Deletion of the transmembrane protein Prom1b in zebrafish disrupts outer-segment morphogenesis and causes photoreceptor degeneration.

Mutations in human prominin 1 (PROM1), encoding a transmembrane glycoprotein localized mainly to plasma membrane protrusions, have been reported to cause retinitis pigmentosa, macular degeneration, and cone-rod dystrophy. Although the structural role of PROM1 in outer-segment (OS) morphogenesis has been demonstrated in Prom1-knockout mouse, the mechanisms underlying these complex disease phenotypes remain unclear. Here, we utilized a zebrafish model to further investigate PROM1's role in the retina. The Prom1 orthologs in zebrafish include prom1a and prom1b, and our results showed that prom1b, rather than prom1a, plays an important role in zebrafish photoreceptors. Loss of prom1b disrupted OS morphogenesis, with rods and cones exhibiting differences in impairment: cones degenerated at an early age, whereas rods remained viable but with an abnormal OS, even at 9 months postfertilization. Immunofluorescence experiments with WT zebrafish revealed that Prph2, an ortholog of the human transmembrane protein peripherin 2 and also associated with OS formation, is localized to the edge of OS and is more highly expressed in the cone OS than in the rod OS. Moreover, we found that Prom1b deletion causes mislocalization of Prph2 and disrupts its oligomerization. We conclude that the variation in Prph2 levels between cones and rods was one of the reasons for the different PROM1 mutation-induced phenotypes of these retinal structures. These findings expand our understanding of the phenotypes caused by PROM1 mutations and provide critical insights into its function.

CERKL gene knockout disturbs photoreceptor outer segment phagocytosis and causes rod-cone dystrophy in zebrafish.

In humans, CERKL mutations cause widespread retinal degeneration: early dysfunction and loss of rod and cone photoreceptors in the outer retina and, progressively, death of cells in the inner retina. Despite intensive efforts, the function of CERKL remains obscure and studies in animal models have failed to clarify the disease mechanism of CERKL mutations. To address this gap in knowledge, we have generated a stable CERKL knockout zebrafish model by TALEN technology and a 7bp deletion in CERKL cDNA that caused the premature termination of CERKL. These CERKL-/- animals showed progressive degeneration of photoreceptor outer segments (OSs) and increased apoptosis of retinal cells, including those in the outer and inner retinal layers. Additionally, we confirmed by immunofluorescence and western-blot that rod degeneration in CERKL-/- zebrafish occurred earlier and was more significant than that in cone cells. Accumulation of shed OSs in the interphotoreceptor matrix was observed by transmission election microscopy (TEM). This suggested that CERKL may regulate the phagocytosis of OSs by the retinal pigment epithelium (RPE). We further found that the phagocytosis-associated protein MERTK was significantly reduced in CERKL-/- zebrafish. Additionally, in ARPE-19 cell lines, knockdown of CERKL also decreased the mRNA and protein level of MERTK, as well as the ox-POS phagocytosis. We conclude that CERKL deficiency in zebrafish may cause rod-cone dystrophy, but not cone-rod dystrophy, while interfering with the phagocytosis function of RPE associated with down-regulation of the expression of MERTK.

CERKL interacts with mitochondrial TRX2 and protects retinal cells from oxidative stress-induced apoptosis.

Mutations in the ceramide kinase-like gene (CERKL) are associated with severe retinal degeneration. However, the exact function of the encoded protein (CERKL) remains unknown. Here we show that CERKL interacts with mitochondrial thioredoxin 2 (TRX2) and maintains TRX2 in the reduced redox state. Overexpression of CERKL protects cells from apoptosis under oxidative stress, whereas suppressing CERKL renders cells more sensitive to oxidative stress. In zebrafish, CERKL protein prominently locates in the outer segment and inner segment of the photoreceptor of the retina. Knockdown of CERKL in the zebrafish leads to an increase of retinal cell death, including cone and rod photoreceptor degeneration. Signs of oxidative damage to macromolecules were also detected in CERKL deficient zebrafish retina. Our results show that CERKL interacts with TRX2 and plays a novel key role in the regulation of the TRX2 antioxidant pathway and, for the first time, provides an explanation of how mutations in CERKL may lead to retinal cell death.

Novel compound heterozygous mutations in MYO7A in a Chinese family with Usher syndrome type 1.

