Stewart A. Bloomfield

Connexin36 Is Essential for Transmission of Rod-Mediated Visual Signals in the Mammalian Retina

To examine the functions of electrical synapses in the transmission of signals from rod photoreceptors to ganglion cells, we generated connexin36 knockout mice. Reporter expression indicated that connexin36 was present in multiple retinal neurons including rod photoreceptors, cone bipolar cells, and AII amacrine cells. Disruption of electrical synapses between adjacent AIIs and between AIIs and ON cone bipolars was demonstrated by intracellular injection of Neurobiotin. In addition, extracellular recording in the knockout revealed the complete elimination of rod-mediated, on-center responses at the ganglion cell level. These data represent direct proof that electrical synapses are critical for the propagation of rod signals across the mammalian retina, and they demonstrate the existence of multiple rod pathways, each of which is dependent on electrical synapses.

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.

The diverse functional roles and regulation of neuronal gap junctions in the retina

Electrical synaptic transmission through gap junctions underlies direct and rapid neuronal communication in the CNS. The diversity of functional roles that electrical synapses have is perhaps best exemplified in the vertebrate retina, in which gap junctions are formed by each of the five major neuron types. These junctions are dynamically regulated by ambient illumination and by circadian rhythms acting through light-activated neuromodulators such as dopamine and nitric oxide, which in turn activate intracellular signalling pathways in the retina.The networks formed by electrically coupled neurons are plastic and reconfigurable, and those in the retina are positioned to play key and diverse parts in the transmission and processing of visual information at every retinal level.

Tracer coupling patterns of the ganglion cell subtypes in the mouse retina

It is now clear that electrical coupling via gap junctions is prevalent across the retina, expressed by each of the five main neuronal types. With the introduction of mutants in which selective gap junction connexins are deleted, the mouse has recently become an important model for studying the function of coupling between retinal neurons. In this study we examined the tracer‐coupling pattern of ganglion cells by injecting them with the gap junction‐permanent tracer Neurobiotin to provide, for the first time, a comprehensive survey of ganglion cell coupling in the wildtype mouse retina. Murine ganglion cells were differentiated into 22 morphologically distinct subtypes based on soma‐dendritic parameters. Most (16/22) ganglion cell subtypes were tracer‐coupled to neighboring ganglion and/or amacrine cells. The amacrine cells coupled to ganglion cells displayed either polyaxonal or wide‐field morphologies with extensive arbors. We found that different subtypes of ganglion cells were never coupled to one another, indicating that they subserved independent electrical networks. Finally, we found that the tracer‐coupling patterns of the 22 ganglion cell populations were largely stereotypic across the 71 retinas studied. Our results indicate that electrical coupling is extensive in the inner retina of the mouse, suggesting that gap junctions play essential roles in visual information processing. J. Comp. Neurol. 512:664–687, 2009.

Convergence and segregation of the multiple rod pathways in mammalian retina

Using a multidisciplinary approach, we demonstrate that three different pathways are responsible for the transmission of rod signals across the mouse retina. Each pathway serves a primarily nonoverlapping range of stimulus intensities, with ganglion cells receiving either segregated or convergent inputs. For both on-center (ON) and off-center (OFF) ganglion cells, the primary rod pathway carries signals with the lowest threshold, whereas the secondary rod pathway is less sensitive by ∼1 log unit. In addition, OFF signaling uses a tertiary rod pathway that is ∼1 log unit less sensitive than the secondary. Although some ganglion cells received rod inputs exclusively from one of the pathways, others showed convergent inputs. Using pharmacological and genetic approaches, we defined classes of ON and OFF ganglion cells for which the scotopic inputs derive only from the primary pathway or from both primary and secondary pathways. In addition, we observed a class of OFF ganglion cell receiving mixed input from primary and tertiary pathways. Interestingly, OFF ganglion cells receiving convergent inputs from all three rod pathways or from the secondary and tertiary pathways together were never observed. Overall, our data show a complex arrangement of convergence and segregation of rod inputs to ganglion cells in the mammalian retina.

Light-induced modulation of coupling between AII amacrine cells in the rabbit retina.

The rod-driven, AII amacrine cells in the mammalian retina maintain homologous gap junctions with one another as well as heterologous gap junctions with on-cone bipolar cells. We used background illumination to study whether changes in the adaptational state of the retina affected the permeabilities of these two sets of gap junctions. To access changes in permeability, we injected single AII amacrine cells with the biotinylated tracer, Neurobiotin, and measured the extent of tracer coupling to neighboring AII cells and neighboring cone bipolar cells. We also measured the center-receptive field size of AII cells to assess concomitant changes in electrical coupling. Our results indicate that in well dark-adapted retinas, AII cells form relatively small networks averaging 20 amacrine cells and covering about 75 microns. The size of these networks matched closely to the size of AII cell on-center receptive fields. However, over most of their operating range, AII cells formed dramatically larger networks, averaging 326 amacrine cells, which corresponded to an increased receptive-field size. As the retina was light adapted beyond the operating range of the AII cells, they uncoupled to form networks comparable in size to those seem in well dark-adapted retinas. Our results, then, indicate that the adaptational state of the retina has a profound effect on the extent of electrical coupling between AII amacrine cells. Although we observed light-induced changes in the number of tracer-coupled cone bipolar cells, these appeared to be an epiphenomenon of changes in homologous coupling between AII amacrine cells. Therefore, in contrast to the robust changes in AII-AII coupling produced by background illumination, our data provided no evidence of a light-induced modulation of coupling between AII cells and on-cone bipolar cells.

