Biomedical subjects

K Kohler

Publications and source records attributed to K Kohler.

At least 19 recordsLinked to original sources

PubMed2001

Combined HPLC-MS and HPLC-NMR on-line coupling for the separation and determination of lutein and zeaxanthin stereoisomers in spinach and in retina.

The determination and unambiguous identification of carotenoid stereoisomers from biological tissues, avoiding isomerization and oxidation due to the extraction process, is still a major challenge. Particularly, the analysis of lutein and zeaxanthin stereoisomers is of great importance, as these are the main constituents of the macula lutea, the central part of the human retina, and act as possible agents in the prevention and treatment of age-related macular degeneration (AMD). By combining a mild and quick extraction technique such as matrix solid-phase dispersion together with high-performance liquid chromatography (HPLC), the extremely light and oxygen sensitive lutein and zeaxanthin stereoisomers are extracted, enriched, and separated directly from the solid plant or tissue samples, excluding preparation of artifacts. HPLC separations are performed with C30 phases due to their enhanced shape selectivity compared to C18 phases and on-line coupled to mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy. By using HPLC-MS with atmospheric pressure chemical ionization, the lutein stereoisomers can be distinguished from the zeaxanthin stereoisomers within one chromatographic run in the upper picogram range, whereas HPLC-NMR coupling allows the unequivocal identification of each stereoisomer with a concentration in the upper nanogram range. This article provides an analytical method for the artifact-free determination of lutein and zeaxanthin stereoisomers directly from the solid biological tissue spinach as a source of carotenoids and retina as the sphere of activity for AMD. In addition, the structures of these stereoisomers were unambiguously elucidated by employing hyphenated analytical techniques.

Chromatography, High Pressure Liquid↗

PubMed2001

Expression of angiotensin-converting enzyme (ACE) in the developing chicken retina.

Angiotensin-converting enzyme (ACE) performs two contrasting enzymatic effects: as part of the renin-angiotensin system it converts angiotensin I into physiologically active angiotensin II, and it inactivates a number of peptides, e.g. substance P. These peptides are well known neurotransmitters in the retina and recently angiotensin II was described in retinal neurons. We therefore investigated a possible involvement of ACE in retinal metabolism by determining the mRNA and protein expression of ACE in the developing and mature chicken retina. ACE-mRNA expression was investigated by RT-PCR in the iris/ciliary body, the choroid, the optic nerve head, pecten, and the retina. Levels of ACE-mRNA were quantified by competitive PCR with heterologous competitor fragments in the retina at different developmental stages. To localize protein expression of ACE in the mature chicken retina an antibody directed against ACE was used. ACE-mRNA was present in all ocular tissues examined. Quantification of ACE-mRNA in avascular retinas of developing chickens revealed small amounts (0.13 attomol microl(-1)) at embryonic day 7 and values of about 0.6 attomol microl(-1)during embryonic days 7-17. ACE-mRNA expression transiently increased ten-fold (7.3 attomol microl(-1)) on postnatal day 1, decreased again to about 1.4 attomol microl(-1)on postnatal day 6, and remained constant thereafter. ACE-immunohistochemistry revealed labeling of photoreceptors, bipolar cells, amacrine cells, and cells in the ganglion cell layer as well as of Müller glia. Our data show that ACE-mRNA is an intrinsic component of the retina and that ACE itself has a widespread but distinct cellular distribution. The transient high expression of ACE-mRNA directly after hatching indicate, that ACE may be involved in fine tuning the neuropeptidergic equipment of the retinal network during the initial phase of visual experience.

Animals↗

PubMed2001

Evaluation of the rhodopsin knockout mouse as a model of pure cone function.

PURPOSE: To determine a time window in the rhodopsin knockout (Rho(-/-)) mouse during which retinal function is already sufficiently developed but cone degeneration is not yet substantial, thus representing an all-cone retina. METHODS: Electroretinograms (ERGs) were obtained from 14 homozygous Rho(-/-) mice and eight C57Bl/6 control mice. The same individuals were tested every 7 days, beginning as early as postnatal day (P)14. The ERG protocols included flash and flicker stimuli, both under photopic and scotopic conditions. Retinal and choroidal morphology was observed in animals of comparable age. RESULTS: Functionally, the developmental phase lasted until postnatal week (PW)3 in both the Rho(-/-) mice and the control animals. During PW4 to 6, the Rho(-/-) mice showed a plateau in ERG parameters with normal or even supernormal cone responses and complete absence of rod contributions. At PW7, there was a marked onset of degeneration, which progressed so that no ERG signals were left at PW13, when the control eyes still had normal ERG responses. Microscopically, cone degeneration paralleled the functional changes, beginning at approximately PW6 and almost complete at PW13, whereas retinal pigment epithelium (RPE) and choroid did not show any abnormalities. CONCLUSIONS: From PW4 to 6, Rho(-/-) mice appear to have normal cone and no rod function. Despite the missing rod outer segment (OS), the structure of retina, RPE, and choroid remained unchanged. Therefore, the Rho(-/-) mice can serve during this age period as a model for pure cone function. Such a model is particularly useful to evaluate rod-cone interaction and to dissect rod- from cone-mediated signaling pathways in vivo.

