ON and OFF starburst amacrine cells are controlled by distinct cholinergic pathways.

Cholinergic signaling in the retina is mediated by acetylcholine (ACh) released from starburst amacrine cells (SACs), which are key neurons for motion detection. SACs comprise ON and OFF subtypes, which morphologically show mirror symmetry to each other. Although many physiological studies on SACs have targeted ON cells only, the synaptic computation of ON and OFF SACs is assumed to be similar. Recent studies demonstrated that gene expression patterns and receptor types differed between ON and OFF SACs, suggesting differences in their functions. Here, we compared cholinergic signaling pathways between ON and OFF SACs in the mouse retina using the patch clamp technique. The application of ACh increased GABAergic feedback, observed as postsynaptic currents to SACs, in both ON and OFF SACs; however, the mode of GABAergic feedback differed. Nicotinic receptors mediated GABAergic feedback in both ON and OFF SACs, while muscarinic receptors mediated GABAergic feedback in ON SACs only in adults. Neither tetrodotoxin, which blocked action potentials, nor LY354740, which blocked neurotransmitter release from SACs, eliminated ACh-induced GABAergic feedback in SACs. These results suggest that ACh-induced GABAergic feedback in ON and OFF SACs is regulated by different feedback mechanisms in adults and mediated by non-spiking amacrine cells other than SACs.

Effects of Progesterone and Other Gonadal Hormones on Glutamatergic Circuits in the Retina.

BACKGROUND: Gonadal hormones function in the retina; however, their targets have not yet been identified. Therefore, the present study examined the effects of progesterone and other gonadal hormones on glutamatergic circuits in the retina. METHODS: Extracellular glutamate concentrations, which correspond to the amount of glutamate released, were examined using an enzyme-linked fluorescent assay system. The activity of glutamatergic synapses between bipolar cells and ganglion cells was investigated using a patch clamp technique. Changes in retinal thickness during pregnancy were assessed using optical coherence tomography (OCT) images. RESULTS: Progesterone and pregnenolone sulfate increased extracellular glutamate concentrations, whereas estrogen and testosterone did not. Progesterone increased the activity of glutamatergic synapses between bipolar cells and ganglion cells. A temporal decrease in the thickness of the peripheral retina was observed in the 1st trimester. CONCLUSIONS: Progesterone, but not estrogen or testosterone, activated glutamate release in the mouse retina. Increases in the concentration of progesterone during pregnancy did not induce any detectable change in retinal thickness.

Starburst amacrine cells form gap junctions in the early postnatal stage of the mouse retina.

Although gap junctional coupling in the developing retina is important for the maturation of neuronal networks, its role in the development of individual neurons remains unclear. Therefore, we herein investigated whether gap junctional coupling by starburst amacrine cells (SACs), a key neuron for the formation of direction selectivity, occurs during the developmental stage in the mouse retina. Neurobiotin-injected SACs coupled with many neighboring cells before eye-opening. The majority of tracer-coupled cells were retinal ganglion cells, and tracer coupling was not detected between SACs. The number of tracer-coupled cells significantly decreased after eye-opening and mostly disappeared by postnatal day 28 (P28). Membrane capacitance (Cm), an indicator of the formation of electrical coupling with gap junctions, was larger in SACs before than after eye-opening. The application of meclofenamic acid, a gap junction blocker, reduced the Cm of SACs. Gap junctional coupling by SACs was regulated by dopamine D1 receptors before eye-opening. In contrast, the reduction in gap junctional coupling after eye-opening was not affected by visual experience. At the mRNA level, 4 subtypes of connexins (23, 36, 43, and 45) were detected in SACs before eye-opening. Connexin 43 expression levels significantly decreased after eye-opening. These results indicate that gap junctional coupling by SACs occurs during the developmental period and suggest that the elimination of gap junctions proceeds with the innate system.

P2X2 receptors supply extracellular choline as a substrate for acetylcholine synthesis.

Acetylcholine (ACh), an excitatory neurotransmitter, is biosynthesized from choline in cholinergic neurons. Import from the extracellular space to the intracellular environment through the high-affinity choline transporter is currently regarded to be the only source of choline for ACh synthesis. We recently demonstrated that the P2X2 receptor, through which large cations permeate, functions as an alternative pathway for choline transport in the mouse retina. In the present study, we investigated whether choline entering cells through P2X2 receptors is used for ACh synthesis using a recombinant system. When P2X2 receptors expressed on HEK293 cell lines were stimulated with ATP, intracellular ACh concentrations increased. These results suggest that P2X2 receptors function in a novel pathway that supplies choline for ACh synthesis.

