Dao-Qi Zhang

Intraretinal signaling by ganglion cell photoreceptors to dopaminergic amacrine neurons

Retinal dopaminergic amacrine neurons (DA neurons) play a central role in reconfiguring retinal function according to prevailing illumination conditions, yet the mechanisms by which light regulates their activity are poorly understood. We investigated the means by which sustained light responses are evoked in DA neurons. Sustained light responses were driven by cationic currents and persisted in vitro and in vivo in the presence of L-AP4, a blocker of retinal ON-bipolar cells. Several characteristics of these L-AP4-resistant light responses suggested that they were driven by melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs), including long latencies, marked poststimulus persistence, and a peak spectral sensitivity of 478 nm. Furthermore, sustained DA neuron light responses, but not transient DA neuron responses, persisted in rod/cone degenerate retinas, in which ipRGCs account for virtually all remaining retinal phototransduction. Thus, ganglion-cell photoreceptors provide excitatory drive to DA neurons, most likely by way of the coramification of their dendrites and the processes of DA neurons in the inner plexiform layer. This unprecedented centrifugal outflow of ganglion-cell signals within the retina provides a novel basis for the restructuring of retinal circuits by light.

Functional Heterogeneity of Retinal Dopaminergic Neurons Underlying Their Multiple Roles in Vision

Dopaminergic neurons play key roles in the CNS, mediating basic mechanisms of vision, movement, motivation, and mood. The most accessible dopaminergic neurons of the vertebrate CNS are the dopaminergic amacrine cells of the retina. Here, we have characterized the intrinsic neural activity, synaptic input, and light responses of retinal dopaminergic neurons in situ, using targeted electrophysiological recordings of fluorescent neurons in TH::RFP (tyrosine hydroxylase gene promoter::red fluorescent protein) transgenic mice. Dopaminergic amacrine cells exhibit two classes of intrinsic bursting in the dark, shaped by inhibitory synaptic inputs, and two classes of light responses, ON-transient and ON-sustained, as well as light-independent activity, tuned to mediate specific dopaminergic functions in vision. The functional heterogeneity revealed in dopaminergic amacrine cells provides a cellular basis for the multiple roles of dopaminergic amacrine neurons in vision and is likely a general property of dopaminergic neurons throughout the CNS.

Melanopsin Mediates Retrograde Visual Signaling in the Retina

The canonical flow of visual signals proceeds from outer to inner retina (photoreceptors→bipolar cells→ganglion cells). However, melanopsin-expressing ganglion cells are photosensitive and functional sustained light signaling to retinal dopaminergic interneurons persists in the absence of rods and cones. Here we show that the sustained-type light response of retinal dopamine neurons requires melanopsin and that the response is mediated by AMPA-type glutamate receptors, defining a retrograde retinal visual signaling pathway that fully reverses the usual flow of light signals in retinal circuits.

M1 ipRGCs Influence Visual Function through Retrograde Signaling in the Retina.

Melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs, with five subtypes named M1–M5) are a unique subclass of RGCs with axons that project directly to many brain nuclei involved in non-image-forming functions such as circadian photoentrainment and the pupillary light reflex. Recent evidence suggests that melanopsin-based signals also influence image-forming visual function, including light adaptation, but the mechanisms involved are unclear. Intriguingly, a small population of M1 ipRGCs have intraretinal axon collaterals that project toward the outer retina. Using genetic mouse models, we provide three lines of evidence showing that these axon collaterals make connections with upstream dopaminergic amacrine cells (DACs): (1) ipRGC signaling to DACs is blocked by tetrodotoxin both in vitro and in vivo, indicating that ipRGC-to-DAC transmission requires voltage-gated Na+ channels; (2) this transmission is partly dependent on N-type Ca2+ channels, which are possibly expressed in the axon collateral terminals of ipRGCs; and (3) fluorescence microscopy reveals that ipRGC axon collaterals make putative presynaptic contact with DACs. We further demonstrate that elimination of M1 ipRGCs attenuates light adaptation, as evidenced by an impaired electroretinogram b-wave from cones, whereas a dopamine receptor agonist can potentiate the cone-driven b-wave of retinas lacking M1 ipRGCs. Together, the results strongly suggest that ipRGCs transmit luminance signals retrogradely to the outer retina through the dopaminergic system and in turn influence retinal light adaptation. SIGNIFICANCE STATEMENT Melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) comprise a third class of retinal photoreceptors that are known to mediate physiological responses such as circadian photoentrainment. However, investigation into whether and how ipRGCs contribute to vision has just begun. Here, we provide convergent anatomical and physiological evidence that axon collaterals of ipRGCs constitute a centrifugal pathway to DACs, conveying melanopsin-based signals from the innermost retina to the outer retina. We further demonstrate that retrograde signals likely influence visual processing because elimination of axon collateral-bearing ipRGCs impairs light adaptation by limiting dopamine-dependent facilitation of the cone pathway. Our findings strongly support the hypothesis that retrograde melanopsin-based signaling influences visual function locally within the retina, a notion that refutes the dogma that RGCs only provide physiological signals to the brain.