PURPOSE: To identify the disease-causing mutation(s) in a Chinese family with autosomal recessive Usher syndrome type 1 (USH1). METHODS: An ophthalmic examination and an audiometric test were conducted to ascertain the phenotype of two affected siblings. The microsatellite marker D11S937, which is close to the candidate gene MYO7A (USH1B locus), was selected for genotyping. From the DNA of the proband, all coding exons and exon-intron boundaries of MYO7A were sequenced to identify the disease-causing mutation(s). Restriction fragment length polymorphism (RFLP) analysis was performed to exclude the alternative conclusion that the mutations are non-pathogenic rare polymorphisms. RESULTS: Based on severe hearing impairment, unintelligible speech, and retinitis pigmentosa, a clinical diagnosis of Usher syndrome type 1 was made. The genotyping results did not exclude the USH1B locus, which suggested that the MYO7A gene was likely the gene associated with the disease-causing mutation(s) in the family. With direct DNA sequencing of MYO7A, two novel compound heterozygous mutations (c.3742G>A and c.6051+1G>A) of MYO7A were identified in the proband. DNA sequence analysis and RFLP analysis of other family members showed that the mutations cosegregated with the disease. Unaffected members, including the parents, uncle, and sister of the proband, carry only one of the two mutations. The mutations were not present in the controls (100 normal Chinese subjects=200 chromosomes) according to the RFLP analysis. CONCLUSIONS: In this study, we identified two novel mutations, c.3742G>A (p.E1248K) and c.6051+1G>A (donor splice site mutation in intron 44), of MYO7A in a Chinese non-consanguineous family with USH1. The mutations cosegregated with the disease and most likely cause the phenotype in the two affected siblings who carry these mutations compound heterozygously. Our finding expands the mutational spectrum of MYO7A.

Phenotypic stability of Pro347Leu rhodopsin transgenic pigs as indicated by photoreceptor cell degeneration.

Rhodopsin (Pro347Leu) transgenic pigs are recognized to be an excellent model for the human disease, retinitis pigmentosa. First published in 1997, the rhodopsin transgenic pigs have been maintained since that time at North Carolina State University by outcrossing hemizygous boars to unrelated sows. Nine generations of outcrossing have been completed. Since the genetic background of these pigs has undoubtedly changed, the question of the current phenotype of the transgenic pigs is relevant for their future use. Age-matched transgenic and non-transgenic eyes were submitted for histological analysis using hematoxylin and eosin staining. Even by 2 weeks of age, significant thinning of the outer nuclear layer of photoreceptors was observed. For ages 3 and 4 weeks, thinning was noted similar to that of 2 weeks of age. By 6 weeks of age outer nuclear layer thinning was greater than that of earlier age. At 11 weeks of age, most of the rods have degenerated leaving only a few layers of cones. In all, the phenotype, based on assessment of photoreceptor degeneration, is similar to that of the first description of the transgenic animals. As such the Pro347Leu rhodopsin transgenic pigs have exhibited phenotypic stability through generations of outcrossing and can be used confidently in future studies of the type of retinal degeneration seen with retinitis pigmentosa.

TLS interaction with NMDA R1 splice variant in retinal ganglion cell line RGC-5.

Translocated in liposarcoma (TLS or FUS) is a multifunctional protein component of the heterogenous ribonuclear complex involved in the splicing of pre-mRNA and the export of fully processed mRNA from the nucleus to the cytoplasm. As we determined that TLS was substantially expressed in the adult retina, we investigated the functions of TLS in a rat retinal ganglion cell (RGC) line RGC-5. TLS was found to be associated with N-methyl-d-aspartate (NMDA) receptor 1 (NR1) and myosinVa (MyoVa) in a calcium-dependent manner. We demonstrated that TLS-associated NR1 could be one of the NR1 alternative splice variants, NR1-4, which was predominantly expressed in RGC-5. The degree of colocalization between TLS and NR1 was significantly decreased by depolarization of RGC-5 cells, indicating that the depolarization-induced Ca(2+)-influx triggered a redistribution of NR1 from the TLS-protein complex. These results suggested that TLS might be involved in a calcium-dependent trafficking of specific NR1 splice variants in RGCs.

Knocking out lca5 in zebrafish causes cone-rod dystrophy due to impaired outer segment protein trafficking.