Animals↗

PubMed2000

Self-induced shapiro effect in semiconductor superlattices

We observe that the oscillatory motion of photoinjected electron-hole pairs in a biased semiconductor superlattice (Bloch oscillation) is accompanied by a coherent quasi-dc current that is generated by the interaction of the carriers with the self-induced oscillating field. It is shown that this novel macroscopic quantum effect, which is a coherent analog of the Shapiro effect observed in Josephson junctions, can be controlled by changing the spectral position of the exciting laser pulse, which in turn determines the amplitude and phase of the wave packet oscillations. It is thereby possible to coherently drive the electrons either downwards or upwards in the potential of the static field.

Journal Article↗

PubMed2000

The distribution and developmental regulation of NMDA receptor subunit proteins in the outer and inner retina of the rat.

In order to investigate whether N-methyl-D-aspartate (NMDA) receptors with distinct pharmacological properties are differentially distributed within the retinal layers, the spatial distribution and temporal regulation of all NMDA receptor subunits was analyzed in parallel on the protein level in the rat retina during development. Immunohistochemistry was performed on retinal sections at different developmental ages between embryonic (E) days 20/21 and the adult stage using specific antibodies against NMDA subunits (NR1, NR2A-D). All NMDA subunits were expressed in the rat retina postnatally but showed different spatial patterns. In particular, and in contrast to previous in situ hybridization studies, labeling of NR2 subunits was observed in horizontal cell bodies and in the outer plexiform layer, indicating that functional NMDA receptors are expressed in this retinal cell type in the rat. Expression of NR2D was restricted to the inner retina and seemed to be involved in neurotransmission within the rod pathway. In the inner plexiform layer (IPL), distinct patterns of labeling were observed for different NMDA subunits. NR1 was found in two bands which can be related to the off- and on-signal pathways, whereas NR2A and NR2B were located in two bands within the off-sublaminae of the IPL. The antibody against NR2C was distributed throughout the whole IPL, and NR2D was expressed exclusively in the innermost part of the IPL where rod bipolar cell terminals terminate. Distinct bands of immunoreactivity in the IPL were observed only from P14 on. In conclusion, there are clear differences in the spatial distribution and temporal expression of NMDA receptor subtypes in the rodent retina. This indicates that specific retinal cells selectively express glutamate receptors composed of different subunit combinations and thus display different pharmacological and kinetic properties.

Animals↗

PubMed2000

Coupled bloch-phonon oscillations in semiconductor superlattices

We investigate coherent Bloch oscillations in GaAs/AlxGa1-xAs superlattices with electronic miniband widths larger than the optical phonon energy. In these superlattices the Bloch frequency can be tuned into resonance with the optical phonon. Close to resonance a direct coupling of Bloch oscillations to LO phonons is observed which gives rise to the coherent excitation of LO phonons. The density necessary for driving coherent LO phonons via Bloch oscillations is about 2 orders of magnitude smaller than the density necessary to drive coherent LO phonons in bulk GaAs. The experimental observations are confirmed by the theoretical description of this phenomenon [A.W. Ghosh et al., Phys. Rev. Lett. 85, 1084 (2000)].

Journal Article↗

PubMed2000

Expression of purinergic receptors in bipolar cells of the rat retina.

P2X receptors are ligand-gated ion channels which are activated by excitatory neurotransmitter ATP. Despite considerable evidence of signaling by extracellular nucleotides in other sensory systems, P2X receptors in the visual system have only rarely been studied, and almost nothing is known about their functional significance in the retina. To determine whether ATP plays a role in the modulation of vertical retinal signal pathways, we examined the expression of P2X receptor mRNA in freshly isolated bipolar cells of the rat retina (Brown Norway, P25) using the single-cell RT-PCR technique. Positive amplification signals were found in about 33% of the bipolar cells for P2X(3), P2X(4) and P2X(5) but not for P2X(7) mRNA. We conclude that at least a subpopulation of bipolar cells in the rat retina expresses ionotropic P2 receptors of the P2X type and that these possibly exert a neuromodulatory influence on information processing in the retina.

