The morphology and topographic distribution of AII amacrine cells in the cat retina

When cat retina is incubated in vitro with the fluorescent dye, 4',6-diamidino-2-phenyl-indole (DAPI), a uniform population of neurons is brightly labelled at the inner border of the inner nuclear layer. The dendritic morphology of the DAPI-labelled cells was defined by iontophoretic injection of Lucifer yellow under direct microscopic control: all the filled cells had the narrow-field bistratified morphology that is distinctive of the AII amacrine cells previously described from Golgi-stained retinae. Although the AII amacrines are principal interneurons in the rod-signal pathway, their density distribution does not follow the topography of the rod receptors, but peaks in the central area like the cone receptors and the ganglion cells. There are some 512 000 AII amacrines in the cat retina and their density ranges from 500 cells per square millimetre at the superior margin to 5300 cells per square millimetre in the centre (retinal area is 450 mm2). The isodensity contours are kite-shaped, particularly at intermediate densities, with a horizontal elongation towards nasal retina. The cell body size and the dendritic dimensions of AII amacrines increase with decreasing cell density. The lobular dendrites in sublamina a of the inner plexiform layer span a restricted field of 16-45 microns diameter, while the arboreal dendrites in sublamina b form a varicose tree of 18-95 microns diameter. The dendritic field coverage of the lobular appendages is close to 1.0 (+/- 0.2) at all eccentricities whereas the coverage of the arboreal dendrites doubles within the first 1.5 mm and then remains constant at 3.8 (+/- 0.7) throughout the periphery.     

Morphology of calretinin and tyrosine hydroxylase-immunoreactive neurons in the pig retina

The morphology of calretinin- and tyrosine hydroxylase-immunoreactive (IR) neurons in adult pig retina was studied. These neurons were identified using antibody immunocytochemistry. Calretinin immunoreactivity was found in numerous cell bodies in the ganglion cell layer. Large ganglion cells, however, were not labeled. In the inner nuclear layer, the regular distribution of calretinin-IR neurons, the inner marginal location of their cell bodies in the inner nuclear layer, and the distinctive bilaminar morphologies of their dendritic arbors in the inner plexiform layer suggested that these calretinin-IR cells were AII amacrine cells. Calretinin immunoreactivity was observed in both A-and B-type horizontal cells. Neurons in the photoreceptor cell layer were not labeled by this antibody. The great majority of tyrosine hydroxylase-IR neurons were located at the innermost border of the inner nuclear layer (conventional amacrines). The processes were monostratified and ran laterally within layer 1 of the inner plexiform layer. Some of the tyrosine hydroxylase-IR neurons were located in the ganglion cell layer (displaced amacrines). The processes of displaced tyrosine hydroxylase-IR amacrine cells were also located within layer 1 of the inner plexiform layer. Some processes of a few neurons were located in the outer plexiform layer. A very low density of neurons had additional bands of tyrosine hydroxylase-IR processes in the middle and deep layers of the inner plexiform layer. The processes of tyrosine hydroxylase-IR neurons extended radially over a wide area and formed large, moderately branched dendritic fields. These processes occasionally had varicosities and formed "dendritic rings". These results indicate that calretinin- and tyrosine hydroxylase-IR neurons represent specific neuronal cell types in the pig retina.     

Neurofibrillar long-range amacrine cells in mammalian retinae

A distinct population of wide-field, unistratified amacrine cells are shown to be selectively stained by using neurofibrillar methods in rabbit and cat retinae. Their cell bodies may be located in the inner nuclear, inner plexiform or ganglion cell layers and they branch predominantly in stratum 2 of the inner plexiform layer. Characteristically, each cell has two or more long-range distal processes which extend for 2-3 mm beyond a more symmetrical, proximal dendritic field of 0.6-0.8 mm diameter. Although the neurofibrillar long-range amacrines account for less than 1 amacrine in 500, they achieve effective coverage of the retina by both the proximal and distal dendrites.     

