Christopher W. Yee

Aberrant activity in retinal degeneration impairs central visual processing and relies on Cx36-containing gap junctions

In retinal degenerative disease (RD), the diminished light signal from dying photoreceptors has been considered the sole cause of visual impairment. Recent studies show a 10-fold increase in spontaneous activity in the RD network, challenging this paradigm. This aberrant activity forms a new barrier for the light signal, and not only exacerbates the loss of vision, but also may stand in the way of visual restoration. This activity originates in AII amacrine cells and relies on excessive activation of gap junctions. However, it remains unclear whether aberrant activity affects central visual processing and what mechanisms lead to this excessive activation of gap junctions. By combining genetic manipulation with electrophysiological recordings of light-induced activity in both living mice and isolated wholemount retina, we demonstrate that aberrant activity extends along retinotectal projections to alter activity in higher brain centers. Next, to selectively eliminate Cx36-containing gap junctions, which are the primary type expressed by AII amacrine cells, we crossed rd10 mice, a slow-degenerating model of RD, with Cx36 knockout mice. We found that retinal aberrant activity was reduced in the rd10/Cx36KO mice compared to rd10 controls, a direct evidence for involvement of Cx36-containing gap junctions in generating aberrant activity in RD. These data provide an essential support for future experiments to determine if selectively targeting these gap junctions could be a valid strategy for reducing aberrant activity and restoring light responses in RD.

Experience-Enabled Enhancement of Adult Visual Cortex Function

We previously reported in adult mice that visuomotor experience during monocular deprivation (MD) augmented enhancement of visual-cortex-dependent behavior through the non-deprived eye (NDE) during deprivation, and enabled enhanced function to persist after MD. We investigated the physiological substrates of this experience-enabled form of adult cortical plasticity by measuring visual behavior and visually evoked potentials (VEPs) in binocular visual cortex of the same mice before, during, and after MD. MD on its own potentiated VEPs contralateral to the NDE during MD and shifted ocular dominance (OD) in favor of the NDE in both hemispheres. Whereas we expected visuomotor experience during MD to augment these effects, instead enhanced responses contralateral to the NDE, and the OD shift ipsilateral to the NDE were attenuated. However, in the same animals, we measured NMDA receptor-dependent VEP potentiation ipsilateral to the NDE during MD, which persisted after MD. The results indicate that visuomotor experience during adult MD leads to enduring enhancement of behavioral function, not simply by amplifying MD-induced changes in cortical OD, but through an independent process of increasing NDE drive in ipsilateral visual cortex. Because the plasticity is resident in the mature visual cortex and selectively effects gain of visual behavior through experiential means, it may have the therapeutic potential to target and non-invasively treat eye- or visual-field-specific cortical impairment.

Increased phosphorylation of Cx36 gap junctions in the AII amacrine cells of RD retina

Retinal degeneration (RD) encompasses a family of diseases that lead to photoreceptor death and visual impairment. Visual decline due to photoreceptor cell loss is further compromised by emerging spontaneous hyperactivity in inner retinal cells. This aberrant activity acts as a barrier to signals from the remaining photoreceptors, hindering therapeutic strategies to restore light sensitivity in RD. Gap junctions, particularly those expressed in AII amacrine cells, have been shown to be integral to the generation of aberrant activity. It is unclear whether gap junction expression and coupling are altered in RD. To test this, we evaluated the expression and phosphorylation state of connexin36 (Cx36), the gap junction subunit predominantly expressed in AII amacrine cells, in two mouse models of RD, rd10 (slow degeneration) and rd1 (fast degeneration). Using Ser293-P antibody, which recognizes a phosphorylated form of connexin36, we found that phosphorylation of connexin36 in both slow and fast RD models was significantly greater than in wildtype controls. This elevated phosphorylation may underlie the increased gap junction coupling of AII amacrine cells exhibited by RD retina.

Disruption in dopaminergic innervation during photoreceptor degeneration.

