AII Amacrine Cells Research Articles

Visual Transmission Deficits in Mice with Targeted Disruption of the Gap Junction Gene Connexin36

In the mammalian retina, rods feed into the cone pathway through electrotonic coupling, and recent histological data suggest the involvement of connexin36 (Cx36) in this pathway. We therefore generated Cx36 null mice and monitored the functional consequences of this deficiency on early visual transmission. The homozygous mutant mice had a normally developed retina and showed no changes in the cellular organization of the rod pathway. In contrast, the functional coupling between AII amacrine cells and bipolar cells was impaired. Recordings of electroretinograms revealed a significant decrease of the scotopic b-wave in mutant animals and an increased cone threshold that is compatible with a distorted, gap junctional transmission between AII amacrine cells and cone bipolar cells. Recordings of visual evoked potentials showed extended latency in mutant mice but unaffected ON and OFF components. Our results demonstrate that Cx36-containing gap junctions are essential for normal synaptic transmission within the rod pathway.

Coupling from AII amacrine cells to ON cone bipolar cells is bidirectional

The AII amacrine cell is a critical interneuron in the rod pathway of the mammalian retina. Rod signals pass into cone pathways by means of gap junctions between AII amacrine cells and ON cone bipolar cells. Filling AII amacrine cells with Neurobiotin produces labeling of cone bipolar cells by means of these gap junctions. However, tracer injections into bipolar cells do not produce labeling of the AII network (Vaney [1997] Invest Ophthalmol Vis Sci. 38:267-273), which suggests that the AII/bipolar gap junctions allow the passage of tracer in only one direction. This mechanism stands in contrast to physiological results, which indicate that light adapted signals can pass from ON cone bipolar cells into the AII network (Xin and Bloomfield [1999] Vis Neurosci. 16:653-665). Here, we report that a variety of ON and OFF bipolar cells are sometimes anomalously coupled to the A-type horizontal cell network. These relatively rare examples do not result from dye injection errors, but seem to represent minor developmental errors. However, this provides a method to obtain Neurobiotin-filled cone bipolar cells without the necessity of impaling them with a microelectrode. Under these conditions, Neurobiotin spreads from ON cone bipolar cells into neighboring AII amacrine cells. The dye-coupled AII amacrine cells, positively identified by double labeling with an antibody against calretinin, were centered around anomalously coupled ON bipolar cells. These results indicate that AII/bipolar cell gap junctions allow tracer coupling in both directions, consistent with previous physiological results. The previous failure to detect the passage of neuronal tracer from injected bipolar cells to AII amacrine cells may reflect electrode damage or perhaps the asymmetrical voltage sensitivity of a heterotypic gap junction.

Rod pathways in the mammalian retina use connexin 36

Many neurons in the mammalian retina are coupled by means of gap junctions. Here, we show that, in rabbit retina, an antibody to connexin 36 heavily labels processes of AII amacrine cells, a critical interneuron in the rod pathway. Image analysis indicates that Cx36 is primarily located at dendritic crossings between overlapping AII amacrine cells. This finding suggests that Cx36 participates in homotypic gap junctions between pairs of AII amacrine cells. Cx36 was also found at AII/cone bipolar contacts, previously shown to be gap junction sites. This finding suggests that Cx36 participates at gap junctions that may be heterotypic. These results place an identified neuronal connexin in the context of a well-defined retinal circuit. The absence of Cx36 in many other neurons known to be coupled suggests the presence of additional unidentified connexins in mammalian neurons. Conversely, Cx36 labeling in other regions of the retina is not associated with AII amacrine cells, indicating some other cell types use Cx36. J. Comp. Neurol. 436:336–350, 2001. © 2001 Wiley-Liss, Inc.