Involvement of the C-terminal domain in cell surface localization and G-protein coupling of mGluR6.

Metabotropic glutamate receptor 6, mGluR6, interacts with scaffold proteins and Gßγ subunits via its intracellular C-terminal domain (CTD). The mGluR6 pathway is critically involved in the retinal processing of visual signals. We herein investigated whether the CTD (residues 840-871) was necessary for mGluR6 cell surface localization and G-protein coupling using mGluR6-CTD mutants with immunocytochemistry, surface biotinylation assays, and electrophysiological approaches. We used 293T cells and primary hippocampal neurons as model systems. We examined C-terminally truncated mGluR6 and showed that the removal of up to residue 858 did not affect surface localization or glutamate-induced G-protein-mediated responses, whereas a 15-amino acid deletion (Δ857-871) impaired these functions. However, a 21-amino acid deletion (Δ851-871) restored surface localization and glutamate-dependent responses, which were again attenuated when the entire CTD was removed. The sequence alignment of group III mGluRs showed conserved amino acids resembling an ER retention motif in the CTD. These results suggest that the intracellular CTD is required for the cell surface transportation and receptor function of mGluR6, whereas it may contain regulatory elements for intracellular trafficking and signaling.

Orai1 Channels Are Essential for Amplification of Glutamate-Evoked Ca2+ Signals in Dendritic Spines to Regulate Working and Associative Memory.

Store-operated Orai1 calcium channels function as highly Ca2+-selective ion channels and are broadly expressed in many tissues including the central nervous system, but their contributions to cognitive processing are largely unknown. Here, we report that many measures of synaptic, cellular, and behavioral models of learning are markedly attenuated in mice lacking Orai1 in forebrain excitatory neurons. Results with focal glutamate uncaging in hippocampal neurons support an essential role of Orai1 channels in amplifying NMDA-receptor-induced dendritic Ca2+ transients that drive activity-dependent spine morphogenesis and long-term potentiation at Schaffer collateral-CA1 synapses. Consistent with these signaling roles, mice lacking Orai1 in pyramidal neurons (but not interneurons) exhibit striking deficits in working and associative memory tasks. These findings identify Orai1 channels as essential regulators of dendritic spine Ca2+ signaling, synaptic plasticity, and cognition.

Interspike intervals within retinal spike bursts combinatorially encode multiple stimulus features.

Neurons in various regions of the brain generate spike bursts. While the number of spikes within a burst has been shown to carry information, information coding by interspike intervals (ISIs) is less well understood. In particular, a burst with k spikes has k-1 intraburst ISIs, and these k-1 ISIs could theoretically encode k-1 independent values. In this study, we demonstrate that such combinatorial coding occurs for retinal bursts. By recording ganglion cell spikes from isolated salamander retinae, we found that intraburst ISIs encode oscillatory light sequences that are much faster than the light intensity modulation encoded by the number of spikes. When a burst has three spikes, the two intraburst ISIs combinatorially encode the amplitude and phase of the oscillatory sequence. Analysis of trial-to-trial variability suggested that intraburst ISIs are regulated by two independent mechanisms responding to orthogonal oscillatory components, one of which is common to bursts with a different number of spikes. Therefore, the retina encodes multiple stimulus features by exploiting all degrees of freedom of burst spike patterns, i.e., the spike number and multiple intraburst ISIs.

Two Types of Cl Transporters Contribute to the Regulation of Intracellular Cl Concentrations in ON- and OFF-type Bipolar Cells in the Mouse Retina.