Light- and electron-microscopic study of substance P-immunoreactive neurons in the guinea pig retina

Substance P (SP) immunoreactivity in the guinea pig retina was studied by light and electron microscopy. The morphology and distribution of SP-immunoreactive neurons was defined by light microscopy. The SP-immunoreactive neurons formed one population of amacrine cells whose cell bodies were located in the proximal row of the inner nuclear layer. A single dendrite emerged from each soma and descended through the inner plexiform layer toward the ganglion cell layer. SP-immunoreactive processes ramified mainly in strata 4 and 5 of the inner plexiform layer. SP-immunoreactive amacrine cells were present at a higher density in the central region around the optic nerve head and at a lower density in the peripheral region of the retina. The synaptic connectivity of SP-immunoreactive amacrine cells was identified by electron microscopy. SP-labeled amacrine cell processes received synaptic inputs from other amacrine cell processes in all strata of the inner plexiform layer and from bipolar cell axon terminals in sublamina b of the same layer. The most frequent postsynaptic targets of SP-immunoreactive amacrine cells were the somata of ganglion cells and their dendrites in sublamina b of the inner plexiform layer. Amacrine cell processes were also postsynaptic to SP-immunoreactive neurons in this sublamina. No synaptic outputs onto the bipolar cells were observed.     

Amacrine cells, displaced amacrine cells and interplexiform cells in the retina of the rat

The amacrine cells in the retina of the rat are described in Golgi-stained whole-mounted retinae. Nine morphologically distinct types of cell were found: one type of diffuse cell, five types of unistratified cell, two types of bistratified cell, and one type of stratified diffuse cell. Measurements show that the largest unistratified cells have a dendritic field 2 mm across. One type of interplexiform cell is also described. Wide-field diffuse amacrine cells and unistratified amacrine cells were found with their somata located in either the inner nuclear layer or the ganglion cell layer. It is clear that there may be an amacrine cell system in the ganglion cell layer of the rat retina.     

Seizure-Related Gene 6 (Sez-6) in Amacrine Cells of the Rodent Retina and the Consequence of Gene Deletion

Background

Seizure-related gene 6 (Sez-6) is expressed in neurons of the mouse brain, retina and spinal cord. In the cortex, Sez-6 plays a role in specifying dendritic branching patterns and excitatory synapse numbers during development.

Methodology/Principal Findings

The distribution pattern of Sez-6 in the retina was studied using a polyclonal antibody that detects the multiple isoforms of Sez-6. Prominent immunostaining was detected in GABAergic, but not in AII glycinergic, amacrine cell subpopulations of the rat and mouse retina. Amacrine cell somata displayed a distinct staining pattern with the Sez-6 antibody: a discrete, often roughly triangular-shaped bright spot positioned between the nucleus and the apical dendrite superimposed over weaker general cytoplasmic staining. Displaced amacrines in the ganglion cell layer were also positive for Sez-6 and weaker staining was occasionally observed in neurons with the morphology of alpha ganglion cells. Two distinct Sez-6 positive strata were present in the inner plexiform layer in addition to generalized punctate staining. Certain inner nuclear layer cells, including bipolar cells, stained more weakly and diffusely than amacrine cells, although some bipolar cells exhibited a perinuclear “bright spot” similar to amacrine cells. In order to assess the role of Sez-6 in the retina, we analyzed the morphology of the Sez-6 knockout mouse retina with immunohistochemical markers and compared ganglion cell dendritic arbor patterning in Sez-6 null retinae with controls. The functional importance of Sez-6 was assessed by dark-adapted paired-flash electroretinography (ERG).

Conclusions

In summary, we have reported the detailed expression pattern of a novel retinal marker with broad cell specificity, useful for retinal characterization in rodent experimental models. Retinal morphology, ganglion cell dendritic branching and ERG waveforms appeared normal in the Sez-6 knockout mouse suggesting that, in spite of widespread expression of Sez-6, retinal function in the absence of Sez-6 is not affected.     

The morphology and topographic distribution of substance-P-like immunoreactive amacrine cells in the cat retina

In cat retinal wholemounts, substance-P-like immunoreactivity (SP-IR) was localized in a distinct population of amacrines whose cell bodies were normally placed in the ganglion cell layer. Although displaced amacrines accounted for 80-95% of the SP-IR amacrines in peripheral retina, this proportion decreased considerably within the area centralis, accounting for 50-80% of the labelled cells at maximum density. The SP-IR cells in both the inner nuclear and ganglion cell layers gave rise to well-defined varicose dendrites of uniform appearance that stratified around 60% depth (S3/S4) of the inner plexiform layer. In addition, sparse fine dendrites in stratum 1 (S1) could sometimes be traced to inner nuclear cells and occasionally to displaced amacrines. The combined SP-IR cell density ranged from less than 50 cells mm-2 in the far periphery to more than 500 cells mm-2 in the area centralis; the maximum density showed little individual variation despite wide differences in the proportion of displaced cells. The 39,000 SP-IR amacrines in a mapped retina had a triangular topographic distribution, with intermediate isodensity lines extending vertically in superior retina and horizontally along both arms of the visual streak. Colocalization experiments established that all SP-IR cells in cat retina showed GABA-like immunoreactivity, and that the SP-IR amacrines were quite distinct from the cholinergic amacrines identified by choline acetyltransferase immunohistochemistry.     

