Synaptic organization of cholinergic amacrine cells in the rhesus monkey retina

In the rhesus monkey retina, choline acetyltransferase (ChAT) immunoreactivity has been used to study the localization and synaptic organization of cholinergic neurons by both light and electron microscopy with peroxidase-antiperoxidase immunohistochemistry. ChAT-containing neurons are a type of amacrine cell with 97.5% of their cell bodies localized to the ganglion cell layer and the remainder in the inner nuclear layer. Their processes arborize in a single narrow band in the inner plexiform layer in a plane dividing the outer two-thirds from the inner one-third of this synaptic region. With electron microscopy, ChAT-immunoreactive amacrine cell processes were observed to be primarily postsynaptic to the diffuse invaginating cone bipolar cells and presynaptic to ganglion cells, although they are both post- and presynaptic to immunohistochemically unlabeled amacrine cell profiles and to ChAT-containing amacrine cell processes as well.     

Localization of neuropeptide Y1 receptor immunoreactivity in the rat retina and the synaptic connectivity of Y1 immunoreactive cells

Neuropeptide Y (NPY), an inhibitory neuropeptide expressed by a moderately dense population of wide-field amacrine cells in the rat retina, acts through multiple (Y1-y6) G-protein-coupled receptors. This study determined the cellular localization of Y1 receptors and the synaptic connectivity of Y1 processes in the inner plexiform layer (IPL) of the rat retina. Specific Y1 immunoreactivity was localized to horizontal cell bodies in the distal inner nuclear layer and their processes in the outer plexiform layer. Immunoreactivity was also prominent in cell processes located in strata 2 and 4, and puncta in strata 4 and 5 of the IPL. Double-label immunohistochemical experiments with calbindin, a horizontal cell marker, confirmed Y1 immunostaining in all horizontal cells. Double-label immunohistochemical experiments, using antibodies to choline acetyltransferase and vesicular acetylcholine transporter to label cholinergic amacrine cell processes, demonstrated that Y1 immunoreactivity in strata 2 and 4 of the IPL was localized to cholinergic amacrine cell processes. Electron microscopic studies of the inner retina showed that Y1-immunostained amacrine cell processes and puncta received synaptic inputs from unlabeled amacrine cell processes (65.2%) and bipolar cell axon terminals (34.8%). Y1-immunoreactive amacrine cell processes most frequently formed synaptic outputs onto unlabeled amacrine cell processes (34.0%) and ganglion cell dendrites (54.1%). NPY immunoreactivity in the rat retina is distributed primarily to strata 1 and 5 of the IPL, and the present findings, thus, suggest that NPY acts in a paracrine manner on Y1 receptors to influence both horizontal and amacrine cells.     

Immunocytochemical analysis of cholinergic amacrine cells in the tiger salamander retina

Cholinergic amacrine cells in the tiger salamander retina were observed for the first time by using antibodies against choline acetyltransferase (ChAT). ChAT-immunoreactive cells were present in the inner nuclear layer (INL) and in the ganglion cell layer (GCL), and the somas of the former population (average diameter = 15.13 microm) were slightly smaller than those of the latter population (average diameter = 16.42 microm). The processes of these cells form two distinct narrow bands in the inner plexiform layer (IPL), one located near 0.2 inner plexiform units (IU) and the other near 0.65-0.7 IU. Soma size, cell density and spatial distribution of ChAT-positive cells were quantitatively analyzed. Our results suggest that cholinergic amacrine cells in the salamander retina are very similar to their counter parts in other species, and they can be used as a model system for studying cholinergic functions in the visual system.     

