Muscarinic acetylcholine receptors in the normal, developing and regenerating newt retinas

Immunoreactivity for m2 and m4 muscarinic acetylcholine receptors (mAChRs) was demonstrated in the adult newt retina. The m2 mAChR was localized to somata on either side of the inner plexiform layer (IPL), especially ganglion cells, and also distributed into two bands within the IPL. The distal band at a depth of 0-15% IPL co-localized with one of two choline acetyltransferase (ChAT) immunoreactive bands, while the proximal band at 85-100% depth did not overlap with either of the ChAT-ir bands. The m4 mAChR was localized to somata closely apposed to either side of the IPL, probably amacrine cell somata, and no immunoreactivity was detectable throughout the IPL. The time course of appearance of the m2 and m4 mAChRs was examined in both developing and regenerating retinas. Like acetylcholinesterase (AChE), the m2 was first detected in somata located at the most proximal level of the retina well before ChAT-ir cholinergic neurons appeared, while the m4 was detected at the time of appearance of ChAT, in both developing and regenerating retinas. When the outer plexiform layer (OPL) began to form, somata in the horizontal cell layer became transiently immunoreactive to the m2. The discrepancy in distribution of the m2 and ChAT in the IPL suggests that mAChR may play a role other than cholinergic neurotransmission. Furthermore, the similarity in time course of appearance of the m2 and m4, as well as other cholinergic system components [4], in both developing and regenerating retinas would suggest that the mechanisms that control neuronal differentiation during retinal development and regeneration are similar.     

Localization of hydroxyindole-O-methyltransferase-like immunoreactivity in photoreceptors and cone bipolar cells in the human retina: a light and electron microscope study

The localization of the melatonin-synthesizing enzyme hydroxyindole-O-methyltransferase (HIOMT) was examined by light and electron microscopic immunocytochemistry in the human retina. HIOMT-like immunoreactivity was observed in the photoreceptor layers and the inner nuclear layer (INL). The immunoreactive cells in the INL were more numerous in the central retina than in the peripheral retina and sent processes to both the outer plexiform and inner plexiform layers. The HIOMT immunoreactivity in the inner plexiform layer (IPL) appeared as punctate terminals in the proximal and distal one-thirds of that layer. At the ultrastructural level, HIOMT-like immunoreactivity was localized to the cytoplasm of rod and cone photoreceptors and to a population of cone bipolar cells. HIOMT-immunoreactive bipolar cell dendrites were observed to make both invaginating and flat synaptic contacts with cone pedicles. No immunoreactive invaginating contacts in rod spherules were observed. HIOMT immunoreactivity was observed in the bipolar cell cytoplasm in the INL, and in the bipolar synaptic terminals in the IPL. These terminals contained synaptic ribbons, which formed synaptic contacts with unlabeled cells in the IPL. HIOMT radioenzymatic assays confirmed the presence of HIOMT in the human retina. Average HIOMT activity of eight donors was determined to be 15.0 pmol/mg protein/hour +/- 7.2 S.D. The ultrastructural localization of HIOMT observed in this study, combined with reports from other laboratories, suggests that the cytoplasm of the photoreceptors and a population of cone bipolar cells may be the sites of melatonin synthesis in the human retina.     

Intricate paths of cells and networks becoming "Cholinergic" in the embryonic chicken retina

Choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) are the decisive enzymatic activities regulating the availability of acetylcholine (ACh) at a given synaptic or nonsynaptic locus. The only cholinergic cells of the mature inner retina are the so‐called starburst amacrine cells (SACs). A type‐I SAC, found at the outer border of the inner plexiform layer (IPL), forms a synaptic subband “a” within the IPL, while a type‐II SAC located at the inner IPL border projects into subband “d.” Applying immunohistochemistry for ChAT and AChE on sections of the chicken retina, we here have revealed intricate relationships of how retinal networks became dominated by AChE or by ChAT reactivities. AChE⁺ cells were first detectable in an embryonic day (E)4 retina, while ChAT appeared 1 day later in the very same cells; at this stage all are Brn3a⁺, a marker for ganglion cells (GCs). On either side of a faint AChE⁺ band, indicating the future IPL, pairs of ChAT⁺/AChE^?/Brn3a^? cells appeared between E7/8. Type‐I cells had increased ChAT and lost AChE; type‐II cells presented less ChAT, but some AChE on their surfaces. Direct neighbors of SACs tended to express much AChE. Along with maturation, subband “a” presented more ChAT but less AChE; in subband “d” this pattern was reversed. In conclusion, the two retinal cholinergic networks segregate out from one cell pool, become locally opposed to each other, and become dominated by either synthesis or degradation of ACh. These “cholinergic developmental divergences” may also have significant physiologic consequences. J. Comp. Neurol., 520:3181–3193, 2012. © 2012 Wiley Periodicals, Inc.     

Hydrolysis of substance P in the rabbit retina: I. Involvement of acetylcholine and acetylcholinesterase. An in vivo study.

The laminar patterns of acetylcholinesterase (AChE) activity and substance P (SP) immunoreactivity within the inner plexiform layer (IPL) of the rabbit retina show striking similarities. Discrete bands of SP-immunoreactivity were seen at 1-7%, 40-48% and 85-95% depth of IPL. AChE activity was present throughout the entire thickness of the IPL with moderately stained bands in each sublamina (3-24% in sublamina a and 62-89% in sublamina b depth IPL). These bands were bordered on both sides by bands of even greater density (in sublamina a 0-3% and 24-34% and in sublamina b 55-62% and 89-100% depth IPL). Cell processes staining for choline acetyltransferase (ChAT) have previously been shown to ramify at 19-24% and 63-79% depth levels. Thus, SP- and ChAT-immunoreactive bands are located in both sublaminae, positioned within regions of moderate AChE activity and flanked by bands with greater AChE activity. This strong morphological correspondence and reported interactions between acetylcholine (ACh), AChE and SP in vitro provide the basis for the present study to determine whether such interactions can be demonstrated in vivo. Retinas infused with ACh showed a 60% average increase in SP-IR as compared with untreated retinas from the same animals. Treatment with diisopropylfluorophosphate (DFP) also resulted in a 56% increase in SP-IR. The ability of ACh to induce increased levels of SP was not inhibited by CoCl2, atropine or mecamylamine, ruling out the possibilities of polysynaptic transmission or involvement of muscarinic or nicotinic receptors. Infusion of ACh did not increase the levels of preprotachykinin-mRNA indicating that the increase in SP-IR is not due to de novo synthesis but rather to inhibition of the enzyme(s) responsible for SP degradation. Whether AChE functions alone or in concert with other enzymes to hydrolyze SP cannot be determined from these experiments but is addressed in a separate study.     

Localization of choline acetyltransferase in the developing and adult turtle retinas

Acetylcholine has important epigenetic roles in the developing retina. In this study, cells that expressed choline acetyltransferase (ChAT), the enzyme that synthesizes acetylcholine, were investigated in embryonic, postnatal, and adult turtle retinas by using immunofluorescence histochemistry. ChAT was present at stage 15 (S15) in cells near the vitreal surface. With the formation of the inner plexiform layer (IPL) at S18, ChAT-immunoreactive (-IR) cells were located in the inner nuclear layer (INL) and the ganglion cell layer (GCL). In the INL, presumed starburst amacrine cells were homogenous in appearance and formed a single row next to the IPL: This pattern was conserved until adulthood. In the GCL, however, there were multiple rows of ChAT-IR cells early in development, and this high density of labeled cells continued during the embryonic stages, until around birth. The high density of ChAT-IR cells in the GCL was due in part to a population of cells that expressed ChAT transiently. In postnatal stages and adult retinas, the presumed starburst amacrine ChAT-IR cells formed two mirror-like rows of homogenous cells on both borders of the IPL. Two cholinergic dendritic strata that were continuous with these cells were observed as early as S18, and their depths in the IPL were relatively stable throughout development. A third population of ChAT-IR cells was observed toward the middle of the INL around S25 and persisted into adulthood. Finally, cells in the outer nuclear layer (ONL) were ChAT-IR during the embryonic stages, were less immunoreactive during the postnatal stages, and were not immunoreactive in the adult retinas.     