Synaptic development in semi-dissociated cultures of rat retina

Cultured neurons provide a simpler and more accessible environment to study the synaptic physiology. However, it is not clear if development of synapses in culture is similar to that in the in vivo condition. We studied the developmental sequence and morphological differentiation of chemical synapses in semi-dissociated rat retinal cultures that consisted of dissociated neurons as well as undissociated retinal aggregates. Synapses were quantified by synaptophysin immunoreactive puncta. During second week of in vitro development the average number of chemical synapses on the cell body decreased while that on the neurites increased significantly. Conventional synapses appeared both in aggregate and in dissociated neurons, with the developmental profile similar to that reported for in vivo retina. In contrast, the development of ribbon synapses was adversely affected by the in vitro microenvironment as suggested by following observations. The ribbon synapses were more frequently found in aggregate than in dissociated neurons, and were not associated with dyadic or triadic synaptic arrangement. The photoreceptor ribbons did not contact a postsynaptic process while bipolar ribbons made single (monadic) synapses. Further, photoreceptor ribbons in dissociated neurons were late to form and took more time to mature as compared to those in the aggregate cultures. Most of the rod bipolar cells, identified by their immunoreactivity to protein kinase C (PKC), had three or more neurites. Unlike in the in vivo retina, the dissociated rod bipolar cells did not show any PKC immunoreactive varicosities, suggesting that they failed to develop a well-differentiated synaptic terminal. Interestingly, we did not find any parvalbumin positive AII amacrine cells that are normally postsynaptic to rod bipolar cells. These results show that the conventional synapses of retina, which are similar to chemical synapses in other parts of the brain, develop normally both in aggregate and dissociated neurons. However, the highly specialized ribbon synapses have more stringent developmental requirements, and their normal development may require the presence of postsynaptic neurons in their close vicinity.

Rod pathways in the mammalian retina use connexin 36

Many neurons in the mammalian retina are coupled by means of gap junctions. Here, we show that, in rabbit retina, an antibody to connexin 36 heavily labels processes of AII amacrine cells, a critical interneuron in the rod pathway. Image analysis indicates that Cx36 is primarily located at dendritic crossings between overlapping AII amacrine cells. This finding suggests that Cx36 participates in homotypic gap junctions between pairs of AII amacrine cells. Cx36 was also found at AII/cone bipolar contacts, previously shown to be gap junction sites. This finding suggests that Cx36 participates at gap junctions that may be heterotypic. These results place an identified neuronal connexin in the context of a well-defined retinal circuit. The absence of Cx36 in many other neurons known to be coupled suggests the presence of additional unidentified connexins in mammalian neurons. Conversely, Cx36 labeling in other regions of the retina is not associated with AII amacrine cells, indicating some other cell types use Cx36.

Rod vision: pathways and processing in the mammalian retina.

Bipolar cells in the mammalian retina are postsynaptic to either rod or cone photoreceptors, thereby segregating their respective signals into parallel vertical streams. In contrast to the cone pathways, only one type of rod bipolar cell exists, apparently limiting the routes available for the propagation of rod signals. However, due to numerous interactions between the rod and cone circuitry, there is now strong evidence for the existence of up to three different pathways for the transmission of scotopic visual information. Here we survey work over the last decade or so that have defined the structure and function of the interneurons subserving the rod pathways in the mammalian retina. We have focused on: (1) the synaptic ultrastructure of the interneurons; (2) their light-evoked physiologies; (3) localization of specific transmitter receptor subtypes; (4) plasticity of gap junctions related to changes in adaptational state; and (5) the functional implications of the existence of multiple rod pathways. Special emphasis has been placed on defining the circuits underlying the different response components of the AII amacrine cell, a central element in the transmission of scotopic signals.

Expression of Neuronal Connexin36 in AII Amacrine Cells of the Mammalian Retina

We have studied the expression pattern of neuronal connexin36 (Cx36) in the mouse and rat retina. In vertical sections of both retinas, a polyclonal antibody directed against Cx36 produced punctate labeling in the inner plexiform layer (IPL). Intense immunoreactivity was localized to the entire OFF sublamina of the IPL, and much weaker staining could be observed in the ON sublamina. Double-labeling experiments in the rat retina with antibodies directed against parvalbumin indicate that Cx36 is expressed on dendrites of AII amacrine cells. Cx36-like immunoreactivity in sublamina a of the IPL did not overlap with lobular appendages or cell bodies of AII amacrine cells. In a mouse retinal slice preparation, AII amacrine and ON cone bipolar cells were intracellularly injected with Neurobiotin and counterstained with antibody against Cx36. Punctate labeling appeared to be in register with dendritic arborization of AII amacrines and cone bipolar cells in the ON sublamina of the IPL. Whereas AII amacrine cells isolated from the rat retina clearly displayed Cx36-like immunoreactivity, isolated ON cone bipolar cells were negative for Cx36. Axon terminals of rod bipolar cells were decorated with Cx36-positive contacts but did not express Cx36 themselves. These results indicate that Cx36 is expressed by AII amacrine cells in homologous and heterologous gap junctions made with AII amacrines and cone bipolar cells, respectively. The heterologous gap junctions appear to be heterotypic, because ON cone bipolar cells do not express Cx36.

