Phosphoinositides play important roles in numerous intracellular membrane pathways. Little is known about the regulation or function of these lipids in rod photoreceptor cells, which have highly active membrane dynamics. Using new assays with femtomole sensitivity, we determined that whereas levels of phosphatidylinositol-3,4-bisphosphate and phosphatidylinositol-3,4,5-trisphosphate were below detection limits, phosphatidylinositol-3-phosphate (PI(3)P) levels in rod inner/outer segments increased more than 30-fold after light exposure. This increase was blocked in a rod-specific knockout of the PI-3 kinase Vps34, resulting in failure of endosomal and autophagy-related membranes to fuse with lysosomes, and accumulation of abnormal membrane structures. At early ages, rods displayed normal morphology, rhodopsin trafficking, and light responses, but underwent progressive neurodegeneration with eventual loss of both rods and cones by twelve weeks. The degeneration is considerably faster than in rod knockouts of autophagy genes, indicating defects in endosome recycling or other PI(3)P-dependent membrane trafficking pathways are also essential for rod survival.

The rod cell has an extraordinarily specialized structure that allows it to carry out its unique function of detecting individual photons of light. Both the structural features of the rod and the metabolic processes required for highly amplified light detection seem to have rendered the rod especially sensitive to structural and metabolic defects, so that a large number of gene defects are primarily associated with rod cell death and give rise to blinding retinal dystrophies. The structures of the rod, especially those of the sensory cilium known as the outer segment, have been the subject of structural, biochemical, and genetic analysis for many years, but the molecular bases for rod morphogenesis and for cell death in rod dystrophies are still poorly understood. Recent developments in imaging technology, such as cryo-electron tomography and super-resolution fluorescence microscopy, in gene sequencing technology, and in gene editing technology are rapidly leading to new breakthroughs in our understanding of these questions. A summary is presented of our current understanding of selected aspects of these questions, highlighting areas of uncertainty and contention as well as recent discoveries that provide new insights. Examples of structural data from emerging imaging technologies are presented.

SUMMARY Loss of primary neuronal inputs inevitably strikes every neural circuit. The deafferented circuit could propagate, amplify, or mitigate input loss, thus affecting the circuit’s output. How the deafferented circuit contributes to the effect on the output is poorly understood because of lack of control over loss of and access to circuit elements. Here, we control the timing and degree of rod photoreceptor ablation in mature mouse retina and uncover compensation. Following loss of half of the rods, rod bipolar cells mitigate the loss by preserving voltage output. Such mitigation allows partial recovery of ganglion cell responses. We conclude that rod death is compensated for in the circuit because ganglion cell responses to stimulation of half of the rods in an unperturbed circuit are weaker than responses after death of half of the rods. The dominant mechanism of such compensation includes homeostatic regulation of inhibition to balance the loss of excitation.

Background: TRPM1 is essential for the light response of retinal depolarizing bipolar cells. Results: Recombinant purified TRPM1 is mostly dimeric, and a low resolution cryo-EM structure is presented. Conclusion: Because most TRP channels function as tetramers, active TRPM1 channels likely require additional partner subunits. Significance: The results suggest a novel paradigm for structure and regulation within the TRP channel family.

The nucleated thrombocytes of non-mammalian vertebrates are partially flattened, ovoid cells morphologically distinct from mammalian platelets, and the extent of their functional equivalence is unknown. To test whether they resemble platelets in having similar F-actin-based post-activation stages, rapid fixation/extraction/labeling methods were developed to reveal cytoskeletal organization in dogfish thrombocytes by confocal microscopy. Unactivated cells contained cortical F-actin plus denser F-actin co-localizing with outer marginal band (MB) microtubules. In the post-activation sequence, determined for the first time by continuous observation of individual thrombocytes following thrombin perfusion, cells rounded and blebbed, spread, and eventually flattened extensively. The MB twisted and then became disorganized, with microtubule bundles remaining centrally located and associated with nuclear clefts. In contrast, F-actin occupied blebs and outward-spreading cytoplasm, initially in spiky projections, then predominantly in stress fibers, and inhibitors of F-actin assembly or myosin ATPase blocked shape changes. Thus, the post-activation stages and cytoskeletal events observed in nucleated thrombocytes were found to parallel those of platelets.

