Neuropeptide Y-like immunoreactive amacrine cells in the retina ofBufo marinus

Neuropeptide Y-like immunoreactive (NPY-LI) amacrine cells of theBufo marinus retina were morphologically characterized, and their retinal distribution was established using immunohistochemistry on retinal wholemount preparations and sectioned material. The somas of NPY-LI amacrine cells were situated in the innermost part of the inner nuclear layer and their dendrites branched primarily in the scleral sublamina of the inner plexiform layer. A subgroup of the NPY-LI cells had dendrites in both the scleral and vitreal sublamina. All immunoreactive cells had large dendritic fields (average 0.5 mm²) that resulted in a high dendritic overlap across the retina. NPY-LI amacrine cells were evenly distributed across the retina, with an average density of 30 cells/mm², although higher densities were observed at regions adjacent to the ciliary margin. The dendritic field size of the NPY-LI cells, together with the previously characterized substance P-like immunoreactive (SP-LI) amacrine cells, indicates that they belong to the class of wide-field amacrine cells. However, unlike the SP-LI neurons whose dendrites branch in the vitreal sublamina of the inner plexiform layer, the dendrites of the majority of the NPY-LI neurons branch in the scleral sublamina.     

Neuropeptide Y- and substance P-like immunoreactive amacrine cells in the retina of the developing Xenopus laevis.

The development of neuropeptide Y-like (NPY-LI) and substance P-like (SP-LI) immunoreactive neurons was studied in retinas of Xenopus laevis from young tadpole through to adult animals. In adult retina these neuropeptides are present in wide-field amacrine cells located in the inner nuclear layer and the ganglion cell layer of the retina. Retinal wholemount preparations and sectioned material showed that immunoreactive cells appeared during early larval life and NPY-LI occurred earlier than SP-LI cells. The primary dendritic branching of NPY-LI neurons appeared from early larval life whilst SP-LI was evident in dendrites from mid-larval stages. In postmetamorphic animals the numbers of immunoreactive cells increased in proportion to retinal area growth with a relatively constant cell density of about 35 cells/mm2 for SP-LI and 45 cells/mm2 for NPY-LI. The maturation of dendritic morphology of both NPY- and SP-LI amacrine cells appeared later in larval development than the appearance of immunoreactivity in cell somas. However, the sequence of expression of NPY- or SP-LI and their dendritic maturation was different for the two classes of amacrine cells. It is suggested that the maturation of dendritic fields of amacrine cells is complete just prior to metamorphosis, consistent with the postmetamorphic onset of electrophysiological features of ganglion cells attributed to amacrine cells.     

Substance P-like immunoreactive amacrine cells in the cat retina

Substance P-like immunoreactivity was localized by immunocytochemical techniques to two subpopulations of amacrine cells in the cat retina. One cell was a unistratified amacrine with processes ramifying within stratum 4 of the inner plexiform layer. The other cell type was a bistratified cell with processes in both stratum 1 (s1) and stratum 4 (s4). Both cell types were seen with their somas displaced to the ganglion cell layer as well as in the conventional amacrine location in the inner nuclear layer. Substance P cells were present in the greatest density within the area centralis and decreased in number toward the periphery. The ratio of amacrine to displaced amacrine cells also decreased peripherally. However, the coverage by immunoreactive fibers in s4 remained three times that seen in s1. Computer-assisted analysis confirmed the location of substance P-containing processes at 5-15% (s1) and 50-70% (s4) depth levels in the inner plexiform layer. A comparison of substance P-like immunoreactivity in light- and dark-adapted cat retinas showed no apparent differences in the distribution of immunoreactivity due to lighting conditions.     

Connectivity of glycine immunoreactive amacrine cells in the cat retina

The synaptic relationships of glycine immunoreactive amacrine cells in the cat retina were studied through the use of postembedding immunogold techniques. Glycine immunoreactive amacrine cells were found to synapse extensively with other amacrines and ganglion cells, particularly in strata 1-3 of the inner plexiform layer. This contrasts with GABA immunoreactive amacrine cells which provide major input to bipolar cells in strata 3-5. Glycine containing amacrine terminals exhibited diversity with respect to the morphology of their synaptic vesicles. The three types of terminals which could be distinguished were characterized by small pleomorphic (32-35 nm), medium-sized flattened (38-45 nm), or larger rounded (48-55 nm) vesicles. Comparison of retinal sections processed for glycine immunoreactivity with adjacent sections stained for GABA reactivity revealed a colocalization of glycine and GABA in 3% of the cells in the amacrine layer and approximately 40% of the cells in the ganglion cell layer. The amacrine terminals in which glycine and GABA were colocalized typically contained the small pleomorphic type of vesicles.     