Comparison of the localization of acetylcholinesterase and non-specific cholinesterase activities in mammalian and avian retians

The localization of acetylcholinesterase and non-specific cholinesterase was studied in the cone retinas of the pigeon and ground squirrel, and in the predominantly rod retinas of the rabbit, rat and cat. The enzymes were localized histochemically using the copper or gold thiocholine method. In the cone retinas, acetylcholinesterase was found in discrete bands in the inner plexiform layer. Cells corresponding in position and morphological detail to amacrine cells were stained for acetylcholinesterase. Cells of the ganglion cell layer that resembled displaced amacrine cells were also stained for acetylcholinesterase, as well as other cells that appeared to be ganglion cells. No stained processes could be followed from these latter cells, however. The horizontal cells were stained for non-specific cholinesterase; these cells were localized to specific areas of the retina, which differed in the two species. In the predominantly rod retinas, there was no staining for non-specific cholinesterase, but there was diffuse staining for acetylcholinesterase throughout the inner plexiform layer. In the rabbit, this staining pattern was not changed by chronic section of the optic nerve. No stained amacrine cells could be seen distinctly in any of the three species with predominantly rod retinas. After an intravenous dose of diisopropyl phosophorofluoridate sufficient to inactivate all the retinal acetylcholinesterase, however, synthesis of new enzyme could be followed in the amacrine cells and their processes. In all five species examined, therefore, the amacrine cells appeared to be the major or sole source of acetylcholinesterase for the inner plexiform layer of the retina. The possible function of this enzyme with regard to synaptic transmission in the retina is discussed.     

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.     

Immunocytochemical localization of L-glutamate decarboxylase, gamma-aminobutyric acid transaminase, cysteine sulfinic acid decarboxylase, aspartate aminotransferase and somatostatin in rat retina

The regional distribution and cellular location of GABA-synthesizing enzyme, L-glutamate decarboxylase (GAD), GABA degrading enzyme, GABA-transaminase (GABA-T), taurine synthesizing enzyme, cysteine sulfinic acid decarboxylase (CSAD), aspartate and glutamate converting enzyme, aspartate aminotransferase (AAT), and somatostatin have been visualized in the rat retina by immunocytochemical methods. GAD immunoreactivity was found to be concentrated in the inner plexiform layer. A moderate to weak staining of GAD was found in the inner nuclear layer. The distribution of GABA-T immunoreactivity was similar to that of GAD with the exception that a weak to moderate staining of GABA-T was also observed in the outer plexiform layer. CSAD immunoreactivity was seen in every layer with the heaviest staining in the inner plexiform layer, and moderate staining in the inner and outer nuclear layers and ganglion cell layer. AAT immunoreactivity was mostly concentrated in the outer nuclear layer; there was weak staining in the inner nuclear layer and inner and outer plexiform layer. Dense somatostatin staining was seen in the inner plexiform layer and moderate staining was present in the inner nuclear layer, outer plexiform layer and ganglion cell layer. These findings suggest that in rat retina, GABA-containing cells occur in some types of amacrine cells only, while taurine and somatostatin appear in both amacrine and horizontal cells. AAT immunoreactivity was primarily associated with the photoreceptor cells suggesting that AAT may be used as a marker for aspartergic/glutamergic cells and their endings in the central nervous system.     

Immunocytochemical localization of l-glutamate decar☐ylase, gamma-aminobutyric acid transaminase, cysteine sulfinic acid decar☐ylase, aspartate aminotransferase and somatostatin in rat retina

The regional distribution and cellular location of GABA-synthesizing enzyme, l-glutamate decar☐ylase (GAD), GABA degradating enzyme, GABA-transaminase (GABA-T), taurine synthesizing enzyme, cysteinesulfinic acid decar☐ylase (CSAD), aspartate and glutamate converting enzyme, aspartate aminotrasferase (AAT), and somatostatin have been visualized in the rat retina by immunocytochemical methods. GAD immunoreactivity was found to be concentrated in the inner plexiform layer. A moderate to weak staining of GAD was found in the inner nuclear layer. The distribution of GABA-T immunoreactivity was similar to that of GAD with the exception that a weak to moderate staining of GABA-T was also observed in the outer plexiform layer. CSAD immunoreactivity was seen in every layer with the heaviest staining in the inner plexiform layer, and moderate staining in the inner and outer nuclear layers and ganglion cell layer. AAT immunoreactivity was mostly concentrated in the outer nuclear layer; there was weak staining in the inner nuclear layer and inner and outer plexiform layer. Dense somatostatin staining was seen in the inner plexiform layer and moderate staining was present in the inner nuclear layer, outer plexiform layer and ganglion cell layer. These findings suggest that in rat retina, GABA-containing cells occur in some types of amacrine cells only, while taurine and somatostatin appear in both amacrine and horizontal cells. AAT immunoreactivity was primarily associated with the photoreceptor cells suggesting that AAT may be used as a marker for aspartegic/glutamergic cells and their endings in the central nervous system.     

