‘Starburst’ amacrine cells and cholinergic neurons: mirror-symmetric ON and OFF amacrine cells of rabbit retina

Golgi-impregnated 'starburst' amacrine cells share significant morphological features with cholinergic neurons in rabbit retina. They are mirror-symmetrical about the a/b (OFF/ON) sublaminar border of the inner plexiform layer. Type a starburst amacrines have cell bodies in the amacrine cell layer and dendrites in sublamina a, while type b cells have their cell bodies in the ganglion cell layer and dendrites in sublamina b of the inner plexiform layer (IPL). The two levels of narrow dendritic stratification are precisely those demonstrated by Masland and Mills for cholinergic amacrine cells. The morphological evidence indicates that the duality of ON and OFF pathways is served separately by type b (displaced) and type a starburst amacrine cells, respectively.     

ON cone bipolar cell axonal synapses in the OFF inner plexiform layer of the rabbit retina

Analysis of the rabbit retinal connectome RC1 reveals that the division between the ON and the OFF inner plexiform layer (IPL) is not structurally absolute. ON cone bipolar cells make noncanonical axonal synapses onto specific targets and receive amacrine cell synapses in the nominal OFF layer, creating novel motifs, including inhibitory crossover networks. Automated transmission electron microscopic imaging, molecular tagging, tracing, and rendering of ～400 bipolar cells reveals axonal ribbons in 36% of ON cone bipolar cells, throughout the OFF IPL. The targets include γ‐aminobutyrate (GABA)‐positive amacrine cells (γACs), glycine‐positive amacrine cells (GACs), and ganglion cells. Most ON cone bipolar cell axonal contacts target GACs driven by OFF cone bipolar cells, forming new architectures for generating ON–OFF amacrine cells. Many of these ON–OFF GACs target ON cone bipolar cell axons, ON γACs, and/or ON–OFF ganglion cells, representing widespread mechanisms for OFF to ON crossover inhibition. Other targets include OFF γACs presynaptic to OFF bipolar cells, forming γAC‐mediated crossover motifs. ON cone bipolar cell axonal ribbons drive bistratified ON–OFF ganglion cells in the OFF layer and provide ON drive to polarity‐appropriate targets such as bistratified diving ganglion cells (bsdGCs). The targeting precision of ON cone bipolar cell axonal synapses shows that this drive incidence is necessarily a joint distribution of cone bipolar cell axonal frequency and target cell trajectories through a given volume of the OFF layer. Such joint distribution sampling is likely common when targets are sparser than sources and when sources are coupled, as are ON cone bipolar cells. J. Comp. Neurol. 521:977–1000, 2013. © 2012 Wiley Periodicals, Inc.     

Pathway-specific maturation, visual deprivation, and development of retinal pathway.

One of the fundamental features of the visual system is the segregation of neural circuits that process increments and decrements of luminance into ON and OFF pathways. In mature retina, the dendrites of retinal ganglion cells (RGCs) in the inner plexiform layer (IPL) of retina are separated into ON or OFF sublamina-specific stratification. At an early developmental stage, however, the dendrites of most RGCs are ramified throughout the IPL. The maturation of RGC ON/OFF dendritic stratification requires neural activities mediated by afferent inputs from bipolar and amacrine cells. The synchronized spontaneous burst activities in early postnatal developing retina regulate RGC dendritic filopodial movements and the maintenance or elimination of dendritic processes. After eye opening, visual experience further remodels and consolidates the retinal neural circuit into mature forms. Several neurotransmitter systems, including glutamatergic, acetylcholinergic, GABAergic, and glycinergic systems, might act together to modulate the RGC dendritic refinement. In addition, both the bipolar cells and cholinergic amacrine cells may provide laminar cues for the maturation of RGC dendritic stratification.     

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)     

Morphology and tracer coupling pattern of alpha ganglion cells in the mouse retina

Alpha cells are a type of ganglion cell whose morphology appears to be conserved across a number of mammalian retinas. In particular, alpha cells display the largest somata and dendritic arbors at a given eccentricity and tile the retina as independent on- (ON) and off-center (OFF) subtypes. Mammalian alpha cells also express a variable tracer coupling pattern, which often includes homologous (same cell type) coupling to a few neighboring alpha cells and extensive heterologous (different cell type) coupling to two to three amacrine cell types. Here, we use the gap junction-permeant tracer Neurobiotin to determine the architecture and coupling pattern of alpha cells in the mouse retina. We find that alpha cells show the same somatic and dendritic architecture described previously in the mammal. However, alpha cells show varied tracer coupling patterns related to their ON and OFF physiologies. ON alpha cells show no evidence of homologous tracer coupling but are coupled heterologously to at least two types of amacrine cell whose somata lie within the ganglion cell layer. In contrast, OFF alpha cells are coupled to one another in circumscribed arrays as well as to two to three types of amacrine cell with somata occupying the inner nuclear layer. We find that homologous coupling between OFF alpha cells is unaltered in the connexin36 (Cx36) knockout (KO) mouse retina, indicating that it is not dependent on Cx36. However, a subset of the heterologous coupling of ON alpha cells and all the heterologous coupling of OFF alpha cells are eliminated in the KO retina, suggesting that Cx36 comprises most of the junctions made with amacrine 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.     