Leber congenital amaurosis (LCA) is the most serious form of inherited retinal dystrophy that leads to blindness or severe visual impairment within a few months after birth. Approximately 1-2% of the reported cases are caused by mutations in the LCA5 gene. This gene encodes a ciliary protein called LCA5 that is localized to the connecting cilium of photoreceptors. The retinal phenotypes caused by LCA5 mutations and the underlying pathological mechanisms are still not well understood. In this study, we knocked out the lca5 gene in zebrafish using CRISPR/Cas9 technology. An early onset visual defect is detected by the ERG in 7 dpf lca5-/- zebrafish. Histological analysis by HE staining and immunofluorescence reveal progressive degeneration of rod and cone photoreceptors, with a pattern that cones are more severely affected than rods. In addition, ultrastructural analysis by transmission electron microscopy shows disordered and broken membrane discs in rods' and cones' outer segments, respectively. In our lca5-/- zebrafish, the red-cone opsin and cone α-transducin are selectively mislocalized to the inner segment and synaptic terminal. Moreover, we found that Ift88, a key component of the intraflagellar transport complex, is retained in the outer segments. These data suggest that the intraflagellar transport complex-mediated outer segment protein trafficking might be impaired due to lca5 deletion, which finally leads to a type of retinal degeneration mimicking the phenotype of cone-rod dystrophy in human. Our work provides a novel animal model to study the physiological function of LCA5 and develop potential treatments of LCA.

Knockout of Nr2e3 prevents rod photoreceptor differentiation and leads to selective L-/M-cone photoreceptor degeneration in zebrafish.

Mutations in the photoreceptor cell-specific nuclear receptor gene Nr2e3 increased the number of S-cone photoreceptors in human and murine retinas and led to retinal degeneration that involved photoreceptor and non-photoreceptor cells. The mechanisms underlying these complex phenotypes remain unclear. In the hope of understanding the precise role of Nr2e3 in photoreceptor cell fate determination and differentiation, we generated a line of Nr2e3 knockout zebrafish using CRISPR technology. In these Nr2e3-null animals, rod precursors undergo terminal mitoses but fail to differentiate as rods. Rod-specific genes are not expressed and the outer segment (OS) fails to form. Formation and differentiation of cone photoreceptors is normal. Specifically, there is no increase in the number of UV-cone or S-cone photoreceptors. Laminated retinal structure is maintained. After normal development, L-/M-cones selectively degenerate, with progressive shortening of OS that starts at age 1 month. The amount of cone phototransduction proteins is concomitantly reduced, whereas UV- and S-cones have normal OS lengths even at age 10 months. In vitro studies show Nr2e3 synergizes with Crx and Nrl to enhance rhodopsin gene expression. Nr2e3 does not affect cone opsin expression. Our results extend the knowledge of Nr2e3's roles and have specific implications for the interpretation of the phenotypes observed in human and murine retinas. Furthermore, our model may offer new opportunities in finding treatments for enhanced S-cone syndrome (ESCS) and other retinal degenerative diseases.

Multifocal electroretinogram in rhodopsin P347L transgenic pigs.

PURPOSE: Neural ectopic rewiring in retinal degeneration such as retinitis pigmentosa (RP) may form functional synapses between cones and rod bipolar cells that cause atypical signal processing. In this study, the multifocal electroretinograms (mfERGs) of a large animal model of RP, the rhodopsin P347L transgenic (Tg) pig, were measured to examine the sources and nature of altered signal processing. METHODS: mfERG responses from a 6-week-old Tg pig were recorded before and after sequential application of tetrodotoxin (TTX), N-methyl-D-aspartate (NMDA), 2-amino-4-phosphonobutyric acid (APB), and cis-2,3-piperidinedicarboylic acid (PDA), to identify contributions to the retinal signal from inner retinal neurons, the ON-pathway, the OFF-pathway, and photoreceptors. The mfERG response contributions from different retinal components of in the Tg eyes were estimated and compared with control data from eyes of age-matched wild-type (WT) pigs. RESULTS: There was a prominent difference in the estimates of the inner retinal response and ON-bipolar cell pathway contribution between the Tg and WT mfERG responses. In particular, the early components of the inner retinal contribution were obviously altered in the Tg mfERG. The inner retinal components at approximately 24 and 40 ms appeared to be inverted. Differences in the estimates of OFF-bipolar cell pathway contributions were minimal. There was no change in cone cell responses in the Tg mfERG. CONCLUSIONS: In Tg retinas, ectopic synapses formed between cones and rod bipolar cells probably altered signal processing of the ON-bipolar cell pathway. In response to the altered visual signal input from the outer retina, signal processing in inner retinal neurons was also modified.