Expression of the dopamine transporter in rat and bullfrog retinas

We examined the expression of the dopamine transporter in rat and bullfrog retinas by immunohistochemistry. In both species, the dopamine transporter was strongly expressed in somata and processes of all dopaminergic amacrine cells. In contrast, no immunoreactivity for dopamine transporter was observed in cholinergic amacrine cells. In rat dopaminergic interplexiform cells, dopamine transporter immunoreactivity was also observed on the ascending processes terminating in the outer plexiform layer. Furthermore, the labeling for dopamine transporter diffusely appeared in both outer and inner plexiform layers. This expression profile of the dopamine transporter suggests that dopamine may be taken up not only in the synapses but also extrasynaptically by dopamine transporter, diffusely distributed in both plexiform layers.     

Immunohistochemical Localization of Dopamine D Receptors in Rat Retina

We have localized the dopamine D1 receptor in rat retina using a subtype-specific monoclonal antibody. Immunolabelling can be detected in the inner and outer plexiform layers and in a number of cells in the inner nuclear layer. In the inner plexiform layer, labelled processes form four distinct horizontal bands and a series of patches. In order further to characterize the labelling pattern of the D₁ receptor antibody, double-labelling experiments were performed with antibodies against population-specific neuronal markers in the retina. Antibodies against tyrosine hydroxylase. choline acetyltransferase, calretinin, calbindin, the glutamate transporter GLT-I, protein kinase C, recoverin and parvalbumin were co-applied with the D, receptor antibody. With these cell markers we demonstrate that horizontal cells, at least three types of cone bipolar cells and a small number of amacrine cells are immunolabelled for the D1 receptor. In the inner plexiform layer, processes labelled by the D1 receptor antibody are co-stratified with processes labelled by the GLT-1 antibody. D₁ receptor-labelled processes are not co-localized with the processes of amacrine cells and ganglion cells labelled by antibodies against tyrosine hydroxylase, choline acetyltransferase or calretinin. Our results indicate that dopamine Dl receptors are localized predominantly to horizontal cells and cone bipolar cells. Furthermore, the spatial disparity between dopaminergic processes and the site of the majority of D1 receptors supports the idea that in the retina dopamine acts as a neuromodulator that diffuses through extracellular space. The localization of D1 receptors to a number of identified cell types enables future physiological work to be directed towards specific synaptic circuits within the retina.     

Cholinergic and GABAergic neurons occur in both the distal and proximal turtle retina.

Turtle retinas were processed immunocytochemically and histochemically to detect the presence of choline acetyltransferase (ChAT), acetylcholinesterase (AChE), and glutamate decarboxylase (GAD). We observed cholinergic and gamma-aminobutyric acid (GABA)ergic neurons in the proximal retina, as expected, and in the distal retina as well. ChAT immunoreactivity in the distal retina was observed within the axons and pedicles of numerous cone photoreceptors, suggesting that a population of turtle cone photoreceptors uses ACh as a neurotransmitter. Type L2 horizontal cells were immunoreactive for GAD, and their dendrites invaginated into cone pedicles. AChE histochemistry revealed processes within the outer plexiform layer which formed a loosely organized lattice. In the proximal retina, labeling for ChAT and GAD was similar to that reported by previous investigators. Processes from ChAT-labeled amacrine cells in the inner nuclear layer formed a stratum within the distal inner plexiform layer (IPL) (at 16-21% relative IPL depth), and processes from ChAT-labeled amacrines in the ganglion cell layer formed a proximal ChAT stratum (at 55-58% relative IPL depth). In addition, six AChE-labeled bands and five GAD-labeled bands were observed within the IPL of stained retinas. Therefore, we determined that the two broadest AChE-labeled bands and the two broadest GAD-labeled bands overlapped the two labeled ChAT strata. The evidence for cholinergic and GABAergic processes in both the inner plexiform layer and the outer plexiform layer, combined with electrophysiological evidence from other investigators, raises the possibility that distal retinal neurons may be involved in the encoding of directional information.     