Neuropeptide Y-like immunoreactive neurons in the human olfactory bulb

Neuropeptide Y-like (NPY) immunoreactivity was localized in the adult human olfactory bulb by the unlabeled antibody enzyme (peroxidase anti-peroxidase; PAP) technique in vibratome sections. The majority of NPY-immunoreactive somata was localized in the white matter surrounding the anterior olfactory nucleus. Immunoreactive neurons were less numerous within the anterior olfactory nucleus and within the olfactory bulb layers. NPY-immunoreactive fibres were present in the white matter, the anterior olfactory nucleus, and in the olfactory bulb layers. Fibres within the white matter were generally aligned in a straight path parallel to the long axis of the olfactory bulb and tract. Fibres within the anterior olfactory nucleus showed no clear orientation and displayed numerous branching points. Coiled plexus of NPY-immunoreactive fibres were present in the glomerular layer of the olfactory bulb. Additional characteristics of the NPY-immunoreactive neurons were studied after decolouring the chromogen and restaining the sections with aldehydefuchsin to demonstrate the presence of lipofuscin granules and also with gallocyanin chrome alum to stain the Nissl substance. This analysis showed that the neurons belong to the class of non-pigmented nerve cells.     

Synaptic analysis of amacrine cells with neuropeptide Y-like immunoreactivity in turtle retina

Neuropeptide Y-like immunoreactivity has been localized previously within three classes of amacrine cells in the turtle retina. We have used the avidin-biotin with horseradish peroxidase technique to label these neurons for examination at the ultrastructural level to answer the following questions. Where are the synaptic contacts of these neurons made? What types of neurons are involved pre- and postsynaptically? What is the intracellular distribution of the immunoreactivity? Processes with neuropeptide Y-like immunoreactivity were located primarily within three regions of the inner plexiform layer: stratum 1, stratum 3, and at the border between strata 4 and 5. In all three regions the processes with neuropeptide Y-like immunoreactivity received synaptic contacts from both unlabeled amacrine and bipolar cells, but the majority of the synaptic input in all three regions was from unlabeled amacrine cells. Processes with neuropeptide Y-like immunoreactivity were presynaptic to unlabeled amacrine cells in all three regions, but also formed contacts onto unlabeled bipolar cells in the region between strata 4 and 5. The immunoreactivity within these cells gave rise to a diffuse reaction product that was distributed throughout the cytoplasm and within large vesicles. This localization of neuropeptide Y-like immunoreactivity within large vesicles suggests that this peptide may play a neuromodulatory role. Such a role would be consistent with previous studies of neuropeptides in the turtle retina.     

GABA-like immunoreactive neurons in the retina of Bufo marinus: evidence for the presence of GABA-containing ganglion cells.

gamma-Aminobutyric acid (GABA)-like immunoreactive (IR) neurons in the retina of the cane toad Bufo marinus were revealed using immunohistochemistry on retinal wholemount preparation and sectioned material. GABA-IR neurons included horizontal, bipolar and amacrine cells in the inner nuclear layer and small to medium sized cells in the ganglion cell layer. A few IR axons were seen in the optic fiber layer of the retina. Following the injection of the carbocyanine dye, DiI into the optic tectum ganglion cells were retrogradely filled. A small population of DiI-filled ganglion cells (2.8%) was found to be GABA-IR. GABA-IR neurons in the ganglion cell layer without DiI label were considered to be displaced amacrine cells of which 45.3% were GABA positive. It is proposed that GABA-containing ganglion cells may form an inhibitory projection to visual centers of the anuran brain.     

Synaptic organization of neurotensin immunoreactive amacrine cells in the chicken retina

Immunohistochemistry was utilized to investigate the light and electron microscopic localization of neurotensinlike immunoreactive (NT) amacrine cells in the chicken retina. The NT cells possess oval cell bodies (7 microns in diameter) that are located in either the second or third tier of cells from the border of the inner nuclear and inner plexiform layers. The processes of such cells extend into the inner plexiform layer where they ramify as a narrow plexus in sublamina 1 and as a broad plexus in sublaminas 3 and 4. Additionally, stained processes are observed occasionally within sublamina 5. At the ultrastructural level, NT-positive somas exhibit a rather dense and evenly distributed peroxidase reaction product throughout their cytoplasm. The nucleus of NT amacrine cells possess a round, unindented nuclear membrane. NT-immunoreactive processes in the inner plexiform layer interact synaptically only with non-NT cells. NT processes receive synaptic input mainly from the processes of amacrine cells and to a lesser degree from bipolar cells. The large majority of NT-stained varicosities form presynaptic contacts onto the processes of amacrine cells, but are also presynaptic to bipolar cell axon terminals. Moreover, each of the above synaptic relationships can be identified in each of sublaminas 1 and 3 to 4 of the inner plexiform layer. In addition, NT processes are presynaptic to processes devoid of synaptic vesicles that may originate from ganglion cells. Finally, NT processes occasionally form synaptic contacts onto somas situated in the most proximal row of the inner nuclear layer.     

