Somatostatin-like immunoreactive material in the rabbit retina: immunohistochemical staining using monoclonal antibodies

Two mouse monoclonal antibodies to somatostatin-14 were used with avidin-biotin-peroxidase immunohistochemical technique to examine the rabbit retina. In agreement with a previous study using a polyclonal anti-serum, a sparse population (about 1,000 per retina) of neurons in the ganglion cell layer are immunoreactive for somatostatin; the vast majority of these cells are inferior to the myelinated fiber bundle. In addition, the monoclonal antibodies disclose a second neuronal population that forms a circumferential band of immunoreactive neurons around the extreme periphery of the retina. The cells in the body of the inferior retina have dendrites that ramify in the inner plexiform layer. Both the circumferential band of cells and the cells in the body of the inferior retina give off axonlike processes that run in the inner plexiform layer and do not enter the optic nerve. These long, straight varicose fibers form a meshwork that covers the entire retina. The superior retina, which contains only rare immunoreactive cell bodies, has a plexus of stained fibers comparable to that of the inferior retina. The circumferential band of cells is relatively resistant to the neurotoxin kainic acid, explaining a previously reported observation that this toxin depletes only about 50% of the content of somatostatin-like immunoreactivity from the rabbit retina. Moreover, the somatostatin immunoreactive neurons are not labeled by the intraocular injection of the fluorescent dye DAPI, which labels the cholinergic displaced amacrine cells of the rabbit retina. These observations imply that somatostatin-like immunoreactivity is localized to two populations of associational ganglion cells, neurons with cell bodies in the ganglion cell layer, the axons of which remain within the retina.     

Critical neuroprotective roles of heme oxygenase‐1 induction against axonal injury‐induced retinal ganglion cell death

Although axonal damage induces significant retinal ganglion cell (RGC) death, small numbers of RGCs are able to survive up to 7 days after optic nerve crush (NC) injury. To develop new treatments, we set out to identify patterns of change in the gene expression of axonal damage‐resistant RGCs. To compensate for the low density of RGCs in the retina, we performed retrograde labeling of these cells with 4Di‐10ASP in adult mice and 7 days after NC purified the RGCs with fluorescence‐activated cell sorting. Gene expression in the cells was determined with a microarray, and the expression of Ho‐1 was determined with quantitative PCR (qPCR). Changes in protein expression were assessed with immunohistochemistry and immunoblotting. Additionally, the density of Fluoro‐gold‐labeled RGCs was counted in retinas from mice pretreated with CoPP, a potent HO‐1 inducer. The microarray and qPCR analyses showed increased expression of Ho‐1 in the post‐NC RGCs. Immunohistochemistry also showed that HO‐1‐positive cells were present in the ganglion cell layer (GCL), and cell counting showed that the proportion of HO‐1‐positive cells in the GCL rose significantly after NC. Seven days after NC, the number of RGCs in the CoPP‐treated mice was significantly higher than in the control mice. Combined pretreatment with SnPP, an HO‐1 inhibitor, suppressed the neuroprotective effect of CoPP. These results reflect changes in HO‐1 activity to RGCs that are a key part of RGC survival. Upregulation of HO‐1 signaling may therefore be a novel therapeutic strategy for glaucoma. © 2014 Wiley Periodicals, Inc.     

Localization of two calcium binding proteins, calbindin (28 kD) and parvalbumin (12 kD), in the vertebrate retina

We used immunocytochemistry to locate two calcium binding proteins, calbindin (CaB) and parvalbumin (PV), in the retina of goldfish, frog, chick, rat, guinea pig, dog, and man. The location of CaB depended on the type of dominant photoreceptor cells in birds and mammals. In cone-dominant retinas such as those of the chick, CaB-like immunoreactivity was found in the cones, cone bipolars, and ganglion cells. Amacrine cells 5-12 microns across were also labeled. In rod-dominant retinas, such as those of the rat, guinea pig, and dog, horizontal cells, small amacrine cells (about 6 microns across), and cells in the ganglion cell layer were labeled. In the human retina, which has both cones and rods in abundance, cones, cone bipolars, ganglion cells, horizontal cells, and small and large amacrine cells were labeled. In the frog and goldfish, the level of CaB-like immunoreactivity was low. In the frog, a few cones, amacrine cells, and cells in the ganglion cell layer were labeled. No immunoreactive structures were seen in the goldfish retina. PV-like immunoreactivity was found in chicks, rats, and dogs. No such immunoreactive structures were seen in the other species. In the chick, only amacrine cells were labeled. In the rat, amacrine cells and several displaced amacrine cells were labeled. In the dog, in addition to amacrine cells and displaced amacrine cells, horizontal cells were strongly labeled. Thus, PV-like immunoreactivity was found in those elements relating to the modulation of the main pathway of the visual transmission system.     

