Anitha Bruun

Uptake of certain possible neurotransmitters into retinal neurons of some mammals

The uptake of glycine, GABA, and β-alanine was studied in rat, guinea-pig, cat, monkey and human retinas. The human retinas were studied in vitro only, the others both in vivo and in vitro. Either in vivo or in vitro [3H]glycine was aceumulated by certain cells which presumably are a type of amacrine cells. Glycine was not significantly metabolized under the conditions of the experiments. There was no significant variation between the species with this amino acid. [3H]GABA was preferentially accumulated by a type of amacrines in vivo. In rats and monkeys there was also a variable uptake into Muller cells already in vivo, and this uptake often disguised the neuronal uptake into amacrines. [3H]GABA in vitro gave predominantly glial uptake in all species except in cats and guinea-pigs where radioactive amacrines were still seen. [3H]β-alanine gave results similar to [3H]GABA. [3H]Glutamic acid produced predominantly glial uptake either in vivo or in vitro. Rods but not cones became somewhat radioactive in vitro. The results emphasize that particularly the GABA uptake varies significantly between different species.

In vitro uptake of ß-alanine into rabbit retinal neurons

SummaryThe uptake of β-alanine into rabbit retina was found to proceed without accumulation of metabolites of β-alanine. In vitro, the β-alanine was taken up into cells with the position of amacrines and also in some ganglion cells, similar to the uptake in vivo. The uptake was found to be saturable, temperaturedependent, and inhibitable with ouabain and 2.4-dinitrophenol, indicating an active uptake mechanism. The Km of the β-alanine uptake was 2.57 × 10−5M. For the isomer, α-alanine, the Km was found to be 1.1 × 10−3M. The uptake was not influenced by aspartic acid, glutamic acid, glycine, taurine, histidine, leucine, or a-alanine (all 10−5M). GABA (10−5M) competitively inhibited the β-alanine uptake by 27%.

Transplantation of full-thickness retina in the rhodopsin transgenic pig.

Purpose To establish the morphology of full-thickness neuroretinal grafts transplanted to hosts with degenerative photoreceptor disease. Methods Twenty rhodopsin transgenic pigs received a neuroretinal sheet from a neonatal normal pig in one eye. Following vitrectomy and retinotomy with bleb formation, the grafts were positioned inside the bleb between the host neuroretina and retinal pigment epithelium. After a survival time of 4 months, eye specimens were studied by light and electron microscopy as well as with immunohistochemical markers. Results One eye developed endophthalmitis in the immediate postoperative period and was terminated. Laminated grafts with correct polarity were found in 13 of the remaining 19 eyes. In most cases, these grafts had well-developed organized photoreceptors with outer segments apposed to the host retinal pigment epithelium. The inner layers of the graft contained mostly Müller cells. Both eyes of the hosts had a reduction of photoreceptor cells in most of the retina, while inner layers remained relatively intact. Conclusions Full-thickness neuroretinal grafts can be transplanted to a large animal host with photoreceptor degeneration. The transplantation procedure is relatively atraumatic to both graft and host tissue, and the grafts survive well for at least 4 months. The graft and host retina does not seem to form extensive neuronal contacts, and future work must be directed at stimulating such activity without disrupting the retinal neuronal organization.

Retinal glial cell immunoreactivity and neuronal cell changes in rats with STZ-induced diabetes

Purpose. To study whether diabetes could influence glial cells, retinal neurons, and pigment epithelial cells and if so, to evaluate whether any changes could be influenced by aminoguanidine (AG) or probucol (PB). Methods. Streptozocin (STZ)-induced diabetic male Wistar rats and age-matched control rats were fed a normal diet, addition of AG in the drinking water (0.5 g/l for diabetic and 1.0 g/l for control rats) or PB in the pellets (1 % w/w) for one or six months. Paraffin embedded retinal sections were incubated in the primary antibodies GFAP, calbindin, RPE65, and Hu, for glial, horizontal, pigment epithelial, and ganglion cells, respectively, and in fluorescent secondary antibodies. Results. One month after STZ injection, GFAP immunoreactivity was sparse, but after six months it was prominent in glial cells in 5/5 diabetic and 1/7 control retinas (p = 0.015). Neither AG, nor PB influenced this immunoreactivity. Numbers of retinal pigment epithelial cells and cells in the ganglion cell layer, were similar at one and six months of diabetes. By time, the number of horizontal cells decreased (p < 0.001) and branching and numbers of their terminals were reduced (p < 0.001). Conclusion. Diabetes for six months resulted in increased glial cell immunoreactivity, and by age, horizontal cell numbers and branching of their terminals decreased, morphological patterns that were unaffected by AG or PB. The numbers of retinal pigment epithelial cells and cells in the ganglion cell layer were unaffected both by age and diabetes.

