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.     

Dendritic co-stratification of ON and ON-OFF directionally selective ganglion cells with starburst amacrine cells in rabbit retina.

The morphology, dendritic branching patterns, and dendritic stratification of retinal ganglion cells have been studied in Golgi-impregnated, whole-mount preparations of rabbit retina. Among a large number of morphological types identified, two have been found that correspond to the morphology of ON and ON-OFF directionally selective (DS) ganglion cells identified in other studies. These cells have been characterized in the preceding paper in terms of their cell body size, dendritic field size, and branching pattern. In this paper, the two kinds of DS ganglion cell are compared in terms of their levels of dendritic stratification. They are compared with each other and also with examples of class III.1 cells, defined in the preceding paper with reference to our previous studies. Studies employing computer-aided, 3D reconstruction of dendritic trees, as well as analysis of a pair of ON DS and ON-OFF DS ganglion cells with overlapping dendritic trees show that the two types of DS ganglion cell partly co-stratify in the middle of sublamina b (stratum 4). The report that some ON DS ganglion cells extend a few dendrites into sublamina a is confirmed. The study of pairs of ON-OFF DS ganglion cells and starburst amacrine cells with overlapping dendritic trees reveals a precise co-stratification of these two cell types, and many points of close apposition of starburst boutons with ON-OFF DS ganglion cell dendrites in both sublaminae of the inner plexiform layer (IPL). This is confirmed by high-resolution light microscopy and by electron microscopy. It is possible to conclude, therefore, that ON DS are also partly co-stratified with type b starburst (cholinergic) amacrine cells, and are apparently also partly co-stratified with type a starburst amacrine cells, when occasional dendrites rise to that level. The co-stratification of the two kinds of DS ganglion cell is consistent with the sharing of some inputs in common, including some cone bipolar cell inputs. The co-stratification of both with starburst amacrine cells agrees with the physiological demonstration of the powerful pharmacological effects upon ON and ON-OFF DS ganglion cells reported for cholinergic agonists. The major difference in the dendritic stratification of bistratified ON-OFF DS ganglion cells and generally unistratified ON DS ganglion cells is consistent with the bisublaminar organization of ON and OFF pathways in the IPL. The problem of occasional branches of ON DS cells in sublamina a is discussed in terms of a threshold for OFF responses.(ABSTRACT TRUNCATED AT 400 WORDS)     

Starburst amacrine cells in cat retina are associated with bistratified, presumed directionally selective, ganglion cells

Starburst amacrine cells of cat retina are similar in form, though more delicate and less profusely branched, when compared to the starburst/cholinergic amacrine cells of rabbit retina, as identified in Golgi preparations. In both species, type a cells branch in the middle of sublamina a of the inner plexiform layer (IPL), but type b (displaced) starburst amacrine cells of cat branch near the a/b sublaminar border (stratum 3) of the IPL, not in the middle of sublamina b (stratum 4), as do those of rabbit. Nevertheless, in each species, this starburst substratum in sublamina b coincides with the sublamina b-level branching of a bistratified ganglion cell, which in rabbit retina shows directionally selective responses. It is proposed that starburst amacrine cells of cat retina are cholinergic and, as in rabbit retina, make selective connections with on-off directionally selective ganglion cells.     

Ectopic retinal ON bipolar cell synapses in the OFF inner plexiform layer: Contacts with dopaminergic amacrine cells and melanopsin ganglion cells

A key principle of retinal organization is that distinct ON and OFF channels are relayed by separate populations of bipolar cells to different sublaminae of the inner plexiform layer (IPL). ON bipolar cell axons have been thought to synapse exclusively in the inner IPL (the ON sublamina) onto dendrites of ON‐type amacrine and ganglion cells. However, M1 melanopsin‐expressing ganglion cells and dopaminergic amacrine (DA) cells apparently violate this dogma. Both are driven by ON bipolar cells, but their dendrites stratify in the outermost IPL, within the OFF sublamina. Here, in the mouse retina, we show that some ON cone bipolar cells make ribbon synapses in the outermost OFF sublayer, where they costratify with and contact the dendrites of M1 and DA cells. Whole‐cell recording and dye filling in retinal slices indicate that type 6 ON cone bipolars provide some of this ectopic ON channel input. Imaging studies in dissociated bipolar cells show that these ectopic ribbon synapses are capable of vesicular release. There is thus an accessory ON sublayer in the outer IPL. J. Comp. Neurol. 517:226‐244, 2009. © 2009 Wiley‐Liss, Inc.     

