The arterially perfused eyecup of the tree squirrel, Sciurus carolinensis: a preparation for intracellular recording from mammalian retinal neurons

The arterially perfused eyecup of the Eastern gray squirrel, Sciurus carolinensis, has been developed for the study of mammalian retinal neurons by the technique of intracellular recording. Particular emphasis is placed in this report on the development of a convenient perfusion chamber. The choice of this animal and the reason for choosing the arterially perfused open eyecup are also discussed. Intracellular recordings were made from all major types of neurons in the squirrel retina. Data are presented here from ganglion cells and bipolar cells.     

Developing neuronal populations of the cat retinal ganglion cell layer

An improved flat-mount procedure demonstrates that the developing ganglion cell layer of the cat retina contains two morphologically distinct populations of presumed neurons at all ages between embryonic day 36 (E36) and adulthood. One population resembles the adult "classical neurons" composing the ganglion cells and bar-cells of Hughes, while the remaining cells, which are smaller and possess much less Nissl substance, presumably correspond to precursors of the adult microneurons. Although the total neuron population of the retinal ganglion cell layer remains quite constant at all studied ages, its component subpopulations alter significantly during prenatal development; some 50% of classical neurons disappear before birth and the microneuron population doubles during the same period. An obvious centroperipheral gradient exists for classical neurons by stage E47, but the microneuron density gradient only becomes apparent at birth. A 2:1 centroperipheral ratio for the total neuron population is also apparent at E47. Centroperipheral neuronal density gradients continue to increase during postnatal growth. Loss of classical neurons during prenatal life as a result of cell death or transformation into microneurons, has been postulated as a mechanism for determining neuron density gradients. Cell death does occur in the ganglion cell population but it is not yet established whether microneurons of the ganglion cell layer originate from ganglion cell transformation or migrate as a differentiated class from the ventricular layer. However, it can be concluded that not all microneurons originate from ganglion cell transformation, because the total loss of classical neurons is less than the increase in microneuron numbers during development. The population magnitudes of both neuronal classes in the ganglion cell layer stabilise after birth. However, it is during the postnatal period that the adult cruciate density topography is achieved by both populations. It is concluded that differential areal growth is the prime mechanism for postnatal cell redistribution.     

An immunocytochemical study of the development of central serotoninergic neurons in the chick embryo

The development of central serotoninergic neurons in the chick embryo has been investigated immunocytochemically by utilizing an antiserum to serotonin (5-HT). Immunoreactive neurons are first detected in the brainstem on embryonic day 4 (E4, stage 23), days earlier than 5-HT systems have been detected previously by biochemical techniques. The earliest 5-HT-containing cells at E4 appear rostral to the pontine flexure, yet by E5, 5-HT neuronal groups are observed throughout the brainstem from just caudal to the mesencephalic flexure to the cervical flexure. During this and subsequent phases of development, two distinct patterns of cellular migration seem to be involved in the formation of the various 5-HT neuronal groups. One pattern involves a ventral migration of 5-HT cells, which appears dependent upon the directional guidance of midline radial processes (formed by floor plate cells) that extend across the neuroepithelium. The other pattern involves a lateral migration of cells, followed by an aggregation and rearrangement of 5-HT neurons into distinct subgroups or clusters. Through these patterns of migration most components of the 5-HT neuronal system can be recognized as early as E12, with the mature organization of the 5-HT cell groups occurring by E17. One unexpected finding was the comparatively late appearance (between E9 and E12) of 5-HT neurons in the paraventricular organ of the hypothalamus. Thus, in comparison to the initial observation of the majority of brainstem 5-HT neurons at E4 to E5, the hypothalamic 5-HT cells appear after a delay of between 5 and 7 days. Such differences illustrate the fact that neurons sharing a common neurotransmitter phenotype do not necessarily share the same developmental timetable for the expression of that particular phenotype, or they may undergo neurogenesis during considerably different periods of embryogenesis.     