Porcine global flash multifocal electroretinogram: possible mechanisms for the glaucomatous changes in contrast response function.

PURPOSE: The aim of this study was to obtain a better understanding of the cellular contributions to the porcine global flash mfERG by using a pharmacologic dissection method, together with the method using variation of stimulus contrast which has been used to demonstrate mfERG changes in human glaucoma. METHODS: Global flash mfERGs with different stimulus-contrast settings (99%, 65%, 49% or 29%) were recorded from 14 eyes of ten 6-week-old Yorkshire pigs in control conditions and after suppression of inner retinal responses with inhalation of isoflurance (ISO), and injections of tetrodotoxin (TTX) and N-methyl-d-aspartic acid (NMDA). ON- and OFF-pathway responses were isolated by injection of 2-amino-4-phosphonobutyric acid (APB) and cis-2,3-piperidinedicarboylic acid (PDA). RESULTS: The porcine global flash mfERG consisted of an early direct component (DC) and a late induced component (IC). ISO and TTX removed inner retinal contributions to the IC; NMDA application further abolished the oscillatory wavelets in the DC and removed the residual IC waveform. The inner retina contributed regular oscillation-like wavelets (W1, W2 and W3) to the DC and shaped the IC. After removing the inner retinal contributions, the porcine global flash mfERG waveform becomes comparable to that obtained with conventional mfERG stimulation. The remaining waveform (smoothed DC) was mainly contributed by the ON- and OFF-bipolar cells as revealed after APB or PDA injection. Photoreceptors contributed a small signal to the leading edge of N1. The characteristic of contrast response function of DC was demonstrated to be contributed by the inner retinal oscillation-like wavelets. CONCLUSION: We believe that the DC of the porcine global flash mfERG is mainly composed of contributions from photoreceptors, and ON- and OFF-bipolar cells, where inner retinal activity partially shaped the DC with superimposed regular wavelets. However, the IC is dominated by inner retinal activity. The contrast response functions of DC consisted of both outer retinal response and oscillation-like wavelets of the inner retinal response. Both contain different characteristics during contrast modulation of the stimulus, where the changes of W2 of the inner retinal response seem independent of contrast modulation. The DC contrast response feature depends mainly on the relative contribution of inner retinal activities; the loss of inner retinal cells may alter the DC contrast response function, making it tend toward linearity.

Effect of cellular senescence on the P2Y-receptor mediated calcium response in trabecular meshwork cells.

PURPOSE: The objectives of this study were to evaluate the potential release of ATP that is mediated by mechanical stress on trabecular meshwork (TM) cells, to identify the specific P2Y receptors mediating the ATP response, and to determine whether cellular senescence might interfere with the P2Y receptor-mediated calcium response, thus contributing to the loss of physiologic TM function in aging and primary open angle glaucoma (POAG). METHODS: Experiments were conducted using primary cultures of porcine TM cells. Cyclic mechanical stretch (10% stretching/second) was generated using the Flexcell system. ATP release and ectoATPase activity induced by mechanical stress were measured using a luciferin/luciferase assay. Replicative senescence was induced by passing the cells 18 times at a 1:2 split ratio and confirmed by the presence of senescence-associated beta-galactosidase (sa-beta-gal) and autofluorescence. For calcium imaging, cells were plated on gelatin-coated coverslips, bathed in calcium Ringer's solution, and loaded with fluo-4 (5 microM) for 1 h. Agonists of P2Y1 (ADP) and P2Y2/P2Y4 (ATP, UTP) receptors at 10 microM or 100 microM concentrations were added to the bathing medium. Relative changes in cytosolic calcium concentration as a function of time were measured by fluorescent microscopy and reported as peak amplitudes of fluo-4 fluorescence normalized to baseline values (deltaF/Fo). RESULTS: Mechanical stress induced an increase in ATP release from TM cells (258%+/-23% at 15 min, 188%+/-11% at 30 min, and 900%+/-203% at 1 h; p<0.017, n=4) as well as an increase in ectoATPase activity present in the extracellular media during the first 15 min of stress (57%+/-15%, p=0.011, n=4). The P2Y receptor agonists listed above induced a concentration-dependent rise in intracellular calcium in the TM cells. The peak amplitude, deltaF/Fo, was 1.07+/-0.12 (n=3) for 10 microM ADP, 2.59+/-0.33 (n=6) for 100 microM ADP, 1.21+/-0.64 (n=12) for 10 microM UTP, 3.22+/-2.0 (n=12) for 100 microM UTP, 0.88+/-0.40 (n=9) for 10 microM ATP, and 1.37+/-0.61 (n=25) for 100 microM ATP. Cells at passage 18 showed significantly lower levels of intracellular calcium induced by ATP (36%), UTP (34%), and ADP (52%) compared to cells at passage 2, independent from any changes in P2Y receptor changes in expression. CONCLUSIONS: The ability to release ATP in response to mechanical stress and the presence of functional P2Y receptors in TM cells suggest a novel mechanism by which TM cells could sense and respond to changes in intraocular pressure (IOP). In addition, the decrease in P2Y receptor-mediated calcium responses observed in senescent TM cells suggests that the disregulation of calcium homeostasis in senescence may contribute to the alterations of the TM in aging and POAG.

