The spatial distribution of excitatory and inhibitory inputs to ganglion cell dendrites in the tiger salamander retina

In response to focal stimuli, ganglion cell dendrites receive excitation over a relatively narrow extent of the inner plexiform layer (IPL). This excitation is embedded in 2 wider lateral inhibitory regions. Here we estimate the lateral dimensions of the inhibitory regions. Ganglion cells were whole-cell patch-clamped and dendrites were identified and located in retinal slices using Lucifer yellow in the pipettes. The spatial distribution of ganglion cell dendritic sensitivity was measured with puffs of transmitter substances applied at different distances along the dendrites. All ganglion cell dendrites were sensitive to glutamate, GABA, and glycine across their full extent. The responses to puffs decreased with lateral distance from the soma and were well fit by Gaussians. The responses to puffs of potassium showed a similar decrement with distance. Since potassium channels are probably uniformly distributed along the dendrites, the similarity in profiles suggests that receptor density is also uniform along the dendrites. The spatial distribution of responses of ganglion cells to excitatory and inhibitory synaptic inputs was measured by depolarizing local populations of bipolar terminals (and subsequently local populations of amacrine cells) with transretinal current (TRC). TRC-stimulating electrodes were displaced laterally, with respect to the ganglion cell soma, to generate response profiles. We estimated the dimensions of the inhibitory and excitatory signals received by the ganglion cells by removing the contributions of their dendrites, the stimulus, and other interneurons from the response profiles. The excitatory signal extended less than 100 microns, the approximate dimensions of the ganglion cell dendrites, and corresponds roughly to the width of the bipolar inputs. The GABAergic signal extended, on average, 253 microns and glycinergic signal extended, on average, 315 microns. These inhibitory signal dimensions correspond to the width of classes of amacrine cell processes measured in other studies.     

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.     

Localization of serotoninlike-immunoreactive amacrine cells in the larval tiger salamander retina

Light microscopic immunocytochemistry was used to study the populations of serotoninlike-immunoreactive cells in the larval tiger salamander retina. Of 1,135 serotonin-immunostained cells observed in transverse cryosections, 87% were identified as amacrine cells, whereas 13% were tentatively designated as displaced amacrine cells. The somas of the vast majority of serotonin-amacrine cells were situated in the innermost cell row of the inner nuclear layer. Only a few serotonin-immunostained amacrine cell somas were observed in the second row of cells from the inner nuclear layer. Serotonin-immunoreactive processes generally appeared as a diffuse plexus distributed evenly throughout all levels of the inner plexiform layer. As determined in whole-mount preparations, serotonin-amacrine cells were divisible into two populations on the basis of the diameters of their somas. Large cells (45%) ranged from 16 to 19 microns in diameter with the vast majority measuring 17-18 microns. Smaller and sometimes less intensely stained cells ranged from 14 to 16 microns in diameter with the large majority measuring 15 microns. The diameters of serotonin-displaced amacrine cells ranged from 19 to 22 microns with the large majority measuring 20 microns in diameter. An examination of whole-mount retinas revealed that serotonin-immunoreactive amacrine and displaced amacrine cells were distributed throughout the center and the periphery of the retina. The density of serotonin-amacrine cells (large and small combined) was calculated to be 173 +/- 4.5 (mean +/- standard error) cells per mm2.     

Localization of tyrosine-hydroxylase-like-immunoreactive amacrine cells in the larval tiger salamander retina

Immunocytochemistry was used to localize the populations of tyrosine-hydroxylase-like (TH)-immunoreactive cells in the tiger salamander retina. Ninety percent of these cells possessed somas that were situated in the innermost cell row of the inner nuclear layer and were classified as amacrine cells. Ten percent of TH-immunoreactive somas were located in the ganglion cell layer and were tentatively designated as those of displaced amacrine cells. The processes of TH-immunoreactive cells ramified most heavily in sublayer 1 of the inner plexiform layer, while a relatively small number of TH-labelled processes distributed in sublayers 3 and 5. Less than 1% of TH-immunoreactive cells in the amacrine cell layer exhibited a short process of somal origin that extended distally toward the outer plexiform layer. However, these processes did not cross the whole of the inner nuclear layer, and no immunolabelling was observed in the outer plexiform layer. An examination of retinal whole-mounts revealed that TH-immunoreactive amacrine and displaced amacrine cells were distributed throughout the center and periphery of the retina. The density of TH-immunolabelled amacrine cells was calculated to be 49 +/- 13 (mean +/- standard error) cells per mm2. The vast majority of TH-immunoreactive amacrine and displaced amacrine cells exhibited a stellate appearance and gave rise to three or more primary dendrites. A few TH-amacrine and displaced amacrine cells possessed two primary dendrites that emerged from opposite sides of their somas. The processes of TH-immunoreactive cells were generally poorly branched and varicose with terminal branches sometimes appearing thin and beaded. Because some TH-immunolabelled processes were very long, there was considerable overlap between the dendritic fields of neighboring TH-cells. Lastly, individual TH-immunoreactive amacrine and displaced amacrine cells were often observed in whole-mounts to provide processes that ramified at more than one level of the inner plexiform layer.     