Development of cholinergic amacrine cells is visual activity-dependent in the postnatal mouse retina

In the present study, we used immunocytochemistry to study the temporal and spatial arrangement of mouse cholinergic amacrine cells during postnatal retinal development under normal light/dark cycles and during visual deprivation. Choline acetyltransferase (ChAT)-immunolabeled cells were detected in the neuroblastic layer (NBL) and in the ganglion cell layer (GCL) at postnatal day 0 (P0). Between P3-5, two characteristic cholinergic bands were clearly identified in the inner plexiform layer (IPL). The signal intensity of somas and processes progressively increased over the first 2 postnatal weeks. Around eye opening at P12, cholinergic neurons were mature-like. This early developmental process was not altered by visual deprivation. After eye opening, the space between the two cholinergic bands increased continuously and the spatial regularity index changed constantly, indicating that the cholinergic neurons possibly underwent refinement during later postnatal development. The changes occurring following eye opening were retarded by visual deprivation. The morphologies of photoreceptors, horizontal cells, recoverin-positive OFF-cone bipolar cells, rod bipolar cells, dopaminergic amacrine cells, and Müller cells appeared normal. Their stratification in the outer plexiform layer (OPL) and the IPL was not affected by visual deprivation. However, glial cells grew vertically across the entire thickness of dark-reared retinas. Our results suggest that the development of cholinergic neurons before eye opening is independent of the lighting conditions. Their development after eye opening is greatly impeded by visual deprivation. This visual activity-dependent phase of development may be a critical period for the maturation and synaptic wiring of cholinergic amacrine cells in the mammalian retina.     

Choline acetyltransferase-immunoreactive neurons in the retina of normal and dark-reared turtle

Visual deprivation alters retinal-ganglion-cell response properties through changes in spontaneous wave-like activity (Sernagor and Grzywacz [1996] Curr Biol 6:1503-1508). This activity depends on cholinergic synaptic transmission in the turtle retina (ibid; Sernagor and Mehta [ 2001] J Anat 199:375-383). We studied the expression of choline acetyltransferase (ChAT) by immunocytochemistry and Western blot in developing retinas of control and dark-reared turtles. At postnatal day 0 (P0), right after hatching, ChAT-immunoreactivity was present in the ganglion cell layer (GCL), in the inner nuclear layer (INL), and in two distinct bands of the inner plexiform layer (IPL). In P14- and P28-control, and P14- and P28-dark-reared retinas, ChAT-immunoreactivity showed similar patterns to those in P0. However, in P14- and P28-dark-reared retinas the density of ChAT-immunoreactive cells was higher in both the INL and GCL than in P14- and P28-control retinas, respectively. Moreover, Western blotting showed that ChAT protein levels were significantly increased in the dark-reared retina compared to those of the control. TUNEL studies indicated that the difference between normal and dark-reared conditions was not due to extra apoptosis in the former. In turn, proliferating-cell nuclear antigen immunocytochemistry showed no extra proliferating cells in the latter. Finally, nearest-neighbor analysis revealed that the denser population of cholinergic cells in dark-reared turtles formed a mosaic as regular as the normal ones in the GCL. Thus, light deprivation increases the expression of ChAT, increasing the apparent density of cholinergic neurons in the developing turtle retina.     

