Ji-Jie Pang

Light-evoked current responses in rod bipolar cells, cone depolarizing bipolar cells and AII amacrine cells in dark-adapted mouse retina.

Light‐evoked excitatory cation current (ΔIC) and inhibitory chloride current (ΔICl) of rod and cone depolarizing bipolar cells (DBCRs and DBCCs) and AII amacrine cells (AIIACs) in dark‐adapted mouse retinal slices were studied by whole‐cell voltage‐clamp recording techniques, and the cell morphology was revealed by Lucifer yellow fluorescence with a confocal microscope. ΔIC of all DBCRs exhibited similar high sensitivity to 500 nm light, but two patterns of ΔICl were observed in DBCRs with slightly different axon morphology. At least two types of DBCCs were identified: one with axon terminals ramified in 70–85% of the depth of the inner plexiform layer (IPL) and DBCR‐like ΔIC sensitivity, whereas the other with axon terminals ramified in 55–75% of IPL depth and much lower ΔIC sensitivity. The relative rod/cone inputs to DBCs and AIIACs were analysed by comparing the ΔIC and ΔICl thresholds and dynamic ranges with the corresponding values of rods and cones. On average, the sensitivity of a DBCR to the 500 nm light is about 20 times higher than that of a rod. The sensitivity of an AIIAC is more than 1000 times higher than that of a rod, suggesting that AIIAC responses are pooled through a coupled network of about 40 AIIACs. Interactions of rod and cone signals in dark‐adapted mouse retina appear asymmetrical: rod signals spread into the cone system more efficiently than cone signals into the rod system. The mouse synaptic circuitry allows small rod signals to be highly amplified, and effectively transmitted to the cone system via rod–cone and AIIAC–DBCC coupling.

Direct rod input to cone BCs and direct cone input to rod BCs challenge the traditional view of mammalian BC circuitry

Bipolar cells are the central neurons of the retina that transmit visual signals from rod and cone photoreceptors to third-order neurons in the inner retina and the brain. A dogma set forth by early anatomical studies is that bipolar cells in mammalian retinas receive segregated rod/cone synaptic inputs (either from rods or from cones), and here, we present evidence that challenges this traditional view. By analyzing light-evoked cation currents from morphologically identified depolarizing bipolar cells (DBCs) in the wild-type and three pathway-specific knockout mice (rod transducin knockout [Trα−/−], connexin36 knockout [Cx36−/−], and transcription factor beta4 knockout [Bhlhb4−/−]), we show that a subpopulation of rod DBCs (DBCR2s) receives substantial input directly from cones and a subpopulation of cone DBCs (DBCC1s) receives substantial input directly from rods. These results provide evidence of the existence of functional rod-DBCC and cone-DBCR synaptic pathways in the mouse retina as well as the previously proposed rod hyperpolarizing bipolar-cells pathway. This is grounds for revising the mammalian rod/cone bipolar cell dogma.

Relative contributions of rod and cone bipolar cell inputs to AII amacrine cell light responses in the mouse retina

AII amacrine cells (AIIACs) are crucial relay stations for rod‐mediated signals in the mammalian retina and they receive synaptic inputs from depolarizing and hyperpolarizing bipolar cells (DBCs and HBCs) as well as from other amacrine cells. Using whole‐cell voltage‐clamp technique in conjunction with pharmacological tools, we found that the light‐evoked current response of AIIACs in the mouse retina is almost completely mediated by two DBC synaptic inputs: a 6,7‐dinitro‐quinoxaline‐2,3‐dione (DNQX)‐resistant component mediated by cone DBCs (DBCCs) through an electrical synapse, and a DNQX‐sensitive component mediated by rod DBCs (DBCRs). This scheme is supported by AIIAC current responses recorded from two knockout mice. The dynamic range of the AIIAC light response in the Bhlhb4−/− mouse (which lacks DBCRs) resembles that of the DNQX‐resistant component, and that of the connexin36 (Cx36)−/− mouse resembles the DNQX‐sensitive component. By comparing the light responses of the DBCCs with the DNQX‐resistant AIIAC component, and light responses of the DBCRs with the DNQX‐sensitive AIIAC component, we obtained the input–output relations of the DBCC→AIIAC electrical synapse and the DBCR→AIIAC chemical synapse. Similar to other glutamatergic chemical synapses in the retina, the DBCR→AIIAC synapse is non‐linear. Its highest voltage gain (approximately 5) is found near the dark membrane potential, and it saturates for presynaptic signals larger than 5.5 mV. The DBCC→AIIAC electrical synapse is approximately linear (voltage gain of 0.92), consistent with the linear junctional conductance found in retinal electrical synapses. Moreover, relative DBCR and DBCC contributions to the AIIAC response at various light intensity levels are determined.

