Biomedical subjects

E Guenther

Publications and source records attributed to E Guenther.

At least 19 recordsLinked to original sources

PubMed2001

Presence of kynurenic acid and kynurenine aminotransferases in the inner retina.

Kynurenine aminotransferases (KATs I and II) are pivotal to the synthesis of kynurenic acid (KYNA), the only known endogenous glutamate receptor antagonist and neuroprotectant. This study is the first to identify KYNA in the rat retina and to examine immunohistochemically the distribution of KAT isoforms. As determined by HPLC, KYNA concentration in the retina was 99.9 +/- 24.6 pmol/g wet wt. Immunohisto- chemical experiments showed that both KATs were present in the retina. KAT I was preferentially localised on Müller cell endfeet while KAT II was expressed in cells within the ganglion cell layer. In conclusion, KYNA is present and synthesised in the inner retina. This may suggest a modulatory role in glutamate-mediated retinal neurotransmission.

Animals↗

PubMed2001

Identification of purinergic receptors in retinal ganglion cells.

P2X receptors are ligand-gated ion channels activated by adenosine triphosphate and expressed in a broad variety of tissues. The present study demonstrates the expression of various types of purinergic P2X receptors in identified retinal ganglion cells (RGCs) of the adult rat retina. Single-cell reverse transcription polymerase chain reaction (SC-RT-PCR) resulted in a positive amplification signal for all P2X receptor subunit mRNAs examined (P2X(3-5), P2X(7)). Immunohistochemistry with P2X(3,4) receptor subunit-specific antibodies showed a labelling of neurons in the ganglion cell layer and inner nuclear layer. Our data suggest that extracellular ATP acts directly on RGCs via several types of P2X receptors and may provide neuromodulatory influences on information processing in the retina.

Adenosine Triphosphate↗

PubMed2001

Electrophysiological properties of rat retinal Müller (glial) cells in postnatally developing and in pathologically altered retinae.

Retinal glial Müller cells are characterized by dominant K(+) conductances. The cells may undergo changes of their membrane currents during ontogeny and gliosis as described in rabbit and man. Although the rat retina is often used in physiological experiments, the electrophysiology of rat Müller cells is less well studied. The aim of the present study was to characterize their membrane currents in postnatal development and in two models of retinal degeneration. Freshly isolated cells were subjected to whole-cell patch clamp recordings. During the first 4 weeks after birth of rats, their Müller cells displayed an increase in all membrane currents, particularly in the inward currents elicited at hyperpolarizing potentials. The decrease of the membrane resistance from more than 760 MOmega to less than 50 MOmega was accompanied by a shift of the zero current potential from about -20 mV to -80 mV, similar as earlier observed in developing rabbit Müller cells. These developmental changes were found in pigmented Brown Norway rats as well as in rats with inherited retinal dystrophy (RCS rats). Moreover, an infection of Lewis rats with the Borna disease virus caused substantial neuroretinal degeneration but did not result in a strong reduction of inward currents and of the zero current potential of the Müller cells. Thus, rat Müller cells fail to change their basic membrane properties in two different models of retinal pathology. This is in contrast to human and rabbit Müller cells, which have been shown to undergo dramatic changes of their membrane physiology in response to retinal diseases and injuries.

Animals↗

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

[Subretinal microphotodiode array as replacement for degenerated photoreceptors?].

A survey is given on the status of developments, concerning a subretinal electronic microphotodiode array that aims at replacing degenerated photoreceptors. Various prototypes have been developed, tested, and implanted in various experimental animals up to 18 months. The fact that electrical responses were recorded from the visual cortex of pigs after electrical stimulation by subretinal electrodes and the fact that responses are also recorded in-vitro in degenerated rat retinae, shows the feasibility of this approach. However, there are a number of open questions concerning the biocompatibility, the long-time stability, and the type of transmitted image to be solved before application in patients can be considered.

Animals↗

PubMed2001

Determination of telomerase activity in squamous cell carcinoma of the penis.

