Light disrupts social memory via a retina-to-supraoptic nucleus circuit.

The formation of social memory between individuals of the opposite sex is crucial for expanding mating options or establishing monogamous pair bonding. A specialized neuronal circuit that regulates social memory could enhance an individual's mating opportunities and provide a parallel pathway for computing social behaviors. While the influence of light exposure on various forms of memory, such as fear and object memory, has been studied, its modulation of social recognition memory remains unclear. Here, we demonstrate that acute exposure to light impairs social recognition memory (SRM) in mice. Unlike sound and touch stimuli, light inhibits oxytocin neurons in the supraoptic nucleus (SON) via M1 SON-projecting intrinsically photosensitive retinal ganglion cells (ipRGCs) and GABAergic neurons in the perinuclear zone of the SON (pSON). We further show that optogenetic activation of SON oxytocin neurons using channelrhodopsin is sufficient to enhance SRM performance, even under light conditions. Our findings unveil a dedicated neuronal circuit through which luminance affects SRM, utilizing a non-image-forming visual pathway, distinct from the canonical modulatory role of the oxytocin system.

External light activates hair follicle stem cells through eyes via an ipRGC-SCN-sympathetic neural pathway.

Changes in external light patterns can alter cell activities in peripheral tissues through slow entrainment of the central clock in suprachiasmatic nucleus (SCN). It remains unclear whether cells in otherwise photo-insensitive tissues can achieve rapid responses to changes in external light. Here we show that light stimulation of animals' eyes results in rapid activation of hair follicle stem cells with prominent hair regeneration. Mechanistically, light signals are interpreted by M1-type intrinsically photosensitive retinal ganglion cells (ipRGCs), which signal to the SCN via melanopsin. Subsequently, efferent sympathetic nerves are immediately activated. Increased norepinephrine release in skin promotes hedgehog signaling to activate hair follicle stem cells. Thus, external light can directly regulate tissue stem cells via an ipRGC-SCN autonomic nervous system circuit. Since activation of sympathetic nerves is not limited to skin, this circuit can also facilitate rapid adaptive responses to external light in other homeostatic tissues.

Degeneration of ipRGCs in Mouse Models of Huntington's Disease Disrupts Non-Image-Forming Behaviors Before Motor Impairment.

Intrinsically photosensitive retinal ganglion cells (ipRGCs), which express the photopigment melanopsin, are photosensitive neurons in the retina and are essential for non-image-forming functions, circadian photoentrainment, and pupillary light reflexes. Five subtypes of ipRGCs (M1-M5) have been identified in mice. Although ipRGCs are spared in several forms of inherited blindness, they are affected in Alzheimer's disease and aging, which are associated with impaired circadian rhythms. Huntington's disease (HD) is an autosomal neurodegenerative disease caused by the expansion of a CAG repeat in the huntingtin gene. In addition to motor function impairment, HD mice also show impaired circadian rhythms and loss of ipRGC. Here, we found that, in HD mouse models (R6/2 and N171-82Q male mice), the expression of melanopsin was reduced before the onset of motor deficits. The expression of retinal T-box brain 2, a transcription factor essential for ipRGCs, was associated with the survival of ipRGCs. The number of M1 ipRGCs in R6/2 male mice was reduced due to apoptosis, whereas non-M1 ipRGCs were relatively resilient to HD progression. Most importantly, the reduced innervations of M1 ipRGCs, which was assessed by X-gal staining in R6/2-OPN4Lacz/+ male mice, contributed to the diminished light-induced c-fos and vasoactive intestinal peptide in the suprachiasmatic nuclei (SCN), which may explain the impaired circadian photoentrainment in HD mice. Collectively, our results show that M1 ipRGCs were susceptible to the toxicity caused by mutant Huntingtin. The resultant impairment of M1 ipRGCs contributed to the early degeneration of the ipRGC-SCN pathway and disrupted circadian regulation during HD progression.SIGNIFICANCE STATEMENT Circadian disruption is a common nonmotor symptom of Huntington's disease (HD). In addition to the molecular defects in the suprachiasmatic nuclei (SCN), the cause of circadian disruption in HD remains to be further explored. We hypothesized that ipRGCs, by integrating light input to the SCN, participate in the circadian regulation in HD mice. We report early reductions in melanopsin in two mouse models of HD, R6/2, and N171-82Q. Suppression of retinal T-box brain 2, a transcription factor essential for ipRGCs, by mutant Huntingtin might mediate the reduced number of ipRGCs. Importantly, M1 ipRGCs showed higher susceptibility than non-M1 ipRGCs in R6/2 mice. The resultant impairment of M1 ipRGCs contributed to the early degeneration of the ipRGC-SCN pathway and the circadian abnormality during HD progression.