Elevated Intraocular Pressure Causes Inner Retinal Dysfunction Before Cell Loss in a Mouse Model of Experimental Glaucoma

PURPOSE We assessed the relationship among intraocular pressure (IOP), histology, and retinal function changes in a mouse model of induced, chronic, mild ocular hypertension. METHODS IOP was elevated experimentally via anterior chamber injection of polystyrene beads and measured twice weekly with a rebound tonometer. Histology was assessed with a combination of neurobiotin (NB) retrograde labeling of retinal ganglion cells (RGCs) and TO-PRO3 staining. Retinal function was assessed with serial dark-adapted electroretinograms (ERGs) optimized for detection of the a-wave, b-wave, and positive and negative scotopic threshold responses (pSTR, nSTR). Comparisons between bead-injected and saline-injected (control) eyes were conducted. RESULTS IOP remained elevated for at least 3 months following a single injection of polystyrene beads. Elevated IOP resulted in a mild, progressive reduction of RGCs, and a mild increase in axial length at 6 and 12 weeks after bead injection. The raw b-wave amplitude was increased shortly after IOP elevation, but the raw a-wave, pSTR, and nSTR amplitudes were unchanged. pSTR and nSTR amplitudes were normalized to the increased b-wave. With this normalization, the pSTR amplitude was decreased shortly after IOP elevation. CONCLUSIONS Polystyrene bead injection results in a mild, chronic elevation of IOP that recapitulates several critical aspects of human ocular hypertension and glaucoma, and results in early changes in retinal electrical function that precede histologic changes. It is possible that glaucoma associated with elevated IOP involves the early disruption of a complex combination of retinal synapses.

How do tonic glutamatergic synapses evade receptor desensitization

Photoreceptor output synapses are the best known tonic chemical synapses in the nervous system, in which glutamate is continuously released in darkness, activating AMPA/kainate receptors in postsynaptic neurons. It has been shown that glutamate receptors in certain types of second‐order retinal cells are largely desensitized in darkness, leading to small postsynaptic currents and reduced response dynamic ranges. Here we show that the tonic glutamatergic synapses between photoreceptors and rod‐dominated hyperpolarizing bipolar cells (HBCRs) in the salamander retina evade postsynaptic receptor desensitization by using (1) multiple invaginating ribbon junctions as releasing sites for low‐frequency, synchronized multiquantal release at each site; and (2) the GluR4 AMPA receptors as the postsynaptic receptors. The multiquantal events exhibit faster decay time than the GluR4 receptor desensitization time constant and therefore self‐desensitization is minimized, and the average inter‐event duration in darkness is much longer than the GluR4 desensitization recovery time and thus mutual desensitization is avoided. Consequently, the HBCRs are not desensitized in darkness, allowing light signals to be encoded by the full operating range of the glutamate‐gated postsynaptic currents. Our study illustrates for the first time how a tonic glutamatergic synapse avoids postsynaptic receptor desensitization, a strategy that may be shared by many other synapses in the nervous system that need extended operation capacity.