Telomerase activity was studied in 51 penile carcinomas, and detected in all samples from 3 patients with verrucous carcinoma, in 85.4% (41/48) of invasive carcinomas, in 81.8% (9/11) of adjacent non-cancerous skin and in 80% (8/10) of adjacent non-cancerous corpus cavernosum. All skin and corpus cavernosum samples from patients with prostatic carcinoma were found to be telomerase negative. Our results indicate a correlation between frequency of telomerase activity and grade of penile carcinoma. The finding of telomerase activity in skin and corpus cavernosum samples adjacent to tumor suggests that unidentified local factors may modulate telomerase activity in normal tissues.

Biomarkers, Tumor↗

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

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

Evidence for P2X(3), P2X(4), P2X(5) but not for P2X(7) containing purinergic receptors in Müller cells of the rat retina.

P2X receptors are ligand-gated ion channels activated by ATP. They are expressed in a broad variety of tissues. To date, eight P2X receptor subunits (P2X(1)-P2X(7), P2XM) have been cloned. In spite of the considerable evidence of signaling by extracellular nucleotides in other sensory systems, only few studies have been undertaken in the retina. In earlier studies, we have demonstrated that there is mRNA expression of the P2X(2-5) and P2X(7) subunits in the rat retina. In the present study, molecular biological methods were used to investigate expression of P2X receptor mRNA in freshly isolated Müller cells (MCs) of the adult rat retina (Brown Norway). A total of 36 MCs was analyzed, employing the single-cell RT-PCR. A positive amplification signal of 11/14 for P2X(3)-mRNA, 5/10 for P2X(4)-mRNA, 3/10 for P2X(5)-mRNA and 0/8 for P2X(7)-mRNA was revealed. Additionally, the astroglial identity of the cells under studied was confirmed in 10 cases by simultaneous amplification of RT-PCR products of glutamine synthetase (GS)- and P2X-mRNA. We conclude that MCs of rat retina express ionotropic P2 receptors, which, in addition to other functions, may play a key role within the recently described long range calcium signaling and the fast direct glia-neuron interactions in the rat retina.

Animals↗

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

Genes and channels: patch/voltage-clamp analysis and single-cell RT-PCR.

Technological advances in electrophysiology and molecular biology in the last two decades have led to great progress in ion channel research. The invention of the patch-clamp recording technique has enabled the characterization of the biophysical and pharmacological properties of single channels. Rapid progress in the development of molecular biology techniques and their application to ion channel research led to the cloning, in the 1980s, of genes encoding all major classes of voltage- and ligand-gated ionic channels. It has become clear that operationally defined channel types represent extended families of ionic channels. Several experimental approaches have been developed to test whether there is a correlation between the detection of particular ion channel subunit mRNAs and the electrophysiological response to a pharmacological or electrical stimulus in a cell. In one method, whole-cell patch-clamp recording is performed on a cell in culture or tissue-slice preparation. The biophysical and pharmacological properties of the ionic channels of interest are characterized and the cytoplasmic contents of the recorded cell are then harvested into the patch pipette. In a variant of this method, the physiological properties of a cell are characterized with a two-electrode voltage clamp and, following the recording, the entire cell is harvested for its RNA. In both methods, the RNA from a single cell is reverse-transcribed into cDNA by a reverse transcriptase and subsequently amplified by the polymerase chain reaction, i.e. by the so-called single-cell/reverse transcription/polymerase chain reaction method (SC-RT-PCR). This review presents an analysis of the results of work obtained by using a combination of whole-cell patch-clamp recording or two-electrode voltage clamp and SC-RT-PCR with emphasis on its potential and limitations for quantitative analysis.

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

Coexpression patterns of mGLuR mRNAs in rat retinal ganglion cells: a single-cell RT-PCR study.