Analysis of experience-regulated transcriptome and imprintome during critical periods of mouse visual system development reveals spatiotemporal dynamics.

Visual system development is light-experience dependent, which strongly implicates epigenetic mechanisms in light-regulated maturation. Among many epigenetic processes, genomic imprinting is an epigenetic mechanism through which monoallelic gene expression occurs in a parent-of-origin-specific manner. It is unknown if genomic imprinting contributes to visual system development. We profiled the transcriptome and imprintome during critical periods of mouse visual system development under normal- and dark-rearing conditions using B6/CAST F1 hybrid mice. We identified experience-regulated, isoform-specific and brain-region-specific imprinted genes. We also found imprinted microRNAs were predominantly clustered into the Dlk1-Dio3 imprinted locus with light experience affecting some imprinted miRNA expression. Our findings provide the first comprehensive analysis of light-experience regulation of the transcriptome and imprintome during critical periods of visual system development. Our results may contribute to therapeutic strategies for visual impairments and circadian rhythm disorders resulting from a dysfunctional imprintome.

Architecture of retinal projections to the central circadian pacemaker.

The suprachiasmatic nucleus (SCN) receives direct retinal input from the intrinsically photosensitive retinal ganglion cells (ipRGCs) for circadian photoentrainment. Interestingly, the SCN is the only brain region that receives equal inputs from the left and right eyes. Despite morphological assessments showing that axonal fibers originating from ipRGCs cover the entire SCN, physiological evidence suggests that only vasoactive intestinal polypeptide (VIP)/gastrin-releasing peptide (GRP) cells located ventrally in the SCN receive retinal input. It is still unclear, therefore, which subpopulation of SCN neurons receives synaptic input from the retina and how the SCN receives equal inputs from both eyes. Here, using single ipRGC axonal tracing and a confocal microscopic analysis in mice, we show that ipRGCs have elaborate innervation patterns throughout the entire SCN. Unlike conventional retinal ganglion cells (RGCs) that innervate visual targets either ipsilaterally or contralaterally, a single ipRGC can bilaterally innervate the SCN. ipRGCs form synaptic contacts with major peptidergic cells of the SCN, including VIP, GRP, and arginine vasopressin (AVP) neurons, with each ipRGC innervating specific subdomains of the SCN. Furthermore, a single SCN-projecting ipRGC can send collateral inputs to many other brain regions. However, the size and complexity of the axonal arborizations in non-SCN regions are less elaborate than those in the SCN. Our results provide a better understanding of how retinal neurons connect to the central circadian pacemaker to synchronize endogenous circadian clocks with the solar day.

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

UNLABELLED: 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. 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.

Recurrent axon collaterals of intrinsically photosensitive retinal ganglion cells.