Correlations between cholinergic neurons and muscarinic m2 receptors in the rat retina

ACETYLCHOLINE is well established as the neurotransmitter of starburst amacrine cells in the vertebrate retina but their function is poorly understood. We compared the distribution of muscarinic m2 receptors in the rat retina with the localization of the starburst cell processes. mAChR2 immunoreactivity appeared in a central band in the inner plexiform layer, which did not co-localize with the processes of the cholinergic amacrine cells. We found co-labelling of VAChT and ChAT making it highly unlikely that there are undetected cholinergic neurons in rat retina. Most mAChR2 receptors were located far from the cholinergic neurons, suggesting that most of them are unlikely to be associated with conventional cholinergic synapses.

Alterations in electroretinograms and retinal morphology in rabbits treated with vigabatrin

Purpose: To determine whether long-term treatment with the anti-epileptic drug vigabatrin causes damage to rabbit retina. Methods: Five rabbits were treated continuously with a daily dose of vigabatrin solution per orally during a period of 1–8 months. Two rabbits receiving water were used as controls. Repeated full-field electroretinograms (every two weeks) were assessed during this period. Vigabatrin serum concentration was repeatedly measured for securing successful drug administration. After termination of treatment the rabbits were sacrificed and the morphology of the sectioned retina was studied. Results: In all rabbits treated with vigabatrin the serum analyses repeatedly demonstrated elevated drug concentration. Full-field electroretinograms demonstrated normal rod function in all treated rabbits, but reduced cone function in two of the five treated rabbits verified by 30 Hz flicker stimulation. Morphologic studies of the sectioned retina demonstrated GFAP immunoactivity of the glial cells localized in the retinal periphery in all five treated rabbits, one of which had staining also in the centrally localized glial cells. The treated rabbits also demonstrated a weaker GAD staining in the IPL and less positive amacrine cells, compared to the controls. Only two treated rabbits had normal GABA staining while three had an enhanced GABA immunoreactivity and undistinguishable fibers in the IPL. In three out of five treated rabbits the Müller cells were short, stubby and fragmented, with swollen endfeet. Conclusion: This study demonstrates changes in histopathology caused by vigabatrin in an animal model, which has not been reported previously. We have found that vigabatrin orally administrated to rabbits does not affect rod function but may reduce cone function in the full-field electroretinogram, which is similar to the previously reported vigabatrin effect on the human ERG. The results indicate that vigabatrin may damage or influence, at least one cell type in the rabbit retina.

NPY-induced neurotransmitter release from the rabbit and chicken retina

Abstract. Neuropeptide Y (NPY) or closely related peptides are present in the retina of certain vertebrates, but their actions are not known. We have therefore studied the NPY‐induced release of [3H]‐GABA, [3H]‐glycine, [3H]‐dopamine, [3H]‐5‐hydroxytryptamine, and [3H]‐choline chloride‐derived radioactivity in the rabbit and chicken retina. NPY affected the release of [3H]‐glycine, [3H]‐dopamine, [3H]‐5‐hydroxytryptamine, and [3H]‐choline chloride‐derived radioactivity in rabbit retina and of [3H]‐GABA, [3H]‐5‐hydroxytryptamine and [3H]‐choline chloride‐derived radioactivity in chicken retina in an energy requiring, NA+K+‐ATPase dependent and calcium dependent manner. Certain related peptides, APP (=avian pancreatic polypeptide), BPP (=bovine pancreatic polypeptide), and PYY (=peptide YY), had variable and less pronounced effects. The results suggest a neurophysiological role in both chicken and rabbit retina for NPY or some related peptide.