Zinc modulation of hemi-gap-junction channel currents in retinal horizontal cells.

Hemi-gap-junction (HGJ) channels of retinal horizontal cells (HCs) function as transmembrane ion channels that are modulated by voltage and calcium. As an endogenous retinal neuromodulator, zinc, which is coreleased with glutamate at photoreceptor synapses, plays an important role in shaping visual signals by acting on postsynaptic HCs in vivo. To understand more fully the regulation and function of HC HGJ channels, we examined the effect of Zn(2+) on HGJ channel currents in bass retinal HCs. Hemichannel currents elicited by depolarization in Ca(2+)-free medium and in 1 mM Ca(2+) medium were significantly inhibited by extracellular Zn(2+). The inhibition by Zn(2+) of hemichannel currents was dose dependent with a half-maximum inhibitory concentration of 37 microM. Compared with other divalent cations, Zn(2+) exhibited higher inhibitory potency, with the order being Zn(2+) > Cd(2+) approximately Co(2+) > Ca(2+) > Ba(2+) > Mg(2+). Zn(2+) and Ca(2+) were found to modulate HGJ channels independently in additivity experiments. Modification of histidine residues with N-bromosuccinimide suppressed the inhibitory action of Zn(2+), whereas modification of cysteine residues had no significant effect on Zn(2+) inhibition. Taken together, these results suggest that zinc acts on HGJ channels in a calcium-independent way and that histidine residues on the extracellular domain of HGJ channels mediate the inhibitory action of zinc.

Functional integrity and modification of retinal dopaminergic neurons in the rd1 mutant mouse: roles of melanopsin and GABA

The progressive loss of rod and cone photoreceptors in human subjects with retinitis pigmentosa causes a gradual decline in vision and can result in blindness. Current treatment strategies for the disease rely on the integrity of inner retinal neurons, such as amacrine cells, that are postsynaptic to photoreceptors. Previous work has demonstrated that a specialized subclass of retinal amacrine cell that synthesizes and releases the key neurotransmitter dopamine remains morphologically intact during the disease; however, the pathophysiological function of these neurons remains poorly understood. Here we examined spontaneous and light-evoked spike activity of genetically labeled dopamine neurons from the retinas of retinal degeneration 1 (rd1) mice. Our results indicated that rd1 dopamine neurons remained functionally intact with preserved spontaneous spiking activity and light-evoked responses. The light responses were mediated exclusively by melanopsin phototransduction, not by surviving cones. Our data also suggested that dopamine neurons were altered during photoreceptor loss, as evidenced by less spontaneous bursting activity and increased light-evoked responses with age. Further evidence showed that these alterations were attributed to enhanced GABA/melanopsin signaling to dopamine neurons during disease progression. Taken together, our studies provide valuable information regarding the preservation and functional modification of the retinal dopamine neuronal system in rd1; this information should be considered when designing treatment strategies for retinitis pigmentosa.

Circadian rhythm of Period1 clock gene expression in NOS amacrine cells of the mouse retina

The vertebrate retina contains self-sustained circadian clocks that broadly influence retinal physiology. In the present study, we have examined the relationship of nitric oxide, GABAergic and glycinergic inner retinal neurons with expression of a reporter for the circadian clock gene Period1 (Per1). Using Per1 : :GFP transgenic mice, we found that 72% of brain nitric oxide synthase (bNOS) expressing amacrine cells (NOS amacrine cells) sampled during the daytime were also immunoreactive for Per1-driven GFP. The number of bright GFP(+) NOS(+) cells was greater at Zeitgeber time (ZT) 10 than at 22, and this pattern persisted in retinas from animals which were placed in constant darkness [Circadian time (CT) 10 vs. 22]. Intensities of GFP-IR for individual NOS amacrine cells were analyzed at ZT4, 10, 16 and 22, with the peak value occurring at ZT10. Similar results were obtained from retinas sampled at CT4, 10, 16 and 22 in constant darkness, indicating that an endogenous circadian clock drives the transcription of the Per1 clock gene within NOS amacrine cells. The predominance of Per1 : :GFP(+) amacrine cells (82%), was immunoreactive to glutamate decarboxylase 65, but no Per1 : :GFP(+) amacrine cells colabeled with a glycine transporter 1 antibody. The results demonstrate circadian rhythms in Per1 promoter activation in nitric oxide (NO) and GABA secreting amacrine cells, and suggest that NO and GABA could be controlled by circadian clock mechanisms in the mammalian retina.