Retinal ganglion cells (RGCs), the output neurons of the retina, have axons that project via the optic nerve to diverse targets in the brain. Typically, RGC axons do not branch before exiting the retina and thus do not provide it with synaptic feedback. Although a small subset of RGCs with intraretinal axon collaterals has been previously observed in human, monkey, cat, and turtle, their function remains unknown. A small, more recently identified population of RGCs expresses the photopigment melanopsin. These intrinsically photosensitive retinal ganglion cells (ipRGCs) transmit an irradiance-coding signal to visual nuclei in the brain, contributing both to image-forming vision and to several nonimage-forming functions, including circadian photoentrainment and the pupillary light reflex. In this study, using melanopsin immunolabeling in monkey and a genetic method to sparsely label the melanopsin cells in mouse, we show that a subgroup of ipRGCs have axons that branch en route to the optic disc, forming intraretinal axon collaterals that terminate in the inner plexiform layer of the retina. The previously described collateral-bearing population identified by intracellular dye injection is anatomically indistinguishable from the collateral-bearing melanopsin cells identified here, suggesting they are a subset of the melanopsin-expressing RGC type and may therefore share its functional properties. Identification of an anatomically distinct subpopulation in mouse, monkey, and human suggests this pathway may be conserved in these and other species (turtle and cat) with intraretinal axon collaterals. We speculate that ipRGC axon collaterals constitute a likely synaptic pathway for feedback of an irradiance signal to modulate retinal light responses.

Early Dysbiosis and Dampened Gut Microbe Oscillation Precede Motor Dysfunction and Neuropathology in Animal Models of Parkinson's Disease.

BACKGROUND: Studies have shown different gut microbiomes in patients with Parkinson's disease (PD) compared to unaffected controls. However, when the gut microbiota shift toward dysbiosis in the PD process remains unclear. OBJECTIVE: We aim to investigate the changes in gut microbiota, locomotor function, and neuropathology longitudinally in PD rodent models. METHODS: Fecal microbiota were longitudinally assessed by sequencing the V4-V5 region of the 16S ribosomal RNA gene in a human mutant α-synuclein over-expressing mouse model of PD, SNCA p.A53T mice, and the non-transgenic littermate controls. The locomotor function, neuronal integrity, and α-synuclein expression in the different brain regions were compared between groups. Human fecal microbiota communities from 58 patients with PD and 46 unaffected controls were also analyzed using metagenomic sequencing for comparison. RESULTS: Compared to non-transgenic littermate controls, the altered gut microbiota of the SNCA p.A53T mice can be detected as early as 2 months old, and the diurnal oscillation of the gut microbiome was dampened throughout PD progression starting from 4 months old. However, neuropathology changes and motor deficits were observed starting at 6 months old. Similar changes in altered gut microbiota were also observed in another PD genetic mouse model carrying the LRRK2 p.G2019S mutation at 2 months old. Among the commonly enriched gut microbiota in both PD genetic mouse models, the abundance of Parabateroides Merdae and Ruminococcus torques were also increased in human PD patients compared to controls. CONCLUSION: These findings revealed the altered gut microbiota communities and oscillations preceding the occurrence of neuropathy and motor dysfunction in the PD process.

Single-laser-based simultaneous four-wavelength excitation source for femtosecond two-photon fluorescence microscopy.

Multicolor labeling of biological samples with large volume is required for omic-level of study such as the construction of nervous system connectome. Among the various imaging method, two photon microscope has multiple advantages over traditional single photon microscope for higher resolution and could image large 3D volumes of tissue samples with superior imaging depth. However, the growing number of fluorophores for labeling underlines the urgent need for an ultrafast laser source with the capability of providing simultaneous plural excitation wavelengths for multiple fluorophores. Here, we propose and demonstrate a single-laser-based four-wavelength excitation source for two-photon fluorescence microscopy. Using a sub-100 fs 1,070-nm Yb:fiber laser to pump an ultrashort nonlinear photonic crystal fiber in the low negative dispersion region, we introduced efficient self-phase modulation and acquired a blue-shifted spectrum dual-peaked at 812 and 960 nm with 28.5% wavelength conversion efficiency. By compressing the blue-shift near-IR spectrum to 33 fs to ensure the temporal overlap of the 812 and 960 nm peaks, the so-called sum frequency effect created the third virtual excitation wavelength effectively at 886 nm. Combined with the 1,070 nm laser source as the fourth excitation wavelength, the all-fiber-format four-wavelength excitation source enabled simultaneous four-color two-photon imaging in Brainbow AAV-labeled (TagBFP, mTFP, EYFP, and mCherry) brain samples. With an increased number of excitation wavelengths and improved excitation efficiency than typical commercial femtosecond lasers, our compact four-wavelength excitation approach can provide a versatile, efficient, and easily accessible solution for multiple-color two-photon fluorescence imaging in the field of neuroscience, biomolecular probing, and clinical applications with at least four spectrally-distinct fluorophores.