Topography of pig retinal ganglion cells

In the present work we analyzed the distribution of retinal ganglion cells (RGCs) in the pig retina. RGCs were retrogradely labeled in vivo by injecting Fluoro-Gold into the optic nerve. RGC density and the distribution of RGCs in terms of soma size were analyzed. Different regions of the porcine retina were identified following analysis of the distribution of RGCs in terms of cell density and soma size: in the central retina, we found a high-density horizontal RGC band lying dorsal to the optic disc. Moreover, in this region, a high proportion of RCGs with small soma size was observed. From the central to the more peripheral retina, we observed a decrease in RGC density, together with a greater presence of RGCs with larger somas. The results of this study should prove to be useful as a foundation for future studies with the porcine retina as a model in ophthalmic research. The study also highlights the necessity to label the RGC population specifically with retrograde tracers in order to quantify precisely alterations in the cell population associated with experimental treatments.     

Asymmetric distribution of retinal ganglion cells in goldfish

The distribution of retinal ganglion cells (RGCs) in goldfish was determined by removing an eye and applying cobaltous-lysine to the optic nerve for 24 hr. This procedure allowed the cobalt label to be in continuous contact with the cut ends of the optic axons and thereby backfilled many RGCs. RGC density was determined across three different sizes of retinae by using fish with different eye sizes. Confirming earlier work, we found that RGC density diminished as retinal area increased. However, irrespective of the retinal size, the density of RGCs was elevated along the temporal boundary between the dorsal and the ventral retina. A conservative estimate indicated that the RGC density in the temporal retina was at least 1.8-2.5 times higher than the mean RGC density of the entire retina. Thus, the goldfish retina does not appear to have a homogeneous distribution of RGCs as was previously considered. Small and large retinae differed with respect to the percentage of cells in the RGC layer that was RGCs. In small retinae, even when the noncobalt-filled cells (glia and displaced amacrine cells) were added to the cobalt-filled RGCs, the density of all cell types was elevated in the temporal retina relative to the remainder of the retina. Furthermore, in small retinae, the percentage of cells in the RGC layer that was RGCs (75%) was constant across the radial and circumferential aspects of the retina. In marked contrast, in medium-large retinae, a homogeneous distribution of cells across the entire retina resulted when the noncobalt-filled cells were added to the cobalt-filled cells. However, the percentage of cells that was cobalt-filled RGCs was significantly greater in the temporal retina (50%) than in the remainder of the retina (35%). In large retinae, as in small retinae, the percentage of cells that was RGCs did not vary as a function of distance from the optic disc. These data suggest that, in the course of retinal maturation, cell density in the temporal retina is elevated initially and then declines subsequently to the level of the surrounding retina. Over time, more displaced to the level of the surrounding retina. Over time, more displaced amacrine cells may be added to the tissue surrounding the temporal retina. Alternatively, more RGCs outside the temporal retina may become displaced amacrine cells. Such events could account for the growth-associated, disproportionate decrease in the percentage of cells that is RGCs in the tissue surrounding the temporal retina.(ABSTRACT TRUNCATED AT 400 WORDS)     

Distribution and synaptic connectivity of neuropeptide Y-immunoreactive amacrine cells in the rat retina