Development of starburst cholinergic amacrine cells in the retina of Tupaia belangeri

"Starburst" cholinergic amacrines specify the response of direction-selective ganglion cells to image motion. Here, development of cholinergic amacrines was studied in the tree shrew Tupaia belangeri (Scandentia) by immunohistochemistry with antibodies against choline acetyltransferase (ChAT) and neurofilament proteins. Starburst amacrines expressed ChAT much earlier than previously thought. From embryonic day 34 (E34) onward, orthotopic and displaced subpopulations segregated from a single cluster of immunoreactive precursor cells. Orthotopic starburst amacrines rapidly took up positions in the inner nuclear layer. Displaced starburst amacrines were first arranged in a monocellular row in the inner plexiform layer, and, with a delay of 1 week, they descended to the ganglion cell layer. Conversely, dendritic stratification of displaced amacrines slightly preceded that of orthotopic ones. Starburst amacrines expressed the medium-molecular-weight neurofilament protein (NF-M) from E34 to postnatal day 11 (P11) and coexpressed alpha-internexin from E36.5 to P11. Consequently, neurofilaments composed of alpha-internexin and NF-M may stabilize developing dendrites of starburst amacrines. During the first 2 postnatal weeks, subpopulations of anti-NF-M-labeled ganglion cells costratified with the preexisting dendritic strata of starburst amacrines in the ON sublamina, OFF sublamina, or both. Hence, anti-NF-M-labeled ganglion cells may include direction-selective ones. Thereafter, NF-M and alpha-internexin proteins disappeared from starburst amacrines, and NF-M immunoreactivity was lost in the dendrites of ganglion cells. Our findings suggest that NF-M and alpha-internexin are important for starburst amacrines and ganglion cells to recognize each other and, thus, contribute to the formation of early developing retinal circuits in the inner plexiform layer.     

Synaptic organization of starburst amacrine cells in rabbit retina: analysis of serial thin sections by electron microscopy and graphic reconstruction

The synaptic organization of starburst amacrine cells was studied by electron microscopy of individual or overlapping pairs of Golgi-impregnated cells. Both type a and type b cells were analyzed, the former with normally placed somata and dendritic branching in sublamina a, and the latter with somata displaced to the ganglion cell layer and branching in sublamina b. Starburst amacrine cells were thin-sectioned horizontally, tangential to the retinal surface, and electron micrographs of each section in a series were taken en montage. Cell bodies and dendritic trees were reconstructed graphically from sets of photographic montages representing the serial sections. Synaptic inputs from cone bipolar cells and amacrine cells are distributed sparsely and irregularly all along the dendritic tree. Sites of termination include the synaptic boutons of starburst amacrine cells, which lie at the perimeter of the dendritic tree in the "distal dendritic zone." In central retina, bipolar cell input is associated with very small dendritic spines near the cell body in the "proximal dendritic zone." The proximal dendrites of type a and type b cells generally lie in planes or "strata" of the inner plexiform layer (IPL), near the margins of the IPL. The boutons and varicosities of starburst amacrine cells, distributed int he distal dendritic zone, lie in the "starburst substrata," which occupy a narrow middle region in each of the two sublaminae, a and b, in rabbit retina. As a consequence of differences in stratification, proximal and distal dendritic zones are potentially subject to different types of input. Type b starburst amacrines do not receive inputs from rod bipolar terminals, which lie mainly in the inner marginal zone of the IPL (stratum 5), but type a cells receive some input from the lobular presynaptic appendages of rod amacrine cells in sublamina a, at the border of strata 1 and 2. There is good correspondence between boutons or varicosities and synaptic outputs of starburst amacrine cells, but not all boutons gave ultrastructural evidence of presynaptic junctions. The boutons and varicosities may be both pre- and postsynaptic. They are postsynaptic to cone bipolar cell and amacrine cell terminals, and presynaptic primarily to ganglion cell dendrites. In two pairs of type b starburst amacrine cells with overlapping dendritic fields, close apposition of synaptic boutons was observed, raising the possibility of synaptic contact between them. The density of the Golgi-impregnation and other technical factors prevented definite resolution of this question. No unimpregnated profiles, obviously amacrine in origin, were found postsynaptic to the impregnated starburst boutons.(ABSTRACT TRUNCATED AT 400 WORDS)     