Chapter 12 Plasticity of AII amacrine cell circuitry in the mammalian retina

Chapter 12 Plasticity of AII amacrine cell circuitry in the mammalian retina

A Series of Biotinylated Tracers Distinguishes Three Types of Gap Junction in Retina

Gap junctions serve many important roles in various tissues, but their abundance and diversity in neurons is only beginning to be understood. The tracer Neurobiotin has revealed many different networks interconnected by gap junctions in retina. We compared the relative permeabilities of five different retinal gap junctions by measuring their permeabilities to a series of structurally related tracers. When large tracers were injected, the staining of coupled cells fell off more rapidly in some networks than others relative to Neurobiotin controls. Three distinctly different permeability profiles were found, suggesting that multiple neuronal connexin types were present. The most permeant to large molecules were gap junctions from A-type horizontal cells. The permeability of gap junctions of two types of amacrine cell were not distinguishable from those from B-type horizontal cells. The lowest permeability was found for gap junctions between cone bipolar cells and the AII amacrine cells to which they are coupled. Because only a single neural connexin type has been identified in retina, our results suggest more types remain to be found. To determine whether the unitary permeability of channels is altered by channel modulators, we reduced permeability with octanol and a cAMP analog. Although net permeability was substantially diminished, the proportion by which it declined was constant across tracer size. This suggests that these agents act only to close channels rather than alter individual channel permeabilities. This tracer series can therefore be used to contrast permeability properties of gap junctions in intact circuits, even at the level of individual channels.

Morphological and physiological properties of the A17 amacrine cell of the rat retina.

In addition to the well-studied AII amacrine cell, there is another amacrine cell type participating in the rod pathway of the mammalian retina. In cat, this cell is called the A17 amacrine cell, and in rabbits, it is called the indoleamine-accumulating amacrine cell (S1 and S2); however, the presence of the corresponding cell type has not yet been described in detail for the rat retina. To this end, we injected amacrine cells with Neurobiotin in vertical retinal slices. After histological processing, we were able to reconstruct the morphology of a wide-field amacrine cell which showed characteristics of A17 and S1/S2 amacrine cells. The rat wide-field amacrine cells exhibited the same stratification pattern, their dendrites bore varicosities and ramified in sublamina 5 of the inner plexiform layer (IPL), and they were dye-coupled to other amacrine cells. To determine whether those amacrine cells shared electrophysiological characteristics as well, we performed whole-cell patch-clamp recordings and examined their voltage-activated currents and neurotransmitter-induced currents. We never observed voltage-gated Na+ currents and spike-like potentials upon depolarization by current injection in these cells. We identified GABA- and glycine-sensitive Cl- currents that could be blocked by bicuculline and strychnine, respectively. We also observed kainate- and AMPA-activated currents, which could be inhibited by the application of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Finally, a 400-ms full-field light stimulus was used to characterize the light responses of A17 amacrine cells. The light ON-induced inward current could be suppressed by the application of 2,3-Dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulphonamide (NBQX), while the majority of the light OFF-induced current was inhibited by bicuculline and reduced to a smaller extent by NBQX. CPP, an NMDA blocker, had no effect on the light response of rat A17 amacrine cells.

Surround inhibition of mammalian AII amacrine cells is generated in the proximal retina.