Tracer coupling pattern of amacrine and ganglion cells in the rabbit retina

We examined the tracer coupling pattern of more than 15 morphological types of amacrine and ganglion cells in the rabbit retina. Individual cells were injected intracellularly with the biotinylated tracer Neurobiotin, which was then allowed to diffuse across gap junctions to label neighboring neurons. We found that homologous and/or heterologous tracer coupling was common for most proximal neurons. In fact, the starburst amacrine cell was the only amacrine cell type that showed no evidence of coupling. The remaining types of amacrine cell were coupled exclusively to other amacrines, either homologously or, more often, through a combination of homologous and heterologous junctions. In only one case did we visualize labeled ganglion cells following injection of Neurobiotin into an amacrine cell. In contrast, injection of Neurobiotin into ganglion cells almost always resulted in the labeling of amacrine cells. Taken together, these results suggest a directionality to the movement of tracer across gap junctions connecting amacrine and ganglion cells. We found that the coupling pattern for a given morphological type of cell was generally stereotypic and consistent across retinas. The notable exceptions to this finding were alpha ganglion cells and cells with morphology corresponding to that of on‐off direction selective ganglion cells. In both cases, individual cells showed either extensive coupling to both amacrine and ganglion cells or no coupling at all. A notable finding was that, in every case, the neighboring cells within a tracer‐coupled array were always within one gap junction of the injected neuron. Furthermore, in many cases, the array formed by the somata of tracer‐coupled cells was almost perfectly coincident with the dendritic arbor of the injected cell. Thus, our results indicate that whereas coupling is extensive within the proximal retina, individual cells partake in coupled networks that are stereotypic and highly circumscribed. J. Comp. Neurol. 383:512‐528, 1997.

DARK- AND LIGHT-INDUCED CHANGES IN COUPLING BETWEEN HORIZONTAL CELLS IN MAMMALIAN RETINA

Retinal horizontal cells exhibit large receptive fields derived from their extensive electrical coupling by means of gap junctions. The conductance of these gap junctions seems to be regulated by dopamine acting through a cAMP‐mediated cascade. There is now abundant evidence that extracellular dopamine levels vary with changes in ambient light intensity, suggesting that changes in the dark/light adaptational state of the retina can modulate coupling between horizontal cells. We studied this question in the mammalian retina by determining the effects of ambient light levels, in the form of changing background light intensity, on the coupling profiles of A‐ and B‐type horizontal cells in the rabbit. Changes in coupling were assessed by measurements of the space constants of the syncytium formed by horizontal cells and the intercellular spread of the biotinylated tracer Neurobiotin. Our results indicate that dark‐adapted horizontal cells show relatively weak coupling. However, presentation of background lights as dim as one‐quarter log unit above rod threshold resulted in increases in both the averaged extent of tracer coupling and space constants of A‐ and B‐type horizontal cells. Coupling expanded further as background light intensities were increased by 1–1.5 log units, after which additional light adaptation brought about an uncoupling of cells. Coupling reached its minimum at light intensities about 3 log units above rod threshold, after which, with further light adaptation, it stabilized at levels close to those seen in dark‐adapted retinas. Our results indicate that electrical coupling between mammalian horizontal cells is modulated dramatically by changes in the adaptational state of the retina: coupling is maximized under dim ambient light conditions and diminishes as the retina is dark or light adapted from this level. J. Comp. Neurol. 405:75–87, 1999.

Comparison of the responses of AII amacrine cells in the dark- and light-adapted rabbit retina.

We studied the light-evoked responses of AII amacrine cells in the rabbit retina under dark- and light-adapted conditions. In contrast to the results of previous studies, we found that AII cells display robust responses to light over a 6-7 log unit intensity range, well beyond the operating range of rod photoreceptors. Under dark adaptation, AII cells showed an ON-center/OFF-surround receptive-field organization. The intensity-response profile of the center-mediated response component followed a dual-limbed sigmoidal function indicating a transition from rod to cone mediation as stimulus intensities were increased. Following light adaptation, the receptive-field organization of AII cells changed dramatically. Light-adapted AII cells showed both ON- and OFF-responses to stimulation of the center receptive field, but we found no evidence for an antagonistic surround. Interestingly, the OFF-center response appeared first following rapid light adaptation and was then replaced gradually over a 1-4 min period by the emerging ON-center response component. Application of the metabotropic glutamate receptor agonist APB, the ionotropic glutamate blocker CNQX, 8-bromo-cGMP, and the nitric oxide donor SNAP all showed differential effects on the various center-mediated responses displayed by dark- and light-adapted AII cells. Taken together, these pharmacological results indicated that different synaptic circuits are responsible for the generation of the different AII cell responses. Specifically, the rod-driven ON-center responses are apparently derived from rod bipolar cell synaptic inputs, whereas the cone-driven ON-center responses arise from signals crossing the gap junctions between AII cells and ON-center cone bipolar cells. Additionally, the OFF-center response of light-adapted AII cells reflects direct synaptic inputs from OFF-center cone bipolar cells to AII dendritic processes in the distal inner plexiform layer.

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 ≈100 μm 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.