Morphology and connectivity of the small bistratified A8 amacrine cell in the mouse retina

Amacrine cells comprise ～30 morphological types in the mammalian retina. The synaptic connectivity and function of a few γ‐aminobutyric acid (GABA)ergic wide‐field amacrine cells have recently been studied; however, with the exception of the rod pathway‐specific AII amacrine cell, the connectivity of glycinergic small‐field amacrine cells has not been investigated in the mouse retina. Here, we studied the morphology and connectivity pattern of the small‐field A8 amacrine cell. A8 cells in mouse retina are bistratified with lobular processes in the ON sublamina and arboreal dendrites in the OFF sublamina of the inner plexiform layer. The distinct bistratified morphology was first visible at postnatal day 8, reaching the adult shape at P13, around eye opening. The connectivity of A8 cells to bipolar cells and ganglion cells was studied by double and triple immunolabeling experiments by using various cell markers combined with synaptic markers. Our data suggest that A8 amacrine cells receive glutamatergic input from both OFF and ON cone bipolar cells. Furthermore, A8 cells are coupled to ON cone bipolar cells by gap junctions, and provide inhibitory input via glycine receptor (GlyR) subunit α1 to OFF cone bipolar cells and to ON A‐type ganglion cells. Measurements of spontaneous glycinergic postsynaptic currents and GlyR immunolabeling revealed that A8 cells express GlyRs containing the α2 subunit. The results show that the bistratified A8 cell makes very similar synaptic contacts with cone bipolar cells as the rod pathway‐specific AII amacrine cell. However, unlike AII cells, A8 amacrine cells provide glycinergic input to ON A‐type ganglion cells. J. Comp. Neurol. 523:1529–1547, 2015. © 2015 Wiley Periodicals, Inc.     

Variability of light-evoked response pattern and morphological characterization of amacrine cells in goldfish retina

Amacrine cells of the goldfish retina were characterized electrophysiologically and subsequently labelled by intracellular injection of horseradish peroxidase. An attempt was made to broaden the electrophysiological classification of the cells. Light-evoked sustained amacrine cell responses were divided into two subtypes depending on colour opponency. Colour-coded responses (red/depolarizing and green/hyperpolarizing) were found to arise in amacrine cells possessing highly polarized dendritic fields; the dendrites were monostratified in the proximal half (sublamina b) of the inner plexiform layer. Non-colour-opponent sustained responses also arose in monostratified units, but the level of dendritic ramification was in sublamina a or b (hyperpolarizing or depolarizing units, respectively). Transient (ON-OFF) responses were associated mainly with bi- or multi-stratified or diffuse amacrine cells. Some variability was observed in the sizes of the dendritic fields in different sublaminae. There was a tendency for units with brisk components of responses to be narrowly stratified in the inner plexiform layer. Some units possessed "distant" dendrites. Several aspects of structure-function correlation in amacrine cells are discussed.     

A novel type of interplexiform amacrine cell in the mouse retina

Mammalian retinas comprise an enormous variety of amacrine cells with distinct properties and functions. The present paper describes a new interplexiform amacrine cell type in the mouse retina. A transgenic mouse mutant was used that expressed the gene for the enhanced green fluorescent protein (EGFP) instead of the coding DNA of connexin45 in several retinal cell classes, among which a single amacrine cell population was most prominently labelled. Staining for EGFP and different marker proteins showed that these amacrine cells are interplexiform: they stratify in stratum S4/5 of the inner plexiform layer and send processes to the outer plexiform layer. These cells were termed IPA-S4/5 cells. They belong to the group of medium-field amacrine cells and are coupled homologously and heterologously to other amacrine cells by connexin45. Immunostaining revealed that IPA-S4/5 cells are GABAergic and express GAT-1, a plasma-membrane-bound GABA transporter possibly involved in non-vesicular GABA release. To characterize the light responses of IPA-S4/5 cells, patch-clamp recordings in retinal slices were made. Consistent with their stratification in the ON sublamina of the inner plexiform layer, cells depolarized in response to light ON stimuli and transiently hyperpolarized in response to light OFF. Responses of cells to green (578 nm) and blue (400 nm) light suggest that they receive input from cone bipolar cells contacting both M- and S-cones, possibly with reduced S-cone input. A new type of interplexiform ON amacrine cell is described, which is strongly coupled and uses GABA but not dopamine as its neurotransmitter.     