Light responses and morphology of bNOS-immunoreactive neurons in the mouse retina

Nitric oxide (NO), produced by NO synthase (NOS), modulates the function of all retinal neurons and ocular blood vessels and participates in the pathogenesis of ocular diseases. To further understand the regulation of ocular NO release, we systematically studied the morphology, topography, and light responses of NOS‐containing amacrine cells (NOACs) in dark‐adapted mouse retina. Immunohistological staining for neuronal NOS (bNOS), combined with retrograde labeling of ganglion cells (GCs) with Neurobiotin (NB, a gap junction permeable dye) and Lucifer yellow (LY, a less permeable dye), was used to identify NOACs. The light responses of ACs were recorded under whole‐cell voltage clamp conditions and cell morphology was examined with a confocal microscope. We found that in dark‐adapted conditions bNOS‐immunoreactivity (IR) was present primarily in the inner nuclear layer and the ganglion cell layer. bNOS‐IR somas were negative for LY, thus they were identified as ACs; nearly 6% of the cells were labeled by NB but not by LY, indicating that they were dye‐coupled with GCs. Three morphological subtypes of NOACs (NI, NII, and displaced) were identified. The cell density, intercellular distance, and the distribution of NOACs were studied in whole retinas. Light evoked depolarizing highly sensitive ON‐OFF responses in NI cells and less sensitive OFF responses in NII cells. Frequent (1–2 Hz) or abrupt change of light intensity evoked larger peak responses. The possibility for light to modify NO release from NOACs is discussed. J. Comp. Neurol. 518:2456–2474, 2010.

Synaptic circuitry mediating light-evoked signals in dark-adapted mouse retina.

Light-evoked excitatory cation current (DeltaIC) and inhibitory chloride current (DeltaICl) of rod and cone bipolar cells and AII amacrine cells (AIIACs) were recorded from slices of dark-adapted mouse retinas, and alpha ganglion cells were recorded from flatmounts of dark-adapted mouse retinas. The cell morphology was revealed by Lucifer yellow fluorescence with a confocal microscope. DeltaIC of all rod depolarizing bipolar cells (DBCRs) exhibited similar high sensitivity to 500 nm light, but two patterns of DeltaICl were observed with slightly different axon morphologies. At least two types of cone depolarizing bipolar cells (DBCCs) were identified: one with axon terminals ramified in 70-85% of IPL depth and DBCR-like DeltaIC sensitivity, and the other with axon terminals ramified in 55-75% of IPL depth and much lower DeltaIC sensitivity. The relative rod/cone inputs to DBCs and AIIACs were analyzed by comparing the DeltaIC and DeltaICl thresholds and dynamic ranges with the corresponding values of rods and cones. On average, the sensitivity of a DBCR to the 500 nm light is about 20 times higher than that of a rod. The sensitivity of an AIIAC is more than 1000 times higher than that of a rod, suggesting that AIIAC responses are pooled through a coupled network of about 40 AIIACs. Interactions of rod and cone signals in dark-adapted mouse retinas appear asymmetrical: rod signals spread into the cone system more efficiently than cone signals into the rod system. The mouse synaptic circuitry allows small rod signals to be highly amplified and effectively transmitted to the cone system via rod/cone and AIIAC/DBCC coupling. Three types of alpha ganglion cells (alphaGCs) were identified. (1) ONGCs exhibits no spike activity in darkness, increased spikes in light, sustained inward DeltaIC, sustained outward DeltaICl of varying amplitude, and large soma (20-25 microm in diameter) with an alpha-cell-like dendritic field about 180-350 microm stratifying near 70% of the IPL depth. (2) Transient OFFalphaGCs (tOFFalphaGCs) exhibit no spike activity in darkness, transient increased spikes at light offset, small sustained outward DeltaIC in light, a large transient inward DeltaIC at light offset, a sustained outward DeltaICl, and a morphology similar to the ONalphaGCs except for that their dendrites stratified near 30% of the IPL depth. (3) Sustained OFFalpha GCs (sOFFalphaGCs) exhibit maintained spike activity of 5-10 Hz in darkness, sustained decrease of spikes in light, sustained outward DeltaIC, sustained outward DeltaICl, and a morphology similar to the tOFFalphaGCs. By comparing the response thresholds and dynamic ranges of alphaGCs with those of the pre-ganglion cells, our data suggest that the light responses of each type of alphaGCs are mediated by different sets of bipolar cells and amacrine cells.