In the retina, ON- and OFF-type bipolar cells are classified by subtype-specific center responses, which are attributed to differences in glutamate receptor subtypes. However, the mechanisms by which ON- and OFF-type bipolar cells generate subtype-specific surround responses remain unclear. One hypothesis for surround responses is that intracellular Cl concentrations ([Cl-]i) are set at different levels to achieve opposite polarities for GABA responses in ON- and OFF-type bipolar cells. Although this hypothesis is supported by previous findings obtained from rod (ON-) type bipolar cells, there is currently no information on OFF-type bipolar cells. In the present study, we examined the distribution and function of the Cl transporters, the Na-K-Cl co-transporter (NKCC1) and K-Cl co-transporter (KCC2), in rod (ON-) and OFF-type bipolar cells using immunohistochemical, in situ hybridization, and electrophysiological methods. Rod (ON-) and OFF-type bipolar cells both expressed NKCC1 and KCC2. However, the functional contribution of NKCC1 and KCC2 to the regulation of [Cl-]i differed between rod (ON-) and OFF-type bipolar cells. Strong NKCC1 activity increased [Cl-]i in rod (ON-) type bipolar cells, while that of KCC2 decreased [Cl-]i in OFF-type bipolar cells. We also confirmed the presence of a [Cl-]i gradient between dendrites and axon terminals in rod (ON-type) bipolar cells. Thus, the subtype-specific control of [Cl-]i is achieved by the activity of NKCC1 relative to that of KCC2 and appears to influence the polarity of surround responses.

Auxiliary Proteins are the Predominant Determinants of Differential Efficacy of Clinical Candidates Acting as AMPA Receptor Positive Allosteric Modulators.

Positive allosteric modulators (PAMs) of AMPA receptors boost cognitive performance in preclinical and clinical studies. Their therapeutic window is narrow, however, and clinical application will likely only occur if greater discrimination in activity is achieved. Toward that end, we compared the modulatory activity of two PAMs recently considered as clinical candidates, LY451395 (mibampator) and PF-04958242/BIIB104, on recombinant and native AMPA receptors (AMPARs). We found that the principle molecular determinant that shaped modulatory activity of both PAMs on deactivation (recombinant) and decay (synaptic) of AMPARs was the auxiliary protein incorporated into the receptor complexes. AMPARs containing the stargazin/γ2 transmembrane AMPAR regulatory protein (TARP) were slowed to a >10-fold degree by both PAMs as compared with those incorporating γ8 TARP. Neither subunit composition nor flip/flop splice variation had substantive effect. Similarly, stargazin/γ2-containing mossy fiber EPSCs in cerebellar granule neurons were slowed to a ∼5-fold greater degree than EPSCs in hippocampal CA1 pyramidal cell neurons, which express the γ8 TARP. LY451395 exhibited greater efficacy than BIIB104 at both synapses. These studies provide insight into the receptor constituents that determine efficacy of sulfonamide PAMs. We conclude that compounds that discriminate between AMPARs complexed with distinct TARPs, and particularly those with lower stargazin/γ2 efficacy such as BIIB104, could act as viable procognitive therapeutics. SIGNIFICANCE STATEMENT: Positive allosteric modulators (PAMs) of AMPA receptors enhance cognitive function in a variety of preclinical models. A clearer understanding of the critical determinants of PAM activity could yield critical insight into pathways to maximize their therapeutic index. Here we show that auxiliary proteins for AMPARs play a major, but thus far underappreciated, role in shaping recombinant and neuronal AMPAR modulation by two clinical candidate PAMs. These data will inform both clinical outcomes as well as future rational development of new modulators.

Dietary Supplementation with Lysine and Threonine Modulates the Performance and Plasma Metabolites of Broiler Chicken.

Here, we investigated whether the optimal threonine (Thr) to lysine (Lys) ratio in high Lys diet improves the growth performance of modern broiler chickens at finisher period and determined the possible mechanism underlying improvement in the growth performance of chickens fed with high Lys or Lys + Thr diet using metabolome analyses. Eighteen 21-day-old chickens housed in individual cages were randomly divided into three groups of six chickens fed with different diets as follows: control diet, high Lys diet (150% Lys content of National Research Council requirement), and high Lys + Thr diet (0.68 of Thr/Lys in high Lys diet). Body weight gain (BWG) increased in chickens receiving high Lys diet as compared with those fed with the control diet (P<0.05); no significant difference was observed in BWG of chickens from high Lys + Thr and high Lys groups. Feed conversion ratio (FCR) was lower in chickens fed with high Lys or high Lys + Thr diet than in those on the control diet. Serotonin concentration increased in the plasma of chickens fed with high Lys diet as compared to those fed with other diets. A negative correlation was observed between plasma serotonin concentration and FCR. These results provide the first evidence on the use of high Lys in broiler diets to reduce FCR during finisher period, which may be associated with change in plasma serotonin concentration. These findings suggest that high Lys content in finisher diet, but not high Thr + Lys diet, may affect the peripheral serotonergic metabolism and improve FCR. Thus, plasma serotonin may serve as a biomarker of FCR in broilers.