A diffuse,invaginating cone bipolar cell in primate retina

A bipolar cell type forming invaginating contacts with many cones was found by light and electron microscopy of Golgi preparations of the rhesus monkey retina. This diffuse, invaginating cone bipolar cell resembles, superficially, rod (“mop”) bipolars, and so may correspond to Polyak's “brush” bipolar. However, it differs from rod bipolars in that its dendrites are finer and they end in a single stratum containing cone pedicles in the outer plexiform layer (OPL). In the inner plexiform layer (IPL) its axon terminal is located sclerad (S₄) to those of rod bipolars (S₅), is thinner, and also more branched, and wider in span than rod bipolar axon terminals. Resectioning of Golgi-impregnated diffuse, invaginating cone bipolars to study their connections in the OPL shows that their dendrites invaginate as many as seven cone pedicles, and terminate as central elements at the ribbon synaptic complex. Thus, the primate retina has multiple (diffuse) and single (midget) cone-contacting bipolar cell pathways in both invaginating as well as flat varieties.     

Differential distribution of hyperpolarization-activated and cyclic nucleotide-gated channels in cone bipolar cells of the rat retina

The hyperpolarization-activated and cyclic nucleotide-gated (HCN) channel isoforms HCN1, HCN2, and HCN4 were localized by immunofluorescence in the rat retina. Double labeling with the vesicular glutamate transporter (VGLUT1) was used to identify bipolar cell axon terminals in the inner retina. The HCN1 channel was localized to two cell types with differing intracellular distributions, insofar as staining was seen in the dendrites of a putative OFF-type cone bipolar cell and in the axon terminals of an ON-type bipolar that ramifies in stratum 3 (s3) of the inner plexiform layer (IPL). Staining for HCN4 was seen in two sets of bipolar axon terminals located in s2 and s3 and positioned between the two bands of choline acetyltransferase (ChAT) staining. The cells that ramify in s2 were identified as type 3 cone bipolar cells and the cells that ramify in s3 cells as a subclass of type 5 cone bipolars. The latter group, designated here as type 5b, exhibit diffuse axon terminals and can be distinguished from the narrowly stratifying type 5a cells. Double labeling showed that type 5b cone bipolar cells express both HCN1 and HCN4 as well as HCN2. Superposition of HCN channel labeling with VGLUT1 staining confirmed the presence of a cone bipolar cell whose terminals ramify in the same stratum of the IPL as type 5b cells but that do not express these HCN channels.     

Immunocytochemical localization of GABAA receptors in goldfish and chicken retinas

A monoclonal antibody (mAb 62-3G1) to the GABAA receptor/benzodiazepine receptor/Cl- channel complex from bovine brain was used with light and electron microscopy in goldfish retina and light microscopy in chicken retina to localize GABAA receptor immunoreactivity (GABAr-IR). GABAr-IR was found in the outer plexiform layer (OPL) in both species, in three broad bands in the inner plexiform layer (IPL) of goldfish, and in seven major bands of the chicken IPL. A small percentage of amacrine cell bodies (composing at least three types) were stained in chicken. In goldfish OPL, GABAr-IR was localized intracellularly and along the plasma membrane of cone pedicles, whereas rod spherules were lightly stained, but always only intracellularly. In chicken, all three sublayers of the OPL were GABAr-IR. The presence of GABAr-IR on photoreceptor terminals is consistent with data indicating feedback from GABAergic horizontal cells to cones. In the goldfish IPL, GABAr-IR was localized to postsynaptic sites of amacrine cell synapses; intracellular staining of processes in the IPL also was observed in presumed "GABAergic" targets. A comparison of GABAr-IR with the distributions of 3H-muscimol uptake/binding, glutamate decarboxylase-IR, GABA-IR, and 3H-GABA uptake in the IPL showed either a reasonable correspondence or mismatch, depending on the marker, species, and lamina within the IPL. The distribution of GABAr-IR in the retina corresponded better with the 3H-muscimol than with 3H-benzodiazepine binding patterns yet overall was in excellent agreement with many other physiological and anatomical indicators of GABAergic function. We suggest that intracellular GABAr-IR represents the biosynthetic and/or degradative pathway of the receptor and we conclude that mAb 62-3G1 is a valid marker of GABAA receptors in these retinas and will serve as a useful probe with which to address the issue of mismatches between the localization of GABAA receptors and indicators of presynaptic GABAergic terminals.     