1. Intracellular recordings were obtained from neurons in the superfused retina-eyecup preparation of the rabbit under dark-adapted conditions. Neurotransmitter agonists and antagonists were applied exogenously via the superfusate to dissect the synaptic pathways pharmacologically and thereby determine those pathways responsible for the generation of the on-centre/off-surround receptive fields of AII amacrine cells. 2. Application of the metabotropic glutamate receptor agonist, APB, reversibly blocked both the on-centre and off-surround responses of AII cells. These data were consistent with the idea that both the centre- and surround-mediated responses are derived from inputs from the presynaptic rod bipolar cells. 3. Whereas rod bipolar cells showed on-receptive fields approximately 100 microm across, we found no evidence for an antagonistic off-surround response using light stimuli which effectively elicited the off-surrounds of AII amacrine cells. These results indicated that the surrounds of AII cells are not derived from rod bipolar cell inputs. 4. Application of the ionotropic glutamate receptor antagonists CNQX or DNQX enhanced the on-centre responses of AII cells but attenuated the off-surround responses. These data indicated that the centre- and surround-mediated responses could not both be derived from signals crossing the rod bipolar-to-AII cell synapse. 5. Application of the glycine antagonist, strychnine, had only minor and variable effects on AII cell responses. However, the GABA antagonists picrotoxin and bicuculline enhanced the on-centre response but attenuated or completely blocked the off-surround response of AII cells. The GABA antagonists had no effect on the responses of horizontal cells indicating that their effects on AII cell responses reflected actions on inner retinal circuitry rather than feedback circuitry in the outer plexiform layer. 6. Application of the voltage-gated sodium channel blocker TTX enhanced the on-centre responses of AII cells but attenuated or abolished their off-surround responses. 7. Taken together, our results suggest that the on-centre responses of AII cells result from the major excitatory drive from rod bipolar cells. However, the surround receptive fields of AII cells appear to be generated by lateral, inhibitory signals derived from neighbouring GABAergic, on-centre amacrine cells. A model is presented whereby the S1 amacrine cells produce the surround receptive fields of AII amacrine cells via inhibitory, feedback circuitry to the axon terminals of rod bipolar cells.

Synaptic connections of DB3 diffuse bipolar cell axons in macaque retina.

In primate retinas, the dendrites of DB3 diffuse bipolar cells are known to receive inputs from cones. The goal of this study was to describe the synaptic connections of DB3 bipolar cell axons in the inner plexiform layer. DB3 bipolar cells in midperipheral retina were labeled with antibodies to calbindin, and their axons were analyzed in serial, ultrathin sections by electron microscopy. Synapses were found almost exclusively at the axonal varicosities of DB3 axon terminals. There were 2.14 synaptic ribbons per varicosity. There were 33 varicosities per DB3 cell, giving an average of 71 ribbons per axon terminal. Because there were 1.5 postsynaptic ganglion cell dendrites per DB3 axonal varicosity, we estimate that there is at least 1 synapse per varicosity onto a parasol ganglion cell dendrite. There were 3.4 input synapses from amacrine cells per axonal varicosity. Among these were feedback synapses to the DB3 bipolar cell axon varicosities, which were made by 47% of the postsynaptic amacrine cell processes. Some of the feedback synapses could be from amacrine cells immunoreactive for cholecystokinin precursor or choline acetyltransferase, because both types of amacrine cells costratify with parasol cells and are known to be presynaptic to bipolar cells. AII amacrine cells were both presynaptic and postsynaptic to DB3 axons, a finding consistent with the large rod input to parasol ganglion cells reported in physiological experiments. DB3 bipolar cell axons also made frequent contacts with neighboring DB3 axons, and gap junctions were always found at these sites.

Neurotrophins and development of the rod pathway: an elementary deduction.

The rodent retina is a particularly attractive model for the study of neuronal developmental processes since considerable neurogenesis, cellular migration, phenotypic differentiation of retinal cell types and synaptogenesis occurs postnatally. In addition, the retina is readily accessible to surgical intervention, pharmacological manipulation, and local suppression of gene expression-tools that can be utilized to study mechanisms underlying the development of retinal neurons and their interconnections that form distinct functional circuits. Here, I review our studies describing the ontogeny of a specific retinal interneuron, the AII amacrine cell, an integral element in the rod (scotopic) pathway. Specifically, we used a number of approaches to examine the potential role of neurotrophic factors on the morphological and neurochemical differentiation of the AII.

Disabled-1 is expressed in type AII amacrine cells in the mouse retina.