Variation in the Phenotype of Photosensitive Cells Produced from Human Fibroblast Cell Lines.

BACKGROUND: Photoreceptors differentiated from somatic cells are a useful tool for transplantation and drug screening. We previously showed that photosensitive cells are differentiated from human fibroblasts by direct reprogramming. In induced pluripotent stem (iPS) cells or embryonic stem (ES) cells, the properties of differentiated cells differ among the source of cell lines. However, whether or not the properties of the photosensitive cells produced by direct reprogramming are controlled by the origin of the cell line remains unknown. METHODS: We compared the morphological and physiological properties of photosensitive cells induced by two fibroblast cell lines. RESULTS: The differentiated cells had larger somas and more primary processes than the non-infected cells in both cell lines. The degree of morphological change was statistically different between the two cell lines. In addition, physiological responses to light differed between the two cell lines. An outward current (photoreceptor-like response) was observed in both cell lines, while an inward current (intrinsically photosensitive retinal ganglion cell-like response) was observed only in one cell line under light stimulation. CONCLUSIONS: These results suggest that photosensitive cells produced from different cell lines by direct reprogramming might express different phenotypes.

Peripherally derived T regulatory and γδ T cells have opposing roles in the pathogenesis of intractable pediatric epilepsy.

The pathophysiology of drug-resistant pediatric epilepsy is unknown. Flow cytometric analysis of inflammatory leukocytes in resected brain tissues from 29 pediatric patients with genetic (focal cortical dysplasia) or acquired (encephalomalacia) epilepsy demonstrated significant brain infiltration of blood-borne inflammatory myeloid cells and memory CD4+ and CD8+ T cells. Significantly, proinflammatory (IL-17- and GM-CSF-producing) γδ T cells were concentrated in epileptogenic lesions, and their numbers positively correlated with disease severity. Conversely, numbers of regulatory T (T reg) cells inversely correlated with disease severity. Correspondingly, using the kainic acid model of status epilepticus, we show ameliorated seizure activity in both γδ T cell- and IL-17RA-deficient mice and in recipients of T reg cells, whereas T reg cell depletion heightened seizure severity. Moreover, both IL-17 and GM-CSF induced neuronal hyperexcitability in brain slice cultures. These studies support a major pathological role for peripherally derived innate and adaptive proinflammatory immune responses in the pathogenesis of intractable epilepsy and suggest testing of immunomodulatory therapies.

Excitatory Synaptic Input to Hilar Mossy Cells under Basal and Hyperexcitable Conditions.

Hilar mossy cells (HMCs) in the hippocampus receive glutamatergic input from dentate granule cells (DGCs) via mossy fibers (MFs) and back-projections from CA3 pyramidal neuron collateral axons. Many fundamental features of these excitatory synapses have not been characterized in detail despite their potential relevance to hippocampal cognitive processing and epilepsy-induced adaptations in circuit excitability. In this study, we compared pre- and postsynaptic parameters between MF and CA3 inputs to HMCs in young and adult mice of either sex and determined the relative contributions of the respective excitatory inputs during in vitro and in vivo models of hippocampal hyperexcitability. The two types of excitatory synapses both exhibited a modest degree of short-term plasticity, with MF inputs to HMCs exhibiting lower paired-pulse (PP) and frequency facilitation than was described previously for MF-CA3 pyramidal cell synapses. MF-HMC synapses exhibited unitary excitatory synaptic currents (EPSCs) of larger amplitude, contained postsynaptic kainate receptors, and had a lower NMDA/AMPA receptor ratio compared to CA3-HMC synapses. Pharmacological induction of hippocampal hyperexcitability in vitro transformed the abundant but relatively weak CA3-HMC connections to very large amplitude spontaneous bursts of compound EPSCs (cEPSCs) in young mice (∼P20) and, to a lesser degree, in adult mice (∼P70). CA3-HMC cEPSCs were also observed in slices prepared from mice with spontaneous seizures several weeks after intrahippocampal kainate injection. Strong excitation of HMCs during synchronous CA3 activity represents an avenue of significant excitatory network generation back to DGCs and might be important in generating epileptic networks.

Novel channel-mediated choline transport in cholinergic neurons of the mouse retina.

