Photoreceptor deficits appear at eye opening in Rs1 mutant mouse models of X-linked retinoschisis.

X-linked retinoschisis (XLRS) is an early onset degenerative retinal disease characterized by cystic lesions in the middle layers of the retina. These structural changes are accompanied by a loss of visual acuity and decreased contrast sensitivity. XLRS is caused by mutations in the gene Rs1 which encodes the secreted protein Retinoschisin 1. Young Rs1-mutant mouse models develop key hallmarks of XLRS including intraretinal schisis and abnormal electroretinograms. The electroretinogram (ERG) comprises activity of multiple cellular generators, and it is not known how and when each of these is impacted in Rs1 mutant mice. Here we use an ex vivo ERG system and pharmacological blockade to determine how ERG components generated by photoreceptors, ON-bipolar, and Müller glial cells are impacted in Rs1 mutants and to determine the time course of these changes. We report that ERG abnormalities begin near eye-opening and that all ERG components are involved.

Broad locations of antigenic regions for anti-TRPM1 autoantibodies in paraneoplastic retinopathy with retinal ON bipolar cell dysfunction.

PURPOSE: Cancer-associated retinal ON bipolar cell dysfunction (CARBD), which includes melanoma-associated retinopathy (MAR), has been reported to be caused by autoantibodies against the molecules expressed in ON bipolar cells, including TRPM1. The purpose of this study was to determine the antigenic regions of the autoantibodies against TRPM1 in the sera of CARBD patients, in whom we previously detected anti-TRPM1 autoantibodies. METHODS: The antigenic regions against TRPM1 in the sera of eight CARBD patients were examined by Western blots using HEK293T cells transfected with the plasmids expressing FLAG-tagged TRPM1 fragments. The clinical course of these patients was also documented. RESULTS: The clinical course differed among the patients. The electroretinograms (ERGs) and symptoms were improved in three patients, deteriorated in one patient, remained unchanged for a long time in one patient, and were not followable in three patients. Seven of the eight sera possessed multiple antigenic regions: two sera contained at least four antigen recognition regions, and three sera had at least three regions. The antigen regions were spread over the entire TRPM1 protein: five sera in the N-terminal intracellular domain, six sera in the transmembrane-containing region, and six sera in the C-terminal intracellular domain. No significant relationship was observed between the location of the antigen epitope and the patients' clinical course. CONCLUSIONS: The antigenic regions of anti-TRPM1 autoantibodies in CARBD patients were present not only in the N-terminal intracellular domain, which was reported in an earlier report, but also in the transmembrane-containing region and in the C-terminal intracellular domain. In addition, the antigenic regions for TRPM1 were found to vary among the CARBD patients examined, and most of the sera had multiple antigenic regions.

Disassembly and rewiring of a mature converging excitatory circuit following injury.

Specificity and timing of synapse disassembly in the CNS are essential to learning how individual circuits react to neurodegeneration of the postsynaptic neuron. In sensory systems such as the mammalian retina, synaptic connections of second-order neurons are known to remodel and reconnect in the face of sensory cell loss. Here we analyzed whether degenerating third-order neurons can remodel their local presynaptic connectivity. We injured adult retinal ganglion cells by transiently elevating intraocular pressure. We show that loss of presynaptic structures occurs before postsynaptic density proteins and accounts for impaired transmission from presynaptic neurons, despite no evidence of presynaptic cell loss, axon terminal shrinkage, or reduced functional input. Loss of synapses is biased among converging presynaptic neuron types, with preferential loss of the major excitatory cone-driven partner and increased connectivity with rod-driven presynaptic partners, demonstrating that this adult neural circuit is capable of structural plasticity while undergoing neurodegeneration.

Arginyltransferase (Ate1) regulates the RGS7 protein level and the sensitivity of light-evoked ON-bipolar responses.

Regulator of G-protein signaling 7 (RGS7) is predominately present in the nervous system and is essential for neuronal signaling involving G-proteins. Prior studies in cultured cells showed that RGS7 is regulated via proteasomal degradation, however no protein is known to facilitate proteasomal degradation of RGS7 and it has not been shown whether this regulation affects G-protein signaling in neurons. Here we used a knockout mouse model with conditional deletion of arginyltransferase (Ate1) in the nervous system and found that in retinal ON bipolar cells, where RGS7 modulates a G-protein to signal light increments, deletion of Ate1 raised the level of RGS7. Electroretinographs revealed that lack of Ate1 leads to increased light-evoked response sensitivities of ON-bipolar cells, as well as their downstream neurons. In cultured mouse embryonic fibroblasts (MEF), RGS7 was rapidly degraded via proteasome pathway and this degradation was abolished in Ate1 knockout MEF. Our results indicate that Ate1 regulates RGS7 protein level by facilitating proteasomal degradation of RGS7 and thus affects G-protein signaling in neurons.