Animals↗

PubMed2000

Direct observation of depolarization shift of the intersubband resonance

We have studied the intersubband resonance of GaAs/AlGaAs multi-quantum-well systems by comparing photon drag and absorption spectra obtained by in-plane photocurrent and photoconduction measurements. The peak absorption at room temperature is found to be blueshifted from the photon drag resonance by as much as 33 cm(-1). We argue that this difference gives directly the depolarization shift, since the resonant photon drag current is driven by the Doppler effect, which is a k-vector dependent single particle process.

Journal Article↗

PubMed2000

[Evaluating the Pro Ton applanation tonometer in rabbits].

PURPOSE: To assess use of the tonometer ProTon for measuring intraocular pressure in rabbits. MATERIALS AND METHODS: Eleven chinchilla-bastard rabbits were measured under standardized conditions with the applanation tonometer ProTon for 62 +/- 15 days. One rabbit was excluded because of intensively defensive reactions. RESULTS: Intraocular pressure in the ten rabbits was 12.15 +/- 3.63 mmHg in the right eye and 12.11 +/- 3.52 mmHg in the left eye. CONCLUSION: Intraocular pressure can be measured reliably over a long period with the applanation tonometer ProTon. It is therefore suitable for determining intraocular pressure in experimentally induced glaucoma in rabbits.

Animals↗

PubMed2000

Cell differentiation, synaptogenesis, and influence of the retinal pigment epithelium in a rat neonatal organotypic retina culture.

This study was focused on the analysis of cell type differentiation and synaptogenesis as well as outer segment formation in an organotypic culture of the neonatal rat retina during a 6-14 day period of in vitro development. Moreover, the effects of the retinal pigment epithelium (RPE) on these processes were investigated. The in vitro development resulted in a retinal architecture and lamination comparable to that of in vivo retinas. The RPE influences the proper alignment of photoreceptors as well as the formation of the outer limiting membrane (OLM), but not processes of cell differentiation, synaptogenesis and inner retinal lamination.

Animals↗

PubMed2000

Developmental expression of a tandemly repeated, glycine- and serine-rich spore wall protein in the microsporidian pathogen Encephalitozoon cuniculi.

Microsporidia are intracellular organisms of increasing importance as opportunistic pathogens in immunocompromised patients. Host cells are infected by the extrusion and injection of polar tubes located within spores. The spore is surrounded by a rigid spore wall which, in addition to providing mechanical resistance, might be involved in host cell recognition and initiation of the infection process. A 51-kDa outer spore wall protein was identified in Encephalitozoon cuniculi with the aid of a monoclonal antibody, and the corresponding gene, SWP1, was cloned by immunoscreening of a cDNA expression library. The cDNA encodes a protein of 450 amino acids which displays no significant similarities to known proteins in databases. The carboxy-terminal region consists of five tandemly arranged glycine- and serine-rich repetitive elements. SWP1 is a single-copy gene that is also present in the genomes of Encephalitozoon intestinalis and Encephalitozoon hellem as demonstrated by Southern analysis. Indirect immunofluorescence and immunoelectron microscopy revealed that SWP1 is differentially expressed during the infection cycle. The protein is absent in replicative meronts until 24 h postinfection, and its expression is first induced in early sporonts at a time when organisms translocate from the periphery to the center of the parasitophorous vacuole. Expression of SWP1 appears to be regulated at the mRNA level, as was shown by reverse transcriptase PCR analysis. Further identification and characterization of stage-specific genes might help to unravel the complex intracellular differentiation process of microsporidia.

Amino Acid Sequence↗

PubMed2000

Distribution and developmental regulation of AMPA receptor subunit proteins in rat retina.

