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The passing of Jack Pettigrew (University of Queensland) and Bogdan Dreher (University of Sydney) within 2 weeks of each other in May 2019 provided the catalyst for documenting the roles that they and their colleagues from the Peter Bishop School played in establishing vision research as a major strength in Australian neuroscience in the second half of the 20th century., (© 2020 Wiley Periodicals, Inc.)

This paper examines the role of inhibition in generating the receptive-field properties of local edge detector (LED) ganglion cells in the rabbit retina. We confirm that the feed-forward inhibition is largely glycinergic but, contrary to a recent report, our data demonstrate that the glycinergic inhibition contributes to temporal tuning for the OFF and ON inputs to the LEDs by delaying the onset of spiking; this delay was more pronounced for the ON inputs (∼ 340 ms) than the OFF inputs (∼ 12 ms). Blocking glycinergic transmission reduced the delay to spike onset and increased the responses to flickering stimuli at high frequencies. Analysis of the synaptic conductances indicates that glycinergic amacrine cells affect temporal tuning through both postsynaptic inhibition of the LEDs and presynaptic modulation of the bipolar cells that drive the LEDs. The results also confirm that presynaptic GABAergic transmission contributes significantly to the concentric surround antagonism in LEDs; however, unlike presumed LEDs in the mouse retina, the surround is only partly generated by spiking amacrine cells.

Visual information is processed in the retina to a remarkable degree before it is transmitted to higher visual centres. Several types of retinal ganglion cells (the output neurons of the retina) respond preferentially to image motion in a particular direction, and each type of direction-selective ganglion cell (DSGC) is comprised of multiple subtypes with different preferred directions. The direction selectivity of the cells is generated by diverse mechanisms operating within microcircuits that rely on independent neuronal processing in individual dendrites of both the DSGCs and the presynaptic neurons that innervate them.

The 15-20 physiological types of retinal ganglion cells (RGCs) can be grouped according to whether they fire to increased illumination in the receptive-field center (ON cells), decreased illumination (OFF cells), or both (ON-OFF cells). The diversity of RGCs has been best described in the rabbit retina, which has three types of ON-OFF RGCs with complex receptive-field properties: the ON-OFF direction-selective ganglion cells (DSGCs), the local edge detectors, and the uniformity detectors. Here we describe a novel type of bistratified ON-OFF RGC that has not been described in either physiological or morphological studies of rabbit RGCs. These cells stratify in the ON and OFF sublaminae of the inner plexiform layer, branching at about 30% and 60% depth, between the ON and OFF arbors of the bistratified DSGCs. Similar to the ON-OFF DSGCs, these cells respond with transient firing to both bright and dark spots flashed in the receptive field but, unlike the DSGCs, they show no directional preference for moving stimuli. We have termed these cells "transient ON-OFF" RGCs. Area-response measurements show that both the ON and the OFF spike responses have an antagonistic receptive-field organization, but with different spatial extents. Voltage-clamp recordings reveal transient excitatory inputs at light ON and light OFF; this excitation is strongly suppressed by surround stimulation, which also elicits direct inhibitory inputs to the cells at light ON and light OFF. Thus the receptive-field organization is mediated both within the presynaptic circuitry and by direct feed-forward inhibition., (Copyright © 2011 Wiley-Liss, Inc.)

Using both NADPH diaphorase and anti-nNOS antibodies, we have identified-from retinal flatmounts-neuronal types in the inner retina of the chicken that are likely to be nitrergic. The two methods gave similar results and yielded a total of 15 types of neurons, comprising 9 amacrine cells, 5 ganglion cells, and 1 centrifugal midbrain neuron. Six of these 15 cell types are ubiquitously distributed, comprising 3 amacrine cells, 2 displaced ganglion cells, and a presumed orthotopic ganglion cell. The remaining nine cell types are regionally restricted within the retina. As previously reported, efferent fibers of midbrain neurons and their postsynaptic partners, the unusual axon-bearing target amacrine cells, are entirely confined to the ventral retina. Also confined to the ventral retina, though with somewhat different distributions, are the "bullwhip" amacrine cells thought to be involved in eye growth, an orthotopic ganglion cell, and two types of large axon-bearing amacrine cells whose dendrites and axons lie in stratum 1 of the inner plexiform layer (IPL). Intracellular fills of these two cell types showed that only a minority of otherwise morphologically indistinguishable neurons are nitrergic. Two amacrine cells that branch throughout the IPL are confined to an equatorial band, and one small-field orthotopic ganglion cell that branches in the proximal IPL is entirely dorsal. These findings suggest that the retina uses different processing on different regions of the visual image, though the benefit of this is presently obscure.

Transient uniformity detectors (UDs) are a unique type of retinal ganglion cell (RGC) whose maintained firing is transiently suppressed by all types of visual stimuli. In this study, we have characterized the dendritic morphology and tracer-coupling pattern of UDs that were labeled by loose-seal electroporation of Neurobiotin following functional identification in the isolated rabbit retina. The UDs have a bistratified dendritic tree, branching near the margins of the inner plexiform layer in stratum 1 (part of the OFF sublamina) and stratum 4/5 (part of the ON sublamina). Characteristically, many of the distal dendrites in the OFF arbor do not terminate there but dive recurrently back to the ON arbor. As a consequence, the ON dendritic arbor is usually twice as large as the OFF dendritic arbor in area. The UDs sometimes show homologous tracer coupling to neighboring RGCs with the same morphology, and from this material, we estimate that the UDs have a threefold dendritic field overlap and a maximum density of ~100 cells/mm2 on the peak visual streak, accounting for ~2% of RGCs in rabbit retina. The UDs also show strong heterologous tracer coupling to a novel type of amacrine cell that costratifies with the ON arbor of the UD. Consistent with their unistratified medium-field morphology, these St4/5 amacrine cells appear to be GABAergic: their somata are immunopositive for GABA but immunonegative for glycine and glycine transporter 1. We compare the dendritic morphology of the UDs to that of other types of bistratified RGCs described in rabbit retina and note that the stratification levels and distinctive recurrent dendrites closely resemble those of the "ON bistratified diving" RGCs. This raises the possibility that there are two types of RGCs with distinctive physiological properties that have almost identical bistratified dendritic morphologies.