Histogenesis of corticotropin releasing factor-like immunoreactive amacrine cells in the rat retina

The histogenesis of amacrine cells expressing corticotropin releasing factor-like immunoreactivity (CRF-LI) was examined in the rat retina by incorporating [3H]thymidine autoradiography and immunohistochemistry. Our results indicate that, during the period of amacrine cell generation, the population of CRF-LI amacrine cells are generated within a window of time from gestational days (ED)-16 to -20; the majority of them are produced by ED-18. No CRF-LI cells have been found to be 'born' postnatally. The histogenetic pattern of these cells follows a center-to-periphery gradient. Furthermore, CRF-LI cells in both the inner nuclear layer and the ganglion cell layer are generated in the same histogenetic wave and follow an identical temporo-spatial pattern. These results are consistent with and extend our previous findings of a differential change in CRF-LI cell density and total cell number during postnatal development.     

Neuropeptide Y-immunoreactive amacrine cells in the retina of the turtle Pseudemys scripta elegans

Antiserum directed against neuropeptide Y selectively labeled certain amacrine cells in the turtle retina. The cell types, sizes, dendritic stratification, regional distribution, and degrees of immunolabeling were examined. The results indicated that three morphologically distinct cell types were labeled: types A, B, and C. Computer rotation of digitized data from camera lucida drawings was used to study dendritic stratification. The type A somata were large (11.5 micron in diameter), well-stained, and located in the third tier of the inner nuclear layer. Type A somata gave rise to well-stained processes which arborized within the inner plexiform layer in strata 1 and 3 and at the border between strata 4 and 5. Processes in stratum 1 were sparse and delicate with small boutons. Processes in stratum 3 were numerous and often coarse, with many large and small boutons. At the border between strata 4 and 5 the processes were frequently numerous but slender, with many small boutons. Occasional immunolabeled processes were found in the ganglion cell layer. The somata of the type B cells were smaller (9.0 micron in diameter) and gave rise to single labeled processes which descended into the inner plexiform layer and divided quickly into two secondary processes. These secondary processes gave rise to lightly labeled dendritic fields which arborized primarily in strata 2 and 4. The type C cells were usually observed at the periphery of the retina and had large somata (12.0 micron in diameter) with simple, but very elongated, dendritic arborizations in strata 1, 4, and 5. Observations also showed that type A and B cells were often found in close proximity to each other and suggested that dendrites of these cells made contact with each other. The labeled neurons were distributed relatively evenly throughout the retina except for the visual streak where they were sparse. This study indicates that neuropeptide Y-like immunoreactivity is found in more than one anatomically distinct class of amacrine cells in the turtle retina.     

Synaptic organization of substance P-like immunoreactive amacrine cells in goldfish retina

A class of amacrine cells in the goldfish retina displays substance P-like immunoreactivity (SPIR). We studied the synaptic organization of SPIR amacrine cells by electron microscopical immunocytochemistry. Amacrine cells showing SPIR have processes which ramify in a very narrow band in layer 3 of the inner plexiform layer. SPIR is restricted to large dense-cored vesicles (DCVs), which are distributed throughout the dendrites. Processes labeled with SPIR contain a mixture of DCVs and numerous small agranular vesicles. Of 88 synaptic contracts analyzed, SPIR processes occurred as the presynaptic element 57 times and as the postsynaptic element 31 times. SPIR processes made synapses upon amacrine and ganglion cell dendrites with equal frequency and received synaptic input from both amacrine and bipolar cells. The stratification of SPIR amacrine cells in proximal sublamina a suggests that their synaptic interactions are restricted to "off" and "on-off" neurons. However, this is in contrast to published electrophysiological data. Possible explanations for this discrepancy are discussed in detail.     