Goalpha labels ON bipolar cells in the tiger salamander retina

By using double-label immunocytochemistry and confocal microscopy, we studied rod and cone synaptic contacts, photoreceptor-bipolar cell convergence, and patterns of axon terminal ramification of ON bipolar cells in the tiger salamander retina. An antibody to recoverin, a calcium-binding protein found in photoreceptors and other retinal neurons in various vertebrates, differentially labeled rods and cones by lightly staining rod cell bodies, axons, and synaptic pedicles and heavily staining cone cell bodies and pedicles. An antibody to G(oalpha) labeled most ON bipolar cells, with axon terminals ramified mainly in strata 6-9 and a minor band in stratum 3 of the inner plexiform layer (IPL). Stratum 10 of the IPL was G(oalpha) negative, and previous studies showed that axon terminals of rod-dominated ON bipolar cells are monostratified in that stratum. The axonal morphology of G(oalpha)-positive cells resembled that of the cone-dominated (DBC(C)) or mixed rod and cone ON (DBC(M)) bipolar cells. The G(oalpha)-positive dendritic processes made close contact with all cone pedicles and superficial contact with some rod pedicles, consistent with the idea that G(oalpha) subunits are present in DBC(C)s and DBC(M)s. The size and density of these cells were analyzed, and their spatial distributions were determined. To our knowledge, this is the first study to characterize photoreceptor inputs and axon terminal morphology of a population of ON bipolar cell with the use of a G(oalpha) antibody as an immunomarker in the salamander retina.     

Cholinergic neurons in the rabbit retina: dendritic branching and ultrastructural connectivity

The structure and synaptic connectivity of rabbit retinal cholinergic neurons have been studied with an immunocytochemical technique for the localization of choline acetyltransferase (ChAT). Cholinergic processes ramified in two narrow strata within the inner plexiform layer; viewed in the plane of the retina, each immunoreactive stratum took the form of a polygonal meshwork with openings of about 20 microns. There was frequently an extensive matching of the patterns of the two strata, such that openings in one meshwork lay directly over openings of similar size in the other meshwork. It is hypothesized that the pattern of branching of these cholinergic processes is a reflection of the branching pattern of ganglion cell dendrites in the cholinergic strata. The proximal cholinergic stratum was examined ultrastructurally, and had the following characteristics: (1) ChAT-immunoreactive processes made large numbers of synaptic contacts with ganglion cell dendrites; often there were many such ganglion cell dendrites running past each other at various angles within the plane of the cholinergic stratum; (2) cholinergic processes were never observed presynaptic to any other type of inner plexiform process; (3) the principal input onto cholinergic processes was provided by bipolar cell axon terminals; (4) ganglion cell dendrites within the cholinergic stratum also received direct bipolar input; and (5) unidentified (i.e. non-cholinergic and probably non-GABAergic) amacrine cell processes were often found that were presynaptic to these same ganglion cell dendrites, and that also formed reciprocal contacts with bipolar axon terminals within these synaptic complexes.     

Spatiotemporal gradients of differentiation of chick retina types I and II cholinergic cells: identification of a common postmitotic cell population.

The chick retina has three types of cholinergic amacrine cells. We have found that Types I and II differentiate from a common population of postmitotic cells temporarily located in the inner plexiform layer (IPL cells). Golgi staining and immunocytochemistry for choline acetyltransferase (ChAT) and gamma-aminobutyric acid (GABA) were used to trace the development and fate of IPL cells. Transformation of the shape of IPL cells into those typical of both conventional amacrine cells and those displaced to the ganglion cell layer are seen. All IPL cells are doubly immunoreactive, for ChAT and GABA, from the time they appear as a cell population within the inner plexiform layer (IPL) until their separation into the two amacrine cell populations. Polarization and early stages of shape differentiation of both types occur while they are in the IPL, starting in the dorsocentral area in the temporal retina and spreading to the rest of the retina. Three spatial gradients of differentiation are observed: from central-to-peripheral, dorsal-to-ventral, and temporal-to-nasal retina. Our findings suggest that the fate of both types of cells in the chick is determined locally, whereas their postmitotic precursors are within the IPL. The presence of GABA and acetylcholine in both types of amacrine cells at early stages of their morphogenesis, well before they have synaptic interactions, suggests a morphogenetic role for these molecules in inner retinal differentiation.     