Choline uptake into the presynaptic terminal of cholinergic neurons is mediated by the high-affinity choline transporter and is essential for acetylcholine synthesis. In a previous study, we reported that P2X2 purinoceptors are selectively expressed in OFF-cholinergic amacrine cells of the mouse retina. Under specific conditions, P2X2 purinoceptors acquire permeability to large cations, such as N-methyl-d-glucamine, and therefore potentially could act as a noncanonical pathway for choline entry into neurons. We tested this hypothesis in OFF-cholinergic amacrine cells of the mouse retina. ATP-induced choline currents were observed in OFF-cholinergic amacrine cells, but not in ON-cholinergic amacrine cells, in mouse retinal slice preparations. High-affinity choline transporters are expressed at higher levels in ON-cholinergic amacrine cells than in OFF-cholinergic amacrine cells. In dissociated preparations of cholinergic amacrine cells, ATP-activated cation currents arose from permeation of extracellular choline. We also examined the pharmacological properties of choline currents. Pharmacologically, α,ß-methylene ATP did not produce a cation current, whereas ATPγS and benzoyl-benzoyl-ATP (BzATP) activated choline currents. However, the amplitude of the choline current activated by BzATP was very small. The choline current activated by ATP was strongly inhibited by pyridoxalphosphate-6-azophenyl-2',4'-sulfonic acid. Accordingly, P2X2 purinoceptors expressed in HEK-293T cells were permeable to choline and similarly functioned as a choline uptake pathway. Our physiological and pharmacological findings support the hypothesis that P2 purinoceptors, including P2X2 purinoceptors, function as a novel choline transport pathway and may provide a new regulatory mechanism for cholinergic signaling transmission at synapses in OFF-cholinergic amacrine cells of the mouse retina.NEW & NOTEWORTHY Choline transport across the membrane is exerted by both the high-affinity and low-affinity choline transporters. We found that choline can permeate P2 purinergic receptors, including P2X2 purinoceptors, in cholinergic neurons of the retina. Our findings show the presence of a novel choline transport pathway in cholinergic neurons. Our findings also indicate that the permeability of P2X2 purinergic receptors to choline observed in the heterologous expression system may have a physiological relevance in vivo.

Physiological contribution of P2X receptors in postreceptoral signal processing in the mouse retina.

ATP activates P2X receptors and acts as a neurotransmitter in the nervous system. We have previously reported that P2X receptors modulate the firing rate of retinal ganglion cells. Since many subtypes of P2X receptors are distributed in the mouse retina, it is likely that the modulatory effects of P2X receptor-mediated signaling can occur at multiple synaptic levels in the retina. In this study, we investigated whether P2X receptors expressed between the photoreceptor layer and the inner nuclear layer in the mouse retina were physiologically functional, by electroretinography (ERG). In the combined rod-cone ERG and the scotopic ERG, intravitreal injection of PPADS, an antagonist of P2X receptors, had no effects on the amplitude of the a-wave, but decreased the amplitude of the b-wave. In the photopic ERG, intravitreal injection of PPADS significantly decreased the amplitude of both the a-wave and the b-wave. In ex vivo recordings, a decrease in the b-wave amplitude was observed at 20µM PPADS, confirming that the inhibition of the b-wave by intravitreal injection of PPADS is due to the inhibition of P2X receptors. Our findings suggest that P2X receptor-mediated signaling has a physiological effect in both the rod and the cone pathways in postreceptoral processing.

In vitro transdifferentiation of human peripheral blood mononuclear cells to photoreceptor-like cells.

Direct reprogramming is a promising, simple and low-cost approach to generate target cells from somatic cells without using induced pluripotent stem cells. Recently, peripheral blood mononuclear cells (PBMCs) have attracted considerable attention as a somatic cell source for reprogramming. As a cell source, PBMCs have an advantage over dermal fibroblasts with respect to the ease of collecting tissues. Based on our studies involving generation of photosensitive photoreceptor cells from human iris cells and human dermal fibroblasts by transduction of photoreceptor-related transcription factors via retrovirus vectors, we transduced these transcription factors into PBMCs via Sendai virus vectors. We found that retinal disease-related genes were efficiently detected in CRX-transduced cells, most of which are crucial to photoreceptor functions. In functional studies, a light-induced inward current was detected in some CRX-transduced cells. Moreover, by modification of the culture conditions including additional transduction of RAX1 and NEUROD1, we found a greater variety of retinal disease-related genes than that observed in CRX-transduced PBMCs. These data suggest that CRX acts as a master control gene for reprogramming PBMCs into photoreceptor-like cells and that our induced photoreceptor-like cells might contribute to individualized drug screening and disease modeling of inherited retinal degeneration.