Dopaminergic amacrine cells (DACs) release dopamine in response to light‐driven synaptic inputs, and are critical to retinal light adaptation. Retinal degeneration (RD) compromises the light responsiveness of the retina and, subsequently, dopamine metabolism is impaired. As RD progresses, retinal neurons exhibit aberrant activity, driven by AII amacrine cells, a primary target of the retinal dopaminergic network. Surprisingly, DACs are an exception to this physiological change; DACs exhibit rhythmic activity in healthy retina, but do not burst in RD. The underlying mechanism of this divergent behavior is not known. It is also unclear whether RD leads to structural changes in DACs, impairing functional regulation of AII amacrine cells. Here we examine the anatomical details of DACs in three mouse models of human RD to determine how changes to the dopaminergic network may underlie physiological changes in RD. By using rd10, rd1, and rd1/C57 mice we were able to dissect the impacts of genetic background and the degenerative process on DAC structure in RD retina. We found that DACs density, soma size, and primary dendrite length are all significantly reduced. Using a novel adeno‐associated virus‐mediated technique to label AII amacrine cells in mouse retina, we observed diminished dopaminergic contacts to AII amacrine cells in RD mice. This was accompanied by changes to the components responsible for dopamine synthesis and release. Together, these data suggest that structural alterations of the retinal dopaminergic network underlie physiological changes during RD. J. Comp. Neurol. 524:1208–1221, 2016.

Leveraging Optogenetic-Based Neurovascular Circuit Characterization for Repair

Optogenetic techniques are a powerful tool for determining the role of individual functional components within complex neural circuits. By genetically targeting specific cell types, neural mechanisms can be actively manipulated to gain a better understanding of their origin and function, both in health and disease. The potential of optogenetics is not limited to answering biological questions, as it is also a promising therapeutic approach for neurological diseases. An important prerequisite for this approach is to have an identified target with a uniquely defined role within a given neural circuit. Here, we examine the retinal neurovascular unit, a circuit that incorporates neurons and vascular cells to control blood flow in the retina. We highlight the role of a specific cell type, the cholinergic amacrine cell, in modulating vascular cells, and demonstrate how this can be targeted and controlled with optogenetics. A better understanding of these mechanisms will not only extend our understanding of neurovascular interactions in the brain, but ultimately may also provide new targets to treat vision loss in a variety of retinal diseases.

Pannexin 1 sustains the electrophysiological responsiveness of retinal ganglion cells

Pannexin 1 (Panx1) forms ATP-permeable membrane channels that play a key role in purinergic signaling in the nervous system in both normal and pathological conditions. In the retina, particularly high levels of Panx1 are found in retinal ganglion cells (RGCs), but the normal physiological function in these cells remains unclear. In this study, we used patch clamp recordings in the intact inner retina to show that evoked currents characteristic of Panx1 channel activity were detected only in RGCs, particularly in the OFF-type cells. The analysis of pattern electroretinogram (PERG) recordings indicated that Panx1 contributes to the electrical output of the retina. Consistently, PERG amplitudes were significantly impaired in the eyes with targeted ablation of the Panx1 gene in RGCs. Under ocular hypertension and ischemic conditions, however, high Panx1 activity permeated cell membranes and facilitated the selective loss of RGCs or stably transfected Neuro2A cells. Our results show that high expression of the Panx1 channel in RGCs is essential for visual function in the inner retina but makes these cells highly sensitive to mechanical and ischemic stresses. These findings are relevant to the pathophysiology of retinal disorders induced by increased intraocular pressure, such as glaucoma.

Atypical Expression and Activation of GluN2A- and GluN2B-Containing NMDA Receptors at Ganglion Cells during Retinal Degeneration

Cellular communication through chemical synapses is determined by the nature of the neurotransmitter and the composition of postsynaptic receptors. In the excitatory synapse between bipolar and ganglion cells of the retina, postsynaptic AMPA receptors mediate resting activity. During evoked response, however, more abundant and sustained levels of glutamate also activate GluN2B-containing NMDA receptors (NMDARs). This phasic recruitment of distinct glutamate receptors is essential for visual discrimination; however, the fidelity of this basic mechanism under elevated glutamate levels due to aberrant activity, a common pathophysiology, is not known. Here, in both male and female mice with retinal degeneration (rd10), a condition associated with elevated synaptic activity, we reveal that changes in synaptic input to ganglion cells altered both composition and activation of NMDARs. We found that, in contrast to wild type, the spontaneous activity of rd10 cells was largely NMDAR-dependent. Surprisingly, this activity was driven primarily by atypical activation of GluN2A -containing NMDARs, not GluN2B-NMDARs. Indeed, immunohistochemical analyses and Western blot showed greater levels of the GluN2A-NMDAR subunit expression in rd10 retina compared to wild type. Overall, these results demonstrate how aberrant signaling leads to pathway-specific alterations in NMDAR expression and function.