Multiple cone pathways are involved in photic regulation of retinal dopamine

Dopamine is a key neurotransmitter in the retina and plays a central role in the light adaptive processes of the visual system. The sole source of retinal dopamine is dopaminergic amacrine cells (DACs). We and others have previously demonstrated that DACs are activated by rods, cones, and intrinsically photosensitive retinal ganglion cells (ipRGCs) upon illumination. However, it is still not clear how each class of photosensitive cells generates light responses in DACs. We genetically isolated cone function in mice to specifically examine the cone-mediated responses of DACs and their neural pathways. In addition to the reported excitatory input to DACs from light-increment (ON) bipolar cells, we found that cones alternatively signal to DACs via a retrograde signalling pathway from ipRGCs. Cones also produce ON and light-decrement (OFF) inhibitory responses in DACs, which are mediated by other amacrine cells, likely driven by type 1 and type 2/3a OFF bipolar cells, respectively. Dye injections indicated that DACs had similar morphological profiles with or without ON/OFF inhibition. Our data demonstrate that cones utilize specific parallel excitatory and inhibitory circuits to modulate DAC activity and efficiently regulate dopamine release and the light-adaptive state of the retina.

Physiological and molecular characterization of connexin hemichannels in zebrafish retinal horizontal cells

Connexin channels mediate electrical synaptic transmission when assembled as cell-to-cell pores at gap junctions and can mediate transmembrane currents when expressed in plasma membranes as hemichannels. They are widely expressed in the vertebrate retina where in electrical synapses they are critical for transmission of visual signals. While the roles of connexins in electrical synapses are well-studied, the function and roles of connexin hemichannels in the nervous system are less well understood. Genetic deletion in zebrafish of connexin (Cx) 55.5 alters horizontal cell feedback to cones, spectral responses, and visual behavior. Here, we have characterized the properties of hemichannel currents in zebrafish retinal horizontal cells and examined the roles of two connexin isoforms, Cx55.5 and Cx52.6, that are coexpressed in these cells. We report that zebrafish horizontal cells express hemichannel currents that conduct inward current at physiological negative potentials and Ca(2+) levels. Manipulation of Cx55.5 and Cx52.6 gene expression in horizontal cells of adult zebrafish revealed that both Cx55.5 and Cx52.6 contribute to hemichannel currents; however, Cx55.5 expression is necessary for high-amplitude currents. Similarly, coexpression of Cx55.5 with Cx52.6 in oocytes increased hemichannel currents in a supra-additive manner. Taken together these results demonstrate that zebrafish horizontal cell hemichannel currents exhibit the functional characteristics necessary to contribute to synaptic feedback at the first visual synapse, that both Cx55.5 and Cx52.6 contribute to hemichannel currents, and that Cx55.5 may have an additional regulatory function enhancing the amplitude of hemichannel currents.

Modulation of A-type potassium currents in retinal horizontal cells by extracellular calcium and zinc.

Extracellular Ca2+ and Zn2+ influence many aspects of retinal function. Here, we examined the effect of external Ca2+ and Zn2+ on potassium channels of retinal horizontal cells. When extracellular Ca2+ was lowered from 3 mM to 0.3 mM, horizontal cell transient outward currents elicited by voltage steps from resting membrane potential (-70 mV) were decreased by approximately 50%, whereas the sustained currents remained unchanged. This effect was due to a hyperpolarizing shift in the steady-state inactivation curve of A-type K+ currents when extracellular Ca2+ concentration was lowered. The mean half inactivation potential of the steady-state inactivation curves was hyperpolarized from -56.3 +/- 4.7 mV in 3 mM Ca2+ to -76.4 +/- 3.9 mV in 0.3 mM Ca2+. Neither the state-steady activation curve nor the kinetics of inactivation was significantly changed in low extracellular Ca2+. The addition of 30 microM Zn2+ restored peak outward currents in 0.3 mM Ca2+. The half inactivation voltages were depolarized from -70 +/- 2.8 mV in 0.3 mM Ca2+ to -56 +/- 2.6 mV in 0.3 mM Ca2+ plus 30 microM Zn2+. Taken together, the results indicate that external Ca2+ and Zn2+ maintain the activity of A-type potassium channels in retinal horizontal cells by influencing the voltage dependence of steady-state inactivation.