Impaired Ca2+ Sensitivity of a Novel GCAP1 Variant Causes Cone Dystrophy and Leads to Abnormal Synaptic Transmission Between Photoreceptors and Bipolar Cells.

Guanylate cyclase-activating protein 1 (GCAP1) is involved in the shutdown of the phototransduction cascade by regulating the enzymatic activity of retinal guanylate cyclase via a Ca2+/cGMP negative feedback. While the phototransduction-associated role of GCAP1 in the photoreceptor outer segment is widely established, its implication in synaptic transmission to downstream neurons remains to be clarified. Here, we present clinical and biochemical data on a novel isolate GCAP1 variant leading to a double amino acid substitution (p.N104K and p.G105R) and associated with cone dystrophy (COD) with an unusual phenotype. Severe alterations of the electroretinogram were observed under both scotopic and photopic conditions, with a negative pattern and abnormally attenuated b-wave component. The biochemical and biophysical analysis of the heterologously expressed N104K-G105R variant corroborated by molecular dynamics simulations highlighted a severely compromised Ca2+-sensitivity, accompanied by minor structural and stability alterations. Such differences reflected on the dysregulation of both guanylate cyclase isoforms (RetGC1 and RetGC2), resulting in the constitutive activation of both enzymes at physiological levels of Ca2+. As observed with other GCAP1-associated COD, perturbation of the homeostasis of Ca2+ and cGMP may lead to the toxic accumulation of second messengers, ultimately triggering cell death. However, the abnormal electroretinogram recorded in this patient also suggested that the dysregulation of the GCAP1-cyclase complex further propagates to the synaptic terminal, thereby altering the ON-pathway related to the b-wave generation. In conclusion, the pathological phenotype may rise from a combination of second messengers' accumulation and dysfunctional synaptic communication with bipolar cells, whose molecular mechanisms remain to be clarified.

Activation of Apoptosis in a ßB1-CTGF Transgenic Mouse Model.

To reveal the pathomechanisms of glaucoma, a common cause of blindness, suitable animal models are needed. As previously shown, retinal ganglion cell and optic nerve degeneration occur in ßB1-CTGF mice. Here, we aimed to determine possible apoptotic mechanisms and degeneration of different retinal cells. Hence, retinae were processed for immunohistology (n = 5-9/group) and quantitative real-time PCR analysis (n = 5-7/group) in 5- and 10-week-old ßB1-CTGF and wildtype controls. We noted significantly more cleaved caspase 3+ cells in ßB1-CTGF retinae at 5 (p = 0.005) and 10 weeks (p = 0.02), and a significant upregulation of Casp3 and Bax/Bcl2 mRNA levels (p < 0.05). Furthermore, more terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL+) cells were detected in transgenic mice at 5 (p = 0.03) and 10 weeks (p = 0.02). Neurofilament H staining (p = 0.01) as well as Nefh (p = 0.02) and Tubb3 (p = 0.009) mRNA levels were significantly decreased at 10 weeks. GABAergic synapse intensity was lower at 5 weeks, while no alterations were noted at 10 weeks. The glutamatergic synapse intensity was decreased at 5 (p = 0.007) and 10 weeks (p = 0.01). No changes were observed for bipolar cells, photoreceptors, and macroglia. We conclude that apoptotic processes and synapse loss precede neuronal death in this model. This slow progression rate makes the ßB1-CTGF mice a suitable model to study primary open-angle glaucoma.

Prion-induced photoreceptor degeneration begins with misfolded prion protein accumulation in cones at two distinct sites: cilia and ribbon synapses.