Altered light responses of single rod photoreceptors in transgenic pigs expressing P347L or P347S rhodopsin.

PURPOSE: Numerous mutations of rhodopsin lead to rod cell death and ultimately to complete blindness, yet little is known about the alterations in the physiology of the light sensors containing the aberrant protein, the rod photoreceptors. METHODS: Suction pipettes were used to record the light responses from single rod photoreceptors isolated from the retinas of transgenic pigs of various ages and at progressive stages of retinal degeneration. RESULTS: We have observed changes in the photoresponse of transgenic porcine rods containing both wild type and mutant rhodospin. Our findings are consistent with the idea that substitutions at position proline 347 of rhodopsin interfere with the inactivation of R*. In addition the level of photoreceptor degeneration is positively correlated with an acceleration and desensitization of the photoresponse to dim flashes. CONCLUSIONS: It appears that the phototransduction cascade, even when initiated by wild type rhodopsin molecules is altered in a way that is progressive with the level of retinal degeneration. A model consistent with our observations introduces the idea of a binding site for the carboxy terminus of rhodopsin on rhodopsin kinase.

RGD peptide-assisted vitrectomy to facilitate induction of a posterior vitreous detachment: a new principle in pharmacological vitreolysis.

PURPOSE: To evaluate a new concept in pharmacological vitreolysis by studying the efficacy of intravitreal RGD peptide-assisted vitrectomy in facilitating the separation of the posterior cortical vitreous from the retinal surface in an animal model. METHODS: Eight rabbits (16 eyes) received an intravitreal injection of 1 or 5 mg of RGD peptide in one eye and either RGE peptide (inactive control) or phosphate buffered saline in the fellow eye. After 24 hours, a pars plana vitrectomy with low aspiration (< or =30 mmHg) was performed in an attempt to create a detachment of the posterior cortical vitreous. A masked observer performed pre- and postoperative indirect ophthalmoscopy and B-scan ultrasonography. Postoperative scanning electron microscopy evaluated the vitreoretinal surface in selected eyes. Two additional rabbits received intravitreal injections of RGD peptide in one eye (1 mg and 5 mg) and 1 mg of RGE peptide in the fellow eye to examine apoptosis of the retinal cells by TUNEL assay. RESULTS: Based on postoperative ultrasound findings, six of the eight rabbits had a greater degree of posterior vitreous detachment in the RGD eye compared to the fellow eye (p = 0.03). The total number and the average number of detached quadrants in the group of RGD peptide eyes was twenty-three and 2.85 respectively compared to seven and 0.85 for the control fellow eyes (p = 0.02). Scanning electron microscopy confirmed the presence of postoperative posterior vitreous detachment. There was no evidence of retinal cell apoptosis in RGD injected eyes. CONCLUSION: RGD peptide-assisted vitrectomy facilitated posterior vitreous detachment in rabbit eyes, suggesting that RGD-containing peptides may prove to be effective adjuncts in producing posterior vitreous separation during vitreous surgery.

A novel rhodopsin point mutation, proline-170-histidine, associated with sectoral retinitis pigmentosa.

Identification and classification of all retinitis pigmentosa (RP) causing mutations contribute to a better understanding of disease variants. In this report we describe a New Zealand family, of European heritage, affected by a sectoral type RP phenotype in association with a novel rhodopsin mutation (proline-170-histidine) in a highly conserved site.

Rhodopsin F45L Allele Does Not Cause Autosomal Dominant Retinitis Pigmentosa in a Large Caucasian Family.