A synaptic antigen (B16) is localized in retinal synaptic ribbons.

This morphological and biochemical study examines the cytoplasmic synaptic determinant recognized by a monoclonal antibody (B16). This antibody was generated by using an immunosuppression protocol that generates antibodies to relatively rare antigens. The B16 antibody labels structures in the brain that are dot-shaped and in the retina that resemble synaptic ribbons in their location, size, developmental emergence, and biochemical composition. The antigen is apparently conserved across species as it is found in retinas from lizards, frogs, fish, birds, mice, rats, rabbits, cats, and monkeys. This paper focuses on observations in the murine retina. Labeling in the outer plexiform layer of the retina is confined to the margin between the outer plexiform layer (OPL) and the outer nuclear layer. The labeled structure resembles a semiellipse or an arc with the open end facing the OPL and the top facing the outer nuclear layer. Overall, the arc is approximately 1 micron in length and less than 0.5 micron thick. Approximately 10% of the labeled arcs occur in a proximal stratum of the OPL and form a planar cluster that resembles a flat plaque parallel to the OPL. Five to ten arcs are found in each plaque. The arcs found within the plaques are approximately 50% smaller than the larger isolated arcs. Counterstaining with peanut agglutinin (PNA), a lectin that recognizes cone photoreceptors and their associated processes, demonstrates that the plaques are associated with the cone pedicles. Animals that have a higher ratio of cones/rods than mice demonstrate a much higher ratio of plaques/isolated arcs in the OPL. The structure labeled in the inner plexiform layer resembles a short bar (0.8 micron long by less than 0.5 micron wide) that is confined to the inner half of the inner plexiform layer in mice. The relative mobility (Mr) of the B16 antigen obtained from mouse retinal and brain tissue is 88 kD, as determined by SDS-PAGE followed by Western blotting. The mouse 88 kD protein is relatively soluble (precipitates at 70% ammonium sulphate) and elutes at a pH of 7.3 from an isoelectric focusing column. It appears that the determinant recognized by the B16 antibody is a previously undescribed synaptic protein that is associated with the synaptic ribbons in photoreceptor and bipolar terminals of most vertebrate retinas.     

Dark-rearing-induced reduction of GABA and GAD and prevention of the effect by BDNF in the mouse retina

Gamma‐aminobutyric acid (GABA) is an important retinal neurotransmitter. We studied the expression of GABA, glutamate decarboxylase 65 (GAD65) and GAD67 by immunocytochemistry and Western blot, in the retinas of control and dark‐reared C57BL/6J black mice. This study asked three questions. First, is visual input necessary for the normal expression of GABA, GAD65 and GAD67? Second, can the retina recover from the effects of dark‐rearing if returned to a normal light–dark cycle? Third, does BDNF prevent the influence of dark‐rearing on the expression of GABA and GAD? At postnatal day 10 (P10), before eye opening, GABA immunoreactivity was present in the ganglion cell layer (GCL), in the innermost rows of the inner nuclear layer (INL) and throughout the inner plexiform layer (IPL) of control and dark‐reared retinas. In P30 control retinas, GABA immunoreactivity showed similar patterns to those at P10. However, in P30 dark‐reared retinas, the density of GABA‐immunoreactive cells was lower in both the INL and GCL than in control retinas. In addition, visual deprivation retarded GABA immunoreactivity in the IPL. Western blot analysis showed corresponding differences in the levels of GAD65 but not of GAD67 expression between control and dark‐rearing conditions. In our study, dark‐rearing effects were reversed when the mice were put in normal cyclic light–dark conditions for 2 weeks. Moreover, dark‐reared retinas treated with BDNF showed normal expression of both GABA and GAD65. Our data indicate that normal expression of GABA and GAD65 is dependent on visual input. Furthermore, the data suggest that BDNF controls this dependence.     