There are two types of direction-selective ganglion cells (DSGCs) identified in the rabbit retina, which can be readily distinguished both morphologically and physiologically. The well characterized ON-OFF DSGCs respond to a broad range of image velocities whereas the less common ON DSGCs are tuned to slower image velocities. This study examined how the synaptic inputs shape the velocity tuning of DSGCs in an isolated preparation of the rabbit retina. The receptive-field properties were mapped by extracellular spike recordings and compared with the light-evoked excitatory and inhibitory synaptic conductances that were measured under voltage-clamp. The synaptic mechanisms underlying the generation of direction selectivity appear to be similar in both cell types in that preferred-direction image motion elicits a greater excitatory input and null-direction image motion elicits a greater inhibitory input. To examine the temporal tuning of the DSGCs, the cells were stimulated with either a grating drifted over the receptive-field centre at a range of velocities or with a light spot flickered at different temporal frequencies. Whereas the excitatory and inhibitory inputs to the ON-OFF DSGCs are relatively constant over a wide range of temporal frequencies, the ON DSGCs receive less excitation and more inhibition at higher temporal frequencies. Moreover, transient inhibition precedes sustained excitation in the ON DSGCs, leading to slowly activating, sustained spike responses. Consequently, at higher temporal frequencies, weaker excitation combines with fast-rising inhibition resulting in lower spike output.

Retinal ganglion cells convey information by increasing their firing in response to an optimal visual stimulus or "trigger feature." However, one class of ganglion cell responds to changes in the visual scene by decreasing its firing. These cells, termed uniformity detectors in the rabbit retina, are encountered only rarely and the synaptic mechanisms underlying their unusual responses have not been investigated. In this study, patch-clamp recordings of uniformity detectors show that the action potentials underlying the maintained firing arise within "complex spikes." Both ON and OFF visual stimuli elicit only inhibitory synaptic input, the immediate effect of which is to suppress the maintained firing. However, this inhibition also alters the properties of the "renascent" spiking by increasing the amplitude of the spikes within each burst, suggesting that the effect may increase the efficacy of spike propagation and transmission.

The visual system of birds includes an efferent projection from a visual area, the isthmo-optic nucleus in the midbrain, back to the retina. Using a combination of anterograde labeling of efferent fibers, reconstruction of dye-filled neurons, NADPH-diaphorase staining, and transmission electron microscopy, we have examined the distribution of efferent fibers and their synaptic structures in the chicken retina. We show that efferent fibers terminate strictly within the ventral retina. In two completely mapped retinas, only 2 fibers from a total of 15,359 terminated in the dorsal retina. The major synapse made by each efferent fiber is with a single efferent target amacrine cell (TC). This synapse consists of 5-25 boutons of 2 microm diameter, each with multiple active zones, pressed into the TC soma or synapsing with a basketwork of rudimentary TC dendrites in the inner nuclear layer (INL). This basketwork, which is sheathed by Muller cell processes, defines a private neuropil in the INL within which TCs were also seen to receive input from retinal neurons. In addition to the major synapse, efferent fibers typically produce several very thin processes that terminate nearby in single small boutons and for which the soma of a local amacrine cell is one of the likely postsynaptic partners. A minority of efferent fibers also give rise to a thicker process, terminating in a strongly diaphorase-positive ball about 5 microm in diameter.

Intracellular sharp-electrode, whole-cell patch clamp and juxtacellular labeling methods have previously been developed for combined analysis of neuronal structure and function. We describe a novel electroporation technique for labeling neurons with Neurobiotin, using patch electrodes in a semi-loose seal configuration (R = 100-300 MOmega) with very small amplitude pulses (50 mV). The addition of 2% Neurobiotin to the intracellular solution in the patch electrode reduces the dielectric membrane breakdown voltage threshold by about threefold. The resulting pore formation allows for (1) the stable recording of spontaneous and light-evoked postsynaptic potentials without significant cytoplasmic washout and (2) the passage of dye without spillover. The efficiency and reliability of the method makes it particularly suitable for the serial recording and labeling of multiple neurons in a small area of tissue.

Visual acuity is limited by the size and density of the smallest retinal ganglion cells, which correspond to the midget ganglion cells in primate retina and the beta-ganglion cells in cat retina, both of which have concentric receptive fields that respond at either light-On or light-Off. In contrast, the smallest ganglion cells in the rabbit retina are the local edge detectors (LEDs), which respond to spot illumination at both light-On and light-Off. However, the LEDs do not predominate in the rabbit retina and the question arises, what role do they play in fine spatial vision? We studied the morphology and physiology of LEDs in the isolated rabbit retina and examined how their response properties are shaped by the excitatory and inhibitory inputs. Although the LEDs comprise only approximately 15% of the ganglion cells, neighboring LEDs are separated by 30-40 microm on the visual streak, which is sufficient to account for the grating acuity of the rabbit. The spatial and temporal receptive-field properties of LEDs are generated by distinct inhibitory mechanisms. The strong inhibitory surround acts presynaptically to suppress both the excitation and the inhibition elicited by center stimulation. The temporal properties, characterized by sluggish onset, sustained firing, and low bandwidth, are mediated by the temporal properties of the bipolar cells and by postsynaptic interactions between the excitatory and inhibitory inputs. We propose that the LEDs signal fine spatial detail during visual fixation, when high temporal frequencies are minimal.