Neuropeptide Y (NPY) is a potent bioactive peptide that is widely expressed in the nervous system, including the retina. Here we show that specific NPY immunoreactivity was localized to amacrine and displaced amacrine cells in the rat retina. Immunoreactive cells had a regular distribution across the retina and an overall cell density of 280 cells/mm(2) in the inner nuclear layer (INL) and 90 cells/mm(2) in the ganglion cell layer (GCL). In the INL, most immunoreactive cells were characterized by small cell bodies and fine processes that appeared to ramify primarily in stratum 1 of the inner plexiform layer (IPL). A few cells in the INL also ramified in stratum 3 of the IPL. In the GCL, small to medium immunoreactive cells appeared to ramify primarily in stratum 5 of the IPL. A few immunoreactive processes, originating from somata in the INL and processes in the IPL, ramified in the OPL. NPY-immunoreactive cells contained GABA immunoreactivity, and some amacrine cells also contained tyrosine hydroxylase immunoreactivity. NPY-immunostained processes were most frequently presynaptic to nonimmunostained amacrine and ganglion cell processes and postsynaptic to nonimmunostained amacrine cell processes and cone bipolar cell axonal terminals. These findings indicate that NPY immunoreactivity is present in two populations of amacrine cells, one located in the INL and the other in the GCL, and that these cells mainly form synaptic contacts with other amacrine cells. These observations suggest that NPY-immunoreactive cells participate in multiple circuits mediating visual information processing in the inner retina.     

In vivo imaging of murine retinal ganglion cells

Current methods for in vivo retinal ganglion cells (RGCs) imaging involve either retrograde or intravitreal injection of chemical or biological tracers, which are invasive and may require repeated injection for serial long-term assessment. We have developed a confocal scanning laser ophthalmoscope technique (blue-light CSLO or bCSLO) to image retinal ganglion cells (RGCs) in mice expressing cyan fluorescent protein under the control of a Thy-1 promoter. Fluorescent spots corresponding to CFP-expressing retinal ganglion cells were discernable with the bCSLO. 96.1+/-2.6% of CFP expressing cells also were retrograde labeled with DiI indicating the bCSLO imaged fluorescent spots are RGCs. The imaging of Thy-1 promoter-driven CFP expression in these mice could serve as a sensitive indicator to reflect the integrity of RGCs, and provides a non-invasive method for longitudinal study of the mechanism of RGC degeneration and the effect of neuroprotective agents.     

AII amacrine cells in the mammalian retina show disabled-1 immunoreactivity

Disabled 1 (Dab1) is an adapter molecule in a signaling pathway, stimulated by Reelin, which controls cell positioning in the developing brain. It has been localized to AII amacrine cells in the mouse and guinea pig retinas. This study was conducted to identify whether Dab1 is commonly localized to AII amacrine cells in the retinas of other mammals. We investigated Dab1-labeled cells in human, rat, rabbit, and cat retinas in detail by immunocytochemistry with antisera against Dab1. Dab1 immunoreactivity was found in certain populations of amacrine cells, with lobular appendages in the outer half of the inner plexiform layer (IPL) and a bushy, smooth dendritic tree in the inner half of the IPL. Double-labeling experiments demonstrated that all Dab1-immunoreactive amacrine cells were immunoreactive to antisera against calretinin or parvalbumin (i.e., other markers for AII amacrine cells in the mammalian retina) and that they made contacts with the axon terminals of the rod bipolar cells in the IPL close to the ganglion cell layer. Furthermore, all Dab1-labeled amacrine cells showed glycine transporter-1 immunoreactivity, indicating that they are glycinergic. The peak density was relatively high in the human and rat retinas, moderate in the cat retina, and low in the rabbit retina. Together, these morphological and histochemical observations clearly indicate that Dab1 is commonly localized to AII amacrine cells and that antiserum against Dab1 is a reliable and specific marker for AII amacrine cells of diverse mammals.     

Melanopsin expressing human retinal ganglion cells: Subtypes,distribution, and intraretinal connectivity

Intrinsically photosensitive retinal ganglion cells (ipRGCs) expressing the photopigment melanopsin belong to a heterogenic population of RGCs which regulate the circadian clock, masking behavior, melatonin suppression, the pupillary light reflex, and sleep/wake cycles. The different functions seem to be associated to different subtypes of melanopsin cells. In rodents, subtype classification has associated subtypes to function. In primate and human retina such classification has so far, not been applied. In the present study using antibodies against N‐ and C‐terminal parts of human melanopsin, confocal microscopy and 3D reconstruction of melanopsin immunoreactive (‐ir) RGCs, we applied the criteria used in mouse on human melanopsin‐ir RGCs. We identified M1, displaced M1, M2, and M4 cells. We found two other subtypes of melanopsin‐ir RGCs, which were named “gigantic M1 (GM1)” and “gigantic displaced M1 (GDM1).” Few M3 cells and no M5 subtypes were labeled. Total cell counts from one male and one female retina revealed that the human retina contains 7283 ± 237 melanopsin‐ir (0.63–0.75% of the total number of RGCs). The melanopsin subtypes were unevenly distributed. Most significant was the highest density of M4 cells in the nasal retina. We identified input to the melanopsin‐ir RGCs from AII amacrine cells and directly from rod bipolar cells via ribbon synapses in the innermost ON layer of the inner plexiform layer (IPL) and from dopaminergic amacrine cells and GABAergic processes in the outermost OFF layer of the IPL. The study characterizes a heterogenic population of human melanopsin‐ir RGCs, which most likely are involved in different functions.     