Melanopsin retinal ganglion cells receive bipolar and amacrine cell synapses

Melanopsin is a novel opsin synthesized in a small subset of retinal ganglion cells. Ganglion cells expressing melanopsin are capable of depolarizing in response to light in the absence of rod or cone input and are thus intrinsically light sensitive. Melanopsin ganglion cells convey information regarding general levels of environmental illumination to the suprachiasmatic nucleus, the intergeniculate leaflet, and the pretectum. Typically, retinal ganglion cells communicate information to central visual structures by receiving input from retinal photoreceptors via bipolar and amacrine cells. Because melanopsin ganglion cells do not require synaptic input to generate light-induced signals, these cells need not receive synapses from other neurons in the retina. In this study, we examined the ultrastructure of melanopsin ganglion cells in the mouse retina to determine the type (if any) of synaptic input these cells receive. Melanopsin immunoreaction product was associated primarily with the plasma membrane of (1) perikarya in the ganglion cell layer, (2) dendritic processes in the inner plexiform layer (IPL), and (3) axons in the optic fiber layer. Melanopsin-immunoreactive dendrites in the inner (ON) region of the IPL were postsynaptic to bipolar and amacrine terminals, whereas melanopsin dendrites stratifying in the outer (OFF) region of the IPL received only amacrine terminals. These observations suggested that rod and/or cone signals may be capable of modifying the intrinsic light response in melanopsin-expressing retinal ganglion cells.     

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.     

The morphological characterization of orientation‐biased displaced large‐field ganglion cells in the central part of goldfish retina

The vertebrate retina has about 30 subtypes of ganglion cells. Each ganglion cell receives synaptic inputs from specific types of bipolar and amacrine cells ramifying at the same depth of the inner plexiform layer (IPL), each of which is thought to process a specific aspect of visual information. Here, we identified one type of displaced ganglion cell in the goldfish retina which had a large and elongated dendritic field. As a population, all of these ganglion cells were oriented in the horizontal axis and perpendicular to the dorsal–ventral axis of the goldfish eye in the central part of retina. This ganglion cell has previously been classified as Type 1.2. However, the circuit elements which synapse with this ganglion cell are not yet characterized. We found that this displaced ganglion cell was directly tracer‐coupled only with homologous ganglion cells at sites containing Cx35/36 puncta. We further illustrated that the processes of dopaminergic neurons often terminated next to intersections between processes of ganglion cells, close to where dopamine D1 receptors were localized. Finally, we showed that Mb1 ON bipolar cells had ribbon synapses in the axonal processes passing through the IPL and made ectopic synapses with this displaced ganglion cell that stratified into stratum 1 of the IPL. These results suggest that the displaced ganglion cell may synapse with both Mb1 cells using ectopic ribbon synapses and OFF cone bipolar cells with regular ribbon synapses in the IPL to function in both scotopic and photopic light conditions.     

The development of retinal ganglion cell dendritic stratification in ferrets.

A signature feature of mature ferret retinal ganglion cells (RGCs) is the stratification of their dendrites within either ON or OFF sublayers of the retinal inner plexiform layer (IPL). Dendritic stratification is achieved through the gradual restriction of RGC dendrites which initially ramify throughout the IPL. We examined the time course of stratification by retrogradely labeling ferret retinas with DiI at various postnatal ages. Stratification of beta and alpha RGC dendrites into either the ON or OFF sublayers of the IPL begins around postnatal day 5, when class-specific morphologies begin to emerge, and is largely completed by eye opening, at the end of the first postnatal month. Our results imply that dendritic stratification of ferret ON and OFF RGCs, as in other mammals, occurs independently of visually driven activity.     