The On-Off direction-selective ganglion cells (DSGCs) in the rabbit retina comprise four distinct subtypes that respond preferentially to image motion in four orthogonal directions; each subtype forms a regular territorial array, which is overlapped by the other three arrays. In this study, ganglion cells in the developing retina were injected with Neurobiotin, a gap-junction-permeable tracer, and the DSGCs were identified by their characteristic type 1 bistratified (BiS1) morphology. The complex patterns of tracer coupling shown by the BiS1 ganglion cells changed systematically during the course of postnatal development. BiS1 cells appear to be coupled together around the time of birth, but, over the next 10 days, BiS1 cells decouple from each other, leading to the mature pattern in which only one subtype is coupled. At about postnatal day 5, before the ganglion cells become visually responsive, each of the BiS1 cells commonly showed tracer coupling both to a regular array of neighboring BiS1 cells, presumably destined to be DSGCs of the same subtype, and to a regular array of overlapping BiS1 cells, presumably destined to be DSGCs of a different subtype. The gap-junction intercellular communication between subtypes of DSGCs with different preferred directions may play an important role in the differentiation of their synaptic connectivity, with respect to either the inputs that DSGCs receive from retinal interneurons or the outputs that DSGCs make to geniculate neurons., (2004 Wiley-Liss, Inc.)

NADPH diaphorase (NADPHd) histochemistry labels two types of nitrergic amacrine cells in the rabbit retina. Both the large ND1 cells and the small ND2 cells stratify in the middle of the inner plexiform layer, and their overlapping processes produce a dense plexus, which makes it difficult to trace the morphology of single cells. The complete morphology of the ND1 amacrine cells has been revealed by injecting Neurobiotin into large round somata in the inner nuclear layer, which resulted in the labelling of amacrine cells whose proximal morphology and stratification matched those of the ND1 cells stained by NADPHd histochemistry. The Neurobiotin-injected ND1 cells showed strong homologous tracer coupling to surrounding ND1 cells, and double-labelling experiments confirmed that these coupled cells showed NADPHd reactivity. The ND1 amacrine cells branch in stratum 3 of the inner plexiform layer, where they produce a sparsely branched dendritic tree of 400-600 microm diameter in ventral peripheral retina. In addition, each cell gives rise to several fine beaded processes, which arise either from a side branch of the dendritic tree or from the tapering of a distal dendrite. These axon-like processes branch successively within the vicinity of the dendritic field before extending, with little or no further branching, for 3-5 mm from the soma in ventral peripheral retina. Consequently, these cells may span one-third of the visual field of each eye, and their spatial extent appears to be greater than that of most other types of axon-bearing amacrine cells injected with Neurobiotin in this study. The morphology and tracer-coupling pattern of the ND1 cells are compared with those of confirmed type 1 catecholaminergic cells, a presumptive type 2 catecholaminergic cell, the type 1 polyaxonal cells, the long-range amacrine cells, a novel type of axon-bearing cell that also branches in stratum 3, and a type of displaced amacrine cell that may correspond to the type 2 polyaxonal cell., (Copyright 2004 Wiley-Liss, Inc.)

The type 1 polyaxonal (PA1) cell is a distinct type of axon-bearing amacrine cell whose soma commonly occupies an interstitial position in the inner plexiform layer; the proximal branches of the sparse dendritic tree produce 1-4 axon-like processes, which form an extensive axonal arbor that is concentric with the smaller dendritic tree (Dacey, 1989; Famiglietti, 1992a,b). In this study, intracellular injections of Neurobiotin have revealed the complete dendritic and axonal morphology of the PA1 cells in the rabbit retina, as well as labeling the local array of PA1 cells through homologous tracer coupling. The dendritic-field area of the PA1 cells increased from a minimum of 0.15 mm2 (0.44-mm equivalent diameter) on the visual streak to a maximum of 0.67 mm2 (0.92-mm diameter) in the far periphery; the axonal-field area also showed a 3-fold variation across the retina, ranging from 3.1 mm2 (2.0-mm diameter) to 10.2 mm2 (3.6-mm diameter). The increase in dendritic- and axonal-field size was accompanied by a reduction in cell density, from 60 cells/mm2 in the visual streak to 20 cells/mm2 in the far periphery, so that the PA1 cells showed a 12 times overlap of their dendritic fields across the retina and a 200-300 times overlap of their axonal fields. Consequently, the axonal plexus was much denser than the dendritic plexus, with each square millimeter of retina containing approximately 100 mm of dendrites and approximately 1000 mm of axonal processes. The strong homologous tracer coupling revealed that approximately 45% of the PA1 somata were located in the inner nuclear layer, approximately 50% in the inner plexiform layer, and approximately 5% in the ganglion cell layer. In addition, the Neurobiotin-injected PA1 cells sometimes showed clear heterologous tracer coupling to a regular array of small ganglion cells, which were present at half the density of the PA1 cells. The PA1 cells were also shown to contain elevated levels of gamma-aminobutyric acid (GABA), like other axon-bearing amacrine cells.