Immunocytochemical staining of cholinergic amacrine cells in rabbit retina

Cholinergic neurons of rabbit retina were labelled with an antibody against choline acetyltransferase, the synthesizing enzyme for acetylcholine. Two populations of cells are immunoreactive. Type a cell bodies lie in the inner nuclear layer (INL), their dendrites branching narrowly in sublamina a of the inner plexiform layer (IPL), while type b cell bodies lie in the ganglion cell layer (GCL) with dendrites branching in sublamina b of the IPL. The irregular networks of clustered immunoreactive dendrites are similar, but not identical, in the two sublaminae. Type b cells are more numerous than type a cells in central retina. No axons were stained. It appears that the immunoreactive neurons are normally placed and displaced starburst/cholinergic amacrine cells.     

Selective synaptic connections in the retinal pathway for night vision

The mammalian retina encodes visual information in dim light using rod photoreceptors and a specialized circuit: rods→rod bipolar cells→AII amacrine cell. The AII amacrine cell uses sign-conserving electrical synapses to modulate ON cone bipolar cell terminals and sign-inverting chemical (glycinergic) synapses to modulate OFF cone cell bipolar terminals; these ON and OFF cone bipolar terminals then drive the output neurons, retinal ganglion cells (RGCs), following light increments and decrements, respectively. The AII amacrine cell also makes direct glycinergic synapses with certain RGCs, but it is not well established how many types receive this direct AII input. Here, we investigated functional AII amacrine→RGC synaptic connections in the retina of the guinea pig (Cavia porcellus) by recording inhibitory currents from RGCs in the presence of ionotropic glutamate receptor (iGluR) antagonists. This condition isolates a specific pathway through the AII amacrine cell that does not require iGluRs: cone→ON cone bipolar cell→AII amacrine cell→RGC. These recordings show that AII amacrine cells make direct synapses with OFF Alpha, OFF Delta and a smaller OFF transient RGC type that co-stratifies with OFF Alpha cells. However, AII amacrine cells avoid making synapses with numerous RGC types that co-stratify with the connected RGCs. Selective AII connections ensure that a privileged minority of RGC types receives direct input from the night-vision pathway, independent from OFF bipolar cell activity. Furthermore, these results illustrate the specificity of retinal connections, which cannot be predicted solely by co-stratification of dendrites and axons within the inner plexiform layer.     

Localization of GAD- and GABA-like immunoreactivity in ground squirrel retina: retrograde labeling demonstrates GAD-positive ganglion cells.

Glutamic acid decarboxylase (GAD)- and gamma-aminobutyric acid (GABA)-like immunoreactivity was examined in the retina of the 13-lined ground squirrel (Spermophilus tridecemlineatus). Labeling was observed in the inner nuclear layer (INL), inner plexiform layer (IPL) and ganglion cell layer (GCL). The immunoreactive cell bodies in the inner third of the INL were 6-13 microns in diameter and, because of their size and location it was considered that these were amacrine cells. Labeling in the IPL was concentrated in 5 bands corresponding to laminae 1a, 1c, 2, 4 and 5. In the GCL a heterogeneous population of neurons exhibited GAD- and GABA-like immunoreactivity. The soma diameters of the GCL cells ranged from 5 to 17 microns. These may represent displaced amacrines and/or ganglion cells. To determine if any of the immunoreactive cells in the GCL were ganglion cells, double labeling experiments were performed using rhodamine latex microspheres ('beads') as retrograde neuronal tracers. Rhodamine beads were injected into the superior colliculus, and retinas with retrogradely labeled ganglion cells were subsequently incubated with the anti-GAD antiserum. These experiments revealed a small population of GAD-positive ganglion cells, setting a lower limit for the total number of GABAergic ganglion cells.     