Neuronal markers in rat retinal grafts

Rat E15 retina was grafted to the retina of adult rat hosts. After varying survival times (1 week-6 months), grafts were stained by immunohistochemistry for neurofilament 160 kDa (NF), HPC-1 (an amacrine cell marker), choline acetyltransferase (ChAT), tyrosine hydroxylase (TH), glutamic acid decarboxylase (GAD) and somatostatin-28 (SS-28). The first differentiating graft amacrine cells (cholinergic and dopaminergic) could be seen 1 week after transplantation (corresponding to postnatal day 1 = P1). The inner plexiform layer of the graft started to differentiate at 2 weeks (corresponding to P8) seen by HPC-1 and GAD staining. ChAT, TH and SS-28 immunostaining revealed an abnormal lamination pattern in the graft inner plexiform layer. Also by 2 weeks, the outer plexiform layers of the graft contained NF-immunoreactive horizontal cells. No NF-stained retinal ganglion cells could be observed in the graft. Five and 7 weeks after grafting, the transplants had obtained the same staining intensity with different markers as the host retina.     

Cholinergic amacrine cells in the developing cat retina

Antiserum directed against the ACh-synthesizing enzyme choline acetyltransferase (ChAT) was used to determine the development of cholinergic amacrine cell distributions in wholemounted kitten retinae. From birth (P0) two populations of cholinergic amacrine cells were immunolabelled; one population was located in the inner nuclear layer (INL) and the other was displaced to the ganglion cell layer (GCL). Dendrites from cholinergic amacrines in the INL stratified in the outer third of the inner plexiform layer (IPL), whilst those from displaced amacrines in the GCL stratified in the centre of the IPL. There was a centroperipheral gradient of development in both populations, and the total number of cholinergic amacrines in the GCL always exceeded that of the INL. The most dramatic increase in the number of amacrines expressing ChAT was between P0 and P5 when the numbers doubled. During the same period there was an increase in the soma size of both populations in the peripheral retina. In the GCL, cholinergic amacrines did not attain adult size until P21 whereas those of the INL reached adult proportions by P5. Overall, between P0 and P21 there was a fivefold increase in the number of immunoreactive cholinergic amacrines in the GCL and a 3.5-fold increase in those of the INL. In general, the distribution, soma size, percentage per layer, and total number of cholinergic amacrines in both populations resembled those of the adult by P21.     

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.     

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.     

Localization of choline acetyltransferase-like immunoreactivity in the embryonic chick retina

Putative sites of acetylcholine synthesis in the retina of the embryonic and posthatched chick were localized immunohistochemically with antisera to choline acetyltransferase; the resultant choline acetyltransferase-like immunoreactivity (ChAT-IR) was compared to demonstrated sites of acetyltransferase (AChE) activity, and changes were followed in localization during development. The results confirmed the early and rapid course of development of the chick's retinal cholinergic system described in previous biochemical and morphological studies. Immunoreactivity was first detected at embryonic day 6.5 in cells close to the retina's vitreal surface. By 8 days it was present in cells in two juxtaposed rows; by the ninth day the two rows were separated and immunoreactivity was evident in two subliminae of the inner plexiform layer. On the tenth day distribution was like that in the posthatched chicken, in type I cholinergic cells in the inner nuclear layer and in type II cells in the ganglion cell layer (Millar et al.: Neurosci. Lett. 61:311-316, '85), and similar to that of most vertebrates. Three days before hatching, a third population of weakly immunoreactive cells (type III cells) appeared within the inner nuclear layer. The onset of localizable ChAT-IR occurred in amacrine cells and in their processes, before the period of synaptogenesis. Acetylcholinesterase activity was localized at an earlier age than ChAT-IR, and at all ages was present in more cells. The results obtained support the view that "displaced" cholinergic amacrine cells begin to differentiate at the same time and in the same retinal region as type I cholinergic cells. Separation of the two groups is a consequence of the ramification of processes of amacrine and ganglion cells rather than a result of the secondary migration of cells between layers.     