Crizotinib-Induced Abnormal Signal Processing in the Retina.

Molecular target therapy for cancer is characterized by unique adverse effects that are not usually observed with cytotoxic chemotherapy. For example, the anaplastic lymphoma kinase (ALK)-tyrosine kinase inhibitor crizotinib causes characteristic visual disturbances, whereas such effects are rare when another ALK-tyrosine kinase inhibitor, alectinib, is used. To elucidate the mechanism responsible for these visual disturbances, the responses to light exhibited by retinal ganglion cells treated with these agents were evaluated using a C57BL6 mouse ex vivo model. Both crizotinib and alectinib changed the firing rate of ON and OFF type retinal ganglion cells. However, the ratio of alectinib-affected cells (15.7%) was significantly lower than that of crizotinib-affected cells (38.6%). Furthermore, these drugs changed the response properties to light stimuli of retinal ganglion cells in some of the affected cells, i.e., OFF cells responded to both ON and OFF stimuli, etc. Finally, the expressions of ALK (a target receptor of both crizotinib and alectinib) and of MET and ROS1 (additional target receptors of crizotinib) were observed at the mRNA level in the retina. Our findings suggest that these drugs might target retinal ganglion cells and that the potency of the drug actions on the light responses of retinal ganglion cells might be responsible for the difference in the frequencies of visual disturbances observed between patients treated with crizotinib and those treated with alectinib. The present experimental system might be useful for screening new molecular target agents prior to their use in clinical trials.

ON-pathway-dominant glycinergic regulation of cholinergic amacrine cells in the mouse retina.

Direction selectivity in the retina has been studied as a model of dendritic computation of neural circuits. Starburst amacrine cells (SACs) have been examined as a model system of dendritic computation as they play a pivotal role in the formation of direction selectivity. Because the difference of anatomical location inside the retina made ON-SACs an easier target to record, the biophysical properties of ON-SACs have been used to predict those of OFF-SACs. In this study, we systematically compared the responses of ON- and OFF-SACs to the two principal neurotransmitters, glycine and glutamate. We found that responses to glycine were significantly larger in ON-SACs than in OFF-SACs. In contrast, ON- and OFF-SACs responded similarly to glutamate. The amplitude of glycine responses in ON-SACs increased after eye opening and the largest amplitude was observed at postnatal day 28. On the other hand, no increase in the amplitude of glycine responses in OFF-SACs was observed until postnatal day 28. Glycine-evoked currents were inhibited by the application of strychnine. Glutamate-evoked currents were mimicked by the application of AMPA or kainite, and responses to N-methyl-d-aspartate were observed in the absence of Mg(2+) block. Glutamate-evoked currents produced an increase in the frequency of GABAergic inhibitory postsynaptic currents. Our results suggest that signal processing in ON-SACs cannot be directly used to understand the properties of OFF-SACs. Therefore fully defining the physiological properties of OFF-SACs will be critical to understanding and modelling direction selectivity in the retina.

Derivation of human differential photoreceptor cells from adult human dermal fibroblasts by defined combinations of CRX, RAX, OTX2 and NEUROD.

Redirecting differentiation of somatic cells by over-expression of transcription factors is a promising approach for regenerative medicine, elucidation of pathogenesis and development of new therapies. We have previously defined a transcription factor combination, that is, CRX, RAX and NEUROD, that can generate photosensitive photoreceptor cells from human iris cells. Here, we show that human dermal fibroblasts are differentiated to photoreceptor cells by the same transcription factor combination as human iris cells. Transduction of a combination of the CRX, RAX and NEUROD genes up-regulated expression of the photoreceptor-specific genes, recoverin, blue opsin and PDE6C, in all three strains of human dermal fibroblasts that were tested. Additional OTX2 gene transduction increased up-regulation of the photoreceptor-specific genes blue opsin, recoverin, S-antigen, CNGB3 and PDE6C. Global gene expression data by microarray analysis further showed that photoreceptor-related functional genes were significantly increased in induced photoreceptor cells. Functional analysis, that is, patch-clamp recordings, clearly revealed that induced photoreceptor cells from fibroblasts responded to light. Both the NRL gene and the NR2E3 gene were endogenously up-regulated in induced photoreceptor cells, implying that exogenous CRX, RAX, OTX2 and NEUROD, but not NRL, are sufficient to generate rod photoreceptor cells.