Morphological characterization of retinal bipolar cells in the marine teleost Rhinecanthus aculeatus

The marine teleost Rhinecanthus aculeatus (Balistidae) has recently been shown to posses trichromatic color vision supported by a retinal combination of double and single cones. Double cones are composed of two members with different spectral sensitivity. It is not known whether a correlation exists between the chromatic wiring of double cones to the inner retina and trichromacy, nor how unmixed, chromatic information is extracted from the two members of the couple. In mammalians, bipolar cells determine color segregation by means of the midget system, central to trichromatic color vision; however, midget bipolar cells have never been described in teleosts. On the basis of its likely importance in transferring chromatic photoreceptor signals to the inner retina, we have morphologically characterized the retinal bipolar cell types of R. aculeatus using DiOlistic staining techniques to verify if an anatomical specialization of this group of cells is required to support trichromatic color vision. Thirteen cell types are described: eight putative OFF types and five putative ON types. Of these, four had axonal boutons ramifying in both sublayers (ON and OFF) of the inner plexiform layer, six had terminals restricted to the OFF layer, and three cell types had terminals restricted to the ON layer. Dendritic arbors of bipolar cells had narrower diameters (5–40 μm) in comparison to bipolar cells of other teleost species; this supports the idea that a low degree of photoreceptor to bipolar convergence is correlated with trichromacy in this retina and possibly with the function of double cones as color receptors. J. Comp. Neurol. 518:3117–3129, 2010. © 2010 Wiley‐Liss, Inc.     

γ-Aminobutyric acid-mediated inhibition at cholinergic synapses formed by cultured retinal neurons

The purpose of this study was to investigate the effect of γ-aminobutyric acid (GABA) on the function of synapses formed by cholinergic neurons derived from the chick retina. We used an experimental culture system in which striated muscle cells served as postsynaptic targets for cholinergic neurons of the retina. This cell culture system permitted the physiological monitoring of acetylcholine release at synapses formed by retinal neurons. By plating a low of density dissociated retinal cells with myotubes, it was possible to study relatively isolated, presynaptic cholinergic neurons. We found that GABA and agonists, muscimol and isoguvacine, inhibited spontaneous transmission at retina-muscle synapses. These inhibitory effects were reversibly blocked by bicuculline, a GABA receptor antagonist. The benzodiazepine, flurazepam, potentiated GABA-mediated inhibition. Overall, our findings suggest a direct inhibitory action of GABA on the cholinergic retinal neurons studied in our cell culture system.     

Ganglion cell dendritic structure and retinal topography in the rat

The dendritic field size, the distribution of the dendrites relative to the cell body, and the overall shape of the dendritic field of type I ganglion cells in the rat retina were analyzed. These features of neuronal structure were related to the topography of the rat retina. As in the cat, the cell bodies of type I ganglion cells are arranged in a nonrandom mosaic. Previous work has demonstrated that the density of type I cells in the rat retina does not covary with the density of all ganglion cells. Type I dendritic field size varies over the retina; the increase in dendritic field size is accounted for better by the decrease in type I density than by the decrease in overall ganglion cell density. The center of the dendritic field of most type I cells is displaced in the plane of the retina from the cell body. Unlike in carnivore retina (Schall and Leventhal: J. Comp. Neurol. 257:149-159, '87), the dendritic fields in the rat are not displaced down the ganglion cell density gradient. Rather, there is a tendency for the dendritic trees, especially in temporal retina, to be displaced toward dorsal retina. Most of the dendritic fields are elongated, but the degree of elongation is less than that observed in carnivore or primate retina. Unlike in carnivore and primate retina (Leventhal and Schall: J. Comp. Neurol. 220:465-475, '83; Schall et al.: Brain Res. 368:18-23, '86), there is no relationship between dendritic tree orientation and position relative to any point on the retina in the rat.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of the change in number of serotonergic neurons in the chick spinal cord during embryonic development

The existence of serotonin (5-HT)-containing neurons in the spinal cord of the chick embryo was examined by anti-5-HT immunocytochemistry. The first immunoreactive cells were observed in embryos at 7 days of incubation (E7) and were initially located within the floor plate of the early spinal cord. By E9, immunostained cells occurred throughout the length of the spinal cord and were frequently encountered in most transverse sections of the cord. When examined at later embryonic ages of E12, 17 and at hatching (E21 or 22), the 5-HT cells became progressively more difficult to find with the advancing age of the embryos. To determine if this population of spinal cord 5-HT neurons actually diminished during development, a detailed quantitative analysis was undertaken to estimate the number of 5-HT cells in the cord of chick embryos at different ages. The results of this investigation demonstrated that the size of the 5-HT neuronal population rose rapidly from E7 and plateaued (at approximately 3500 neurons) between E9 and E12. As anticipated, the number of 5-HT cells at E17 decreased at all cord levels. Surprisingly, however, the number of spinal cord 5-HT neurons at hatching increased (depending on the cord level) either back to, or above, the counts estimated for the earlier ages of E9 and E12. Therefore, cells expressing the 5-HT phenotype in the spinal cord of the chick embryo persist throughout the period of embryonic development, rather than appear transiently.(ABSTRACT TRUNCATED AT 250 WORDS)     