SOMATOSTATIN-LIKE IMMUNOREACTIVE CELLS IN THE GROUND SQUIRREL RETINA

Immunocytochemical techniques were employed to locate somatostatin (SS)-containing cells in the retina of the 13-lined ground squirrel (Spermophilus tridecemlineatus). In normal retinas immunostain was limited to neuronal processes, yet distinctly labeled somata were detected in retinas of animals pretreated with colchicine. Labeled cell bodies were located in the outermost and innermost portions of the inner nuclear layer (INL) and in the ganglion cell layer (GCL). The largest population of SS-like immunoreactive neurons was found in the innermost INL. These cells were identified as small and medium sized amacrine cells whose soma diameters ranged from 4 to 14μm. A smaller population of immunoreactive cells was observed in the outermost region of the INL. These cells, presumptive horizontal cells, were found mainly in peripheral regions of the retina. Immunoreactive cells in the GCL were of two types: displaced amacrines, and retinal ganglion cells. SS-positive axons in the optic fiber layer suggest that some of the immunoreactive GCL neurons were ganglion cells, and it is our opinion that these cells belong to a class of associational ganglion cells previously identified in other species.     

Morphology and synaptic connectivity of nitric oxide synthase-immunoreactive neurons in the guinea pig retina

Immunocytochemical methods with an antiserum against neuronal nitric oxide synthase (NOS) were applied to identify the morphology and synaptic connectivity of NOS-like immunoreactive neurons in the guinea pig retina. In the present study, two types of amacrine cells were labeled with anti-NOS antisera. Type 1 cells had large somata located in the inner nuclear layer (INL) with long, sparsely branched processes ramifying mainly in stratum 3 of the inner plexiform layer (IPL). The somata of type 2 cells (smaller diameters) were located in the INL. Some displaced amacrine cells in the ganglion cell layer were labeled. The soma size of the displaced amacrine cells was similar to that of the type 2 amacrine cells. However, processes originating from type 2 amacrine cells and displaced amacrine cells stratified mainly in strata 1 and 5, respectively. Some cone bipolar cells were weakly NOS-immunoreactive. The synaptic connectivity of NOS-like immunoreactive amacrine cells was identified in the IPL by electron microscopy. NOS-labeled amacrine cell processes received synaptic input from other amacrine cell processes and bipolar cell axon terminals in all strata of the IPL. The most frequent postsynaptic targets of NOS-immunoreactive amacrine cells were other amacrine cell processes. Cone bipolar cells were postsynaptic to NOS-labeled amacrine cells in all strata of the IPL. Labeled amacrine cells synapsing onto ganglion cells were found only in sublamina b. A few synaptic contacts were observed between labeled cell processes. In the outer plexiform layer, dendrites of labeled bipolar cells made basal contact with cone pedicles or formed a synaptic triad opposed to a synaptic ribbon of cone pedicles.     

Neuropeptide Y (NPY) immunoreactive neurons in the retina of different species

Summary Neurons displaying Neuropeptide Y (NPY) immunoreactivity were found among amacrine cells in the retina of baboon, pig, cat, pigeon, chicken, frog, trout, carp and goldfish. The immunoreactive cell bodies were located in the middle and the innermost cell rows of the inner nuclear layer with processes forming one, two or three more or less well-defined sublayers in the inner plexiform layer. The location and the density of the sublayers varied with the species investigated. In the frog retina, bipolar-like cell bodies were found in the middle of the inner nuclear layer as well as sparsely occurring ovoid cell bodies in the ganglion cell layer. Like the amacrine cells, these cells emitted processes ramifying in three sublayers in the inner plexiform layer.     

Neuropeptide Y (NPY) immunoreactive neurons in the retina of different species

Neurons displaying Neuropeptide Y (NPY) immunoreactivity were found among amacrine cells in the retina of baboon, pig, cat, pigeon, chicken, frog, trout, carp and goldfish. The immunoreactive cell bodies were located in the middle and the innermost cell rows of the inner nuclear layer with processes forming one, two or three more or less well-defined sublayers in the inner plexiform layer. The location and the density of the sublayers varied with the species investigated. In the frog retina, bipolar-like cell bodies were found in the middle of the inner nuclear layer as well as sparsely occurring ovoid cell bodies in the ganglion cell layer. Like the amacrine cells, these cells emitted processes ramifying in three sublayers in the inner plexiform layer.     