Neuropeptide Y-like immunoreactive amacrine cells in the retina of Bufo marinus

Neuropeptide Y-like immunoreactive (NPY-LI) amacrine cells of the Bufo 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.     

Structure-function correlation in transient amacrine cells of goldfish retina: basic and multifractal analyses of dendritic trees in distinct synaptic layers.

Amacrine cells generating light-evoked transient ON-OFF responses were stained by intracellular injection of horseradish peroxidase after determining their input-output (voltage response vs. light intensity) profiles. Ten cells specifically having bistratified dendritic trees were analyzed. The cross-sectional area of the dendrites in each sublamina (a and b) of the inner plexiform layer was initially measured. Although some variability was observed, there was no statistically significant overall difference in the cross-sectional areas of the dendritic trees in sublaminae a and b. Also, the amplitudes of the ON and OFF responses, generated by a midrange criterion stimulus, could not be correlated with the cross-sectional areas or the number of branches of the dendrites in sublaminae b and a, respectively. On the other hand, determination of the generalized fractal spectra revealed that the negative (up to -3) and zero-order fractal dimensions of the dendritic trees in sublamina a were consistently higher than those for sublamina b. Furthermore, there was a positive correlation between response amplitude and some part of the generalized fractal dimension in the respective parts of the dendritic trees. It is concluded that dendritic tree characteristics differ in the two halves of the inner plexiform layer and that these can be related to the cells' light-evoked response amplitudes. Furthermore, generalized fractal analysis appears to be a useful method for correlating structure and function in retinal amacrine cells with complex dendritic trees.     

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.     

Identification and morphological classification of horizontal, bipolar, and amacrine cells within the zebrafish retina

Horizontal, bipolar, and amacrine cells in the zebrafish retina were morphologically characterized using DiOlistic techniques. In this method, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-coated microcarriers are shot at high speed onto the surfaces of living retinal slices where the DiI then delineates axons, somata, and dendrites of isolated neurons. Zebrafish retinal somata were 5-10 microm in diameter. Three horizontal cell types (HA-1, HA-2, and HB) were identified; dendritic tree diameters averaged 25-40 microm. HA somata were round. Cells classified as HA-2 were larger than HA-1 cells and possessed an axon. HB somata were flattened, without an axon, although short fusiform structure(s) projected from the soma. Bipolar cells were separated into 17 morphological types. Dendritic trees ranged from 10 to 70 microM. There were six B(on) types with axon boutons only in the ON sublamina of the inner plexiform layer (IPL), and seven B(off) types with axon boutons or branches only in the OFF sublamina. Four types of bistratified bipolar cells displayed boutons in both ON and OFF layers. Amacrine cells occurred in seven types. A(off) cells (three types) were monostratified and ramified in the IPL OFF sublamina. Dendritic fields were 60-150 microM. A(on) pyriform cells (three types) branched in the ON sublamina. Dendritic fields were 50-170 microM. A(diffuse) cells articulated processes in all IPL strata. Dendritic fields were 15-90 microM. These findings are important for studies examining signal processing in zebrafish retina and for understanding changes in function resulting from mutations and perturbations of retinal organization.     

Choline acetyltransferase is expressed by non-starburst amacrine cells in the ground squirrel retina

We have used immunostaining techniques to reveal a new type of amacrine cell that is immunoreactive for choline acetyltransferase (ChAT), the acetylcholine synthesizing enzyme, in the Ground Squirrel (Spermophilus beecheyi) retina. Cryostat sections and double immunostained wholemount preparations were examined by confocal microscopy. This new ChAT type III cell is distinct in morphology and neurotransmitter content from the well know 'starburst' amacrine cells (types I and II) that are so well represented in the ground squirrel retina [J. Comp. Neurol. 365 (1996) 173-216]. The type III cell colocalizes glycine with the acetylcholine and does not appear to be GABAergic or exhibit calcium-binding proteins like the well-known starburst type. As well, type III cells do not occur as a mirror-symmetric pair with normally placed and displaced varieties. The type III cell is probably a small field amacrine type branching broadly in upper sublamina b of the inner plexiform layer, and is most likely A6 of the Ground Squirrel retina [J. Comp. Neurol. 365 (1996) 173-216]. Type III cells are ideally placed in the architecture of the Ground Squirrel retina to influence ON directionally selective ganglion cell types.     