Morphological identification and systematic classification of mammalian retinal ganglion cells. I. Rabbit retinal ganglion cells

Retinal ganglion cells (RGCs) convey visual signals to 50 regions of the brain. For reasons of interest and convenience, they constitute an excellent system for the study of brain structure and function. There is general agreement that, absent a complete “parts list,” understanding how the nervous system processes information will remain an elusive goal. Recent studies indicate that there are 30–50 types of ganglion cell in mouse retina, whereas only a few years ago it was still written that mice and the more visually oriented lagomorphs had less than 20 types of RGC. More than 30 years ago, I estimated that rabbits have about 40 types of RGC. The present study indicates that this number is much too low. I have employed the old but powerful method of Golgi-impregnation to rabbit retina, studying the range of component neurons in this already well-studied retinal system. Close quantitative and qualitative analyses of 1,142 RGCs in 26 retinas take into account cell body and dendritic field size, level(s) of dendritic stratification in the retina's inner plexiform layer, and details of dendritic branching. Ninety-one morphologies are recognized. Of these, at least 32 can be correlated with physiologically studied RGCs, dye-injected for morphological analysis. It is unlikely that rabbits have 91 types of RGC, but is argued here that this number lies between 60 and 70. The present study provides a “yardstick” for measuring the output of future molecular studies that may be more definitive in fixing the number of RGC types in rabbit retina.     

Distribution of Purkinje cell-specific Zebrin-II/aldolase C immunoreactivity in the mouse,rat, rabbit,and human retina

The developmental, genetic, and biochemical similarities that have been observed between the cerebellum and retina form the basis for ongoing investigations into retinal expression of cerebellar-specific proteins. We have examined the mouse, rat, rabbit, and human retina for expression of a protein that is present in parasagittal Purkinje cell strips and that is recognized by the antibody Zebrin-II. This protein has recently been identified as a member of the aldolase C isoenzymes. Western blotting and immunocytochemistry have been used. The monoclonal antibody Zebrin-II recognized a prominent 36 kDa protein band on immunoblots of both the cerebellum and the retina of the examined species. Immunocytochemistry showed that, in the three nonhuman species, cells were stained in the ganglion cell layer (GCL). In addition, in the mouse and rabbit, cells in the inner nuclear layer (INL) were also labeled. Except for the visual streak, there were more immunopositive cells in the rabbit GCL and INL than in corresponding areas of the mouse retina. In the human, in contrast to the other species, the photoreceptor cell layer was strongly aldolase C immunoreactive. In the h all species except for the rat, the photoreceptor inner segments also displayed a weak labeling. The results show that this aldolase C isoenzyme is another protein that is selectively expressed by the cerebellum: and retina. Furthermore, the retinal expression is species specific, and this pattern seems to show a good correlation with the oxygenation level of the individual compartments. The indication that this aldolase C isoenzyme has specific developmental functions in the retina provides additional clues for our understanding of cerebellar organization. © 1994 Wiley-Liss, Inc.     

Study of the role of the low-affinity neurotrophin receptor p75 in naturally occurring cell death during development of the rat retina

The aim of this study was to examine the tempo-spatial expression of low-affinity neurotrophin receptor p75, or p75(NTR), and its role in the induction of retinal ganglion cell (RGC) apoptosis in the rat retina during development. The cellular distribution of p75 in the retina was demonstrated with immunohistochemistry and double-immunofluorescent staining. Apoptosis in the developing rat retina was detected by DNA gel electrophoresis, and the number of RGCs undergoing apoptosis was estimated by terminal deoxyribonucleotidyl-mediated dUTP-digoxigenin nick end labeling (TUNEL). To localize p75 on apoptotic RGCs, p75 immunofluorescence and TUNEL fluorescent staining was performed on sections with Fluoro-Gold-prelabeled RGCs. p75 immunoreactivities were not detected either on the RGCs or TUNEL-positive cells, whereas Müller cell processes were p75 immunopositive. Thus, it was most unlikely that p75 induced apoptosis of RGCs in the rat retina.     