Accumulation of misfolded host proteins is central to neuropathogenesis of numerous human brain diseases including prion and prion-like diseases. Neurons of retina are also affected by these diseases. Previously, our group and others found that prion-induced retinal damage to photoreceptor cells in mice and humans resembled pathology of human retinitis pigmentosa caused by mutations in retinal proteins. Here, using confocal, epifluorescent and electron microscopy we followed deposition of disease-associated prion protein (PrPSc) and its association with damage to critical retinal structures following intracerebral prion inoculation. The earliest time and place of retinal PrPSc deposition was 67 days post-inoculation (dpi) on the inner segment (IS) of cone photoreceptors. At 104 and 118 dpi, PrPSc was associated with the base of cilia and swollen cone inner segments, suggesting ciliopathy as a pathogenic mechanism. By 118 dpi, PrPSc was deposited in both rods and cones which showed rootlet damage in the IS, and photoreceptor cell death was indicated by thinning of the outer nuclear layer. In the outer plexiform layer (OPL) in uninfected mice, normal host PrP (PrPC) was mainly associated with cone bipolar cell processes, but in infected mice, at 118 dpi, PrPSc was detected on cone and rod bipolar cell dendrites extending into ribbon synapses. Loss of ribbon synapses in cone pedicles and rod spherules in the OPL was observed to precede destruction of most rods and cones over the next 2-3 weeks. However, bipolar cells and horizontal cells were less damaged, indicating high selectivity among neurons for injury by prions. PrPSc deposition in cone and rod inner segments and on the bipolar cell processes participating in ribbon synapses appear to be critical early events leading to damage and death of photoreceptors after prion infection. These mechanisms may also occur in human retinitis pigmentosa and prion-like diseases, such as AD.

LIM Homeobox 4 (lhx4) regulates retinal neural differentiation and visual function in zebrafish.

LIM homeobox 4 (LHX4) is expressed in the photoreceptors (PRs) of the outer nuclear layer (ONL) and bipolar cells (BCs) of the inner nuclear layer (INL) in mouse and chicken retina. It regulates the subtype-specific development of rod BCs and cone BCs in the mouse retina. However, no report has been published on its expression and function in the zebrafish retina. In this study, we assessed the expression of Lhx4 using in situ hybridization (ISH) technique and explored its role in zebrafish (Danio rerio) retinal development via morpholino (MO) technology. We found that the expression of lhx4 in the zebrafish retina begins 48 h post-fertilization (hpf) and is continuously expressed in the ONL and INL. A zebrafish model constructed with lhx4 knockdown in the eyes through vivo-MO revealed that: lhx4 knockdown inhibits the differentiation of Parvalbumin+ amacrine cells (ACs) and Rhodopsin+ rod photoreceptors (RPs), enhances the expression of visual system homeobox 2 (vsx2); and damages the responses of zebrafish to light stimulus, without affecting the differentiation of OFF-BCs and rod BCs, and apoptosis in the retina. These findings reveal that lhx4 regulates neural differentiation in the retina and visual function during zebrafish embryonic development.

LIM-Homeodomain Transcription Factor LHX4 Is Required for the Differentiation of Retinal Rod Bipolar Cells and OFF-Cone Bipolar Subtypes.

Retinal bipolar cells (BCs) connect with photoreceptors and relay visual information to retinal ganglion cells (RGCs). Retina-specific deletion of Lhx4 in mice results in a visual defect resembling human congenital stationary night blindness. This visual dysfunction results from the absence of rod bipolar cells (RBCs) and the loss of selective rod-connecting cone bipolar cell (CBC) subtypes and AII amacrine cells (ACs). Inactivation of Lhx4 causes the apoptosis of BCs and cell fate switch from some BCs to ACs, whereas Lhx4 overexpression promotes BC genesis. Moreover, Lhx4 positively regulates Lhx3 expression to drive the fate choice of type 2 BCs over the GABAergic ACs. Lhx4 inactivation ablates Bhlhe23 expression, whereas overexpression of Bhlhe23 partially rescues RBC development in the absence of Lhx4. Thus, by acting upstream of Bhlhe23, Prdm8, Fezf2, Lhx3, and other BC genes, Lhx4, together with Isl1, could play essential roles in regulating the subtype-specific development of RBCs and CBCs.

Dose-dependent regulation of horizontal cell fate by Onecut family of transcription factors.

Genome duplication leads to an emergence of gene paralogs that are essentially free to undergo the process of neofunctionalization, subfunctionalization or degeneration (gene loss). Onecut1 (Oc1) and Onecut2 (Oc2) transcription factors, encoded by paralogous genes in mammals, are expressed in precursors of horizontal cells (HCs), retinal ganglion cells and cone photoreceptors. Previous studies have shown that ablation of either Oc1 or Oc2 gene in the mouse retina results in a decreased number of HCs, while simultaneous deletion of Oc1 and Oc2 leads to a complete loss of HCs. Here we study the genetic redundancy between Oc1 and Oc2 paralogs and focus on how the dose of Onecut transcription factors influences abundance of individual retinal cell types and overall retina physiology. Our data show that reducing the number of functional Oc alleles in the developing retina leads to a gradual decrease in the number of HCs, progressive thinning of the outer plexiform layer and diminished electrophysiology responses. Taken together, these observations indicate that in the context of HC population, the alleles of Oc1/Oc2 paralogous genes are mutually interchangeable, function additively to support proper retinal function and their molecular evolution does not follow one of the typical routes after gene duplication.