There is mounting evidence that zinc release from glutamatergic nerve terminals serves as a neuromodulator at synaptic sites within the retina and CNS. However, it has not been possible to reliably measure the concentration of zinc co-released with glutamate in the confines of the synaptic cleft. Thus, much of the evidence supporting this view derives from electrophysiological studies showing the modulatory effects of exogenous zinc on the membrane currents of ligand-and voltagegated channels. In the present study, we took advantage of the unique properties of the glutamatergic photoreceptor terminal to demonstrate a feedback signal mediated by endogenous zinc at the synaptic sites from which it is discharged. Through its ability to block voltage-gated calcium channels in the photoreceptor terminal, zinc suppresses the radial dark current of the visual cell, and reduces its release of glutamate. It follows that chelation of extracellular zinc, e.g., with histidine, will lead to an increase both in the dark current and in the release of glutamate, changes that result in an enhancement of the light-evoked a-wave of the ERG and can account for the b-wave enhancement observed previously after zinc chelation when inner retinal responses were not blocked by aspartate. Keywords skate; retina; rod photoreceptors; zinc; feedback Zinc is unquestionably one of the most ubiquitous trace elements in biological systems, and it is now universally acknowledged that zinc is indispensable to all living organisms: (i) zinc serves as an integral and essential component of scores of enzymes, (ii) it participates in a wide variety of metabolic functions, and (iii) it plays a significant role in translation and transcription of the genetic message. Moreover, zinc is indispensable to the growth and development of all forms of life (cf. reviews by Vallee, 1988;O'Halloran, 1993), and the serious consequences of zinc deficiency have been well documented (cf. Smith et al., 1973;Leopold, 1978;Hambidge, 1981;Krebs et al., 2000;Di Cello et al., 2005;Olmez et al., 2007). With the advent of a range of sensitive detection methods (cf. Danscher et al., 1985;Frederickson et Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. 1982, 1987Christensen et al., 1992;Simons, 1993;Thompson et al., 2002), zinc was shown to be present in virtually every tissue of the body where it exists primarily complexed with proteins that serve both metabolic reactions and structural functions. Although there is evidence for the presence of free or loosely bound Zn 2+ ions within the cytoplasm and body fluids (Frederickson, ...

The transient receptor potential channel TRPM1 is required for synaptic transmission between photoreceptors and the ON subtype of bipolar cells (ON-BPC), mediating depolarization in response to light. TRPM1 is present in the somas and postsynaptic dendritic tips of ON-BPCs. Monoclonal antibodies generated against full-length TRPM1 were found to have differential labeling patterns when used to immunostain the mouse retina, with some yielding reduced labeling of dendritic tips relative to the labeling of cell bodies. Epitope mapping revealed that those antibodies that poorly label the dendritic tips share a binding site (N2d) in the N-terminal arm near the transmembrane domain. A major splice variant of TRPM1 lacking exon 19 does not contain the N2d binding site, but quantitative immunoblotting revealed no enrichment of this variant in synaptsomes. One explanation of the differential labeling is masking of the N2d epitope by formation of a synapse-specific multiprotein complex. Identifying the binding partners that are specific for the fraction of TRPM1 present at the synapses is an ongoing challenge for understanding TRPM1 function.

The mammalian retina consists of multiple cell layers including photoreceptor cells, which are light sensing neurons that play essential functions in the visual process. Previously, we identified mutations in SPATA7, encoding spermatogenesis associated protein 7, in families with Leber Congenital Amaurosis (LCA) and juvenile Retinitis Pigmentosa (RP), and showed that Spata7 null mice recapitulate the human disease phenotype of retinal degeneration. SPATA7 is expressed in the connecting cilium of photoreceptor (PR) cells in the mouse retina, as well as in retinal pigment epithelium (RPE) cells, but the functional role of Spata7 in the RPE remains unknown. To investigate whether Spata7 is required in PRs, the RPE, or both, we conditionally knocked out Spata7 in photoreceptors and RPE cells using Crx-Cre and Best1-Cre transgenic mouse lines, respectively. In Spata7 photoreceptor-specific conditional (cKO) mice, both rod and cone photoreceptor dysfunction and degeneration is observed, characterized by progressive thinning of the outer nuclear layer and reduced response to light; however, RPE-specific deletion of Spata7 does not impair retinal function or cell survival. Furthermore, our findings show that both Rhodopsin and RPGRIP1 are mislocalized in the Spata7; Crx-Cre cKO mice, suggesting that loss of Spata7 in photoreceptors alone can result in altered trafficking of these proteins in the connecting cilium. Together, our findings suggest that loss of Spata7 in photoreceptors alone is sufficient to cause photoreceptor degeneration, but its function in the RPE is not required for photoreceptor survival; therefore, loss of Spata7 in photoreceptors alters both rod and cone function and survival, consistent with the clinical phenotypes observed in LCA and RP patients with mutations in SPATA7.