Immunohistochemical analysis of the developing inner plexiform layer in postnatal rat retina

PURPOSE: To investigate the development from early postnatal life to adulthood of neural cell processes that establish the circuitry of the inner plexiform layer (IPL). Emphasis was focused on the ontogeny of subsets of cGMP- and protein kinase C (PKC)immunoreactive amacrine and bipolar cells. METHODS: Paraformaldehyde-fixed postnatal and adult retinas were used for light microscopic analysis of immunohistochemical labeling of cryo-sections. Synthesis of cGMP in neural structures was achieved by means of an in vitro stimulation with a well-established nitric oxide donor. RESULTS: In vitro stimulation of postnatal and mature retina with the nitric oxide donor results in NO-activated cGMP synthesis in subsets of bipolar and amacrine cells. NO-activated cGMP immunoreactivity is expressed in specific cell populations during the first postnatal week. Other cell subsets, consisting of amacrine cells and rod bipolar cells, express PKC immunoreactivity during postnatal development. An increasing number of rod bipolar cells start to exhibit cGMP labeling after eye opening, and a colocalization with PKC is established in adult retinas. Processes from these cell populations terminate in several sublaminas in the developing IPL, but cGMP- and PKC-labeled terminals appear to be confined to ON-lamina as the retina matures. CONCLUSIONS: The development of cGMP- and PKC-labeled fibers within the IPL appears to be in concert with events of neural differentiation and synaptogenesis. These results suggest that the nitric oxide/cGMP signaling pathway and PKC may participate in activity-dependent processes during development that establish the mature circuitry of synaptic contacts within the IPL. The presence of cGMP in mature rod bipolar cells suggests a role in the signal transduction of rod bipolar cell-AII amacrine cell pathway.     

Distribution of GABA-immunoreactive amacrine cell synapses in the inner plexiform layer of macaque monkey retina

The distribution patterns of GABA immunoreactive (+) and immunonegative (-) amacrine cell synapses and profiles in the inner plexiform layer (IPL) were analyzed in three macaque monkey retinas using postembedding electron-microscopic (EM) immunogold cytochemistry. Synapses and profiles were counted at 5% intervals throughout the IPL depth in three EM montages (total area = 6509 microns 2), with 0% depth at the inner nuclear layer/IPL border. Nearly 70% of all amacrine synapses were GABA+, and they contacted all major classes of neurons that arborize in the IPL: bipolars (45%), ganglion cells (25%), and GABA+ (20%) and GABA- (10%) amacrines. A major relationship was seen between GABA+ amacrine processes and bipolar terminals: 76% of all amacrine-to-bipolar synapses were GABA+, and 82% of bipolar output dyads contained at least one GABA+ amacrine. GABA+ amacrine profiles (N = 2455) were concentrated in three wide bands at IPL depths of 0-25%, 40-60%, and 75-100%, corresponding to the dense bands seen with light-microscopic immunocytochemistry. In contrast, GABA+ amacrine synapses (N = 1081) were distributed evenly throughout the IPL depth, rather than being confined to the three dense bands. GABA- amacrine synapses (N = 516) were concentrated at 40% and 60% depths. Each category of amacrine output synapses had a characteristic pattern of stratification in the IPL. GABA+ amacrine-to-bipolar synapses occurred throughout the IPL but were most frequent at 20% and 80% IPL depths, where the dendrites of midget cone bipolars arborize (Polyak, 1941). In contrast, GABA+ amacrine-to-ganglion cell synapses were concentrated at 30% and 70% IPL depths, near the dendritic arborizations of parasol ganglion cells (Watanabe & Rodieck, 1989). GABA+ synapses onto bipolars and amacrines were also concentrated at the level of rod bipolar terminals. GABA+ amacrines must play significant but different roles in ON and OFF midget and parasol pathways as well as the rod pathway.     

Localization of neuroactive substances in the vertebrate retina: evidence for lamination in the inner plexiform layer

The inner plexiform layer (IPL) of the vertebrate retina is a multilaminated region containing bipolar, amacrine, interplexiform and ganglion cell processes. Immunohistochemical methods clearly demonstrate the existence of various neuroactive substances in distinct morphological types of amacrine cells. Sufficient detail in the staining consistently delineates processes containing these neuroactive substances in multiple laminae of the IPL. Thus, the histochemical approach provides a powerful tool for the analysis of the laminar organization of the IPL and can also be used to further clarify the functional role of various transmitter and peptide substances in the retina.     

Nitric oxide-producing cells project from retinal grafts to the inner plexiform layer of the host retina.