PURPOSE: Eight different subunits of metabotropic glutamate receptors (mGluRs) are known to date. mGluRs have been linked to an extensive list of neuromodulatory effects, depending on which intracellular or membrane-bound effector system is activated. Activation of mGluRs can influence neuronal activity and can result in changes of intracellular Ca2+ homeostasis-that is, changes in factors that are known to be crucial for cellular differentiation and cell death. Because mGluRs are known in modulating both intracellular and intercellular activities, this study was designed to determine which types of mGluRs are coexpressed in a neuron and whether distinct coexpression patterns can be found that reflect the different physiological requirements of a neuron at different stages of development and to learn whether neuronal injury results in adaptive changes of mGluR expression. METHODS: Juvenile and adult rat retinal ganglion cells (RGCs) and adult RGCs after axotomy were analyzed for their gene expression pattern of mGluRs by single-cell reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Adult RGCs predominantly expressed one or two different mGluR mRNAs, whereas juvenile RGCs coexpressed two and more. mGluR3, -5, and -7 mRNAs were found more frequently in juvenile than in adult RGCs. mGluR6 was detected in juvenile RGCs in low abundance but never in adult RGCs. However, mGluR6 was expressed in adult RGCs after axotomy. mGluR1 and -7 were also found more frequently in axotomized RGCs than in the adult control group. CONCLUSIONS: All types of mGluR mRNAs are expressed in RGCs. This is in contrast with previous in situ hybridization and immunohistochemical studies in which expression of mGluR3, -5, and -6 was not reported. The expression of some mGluR mRNAs seems to be developmental, although no distinct copatterns were found.

Aging↗

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

Voltage-activated calcium currents in rat retinal ganglion cells in situ: changes during prenatal and postnatal development.

Voltage-activated calcium currents (ICa) are one way by which calcium influx into neurons is mediated. To investigate changes in kinetic properties of ICa during neuronal development and to correlate possible kinetic changes with specific differentiation processes, the ICa of retinal ganglion cells (RGCs) was recorded with the perforated patch-clamp technique in rat retinal slices and in whole mounts at different prenatal and postnatal stages. ICa density increased between embryonic day (E) 20 and the adult stage, paralleled by a shift in activation of the omega-conotoxin GVIA-sensitive ICa toward more negative membrane potentials. Furthermore, developmental alterations were observed in ICa inactivation rate during a 120 msec test pulse and in steady-state inactivation of ICa. The most striking feature in ICa kinetics was a transient slowing of calcium current deactivation, which peaked at postnatal day (P)3-5 and affected all ICa subtypes. Although the shift in activation and the decreased inactivation rate of ICa can be explained by differential regulation of distinct calcium channel subtypes, it is more likely that a more general alteration of the cells' functional state was the underlying factor in alterations in steady-state inactivation and current deactivation of ICa. Alterations in the omega-conotoxin GVIA-sensitive and the toxin-resistant currents temporarily coincide with dendritic differentiation, and it is tempting to speculate about their role in network formation in the inner retina. In contrast, alterations in steady-state inactivation and current deactivation may be involved in the regulation of RGC survival, because they occur during the period of programmed cell death in the ganglion cell layer. In conclusion, distinct time windows of alterations in calcium channel properties were found, and this study has provided a basis for performing functional assays to clarify in detail the developmental process to which these alterations are related.

Aging↗

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

Maturation of intrinsic membrane properties in rat retinal ganglion cells.

In view of the prominent role of voltage-activated conductances in both neuronal differentiation and signal transmission, the present study describes developmental alterations of ion channel properties during the functional maturation of rat RGCs, i.e., between embryonic day 15 when RGCs start to differentiate (E15) and postnatal day 35 (P35) when the retina is fully developed and the animals had already visual input for about 2 weeks. While the sodium system seems to reach maturity already at the end of the second postnatal week, significant alterations in the potassium system were found only from postnatal day 10 on. The functional implications of these alterations are discussed.

Action Potentials↗

PubMed1999

Long-term survival of retinal cell cultures on retinal implant materials.

The aim of the present study was to evaluate cell adhesion and cell survival of mammalian retinal neurons on different materials used for the production of multi-photodiode arrays (MPDAs) intended for implantation in the subretinal space of patients suffering from progressive photoreceptor cell loss. The survival rates of different types of retinal neurons and glia cells were monitored by conventional histochemical techniques and immunocytochemistry up to 4 weeks. Whereas most of the materials tested showed good biocompatibility, cell survival of retinal glia and neurons was markedly reduced on titanium nitride (TiN), especially for culturing periods longer than 2 weeks. The effect was not mediated by diffusible factors released from TiN material. In conclusion, most of the materials tested in this study are suitable for the production of functional MPDAs and no complications are to be expected from long-term implantations of them in the subretinal space.

Animals↗