Synaptic inputs onto small bistratified (blue‐ON/yellow‐OFF) ganglion cells in marmoset retina

The inner plexiform layer of the retina contains functional subdivisions, which segregate ON and OFF type light responses. Here, we studied quantitatively the ON and OFF synaptic input to small bistratified (blue‐ON/yellow‐OFF) ganglion cells in marmosets (Callithrix jacchus). Small bistratified cells display an extensive inner dendritic tier that receives blue‐ON input from short‐wavelength‐sensitive (S) cones via blue cone bipolar cells. The outer dendritic tier is sparse and is thought to receive yellow‐OFF input from medium (M)‐ and long (L)‐wavelength‐sensitive cones via OFF diffuse bipolar cells. In total, 14 small bistratified cells from different eccentricities were analyzed. The cells were retrogradely labeled from the koniocellular layers of the lateral geniculate nucleus and subsequently photofilled. Retinal preparations were processed with antibodies against the C‐terminal binding protein 2, the AMPA receptor subunit GluR4, and/or gephyrin to identify bipolar and/or amacrine input. The results show that the synaptic input is evenly distributed across the dendritic tree, with a density similar to that reported previously for other ganglion cell types. The population of cells showed a consistent pattern, where bipolar input to the inner tier is about fourfold greater than bipolar input to the outer tier. This structural asymmetry of bipolar input may help to balance the weight of cone signals from the sparse S cone array against inputs from the much denser M/L cone array. J. Comp. Neurol. 517:655–669, 2009. © 2009 Wiley‐Liss, Inc.     

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.     

Glycine receptor-mediated synaptic transmission regulates the maturation of ganglion cell synaptic connectivity

It is well documented that neuronal activity is required for the developmental segregation of retinal ganglion cell (RGC) synaptic connectivity with ON and OFF bipolar cells in mammalian retina. Our recent study showed that light deprivation preferentially blocked the developmental RGC dendritic redistribution from the center to sublamina a of the inner plexiform layer (IPL). To determine whether OFF signals in visual stimulation are required for OFF RGC dendritic development, the light-evoked responses and dendritic stratification patterns of RGCs in Spastic mutant mice, in which the OFF signal transmission in the rod pathway is largely blocked due to a reduction of glycine receptor (GlyR) expression, were quantitatively studied at different ages and rearing conditions. The dendritic distribution in the IPL of these mice was indistinguishable from wildtype controls at the age of postnatal day (P)12. However, the adult Spastic mutants had altered RGC light-evoked synaptic inputs from ON and OFF pathways, which could not be mimicked by pharmacologically blocking of glycinergic synaptic transmission on age-matched wildtype animals. Spastic mutation also blocked the developmental redistribution of RGC dendrites from the center to sublamina a of the IPL, which mimicked the effects induced by light deprivation on wildtype animals. Moreover, light deprivation of the Spastic mutants had no additional impact on the RGC dendritic distribution and light response patterns. We interpret these results as that visual stimulation regulates the maturation of RGC synaptic activity and connectivity primarily through GlyR-mediated synaptic transmission.     

Morphological and physiological properties of enhanced green fluorescent protein (EGFP)‐expressing wide‐field amacrine cells in the ChAT‐EGFP mouse line

Mammalian retinas comprise a variety of interneurons, among which amacrine cells represent the largest group, with more than 30 different cell types each exhibiting a rather distinctive morphology and carrying out a unique function in retinal processing. However, many amacrine types have not been studied systematically because, in particular, amacrine cells with large dendritic fields, i.e. wide‐field amacrine cells, have a low abundance and are therefore difficult to target. Here, we used a transgenic mouse line expressing the coding sequence of enhanced green fluorescent protein under the promoter for choline acetyltransferase (ChAT‐EGFP mouse) and characterized a single wide‐field amacrine cell population monostratifying in layer 2/3 of the inner plexiform layer (WA‐S2/3 cell). Somata of WA‐S2/3 cells are located either in the inner nuclear layer or are displaced to the ganglion cell layer and exhibit a low cell density. Using immunohistochemistry, we show that WA‐S2/3 cells are presumably GABAergic but may also release acetylcholine as their somata are weakly positive for ChAT. Two‐photon‐guided patch‐clamp recordings from intact retinas revealed WA‐S2/3 cells to be ON‐OFF cells with a homogenous receptive field even larger than the dendritic field. The large spatial extent of the receptive field is most likely due to the extensive homologous and heterologous coupling among WA‐S2/3 cells and to other amacrine cells, respectively, as indicated by tracer injections. In summary, we have characterized a novel type of GABAergic ON‐OFF wide‐field amacrine cell which is ideally suited to providing long‐range inhibition to ganglion cells due to its strong coupling.     