Neural elements in the pineal complex of the frog, Rana esculenta , II: GABA-immunoreactive neurons and FMRFamide-immunoreactive efferent axons

The photosensory pineal complex of anurans comprises an extracranial part, the frontal organ, and an intracranial part, the pineal organ proper. Although the pineal organ functions mainly as a luminosity detector, the frontal organ may monitor the relative proportions of short and intermediate/long wavelengths in the ambient illumination. The major pathway of information processing in the pineal and frontal organs is the photoreceptor to ganglion cell synapse. It is not known whether interneurons form part of the neural circuitry. In the present study, we demonstrate GABA-immunoreactive (GABA-IR) neurons in the pineal and frontal organs of the frog, Rana esculenta. No GABA-IR axons were observed in the pineal nerve between the frontal and pineal organs, or in the pineal tract that connects the pineal complex with the brain. The GABA-IR neurons differed in morphology from centrally projecting neurons visualized by retrograde labeling with horseradish peroxidase. Thus, we suggest that the GABA-IR neurons in the pineal and frontal organs represent local interneurons. Axons of central origin, immunoreactive with a sensitive antiserum against the tetrapeptide Phe-Met-Phe-Arg-NH2 (FMRFamide), were observed in the intracranial portion of the photosensory pineal organ. The immunoreactive axons enter the caudal pole of the pineal organ via the posterior commissure. The largest density of axons was observed in the caudal part, while fewer axons were detected in the rostral portion. The uneven distribution of the FMRFamide-immunoreactive axons may be related to the distribution of different types of intrapineal neurons. FMRFamide-immunoreactive varicose axons were observed in the extracranial frontal organ. A central innervation of the pineal organ, previously known exclusively from amniotes, is probably not per se linked with the evolutionary transition of the pineal organ from a directly photosensory organ to a neuroendocrine organ. It could rather represent a centrifugal input to a sensory system which has been retained when the directly sensory functions have changed, during phylogeny, to neuroendocrine functions.

Growth of postnatal rat retina in vitro. Development of neurotransmitter systems.

In this study, we demonstrate that explanted neonatal rat retina can be maintained in culture for periods up to 3 weeks. The cultured retinas displayed a distinct layering that was almost identical to litter-matched retinas of the same age, but the majority of the ganglion cells did not survive and photoreceptor outer segments did not develop properly. Distinct synaptophysin immunoreactivity was expressed in both the inner and outer plexiform layers of cultured retina and the pattern mimicked that one observed in vivo. After 2-3 weeks in vitro, the inner retina expressed immunoreactivities to various components of the cholinergic and nitrergic transmitter systems, including nitric oxide activated cyclic GMP immunoreactivity. The investigated cell populations displayed similar distribution patterns as in situ, but morphological differences appeared in vitro. Such differences were mainly observed as irregularities in the arborization patterns in the inner part of the inner plexiform layer. We suggest that these discrepancies may arise as a result of reduced ganglion cell survival. Our observations demonstrate that some neurotransmitter systems develop in vitro and their neural circuitry appears similar to the in vivo situation. The presence of synapses, receptor proteins and transmitter substances implies that neural communication can occur in cultured retinas.

Gap junction protein connexin43 is heterogeneously expressed among glial cells in the adult rabbit retina.

The immunohistochemical distribution and ultrastructural immunolocalization of connexin43 (Cx43) in the neural retina of the rabbit was investigated. Cx43 immunolabeling appeared in the form of distinct puncta distributed on different kinds of glial cells and exclusively in the myelinated fiber region of the neural retina. Double‐label immunohistochemistry showed that the most obvious Cx43 labeling occurred at processes of glial fibrillary acidic protein‐positive astrocytes and on vimentin‐positive Müller cells. Cx43‐immunoreactive puncta were also evident on cell bodies and processes of 2′‐3′‐cyclic nucleotide phosphodiesterase‐labeled oligodendrocytes. As shown by electron microscopy, immunoreactivity to Cx43 was restricted to gap junctions among the macroglial cell population. The homologous interastrocytic and Müller cell‐to‐Müller cell, as well as the heterologous astrocyte‐to‐Müller cell and astrocyte/Müller‐to‐oligodendrocyte gap junctions were symmetrically labeled. Our results indicate a specific expression of Cx43 at gap junctions between macroglial cells located in the myelinated streak. The extensive Cx43 immunolabeling suggests a substantial amount of gap junctional coupling that establishes a macroglial syncytium. J. Comp. Neurol. 407:395–403, 1999.