The organization of several laminated structures in the brain is controlled by a signaling pathway activated by Reelin, a large glycoprotein secreted by pioneer neurons in the developing brain. Reelin binds to transmembrane receptors, including VLDLR and ApoER2, and stimulates tyrosine phosphorylation of Disabled-1 (Dab1), which associates with an NPxY motif present in the cytoplasmic domain of the receptors. Disruption of reelin, dab1, or both the vldr and apoer2 genes results in similar cell positioning defects in laminated brain regions including the cerebellum, hippocampus, and cerebral cortex. Although retinal ganglion cells express reelin during development, there is no obvious disruption of cell positioning in the retina of reeler mice. Here, we examine the expression pattern of Dab1 as a first step toward understanding the function of the Reelin signaling pathway in neural retina. Immunohistochemical analysis of the adult retina revealed that Dab1 is expressed in a specific type of amacrine cell. These cells display a narrow dendritic field and they project to two distinct sublaminae within the inner plexiform layer. Dab1 co-localizes with the high-affinity glycine transporter, indicating that these amacrine cells are glycinergic. Cells that express Dab1 are surrounded by dopaminergic fibers originating from wide-field amacrine cells. These features are characteristic of type AII amacrine cells described in other mammalian species. Analysis of the retina at several stages of development revealed that Dab1 is expressed shortly after birth during the time at which AII amacrine cells extend neurites and form synaptic connections in the inner retina. This raises the possibility that the Reelin/Dab1 signaling pathway contributes to formation of intraretinal circuitry in the neural retina.

Parvalbumin immunoreactivity is enhanced by brain-derived neurotrophic factor in organotypic cultures of rat retina.

The rodent retina undergoes considerable postnatal neurogenesis and phenotypic differentiation, and it is likely that diffusible neurotrophic factors contribute to this development and to the subsequent formation of functional retinal circuitry. Accordingly, perturbation of specific neurotrophin ligand-receptor interactions has provided valuable information as to the fundamental processes underlying this development. In the present studies we have built upon our previous observation that suppression of expression of trk(B), the high-affinity receptor for brain-derived neurotrophic factor (BDNF), in the postnatal rat retina results in the alteration of a specific interneuron in the rod pathway-the parvalbumin (PV)-immunoreactive AII amacrine cell. Here, we isolated retinas from newborn rats and maintained them in organotypic culture for up to 14 days (approximating the time of eye opening, in vivo) in the presence of individual neurotrophins [BDNF or nerve growth factor (NGF)]. We then examined histological sections of cultures for PV immunoreactivity. In control cultures, only sparse PV-immunostained cells were observed. In cultures supplemented with NGF, numerous lightly immunostained somata were present in the inner nuclear layer (INL) at the border of the inner plexiform layer (IPL). Many of these cells had rudimentary dendritic arborizations in the IPL. Cultures supplemented with BDNF displayed numerous well-immunostained somata at the INL/IPL border that gave rise to elaborate dendritic arborizations that approximated the morphology of mature AII amacrine cells in vivo. These observations indicate that neurotrophins have specific effects upon the neurochemical and, perhaps, morphological differentiation of an important interneuron in a specific functional retinal circuit.

Gap junctions between AII amacrine cells and calbindin-positive bipolar cells in the rabbit retina.

Electrical synapses or gap junctions occur between many retinal neurons. However, in most cases, the gap junctions have not been visualized directly. Instead, their presence has been inferred from tracer spread throughout the network of cells. Thus, tracer coupling is taken as a marker for the presence of gap junctions between coupled cells. AII amacrine cells are critical interneurons in the rod pathway of the mammalian retina. Rod bipolar cell output passes to AII amacrine cells, which in turn make conventional synapses with OFF cone bipolar cells and gap junctions with ON cone bipolar cells. Injections of biotinylated tracers into AII amacrine cells reveals coupling between the AII amacrine cell network and heterologous coupling with a variety of ON cone bipolar cells, including the calbindin-positive cone bipolar cell. To directly visualize gap junctions in this network, we prepared material for electron microscopy that was double labeled with antibodies to calretinin and calbindin to label AII amacrine cells and calbindin-positive cone bipolar cells, respectively. AII amacrine cells were postsynaptic to large vesicle-laden rod bipolar terminals, as previously reported. Gap junctions were identified between AII amacrine cells and calbindin-positive cone bipolar cell terminals identified by the presence of immunostaining and ribbon synapses. This represents direct confirmation of gap junctions between two different yet positively identified cells, which are tracer coupled, and provides additional evidence that tracer coupling with Neurobiotin indicates the presence of gap junctions. These results also definitively establish the presence of gap junctions between AII amacrine cells and calbindin bipolar cells which can therefore carry rod signals to the ON alpha ganglion cell.