Signal transmission from cones to amacrine cells in dark- and light-adapted tiger salamander retina

Amacrine cells (ACs) are third-order interneurons in the retina that mediate antagonistic surround inputs to retinal ganglion cells and motion-related signals in the inner retina. Previous studies have revealed that rod-to-AC signals in dark-adapted retina are mediated by a nonlinear high-gain synaptic pathway. In this study, we investigated how cone signals are transmitted to ACs under dark- and light-adapted conditions. By using the spectral subtraction method, we found that the voltage gain of the cone-AC synaptic pathway in dark-adapted salamander retina (GD) is between 28 and 72, which is about one order of magnitude lower than the voltage gain of the rod-AC pathway. This suggests that, in darkness, rod signals are more efficiently transmitted to the ACs than cone signals. The voltage gain of the cone-AC synaptic pathway in the presence of 500 nm/-2.4 background light, GL, ranges between 28 and 56. Linear regression analysis indicates that GD and GL are strongly, positively, and linearly correlated. The average GL/GD ratio is 0.73, suggesting that, on average, GL in any given AC is about 73% of GD. This adaptation-induced change in cone-AC voltage gain exemplifies use-dependent modulations of synaptic transmission in the retina, and possible mechanisms underlying light-mediated alterations of retinal synaptic function are discussed.     

Double-label analyses demonstrating the non-coexistence of enkephalin and glycine in amacrine cells of the larval tiger salamander retina

Enkephalin immunocytochemistry was combined with either glycine immunocytochemistry or autoradiography of high-affinity glycine uptake to examine for colocalization of enkephalin and glycine in amacrine cells of the larval tiger salamander retina. A total of 995 enkephalin-immunoreactive amacrine cells were visualized in double-label preparations. None of the enkephalin-labelled cells was observed to co-label for markers of glycinergic activity.     

Goalpha labels ON bipolar cells in the tiger salamander retina

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

Differential synaptic organization of GABAergic bipolar cells and non-GABAergic (glutamatergic) bipolar cells in the tiger salamander retina

The synaptic organizations of gamma-aminobutyric acid-immunoreactive (GABA-IR, GABAergic) and non-GABA-IR (non-IR, glutamatergic) bipolar cells in salamander retina were compared by postembedding immunoelectron microscopy. A total of 238 presynaptic bipolar cell synapses were studied; 61 were GABA-IR and 177 were non-IR. Both groups were similar in that (1). they made asymmetrical ribbon synapses as well as asymmetrical non-ribbon synapses; (2). they made ribbon synapses at dyads, triads, and monads; and (3). the vast majority of ribbon synapses ( approximately 90%) were with dyads. The differences were that synapses of GABA-IR bipolar cells had a higher proportion of (1). direct contact with ganglion cells, (2). non-ribbon synapses, (3). output to GABA-IR amacrine cells, and (4). output in sublamina a. Overall, the output of GABA-IR ribbons was equally split between amacrine and ganglion cell processes, whereas for non-IR ribbons, it was approximately 2:1 in favor of amacrine cells. The ribbon:non-ribbon synapse ratio was approximately 1.2:1 (33:28) for GABA-IR but approximately 2:1 (118:59) for non-IR terminals. Thus, GABA-IR bipolar cells made more direct contacts with ganglion cells and used a higher proportion of non-ribbon synapses. GABA-IR dyads were more likely to contact GABA-IR amacrine profiles (52% vs. 38%). Finally, GABA-IR ribbon synapses were more common in sublamina a than sublamina b (2:1), whereas non-IR synapses were equally present in sublaminas a and b. This differential targeting of ganglion cells and amacrine cells in the OFF vs. ON layers indicates a difference in the role of bipolar cells in the generation of receptive field properties, depending on whether or not they use GABA as well as glutamate for their transmitter.     

A double-label study demonstrating that all serotonin-like immunoreactive amacrine cells in the larval tiger salamander retina express GABA-like immunoreactivity.

