural stem cells seeded within different regions of the adult central nervous system (CNS). Recently, this possibility has received an additional boost from reports that glia, the support cells of the CNS, might provide a source of neural regeneration. We review some of our findings that Müller glia in the chicken retina are a source of proliferating progenitors that can generate neurons. These Müller cells are fully differentiated glial cells that serve functions ascribed to this cell type. In response to damage or exogenous growth factors, Müller glia dedifferentiate, proliferate, express combinations of transcription factors normally expressed by embryonic retinal progenitors, and produce new neurons and glia. In light of these data, the potential of Müller glia as a source of neural regeneration in the retina of nonavian species, namely humans, seems an avenue of investigation that warrants serious consideration.

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PubMed2002

Exogenous growth factors stimulate the regeneration of ganglion cells in the chicken retina.

Recent reports have found that the posthatch chicken retina has the capacity for neuronal regeneration. The purpose of this study was to test whether the types of cells destroyed by neurotoxic lesions influence the types of cells that are regenerated, and whether exogenous growth factors stimulate neural regeneration in the chicken retina. N-methyl-D-aspartate (NMDA) was used to destroy amacrine and bipolar cells; kainate was used to destroy bipolar, amacrine, and ganglion cells; colchicine was used to selectively destroy ganglion cells. Following toxin-induced damage, bromo-deoxyuridine was used to label proliferating cells. In some animals, growth factors were injected into the vitreous chamber of the eye. We found that the proliferation of cells within the retina was stimulated by toxin-induced cell loss, and by insulin and FGF2. After either kainate- or colchicine-induced retinal damage, some of the newly generated cells expressed markers and had the morphology of ganglion cells. The combination of insulin and FGF2 stimulated the regeneration of ganglion cells in kainate- and colchicine-treated retinas. We conclude that exogenous growth factors can be used to stimulate neural regeneration in the retina. We propose that the type of neuron destroyed in the retina may allow or promote the regeneration of that neuronal type.

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PubMed2002

Insulin and fibroblast growth factor 2 activate a neurogenic program in Müller glia of the chicken retina.

We have reported previously that neurotoxic damage to the chicken retina causes Müller glia to dedifferentiate, proliferate, express transcription factors common to retinal progenitors, and generate new neurons and glia, whereas the majority of newly produced cells remain undifferentiated (Fischer and Reh, 2001). Because damaged retinal cells have been shown to produce increased levels of insulin-related factors and FGFs, in the current study we tested whether intraocular injections of growth factors stimulate Müller glia to proliferate and produce new neurons. We injected growth factors and bromodeoxyuridine into the vitreous chamber of the eyes of chickens and assayed for changes in glial phenotype and proliferation within the retina. Although insulin or FGF2 alone had no effect, the combination of insulin and FGF2 caused Müller glia to coexpress transcription factors common to retinal progenitors (Pax6 and Chx10) and initiated a wave of proliferation in Müller cells that began at the retinal margin and spread into peripheral regions of the retina. Most of the newly formed cells remain undifferentiated, expressing Pax6 and Chx10, whereas some differentiate into Müller glia, and a few differentiate into neurons that express the neuronal markers Hu or calretinin. There was no evidence of retinal damage in eyes treated with insulin and FGF2. We conclude that the combination of insulin and FGF2 stimulated Müller glia to dedifferentiate, proliferate, and generate new neurons. These findings imply that exogenous growth factors might be used to stimulate endogenous glial cells to regenerate neurons in the CNS.

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PubMed2002

Exogenous growth factors induce the production of ganglion cells at the retinal margin.

Neural progenitors at the retinal margin of the post-hatch chicken normally produce amacrine and bipolar cells, but not photoreceptor or ganglion cells. The purpose of this study was to test whether exogenous growth factors influence the types of cells produced by progenitors at the retinal margin. We injected insulin, FGF2 or a combination of insulin and FGF2 into the vitreous chamber of post-hatch chickens. To assay for growth factor-induced changes at the retinal margin, we used in situ hybridization and immunocytochemistry on cryosections. One day after the final injection, we found that insulin alone stimulated the addition of cells to the retinal margin, but this was not further increased when FGF2 was applied with insulin. Insulin alone increased the number of cells in the progenitor zone that expressed neurofilament, and this was further increased when FGF2 was applied with insulin. These neurofilament-expressing cells in the progenitor zone included differentiating neurons that expressed Islet1 or Hu. Four days after the final dose of growth factor, we found that the production of ganglion cells was induced by co-injection of insulin and FGF2, but not by either insulin or FGF2 alone. We conclude that the types of cells produced by progenitors at the retinal margin can be altered by exogenous growth factors and that normally the microenvironment imposes limitations on the types of neurons produced.

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