Isoform‐specific localization of Nogo protein in the optic pathway of mouse embryos

Expression of Nogo protein was investigated in the optic pathway of embryonic mice by using isoform‐specific antibodies Bianca and 11C7, which recognize Nogo‐A/B and Nogo‐A, respectively. Our previous reports from using antibody N18 have shown that Nogo is localized on the radial glia in the retina and at the midline of the ventral diencephalon in mouse embryos during the ingrowth of retinal ganglion cells (RGCs) axons. This glial‐specific localization is markedly different from findings in other studies. This study showed Nogo‐A/B primarily on radial glia in the retina at E13 and then later on retinal ganglion cells and axons at E14 and E15, whereas Nogo‐A was expressed preferentially by RGCs and their axons. In the ventral diencephalon, Nogo‐A/B was expressed strongly on radial glia, particularly in those located in the midline region of the chiasm but also on RGC axons. In Nogo‐A knockout embryos, the isoform Nogo‐B (revealed by Bianca) was observed on radial glia in the ventral diencephalon and on RGCs and their axons. We concluded that Nogo‐A is localized on the ganglion cells and retinal axons, whereas Nogo‐B is expressed by the radial glia in the optic pathway. Nogo‐B may play an important role in guiding axon growth in decisive regions of the visual pathway, which include the optic disc and the optic chiasm. J. Comp. Neurol. 524:2322–2334, 2016. © 2016 Wiley Periodicals, Inc.     

Cellular localization of cyclooxygenase-1 and cyclooxygenase-2 in the normal mouse,rat, and human retina

Prostaglandins, synthesized by cyclooxygenase (COX), regulate diverse neurophysiological actions such as regulation of autonomic responses, transmission of pain, generation of fever, control of sleep-wake cycle, synaptic signaling, and cross-talk between neurons and glia in the central nervous system. Although prostaglandins have been widely studied in the anterior segment tissues of the eye, relatively little is known about prostaglandins in the neural retina. By using immunohistochemistry, we have compared the cellular expression and localization of COX-1 and COX-2 in the normal mouse, rat, and human retina. In the normal mouse retina, COX-1 immunoreactivity is present in the outer segments of photoreceptor cells, horizontal cells, microglia, retinal ganglion cells, and displaced amacrine cells. In the normal rat retina, COX-1 immunoreactivity is present in microglia, retinal ganglion cells, and displaced amacrine cells. In the normal human retina, COX-1 immunoreactivity is present in microglia, astrocytes, retinal ganglion cells, and displaced amacrine cells. In the normal mouse and rat retina, COX-2 immunoreactivity is present in processes of the outer plexiform layer and in certain amacrine cells and retinal ganglion cells. In the normal human retina, COX-2 immunoreactivity is only present in processes of the outer plexiform layer. These results suggest that prostaglandins, synthesized by COX-1 or COX-2, may contribute to normal physiological and homeostatic functions in the retina.     

A novel type of complex ganglion cell in rabbit retina

The 15-20 physiological types of retinal ganglion cells (RGCs) can be grouped according to whether they fire to increased illumination in the receptive-field center (ON cells), decreased illumination (OFF cells), or both (ON-OFF cells). The diversity of RGCs has been best described in the rabbit retina, which has three types of ON-OFF RGCs with complex receptive-field properties: the ON-OFF direction-selective ganglion cells (DSGCs), the local edge detectors, and the uniformity detectors. Here we describe a novel type of bistratified ON-OFF RGC that has not been described in either physiological or morphological studies of rabbit RGCs. These cells stratify in the ON and OFF sublaminae of the inner plexiform layer, branching at about 30% and 60% depth, between the ON and OFF arbors of the bistratified DSGCs. Similar to the ON-OFF DSGCs, these cells respond with transient firing to both bright and dark spots flashed in the receptive field but, unlike the DSGCs, they show no directional preference for moving stimuli. We have termed these cells "transient ON-OFF" RGCs. Area-response measurements show that both the ON and the OFF spike responses have an antagonistic receptive-field organization, but with different spatial extents. Voltage-clamp recordings reveal transient excitatory inputs at light ON and light OFF; this excitation is strongly suppressed by surround stimulation, which also elicits direct inhibitory inputs to the cells at light ON and light OFF. Thus the receptive-field organization is mediated both within the presynaptic circuitry and by direct feed-forward inhibition.