Acetylcholine-synthesizing amacrine cells: identification and selective staining by using radioautography and fluorescent markers

The fluorescent DNA stain 4,6,diamidino-2-phenylindole (DAPI) was applied to the cut axons of the rabbit optic tract, from which it was retrogradely transported to the retinal ganglion cell bodies. The labelled retinas were isolated from the eye and maintained in vitro in the presence of [3H]choline. They were then quick-frozen, freeze-dried, vacuum-embedded, and radioautographed on dry emulsion for identification of the acetylcholine-synthesizing cells. Inspection of the radioautographs by fluorescence microscopy showed the two labels not to co-exist: the cells that contained the transported fluorescence did not contain radioactive acetylcholine. In other animals the optic nerve was sectioned, causing retrograde degeneration of a large fraction of the ganglion cells. A population of small, round neurons in the ganglion cell layer was spared. These retinas synthesized [3H]acetylcholine at the same rate as control tissues; and radioautography showed an identical distribution of the acetylcholine-synthesizing cells. We conclude that the acetylcholine-synthesizing neurons of the ganglion cell layer are displaced amacrine cells. When DAPI was injected intraocularly instead of being applied to the optic tract, a regular mosaic of neurons in the ganglion cell layer was selectively stained, and two bands of fluorescence were observed in the inner plexiform layer, at the level where two bands of radioactive acetylcholine were observed in radioautographs. Quantitative analysis showed that the DAPI-stained cells were the same size as those that survive optic nerve section. Like the acetylcholine-synthesizing cells, they appear to be displaced amacrines; when wheatgerm agglutinin labelled by Evans blue was applied to the optic tract and DAPI was injected intraocularly, the red fluorescence of Evans blue and the blue fluorescence of DAPI accumulated in different cells. When DAPI was injected intraocularly and radioautography for acetylcholine was carried out, the cells brightly labelled by DAPI were found to have synthesized acetylcholine. We conclude that topically applied DAPI selectively labels the acetylcholine-synthesizing neurons of the ganglion cell layer. The distribution of the acetylcholine-synthesizing cells was established by counting the DAPI-labelled cells in whole-mounts.(ABSTRACT TRUNCATED AT 400 WORDS)     

Colocalization of GLUT3 and choline acetyltransferase immunoreactivity in the rat retina

Toward elucidating the functional aspects ofGLUT3, a primary neuronal glucose transporter isoform in the vertebrate central nervous system, this study examined its expression in cholinergic amacrine cells made identifiable by the presence of acetylcholine-synthesizing enzyme, choline acetyltransferase (ChAT), in the rat retina. Double-immunofluorescence staining of adult rat retinal tissue with anti-GLUT3 and anti-ChAT antibodies revealed characteristic stratified GLUT3 immunoreactivity (GLUT3-IR) in the inner plexiform layer (IPL) that was identical to the arborization pattern of ChAT-positive neuronal processes there. In addition, approximately 30-50% of intensely GLUT3-immunoreactive cell bodies in the inner nuclear layer and ganglion cell layer showed ChAT-IR, while the majority of ChAT-positive cell bodies were also intensely GLUT3 immunoreactive. Analysis at the cellular level using retinal cells in culture revealed similar findings. These results collectively indicate that cholinergic amacrine cells constitute the major component of GLUT3-expressing cells in the rat retina. It is expected that the link demonstrated here between GLUT3 expression and cholinergic amacrine cell population will provide clues for further analyzing GLUT3 function in the retina.     