Direction-selective retinal ganglion cells (DSGCs) respond to image motion in a "preferred" direction but not the opposite "null" direction. Extracellular spike recordings from rabbit DSGCs suggested that the key mechanism underlying the directional responses is spatially offset inhibition projecting in the null direction. Recent patch-clamp recordings have shown that this inhibition, which acts directly on the DSGC, is already direction selective. Dual recordings established that the inhibition arises from starburst amacrine cells (SBACs) located on the null side of the DSGC but not from those on the preferred side. Thus, for each radially symmetric SBAC, processes pointing in different directions would provide the null-direction inhibition to subtypes of DSGCs with different preferred directions. Ca2+ imaging revealed that the SBAC terminal processes respond more strongly to image motion away from the soma than towards the soma, therefore accounting for the direction selectivity of the inhibitory input to the DSGCs.

The synaptic conductance of the On-Off direction-selective ganglion cells was measured during visual stimulation to determine whether the direction selectivity is a property of the circuitry presynaptic to the ganglion cells or is generated by postsynaptic interaction of excitatory and inhibitory inputs. Three synaptic asymmetries were identified that contribute to the generation of direction-selective responses: (1) a presynaptic mechanism producing stronger excitation in the preferred direction, (2) a presynaptic mechanism producing stronger inhibition in the opposite direction, and (3) postsynaptic interaction of excitation with spatially offset inhibition. Although the on- and off-responses showed the same directional tuning, the off-response was generated by all three mechanisms, whereas the on-response was generated primarily by the two presynaptic mechanisms. The results indicate that, within a single neuron, different strategies are used within distinct dendritic arbors to accomplish the same neural computation.

The neuronal circuitry underlying the generation of direction selectivity in the retina has remained elusive for almost 40 years. Recent studies indicate that direction selectivity may be established within the radial dendrites of 'starburst' amacrine cells and that retinal ganglion cells may acquire their direction selectivity by the appropriate weighting of excitatory and inhibitory inputs from starburst dendrites pointing in different directions. If so, this would require unexpected complexity and subtlety in the synaptic connectivity of these CNS neurons.

Direction-selective ganglion cells (DSGCs) in the retina respond strongly when stimulated by image motion in a preferred direction but are only weakly excited by image motion in the opposite null direction. Such coding represents an early manifestation of complex information processing in the visual system, but the cellular locus and the synaptic mechanisms have yet to be elucidated. We recorded the synaptic activity of DSGCs using strategies to observe the asymmetric inhibitory inputs that underlie the generation of direction selectivity. The critical nonlinear interactions between the excitatory and inhibitory inputs took place postsynaptically within the dendrites of the DSGCs.

The cholinergic amacrine cells in the rabbit retina slowly accumulate glycine to very high levels when the tissue is incubated with excess sarcosine (methylglycine), even though these cells do not normally contain elevated levels of glycine and do not express high-affinity glycine transporters. Because the sarcosine also depletes the endogenous glycine in the glycine-containing amacrine cells and bipolar cells, the cholinergic amacrine cells can be selectively labeled by glycine immunocytochemistry under these conditions. Incubation experiments indicated that the effect of sarcosine on the cholinergic amacrine cells is indirect: sarcosine raises the extracellular concentration of glycine by blocking its re-uptake by the glycinergic amacrine cells, and the excess glycine is probably taken-up by an unidentified low-affinity transporter on the cholinergic amacrine cells. Neurobiotin injection of the On-Off direction-selective (DS) ganglion cells in sarcosine-incubated rabbit retina was combined with glycine immunocytochemistry to examine the dendritic relationships between the DS ganglion cells and the cholinergic amacrine cells. These double-labeled preparations showed that the dendrites of the DS ganglion cells closely follow the fasciculated dendrites of the cholinergic amacrine cells. Each ganglion cell dendrite located within the cholinergic strata is associated with a cholinergic fascicle and, conversely, there are few cholinergic fascicles that do not contain at least one dendrite from an On-Off DS cell. It is not known how the dendritic co-fasciculation develops, but the cholinergic dendritic plexus may provide the initial scaffold, because the dendrites of the On-Off DS cells commonly run along the outside of the cholinergic fascicles., (Copyright 2000 Wiley-Liss, Inc.)

The regulation of electrical coupling between retinal neurons appears to be an important component of the neuronal mechanism of light adaptation, which enables the retina to operate efficiently over a broad range of light intensities. The information about the ambient light conditions has to be transmitted to the neuronal network of the retina and previous evidence has indicated that dopamine is an important neurochemical signal. In addition, recent studies suggest that another important chemical signal is retinoic acid, which is a light-correlated byproduct of the phototransduction cycle. This review summarizes the latest findings about the effects of dopamine and retinoic acid on gap junctional coupling in the retinas of mouse, rabbit and fish.

Some of the best evidence that different types of gap junction proteins (connexins) interact with each other in vivo has been found in the eye. This review focuses on three diverse ocular tissues that may contain heterotypic or heteromeric gap junction channels. Each of the tissues uses gap junctions in a superlative fashion: The crystalline lens has an exceptionally high density of gap junctions; the ciliary body expresses a surprising variety of connexins; the neural retina shows remarkable specificity in the patterns of intercellular coupling.

Horizontal cells in an isolated wholemount preparation of the mouse retina were injected with Lucifer yellow and neurobiotin to characterize both the pattern of gap junctional connectivity and its regulation by dopamine. The injected horizontal cells had a uniform morphology of a round cell body, a compact dendritic tree, and an axon, which could sometimes be traced to an expansive terminal system. The dendro-dendritic gap junctions between neighboring cells mediated both weak Lucifer yellow dye coupling and strong neurobiotin tracer coupling. The extent of the tracer coupling was decreased by either exogenous dopamine (100 microM) or cyclic adenosine monophosphate (cAMP) analogs and was significantly increased by the D1 antagonist SCH 23390 (10 microM). These results provide the first evidence in the mammalian retina that the gap junctions between horizontal cells are endogenously regulated by dopamine, which acts through D1 receptors to increase the intracellular cAMP. It has been proposed that the gap junctional coupling between horizontal cells is mediated by connexin 32 (Cx32), but the pattern and dopaminergic regulation of horizontal cell coupling were unaffected in Cx32-knockout mice, ruling out the possible involvement of Cx32. Every tracer-coupled horizontal cell showed calbindin immunoreactivity, and vice versa, providing strong evidence that the horizontal cells in the mouse retina comprise a single cell type. Like the axonless horizontal cells in other mammalian retinas, the axon-bearing horizontal cells in the mouse retina are coupled by gap junctions that are permeable to Lucifer yellow and dopamine sensitive, suggesting that the mouse horizontal cells have hybrid properties to compensate for the absence of axonless horizontal cells.