A previous study localized serotonin-like immunoreactivity to amacrine cell populations in the larval tiger salamander retina. The present double-label immunocytochemical analysis of the tiger salamander retina was performed to determine if gamma-aminobutyric acid (GABA)-like immunoreactivity is expressed by serotonin-immunoreactive amacrine cells. More than 3,000 serotonin-amacrine cells were observed in double-label preparations, and all were found to express GABA-like immunoreactivity. This finding extends previous studies of serotonin-GABA coexistence in the retina by providing the first report of the co-localization of endogenous serotonin and GABA-like compounds in a retinal neuron.     

ON ganglion cells are intrinsically photosensitive in the tiger salamander retina

Intrinsically photosensitive retinal ganglion cells (ipRGCs) have been well characterized in mammalian systems, both morphologically and electrophysiologically. They show slow, sustained responses to bright light in the absence of photoreceptor‐based input, mediated by the photopigment melanopsin. Only one mammalian melanopsin gene is expressed in a small fraction of the retinal ganglion cell population, but there are two genes for melanopsin among nonmammalian vertebrates that are widely expressed in a variety of retinal and extraretinal cell types, along with other photosensitive pigments. The current study provides an electrophysiological study of ipRGCs in the larval tiger salamander (Ambystoma tigrinum), a nonmammalian vertebrate with a well‐characterized retina. The results show that the ipRGC population is equivalent to the ON ganglion cell population in the tiger salamander retina. This sheds light on the evolutionary trajectory and functional significance of intrinsic photosensitivity through the vertebrate lineage and also affects our understanding of ON cell activity and development. We have characterized the nature of the intrinsic responses of the ON cell population, compared intrinsic and synaptically based receptive fields, and quantified the spectrum of the intrinsic activity. A wider action spectrum of intrinsic photosensitivity was obtained than would be expected for a single opsin photopigment, suggesting the expression of multiple photopigments in the salamander ipRGC. J. Comp. Neurol., 2012. © 2011 Wiley Periodials, Inc.     

Wide-field amacrine cell inputs to ON parasol ganglion cells in macaque retina

Parasol cells are one of the major types of primate retinal ganglion cells. The goal of this study was to describe the synaptic inputs that shape the light responses of the ON type of parasol cells, which are excited by increments in light intensity. A connectome from central macaque retina was generated by serial blockface scanning electron microscopy. Six neighboring ON parasol cells were reconstructed, and their synaptic inputs were analyzed. On average, they received 21% of their input from bipolar cells, excitatory local circuit neurons receiving input from cones. The majority of their input was from amacrine cells, local circuit neurons of the inner retina that are typically inhibitory. Their contributions to the neural circuit providing input to parasol cells are not well-understood, and the focus of this study was on the presynaptic wide-field amacrine cells, which provided 17% of the input to ON parasol cells. These are GABAergic amacrine cells with long, relatively straight dendrites, and sometimes also axons, that run in a single, narrow stratum of the inner plexiform layer. The presynaptic wide-field amacrine cells were reconstructed, and two types were identified based on their characteristic morphology. One presynaptic amacrine cell was identified as semilunar type 2, a polyaxonal cell that is electrically coupled to ON parasol cells. A second amacrine was identified as wiry type 2, a type known to be sensitive to motion. These inputs likely make ON parasol cells more sensitive to stimuli that are rapidly changing outside their classical receptive fields.     

Electrical coupling, receptive fields, and relative rod/cone inputs of horizontal cells in the tiger salamander retina

Light responses, dendritic/axonal morphology, receptive field diameters, patterns of dye coupling, and relative rod/cone inputs of various types of horizontal cells (HCs) were studied using intracellular recording and Lucifer yellow/neurobiotin dye injection methods in the flatmount tiger salamander retina. Three physiologically and morphologically distinct types of HC entities were identified. 1) The A-type HCs are somas that do not bear axons, with average (+/-SE) soma diameters of 20.01 +/- 0.59 microm, relatively sparse and thick dendrites, and they resemble the A-type HC in mammals. The average receptive field diameter of these cells is 529.6 +/- 10.87 microm and they receive inputs predominantly from cones. 2) The B-type HCs are broad-field somas that bear thin and long axons, with average soma diameters of 17.67 +/- 0.38 microm, thinner dendrites of higher density, and they resemble the B-type HC in mammals. The average receptive field diameter of these cells is 1,633.55 +/- 37.34 microm and they receive mixed inputs from rods and cones. 3) The B-type HC axon terminals are broad-field, coarse axon terminal processes and they resemble the B-type HC axon terminal in rabbits. The average receptive field diameter of these axon terminals is 1,291.67 +/- 24.02 microm and they receive mixed inputs from rods and cones. All these types of HC are dye-coupled with adjacent HCs of the same type. Additionally, B-type HCs and axon terminals are dye-coupled with subpopulations of bipolar cells whose axon terminals ramify in the proximal half of the inner plexiform layer, raising the possibility that these HCs may send feedforward antagonistic surround responses to depolarizing bipolar cells through electrical synapses.     