Characterization of neuropeptide Y-expressing cells in the mouse retina using immunohistochemical and transgenic techniques

The amacrine cells of the retina are a complex family of interneurons. They are made up of numerous subgroups, each with different morphologic and/or biochemical properties and each presumably serving a different function. In this study, we characterized one subgroup, defined by its expression of a peptide, neuropeptide Y (NPY). The cells were identified using antibodies to NPY and characterized using a transgenic mouse line that expressed the reporter enzyme, beta-galactosidase, in the NPY-immunoreactive (NPY-IR) cells. We found that NPY-IR cells were present in two layers, the inner nuclear layer (INL) and the ganglion cell layer (GCL). The cells in both layers were densely distributed, with those in the INL having a mean density of 1452 +/- 65 cells/mm(2), and those in the GCL having a mean density of 644 +/- 41 cells/mm(2). The cells in the INL extended their processes in the sublamina of the inner plexiform layer (IPL) closest to the INL/IPL border, the presumptive OFF sublamina, and the cells in the GCL extended their processes in the sublamina near the GCL/IPL border, the presumptive ON sublamina. Both populations of cells were immunoreactive to a GABA transporter and, thus, likely GABAergic. The high density of these cells suggests that they play a prominent role in IPL processing. The location of their processes suggests that one population acts in the pathway that mediates OFF responses, and the other in the pathway that mediates ON responses, and their expression of a GABA marker indicates that their actions are likely inhibitory.     

Amacrine cells in the tiger salamander retina: morphology, physiology, and neurotransmitter identification.

Amacrine cells of the vertebrate retina comprise multiple neurochemical types. Yet details of their electrophysiological and morphology properties as they relate to neurotransmitter content are limited. This issue of relating light responsiveness, dendritic projection, and neurotransmitter content has been addressed in the retinal slice preparation of the tiger salamander. Amacrine cells were whole-cell clamped and stained with Lucifer yellow (LY), then processed to determine their immunoreactivity (IR) to GABA, glycine, dopamine or tyrosine hydroxylase (TOH), and glucagon antisera. Widefield, ON-OFF amacrine cells were glycine-IR. The processes of these cells extended laterally in the inner plexiform layer (IPL) from 250-600 microns. They were either multistratified in the IPL or monostratified near the IPL midline. Three multistratified ON-OFF narrowfield glycine-IR cells also were found. Four types of ON amacrine cells were found to be GABA-IR; all types had their processes concentrated in the proximal IPL (sublamina b). Type I cells were narrowfield (approximately 100 microns) with a compact projection. Type II cells were widefield (220-300 microns) with a sparse projection. Type III cells had an asymmetrical projection and varicose processes. Type IV cells were pyriform and monostratified in sublamina b. One narrowfield ON-OFF amacrine cell, with processes broadly distributed in the middle of the IPL, was GABA-IR. This cell appeared similar to an ON-OFF cell that was glycine-IR and may comprise a type in which GABA and glycine colocalize. Another class of amacrine cell, with processes forming a major plexus along the distal border of the IPL and a lesser plexus in the proximal IPL, produced slow responses at light ON and OFF; these cells were dopamine/TOH-IR. A narrowfield class of transient ON-OFF amacrine cell, with processes ramifying throughout both sublaminae a and b of the IPL, were glucagon-IR; these cells appeared to be dye-coupled at the soma. We have shown that, with respect to GABA, glycine, dopamine, and glucagon, salamander amacrine cells fall into rather discrete groups on the basis of ramification patterns in the IPL and responses to photic stimulation. The physiological, structural, and neurochemical diversity of amacrine cells is indicative of multiple and complex roles in retinal processing.     

Polyaxonal amacrine cells of rabbit retina: morphology and stratification of PA1 cells.

Polyaxonal amacrine cells are a new class of amacrine cell bearing one to six branching, axon-like processes, closely resembling the axons of Golgi type II cells found elsewhere in the central nervous system. Of the four types of polyaxonal amacrine cell that we have recognized in rabbit retina, three have been described previously in brief communications, and one is the subject of this paper. Type 1 polyaxonal (PA1) amacrine cells have larger cell bodies than most amacrine cells in Golgi preparations, averaging about 13 microns in diameter. These are typically positioned interstitially in the middle of the inner plexiform layer (IPL), although some are also found in the amacrine and ganglion cell layers. Axons and dendrites are broadly stratified in the middle of the IPL, in the vicinity of the a/b sublaminar border. Sparsely branching dendrites have a conventional appearance, branching at a narrow angle, and giving rise to smaller daughter branches, which taper gradually toward their termination. An unusual feature of the dendrites is the zig-zag course of some terminal branches. Clusters of small, pedunculated spines are common on proximal dendrites, and spines are virtually absent on axons. Axons emerge from proximal dendrites within 50 microns of the soma, and more rarely from the soma, in a tapering initial segment, commonly interrupted by one or two large swellings. Subsequent branching is at a wide angle, and the fine caliber is maintained in the transition from parent to daughter branches. The uniform thickness of the axonal branches is interrupted at intervals by boutons en passant. Although the extent of the dendritic tree is large, exceeding 500 microns in radial extent from the cell body, for cells a few millimeters distant from the visual streak, the axonal tree is much larger, and its radial extent is measured in millimeters. PA1 amacrine cells are believed to be polarized in their functional organization, with a primarily recipient dendritic tree and a primarily transmissive axonal tree. PA1 amacrine cells co-stratify with nab cone bipolar cells and with certain small tufted amacrine and ganglion cells at the a/b sublaminar border. The co-stratification of both axons and dendrites at the a/b sublaminar border of the IPL suggests that PA1 amacrine cells are important modulators of neural activity in the middle of the IPL, affecting both ON and OFF responses, and perhaps ON-OFF cells selectively.     