Dual GRIN lens two-photon endoscopy for high-speed volumetric and deep brain imaging.

Studying neural connections and activities in vivo is fundamental to understanding brain functions. Given the cm-size brain and three-dimensional neural circuit dynamics, deep-tissue, high-speed volumetric imaging is highly desirable for brain study. With sub-micrometer spatial resolution, intrinsic optical sectioning, and deep-tissue penetration capability, two-photon microscopy (2PM) has found a niche in neuroscience. However, the current 2PM typically relies on a slow axial scan for volumetric imaging, and the maximal penetration depth is only about 1 mm. Here, we demonstrate that by integrating a gradient-index (GRIN) lens and a tunable acoustic GRIN (TAG) lens into 2PM, both penetration depth and volume-imaging rate can be significantly improved. Specifically, an ∼ 1-cm long GRIN lens allows imaging relay from any target region of a mouse brain, while a TAG lens provides a sub-second volume rate via a 100 kHz ∼ 1 MHz axial scan. This technique enables the study of calcium dynamics in cm-deep brain regions with sub-cellular and sub-second spatiotemporal resolution, paving the way for interrogating deep-brain functional connectome.

Lysophosphatidic acid receptors 2 and 3 regulate erythropoiesis at different hematopoietic stages.

Hematopoiesis, the complex developmental process that forms blood components and replenishes the blood system, involves multiple intracellular and extracellular mechanisms. We previously demonstrated that lysophosphatidic acid (LPA), a lipid growth factor, has opposing regulatory effects on erythrocyte differentiation through activation of LPA receptors 2 and 3; yet the mechanisms underlying this process remain unclear. In this study, LPA2 is observed that highly expressed in common myeloid progenitors (CMP) in murine myeloid cells, whereas the expression of LPA3 displaces in megakaryocyte-erythroid progenitors (MEP) of later stage of myeloid differentiation. Therefore, we hypothesized that the switching expression of LPA2 and LPA3 determine the hematic homeostasis of mammalian megakaryocytic-erythroid lineage. In vitro colony-forming unit assays of murine progenitors reveal that LPA2 agonist GRI reduces the erythroblast differentiation potential of CMP. In contrast, LPA3 agonist OMPT increases the production of erythrocytes from megakaryocyte-erythrocyte progenitor cells (MEP). In addition, treatment with GRI reduces the erythroid, CMP, and MEP populations in mice, indicating that LPA2 predominantly inhibits myeloid differentiation at an early stage. In contrast, activation of LPA3 increases the production of terminally differentiated erythroid cells through activation of erythropoietic transcriptional factor. We also demonstrate that the LPA3 signaling is essential for restoration of phenylhydrazine (PHZ)-induced acute hemolytic anemia in mice and correlates to erythropoiesis impairment of Hutchinson-Gilford progeria Symptom (HGPS) premature aging expressed K562 model. Our results reveal the distinct roles of LPA2 and LPA3 at different stages of hematopoiesis in vivo, providing potentiated therapeutic strategies of anemia treatment.

Transcriptome Analysis of Dnmt3l Knock-Out Mice Derived Multipotent Mesenchymal Stem/Stromal Cells During Osteogenic Differentiation.