AMPA-selective glutamate receptor subunits GluR2 and GluR4 in the cat retina: an immunocytochemical study.

AMPA-selective glutamate receptors play a major role in glutamatergic neurotransmission in the retina and are expressed in a variety of neuronal subpopulations. In the present study, immunocytochemical techniques were used to visualize the distribution of GluR2 and GluR4 subunits in the cat retina. Results were compared with previous localizations of GluR1 and GluR2/3. Staining for GluR2 was limited to a small number of amacrine and ganglion cells whereas GluR4 staining was present in A-type horizontal cells, many amacrine cells including type AII amacrine cells, and the majority of the cells in the ganglion cell layer. Analysis of synaptic relationships in the outer plexiform layer showed the GluR4 subunit to be concentrated at the contacts of cone photoreceptors with A-horizontal cells. In the inner plexiform layer, both GluR2 and GluR4 were postsynaptic to cone bipolar cells at dyad contacts although GluR2 staining was limited to one of the postsynaptic elements whereas GluR4 immunoreactivity was often seen in both postsynaptic elements. Unlike GluR2, GluR4 was also postsynaptic to rod bipolar cells where it could be visualized in processes of AII amacrine cells. The data indicate that GluR3 and GluR4 subunits are colocalized in a number of cell types including A-type horizontal cells, AII amacrine cells, and alpha ganglion cells, but whether they are combined in the same multimeric receptors remains to be determined.

Antibody to calretinin stains AII amacrine cells in the rabbit retina: Double-label and confocal analyses

The AII or rod amacrine cell is a critical interneuron in the rod pathway of mammalian retinae. In this report, it is shown that commercially available antibodies to the calcium binding protein calretinin may be used to label the population of AII amacrine cells selectively. Calretinin-positive amacrine cells had the morphological attributes of AII amacrine cells. Double-labeling procedures showed that calretinin-positive somata were surrounded by dopaminergic varicosities and that calretinin-positive dendrites enclosed rod bipolar terminals, both as previously described for AII amacrine cells. By analyzing the surrounding kernel for each labeled pixel in the rod bipolar image, it is shown here that AII processes are adjacent to rod bipolar terminals at a level that far exceeds the random overlap present in images in which one label was rotated out of phase. Such a spatial relationship is indicative of synaptic connections, as well described for rod bipolar input to AII amacrine cells. AII amacrine cells also were double-labeled for calretinin and parvalbumin; however, a scattergram analysis of red versus green intensity showed that the parvalbumin antibody stained additional unidentified amacrine cells. In conclusion, at the appropriate dilution, calretinin antibodies are a useful marker for AII amacrine cells in the rabbit retina.

Antibody to calretinin stains AII amacrine cells in the rabbit retina: Double‐label and confocal analyses

Antibody to calretinin stains AII amacrine cells in the rabbit retina: Double‐label and confocal analyses

Transmitter‐specific input to OFF‐alpha ganglion cells in the cat retina

The synaptic input to OFF-center alpha ganglion cells in the cat retina was analyzed by electron microscopic reconstruction to quantify the bipolar and amacrine cell input and to determine the neurotransmitter content of the presynaptic cells. Cone bipolar cells were found to comprise 11% of the total input with their dyad synapses distributed across the dendritic tree. The remaining contacts were conventional synapses indicative of amacrine cells; postembedding immunogold labeling was used to characterize these cells as either GABA- or glycine-immunoreactive. Results showed the amacrine input to be equally divided between GABA and glycinergic contacts at each order of dendritic branching of the alpha cells. Among the GABA-positive neurons were A19 amacrine cells, the processes of which are characterized by a dense array of neurotubules. A major source of glycinergic input was from lobular appendages of AII amacrine cells with lesser contributions from other glycine-positive amacrine cells. The physiological role(s) of these amino acids must be interpreted in view of the multiple subpopulations of amacrine cells, which provide input to OFF-alpha cells, and the diversity in receptors at their synapses. Anat Rec 255:363–373, 1999. © 1999 Wiley-Liss, Inc.