We have characterized a distinctive type of bistratified amacrine cell in the rabbit retina at both the single cell and population levels. These cells correspond to the "fountain" amacrine cells recently identified by MacNeil and Masland (1998). The fountain cells can be distinguished in superfused retinal wholemounts labeled with nuclear dyes, thus enabling them to be targeted for intracellular injection with Neurobiotin. This revealed that the primary dendrites ascend steeply to sublamina b of the inner plexiform layer, where they form an irregular arbor at the border of strata 4 and 5. These dendrites then give rise to multiple varicose processes that descend obliquely to sublamina a, where they form a more extensive arbor in stratum 1. The fountain amacrine cells show strong homologous tracer coupling when injected with Neurobiotin, and this has enabled us to map their density distribution across the retina and to examine the dendritic relationships between neighboring cells. The fountain amacrine cells range in density from 90 to 360 cells/mm2 and they account for 1.5% of the amacrine cells in the rabbit retina. The thick tapering dendrites in sublamina b form highly territorial arbors that tile the retina with minimal overlap, whereas the thin varicose processes intermingle in sublamina a. The fountain cells are immunopositive for y-aminobutyric acid and immunonegative for glycine. We further propose that these cells are homologous to the substance P-immunoreactive (SP-IR) amacrine cells in the cat retina and that they may account for a subset of the SP-IR amacrine cells in the rabbit retina.

We have characterized a distinctive type of bistratified amacrine cell in the rabbit retina at both the single cell and population levels. These cells correspond to the "fountain" amacrine cells recently identified by MacNeil and Masland (1998). The fountain cells can be distinguished in superfused retinal wholemounts labeled with nuclear dyes, thus enabling them to be targeted for intracellular injection with Neurobiotin. This revealed that the primary dendrites ascend steeply to sublamina b of the inner plexiform layer, where they form an irregular arbor at the border of strata 4 and 5. These dendrites then give rise to multiple varicose processes that descend obliquely to sublamina a, where they form a more extensive arbor in stratum 1. The fountain amacrine cells show strong homologous tracer coupling when injected with Neurobiotin, and this has enabled us to map their density distribution across the retina and to examine the dendritic relationships between neighboring cells. The fountain amacrine cells range in density from 90 to 360 cells/mm2 and they account for 1.5% of the amacrine cells in the rabbit retina. The thick tapering dendrites in sublamina b form highly territorial arbors that tile the retina with minimal overlap, whereas the thin varicose processes intermingle in sublamina a. The fountain cells are immunopositive for gamma-aminobutyric acid and immunonegative for glycine. We further propose that these cells are homologous to the substance P-immunoreactive (SP-IR) amacrine cells in the cat retina and that they may account for a subset of the SP-IR amacrine cells in the rabbit retina.

In the retina, all-trans retinoic acid (at-RA) could function as a light signal because its production increases with the level of illumination. Given the well-established effects of retinoic acid on cell coupling in other tissues, it is possible that the changing levels of at-RA modulate the gap junctional permeability between retinal neurons. This study examines the effects of retinoic acid on horizontal cell coupling, which is known to be modulated by the ambient light level. Single horizontal cells were injected under visual control with either Neurobiotin (mouse retina) or Lucifer yellow (rabbit retina) and the extent of tracer coupling or dye coupling was used to monitor the gap junctional permeability. In the mouse retina, the injection of Neurobiotin revealed a network of approximately 150-250 tracer-coupled horizontal cells. The tracer coupling was completely abolished by incubating the retina in 150 microM at-RA for 35 min. In the rabbit retina, the injection of Lucifer yellow into A-type horizontal cells revealed networks of approximately 15-30 dye-coupled horizontal cells. Incubation in 150 microM at-RA reduced the dye coupling within 12 min and complete uncoupling was achieved after 35 min. The uncoupling effects of at-RA in the mouse and rabbit retinas were concentration- and time-dependent and they were reversible after washout. The coupling was not affected by either the 9-cis form of retinoic acid or by at-RA that had been isomerized by intensive light. The uncoupling effect of at-RA persisted following treatment with a D1 receptor antagonist and thus was dopamine-independent. This study has established that at-RA is able to modulate the gap junctional permeability between horizontal cells in the mammalian retina, where its light-dependent release has already been demonstrated.

The retina is a model system for studying gap junctional intercellular communication in the CNS. The cellular coupling can be graphically visualized in retinal whole mounts by injecting small cationic tracers into microscopically identified neurons; the pattern of tracer coupling shown by each type of retina neuron is highly stereotyped, with many types of amacrine cells and ganglion cells showing complex patterns of both homologous and heterologous coupling. Parallel physiological studies have demonstrated that the gap junctions can be modulated dynamically by neurotransmitters and by the level of ambient illumination. Taken together, the numerous structural and functional studies on gap junctions in the retina provide powerful support for the concept that electrical synapses are complex components of neuronal circuits, having many of the attributes normally ascribed to chemical synapses.