Rod bipolar cells dysfunction occurs before ganglion cells loss in excitotoxin-damaged mouse retina.

Progressive degeneration of retinal ganglion cells (RGCs) will cause a blinding disease. Most of the study is focusing on the RGCs itself. In this study, we demonstrate a decline of the presynaptic rod bipolar cells (RBCs) response precedes RGCs loss and a decrease of protein kinase Cα (PKCα) protein expression in RBCs dendrites, using whole-cell voltage-clamp, electroretinography (ERG) measurements, immunostaining and co-immunoprecipitation. We present evidence showing that N-methyl D-aspartate receptor subtype 2B (NR2B)/protein interacting with C kinase 1 (PICK1)-dependent degradation of PKCα protein in RBCs contributes to RBCs functional loss. Mechanistically, NR2B forms a complex with PKCα and PICK1 to promote the degradation of PKCα in a phosphorylation- and proteasome-dependent manner. Similar deficits in PKCα expression and response sensitivity were observed in acute ocular hypertension and optic never crush models. In conclusion, we find that three separate experimental models of neurodegeneration, often used to specifically target RGCs, disrupt RBCs function prior to the loss of RGCs. Our findings provide useful information for developing new diagnostic tools and treatments for retinal ganglion cells degeneration disease.

Mice deficient in the lysosomal enzyme palmitoyl-protein thioesterase 1 (PPT1) display a complex retinal phenotype.

Neuronal ceroid lipofuscinosis (NCL) type 1 (CLN1) is a neurodegenerative storage disorder caused by mutations in the gene encoding the lysosomal enzyme palmitoyl-protein thioesterase 1 (PPT1). CLN1 patients suffer from brain atrophy, mental and motor retardation, seizures, and retinal degeneration ultimately resulting in blindness. Here, we performed an in-depth analysis of the retinal phenotype of a PPT1-deficient mouse, an animal model of this condition. Reactive astrogliosis and microgliosis were evident in mutant retinas prior to the onset of retinal cell loss. Progressive accumulation of storage material, a pronounced dysregulation of various lysosomal proteins, and accumulation of sequestosome/p62-positive aggregates in the inner nuclear layer also preceded retinal degeneration. At advanced stages of the disease, the mutant retina was characterized by a significant loss of ganglion cells, rod and cone photoreceptor cells, and rod and cone bipolar cells. Results demonstrate that PPT1 dysfunction results in early-onset pathological alterations in the mutant retina, followed by a progressive degeneration of various retinal cell types at relatively late stages of the disease. Data will serve as a reference for future work aimed at developing therapeutic strategies for the treatment of retinal degeneration in CLN1 disease.

Progressive myoclonic epilepsy-associated gene Kctd7 regulates retinal neurovascular patterning and function.

Neuron function relies on and instructs the development and precise organization of neurovascular units that in turn support circuit activity. However, our understanding of the molecular cues that regulate this relationship remains sparse. Using a high-throughput screening pipeline, we recently identified several new regulators of vascular patterning. Among these was the potassium channel tetramerization domain-containing protein 7 (KCTD7). Mutations in KCTD7 are associated with progressive myoclonic epilepsy, but how KCTD7 regulates neural development and function remains poorly understood. To begin to identify such mechanisms, we focus on mouse retina, a tractable part of the central nervous system that contains precisely ordered neuron subtypes supported by a trilaminar vascular network. We find that deletion of Kctd7 induces defective patterning of the adult retina vascular network, resulting in increased branching, vessel length, and lacunarity. These alterations reflect early and specific defects in vessel development, as emergence of the superficial and deep vascular layers were delayed. These defects are likely due to a role for Kctd7 in inner retina neurons. Kctd7 is absent from vessels but present in neurons in the inner retina, and its deletion resulted in a corresponding increase in the number of bipolar cells in development and increased vessel branching in adults. These alterations were accompanied by retinal function deficits. Together, these data suggest that neuronal Kctd7 drives growth and patterning of the vasculature and that neurovascular interactions may participate in the pathogenesis of KCTD7-related human diseases.