PURPOSE: To learn more about a possible functional role of alpha-amino-3-hydroxy-5-methyl-4-isoxasole-propionate (AMPA) receptors in retinal development, the spatial distribution and temporal regulation of all AMPA receptor subunit proteins was studied in rats. METHODS: Immunohistochemistry was performed on retinal sections between embryonic days (E)20 and E21 and the adult stage by using specific antibodies against AMPA subunits GluR1 to 4. RESULTS: All AMPA subunits were expressed in the ganglion cell layer from E21 on. In the inner plexiform layer (IPL), discernible bands of labeling appeared at distinct retinal ages for the different subunits. GluR1 immunoreactivity (IR) was concentrated in two broad bands by postnatal day (P)3, whereas three bands were visible beginning on P9. Two bands were located in a region of the IPL where off-cells terminate, and one band was found in the innermost part of the IPL where on-cells terminate. In contrast, two bands of GluR2/3- and GluR4-IR in the IPL were only discernible beginning on P14 and seemed to be located between the bands of GluR1-IR. GluR2/3 and GluR4 were observed both in horizontal cells and in the outer plexiform layer from early developmental stages on. GluR1 was not found in the outer retina, indicating that horizontal and bipolar cell processes in the rat express AMPA receptors composed of subunits GluR2 to 4. Double-labeling experiments with cell-specific markers revealed the expression of subunits GluR1 to 4 in cholinergic and AII amacrine cells. CONCLUSIONS: AMPA receptors are expressed before synapse formation, indicating a role not only in fast signal transmission but also in the establishment of inner retinal circuits. The differences in spatial and temporal subunit expression suggest that different retinal cell types selectively express distinct types of AMPA receptors during development of the rat retina.

Animals↗

PubMed1999

Genetic disorders of vision revealed by a behavioral screen of 400 essential loci in zebrafish.

We examined optokinetic and optomotor responses of 450 zebrafish mutants, which were isolated previously based on defects in organ formation, tissue patterning, pigmentation, axon guidance, or other visible phenotypes. These strains carry single point mutations in >400 essential loci. We asked which fraction of the mutants develop blindness or other types of impairments specific to the visual system. Twelve mutants failed to respond in either one or both of our assays. Subsequent histological and electroretinographic analysis revealed unique deficits at various stages of the visual pathway, including lens degeneration (bumper), melanin deficiency (sandy), lack of ganglion cells (lakritz), ipsilateral misrouting of axons (belladonna), optic-nerve disorganization (grumpy and sleepy), inner nuclear layer or outer plexiform layer malfunction (noir, dropje, and possibly steifftier), and disruption of retinotectal impulse activity (macho and blumenkohl). Surprisingly, mutants with abnormally large or small eyes or severe wiring defects frequently exhibit no discernible behavioral deficits. In addition, we identified 13 blind mutants that display outer-retina dystrophy, making this syndrome the single-most common cause of inherited blindness in zebrafish. Our screen showed that a significant fraction (approximately 5%) of the essential loci also participate in visual functions but did not reveal any systematic genetic linkage to particular morphological traits. The mutations uncovered by our behavioral assays provide distinct entry points for the study of visual pathways and set the stage for a genetic dissection of vertebrate vision.

Albinism↗

PubMed1999

Selective loss of cone function in mice lacking the cyclic nucleotide-gated channel CNG3.

Two types of photoreceptors, rods and cones, coexist in the vertebrate retina. An in-depth analysis of the retinal circuitry that transmits rod and cone signals has been hampered by the presence of intimate physical and functional connections between rod and cone pathways. By deleting the cyclic nucleotide-gated channel CNG3 we have generated a mouse lacking any cone-mediated photoresponse. In contrast, the rod pathway is completely intact in CNG3-deficient mice. The functional loss of cone function correlates with a progressive degeneration of cone photoreceptors but not of other retinal cell types. CNG3-deficient mice provide an animal model to dissect unequivocally the contribution of rod and cone pathways for normal retinal function.

Animals↗

PubMed1999

Can subretinal microphotodiodes successfully replace degenerated photoreceptors?

The idea of implanting microphotodiode arrays as visual prostheses has aroused controversy on its feasibility from the moment it appeared in print. We now present results which basically support the concept of replacing damaged photoreceptors with subretinally implanted stimulation devices. Network activity in degenerated rat retinae could be modulated through local electrical stimulation in vitro. We also investigated the long term stability and biocompatibility of the subretinal implants and their impact on retinal physiology in rats. Ganzfeld electroretinograms and histology showed no significant side effect of subretinal implants on retinal function or the architecture of the inner retina.

Animals↗

PubMed1999

Effects of brain-derived neurotrophic factor on the development of NADPH-diaphorase/nitric oxide synthase-positive amacrine cells in the rodent retina.