Although all bipolar cells in the retina probably use the excitatory transmitter glutamate, approximately half of the cone bipolar cells also contain elevated levels of the inhibitory transmitter glycine. Some types of cone bipolar cells make heterologous gap junctions with rod amacrine cells, which contain elevated levels of glycine, leading to the hypothesis that the bipolar cells obtain their glycine from amacrine cells. Experimental support for this hypothesis is now provided by three independent lines of evidence. First, the glycine transporter GLYT1 is expressed by the glycine-containing amacrine cells but not by the glycine-containing bipolar cells, suggesting that only the amacrine cells are functionally glycinergic. Second, the gap-junction blocker carbenoxolone greatly reduces exogenous 3H-glycine accumulation into the bipolar cells but not the amacrine cells. Moreover, when the endogenous glycine stores in both cell classes are depleted by incubating the retina with a glycine-uptake inhibitor, carbenoxolone blocks the subsequent glycine replenishment of the bipolar cells but not the amacrine cells. Third, intracellular injection of rod amacrine cells with the gap-junction permeant tracer Neurobiotin secondarily labels a heterogenous population of cone bipolar cells, all of which show glycine immunoreactivity. Taken together, these findings indicate that the elevated glycine in cone bipolar cells is not derived by high-affinity uptake or de novo synthesis but is obtained by neurotransmitter coupling through gap junctions with glycinergic amacrine cells. Thus transmitter content may be an unreliable indicator of transmitter function for neurons that make heterologous gap junctions.

Gap-junction coupling between retinal neurons of the same (homologous) or different (heterologous) type can be modulated dynamically by neurotransmitters and by the level of ambient illumination. The homologous coupling of both horizontal cells and rod (AII) amacrine cells is reduced by dopamine, but there appear to be cell and species differences in how the ambient illumination interacts with the dopaminergic system to modulate the coupling. Both the homologous coupling of horizontal cells and the heterologous coupling between AII amacrine cells and cone bipolar cells are reduced by nitric oxide, but it is not known if changes in illumination modulate coupling through an endogenous nitrergic mechanism. The heterologous coupling between rod and cone photoreceptors is modulated by illumination, but the endogenous mechanism has not been identified., (Copyright 1998 Academic Press.)

Direction selectivity and orientation selectivity were examined in the direction-selective (DS) and orientation-selective (OS) ganglion cells in the rabbit retina. Spike activities were recorded in vivo using tungsten-in-glass electrodes. Three types of visual stimuli (flashing slit, moving edges behind a slit, and whole-field drifting gratings) were used to distinguish these attributes. Fourier analysis was performed on data obtained using drifting gratings as proposed by Worgotter and Eysel (1987). Results from both angular and Fourier (polar angle frequency) domains were consistent. DS cells had strong directional components and varying strength of orientational components; they also had intact inhibitory surrounds. The phase of the biased orientation did not have a consistent relationship with the preferred direction. OS cells had predominant orientational components and very weak directional components. We conclude that the orientation bias does not contribute to the generation of direction selectivity, and that the mechanism of this bias of DS cells is rather different from the mechanism that generates orientation selectivity in rabbit OS cells. There are indications that it is similar to the mechanism shown to cause orientation bias in cat concentric cells.

In the rabbit retina, the nuclear dye, 4,6,diamidino-2-phenylindole (DAPI), selectively labels a third type of amacrine cell, in addition to the previously characterized type a and type b cholinergic amacrine cells. In this study, these "DAPI-3" amacrine cells have been characterized with respect to their somatic distribution, dendritic morphology, and neurotransmitter content by combining intracellular injection of biotinylated tracers with wholemount immunocytochemistry. There are about 100,000 DAPI-3 amacrine cells in total, accounting for 2% of all amacrine cells in the rabbit retina, and their cell density ranges from about 130 cells/mm2 in far-peripheral retina to 770 cells/mm2 in the visual streak. The thin varicose dendrites of the DAPI-3 amacrine cells form a convoluted dendritic tree that is symmetrically bistratified in S1/S2 and S4 of the inner plexiform layer. Tracer coupling shows that the DAPI-3 amacrine cells have a fivefold dendritic-field overlap in each sublamina, with the gaps in the arborization of each cell being occupied by dendrites from neighboring cells. The DAPI-3 amacrine cells consistently show the strongest glycine immunoreactivity in the rabbit retina and they also accumulate exogenous [3H]-glycine to a high level. By contrast, the AII amacrine cells, which are the best characterized glycinergic cells in the retina, are amongst the most weakly labelled of the glycine-immunopositive amacrine cells. The DAPI-3 amacrine cells costratify narrowly with the cholinergic amacrine cells and the On-Off direction-selective ganglion cells, suggesting that they may play an important role in movement detection.

In this study, we show that specific antibodies can be raised against paraformaldehyde conjugates of amino acids, including the neurotransmitters glycine, gamma-amino-butyric acid and glutamate, and a non-neuroactive amino acid, glutamine. These antibodies against paraformaldehyde conjugates specifically detect the above amino acids in paraformaldehyde-fixed tissues. The penetration of antibodies into paraformaldehyde-fixed tissues is much superior to the penetration of antibodies into glutaraldehyde-fixed tissues; hence good labeling can be observed through the depth of the tissues. Unlike glutaraldehyde, fixation with paraformaldehyde does not give rise to high levels of tissue autofluorescence and, thus, these antibodies are very effective for immunofluorescence studies. Furthermore we suggest that the ability of these antibodies to detect amino acids in paraformaldehyde-fixed tissues will permit their use in situations where it is necessary to detect other other fixation-sensitive antigens, such as neurotransmitter receptors and transporters.