Immunocytochemical analysis of GABA-positive and calretinin-positive horizontal cells in the tiger salamander retina

By using immunocytochemical techniques, we demonstrate that there are two distinct, nonoverlapping populations of horizontal cells (HCs) in the tiger salamander retina: GABA-positive cells account for about 72% and GABA-negative (calretinin-positive) cells account for 28% of the total HC somas. The calretinin-positive HCs have relatively sparse and thick dendrites: soma diameter of 19.72 +/- 0.29 microm, and soma density of 140 +/- 13 cells/mm(2), morphological features very much like the A-type HCs described in the accompanying article. The GABA-positive HCs have thinner dendritic and coarse axon-terminal-like processes of higher density: soma diameter of 18 +/- 0.18 microm, and soma density of 364 +/- 18 cells/mm(2), features that very much resemble the B-type HCs and B-type HC axon terminals in the accompanying article. By using double and triple immunostaining techniques we found that only 18% of the non-GABAergic HC dendritic clusters contact rods, whereas the remaining 82% of the dendritic clusters contact cones. This is consistent with the physiological finding in the accompanying article that the A-type HCs are cone-dominated. On the other hand, 32% of GABAergic HC dendrites contact rod pedicles and 68% contact cone pedicles, consistent with the physiological finding that B-type HCs and B-type HC axon terminals receive mixed rod/cone inputs. Detailed confocal microscope analysis shows that 4% rods, 6% principal double cones/single cones, and 100% accessory double cones contact calretinin-positive HCs, and 79% rods, 100% principal double cones, 14% accessory double cones, and 82% single cones contact GABAergic HCs. These results suggest that GABAergic and non-GABAergic HC input/output synapses differ and they may mediate different functional pathways in the outer retina.     

Vesicle-associated membrane protein isoforms in the tiger salamander retina

Vesicle associated membrane protein (VAMP; also known as synaptobrevin) is a key component of the core complex needed for docking and fusion of synaptic vesicles with the presynaptic plasma membrane. Recent work indicates that the precise complement of presynaptic proteins associated with transmitter release and their isoforms vary among synapses, presumably conferring specific functional release properties. The retina contains two types of vesicular synapses with distinct morphologic, functional, and biochemical characteristics: ribbon and conventional synapses. Although the precise complement of presynaptic proteins is known to differ between conventional and ribbon synapses and among conventional synapses, the distribution of VAMP isoforms among retinal synapses has not been determined. The expression and localization of VAMP isoforms in the salamander retina, a major model system for studies of retinal circuitry, was examined by using immunocytochemical and immunoblotting methods. Both methods indicated that at least two VAMP isoforms were expressed in salamander retina. One isoform, recognized by an immunoglobulin M antibody that recognizes both mammalian VAMP-1 and VAMP-2, was associated with photoreceptor and bipolar cell terminals as well as many conventional synapses, and probably corresponds to mammalian VAMP-2. A different VAMP isoform associated with a subset of amacrine cells, was recognized only by antibodies directed against the N-terminus of mammalian VAMP-2. An antiserum directed against the N-terminus of mammalian VAMP-1 did not specifically recognize any salamander VAMPs in either immunocytochemical or immunoblotting experiments. Heterogeneous distribution of VAMP isoforms among conventional retinal synapses was confirmed by double labeling for synapsin I, a marker for conventional synapses. These studies indicate that VAMP isoforms are expressed heterogeneously among retinal synapses but cannot account for the differences in transmitter release characteristics at ribbon and conventional synapses. These results also corroborate previous studies in Xenopus indicating that the N-terminus of nonmammalian VAMP isoforms differs from their mammalian counterparts.     