Morphology and immunoreactivity of retrogradely double-labeled ganglion cells in the mouse retina.

PURPOSE To examine the specificity and reliability of a retrograde double-labeling technique that was recently established for identification of retinal ganglion cells (GCs) and to characterize the morphology of displaced (d)GCs (dGs). METHODS A mixture of the gap-junction-impermeable dye Lucifer yellow (LY) and the permeable dye neurobiotin (NB) was applied to the optic nerve stump for retrograde labeling of GCs and the cells coupled with them. A confocal microscope was adopted for morphologic observation. RESULTS GCs were identified by LY labeling, and they were all clearly labeled by NB. Cells coupled to GCs contained a weak NB signal but no LY. LY and NB revealed axon bundles, somas and dendrites of GCs. The retrogradely identified GCs numbered approximately 50,000 per retina, and they constituted 44% of the total neurons in the ganglion cell layer (GCL). Somas of retrogradely identified dGs were usually negative for glycine, ChAT (choline acetyltransferase), bNOS (brain-type nitric oxidase), GAD (glutamate decarboxylase), and glial markers, and occasionally, they were weakly GABA-positive. dGs averaged 760 per retina and composed 1.7% of total GCs. Sixteen morphologic subtypes of dGs were encountered, three of which were distinct from known GCs. dGs sent dendrites to either sublaminas of the IPL, mostly sublamina a. CONCLUSIONS The retrograde labeling is reliable for identification of GCs. dGs participate in ON and OFF light pathways but favor the OFF pathway. ChAT, bNOS, glycine, and GAD remain reliable AC markers in the GCL. GCs may couple to GABAergic ACs, and the gap junctions likely pass NB and GABA.

Cross-talk between ON and OFF channels in the salamander retina: indirect bipolar cell inputs to ON-OFF ganglion cells.

It has been widely accepted that ON and OFF channels in the visual system are segregated with little cross-communication, except for the mammalian rod bipolar cell-AII amacrine cell-ganglion cell pathway. Here, we show that in the tiger salamander retina the light responses of a subpopulation of ON-OFF ganglion cells are mediated by crossing the ON and OFF bipolar cell pathways. Although the majority of ON-OFF ganglion cells (type I cells) receive direct excitatory inputs from depolarizing and hyperpolarizing bipolar cells (DBCs and HBCs), about 5% (type II cells) receive indirect excitatory inputs from DBCs and 20% (type III cells) receive indirect excitatory inputs from HBCs. These indirect bipolar cell inputs are likely to be mediated by a subpopulation of amacrine cells that exhibit transient hyperpolarizing light responses (AC(H)s) and make GABAergic/glycinergic synapses on DBC or HBC axon terminals. GABA and glycine receptor antagonists enhanced the ON and OFF excitatory cation current (DeltaI(C)) in type I ganglion cells, but completely suppressed the ON DeltaI(C) mediated by DBCs in type II cells and the OFF DeltaI(C) mediated by HBCs in types III cells. Dendrites of type I cells ramify in both sublamina A and B, type II cells exclusively in sublamina A, and type III cells exclusively in sublamina B of the inner plexiform layer. These results demonstrate that indirect, amacrine cell-mediated bipolar cell-ganglion cell synaptic pathways exist in a non-mammalian retina, and that bidirectional cross-talk between ON and OFF channels is present in the vertebrate retina.

Rod, M‐cone and M/S‐cone inputs to hyperpolarizing bipolar cells in the mouse retina

Non‐technical summary  ON and OFF pathways are major information processing channels of the visual system, and they begin at the retinal bipolar cell level. Here we show a systematic study correlating light response characteristics and cell morphology of OFF (hyperpolarizing) bipolar cells (HBCs) in the mouse retina. Results from this study provide a description of how rods, M‐cones and M/S‐cones mediate responses in three major types of HBCs, and how synapses in the OFF pathway are interconnected to serve various visual tasks.