Horizontal cells mediate lateral inhibition in the outer retina, and this process is dependent on electrical coupling through gap junctions, giving rise to receptive fields that are much wider than the dendritic fields. This study on rabbit retina shows that the permeability of the gap junctions between A-type horizontal cells, as assessed by Lucifer yellow dye coupling, is modulated by dopamine through a D1 receptor linked to adenylate cyclase. Both exogenously applied dopamine and endogenously released dopamine uncoupled the horizontal cells, but the effect was pH-gated whereby it occurred only at an extracellular pH 7.2 +/- 0.05. The horizontal cells also uncoupled in acidic media (pH 7.0 or below) in the absence of dopamine. Our results show that horizontal cell coupling in the mammalian retina is regulated by both dopamine and pH. Given that the pH in the outer retina varies with the metabolic activity of the photoreceptors, these results suggest that ambient light conditions could gate the activity of neurotransmitters through pH-sensitive mechanisms.

We examined the spatial organization of the putative dopaminergic amacrine cells in the chicken retina and how this organization was affected by myopic eye enlargement. Myopia was produced by monocular lid suture for 4-7 months from hatching. Dopaminergic amacrine cells (TH-IR) were labelled by tyrosine hydroxylase immunohistochemistry. The somata of the TH-IR cells were usually located at the inner border of the inner nuclear layer; they gave rise to a dense plexus in stratum 1 (S1) of the inner plexiform layer, to a sparse plexus in stratum 3 (S3), and to short spiny dendrites at the border of strata 4 and 5 (S4/S5). The long thin processes in S1 and S3 could seldom be traced to their cell of origin, whereas the S4/S5 dendrites formed discrete fields that tiled the retina with little overlap. Lid suture resulted in retinal expansion of between 25-70%, but the total number of TH-IR amacrine cells was unaltered. Per retina, there were about 4700 TH-IR amacrine cells which showed a 3:1 density gradient from central to peripheral retina. The size of the S4/S5 dendritic fields increased proportionately in the expanded retinae, thus maintaining their coverage across the retina. The increase was achieved through scaled growth of the S4/S5 dendrites, involving both terminal and non-terminal dendrites. These findings suggest that the expansion of retinal neurons during myopia occurred through normal, albeit excessive, growth mechanisms.

Gap junctions permit the passage of ions and small molecules between cells, thereby providing a basis for direct intercellular communication. In the rabbit retina, the low molecular weight dyes Lucifer yellow and biocytin passed readily from astrocytes into adjacent astrocytes, oligodendrocytes, and Müller cells. However, the dyes rarely passed from either oligodendrocytes or Müller cells into astrocytes. Unidirectional passage of dye suggests the presence of an asymmetric barrier to the movement of molecules through heterologous gap junctions and indicates the potential for a hierarchy of command between interconnected cells.

In most vertebrate retinae, horizontal cells of the same functional type are homologously coupled through gap junctions. However, ultrastructural, physiological and dye-coupling studies have shown that the axon terminals of horizontal cells in mammalian retinae are not connected by gap junctions. In this study, intracellular injection of the junction-permeant tracer, Neurobiotin, combined with photochromic intensification of the diaminobenzidine (DAB) reaction product, has revealed that the B-type horizontal cells in rabbit retina show strong tracer coupling, both between the dendritic trees and between the axonal arborizations. These findings establish that the coupling pattern of axon-bearing horizontal cells in a mammalian retina in qualitatively similar to the coupling patterns of axon-bearing horizontal cells in non-mammalian retinae.

The diaminobenzidine (DAB) reaction product can be greatly intensified by incubating the reacted tissue in either nitro blue tetrazolium or tetranitro blue tetrazolium and then exposing the tissue to strong light. Epi-illumination through a microscope objective enables the photochromic intensification to be carried out under direct visual control, with optimal intensification taking only 10-30 s through a 20x objective. Alternatively, the whole preparation can be intensified in a few minutes by passing it back and forth under a fibre light guide. The method can be used to intensify cells that have been labelled either by immunoperoxidase techniques or with intracellular tracers such as horseradish peroxidase and neurobiotin.

The cellular morphology and topographic distribution of the rod bipolar cells in the rabbit retina have been investigated by selective labelling with protein kinase C-immunohistochemistry (Negishi et al., Neurosci, Lett. 94:247-252, 1988) and by Lucifer Yellow injection of microscopically identified cells in a superfused retinal preparation. The distribution of the rod bipolar cells parallels that of their input neurons, the rod photoreceptors, in that the rod bipolars reach maximum densities of 5,000-7,000 cells/mm2 on the inferior and superior flanks of the visual streak, dropping to slightly lower densities at the peak visual streak. The centre-to-periphery density gradient of the rod bipolars is about 2.5:1, and the density ratio of rods to rod bipolars shows little variation across the retina, ranging from 43:1 in superior retina to 58:1 in inferior retina. The dendritic field area of the rod bipolar cells increases from 600 microns2 on the visual streak to 1,200 microns2 in the far-superior retina, with each point on the retina overlapped by 2.5-3.5 dendritic fields. The axonal field area of the rod bipolar cells increases from about 100 microns2 at the peak visual streak to about 250 microns2 at the retina edge, and the axonal field coverage ranges from 0.55 in the visual streak to about 0.8 in peripheral retina. Although there appear to be gaps in the local array of rod bipolar somata, these areas are covered by the axonal arbours of neighbouring rod bipolar cells.