Taurine uptake by chick embryo retinal neurons and glial cells in purified culture

The properties and cellular distribution of a high-affinity uptake mechanism for taurine have been investigated using separate populations of purified chick embryo neural retina neurons and glia. Purified neuronal monolayers, cultured in serum-free medium, were incubated in radioactive taurine under different conditions and studied autoradiographically and biochemically. Labeling with radioactive taurine was detected in the perikaryon of most of the neurons present in the cultures. Neuronal uptake occurred by means of a high-affinity mechanism which was completely inhibited at low temperatures or in the absence of sodium ions. The uptake was linear for at least 1 hr and, as is the case in vivo, could be inhibited by gamma-aminobutyric acid (GABA) or beta-alanine. Incubation in ouabain, glutamate, or high K+ concentrations failed to cause any increase in the amount of taurine released by neurons preloaded with the radioactive amino acid. The rather wide-spread distribution of high-affinity taurine uptake was confirmed using separate retinal cultures rich in glial cells. Practically 100% of the glial cells appeared labeled after incubation in 10(-7) M [3H] taurine, and this uptake was also inhibited by low-temperature, Na+-free medium, GABA, or beta-alanine. Several pieces of evidence indicate that high-affinity taurine uptake coexists with uptake mechanisms for other amino acids, such as GABA, glutamate, and aspartate, in retinal neurons as well as glial cells. These in vitro populations offer a promising experimental system for the investigation of the effects of taurine on retinal cells.     

Morphological and neurochemical characterization of individual retinal neurons: a combined Golgi and autoradiographic technique

A procedure for combining morphological analysis of Golgi-impregnated neurons with the autoradiographic identification of neurotransmitter substances in the same neurons is described.     

Ultrastructure of retinal ganglion cell death after axotomy in chick embryos

Axotomy often leads to neuronal death, which occurs after a particularly short delay in immature animals. Tectal lesions were made in embryonic day (E) 12 chick embryos, thereby axotomizing the retinal ganglion cells of the contralateral eye, which then died within 3 days. We here describe the ultrastructural changes in the axotomized ganglion cells. The main changes were nuclear invagination and type 3B (cytoplasmic type) cell death characterized by dilation of the perinuclear space, endoplasmic reticulum, and Golgi apparatus. However, nuclear invagination was never seen in type 3B dying cells. All the axotomy-induced retinal ganglion cell death appears to have been of type 3B; apoptosis was not induced by axotomy, as was confirmed by additional light microscopic experiments showing that it did not increase the frequency of apoptotic markers revealed by terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (the TUNEL method) labeling and immunoreactivity for activated caspase-3. However, the latter methods did show small numbers of apoptotic cells dying naturally even in control retinas. After the death of the axotomized ganglion cells, they were phagocytosed mainly in Müller processes. The present findings open up the chick tectal lesion model as a system for analyzing type 3B neuronal death in vivo.     

Development of synapses between chick retinal neurons in dispersed culture

Morphological criteria allow several kinds of synapse to be recognized in the vertebrate retina. It is, however, not presently known if, or how, these morphological differences reflect physiological distinctions. Since a proper investigation of synaptic physiology in the intact retina is compromised by technical difficulties, we have examined dispersed cultures to discover if they are likely to provide a more tractable physiological preparation. The chief question addressed here concerns the extent to which normal synaptic development takes place in the impoverished conditions of dispersed cell culture. Cultures were established from embryonic day 8 chick retina and fixed for microscopy on embryonic equivalent (E.E.) days 12, 14, 16, and 18. Neuronal processes appeared shortly after plating and continued to increase in number and extent through E.E. 16. Cone cells were recognizable by virtue of their distinctive oil droplets. Two classes of cone could be distinguished on the basis of the density of their cytoplasmic staining. Presynaptic ribbons could be observed in cone cells on E.E. 12, but characteristic dyad and triad postsynaptic organization was seldom present at this stage nor was it often observed at subsequent times. An increase in the number of ribbon synapses in culture was seen on E.E. 18. These synapses may represent those of bipolar cells. Conventional synapses were found at all times examined but the number of these increased greatly between E.E. 14 and 16. Of these conventional synapses, we found some whose anatomy was characteristic of synapses made by amacrine cells as well as some whose anatomy was characteristic of synapses made by bipolar cells.     

