immunostaining-whole-mount-retinas-with-clarity-tissue-clearing

Immunostaining of Whole-Mount Retinas with the CLARITY Tissue Clearing Method

The tissue hydrogel delipidation method (CLARITY), originally developed by the Deisseroth laboratory, has been modified and widely used for immunostaining ...

Oakland University

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.

Melanopsin mediates retrograde visual signaling in the retina.

Impairment of visual function has been detected in the early stage of diabetes but the underlying neural mechanisms involved are largely unknown. Morphological and functional alterations of retinal ganglion cells, the final output neurons of the vertebrate retina, are thought to be the major cause of visual defects in diabetes but direct evidence to support this notion is limited. In this study we investigated functional changes of retinal ganglion cells in a type 1-like diabetic mouse model. Our results demonstrated that the spontaneous spiking activity of ON-type retinal ganglion cells was increased in streptozotocin-diabetic mice after 3 to 4 months of diabetes. At this stage of diabetes, no apoptotic signals or cell loss were detected in the ganglion cell layer of the retina, suggesting that the functional alterations in ganglion cells occur prior to massive ganglion cell apoptosis. Furthermore, we found that the increased activity of ON-type ganglion cells was mainly a result of reduced inhibitory signaling to the cells in diabetes. This novel mechanism provides insight into how visual function is impaired in diabetic retinopathy. 

Hyperactivity of ON-type retinal ganglion cells in streptozotocin-induced diabetic mice.

Retinal dopamine deficiency is a potential cause of myopia and visual deficits in retinopathy of prematurity (ROP). We investigated the cellular mechanisms responsible for lowered levels of retinal dopamine in an oxygen-induced retinopathy (OIR) mouse model of ROP.

Vulnerability of Dopaminergic Amacrine Cells to Chronic Ischemia in a Mouse Model of Oxygen-Induced Retinopathy.

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.

Multiple cone pathways are involved in photic regulation of retinal dopamine.

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 Ca(2+) 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.

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

In the vertebrate retina, dopamine is synthesized and released by a specialized type of amacrine cell, the dopaminergic amacrine cell (DAC). DAC activity is stimulated by rods, cones, and melanopsin-expressing intrinsically photosensitive retinal ganglion cells upon illumination. However, the relative contributions of these three photoreceptor systems to the DAC light-induced response are unknown. Here we found that rods excite dark-adapted DACs across a wide range of stimulation intensities, primarily through connexin-36-dependent rod pathways. Similar rod-driven responses were observed in both ventral and dorsal DACs. We further found that in the dorsal retina, M-cones and melanopsin contribute to dark-adapted DAC responses with a similar threshold intensity. In the ventral retina, however, the threshold intensity for M-cone-driven responses was two log units greater than that observed in dorsal DACs, and melanopsin-driven responses were almost undetectable. We also examined the DAC response to prolonged adapting light and found such responses to be mediated by rods under dim lighting conditions, rods/M-cones/melanopsin under intermediate lighting conditions, and cones and melanopsin under bright lighting conditions. Our results elucidate the relative contributions of the three photoreceptor systems to DACs under different lighting conditions, furthering our understanding of the role these cells play in the visual system.

Mapping physiological inputs from multiple photoreceptor systems to dopaminergic amacrine cells in the mouse retina.

The neuropeptides orexin-A and orexin-B are widely expressed in the vertebrate retina; however, their role in visual function is unclear. This study investigates whether and how orexins modulate signal transmission to dopaminergic amacrine cells (DACs) from both outer retinal photoreceptors (rods and cones) and inner retinal photoreceptors (melanopsin-expressing intrinsically photosensitive retinal ganglion cells [ipRGCs]).

Orexin-A Suppresses Signal Transmission to Dopaminergic Amacrine Cells From Outer and Inner Retinal Photoreceptors.

Recently, a line of evidence has demonstrated that the vertebrate retina possesses a novel retrograde signaling pathway. In this pathway, phototransduction is initiated by the photopigment melanopsin, which is expressed in a small population of retinal ganglion cells. These ganglion cell photoreceptors then signal to dopaminergic amacrine cells (DACs) through glutamatergic synapses, influencing visual light adaptation. We have previously demonstrated that in Mg-containing solution, α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptors mediate this glutamatergic transmission. Here, we demonstrate that removing extracellular Mg enhances melanopsin-based DAC light responses at membrane potentials more negative than -40 mV. Melanopsin-based responses in Mg-free solution were profoundly suppressed by the selective N-methyl-D-aspartate (NMDA) receptor antagonist D-AP5. In addition, application of NMDA to the retina produced excitatory inward currents in DACs. These data strongly suggest that DACs express functional NMDA receptors. We further found that in the presence of Mg, D-AP5 reduced the peak amplitude of melanopsin-based DAC responses by ~70% when the cells were held at their resting membrane potential (-50 mV), indicating that NMDA receptors are likely to contribute to retrograde signal transmission to DACs under physiological conditions. Moreover, our data show that melanopsin-based NMDA-receptor-mediated responses in DACs are suppressed by antagonists specific to either the NR2A or NR2B receptor subtype. Immunohistochemical results show that NR2A and NR2B subunits are expressed on DAC somata and processes. These results suggest that DACs express functional NMDA receptors containing both NR2A and NR2B subunits. Collectively, our data reveal that, along with AMPA receptors, NR2A- and NR2B-containing NMDA receptors mediate retrograde signal transmission from ganglion cell photoreceptors to DACs.

NMDA Receptors Contribute to Retrograde Synaptic Transmission from Ganglion Cell Photoreceptors to Dopaminergic Amacrine Cells.

The neuromodulator dopamine plays an important role in light adaptation for the visual system. Light can stimulate dopamine release from dopaminergic amacrine cells (DACs) by activating three classes of photosensitive retinal cells: rods, cones, and melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs). However, the synaptic mechanisms by which these photoreceptors excite DACs remain poorly understood. Our previous work demonstrated that α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptors contribute to light regulation of DAC activity. AMPA receptors are classified into Ca-permeable and Ca-impermeable subtypes. We sought to identify which subtype of AMPA receptors is involved in light regulation of DAC activity.

Expression of GluA2-containing calcium-impermeable AMPA receptors on dopaminergic amacrine cells in the mouse retina.

Correlated spontaneous activity in the developing retina (termed "retinal waves") plays an instructive role in refining neural circuits of the visual system. Depolarizing (ON) and hyperpolarizing (OFF) starburst amacrine cells (SACs) initiate and propagate cholinergic retinal waves. Where cholinergic retinal waves stop, SACs are thought to be driven by glutamatergic retinal waves initiated by ON-bipolar cells. However, the properties and function of cholinergic and glutamatergic waves in ON- and OFF-SACs still remain poorly understood. In the present work, we performed whole-cell patch-clamp recordings and Ca imaging from genetically labeled ON- and OFF-SACs in mouse flat-mount retinas. We found that both SAC subtypes exhibited spontaneous rhythmic depolarization during cholinergic and glutamatergic waves. Interestingly, ON-SACs had wave-induced action potentials (APs) in an age-dependent manner, but OFF-SACs did not. Simultaneous Ca imaging and patch-clamp recordings demonstrated that, during a cholinergic wave, APs of an ON-SAC appeared to promote the dendritic release of acetylcholine onto neighboring ON- and OFF-SACs, which enhances their Ca transients. These results advance the understanding of the cellular mechanisms underlying correlated spontaneous activity in the developing retina.

Dao-Qi Zhang

Eye Research Institute

Oakland University

Immunostaining of Whole-Mount Retinas with the CLARITY Tissue Clearing Method