Multipotent mesenchymal stem/stromal cells (MSCs) exhibit great potential for cell-based therapy. Proper epigenomic signatures in MSCs are important for the maintenance and the subsequent differentiation potential. The DNA methyltransferase 3-like (DNMT3L) that was mainly expressed in the embryonic stem (ES) cells and the developing germ cells plays an important role in shaping the epigenetic landscape. Here, we report the reduced colony forming ability and impaired in vitro osteogenesis in Dnmt3l-knockout-mice-derived MSCs (Dnmt3l KO MSCs). By comparing the transcriptome between undifferentiated Dnmt3l KO MSCs and the MSCs from the wild-type littermates, some of the differentially regulated genes (DEGs) were found to be associated with bone-morphology-related phenotypes. On the third day of osteogenic induction, differentiating Dnmt3l KO MSCs were enriched for genes associated with nucleosome structure, peptide binding and extracellular matrix modulation. Differentially expressed transposable elements in many subfamilies reflected the change of corresponding regional epigenomic signatures. Interestingly, DNMT3L protein is not expressed in cultured MSCs. Therefore, the observed defects in Dnmt3l KO MSCs are unlikely a direct effect from missing DNMT3L in this cell type; instead, we hypothesized them as an outcome of the pre-deposited epigenetic signatures from the DNMT3L-expressing progenitors. We observed that 24 out of the 107 upregulated DEGs in Dnmt3l KO MSCs were hypermethylated in their gene bodies of DNMT3L knock-down ES cells. Among these 24 genes, some were associated with skeletal development or homeostasis. However, we did not observe reduced bone development, or reduced bone density through aging in vivo. The stronger phenotype in vitro suggested the involvement of potential spreading and amplification of the pre-deposited epigenetic defects over passages, and the contribution of oxidative stress during in vitro culture. We demonstrated that transient deficiency of epigenetic co-factor in ES cells or progenitor cells caused compromised property in differentiating cells much later. In order to facilitate safer practice in cell-based therapy, we suggest more in-depth examination shall be implemented for cells before transplantation, even on the epigenetic level, to avoid long-term risk afterward.

Mapping Central Projection of Oxytocin Neurons in Unmated Mice Using Cre and Alkaline Phosphatase Reporter.

Oxytocin, a neuropeptide and peptide hormone, is produced by neurons in the hypothalamus and released by the posterior pituitary to control breastfeeding and labor. Recent studies have revealed that oxytocin in the central nervous system is also involved in modulating social interaction. To understand the potential role and innervation pattern of oxytocin neurons before sexual interaction, here we used transgenic mice which have the Cre recombinase under the control of an endogenous oxytocin promoter and Cre-dependent human placental alkaline phosphatase (AP) reporter to label the oxytocin neurons in the naive mouse brain. Since AP is located on the membrane of oxytocin neurons, AP histochemistry staining enabled us to observe the fine axonal terminals and the innervation pattern of oxytocin neurons in the thick serial coronal brain slices. Here we show that the number of AP-labeled cells varies with staining reaction time and ranges from 30% of the oxytocin immune-positive cell count to slightly higher than the oxytocin immune-positive cell count. Using AP staining with extended reaction time, which may not label all oxytocin neurons, we confirmed many innervation targets of oxytocin neurons from the anterior olfactory nucleus, some cortex regions, the limbic system, the hypothalamus, and the hindbrain, while the cell bodies were exclusively located in the hypothalamus and the bed nucleus of the stria terminalis. Finally, we observe some individual variance at the olfactory area, isocortex, striatum, paraventricular nucleus of thalamus, locus coeruleus, and Barrington's nucleus.

An Index Combining Lost and Remaining Nerve Fibers Correlates with Pain Hypersensitivity in Mice.

Multiple peripheral nerves are known to degenerate after nerve compression injury but the correlation between the extent of nerve alteration and pain severity remains unclear. Here, we used intravital two-photon fluorescence microscopy to longitudinally observe changes in cutaneous fibers in the hind paw of Nav1.8-Cre-tdTomato mice after chronic constriction injury (CCI). Results showed that the CCI led to variable loss of the skin nerve plexus and intraepidermal nerve fibers. The timing of Nav1.8 nerve fiber loss correlated with the development of mechanical hypersensitivity. We compared a scoring approach that assessed whole-paw nerve degeneration with an index that quantified changes in the nerve plexus and terminals in multiple small regions of interest (ROI) from intravital images of the third and fifth toe tips. We found that the number of surviving nerve fibers was not linearly correlated with mechanical hypersensitivity. On the contrary, at 14 days after CCI, the moderately injured mice showed greater mechanical hypersensitivity than the mildly or severely injured mice. This indicates that both surviving and injured nerves are required for evoked neuropathic pain. In addition, these two methods may have the estimative effect as diagnostic and prognostic biomarkers for the assessment of neuropathic pain.