Choline acetyltransferase-immunoreactive neurons in the retina of normal and dark-reared turtle

Visual deprivation alters retinal-ganglion-cell response properties through changes in spontaneous wave-like activity (Sernagor and Grzywacz [1996] Curr Biol 6:1503-1508). This activity depends on cholinergic synaptic transmission in the turtle retina (ibid; Sernagor and Mehta [ 2001] J Anat 199:375-383). We studied the expression of choline acetyltransferase (ChAT) by immunocytochemistry and Western blot in developing retinas of control and dark-reared turtles. At postnatal day 0 (P0), right after hatching, ChAT-immunoreactivity was present in the ganglion cell layer (GCL), in the inner nuclear layer (INL), and in two distinct bands of the inner plexiform layer (IPL). In P14- and P28-control, and P14- and P28-dark-reared retinas, ChAT-immunoreactivity showed similar patterns to those in P0. However, in P14- and P28-dark-reared retinas the density of ChAT-immunoreactive cells was higher in both the INL and GCL than in P14- and P28-control retinas, respectively. Moreover, Western blotting showed that ChAT protein levels were significantly increased in the dark-reared retina compared to those of the control. TUNEL studies indicated that the difference between normal and dark-reared conditions was not due to extra apoptosis in the former. In turn, proliferating-cell nuclear antigen immunocytochemistry showed no extra proliferating cells in the latter. Finally, nearest-neighbor analysis revealed that the denser population of cholinergic cells in dark-reared turtles formed a mosaic as regular as the normal ones in the GCL. Thus, light deprivation increases the expression of ChAT, increasing the apparent density of cholinergic neurons in the developing turtle retina.     

Immunohistochemical localization of chick retinal 24 kdalton protein (visinin) in various vertebrate retinae

Antiserum against a protein (24,000 daltons, visinin) of chick retina has been provided for immunohistochemical study on the localization of visinin in chick retinae during development, as well as in various vertebrate retinae. The photoreceptor cells were stained with anti-visinin serum from 7th day embryonic retinae and its intensity was gradually increased with embryonic age. In addition, visinin-like immunoreactivity was found in some kinds of amacrine and displaced amacrine cells from 11th-day embryonic retinae. When human, cat, frog and carp retinae which contain both rods and cones were examined, staining of cone cells was clearly observed in the photoreceptor cell layer, but not in the rods. Furthermore visinin-like immunoreactivity was barely detectable in the photoreceptor cells of bovine, rat and mouse retinae containing mostly rod cells. These results suggest that visinin is mainly located in the cone cells in various vertebrate retinae and is a good marker for the cone cells.     

Morphological impairments in retinal neurons of the scotopic visual pathway in a monkey model of Parkinson's disease

Physiological abnormalities resulting from death of dopaminergic neurons of the central nervous system in Parkinson's disease also extend to the retina, resulting in impaired visual functions. In both parkinsonian patients and animal models, low levels of dopamine and loss of dopaminergic cells in the retina have been reported. However, the morphology and connectivity of their postsynaptic neurons, the amacrine cells, have not been analyzed. Here we report, with macaques chronically treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) as a model of Parkinson's disease, that morphological impairments in dopaminergic retinal neurons and their plexus in the inner retina are accompanied by an immunoreactivity decrease in gamma-aminobutyric acidergic and glycinergic amacrine cells. Especially deteriorated were AII amacrine cells, the main neuronal subtype postsynaptic to dopaminergic cells, which exhibited a marked loss of lobular appendages and dendritic processes. Concomitantly, electrical synapses among AII cells, as well as chemical synapses between these and rod bipolar cells, were highly deteriorated in parkinsonian monkeys. These results highlight that the scotopic visual pathway is severely impaired in the parkinsonian condition and provide a morphological basis for a number of abnormalities found in electrophysiological and psychophysical trials in Parkinson's disease patients and animal models.     