Amacrine neurons expressing nitric oxide synthase (NOS) contain brain-derived neurotrophic factor (BDNF) receptors and respond to exogenous BDNF [Klöcker, N., Cellerino, A. & Bähr, M. (1998) J. Neurosci., 18, 1038-1046]. We analysed the effects of BDNF on the development of neurons which express NOS in the mouse and rat retina. Rat pups received a total of three intraocular injections of BDNF at intervals of 48 h, starting at postnatal day 16 (P16), and were killed at P22. The retinas were stained for NADPH-diaphorase, a histological marker of NOS. NOS-expressing neurons were found in both the inner nuclear layer (INL) and the ganglion cell layer (GCL). Two classes of NOS-expressing neurons, type I and type II, had already been distinguished in the INL [Koistinaho, J. & Sagar, S.M. (1995) In Osborne, N.N. & Chader, G.J. (eds), Progress in Retinal and Eye Research, Vol. 15. Oxford University Press, pp. 69-87] and a third one in the GCL. Up-regulation of NADPH-diaphorase activity was observed after BDNF treatment. The number of type I neurons remained stable, whereas the number of type II neurons and NOS-positive neurons in the GCL increased significantly (P < 0.001). Type I and type II neurons were significantly larger in BDNF-treated retinas. Double-labelling experiments revealed that BDNF induces NADPH-diaphorase in dopaminergic neurons and amacrine cells displaced to the GCL, but not in retinal ganglion cells. In mice homozygous for a null mutation of the bdnf gene, the intensity of NADPH-diaphorase labelling in both somata and processes was reduced, but the number of labelled neurons was not dramatically reduced. These findings indicate that BDNF regulates the neurotransmitter phenotype of NOS-expressing amacrine neurons under physiological conditions, but is not required for their survival.

Animals↗

PubMed1999

Angiotensin II receptor subtype gene expression and cellular localization in the retina and non-neuronal ocular tissues of the rat.

In addition to its function as a peripheral hormone, angiotensin II (AngII) has been shown to act as a neuromodulator in various brain regions. AngII effects are mediated by two major AngII receptor subtypes, AT1 and AT2, and different AT1 receptor isoforms AT1A and AT1B are described in rat brains. The purpose of the present study was to analyse the expression pattern of AT receptors in different parts of the rat eye with special emphasis on the retina. Specific primers were constructed and the gene expression of AngII receptor subtypes was investigated by means of reverse transcription-polymerase chain reaction (RT-PCR). An antibody was used for cellular localization of AT1 receptor in the retina. AT2 receptor mRNA was localized by in situ hybridization (ISH). We examined the retinas of different developmental stages as well as non-neuronal ocular tissues, e.g. choroid and anterior uveal tract of rats (Brown Norway and Wistar strain), for the gene expression of AT receptors. Our results show that AT1A and AT2 mRNAs are expressed in rat choroid, iris/ciliary body and retinas, whereas AT1B mRNA is not expressed in the retina but in all other ocular tissues under investigation. AT1 receptor immunohistochemistry of the retina showed strong labelling in the ganglion cell layer (GCL), and some cells in the inner nuclear layer (INL), suggesting putative ganglion cell but also amacrine cell labelling. In the retina, ISH for AT2 mRNA revealed labelling in the GCL and a faint labelling in the inner nuclear layer. No AT2 ISH-signal was found in the other ocular tissues. These data suggest that there is a specific distribution pattern of AT receptors in rat ocular tissues, especially in the retina. The expression of AT receptors on retinal ganglion cells confirms the AngII action on these cell types and supports the role of AngII as a retinal neurotransmitter or neuromodulator.

Animals↗

PubMed1998

Reversed-phase high-performance liquid chromatographic identification of lutein and zeaxanthin stereoisomers in bovine retina using a C30 bonded phase.

An efficient reversed-phase high-performance liquid chromatographic assay with ultraviolet detection at 450 nm for simultaneous determination of lutein and zeaxanthin stereoisomers in bovine retina is described. The procedure involves rapid and careful one-step hexane extraction of the carotenoids from the homogenized liquid and enrichment by on-line solid phase extraction on a polystyrene cartridge. The substances were eluted at a flow rate of 1 ml/min with acetone-water (85:15, v/v) on a C30 reversed-phase column. C30 phases exhibit superior shape selectivity for the separation of carotenoid stereoisomers compared with conventional C18 phases. For comparison a mixture of iodine-isomerized lutein and zeaxanthin standards was separated.

Animals↗