Immunocytochemical and autoradiographic localization of GABA system in the vertebrate retina

Summary The localization of -aminobutyric acid (GABA) neurons in the goldfish and the rabbit retina has been studied by immunocytochemical localization of the GABA-synthesizing enzyme L-glutamate decarboxylase (GAD, L-glutamate 1-carboxy-lase, EC 4.1.1.15) and by [³H] GABA uptake autoradiography. In the goldfish retina, GAD is localized in some horizontal cells (H1 type), a few amacrine cells and sublamina b of the inner plexiform layer. Results from immunocytochemical studies of GAD-containing neurons and autoradiographic studies of GABA uptake reveals a marked similarity in the labeling pattern suggesting that in goldfish retina, the neurons which possess a high-affinity system for GABA uptake also contain significant levels of GAD. In the rabbit retina, when Triton X-100 was included in immunocytochemical incubations with a modified protein A-peroxidase-antiperoxidase method, reaction product was found in four broad, evenly spaced laminae within the inner plexiform layer. In the absence of the detergent, these laminae were seen to be composed of small, punctate deposits. When colchicine was injected intravitreally before glutamate decarboxylase staining, cell bodies with the characteristic shape and location of amacrine cells were found to be immunochemically labeled. Electron microscopic examination showed that these processes were presynaptic to ganglion cell dendrites (infrequently), amacrine cell telodendrons, and bipolar cell terminals. Often, bipolar cell terminals were found which were densely innervated by several GAD-positive processes. No definite synapses were observed in which a GAD-positive process represented the postsynaptic element. In autoradiographic studies by intravitreal injection of [³H] GABA a diffuse labeling of the inner plexiform layer and a dense labeling of certain amacrine cell bodies in the inner nuclear layer was observed. Both immunocytochemical and autoradiographic results support the notion that certain, if not all, amacrine cells use GABA as their neurotransmitter.     

Localization of CD15 immunoreactivity in the rat retina

Using immunocytochemistry, we have investigated the localization of CD15 in the rat retina. In the present study, two types of amacrine cell in the inner nuclear layer (INL) and some cells in the ganglion cell layer were labeled with anti-CD15 antisera. Type 1 amacrine cells have large somata located in the INL, with long and branched processes ramifying mainly in stratum 3 of the inner plexiform layer (IPL). Type 2 cells have a smaller soma and processes branching in stratum 1 of the IPL. A third population showing CD15 immunoreactivity was a class of displaced amacrine cells in the ganglion cell layer. The densities of type 1 and type 2 amacrine cells were 166/mm(2) and 190/mm(2) in the central retina, respectively. The density of displaced amacrine cells was 195/mm(2). Colocalization experiments demonstrated that these CD15-immunoreactive cells exhibit gamma-aminobutyric acid and neuronal nitric oxide synthase (nNOS) immunoreactivities. Thus, the same cells of the rat retina are labeled by anti-CD15 and anti-nNOS antisera and these cells constitute a subpopulation of GABAergic amacrine cells.     

Localization of glutamate in the human retina during early prenatal development

In this study we have localized glutamate (GLU) in fetal (14–25 weeks gestation, Wg) human retinas by immunohistochemistry. At 14 Wg, GLU-immunoreactivity (IR) was localized only in the central part of retina, showing a prominently labelled nerve fiber layero A few ganglion cells and displaced amacrine cells were very weakly labelled. At 17 Wg, GLU was localized conspicuously in many ganglion cells, displaced amacrine cells, some amacrine cells and the prospective photoreceptor cell bodies in the neuroepithelial layero With progressive development at 20 and 25 Wg, the IR for GLU was found additionally in the Müller cell endfeet, some bipolar cells as well as in the horizontal cells that were aligned in a row along the outer border of the inner nuclear layer of the central retinao The photoreceptor cell bodies in the outer nuclear layer were also prominently immunopositive for GLU. The developmental distribution of GLU in the human retina tends to indicate that it plays an important role in the differentiation and maturation of retinal neurons.     

Laminar Distributions of Choline Acetyltransferase and Acetylcholinesterase Activities in the Inner Plexiform Layer of Rat Retina

Choline acetyltransferase and acetylcholinesterase activities were measured in samples taken at 7-micron increments through the inner plexiform layer of rat retina. These enzyme activities were not uniformly distributed through the depth of the inner plexiform layer. Peaks of choline acetyltransferase activity occurred at about one-third and peaks of acetylcholinesterase activity at about one-fifth of the depth into the inner plexiform layer from either side. The positions of the two peaks of choline acetyltransferase activity most likely correspond to the locations of processes from cholinergic amacrine somata in the inner nuclear layer, which spread in sublamina a, and processes from cholinergic amacrine somata "displaced" in the ganglion cell layer which spread in sublamina b of the inner plexiform layer. The peaks of acetylcholinesterase activity may in addition correspond to the processes of cholinoceptive amacrine and ganglion cells. The magnitudes of choline acetyltransferase and acetylcholinesterase activities are as high as found anywhere in rat brain, emphasizing the important role of cholinergic mechanisms in visual processing through the rat inner plexiform layer.