Synaptogenesis in the retina of the cat

We have studied the development of synapses in the retina of the cat from E(embryonic day)21 to adulthood. The inner plexiform layer (IPL) could be distinguished by E36, but at this age no synapses had formed, although compact processes had formed in the IPL and membrane specialisations had developed in adjacent processes. Conventional synapses form in the IPL from E45 and become increasingly numerous and differentiated over subsequent weeks. Extracellular space and cellular debris were prominent during the formation of these synapses. The conventional synapses appear to form principally between amacrine cells until E56, when ganglion cell dendrites could be identified as postsynaptic processes. Ribbon synapses characteristic of bipolar cells were identified around birth, suggesting that bipolar cells do not form synapses until that age. The outer plexiform layer (OPL) could be distinguished in central retina at E56. Extracellular space, debris of degenerating cells and mounds of agranular vesicles were prominent at this age but synapses were not observed until E59, when cone pedicles formed ribbon synapses onto horizontal cell processes. The first synapses clearly formed by spherules, also onto horizontal cells, were seen at E62. The central process of the postsynaptic triad, considered to be the dendrite of a bipolar cell, was first observed in both cone pedicles and rod spherules around birth, again suggesting that bipolar cells do not enter into synaptic arrangements until that age. Synaptogenesis in the OPL shows a strong centro-peripheral gradient; its initial stages were observed centrally in the late E50's but synapse formation was not complete in the retinal periphery until P(postnatal day)7 or later. We could not detect a centro-peripheral gradient in the formation of conventional synapses in the IPL, but the formation of ribbon synapses in this layer began centrally at birth and in the mid-periphery at P5. In summary, the first synapses to form in the retina are those which spread information laterally within the plexiform layers, between amacrine cells and from receptor to horizontal cells. The cells which carry information centrally, in particular bipolar cells, enter into synaptic arrangements considerably later. Further, retinal cells seem to form synapses in a distinct sequence: first amacrines, then receptors and lastly bipolar cells.     

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.     

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.     

Distribution of immunoreactivity to protein kinase C in the turtle retina

Immunocytochemical staining procedures using the HRP-complexed antibody to protein kinase C (PKC) have been carried out on the turtle retina. Wholemounts and frozen sections of retina have been studied by light microscopy to evaluate PKC immunoreactivity after stimulation of the retina with light and neurotransmitters known to be active in the vertebrate retina. The most dramatically stained sites are cone synaptic pedicles and bipolar cells under all conditions. Ganglion cells stain weakly under certain conditions. Applying the antibody to a 'control' retina under dark adapted conditions results in uniform background staining of both hyperpolarizing and depolarizing bipolar pathways, while stimulating the retina with K+ under dim light conditions results in discretely stained bipolar cells and a prominent band of staining in stratum 4 of the inner plexiform layer. Stronger stimulation of bipolar cells with their terminals contributing to strata 3 and 4 and the continuous dominant band in stratum 4 can be elicited with incubation of the retina in neurotransmitter agonists, GABA and dopamine. Incubation with dopamine, in particular, brings out the putative dopaminergic amacrine cell. The only condition in which a strong band in stratum 2 can be demonstrated is under stimulation with a flashing bar of spot of light. Thus K+ and neurotransmitter stimulation elicit PKC staining in neurons contributing to the ON or depolarizing sublamina of the IPL, while intermittent flashing light stimulus is required to elicit PKC staining in the OFF or hyperpolarizing sublamina of the IPL.