AII amacrine cells, which are the third-order neurons in the rod pathway, can be differentially labelled in rabbit retina by injecting Nuclear Yellow into the posterior chamber. Under ultraviolet excitation, the labelled retina appears strongly metachromatic, with the AII nuclei fluorescing silvery-yellow and the nuclei of other amacrine cells fluorescing blue. Labelled AII cells were injected with Lucifer Yellow under direct microscopic control in a superfused retinal preparation, and the dye was later photoconverted to an opaque reaction product. Rabbit AII amacrines, which number about 525,000 cells, reach a maximum density of 2,500-3,000 cells/mm2 on the peak visual streak, dropping to 400-500 cells/mm2 at the superior margin. These narrow-field amacrines have a bistratified dendritic morphology, with distinctive "lobular appendages" in sublamina a of the inner plexiform layer and wider ranging "arboreal dendrites" in sublamina b. Although the lobular field area increases 10-fold from the visual streak to the far periphery, the lobular field coverage is almost uniform across the retina, averaging 1.0 in inferior retina and 0.8 in superior retina. The dendritic field area of the arboreal dendrites also increases with eccentricity from the visual streak, but there are pronounced differences between inferior and superior retina. The arboreal fields are 2 to 3 times larger than the lobular fields throughout the inferior retina but up to 15 times larger in the superior retina. The arboreal field overlap is only 1.8 at the peak visual streak, increasing slightly to about 2.4 over most of the inferior retina; the overlap increases sharply in the superior retina, however, reaching values of 10 or more in the far periphery. Both the lobular and arboreal fields of AII cells are spaced more regularly than the somata, thus covering apparent gaps in the somatic array. An analysis of the potential convergence and divergence between rod bipolar cells and AII amacrine cells in the rabbit retina indicates that the neuronal architecture of the rod circuit is not organized in a uniform module that is simply scaled-up from central to peripheral retina. Moreover, peripheral fields in the superior and inferior retina that have equivalent densities of interneurons show markedly different rod bipolar----AII amacrine convergence ratios, with the result that many more rod photoreceptors converge on an AII amacrine cell in the superior retina than in the inferior retina.(ABSTRACT TRUNCATED AT 400 WORDS)

Mammalian retinae have a well-defined neuronal pathway that serves rod vision. In rabbit retina, the different populations of interneurons in the rod pathway can be selectively labeled, either separately or in combination. The rod bipolar cells show protein kinase C immunoreactivity; the rod (AII) amacrine cells can be distinguished in nuclear-yellow labeled retina; the rod reciprocal (S1 & S2) amacrine cells accumulate serotonin; and the dopaminergic amacrine cells show tyrosine-hydroxylase immunoreactivity. Furthermore, intracellular dye injection of the microscopically identified interneurons enables whole-population and single-cell studies to be combined in the same tissue. Using this approach, we have been able to analyze systematically the neuronal architecture of the rod circuit across the rabbit retina and compare its organization with that of the rod circuit in central cat retina. In rabbit retina, the rod interneurons are not organized in a uniform neuronal module that is simply scaled up from central to peripheral retina. Moreover, peripheral fields in superior and inferior retina that have equivalent densities of each neuronal type show markedly different rod bipolar to AII amacrine convergence ratios, with the result that many more rod photoreceptors converge on an AII amacrine cell in superior retina. In rabbit retina, much of the convergence in the rod circuit occurs in the outer retina whereas, in central cat retina, it is more evenly distributed between the inner and outer retina.

This study demonstrates that the junctional connections between rod-signal interneurons in mammalian retina can be visualized by tracer coupling, following intracellular injection of the biotinylated compounds, biocytin and Neurobiotin. In addition, many other types of retinal neurons -including B-type horizontal cells and several types of retinal ganglion cells-show specific patterns of tracer coupling, usually to cells of the same neuronal type but occasionally to cells of other neuronal classes. These findings suggest that electronic transmission occurs commonly throughout the retina and, consequently, diverse types of retinal neurons may form functional networks of coupled cells.

This study has shown that the retinae of Prototherian (egg-laying) mammals possess two neuronal types that are present in non-mammalian retinae, but absent or morphologically different in the retinae of Eutherian (placental) mammals. First, endogenous serotonin-like immunoreactivity has been localized in a population of presumptive amacrine cells in the platypus retina, the first such report in a mammalian retina. Second, the protein kinase C-immunoreactive (PKC-IR) bipolar cells in the echidna retina appear similar to the PKC-IR bipolars in the chicken retina, in that their dendrites give rise to a Landolt's club and their axons are multistratified. By contrast, the PKC-IR rod bipolar cells in the rabbit and in the brushtail possum, a Metatherian (marsupial) mammal, have no Landolt's clubs and their axons form terminal lobes in the innermost stratum of the inner plexiform layer.

The rabbit retinal ganglion cell layer contains a soma population which is morphologically distinct from the demonstrated ganglion cells. These "coronate cells" (Vaney, '80) have many features in common with classical neurons and are qualitatively different from typical glial cells. Detailed investigation by specific staining, ribonuclease treatment, and electron microscopy indicates that coronate cells are microneurons. The coronate cells contain somatic subsurface cisternae typical of rabbit amacrine cells, and are similar in appearance to some neurons of the amacrine cell layer. It is suggested that they represent, at least in part, a population of "displaced" amacrine cells.

In the ganglion cell layer of the rabbit retina, the inhibitory transmitter gamma-aminobutyric acid (GABA) and its analogues are accumulated by neurons that appear to match in size and number the population of displaced amacrine cells that synthesize the excitatory transmitter acetylcholine. In this double-label study, we have established directly that the cholinergic amacrine cells, selectively stained with diamidino-phenylindole, are strongly immunoreactive with GABA antisera. The coexistence of two classical transmitters, one excitatory and the other inhibitory, in this defined neuronal population, suggests that stimulation of the cholinergic amacrines may give rise to complex responses in their target neurons.