Lysophosphatidic acid receptor LPA3 prevents oxidative stress and cellular senescence in Hutchinson-Gilford progeria syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare laminopathy that produces a mutant form of prelamin A, known as Progerin, resulting in premature aging. HGPS cells show morphological abnormalities of the nuclear membrane, reduced cell proliferation rates, accumulation of reactive oxygen species (ROS), and expression of senescence markers. Lysophosphatidic acid (LPA) is a growth factor-like lipid mediator that regulates various physiological functions via activating multiple LPA G protein-coupled receptors. Here, we study the roles of LPA and LPA receptors in premature aging. We report that the protein level of LPA3 was highly downregulated through internalization and the lysosomal degradation pathway in Progerin-transfected HEK293 cells. By treating Progerin HEK293 cells with an LPA3 agonist (OMPT, 1-Oleoyl-2-O-methyl-rac-glycerophosphothionate) and performing shRNA knockdown of the Lpa3r transcript in these cells, we showed that LPA3 activation increased expression levels of antioxidant enzymes, consequently inhibiting ROS accumulation and ameliorating cell senescence. LPA3 was shown to be downregulated in HGPS patient fibroblasts through the lysosomal pathway, and it was shown to be crucial for ameliorating ROS accumulation and cell senescence in fibroblasts. Moreover, in a zebrafish model, LPA3 deficiency was sufficient to cause premature aging phenotypes in multiple organs, as well as a shorter lifespan. Taken together, these findings identify the decline of LPA3 as a key contributor to the premature aging phenotypes of HGPS cells and zebrafish.

Somatostatin peptides produce multiple effects on gating properties of native cone photoreceptor cGMP-gated channels that depend on circadian phase and previous illumination.

A subpopulation of avian amacrine cells expresses somatostatin-14 (SS14) and somatostatin-28 (SS28), which provide a potential efferent limb for light-dependent regulation of photoreceptors. Here, we demonstrate that SS14 and SS28 modulate cone photoreceptor cGMP-gated channels (CNGCs) through multiple mechanisms. In chicken cones cultured in constant darkness for 2 d after previous entrainment to light-dark (LD) cycles or in cells maintained in LD, application of 100 nm SS14 or 100 nm SS28 for either 15 min or 2 h caused a decrease in the sensitivity of CNGCs to cGMP during the night, at circadian time 16 (CT16)-CT20 or zeitgeber time 16 (ZT16)-ZT20. SS14 had no effect during the day (CT4-CT8 or ZT4-ZT8). These effects persist in cells pretreated with pertussis toxin (PTX) and, like dopamine, may work to reinforce long-term circadian fluctuations in CNGCs driven by oscillators within the photoreceptors themselves. In contrast, a 15 min exposure to SS28 caused a seemingly paradoxical increase in the sensitivity of CNGCs to cGMP during the early day (ZT4-ZT6), but only in cones maintained in LD. This effect of SS28 desensitizes rapidly, is blocked by pretreatment with PTX, and is selectively mimicked by the cyclohexapeptide agonist MK-678. This transient response also requires activation of phospholipase C and protein kinase C. The transient response to SS28 may play a role in photoreceptor adaptation to rapid changes in ambient illumination. These data also show that photoreceptor responses to at least some peptide neurotransmitters depend on the previous history of light exposure.

RBFOX3/NeuN is dispensable for visual function.