PURPOSE: Amacrine cells expressing nitric oxide synthase (NOS) are seen in normal retinas and retinal grafts to extend long processes, which can be followed for long distances. Taking advantage of the morphologic features of these cells, the present study examined whether graft-host connections involve cells capable of producing nitric oxide, a recognized retinal neuromodulatory compound. METHODS: Embryonic day 15 rabbit retinas were transplanted to the subretinal space of adult rabbits. The localization of the neuronal form of NOS was assessed by immunocytochemistry in grafts that had reached the equivalent ages of postnatal days 5, 12, 20, 45, 90, and 102. RESULTS: NOS-containing cells and processes were seen in all the transplants. Processes were found to project mainly toward areas within the graft. Yet, at all survival times examined, single immunolabeled fibers could be seen to cross the graft- host border. In fortuitous cases, it was possible to establish that the bridging fiber originated in the graft. Further, bridging fibers were seen to reach the NOS-immunolabeled host inner plexiform layer. CONCLUSIONS: Graft NOS-containing cells are not only capable of projecting into the host but also of reaching the appropriate target for NOS-containing fibers within the host retina. This indicates that at least some graft-host connections are established by graft cells that retain their ability to synthesize a modulatory compound and which potentially could contact their partner cells in the host retina.     

Cones regenerate from retinal stem cells sequestered in the inner nuclear layer of adult goldfish retina

PURPOSE: To determine whether retinal progenitor cells in the inner nuclear layer give rise to regenerated cones after laser ablation of photoreceptors in adult goldfish retina. METHODS: Using a technique developed previously in this laboratory, photoreceptors in the retina of adult goldfish were ablated with an argon laser. The mitotic marker, bromodeoxyuridine, was used to label proliferating and regenerated cells, which were identified with cell-specific markers. RESULTS: Cells proliferating locally within lesion included microglia, Müller glia, and retinal progenitors in the inner nuclear layer (INL). The nuclei of both Müller glia and associated retinal progenitors migrated from the inner to the outer nuclear layer. The proliferating retinal progenitors, which express Notch-3 and N-cadherin, regenerated cone photoreceptors and then rod photoreceptors. CONCLUSIONS: Previous work has demonstrated that photoreceptors in the goldfish retina regenerate selectively after laser ablation, but the source of regenerated cones has not been identified. The results reported here provide support for the existence of retinal stem cells within the adult fish retina that are capable of regenerating cone photoreceptors. The data also support the involvement of Müller glia in the production of regenerated cones.     

Lateral actions at the inner plexiform layer of the carp retina: effects of turning windmill pattern stimulus

Lateral action from amacrine to ganglion cells was studied in the isolated carp retina by using a truncated windmill pattern (TWP). About 25% of ganglion cells of both "on" and "off" center types were suppressed or enhanced in firing activity in response to TWP turning. The suppressed cells were more sensitive to slow turning velocities of TWP than the enhanced cells. In the "on-off" type amacrine cells, a steady depolarizing or hyperpolarizing component (less than several mV) was maintained by stationary TWP, while the cells were exclusively depolarized by turning TWP at a wide range of velocities. These results suggest that individual responses of ganglion cells induced by both stationary and turning TWP are depending on a balance between two factors: the polarizing direction of steady components of the "on-off" amacrine cells and the polarizing direction of ganglion cells synaptically produced by the amacrine cells.     

Morphology of bipolar cells and their participation in spatial organization of the inner plexiform layer of jack mackerel retina

Morphology of bipolar cells in the jack mackerel retina [Trachurus mediterraneus ponticus (Aleev)] was investigated by the Golgi method. Eight types of bipolar cells are described. It is the first time that cells with an unbranched main dendrite are found in fish retina. It is shown that the inner plexiform layer of the jack mackerel retina contains regular lattices, located at 5 levels and conserted in a characteristic way with the cone mosaic. These lattices are formed by swellings of bipolar cell axons. It is shown that only bipolar cells with small dendritic aborizations (less than or equal to 14 micron dia) take part in this organization.     

Alpha2 adrenergic receptor-mediated modulation of cytosolic Ca++ signals at the inner plexiform layer of the rat retina

PURPOSE: Compelling evidence suggests that alpha2 agonists, such as brimonidine, protect retinal ganglion cells (RGCs) from injury in a wide range of animal models. However, the mechanism of action for this protection and the physiological role of the alpha2 adrenergic system in the retina is not well understood. A major goal of this work was to explore the role of the alpha2 adrenergic system in the modulation of cytosolic Ca(2+) signaling at retinal synaptic layers, particularly the inner plexiform layer (IPL), where communication between RGCs and their presynaptic cells takes place. METHODS: Functional Ca(2+) imaging at the inner plexiform layer (IPL) and outer plexiform layer (OPL) of living rat retinal slices was conducted with a high-speed confocal system. The relative changes of cytosolic free Ca(2+) were monitored with the fluorescent Ca(2+) dye fluo-4. The Ca(2+) signal was elicited by membrane depolarization produced by a high K(+) (40 mM) Ringer solution that was delivered rapidly and briefly to the test regions of the retinal slice by a custom-made multichannel local perfusion system. RESULTS: A brief application (8 seconds) of high K(+) Ringer elicited a robust cytosolic Ca(2+) increase at the IPL and OPL. In both cases, this Ca(2+) signal was eliminated by nimodipine, a selective L-type voltage-gated Ca(2+)-channel blocker, or when the extracellular Ca(2+) in the Ringer was replaced with equal molar EGTA. At IPL, the Ca(2+) signal was also suppressed in a dose-dependent manner by brimonidine and other alpha2 receptor agonists, such as medetomidine. The suppressive action of brimonidine and medetomidine was completely blocked by classic alpha2 receptor antagonists, such as yohimbine, rauwolscine, and atipamezole. Interestingly, the alpha2 receptor agonists had no effect on the high K(+) Ringer-elicited cytosolic Ca(2+) signal at OPL. Blocking the N-methyl-d-aspartate (NMDA) type of ionotropic glutamate receptor with D-AP5 attenuated this high K(+)-elicited Ca(2+) signal by approximately 20% at IPL. D-AP5 had no effect on the Ca(2+) signal at OPL. CONCLUSIONS: These findings provide the first direct evidence of alpha2 receptor-mediated modulation of L-type Ca(2+) channel activity in the CNS (the retina is part of the CNS). This alpha2 modulation appears to occur at the IPL but not at the OPL of the retina. These findings suggest that a physiological function of the retinal alpha2 system is the regulation of synaptic transmission at IPL and that brimonidine and other alpha2 agonists may protect RGCs under disease conditions by preventing abnormal elevation of cytosolic free Ca(2+) either in RGCs, in their presynaptic cells, or in both.     