Characterization of secretagogin‐immunoreactive amacrine cells in marmoset retina

The retina contains at least 30 different types of amacrine cells but not many are well characterized. In the present study the calcium‐binding protein secretagogin was localized in a population of regular and displaced amacrine cells in the retina of the common marmoset Callithrix jacchus. Irrespective of their soma location, the dendrites of secretagogin amacrine cells occupy strata 2, 3, and 4 of the inner plexiform layer, between the two bands formed by cholinergic amacrine cells. Segretagogin amacrine cells are also immunopositive to antibodies against glutamic acid decarboxylase, suggesting that they use γ‐aminobutyric acid (GABA) as their neurotransmitter. The spatial density of secretagogin amacrine cells decreases from a peak of about 400 cells/mm² near 1 mm eccentricity to less than 100 cells/mm² in peripheral retina; these densities account for about 1% of amacrine cells in the inner nuclear layer and for up to 27% of displaced amacrine cells. The cell bodies form a regular mosaic, suggesting that they constitute a single amacrine cell population. Secretagogin cells have varicose dendrites, which are decorated with small spines. Intracellular injection of DiI into secretagogin cells revealed an average dendritic field diameter of 170 μm and an average coverage factor of 3.2. In summary, secretagogin cells in marmoset retina are medium‐field amacrine cells that share their stratification pattern with narrow‐field amacrine cells and their neurotransmitter with wide‐field amacrine cells. They may mediate spatial inhibition spanning the centralmost (on and off) bands of the inner plexiform layer. J. Comp. Neurol. 522:435–455, 2014. © 2013 Wiley Periodicals, Inc.     

Substance-P-like immunoreactive amacrine cells in the adult and the developing rat retina.

Substance-P-like immunoreactivity (SP-LI) cells in the Long-Evans rat retina were investigated by combining immunohistochemistry with [3H]thymidine autoradiography. Two subpopulations of SP-LI amacrine cells, with cell bodies in either the proximal portion of the inner nuclear layer (INL) or the ganglion cell layer (GCL), were identified based on morphology, pattern of distribution and development. In the INL, SP-LI cells were found scattered throughout the retina. However, in the GCL, they were limited to the superio-temporal region. Such a contrast in distribution specific to nuclear layers was present upon first detection of SP-LI amacrine cells and persisted throughout development. Birthdating revealed a temporal lag in the histogenesis of SP-LI cells situated in the GCL relative to that in the INL, suggesting that the two subpopulations developed separately. Overall, unique anatomical features of the SP-LI amacrine cells in the rat retina were observed which could only have been uncovered through detailed analyses in the adult as well as during postnatal development.     

Large serotonin-like immunoreactive amacrine cells in the retina of developing Xenopus laevis.

The earliest appearance of serotonin-like immunoreactivity (SLI) in different cell types and the development of large SLI amacrine cells were studied in the retina of Xenopus laevis from stage 33/34 to adult. Intense SLI was first found in the somas of large amacrine cells at stage 39. The somas of small amacrine cells showed weak SLI at stage 41, followed by bipolar cells at stage 43. The number of large SLI amacrine cells in the inner nuclear layer of the retina increased from 57 at stage 40 to 774 in adult. Over the same period, retinal area increased from 0.19 mm2 to 24.57 mm2 with an accompanying decrease of cell density from 301/mm2 to 32/mm2. in adult animals large SLI amacrine cells were non-uniformly distributed. Peak cell density of 50-60/mm2 was located in the center of the ventrotemporal quadrant and a trough of 8-15/mm2 in the dorsal periphery of the retina. Peak cell density region of the adult retina corresponded to part of the retina formed at early developmental stages where the rate of cell generation of large SLI amacrine cells was higher. These observations indicate that (1) SLI is expressed first by large amacrine cells, followed by small amacrine and bipolar cells; (2) large SLI amacrine cells are generated continuously throughout life, (3) the non-uniform retinal distribution of large cells results from a spatio-temporally differential cell generation at the ciliary margin.     

Postnatal development of corticotropin releasing factor-like immunoreactive amacrine cells in the rat retina

The postnatal development of the corticotropin releasing factor-like immunoreactive (CRF-LI) amacrine cells was investigated in the Long-Evans rat retina. The pattern of development of CRF-LI cells was studied by immunohistochemistry, their cell number and density throughout the first two weeks of postnatal development were analyzed, and correlative measures of CRF-LI content were obtained using radioimmunoassay (RIA). The overall pattern of CRF-LI development, as revealed by either method, is characterized initially by faint staining and low content, respectively, which began to increase in staining intensity and content until a peak was reached around postnatal day (PD)-15, the time of eye opening. In determining cell number and density, emphasis was placed on the relationship between the development of CRF-LI neurons in the inner nuclear layer (INL) and that in the ganglion cell layer (GCL). Such quantitative analyses revealed a series of dynamic shifts in the distribution of CRF-LI cell density in both a horizontal orientation and a vertical orientation prior to PD-15. Horizontally, the shift involved a center-to-periphery density gradient which disappeared progressively as the retina matured. Vertically, a reciprocal change in total cell number occurred; the number of CRF-LI cells in the INL decreased while that in the GCL increased. These changes stabilized by PD-15 and, by PD-19, the CRF-LI cells appeared morphologically mature.     