PURPOSE: To ascertain the potential pathogenicity of a retinitis pigmentosa (RP)-causing RHO F45L allele in a family affected by congenital achromatopsia (ACHM). METHODS: Case series/observational study that included two patients with ACHM and 24 extended family members. Molecular genetic analysis was performed to identify RHO F45L carrier status in the family and a control population. An adaptive optics scanning light ophthalmoscope (AOSLO) was used to image the photoreceptor mosaic and assess rod and cone structure. Spectral domain optical coherence tomography (SD-OCT) was used to examine retinal lamination. Comprehensive clinical testing included acuity, color vision, and dilated fundus examination. Electroretinography was used to assess rod and cone function. RESULTS: Five carriers of the RHO F45L allele alone (24-80 years) and three carriers in combination with a heterozygous CNGA3 mutant allele (10-64 years) were all free of the classic symptoms and signs of RP. In heterozygous carriers of both mutations, SD-OCT showed normal retinal thickness and intact outer retinal layers; rod and cone densities were within normal limits on AOSLO. The phenotype in two individuals affected with ACHM and harboring the RHO F45L allele was indistinguishable from that previously reported for ACHM. CONCLUSIONS: The RHO F45L allele is not pathogenic in this large family; hence, the two ACHM patients would unlikely develop RP in the future. TRANSLATIONAL RELEVANCE: The combined approach of comprehensive molecular analysis of individual genomes and noninvasive cellular resolution retinal imaging enhances the current repertoire of clinical diagnostic tools, giving a substantial impetus to personalized medicine.

Identification of variants in CNGA3 as cause for achromatopsia by exome sequencing of a single patient.

OBJECTIVE: To report disease-causing mutations in the cyclic nucleotide-gated channel α 3 gene (CNGA3) identified by exome sequencing and bioinformatics filtering in a single patient. METHODS: The entire protein-coding sequence of a patient with a retinal disease was enriched by in-solution targeted capture and massively parallel sequenced at 50-fold coverage. The assembled sequence was compared with databases of normal genomic sequences to identify nonsynonymous variants, which were further filtered (1) with a prioritization of genes associated with retinal diseases, (2) according to the likelihood of variant damage to protein function, (3) following the predictions of a recessive model, and (4) against common polymorphisms observed in normal genomes. Clinical evaluation and segregation analysis of the mutant alleles in the patient's family were performed; mutations were excluded in healthy controls. RESULTS: Analysis yielded a molecular diagnosis of achromatopsia. Two compound heterozygous mutations were identified in CNGA3 of this patient, c.829C>T p.R277C and c.1580T>G p.L527R; they were not observed in the normal population and cosegregated with the phenotype of achromatopsia in the patient's family. CONCLUSION: These mutations are the cause of achromatopsia in this family. CLINICAL RELEVANCE: The key advantages of massively parallel sequencing over linkage mapping and cloning are highlighted by (1) the small sample size required for successful analysis and (2) the rapid and high-throughput manner in which the mutations are identified. This new tool will likely have major effects on the management and research of rare genetic eye diseases in the new era of personalized genomic medicine.

Pharmacologically defined components of the normal porcine multifocal ERG.

Multifocal electroretinograms (mfERG) from isoflurane anesthetized pigs were recorded and sequential application of TTX, NMDA, APB and PDA were used to identify contributions to the mfERG from inner retinal neurons, ON-pathway, OFF-pathway and photoreceptors. The cellular origins of the first-order kernel (K1) and the first slice of the second-order kernel (K2.1) porcine mfERG are contributed from both inner and outer retina. For the K1 waveform, the n1 involved responses of cone photoreceptors and OFF-bipolar cells. The leading edge of p1 is dominated by ON-bipolar cell depolarization. The rear edge of p1, n2 and p2 are dominated by ON-bipolar activities and shaped by the activities of OFF-bipolar cells and retinal cells with NMDAr and voltage-gated sodium channels other than ganglion cells. The p3 is mainly inner retinal activities. For the K2.1 waveform, the p1 and n1 are the summation of activities of ON-, OFF-bipolar cells and retinal cells rich in NMDAr and voltage-gated sodium channels other than ganglion cells. The p2 seems to be related to the ganglion cells. Better understanding of the cellular origins of the normal porcine mfERG will be useful for comparing and defining the functional changes that may occur in diseased retinas.