Parvalbumin-immunoreactive neurons in the inner nuclear layer of zebrafish retina

The purpose of this investigation is to characterize parvalbumin-immunoreactive (IR) neurons in the inner nuclear layer (INL) of zebrafish retina through immunocytochemistry, quantitative analysis, and confocal microscopy. In the INL, parvalbumin-IR neurons were located in the inner marginal portion of the INL. On the basis of dendritic stratification in the inner plexiform layer (IPL), at least two types of amacrine cells were IR for parvalbumin. The first one formed distinctive laminar tiers within s4 (PVs4) of the IPL, and the second within s5 (PVs5). The average number of PVs4 cells was 8263 cells per retina (n = 3), and the mean density was 1671 cells/mm². The average number of PVs5 cells was 1037 cells per retina (n = 3), and the mean density was 210 cells/mm². Quantitatively, 88.9% of anti-parvalbumin labeled neurons were PVs4 cells and 11.1% were PVs5 cells. Their density was highest in the midcentral region of the ventrotemporal retina and lowest in the periphery of the dorsonasal retina. The average regularity index of the PVs4 cell mosaic was 4.09, while the average regularity index of the PVs5 cell mosaic was 3.46. No parvalbumin-IR cells expressed calretinin or disabled-1, markers for AII amacrine cells, in several animals. These results indicate that parvalbumin-IR neurons in zebrafish are limited to specific subpopulations of amacrine cells and the expressional pattern of parvalbumin may not correspond to AII amacrine cells in several other animals. Their distribution suggests that parvalbumin-IR neurons are mainly involved in ON pathway information flow.     

Electrical feedback mechanism in the processing of signals in the outer plexiform layer of the retina

In any chemical synapse there is an electrical feedback initiated by the electrical current generated by the postsynaptic neurone. We argue that this feedback can be rather effective in the case of invaginating synapses in the retina. A model of a dyad synapse (cone-horizontal cell-bipolar cell) is developed, in which the transfer function between the cone and bipolar cell is modulated by the synaptic current of the horizontal cell. This modulation originates by the change of the potential drop along the intercellular gap between the cone and the horizontal cell. The model takes into account the electrical coupling between the horizontal cells as well as the nonlinearity of their nonsynaptic (somatic) membrane. The model reproduces qualitatively the steady-state responses of an hyperpolarizing bipolar cell to the light spot and an annulus. It gives also the adaptational (with a light background) shift of the cone-bipolar cell transfer function. The model can be applied to depolarizing bipolar cells and to C-type horizontal cells.     

Quantitative aspects of synaptic ribbon formation in the outer plexiform layer of the developing cat retina

The development of synaptic ribbons in rod and cone photoreceptor terminals of the cat retina was studied using quantitative electron microscopy. At the region of the area centralis, synaptic ribbon profiles are initially recognized at PCD (postconception day) 59. Synaptic ribbon density increases rapidly, reaching a peak of 0.55 ribbons/micron 3 at PCD 68 (postnatal day 3) and maintains approximately that value for an additional 8 d. Following PCD 76, ribbon density begins to decrease, to 0.37 ribbons/microns 3 at PCD 82 and 0.25 ribbons/microns 3 at PCD 102. Although ribbon density drops by approximately 50% during this 39-d period, the outer plexiform layer (OPL) volume at the area centralis increases by about 20%. Ribbon density continues to decrease gradually over a protracted period to reach a final adult value of 0.11-0.14 ribbons/microns 3. During the period of high ribbon density, rod spherules with two, or even three ribbon profiles, were routinely observed. In contrast, in the adult, spherules with more than one ribbon profile are only rarely encountered. During development, the length of synaptic ribbon profiles increases from a mean of 0.22 microns at PCD 62 to the 0.47 microns mean length found in the adult.