Immunocytochemical localization of polyamines in the tiger salamander retina

The polyamines spermine and spermidine are present in neural tissue, but their functions there are not well understood. Recent work suggests that the NMDA subtype of glutamate receptors, other glutamate receptor subtypes, and certain K⁺-channels, are neural targets for polyamines. To better understand the neuron-specific roles of polyamines, we have developed antibodies that interact with spermine and spermidine in aldehyde-fixed tissue and used these antibodies in immunocytochemical studies to determine the cellular localization of these polyamines in the tiger salamander retina. The affinity-purified, polyclonal antibodies were highly specific for spermine and spermidine, exhibiting < 1 % cross reactivity with putrescine, and virtually no cross-reactivity with GABA, arginine, lysine, or glutaraldehyde. Polyamine labeling was most abundant in cells in the inner half of the inner nuclear layer and in the ganglion cell layer. Some cells in the outer half of the inner nuclear layer are labeled, and there was some labeling in both synaptic layers. Double-labeling experiments indicated (1) all GABAergic amacrine cells were polyamine-positive; and (2) all ganglion cells (identified by back-filling after microinjections of rhodamine in the optic nerve) were polyamine-positive. These results are consistent with a role for polyamines as modulators of NMDA receptor function and channel function in the inner retina.     

High-voltage-activated calcium channels in Muller cells acutely isolated from tiger salamander retina

Muller cells mediate retinal function by stabilizing the ionic environment and signal glial network activity via calcium waves. Using whole-cell patch clamp recording, we describe a high-voltage-activated, slowly inactivating Ca channel current in isolated salamander Muller cells that has unusual pharmacological properties. The Ca channel current has an activation midpoint of approximately -8 mV and an inactivation midpoint of approximately -26 mV in 10 mM Ba2+. The time constant for inactivation is approximately 380 ms at potentials positive to zero. The current is blocked by Cd2+ with an EC50 of <100 nM. nisoldipine (10 microM) blocks approximately 50%, while nifedipine (1 microM), diltiazem (20 microM), and verapamil (50 microM) each block one-third of the current. In contrast to its typical actions, BayK 8644 blocks the current by approximately 25%. Blockers of other Ca channel subtypes were also tested: omega-agatoxin IVA (200 nM) blocked only 13% of the Ca channel current, while omega-conotoxin GVIA (1 microM) blocked 84% of the current. Immnohistochemistry supported the presence of alpha1A, alpha1B, alpha1C, and alpha1D Ca channel subunits. Mapping of dihydropyridine-binding sites with DM-BODIPY revealed a distribution of channels over the entire membrane of the Muller cell with a higher density at the apical region. Overall, these observations suggest either the presence of a mix of L- and N-type Ca channels or a single, unconventional HVA Ca channel subtype sharing L- and N-type Ca channel characteristics.     

Distribution and synaptic connectivity of neuropeptide Y-immunoreactive amacrine cells in the rat retina

Neuropeptide Y (NPY) is a potent bioactive peptide that is widely expressed in the nervous system, including the retina. Here we show that specific NPY immunoreactivity was localized to amacrine and displaced amacrine cells in the rat retina. Immunoreactive cells had a regular distribution across the retina and an overall cell density of 280 cells/mm(2) in the inner nuclear layer (INL) and 90 cells/mm(2) in the ganglion cell layer (GCL). In the INL, most immunoreactive cells were characterized by small cell bodies and fine processes that appeared to ramify primarily in stratum 1 of the inner plexiform layer (IPL). A few cells in the INL also ramified in stratum 3 of the IPL. In the GCL, small to medium immunoreactive cells appeared to ramify primarily in stratum 5 of the IPL. A few immunoreactive processes, originating from somata in the INL and processes in the IPL, ramified in the OPL. NPY-immunoreactive cells contained GABA immunoreactivity, and some amacrine cells also contained tyrosine hydroxylase immunoreactivity. NPY-immunostained processes were most frequently presynaptic to nonimmunostained amacrine and ganglion cell processes and postsynaptic to nonimmunostained amacrine cell processes and cone bipolar cell axonal terminals. These findings indicate that NPY immunoreactivity is present in two populations of amacrine cells, one located in the INL and the other in the GCL, and that these cells mainly form synaptic contacts with other amacrine cells. These observations suggest that NPY-immunoreactive cells participate in multiple circuits mediating visual information processing in the inner retina.     

An analysis of inputs to ON-OFF amacrine cells in the carp retina with kynurenic acid.

Amacrine cell inputs were studied in the carp retina. Responses to light of bipolar and amacrine cells in off-pathways were suppressed by 0.5 mM kynurenic acid (Kyn), while those in on-pathways were not. In ON-OFF amacrine cells, the sustained potential level during illumination shifted in the depolarizing direction. The depolarizing response elicited in off-center bipolar cells by transretinal current was also suppressed, but those elicited in amacrine cells were not. The results indicated that off-pathways are selectively suppressed by 0.5 mM Kyn at the level of receptor-bipolar synapses and demonstrated that sustained levels of ON-OFF amacrine responses are determined by a balance of inputs from on- and off-center bipolar cells.