Diminished apoptosis in hypoxic porcine retina explant cultures through hypothermia.

Simulation of hypoxic processes in vitro can be achieved through cobalt chloride (CoCl2), which induces strong neurodegeneration. Hypoxia plays an important role in the progression of several retinal diseases. Thus, we investigated whether hypoxia can be reduced by hypothermia. Porcine retinal explants were cultivated for four and eight days and hypoxia was mimicked by adding 300 µM CoCl2 from day one to day three. Hypothermia treatment (30 °C) was applied simultaneously. Retinal ganglion, bipolar and amacrine cells, as well as microglia were evaluated via immunohistological and western blot analysis. Furthermore, quantitative real-time PCR was performed to analyze cellular stress and apoptosis. In addition, the expression of specific marker for the previously described cell types were investigated. A reduction of ROS and stress markers HSP70, iNOS, HIF-1α was achieved via hypothermia. In accordance, an inhibition of apoptotic proteins (caspase 3, caspase 8) and the cell cycle arrest gene p21 was found in hypothermia treated retinae. Furthermore, neurons of the inner retina were protected by hypothermia. In this study, we demonstrate that hypothermia lowers hypoxic processes and cellular stress. Additionally, hypothermia inhibits apoptosis and protects neurons. Hence, this seems to be a promising treatment for retinal neurodegeneration.

Fully Integrated Light-Sensing Stimulator Design for Subretinal Implants.

This paper presents a fully integrated photodiode-based low-power and low-mismatch stimulator for a subretinal prosthesis. It is known that a subretinal prosthesis achieves 1600-pixel stimulators on a limited single-chip area that is implanted beneath the bipolar cell layer. However, the high-density pixels cause high power dissipation during stimulation and high fabrication costs because of special process technologies such as the complementary metal-oxide semiconductor CMOS image sensor process. In addition, the many residual charges arising from the high-density pixel stimulation have deleterious effects, such as tissue damage and electrode corrosion, on the retina tissue. In this work, we adopted a switched-capacitor current mirror technique for the single-pixel stimulator (SPStim) that enables low power consumption and low mismatch in the subretinal device. The customized P+/N-well photodiode used to sense the incident light in the SPStim also reduces the fabrication cost. The 64-pixel stimulators are fabricated in a standard 0.35-µm CMOS process along with a global digital controller, which occupies a chip area of 4.3 × 3.2 mm² and are ex-vivo demonstrated using a dissected pig eyeball. According to measured results, the SPStim accomplishes a maximum biphasic pulse amplitude of 143 µA, which dissipates an average power of 167 µW in a stimulation period of 5 ms, and an average mismatch of 1.12 % between the cathodic and anodic pulses.

CLINICAL COURSE OF PARANEOPLASTIC RETINOPATHY WITH ANTI-TRPM1 AUTOANTIBODY IN JAPANESE COHORT.

PURPOSE: To report the clinical course of eyes with paraneoplastic retinopathy caused by an autoantibody against transient receptor potential cation channel, subfamily M, member 1 (TRPM1). METHODS: Ten paraneoplastic retinopathy patients with retinal ON-bipolar cell dysfunction, including six melanoma-associated retinopathy, from eight institutions in Japan were evaluated for the presence of an anti-TRPM1 antibody. The results of ophthalmic examinations and the presence of anti-TRPM1 antibody were analyzed. RESULTS: Five patients were positive for the anti-TRPM1 antibody. These patients had similar clinical findings in both eyes at the time of diagnosis; relatively preserved best-corrected visual acuity, absence of fundus and optical coherence tomography abnormalities, and specific abnormalities of the electroretinography (ERG); and negative-type ERGs with bright stimulus flashes. One patient whose retinal ON-bipolar cells remained dysfunctional for the entire testing period, although the anti-TRPM1 antibody had disappeared. On the other hand, the ERGs recovered in 2 cases within 2 years after the onset. One case progressed to additional impairment of the photoreceptors with deterioration of ERGs. One case died and the clinical course was unavailable. CONCLUSION: Paraneoplastic retinopathy patients with retinal ON-bipolar cell dysfunction possess autoantibodies against TRPM1 at the onset of the disease process; however, the clinical course of these eyes can be different.

Tmem30a deficiency leads to retinal rod bipolar cell degeneration.