Long-term treatment of the developing retina with the metabotropic glutamate agonist APB induces long-term changes in the stratification of retinal ganglion cell dendrites

The gradual restriction of initially multistratified retinal ganglion cell (RGC) dendrites into ON and OFF sublaminae of the inner plexiform layer (IPL) can be effectively blocked by treating the developing retina with 2-amino-4-phosphonobutyrate (APB), the metabotropic glutamate agonist, or by light deprivation. Previous studies have focused on the short-term consequences of such manipulations, so the long-term effects of arresting dendritic stratification on the structural development of RGCs are as yet unknown. In the present study, we have addressed this issue by performing a morphological analysis of alpha RGCs labeled by retrograde transport of horseradish peroxidase injected into the dorsal lateral geniculate nucleus of adult cats that received monocular injections of APB from postnatal (P) day 2 until P30. A large proportion of the alpha cells in the APB-treated eye (44%) were found to have multistratified dendrites that terminated in both the ON and OFF sublaminae of the IPL. The dendritic arborization pattern in the sublaminae of the IPL of these cells was asymmetric, showing a variety of forms. Immunolabeling of retinal cross-sections showed that mGLUR6 receptors appeared normal in density and location, while qualitative observation suggested an increase in the axonal arborization of rod bipolar cells. These findings indicate that long-term treatment of the neonatal retina with APB induces a long- lasting structural reorganization in retinal circuitry that most likely accounts for some of the previously described changes in the functional properties of RGCs.     

A comparison of the proximal negative response and ganglion cell responses to sinusoidal flicker

In the mudpuppy retina, sinusoidal light stimulation of the proximal negative response (PNR) demonstrates two main components whose phases are essentially the same as those of the spike discharges of ON/OFF ganglion cells. Oscillations superimposed upon these components are synchronized with the spike discharges of ON/OFF cells, but not of ON or OFF cells. Intracellular recordings from ON/OFF ganglion cells reveal slow potentials with nearly identical waveforms and phases to those of the PNR. These results, together with previously published comparisons between the flesh-evoked PNR and amacrine cell responses, support the suggestion that the PNR arises from activity in the on/off system (amacrine and/or ganglion cells).     

Connexin36 mediates gap junctional coupling of alpha-ganglion cells in mouse retina

Alpha-ganglion cells are present in all vertebrate retinae and are subdivided into ON and OFF types according to their level of dendritic ramification within the inner plexiform layer. They have large dendritic fields and usually a good responsiveness to moving stimuli. They were the first ganglion cells in which tracer coupling was observed, suggesting the presence of gap junctions composed of unknown connexins. Here we show that ON-alpha-ganglion cells in the mouse retina are coupled to amacrine cells, whereas OFF-alpha-ganglion cells are coupled to other OFF-alpha-ganglion cells and to amacrine cells. These tracer coupling patterns were completely absent in mice deficient in connexin36 (Cx36). The expression of Cx36 protein in alpha-ganglion cells but not in coupled amacrine cells was confirmed in mice in which the Cx36 coding DNA was replaced by the lacZ reporter gene. The dendritic localization and the distribution pattern of Cx36 patches, analyzed in mice in which the enhanced green fluorescent protein (EGFP) was linked to the C-terminal region of the Cx36 protein, revealed a rather small number of fluorescent plaques and different patterns for ON- and OFF-alpha-ganglion cells. Furthermore, tracer coupling between OFF-alpha-ganglion cells could be inhibited by quinine, a gap junctional blocker with a slight preference for gap junctions formed by Cx36. These data strongly suggest that Cx36 gap junction channels are functional not only in interneurons but also in output neurons of the retina and are responsible for distinct coupling patterns of ganglion cells.