Ontogeny of cholinergic amacrine cells in the opossum (Didelphis aurita) retina

Immunocytochemistry for choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine, was used to determine the onset and to follow the maturation of the cholinergic cells in the retina of a marsupial, the South American opossum (Didelphis aurita). ChAT-immunoreactivity was first detected in amacrine cells in the ganglion cell layer by postnatal day 15 (P15) and in the inner nuclear layer by P35. Much later, at P50 a second sub-population of ChAT-immunoreactive cell bodies was evident in the inner nuclear layer. Processes from ChAT-immunoreactive amacrine cells were detected in the two bands of the inner plexiform layer before synaptogenesis. In the adult retina, these two bands correspond to sublamina 2 and 4 of the inner plexiform layer. In flat whole-mounted preparations, cholinergic cell density was 263±13 cells/mm[2] in the ganglion cell layer and it was estimated a total of 24,000 cholinergic neurons. ChAT-immunoreactive somata showed a random pattern of distribution.     

Characterization of small-field bistratified amacrine cells in macaque retina labeled by antibodies against synaptotagmin-2

Macaque retinae were immunostained with monoclonal antibodies directed against the protein synaptotagmin‐2 (Syt2). Syt2 was localized in a population of small‐field amacrine cells, whose cell bodies formed a regular mosaic within the inner nuclear layer, indicating they represent a single amacrine cell type. The labeled amacrine cells had a bistratified appearance with a dense dendritic plexus in the OFF‐layer and only a few lobular processes extending into the ON‐layer of the inner plexiform layer, similar to A8 amacrine cells described in cat and human retina. Syt2‐labeled cells were immunoreactive for glycine but lacked immunoreactivity for γ‐aminobutyric acid (GABA), suggesting they use glycine as their neurotransmitter. The density of these cells increases from ～200/mm² in peripheral retina to ～1,400/mm² in central retina. Their bipolar cell input was studied by immunolabeling experiments using various bipolar cell markers combined with CtBP2, a marker of presynaptic ribbons. Our data show that Syt2‐labeled amacrine cells receive input from both OFF and ON cone bipolar cells, as well as from rod bipolar cells. The OFF input is dominated by the diffuse bipolar cell DB1 (44%) and the OFF midget bipolar cell (38%). Here we describe a population of bistratified small‐field amacrine cells closely resembling A8 amacrine cells and their cone‐dominated bipolar cell input. J. Comp. Neurol. 521:709–724, 2013. © 2012 Wiley Periodicals, Inc.     

Characterization of an amacrine cell type of the mammalian retina immunoreactive for vesicular glutamate transporter 3

Immunocytochemical staining of vertical sections through rat, mouse, and macaque monkey retinae with antibodies against the vesicular glutamate transporter vesicular glutamate transporter 3 (vGluT3) showed a sparse population of amacrine cells. The labeled cells had similar appearances in the three species and probably represent homologous types. They were studied in detail in the rat retina. The thin varicose dendrites of vGluT3 amacrine cells formed a convoluted dendritic tree of approximately 100 microm in diameter that was bistratified in the center of the inner plexiform layer. The dendrites of vGluT3 cells were squeezed between the two strata of cholinergic dendrites. The density of vGluT3 cells was measured in retinal wholemounts and increased from 200/mm2 in peripheral retina to 400/mm2 in central retina, accounting for about 1% of all amacrine cells in the rat retina. The vGluT3 cells had a two- to threefold dendritic overlap, and their cell bodies formed a regular mosaic, suggesting they represent a single type of amacrine cell. The vGluT3 amacrine cells expressed glycine and glycine transporter 1 (GlyT1) but not the vesicular glycine transporter (vesicular inhibitory amino acid transporter). They also expressed glutamate; hence, there is the possibility that, comparable to cholinergic amacrine cells, they are "dual transmitter" amacrine cells. The synaptic input of vGluT3 cells was studied by electron microscopy. They received input from bipolar cells at ribbon synapses and from other amacrine cells at conventional synapses. The types of bipolar cells possibly involved with vGluT3 cells were demonstrated by double labeling sections for vGluT3 and the calcium-binding protein CaB5. The axon terminals of type 3 and 5 bipolar cells costratified with vGluT3 dendrites, and it is possible that vGluT3 cells have ON and OFF light responses.     