Colocalization of immunoreactive substance P and neurotensin in amacrine cells of the goldfish retina

Using an immunohistochemical double-label technique, neurotensin and substance P immunoreactivity were localized to amacrine cells in the goldfish retina. Both peptides were found in a single population of unistratified amacrine cells branching in sublamina 3 of the inner plexiform layer. A monostratified amacrine cell branching in sublamina 1 contained only substance P immunoreactivity and a bistratified cell branching in sublaminae 1 and 3 contained only neurotensin immunoreactivity.     

Localization of immunoreactive cholecystokinin precursor to amacrine cells and bipolar cells of the macaque monkey retina

We used antisera that recognized precursors of the neuropeptide cholecystokinin extended at the carboxyl terminus in an immunocytochemical study of the macaque retina. A subpopulation of bipolar cells with long, obliquely oriented dendrites was labeled. Their axons terminated exclusively in the fifth stratum of the inner plexiform layer, where they contacted processes of amacrine and ganglion cells. Based on their morphology, these cells appeared to be the type that contacts short-wavelength cones selectively. Two types of amacrine cells were also labeled, and processes from both types formed dense plexuses in the second and fourth strata of the inner plexiform layer. The majority of their synaptic connections were with other amacrine cells, but they had more contacts with bipolar cell axons and retinal ganglion cell dendrites than any other peptidergic cells in the macaque retina. We studied extracts of macaque retina with gel-filtration chromatography and radioimmunoassays to confirm our immunohistochemical results. We found cholecystokinin octapeptide and other immunoreactive forms that were amidated at their carboxyl termini and were therefore likely to be biologically active. Unlike most other regions of the CNS, however, the retina had relatively low concentrations of amidated forms, and forms with extended carboxyl termini that are presumably their precursors were far more abundant. These findings suggest that the rate of cholecystokinin synthesis in the retina is quite high, as we would expect if the peptide were found in tonically active neurons.     

Dopaminergic amacrine cells in the cat retina

Autoradiographic studies of cat retina showed an accumulation of [³H]dopamine in a subpopulation of amacrine cells whose process ramify in the outermost stratum of the inner plexiform layer. Dendrites of these cells are characterized by numerous varicosities measuring up to 2 μm in diameter which are connected by fine intervaricose segments. The dopaminergic amacrine cells are presynaptic to other labeled cells and to unlabeled amacrine populations but not to bipolar or ganglion cells. [³H]Dopamine-labeled processes provide extensive synaptic input to the somata and lobular appendages of type AII amacrine cells. This relationship suggests that dopaminergic amacrine cells may play an important role in the regulation of rod pathways in the cat retina.     

ON-OFF amacrine cells in cat retina

We studied the morphology, photic responses, and synaptic connections of ON-OFF amacrine cells in the cat retina by penetrating them with intracellular electrodes, staining them with horseradish peroxidase, and examining them with the electron microscope. In a sample of seven cells, we found two different morphological types: the A19, which ramifies narrowly in stratum 2 (sublamina a ) of the inner plexiform layer, and the A22, which ramifies mostly in stratum 4 (sublamina b ) but extends some dendrites to sublamina a . Both of these cell types have axon-like processes that extend >800 μm from the conventional dendritic arbor. ON-OFF amacrine cells in our sample had receptive fields (1.7 ± 0.3 mm diameter) that were broader than their dendritic arbors (425 ± 35 μm diameter) and that extended over the region of axon-like processes. In addition, we found many features in common with ON-OFF amacrine cells in poikilotherm vertebrates: a broad receptive field without surround antagonism, two sizes of spike-like events, narrow dynamic range (1 log unit intensity), and excitatory postsynaptic potentials at light on and light off. Two A19 amacrine cells were examined in the electron microscope: most synaptic inputs (93 and 76%, respectively) to either cell were from amacrine cells, with minor inputs from cone bipolar cells. Synaptic outputs were to bipolar, amacrine, and ganglion cells, including the OFF-α cell. © 1996 Wiley-Liss, Inc.