Phospholipids are asymmetrically distributed across the mammalian plasma membrane, with phosphatidylserine (PS) and phosphatidylethanolamine concentrated in the cytoplasmic leaflet of the membrane bilayer and phosphatidylcholine in the exoplasmic leaflet. This asymmetric distribution is dependent on a group of P4 ATPases called PS flippases. The proper transport and function of PS flippases require a ß-subunit transmembrane protein 30A (TMEM30A). Disruption of PS flippases leads to several human diseases. Tmem30a is essential for photoreceptor survival. However, the roles of Tmem30a in the retinal rod bipolar cells (RBC) remain elusive. To investigate the role of Tmem30a in the RBCs, we generated a RBC-specific Tmem30a knockout (cKO) mouse model using PCP2-Cre line. The Tmem30a cKO mice exhibited defect in RBC function and progressive RBC death. PKCα staining of retinal cryosections from cKO mice revealed a remarkable dendritic sprouting of rod bipolar cells during the early degenerative process. Immunostaining analysis of PSD95 and mGluT6 expression demonstrated that rod bipolar cells in Tmem30a cKO retinas exhibited aberrant dendritic sprouting as a result of impaired synaptic efficacy, which implied a crucial role for Tmem30a in synaptic transmission in the retina. In addition, loss of Tmem30a led to reactive gliosis with increased expression of glial fibrillary acidic protein and CD68. TUNEL staining suggested that apoptotic cell death occurred in the retinal inner nuclear layer (INL). Our data show that loss of Tmem30a in RBCs results in dendritic sprouting of rod bipolar cells, increased astrogliosis and RBC death. Taken together, our studies demonstrate an essential role for Tmem30a in the retinal bipolar cells. Cover Image for this issue: doi: 10.1111/jnc.14492.

LRRTM4-C538Y novel gene mutation is associated with hereditary macular degeneration with novel dysfunction of ON-type bipolar cells.

The macula is a unique structure in higher primates, where cone and rod photoreceptors show highest density in the fovea and the surrounding area, respectively. The hereditary macular dystrophies represent a heterozygous group of rare disorders characterized by central visual loss and atrophy of the macula and surrounding retina. Here we report an atypical absence of ON-type bipolar cell response in a Japanese patient with autosomal dominant macular dystrophy (adMD). To identify a causal genetic mutation for the adMD, we performed whole-exome sequencing (WES) on four affected and four-non affected members of the family for three generations, and identified a novel p.C538Y mutation in a post-synaptic gene, LRRTM4. WES analysis revealed seven rare genetic variations in patients. We further referred to our in-house WES data from 1360 families with inherited retinal diseases, and found that only p.C538Y mutation in LRRTM4 was associated with adMD-affected patients. Combinatorial filtration using public database of single-nucleotide polymorphism frequency and genotype-phenotype annotated database identified novel mutation in atypical adMD.

Delayed bipolar and ganglion cells neuroretinal processing in regular cannabis users: The retina as a relevant site to investigate brain synaptic transmission dysfunctions.

Cannabis use is widespread worldwide, but the impact of smoking cannabis regularly on brain synaptic transmission has only been partially elucidated. The retina is considered as an easy means of determining dysfunction in brain synaptic transmission. The endocannabinoid system is involved in regulating retinal synaptic transmission, which might also be affected by tobacco. Previous preliminary results have shown impairments in retinal ganglion cell response in cannabis users. Here, we test the extent to which earlier retinal levels-bipolar cells and photoreceptors-are affected in cannabis users, i.e. by the association of tobacco and cannabis. We recorded pattern (PERG) and flash (fERG) ERG in 53 regular cannabis users and 29 healthy controls. Amplitude and peak time of P50 and N95 (PERG) and of a- and b-waves (fERG) were evaluated. Cannabis users showed a significant increase in PERG N95 peak time and in fERG light-adapted 3.0 b-wave peak time, compared with controls (p = 0.0001 and p = 0.002, respectively; Mann-Whitney U test). No significant difference was found between the groups in terms of wave amplitude (p = 0.525 and p = 0.767 for the N95 and light-adapted 3.0 b-wave amplitude respectively; Mann-Whitney U test). The results demonstrated delayed ganglion and bipolar cell responses in cannabis users. These results reflect a delay in the transmission of visual information from the retina to the brain. This retinal dysfunction may be explained by an effect of cannabis use on retinal synaptic transmission. Main limitations of these results concern tobacco and alcohol use that differed between groups. The consequences of these anomalies on visual perception along with the molecular mechanisms underlying this retinal dysfunction should be explored in future human and animal studies.