Cone signals in monostratified and bistratified amacrine cells of adult zebrafish retina

Strata within the inner plexiform layer (IPL) of vertebrate retinas are suspected to be distinct signaling regions. Functions performed within adult zebrafish IPL strata were examined through microelectrode recording and staining of stratified amacrine types. The stimulus protocol and analysis discriminated the pattern of input from red, green, blue, and UV cones as well as the light‐response waveforms in this tetrachromatic species. A total of 36 cells were analyzed. Transient depolarizing waveforms at ON and OFF originated with bistratified amacrine types, whose dendritic planes branched either in IPL sublaminas a & b, or only within sublamina a. Monophasic‐sustained depolarizing waveforms originated with types monostratified in IPL s4 (sublamina b). OFF responses hyperpolarized at onset, depolarized at offset, and in some cases depolarized during mid‐stimulus. These signals originated with types monostratified in s1 or s2 (sublamina a). Bistratified amacrines received depolarizing signals only from red cones, at both ON and OFF, while s4 stratified ON cells combined red and green cone signals. The s1/s2 stratified OFF cells utilized hyperpolarizing signals from red, red and green, or red and blue cones at ON, but only depolarizing red cone signals at OFF. ON and OFF depolarizing transients from red cones appear widely distributed within IPL strata. “C‐type” physiologies, depolarized by some wavelengths, hyperpolarized by others, in biphasic or triphasic spectral patterns, originated with amacrine cells monostratified in s5. Collectively, cells in this stratum processed signals from all cone types. J. Comp. Neurol. 525:1532–1557, 2017. © 2016 Wiley Periodicals, Inc.     

OFF-cholinergic-pathway-selective localization of P2X2 purinoceptors in the mouse retina

It is known that, in the retina, extracellular adenosine triphosphate (ATP) inhibits acetylcholine (ACh) release from cholinergic neurons, but the types of purinoceptors on cholinergic neurons have not been examined. In the present work, we immunohistochemically examined the distribution of the purinoceptors P2X1, P2X2, P2X4, and P2X7 in relation to the cholinergic system of the retina in wild-type mice and transgenic mice expressing green fluorescent protein (GFP). Immunoreactivity for P2X2 was very strong in sublamina a of the inner plexiform layer but very weak in sublamina b of the inner plexiform layer of the retina. Immunoreactivity for P2X2 was colocalized with that for choline acetyltransferase (ChAT). When transgenic mice were treated with the immunotoxin-mediated cell-targeting technology to ablate cholinergic amacrine cells selectively, immunoreactivity for P2X2 and the signals for GFP disappeared in parallel and selectively in the OFF pathway. The distribution of immunoreactivity for P2X1, P2X4, and P2X7 differed from that of ChAT immunoreactivity. The selective distribution of P2X2 purinergic receptors in OFF-type cholinergic amacrine cells indicates that the P2X2 purinergic signaling systems in the ON and OFF pathways of the inner plexiform layer of the mouse retina are functionally different. The distribution of P2X2 purinoceptors may be responsible for the selective regulation of ACh release in the OFF pathway.     

Cytochemical identification of cholinergic amacrine cells in cat retina

Golgi studies of cat retina have revealed the presence of matching subpopulations of starburst-like amacrine and displaced amacrine cells that are morphologically similar to the cholinergic cells of rabbit retina. The displaced amacrines appear identical with the A14 cells described by Kolb et al. (Kolb, Nelson, and Mariani: Vision Res. 21:1081-1114, 1981). In order to determine whether these cells may be cholinergic, we carried out autoradiography to localize newly synthesized (3H)acetylcholine and immunocytochemistry to demonstrate the distribution of choline acetyltransferase. Autoradiographs showed labeling in somas of both amacrine and displaced amacrine cells. Choline acetyltransferase was found in amacrine cells that ramify in sublamina a of the inner plexiform layer and in displaced amacrine cells ramifying in sublamina b. The pattern of cholinergic neurons in the cat is similar to that in other vertebrates and suggests that acetylcholine may play an important and consistent role in retinal function.