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.     

Observations on the synaptic organization of the retina of the albino rat: a light and electron microscopic study

An electron microscopic study of the retina of the albino rat, with particular emphasis on the synaptic organization of the inner and outer plexiform layers, has been correlated with specimens impregnated with a modified Golgi technique. The central element of the photoreceptor “triad” in the outer plexiform layer is a bipolar cell dendrite. Two types of synaptic contacts were observed in the inner plexiform layer, the “dyad” ribbon synapse and the conventional synapse. The postsynaptic elements of the “dyad” consisted of an amacrine process and a ganglion cell dendrite. Conventional synapses were made by amacrine processes which were usually presynaptic to bipolar terminals. Reciprocal synapses between processes making ribbon synapses and those making conventional synapses were seen. Golgi technique revealed the presence of two types of bipolar cells, three types of amacrine cells, and one type each of horizontal and ganglion cell. These findings are discussed in relation to reported receptive field organization.     

Tyrosine hydroxylase immunoreactivity in the rhesus monkey retina reveals synapses from bipolar cells to dopaminergic amacrine cells

The synaptic organization of dopamine-containing amacrine cells in the rhesus monkey retina was studied using immunohistochemistry of tyrosine hydroxylase (TH), the rate-limiting enzyme in the catecholamine synthetic pathway. Cell bodies of the TH-containing neurons were primarily in the innermost tier of the inner nuclear layer. Their synaptic processes, confined to the outermost stratum of the inner plexiform layer, contained mostly small, clear vesicles and were presynaptic to unlabeled amacrine cell processes and cell bodies at junctions that were symmetrical. Synapses onto the TH-immunoreactive neurons were from bipolar cell axon terminals, nonimmunoreactive amacrine cell processes, and other TH-containing amacrine cells in a decreasing order of predominance. The bipolar cells were presynaptic to the TH-containing neuronal processes at ribbon synapses. The size, structure, and position of the bipolar cell axon terminals, which, like the TH-reactive processes, were narrowly confined to the outermost stratum of the inner plexiform layer, indicate that they are recently described giant bistratified bipolar cells. The identification of this bipolar cell input now provides evidence for a pathway from the outer plexiform layer to dopaminergic amacrine cells in the inner plexiform layer via a type of cone bipolar cell.     

Fluorescence and electron microscopical observations on the amine-accumulating neurons of the cebus monkey retina

The organization of the Cebus monkey regina was analysed after the intraocular injection of 5,6-dihydroxytryptamine. This amine was taken up not only by the previously known dopaminergic neurons, but also by a set of indoleamine-accumulating neurons, whose processes are confined to the inner plexiform layer. The synaptic contacts of the dopaminergic neurons were analysed in the electron microscope after the processes of the indoleamine-accumulating neurons were destroyed by the intravitreal injection of the neurotoxic indoleamine, 5,7-dihydroxytryptamine. The subsequent injection of 5,6-dihydroxytryptamine induces certain changes in the dopaminergic neurons which accumulate the substance: electron-dense cores appear in the synaptic vesicles, and increased electron-density of mitochodrial and cellular membranes is often observed. The dopaminergic neurons were found to be presynaptic to amacrine cell perikarya and processes in the inner plexiform layer. In the outer plexiform layer they were presynaptic to both bipolar and horizontal cells, but they did not contact photoreceptors. The dopaminergic neurons received synapses only in the inner plexiform layer, from amacrine cell processes. It is inferred that in Cebus most dopaminergic neurons belong to a special class of retinal neuron, the interplexiform cells, which appear to transmit information centrifugally within the retina, from the inner to the outer plexiform layers. There are considerable similarities between the synaptology of the dopaminergic interplexiform neurons in the Cebus monkey and the goldfish retina, and the function of interplexiform neurons may therefore be similar in these two species.     

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.