Coexistence of high-affinity uptake mechanisms for putative neurotransmitter molecules in chick embryo retinal neurons in purified culture

We report here that high-affinity uptake mechanisms for two or three putative neurotransmitter molecules coexist in many of the neurons present in glia-free, purified neuronal monolayers from chick embryo retina. Replicate cultures were incubated with the tritiated forms of the amino acids gamma-aminobutyric acid (GABA), taurine (TAU) and aspartate (ASP) either individually or in binary or tertiary combinations. Incubation conditions were those typically used to analyze high-affinity uptake mechanisms. At the end of the incubation period the cultures were either lysed in water for measurement of intracellular radioactivity by liquid scintillation counting, or fixed in glutaraldehyde and prepared for autoradiography. Our results indicate that there is coexistence of uptake mechanisms for putative neurotransmitters in retinal cells. Biochemical measurements showed that, at the concentrations used in these experiments, two or more radioactive amino acids could be taken up simultaneously by the cultured populations without extensive inhibition. Moreover, the percentage of cells that appeared autoradiographically labeled in cultures exposed to two or more radioactive amino acid was less than the sum of the percentages of labeled cells when each amino acid was applied individually. Numerical analysis of the autoradiographs was carried out to determine the percentage of cells that could take up only one, only two, or the three putative amino acid neurotransmitters under investigation. This analysis showed that approximately 20% of the neurons have only the mechanism for TAU, whereas very few if any neurons have high-affinity uptake mechanisms for GABA alone or ASP alone. Our experiments have identified populations of cells that take up GABA and TAU (but not ASP) or ASP and TAU (but not GABA). Interestingly, we have not seen any neurons that can take up ASP and GABA and cannot take up TAU, although as many as 50% of the neurons can take up ASP, GABA, and TAU simultaneously. By showing simultaneous uptake of different putative neurotransmitters within individual neurons, our results emphasize the concept that the presence in a given neuron of a high-affinity uptake mechanism for a particular molecule is not a sufficient criterion to assign a neurotransmitter function to that molecule in that neuron.     

Light activation of dopaminergic neurons in rat retina is mediated through photoreceptors

The effect of light on retinal dopamine turnover was studied in the RCS mutant strain of rats with inherited retinal dystrophy and in age-matched controls. Retinal dopamine and DOPAC were decreased in the 60-day-old RCS rats with complete photoreceptor degeneration killed in the light phase of the light-dark cycle. After 24 h dark adaptation, exposure to light markedly increased retinal DOPAC and dopamine in controls but had no effect in 70-day-old RCS dystrophic animals. Findings indicate that light activates retinal dopaminergic neurons via photoreceptor stimulation and not directly.     

Detection of localized retinal malfunction in retinal degeneration model using a multielectrode array system

Light stimulation inhibits the retinal dark current through phototransduction signals in the photoreceptors. Electroretinography (ERG) detects the blockage of the dark current as the a‐wave of the ERG. However, standard ERGs represent the summed neural activity of the retina, and information on localized functions cannot be obtained. In this study, we used a multielectrode array (MEA) system and directly recorded the focal activities of the photoreceptors of the retina. Retinas were isolated from dark‐adapted rodents and were draped over the electrode array with vitreal surface of the retina on the electrode array. After light stimulation, negative waves were recorded from each electrode. Adding aminobutyric acid, a selective agonist of mGluR6 expressed on ON‐bipolar cells, to the media did not block the generation of the responses. The amplitude of the response increased with increasing retinal development. When the retina was locally injured, light‐elicited responses were diminished only in the injured areas of the retina. Retinas isolated from rats given N‐methyl‐N‐nitrosourea (MNU) were also tested. In central retinas of MNU‐treated rats, the responses were progressively decreased following injection of MNU. In contrast, in the peripheral retinas, amplitude of the responses was relatively retained, consistent with the retinal thickness observed by immunohistochemistry. In conclusion, light‐evoked responses were recorded with the MEA system. The MEA system was useful for detecting subtle and focal activation of photoreceptors. This spatial information should be valuable in investigating local functional recovery in therapeutically treated areas, such as in gene transfer or cell transplantation. © 2009 Wiley‐Liss, Inc.     

Differential expression of acetylcholinesterase molecular forms in neural retina and retinal pigmented epithelium during chick development

Neural retina (NR) and retinal pigmented epithelium (RPE) were used as a model for studying acetylcholinesterase (AChE) expression in neuronal and non-neuronal tissue during development. In chick embryo retina, increasing AChE activity appeared from day 7 to hatching, first in NR and then in RPE. NR contained 3 main AChE forms, 11.3S, 6.5S and 4.5S, resolved by sucrose density gradient centrifugation. An additional 19S form was exclusively detected in RPE whatever the extraction procedure followed. During differentiation, the proportions of AChE molecular form changed until they reached a steady state characteristics of mature neural retina, whereas in RPE, patterns of AChE molecular forms did not change significantly during development. Thus, 19S AChE appeared to be characteristic of non-neuronal retinal tissue. The expression of the AChE molecular forms in NR and RPE, and particularly the 19S form, was independent of the presence of the lens during retinal differentiation.