RBFOX3/NeuN is a neuronal splicing regulator involved in neural circuitry balance, as well as neurogenesis and synaptogenesis. Rbfox3 is expressed in neurons; however, in the retina, expression is restricted to cells in the ganglion cell layer and some cells of the inner nuclear layer. Rbfox3 is expressed in a layer-specific manner in the retina, which implies a functional role, however, the role of RBFOX3 in the retina is unknown. Rbfox3 homozygous knockout (Rbfox3-/-) mice exhibit deficits in visual learning; therefore, understanding the role of RBFOX3 in the retina is critical for interpreting behavioral results. We found Rbfox3 expression was developmentally regulated in the retina and specifically expressed in ganglion cells, amacrine cells and horizontal cells of the retina. We demonstrate deletion of Rbfox3 resulted in a reduction in the thickness of the inner plexiform layer of the retina, where synapses are formed. Number of ganglion cells and amacrine cells is normal with loss of Rbfox3. Innervation of retinal ganglion cells into their targeted brain regions is normal in Rbfox3-/- mice. Importantly, Rbfox3-/- mice displayed normal non-image and image forming functions. Taken together, our results suggest RBFOX3 is dispensable for visual function.

Apoptosis regulates ipRGC spacing necessary for rods and cones to drive circadian photoentrainment.

The retina consists of ordered arrays of individual types of neurons for processing vision. Here, we show that such order is necessary for intrinsically photosensitive retinal ganglion cells (ipRGCs) to function as irradiance detectors. We found that during development, ipRGCs undergo proximity-dependent Bax-mediated apoptosis. Bax mutant mice exhibit disrupted ipRGC spacing and dendritic stratification with an increase in abnormally localized synapses. ipRGCs are the sole conduit for light input to circadian photoentrainment, and either their melanopsin-based photosensitivity or ability to relay rod/cone input is sufficient for circadian photoentrainment. Remarkably, the disrupted ipRGC spacing does not affect melanopsin-based circadian photoentrainment but severely impairs rod/cone-driven photoentrainment. We demonstrate reduced rod/cone-driven cFos activation and electrophysiological responses in ipRGCs, suggesting that impaired synaptic input to ipRGCs underlies the photoentrainment deficits. Thus, for irradiance detection, developmental apoptosis is necessary for the spacing and connectivity of ipRGCs that underlie their functioning within a neural network.

Intrinsically photosensitive retinal ganglion cells: many subtypes, diverse functions.

For decades, rods and cones were thought to be the only photoreceptors in the mammalian retina. However, a population of atypical photoreceptive retinal ganglion cells (RGCs) expresses the photopigment melanopsin and is intrinsically photosensitive (ipRGCs). These ipRGCs are crucial for relaying light information from the retina to the brain to control circadian photoentrainment, pupillary light reflex, and sleep. ipRGCs were initially described as a uniform population involved solely in signaling irradiance for non-image forming functions. Recent work, however, has uncovered that ipRGCs are unexpectedly diverse at the molecular, cellular and functional levels, and could even be involved in image formation. This review summarizes our current understanding of the diversity of ipRGCs and their various roles in modulating behavior.

Melanopsin-expressing retinal ganglion-cell photoreceptors: cellular diversity and role in pattern vision.

Using the photopigment melanopsin, intrinsically photosensitive retinal ganglion cells (ipRGCs) respond directly to light to drive circadian clock resetting and pupillary constriction. We now report that ipRGCs are more abundant and diverse than previously appreciated, project more widely within the brain, and can support spatial visual perception. A Cre-based melanopsin reporter mouse line revealed at least five subtypes of ipRGCs with distinct morphological and physiological characteristics. Collectively, these cells project beyond the known brain targets of ipRGCs to heavily innervate the superior colliculus and dorsal lateral geniculate nucleus, retinotopically organized nuclei mediating object localization and discrimination. Mice lacking classical rod-cone photoreception, and thus entirely dependent on melanopsin for light detection, were able to discriminate grating stimuli from equiluminant gray and had measurable visual acuity. Thus, nonclassical retinal photoreception occurs within diverse cell types and influences circuits and functions encompassing luminance as well as spatial information.